key: cord- -uvfppirt authors: gornati, laura; zanoni, ivan; granucci, francesca title: dendritic cells in the cross hair for the generation of tailored vaccines date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: uvfppirt vaccines represent the discovery of utmost importance for global health, due to both prophylactic action to prevent infections and therapeutic intervention in neoplastic diseases. despite this, current vaccination strategies need to be refined to successfully generate robust protective antigen-specific memory immune responses. to address this issue, one possibility is to exploit the high efficiency of dendritic cells (dcs) as antigen-presenting cells for t cell priming. dcs functional plasticity allows shaping the outcome of immune responses to achieve the required type of immunity. therefore, the choice of adjuvants to guide and sustain dcs maturation, the design of multifaceted vehicles, and the choice of surface molecules to specifically target dcs represent the key issues currently explored in both preclinical and clinical settings. here, we review advances in dcs-based vaccination approaches, which exploit direct in vivo dcs targeting and activation options. we also discuss the recent findings for efficient antitumor dcs-based vaccinations and combination strategies to reduce the immune tolerance promoted by the tumor microenvironment. introduction vaccines represent one of the most effective copernican revolutions for humankind and world health. this innovative discovery by edward jenner in the late years of the xviii century allowed for control or complete eradication of infectious diseases as smallpox ( ) and rinderpest virus ( ) ( ) . this immunization strategy posed the bases for current remarkable therapeutic approaches against not only infections but also cancer. in evolutionary terms, pathogens have acquired the capability to circumvent the immune system with several evasion mechanisms, revised elsewhere ( ) , that prevent pathogen clearance and the establishment of immune memory. vaccines represent the unique tool we have to impede pathogen spread; therefore, the urgent need for efficient vaccines is as relevant as before. mycobacterium tuberculosis, which causes tuberculosis, is currently one of the most feared infectious agent due to its capability to evade the immune system, leading to death of more than one million of people per year. unbelievably, the only licensed vaccine against mycobacterium tuberculosis is bacillus calmette-guérin (bcg) conceived about years ago. nonetheless, bcg has displayed some degree of inefficacy in humans, thus raising the need for new tailored vaccination strategies that are currently under investigation ( ) . moreover, every year, new cases of human immunodeficiency virus (hiv) infections lead to the necessity of a vaccine to control and prevent the spread of the virus. up to now, vaccines against hiv have not passed phase ii clinical trials due to poor protection conferred, requiring revision of delivered antigens (ags) and strategy to improve t cell response ( ) . moreover, the recent outbreaks of ebola virus and zika virus infections clearly demonstrate that still nowadays more than few infectious diseases need to be overwhelmed, as reported by the world health organization. on the other hand, vaccines represent also a therapeutic tool against cancer. one of the hallmarks of cancer is the capability of tumor cells to evade immune-mediated destruction ( ) by promoting a tolerant milieu. therefore, the immune system has to be pushed to respond specifically and robustly against tumors cells. to address this purpose, it is becoming more and more evident that dendritic cells (dcs) stand out as a potent tool in our hands, being the mediators of cellular and humoral responses ( ) . dcs have been discovered in by r. steinman and z. cohn that divided phagocytic cells (discovered by e. metchnikoff in ) in macrophages and dcs on the basis of different effector functions: microbial scavenging activities for macrophages and antigen-presenting function for dcs ( , ) . since then, dcs have emerged as the most potent antigen-presenting cells capable of shaping adaptive responses both during infections and cancer. moreover, the broad spectrum of dcs activation makes them suitable for fine shifting of the type of response the context needs. taking advantage of new adjuvants, innovative ags-delivery carriers and targeting strategies, it is now feasible to optimize the activation and ag presentation processes by the specific dcs subset that is the most effective in the initiation of the adaptive response needed in a given context. here, we discuss the diverse phenotypical and functional properties of dcs subtypes that are exploited by recently developed vaccine approaches, dealing with advances in the use of ags, adjuvants, carriers and dcs-expressed molecules, object of targeting. dendritic cells are the primary professional antigen-presenting cells (apcs) that reside in both lymphoid and non-lymphoid organs ( ) ( ) ( ) . dcs encompass several heterogeneous subsets whose subdivision relies on ontogeny, expression of surface-receptors, and transcription factors ( ) ( ) ( ) . much effort has been done in the identification and characterization of tissue-specific dc subsets to unravel the correlation between phenotype, localization, and functional properties, both in health and disease. initially, dcs have been classified into conventional dcs (cdcs) and plasmacytoid dcs (pdcs). briefly, cdcs prime naïve t cells and orchestrate ag-specific adaptive responses, while pdcs intervene during viral infections producing type i interferons (ifns). advanced approaches have extremely pushed our understanding of dc biology, resulting in a recent readapted taxonomy ( , , ) . indeed, villani and colleagues identify six subsets of dcs and monocytes in human (figure ): dc (clec a + cd + dcs), dc and dc (cd c + dcs), dc (fcgr a/cd + dcs), dc (axl + siglec + dcs) and dc (pdcs). dc represent the cross-presenting cd + /bdca + dcs while d and d correspond to cdcs displaying antigen uptake and processing capabilities. dc seem to be more prone to respond to viruses and are phenotypically close to monocytes. dc represent a newly defined subset that share features with both pdcs and cdcs, even though they appear to be functionally different from pdcs and more similar to cdcs. indeed, dc localize in t cell zone of tonsils, probably promoting fast adaptive immunity. due to this fine clustering, dc correspond to a more pure pdcs population ( ) . this precise classification opens the way for a more accurate view of dcs role in pathologies and provides cues for more specific targeting in immunotherapies. indeed, it is reasonable to assume that this extreme phenotypical diversity correlates with different intrinsic functional properties of dcs, as emerged in villani's work ( , , ) . in addition, environmental cues dictate dc activation and drive specific t cell responses ( , ) . indeed, dcs display a plethora of pattern recognition receptors (prr) that are specifically bound by microbe-or damage-associated molecular pattern (pamp and damp, respectively) ( ) . upon receptors engagement in peripheral tissues, the transduction signals lead to dc maturation with the upregulation of co-stimulatory molecules (referred to as "signal ") and the pivotal chemokine receptor ccr that allows dcs migration through afferent lymphatic vessels to the draining lymph node (ln) ( ) ( ) ( ) . in parallel, dcs mediate ag proteolysis to present intracellular peptides on major histocompatibility complex (mhc) class i to cd + t cells and exogenous peptides on mhc ii to cd + t cells (referred as "signal "). dcs can present exogenous ags on mhc class i through the so-called cross-presentation, allowing them to induce cd + cytotoxic t lymphocytes (ctls) against viruses and tumor cells. indeed, once in the ln, mature dcs encounter cognate naïve t cells and initiate adaptive responses ( ) . in the absence of maturation, as in steady-state conditions, the ag presentation and consequent migration to ln promote peripheral tolerance via t cell anergy or regulatory t cell formation ( ) ( ) ( ) . depending on the receptors engaged, dcs display different maturation states and produce different inflammatory mediators (often referred to as "signal ") that impact on the following cellular and humoral responses. the three signals released by dcs drive t helper (th) cell differentiation. briefly, dcs educate cd + t cells against intracellular bacteria by promoting their polarization into ifn-γ-producing th type (th ) cells. upon infection by multicellular parasites, dcs, with the help of basophils, polarize cd + t cells into th type (th ) cells that produce mainly il- . for specialized mucosal and skin immunity, dcs drive the activation of th type (th ) ( ) . thus, polarization of t cells is a crucial event that provides mechanisms specifically orchestrated to restore physiological homeostasis. dcs undergo apoptosis once they have fulfilled their functions. the rapid dc turnover after activation is necessary to avoid excessive t cell activation ( ) and to maintain self-tolerance ( , ) . t lymphocyte activation culminates with the establishment of the immunological memory, providing the host with t cells more prone and efficient in responding to a reinfection by the same pathogen or upon tumor relapses ( ) . besides, dcs are key players in humoral responses too. indeed, they directly interact with b cells and indirectly support them by activating cd + t cells, leading to humoral memory. all these notions strengthen the idea that dcs represent an optimal target for immunotherapies and vaccines, acting at the interface of innate and adaptive immunity. to harness robust responses through dc-targeting vaccinations, dc maturation is essential. adjuvants become compulsory complement of inactivated or subunit vaccines that may promote suboptimal responses. furthermore, they improve dc migration, ag availability, and specific targeting. although it seems clear that immunization could benefit from adjuvant uses, the solely adjuvant licensed in clinics, until recently, was alum ( ) . despite alum has been used in vaccination practice since the beginning of the last century, the mechanism through which it activates innate immunity for the subsequent activation of adaptive immune responses remains elusive. the adjuvant properties of alum were initially attributed to the activation of nlrp inflammasome ( , ) ; nevertheless, further studies have clearly shown the dispensability of nlrp and caspase- for the generation of responses in the presence of this adjuvant ( , ) . tlr signaling is also dispensable for alum adjuvanticity ( ) as well as mast cells, eosinophils, or macrophages ( ) . recently, it has been proposed that upon contact with alum, dcs produce il- through the activation of src and syk kinases, ca + mobilization, and nfat nuclear translocation. il- , in turn, is required for optimal t cell priming, activation, and antibody production ( ) . in addition to alum, other chemical adjuvants have been tested in preclinical models, showing a clear heterogeneity in the responses driven by different adjuvants, independently of the ag ( ) . this underlies the need of deepening our knowledge on these powerful tools to drive immune responses. indeed, mf , an oil-in-water emulsion adjuvant, that allows long-lasting ag retention in draining ln and enhanced ag uptake by ln-resident dcs, promotes robust humoral responses via follicular dc activation ( ) and cd + t cell immunity induction ( ) . conversely, ic adjuvant, which consists of an antibacterial peptide and a synthetic oligodeoxynucleotide (odn), elicits ifn-β release by human dcs via engagement of endosomal tlrs supporting immunity against intracellular pathogens and cancer ( ) . in the last decades, attention has been focused on tlr ligands as adjuvants. currently, several compounds are under investigation: pam csk , pam csk , or analogs as tlr / or tlr / ligands ( , ) , poly(i:c) and similar compounds acting on tlr ( , ) , tlr agonists ( ), flagellin acting on tlr ( ), imiquimod and other tlr ligands ( , ) , tlr agonists ( ), cpg odn binding tlr ( , ) . due to the possible reactogenicity that may be induced by administering tlr agonists, some compounds are chemically modified to reduce toxicity or are delivered specifically to the dc subsets of interest, avoiding tlr ligand dissemination. monophosphoryl lipid a, a low-toxicity molecule derived from lipopolysaccharide (lps), displays promising effects for vaccine design ( ) even though it promotes terminal differentiation of cd + cells, leading to reduced memory protection ( ) . another lps-analog is -acyl lipid a that has emerged as potent inducer of ifn-γ-mediated ag-specific responses when co-delivered with poorly immunogenic tumor ags ( ) . to improve the effectiveness and strength of immunity, in addition to the efficiency of apc, activation and ag processing and presentation of other aspects should be taken into account. the importance of dc-derived il- in the activation of adaptive responses has been shown not only in alum-driven immune responses and in mouse models of infections ( , ) but also in tests of human t cell priming in the presence of activatory dcs. during the first few hours after interaction with t cells, activatory monocyte-derived dcs (modcs stimulated with the cytokine cocktail, tnf-α, il- , il- β, and pge ) produce il- and cd ( ) . dc-derived il- is, in turn, trans-presented to t cells at the immunological synapse via cd . since naïve t cells start to express cd only many hours after ag encounter, the dc-mediated presentation of the il- /cd complex is indispensable for an efficient t cell priming ( ) . it has been proposed that this is the reason why approved therapies based on the use of anti-cd antibodies to avoid the acute phases of autoimmune diseases, or acute rejection of kidney, heart, and hand transplants, are so efficient in interfering with t cell priming or t cell reactivation ( ) . since il- is produced in nfatdependent manner to improve the adjuvanticity of prr agonists for vaccination purposes, the capacity of selected prr agonists to induce nfat signaling pathway activation and il- production should be considered. many prr ligands have been shown to activate the nfat transcription factor family members in innate immune cells ( ) . the nfat pathway is activated in neutrophils, macrophages, and dcs in response to curdlan ( , ) , it is also activated in dcs in response to lps ( ) downstream of cd , it is activated in response to mannose-capped lipoarabinomannan (man-lam), a major lipoglican of mycobacterium tuberculosis ( ) , and downstream of tlr in response to β-glucan bearing fungi ( ) . the production of il- by innate immune cells during inflammatory responses is relevant not only for an efficient t cell priming but also for the skewing of t cell activation toward type i responses. in mice, dc-derived il- is one of the cytokines required to elicit ifn-γ production from nk cells both in lpsmediated inflammatory conditions and during fungal infections ( ) ( ) ( ) . ifn-γ potently activates macrophages and favors th commitment of cd + t cells. therefore, early ifn-γ release by nk cells is not only crucial for controlling a variety of primary bacterial and fungal infections but also for the induction of type i immunity and memory, fundamental for the protection against bacterial, fungal, and viral infections and in antitumor immune therapies. another important reason for considering the capacity to activate the nfat pathway in adjuvant selection tests is represented by the fact that nfats regulate also the production of the prostanoid pge by activated dcs ( ) . pge promotes activated dc migration ( ) and sustains vasodilation and local edema formation during the inflammatory process. this is particularly relevant for vaccination purposes since the increase of the interstitial pressure generated by the edema forces the fluids into the afferent lymphatics and favors a first wave of antigen arrival to the draining ln ( ) . intriguingly, ln drainage of proteins or antigens occurs very rapidly after subcutaneous, intradermal, and intramuscular immunization ( ) ( ) ( ) , thus permitting an extremely fast uptake by phagocytes strategically localized in close proximity to the subcapsular sinus or lymphatic sinus of draining ln ( ) ( ) ( ) ( ) . antigen-presenting cells in ln then maintain the homeostasis of ln themselves and activate adaptive immune responses. in the last decades, the long-held paradigm of migratory dcs, resident in peripheral tissues as the skin, as unique apcs involved in t cell immunity has dramatically changed. indeed, cd + subcapsular sinus macrophages, medullary macrophages, and ln-resident dcs are ln sentinels that avoid excessive pathogen dissemination ( , ) and mediators of immune responses ( ) . concerning migratory dcs and considering the skin, which represents the site of utmost importance for vaccination strategies due to the ease accessibility and the extremely high presence of dcs, skin-resident dcs have been subdivided into epidermalresident langerhans cells (lcs), which are langerin + and two diverse subsets of dermal (d)dcs: langerin + cd + and langerin − cd − ( , ) . upon infection, ddcs migrate to the ln within - h while lcs within - h, supporting long-lasting ag-presentation. several works reveal the intrinsic differences between the two subsets in inducing th or ctl responses, due to the particular cross-presenting capabilities of cd + ddcs, for instance ( , , ) . once in the ln, whose strategical architecture enhances the probability of encounter between migratory dcs and cognate naïve t cell, adaptive immunity is initiated. of note, ln-resident dcs are sufficient to promote early adaptive responses independently of migratory dcs when pathogens or antigens directly access the lymphatic conduits ( , , ) . in antiviral responses, cd α + ln-resident dcs play a crucial role, thanks to their intrinsic capability of cross-presentation to cd + ctl ( , ) that may be supported by pdcs ( ) . in herpes simplex virus (hsv) skin infection, cd α + ln-resident dcs uptake cargo-antigens, ferried by skinresident migratory dcs in order to elicit ctl ( ) . indeed, lcs and ddcs synergize with cd α + ln-resident dcs, which stand out as the most potent ctl inducers, preferentially sustaining cd + th responses both in influenza ( ) and hsv cutaneous infections ( ) . in addition to cd α + ln-resident dcs, cd + ddcs display intrinsic capability of cross-presentation, as their human counterpart, clec a + cd + dcs ( ) ( ) ( ) ( ) ( ) . besides, some authors demonstrated that blocking dc migration from the skin hinders cd + t cell activation in response to subcutaneous bacterial ( ) or soluble antigen challenge ( ) . ablating langerin + ddcs reduced t cell immunity strength, corroborating the notion that migratory dc complement ln-resident dc effects on adaptive responses ( ) ( ) ( ) . nonetheless, the roles of lcs in activating t cells are still uncertain, probably due to the controversial functional properties of this innate subset ( ) ( ) ( ) . despite this, the synergic effects of ln resident and migratory dcs seem to be undoubted ( , ) . indeed, allenspach and colleagues reported that ag presentation by ln-resident dcs few hours after the infection is required to entrap ag-specific t cells in the draining ln and to favor an optimal activation of t cells by migratory dcs that arrive at the ln many hours later ( ) . it emerges, therefore, that another aspect to be considered for the identification of efficacious adjuvants concerns the type of dc subset to be targeted and the consequential effects that adjuvants imprint on that subset. adjuvants play a pivotal role in determining tissue-resident dc mobility to draining ln and efficiency of t cell polarization. indeed, ddcs acquire mobility after subcutaneous injection of th -specific adjuvants as cpg and lps, but not with th -specific ones, as papain, or following contact sensitization with dibutyl phthalate and acetone. moreover, ddcs are sufficient to promote th and th responses, while lcs are only supportive of th ( ) . this evidence underscore that, in addition to the polarizing capabilities of adjuvants, also the targeted dc subset must be considered to elicit specific adaptive immunity. indeed, antonialli and colleagues reported differential immune responses when cd α + and cd α − dcs were targeted with the same ag and adjuvant, either cpg odn or flagellin ( ) . in addition, to enhance the efficacy of vaccination, the coincident delivery of ag and prr adjuvants to apcs plays a crucial role. encouraging evidence highlights the importance of conjugation of ag with prr adjuvants, since it improves ag uptake, humoral and cellular responses when compared to vaccination with ag codelivered with free tlr ligands ( ) . these findings strengthen the notion that adjuvants are formidable chiefs in shaping immune responses and must be selected for the outcomes they promote, in chemical association to the ag of interest. the traditional vaccination approaches consisted in the adminis tration of live or attenuated micro-organisms. up to now, several innovative strategies have emerged to address the need for efficient vaccines, especially against diseases that are critical to treat, as cancer and the infectious diseases already mentioned. the main purpose is to convey ag, adjuvant, and targeting-molecule in a unique compound to increase the efficacy of the ag-specific immune response. to address this issue, different approaches have been explored or are currently under investigation, as shown in figure . recombinant antibody (rab) represents a feasible option. this approach exploits the possibility to chemically fuse peptide ag, adjuvant, and targeting-molecule to ab to tailor dcs targeting ( ) ( ) ( ) . in addition to rab, single-chain fragments variable (scfv) revealed to be an appealing strategy due to their reduced size and enhanced infiltration into tissues, as in solid tumors ( ) . other approaches involve the use of nano-carries as vehicles. the most promising solution to target phagocytes is indeed the use of particulate materials ( , ) . nanoparticles (nps) are the best candidates as delivery system, since they can be manipulated to efficiently and predominantly target phagocytes. this is possible, thanks to the versatility of nps due to: (i) the large amounts of existing different nanomaterials; (ii) the possibility to adjust their size, morphology, and deformability with great precision; (iii) the possibility to load virtually any different type of drug molecules ( ) . viral vectors-based vaccines or virus-like particles rely on the intrinsic capability of viruses to infect cells and exploit their protein-encoding machinery, allowing expression in the cytosol of the engineered plasmid-genes, as ag, costimulatory molecules, cytokines, and adjuvants, providing the bases for strong ctl induction ( ) . on the other hand, naked dna can be directly injected or conjugated to nano-carriers to favor specific targeting. the easy designing of nano-carriers-based vaccines along with their multi-component loading feature improve targeting of specific subsets ( ) and shape immune responses ( , ) , favoring their application in several fields. in a cancer setting, nano-carriers allow to avoid killing of healthy cells, by delivering tumor ags or dna encoding these peptides to apcs, inducing specific antitumor responses. indeed, nps allow endocytosis and mhc presentation on both class i and ii ( ) eliciting broad adaptive immunity, even against cancer cells. rosalia and colleagues designed a polymer-based biodegradable poly(lactic-co-glycolic acid) plga nps loaded with ag, pam csk , and poly(i:c) and coated with an agonistic αcd -monoclonal ab (np-cd ). this multi-functional strategy resulted in efficient and selective delivery of nps to dcs in vivo upon s.c. injection and induced priming of cd + t cells against tumor associated ags, increasing tumor-bearing mice survival ( ) . plga nps carrying the poorly immunogenic melanoma-derived antigen tyrosinase-related protein along with -acyl lipid a, manage in breaking the immunotolerance acting against tumor-antigens. indeed, administration of the abovementioned nps resulted in antigen-specific cd + ctl responses, characterized by ifn-γ production and increase of pro-inflammatory cytokines in the tumor microenvironment (tme) ( ) . another nano-carrier-based approach relies on liposome, self-assembled vesicles composed by lipid bilayers with high functionalizing properties. besides, maji and colleagues reported that after uptake by dcs, cationic liposomes localize in endosomal compartments that allow ag presentation preferentially on mhc i but do not exclude mhc ii ag presentation ( ), suggesting a crucial role in antitumor or antiviral immunity supported by th responses. in addition to the use of nps, targeting dc-specific receptors has become an attractive strategy for vaccine development due to the enforced efficiency of immune responses when compared to generic-delivering approaches. here, we report the more characterized dcs receptors, currently under investigation in the scenario of tailored-vaccination, as shown in table . clec a or dngr is a c-type lectin receptor that mediates endocytosis, but not phagocytosis, with low ph endosomes promoting the drift toward cross-presentation. importantly, clec a binding of antigens induces antigen presentation on both mhc i (cross-presentation) and mhc ii. it is highly and specifically expressed on cd c + cd + xcr + cdcs and cd + cd − monocytes in human and in murine pdcs and xcr + cd a + ln resident but not cd + xcr + migrating dcs ( , ) . indeed, cd + xcr + dcs constitute the human counterpart of cd α + xcr + murine dcs ( ) . they share xcr , the receptor of xcl . xcl is released by activated t cells and the axis xcr -xcl is necessary for robust ctl responses ( ) . cd + xcr + dcs are the main cross-presenting dcs in human, thus they appear promising for ctl-mediated responses, in tumors and viral infections ( ) . this specific subset is characterized by the expression of tlr that may be exploited to fully activate clec a + xcr + dcs since antibody binding of clec a leads to its rapid internalization but not tlr-pathway activation, preventing pro-inflammatory cytokine production and full maturation of dcs ( ) . conversely, caminschi and li independently demonstrated the potentiality of targeting clec a that resulted in enhanced humoral immunity independently of trif-myd or tlr pathway, even in the absence of adjuvants ( , ) . targeting clec a induces enhanced cd + t cell proliferation in vivo, which supports b cell immunity, when compared to the targeting of another endocytic receptor, discussed later, dec- , independently of the use of adjuvants as cpg ( ) . some years later, different authors demonstrated that this strong humoral response is endorsed by the establishment of follicular t helper cells memory, even upon vaccination with glycoprotein d of hsv, both in mice and non-human primates ( , , ) . these promising results were confirmed also in a human in vitro setting, on cd + dcs ( ) . finally, the efficacy of targeting clec a has been evaluated in the delivery of poorly immunogenic virus-derived antigens. park and colleagues managed in conferring specific humoral response, protective upon reinfection ( ) . thus, exploiting the specific expression of this receptor on the most specialized dcs in cross-presentation in combination with tlr ligands, will enhance antiviral and anticancer responses ( ), combined with robust humoral immunity. dec- or cd is a kda endocytic receptor that has a cysteine-rich domain, a fibronectin type ii domain, and c-type lectin-like domains, as well as an internalization sequence in its cytoplasmic tail ( ) . thus, it mediates cross-presentation through clathrin-and dynamin-dependent receptor-mediated endocytosis. indeed, it is expressed by the most professional cross-presenting dcs, the cd α + dcs subtype, while cd α − dcs display very low level of this receptor. in addition, dec- is found on dermal/interstitial dcs and lcs ( ), thus guaranteeing ag delivery to both skin-resident and ln-resident professional apcs. in humans, dec- is shared among cdcs, monocytes, and b cells, while pdcs, granulocytes, nk cells, and t lymphocytes express low levels of this receptor ( ) . in addition, dec- regulates molecule recycling through late endosomes, promoting also mhc ii presentation to cd + t cells in lcs ( ) . steinman and nussenzweig have addressed this molecule to improve vaccine efficacy since ( ) . by taking advantage of anti-dec- rab conjugated to ova peptide, they demonstrated that s.c. injections of this compound lead to a strong ifn-γ and il- mediated immunity only when dcs activation was supported by αcd mab, otherwise, tolerance against the ova peptide occurs ( ) . indeed, diversely from prr agonists, antibody crosslinking the dec- does not induce dcs maturation ( ) . furthermore, few years later, the combined strategy of anti-dec- and αcd was reported to confer protection against melanoma and intranasal influenza infection ( ) . in a viral setting, anti-dec- rab chemically coupled with hiv p gag protein tested in vitro on blood cells derived by hiv-infected donors has revealed efficient expansion of ifn-γ-producing cd + t lymphocytes ( ) from all the different donors. this indicated that dcs and cd can lead to the generation of different peptides from a single protein. moreover, vaccines based on the filamentous bacteriophage fd presenting an αdec- scfv, efficiently induce dcs maturation via the activation of the tlr -myd pathway ( ) , without adjuvants and further elicit potent antitumor responses when compared to non-tailored ag delivery ( ) . intriguingly, dec- , orphan of a specific ligand, has been proven to be necessary for cpg uptake and eventual dc activation ( ) . cd is a molecule belonging to the tnf receptor family, expressed by several cell types and among these, dcs. it has emerged as a receptor for the human chaperone heat shock protein (hsp) that mediates the internalization of peptides bound to hsp itself ( ) . moreover, upon activation, t cell transiently expresses cd l allowing cross-linking of cd on dcs and completing their maturation. from these notions, cd appeared an interesting molecule to target for dc-based vaccination strategies. indeed, by engineering antibody chemical structure, schjetne and colleagues demonstrated the efficacy of cd engagement conferring protection against myeloma-and lymphoma-derived ags ( ) . moreover, through the co-administration of two dna-based vaccines encoding either cd and the foot-and-mouth disease-derived ags, the transient increase of endogenous αcd antibodies allows an efficacious dcs activation and an efficient development of ag-specific t cell immunity, if compared to the administration of dna encoding ags alone ( ) . further promising results have been obtained in a vaccine against cyclin-d that is overexpressed by mantle cell lymphoma (mcl). thanks to algorithm analysis, chen and colleagues identified three cyclin-d -derived peptides that efficiently bind to mhc class i of dcs, potentially overexpressed in all mcl patients. by generating a rab targeting cd , they efficiently delivered these tumor associated ags to dcs and mounted ifn-γ-specific t cell responses in patients-derived peripheral blood mononuclear cells ( ) . thus, cd represents a specific dc-targeting molecule that has been used in combination with other targeting approaches to support specific dcs activation, avoid tolerance, and induce robust t cell immunity ( , ) . when evaluating vaccination strategies for cancer patients, it is compulsory to take into account one of the hallmarks of cancer: avoiding immune destruction by promoting tolerance and disarming the immune system ( ) . the orchestration of antitumor responses involves multiple protagonists and mediators, among these, cytotoxic t cells and nk cells, whose activation is supported by dcs ( ) . furthermore, dcs-based vaccines has emerged as more efficient in promoting t cell immunity if compared to peptide-based vaccination approaches ( ) . thus, much effort has been made to improve strategies of dcs-based vaccination in neoplastic diseases, to ameliorate the prognosis or eradicate both primary tumor and metastases. up to now, two different approaches have been addressed: ex vivo generation of autologous pulsed dcs and direct in vivo targeting of dcs, as previously discussed. the former strategy provides a better control of the maturation and activation state of dcs and a specific load of the ag to the selected dcs subset. despite this, intense work is needed to generate this vaccine, since it is personalized for each patient and only few subsets of dcs are feasibly generated in vitro or collected ex vivo, limiting the access of ags to other more functionally driven subsets. diversely, the in vivo targeting methods allow the generation of large amount of vaccine in a one-step procedure, and the targeting of diverse dcs subsets in their natural environment. once the dcs-based vaccine is generated, the efficacy of antitumoral responses has to be evaluated. it is mainly related to (i) the capability to establish specific antitumor-associated ag (taa) immunity and (ii) the overcome of the tolerogenic status promoted by the tme. to select highly immunogenic ags, multiple solutions have been tested: whole tumor lysate or killed tumor cells, synthetic long peptides (slps), full length proteins, transfection or electroporation with dna or mrna coding for taa, transduction with lentiviral vectors and neoantigens. the availability of an elevated number of antigens through the incubation of dcs with whole tumor lysates or autologous tumor cells allows the presentation of multiple epitopes, loaded on both mhc class i or ii, which leads to th and cytotoxic responses. indeed, several clinical trials are currently evaluating the benefits obtained by using this approach (nct ; nct ; nct ). slps are - aa long peptides cross-presented by dcs ( ), currently under investigation in both preclinical and clinical setting. compared to short synthetic peptides, the use of slps lacks the necessity to know the patients' hla haplotype, thus permitting their full exploitation in a larger cohort of people. moreover, slps administration to dcs leads to an enhanced cd + t cells activation since, once engulfed, they rapidly escape from the endolysosome to follow the path of mhc class i presentation, fundamental in antitumor responses. indeed, slps and dcs-based vaccines are showing promising results in terms of safety and immunogenicity, in both preclinical and clinical settings ( ) . they have gained attention in the context of human papilloma virus cervical ( ) , ovarian ( ) , and colorectal cancer ( , ) , displaying immunogenic capacities, in terms of antibody production and cd + and cd + t cell activation, when delivered with adjuvants, as poly iclc, montanide-isa- (nct ), and ifnα. when comparing slps and full length proteins, it has emerged that dcs process slps better that full length protein, due to the slower processing route the latter display ( ) . concerning transfection or electroporation of dcs with mrna or dna encoding, not only for taa but also for costimulatory molecules and cytokines, to enforce adaptive immunity, has proven to be efficacious in inducing antitumor cd + and cd + t cells expansion, mediated by dcs targeting ( ) . a similar approach regards in vivo lentiviral transduction of dcs, which displays versatility for gene delivery and efficient transduction for non-dividing cells, as dcs. indeed, bryson et al. conceived a multifunctional vaccine composed by a modified lentivirus, whose glycoproteins can directly target dc-sign on dcs, loaded with breast cancer ags, alpha lactalbumin, and erb-b receptor tyrosine kinase . single injections of the compound provided tumor self-ags-specific cd + t cell immunity, reducing tumor growth ( ) . despite the improvements derived by these advanced strategies, in the last years, neoantigens are becoming more and more appealing ( ) . during tumor progression, cancer cells give rise to neoantigens, novel ags different from the self-tumor ags, derived by the tumor-specific mutations. therefore, prediction tools, rna mutanome, and deep-sequencing have allowed the identification of specific non-self-ags that are fundamental in strong t cell immunity ( ) ( ) ( ) . indeed, several clinical trials are currently investigating the potential of neoantigens (nct ; nct ; nct ; nct ; nct ). as emerged, different strategies of ags selection have been explored and, even though one strategy may result in a more enforced antitumor immunity if compared to another, still the issue of the tme negative influence on the immune system has to be faced. indeed, the tme actively suppresses the activation of the immune system. tumor cells secrete immunosuppressive cytokines, as vascular endothelial growth factor ( , ) , macrophage colony-stimulating factor ( ), transforming growth factor β (tgf-β) ( ) , and il- ( , ) . even though some of these cytokine display controversial roles, depending on the pathological context, they generally promote dcs tolerogenicity, by limiting their activation and increasing their expression of pro-tumor molecules, such as programmed cell death (pd- ) and indoleamine , -dioxygenase (ido). therefore, tolerogenic dcs lead to t cells anergy, tregs expansion, and th responses inhibition. phenotypical characterization of immune cells isolated from breast cancer patients, highlighted the functional alteration in dcs, t, and nk cells in promoting antitumor responses ( ) . furthermore, tumor cells retain dcs into the tme, preventing their migration to draining lns and promoting metastatization ( ) . to address this issue, some ex vivo generated dcs-based vaccines are directly administered intranodally, as for the cd c + dcs pulsed with hla-a . -restricted tumor peptides administered to patients with stage iv melanoma (nct ), which generated tumor-specific cd + t cells responses and further improvement of survival ( ) . to reduce the tolerogenic influence of the tme on dcs, the positive role of gm-csf in improving dcs survival and responsiveness is currently exploited in some clinical trials like a phase i/ii trial with a dc/tumor cell fusion vaccine administered in association with gm-csf to treat renal cancer (nct ). similarly, others are focusing their attention on fms-like tyrosine kinase -ligand (flt l), another crucial dcs growth factor, in combination with other compounds (nct ; nct ; nct ; nct ). flt l has, indeed, been shown to increase the efficacy of proteins-and rna-based vaccines, due to a maturation effect on dcs ( ) ( ) ( ) . additional efforts made to counteract the tolerogenic influence of the tme include the use of pd- and ido inhibitors. co-administration of anti-pd- molecules increases the efficacy of dcs-based vaccines, in terms of enforced intratumoral cd + t cell responses and trafficking of cd + memory t cells, as observed in a preclinical model of glioblastoma ( ) . in parallel, several clinical trials are aiming at evaluating the efficacy of dcs-based vaccines combined with anti-pd- agents (nct ; nct ; nct ). the other tolerogenic marker addressed in cancer immunotherapy and dcs-based vaccine is ido. indeed, silencing approaches to reduce the expression of ido in dcs for vaccination in preclinical models, have resulted in decreased t cell apoptosis, reduced numbers of tregs, decreased tumor size when compared to mice that had received ags-loaded dcs without ido silencing ( ) . ido inhibitors in dcs vaccination are currently being tested in phase ii clinical trials (nct ; nct ). all these approaches have explored different scenarios to evaluate the more efficient therapeutic combination that seems to move toward personalized vaccinations for cancer patients. in this review, we have underscored the crucial role of dcs in orchestrating immune responses and; therefore, the great interest in targeting these cells in novel vaccination strategies. we have reported examples of different approaches aimed at amplifying the efficiency of immunizations against cancer or infectious diseases. indeed, the urgent need of vaccines is as relevant as before because of newly emerging diseases with ineffective current therapies. deepen the mechanisms underlying these pathologies may provide cues on the more appropriate design of vaccines and by merging diverse tailoring strategies we could enforce the immune system. as a matter of fact, it is suggested to act on different fronts when designing new vaccines, since several factors must be considered: (i) targeting dc subsets specialized in initiating the desired cellular or humoral immunity/memory; (ii) adjuvants that strengthen and drive t and b cell responses; (iii) fine and optimized selection of the immunogenic ags to drive enforced responses; (iv) novel strategies to convey ags and adjuvants to dcs; (v) route of administration. starting from these notions, in the last decades, enormous efforts have been made to tailor vaccination strategies. new technologies as well as recent advances have allowed extreme flexibility in designing vaccines and shaping the following outcomes. nowadays, researchers do have smart tools to manipulate immune responses with prophylactic or therapeutic vaccinations. the abovementioned findings pave the way for possible therapeutic approaches, theoretically applicable to all pathological contexts. despite this encouraging evidence, several limitations or issues still have to be overcome. indeed, more than a few vaccines do not pass phases i of clinical trials either for toxicity issues and lack of immunogenicity in some individuals. what is missing? part of the answer to this question could sit on human genetics and population variability. syngeneic animal models are ideal settings in which the systems are pushed although they constitute a necessary and useful step preceding clinical trials. moreover, when translating vaccine testing from in vivo experiments on animals to ex vivo on human cells, often the opted choice are blood human cells, while in most of the cases vaccines will be administered in the skin, having a complete different dcs-based milieu ( ) . crucially, idoyaga and colleagues dissected the interindividual variability in skin-resident dcs, stressing the need of shedding light on the effects that genetics and environment imprint on dcs. it is compulsory to decode the complex scenario of human diversity to provide personalized therapies with increased efficacy. in the omics era, systems biology and computational modeling integrate huge data-sets to address the urgent need of information on the global behavior. indeed, genome-wide association studies have provided insights into human genetics variants associated to the immunogenicity of vaccines ( , ) . therefore, integration of "wet" evidence and "dry" notions may fasten the designing process and provide both efficient vaccine strategies and their predictive efficacy. all authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication. eradicating infectious disease: can we and should we? front immunol anti-immunology: evasion of the host immune system by bacterial and viral pathogens why don't we have an effective tuberculosis vaccine yet? conserved hiv epitopes for an effective hiv vaccine hallmarks of cancer: the next generation dendritic cells and the control of immunity identification of a novel cell type in peripheral lymphoid organs of mice. i. morphology, quantitation, tissue distribution identification of a novel cell type in peripheral lympoid organs of mice. ii. functional properties in vitro dendritic cells display subset and tissue-specific maturation dynamics over human life unsupervised high-dimensional analysis aligns dendritic cells across tissues and species lymphoid organ-resident dendritic cells exhibit unique transcriptional fingerprints based on subset and site single-cell rna-seq reveals new types of human blood dendritic cells, monocytes, and progenitors human lymphoid organ dendritic cell identity is predominantly dictated by ontogeny, not tissue microenvironment the dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting high-dimensional phenotypic mapping of human dendritic cells reveals interindividual variation and tissue specialization mapping the human dc lineage through the integration of high-dimensional techniques development and function of dendritic cell subsets characterization of resident and migratory dendritic cells in human lymph nodes candida albicans morphology and dendritic cell subsets determine t helper cell differentiation skin-resident murine dendritic cell subsets promote distinct and opposing antigen-specific t helper cell responses pattern recognition receptors and inflammation ccr and irf -dependent dendritic cells regulate lymphatic collecting vessel permeability lymph node homing of t cells and dendritic cells via afferent lymphatics afferent lymph-derived t cells and dcs use different chemokine receptor ccr -dependent routes for entry into the lymph node and intranodal migration distinct dendritic cell populations sequentially present antigen to cd t cells and stimulate different aspects of cell-mediated immunity migratory, and not lymphoid-resident, dendritic cells maintain peripheral self-tolerance and prevent autoimmunity via induction of itreg cells dendritic cells induce peripheral t cell unresponsiveness under steady state conditions in vivo tolerogenic dendritic cells th cells in mucosal immunity and tissue inflammation cd regulates the dendritic cell life cycle after lps exposure through nfat activation dendritic cell apoptosis in the maintenance of immune tolerance elimination of antigen-presenting cells and autoreactive t cells by fas contributes to prevention of autoimmunity most microbe-specific naïve cd + t cells produce memory cells during infection alum: an old dog with new tricks cutting edge: alum adjuvant stimulates inflammatory dendritic cells through activation of the nalp inflammasome cutting edge: inflammasome activation by alum and alum's adjuvant effect are mediated by nlrp the nlrp inflammasome is critical for aluminum hydroxide-mediated il- β secretion but dispensable for adjuvant activity dna released from dying host cells mediates aluminum adjuvant activity adjuvantenhanced antibody receptor signaling alum induces innate immune responses through macrophage and mast cell sensors, but these sensors are not required for alum to act as an adjuvant for specific immunity the syk-nfat-il- pathway in dendritic cells is required for optimal sterile immunity elicited by alum adjuvants different human vaccine adjuvants promote distinct antigen-independent immunological signatures tailored to different pathogens vaccine adjuvant mf promotes retention of unprocessed antigen in lymph node macrophage compartments and follicular dendritic cells vaccine adjuvant mf promotes the intranodal differentiation of antigen-loaded and activated monocyte-derived dendritic cells the two-component adjuvant ic ® boosts type i interferon production of human monocyte-derived dendritic cells via ligation of endosomal tlrs pegylation of a tlr -agonist-based vaccine delivery system improves anti gen trafficking and the magnitude of ensuing antibody and cd +t cell responses the tlr / ligand pam csk is a th polarizing adjuvant in leishmania major and brugia malayi murine vaccine models defined tlr -specific adjuvant that induces nk and ctl activation without significant cytokine production in vivo using tlr ligands as a combination adjuvant induces qualitative changes in t cell responses needed for antiviral protection in mice the science of vaccine adjuvants: advances in tlr ligand adjuvants functional properties of flagellin as a stimulator of innate immunity novel adjuvant alum-tlr significantly potentiates immune response to glycoconjugate vaccines vaccine composition formulated with a novel tlr -dependent adjuvant induces high and broad protection against staphylococcus aureus a formulated tlr / agonist is a flexible, highly potent and effective adjuvant for pandemic influenza vaccines a tlr agonist enhances the anti-tumor immunity of peptide and lipopeptide vaccines via different mechanisms a dual tlr agonist adjuvant enhances the immunogenicity and protective efficacy of the tuberculosis vaccine antigen id the toll-like receptor agonist monophosphoryl lipid a augments innate host resistance to systemic bacterial infection tlr ligands lipopolysaccharide and monophosphoryl lipid a differentially regulate effector and memory cd + t cell differentiation co-delivery of cancer-associated antigen and toll-like receptor ligand in plga nanoparticles induces potent cd +t cell-mediated anti-tumor immunity inducible il- production by dendritic cells revealed by global gene expression analysis regulation and dysregulation of innate immunity by nfat signaling downstream of pattern recognition receptors (prrs) a role for interleukin- trans-presentation in dendritic cell-mediated t cell activation in humans, as revealed by daclizumab therapy immunity and cancer: a transcription factor comes of age dectin- stimulation by candida albicans yeast or zymosan triggers nfat activation in macrophages and dendritic cells calcineurin regulates innate antifungal immunity in neutrophils dectin- is a direct receptor for mannose-capped lipoarabinomannan of mycobacteria phagocytosis-dependent activation of a tlr -btk-calcineurin-nfat pathway co-ordinates innate immunity to aspergillus fumigatus skin infections are eliminated by cooperation of the fibrinolytic and innate immune systems il- cis presentation is required for optimal nk cell activation in lipopolysaccharide-mediated inflammatory conditions a contribution of mouse dendritic cell-derived il- for nk cell activation cd and nfat mediate lipopolysaccharide-induced skin edema formation in mice prostaglandin e is generally required for human dendritic cell migration and exerts its effect via ep and ep receptors a spatially-organized multicellular innate immune response in lymph nodes limits systemic pathogen spread capture of influenza by medullary dendritic cells via sign-r is essential for humoral immunity in draining lymph nodes conduits mediate transport of low-molecular-weight antigen to lymph node follicles strategically localized dendritic cells promote rapid t cell responses to lymph-borne particulate antigens histo-cytometry: a method for highly multiplex quantitative tissue imaging analysis applied to dendritic cell subset microanatomy in lymph nodes subcapsular sinus macrophages prevent cns invasion on peripheral infection with a neurotropic virus the conduit system transports soluble antigens from the afferent lymph to resident dendritic cells in the t cell area of the lymph node lymph node macrophages restrict murine cytomegalovirus dissemination subcapsular sinus macrophages limit acute gammaherpesvirus dissemination tracking and quantification of dendritic cell migration and antigen trafficking between the skin and lymph nodes dynamics and function of langerhans cells in vivo: dermal dendritic cells colonize lymph node areasdistinct from slower migrating langerhans cells lymph-node resident cd a+ dendritic cells capture antigens from migratory malaria sporozoites and induce cd + t cell responses lymph node resident rather than skin-derived dendritic cells initiate specific t cell responses after leishmania major infection cross-presentation, dendritic cell subsets, and the generation of immunity to cellular antigens cross-presentation of cell-associated antigens by mouse splenic dendritic cell populations cd +t cells orchestrate pdc-xcr +dendritic cell spatial and functional cooperativity to optimize priming migratory dendritic cells transfer antigen to a lymph node-resident dendritic cell population for efficient ctl priming multiple dendritic cell populations activate cd + t cells after viral stimu lation cross-presentation of viral and self antigens by skin-derived cd + dendritic cells imaging of the cross-presenting dendritic cell subsets in the skin-draining lymph node resident cd + and migratory cd + dendritic cells control cd t cell immunity during acute influenza infection human tissues contain cd hicross-presenting dendritic cells with functional homology to mouse cd +nonlymphoid dendritic cells peripheral cd + dendritic cells form a unified subset developmentally related to cd α + conventional dendritic cells selective expression of the chemokine receptor xcr on cross-presenting dendritic cells determines cooperation with cd + t cells ccr -dependent recruitment of blood phagocytes is necessary for rapid cd t cell responses to local bacterial infection expression of the self-marker cd on dendritic cells governs their trafficking to secondary lymphoid organs tumor immunotherapy by epicutaneous immunization requires langerhans cells langerin expressing cells promote skin immune responses under defined conditions langerin + dermal dc, but not langerhans cells, are required for effective cd -mediated immune responses after skin scarification with vaccinia virus langerhans cells: not your average dendritic cell langerhans cells suppress contact hypersensitivity responses via cognate cd interaction and langerhans cell-derived il- the balance between immunity and tolerance: the role of langerhans cells robust anti-viral immunity requires multiple distinct t cell-dendritic cell interactions migratory and lymphoid-resident dendritic cells cooperate to efficiently prime naive cd t cells selective and site-specific mobilization of dermal dendritic cells and langerhans cells by th -and th -polarizing adjuvants cpg oligodeoxinucleotides and flagellin modulate the immune response to antigens targeted to cd α+and cd α-conventional dendritic cell subsets cd -targeted dendritic cell delivery of plga-nanoparticle vaccines induce potent anti-tumor responses intensified and protective cd + t cell immunity in mice with anti-dendritic cell hiv gag fusion antibody vaccine antigen targeting to dendritic cells elicits long-lived t cell help for antibody responses in vivo targeting of antigens to maturing dendritic cells via the dec- receptor improves t cell vaccination antibody constructs in cancer therapy: protein engineering strategies to improve exposure in solid tumors magic bullets" for targeting the immune system drug nanocarriers to treat autoimmunity and chronic inflammatory diseases nanoparticles and innate immunity: new perspectives on host defence overall survival analysis of a phase ii randomized controlled trial of a poxviral-based psa-targeted immunotherapy in metastatic castrationresistant prostate cancer a novel antigen delivery system induces strong humoral and ctl immune responses vaccine nanocarriers: coupling intracellular pathways and cellular biodistribution to control cd vs cd t cell responses synthetic vaccine nanoparticles target to lymph node triggering enhanced innate and adaptive antitumor immunity plga microspheres for improved antigen delivery to dendritic cells as cellular vaccines a lipid based antigen delivery system efficiently facilitates mhc class-i antigen presentation in dendritic cells to stimulate cd +t cells superior antigen cross-presentation and xcr expression define human cd c + cd + cells as homologues of mouse cd + dendritic cells clec a is a novel activation c-type lectin-like receptor expressed on bdca + dendritic cells and a subset of monocytes characterization of human dngr- + bdca + leukocytes as putative equivalents of mouse cd α + dendritic cells the role of xcr and its ligand xcl in antigen cross-presentation by murine and human dendritic cells the c-type lectin receptor clec a mediates antigen uptake and (cross-) presentation by human blood bdca + myeloid dendritic cells antibodies targeting clec a promote strong humoral immunity without adjuvant in mice and non-human primates the dendritic cell subtype restricted c-type lectin clec a is a target for vaccine enhancement targeting antigen to mouse dendritic cells via clec a induces potent cd t cell responses biased toward a follicular helper phenotype targeting antigen to clec a primes follicular th cell memory responses capable of robust recall evolution of b cell responses to clec a-targeted antigen targeting clec a delivers antigen to human cd + dc for cd + and cd +t cell recognition enhancing vaccine antibody responses by targeting clec a on dendritic cells tumor therapy in mice via antigen targeting to a novel, dc-restricted c-type lectin the receptor dec- expressed by dendritic cells and thymic epithelial cells is involved in antigen processing cd (dec- ): a recognition receptor for apoptotic and necrotic self expression of human dec- (cd ) multilectin receptor on leukocytes the dendritic cell receptor for endocytosis, dec- , can recycle and enhance antigen presentation via major histocompatibility complex class ii -positive lysosomal compartments a monoclonal antibody to the dec- endocytosis receptor on human dendritic cells efficient targeting of protein antigen to the dendritic cell receptor frontiers in immunology | www.frontiersin.org in the steady state leads to antigen presentation on major histocompatibility complex class i products and peripheral cd + t cell tolerance dec- mediates antigen uptake and presentation by both resting and activated human plasmacytoid dendritic cells dec- receptor on dendritic cells mediates presentation of hiv gag protein to cd + t cells in a spectrum of human mhc i haplotypes antigen delivery by filamentous bacteriophage fd displaying an anti-dec- single-chain variable fragment confers adjuvanticity by triggering a tlr -mediated immune response vaccination with filamentous bacteriophages targeting dec- induces dc maturation and potent anti-tumor t-cell responses in the absence of adjuvants dec- is a cell surface receptor for cpg oligonucleotides cd , an extracellular receptor for binding and uptake of hsp -peptide complexes delivery of antigen to cd induces protective immune responses against tumors cd -expressing plasmid induces anti-cd antibody and enhances immune responses to dna vaccination a novel vaccine for mantle cell lymphoma based on targeting cyclin d to dendritic cells via cd hematology & oncology prolonged contact with dendritic cells turns lymph node-resident nk cells into anti-tumor effectors peptide-pulsed dendritic cells have superior ability to induce immune-mediated tissue destruction compared to peptide with adjuvant superior induction of anti-tumor ctl immunity by extended peptide vaccines involves prolonged, dc-focused antigen presentation dendritic cells process synthetic long peptides better than whole protein, improving antigen presentation and t-cell activation vaccination against hpv- oncoproteins for vulvar intraepithelial neoplasia phase i trial of overlapping long peptides from a tumor self-antigen and poly-iclc shows rapid induction of integrated immune response in ovarian cancer patients addition of interferon-α to the p -slp v vaccine results in increased production of interferon-c in vaccinated colorectal cancer patients: a phase i/ii clinical trial induction of p -specific immunity by a p synthetic long peptide vaccine in patients t reated for metastatic colorectal cancer vaccination with mrna-electroporated dendritic cells induces robust tumor antigen-specific cd + and cd + t cells responses in stage iii and iv melanoma patients breast cancer vaccines delivered by dendritic cell-targeted lentivectors induce potent antitumor immune responses and protect mice from mammary tumor growth neoantigens in cancer immunotherapy personalized cancer vaccine effectively mobilizes antitumor t cell immunity in ovarian cancer personalized rna mutanome vaccines mobilize poly-specific therapeutic immunity against cancer an immunogenic personal neoantigen vaccine for patients with melanoma vascular endothelial growth factor impairs the functional ability of dendritic cells through id pathways vascular endothelial growth factor inhibits the function of human mature dendritic cells mediated by vegf receptor- high co-expression of il- and m-csf correlates with tumor progression and poor survival in lung cancers tumor-derived tgf-β reduces the efficacy of dendritic cell/tumor fusion vaccine serum il- predicts worse outcome in cancer patients: a meta-analysis il- : master switch from tumor-promoting in flammation to antitumor immunity immune cell dysfunctions in breast cancer patients detected through whole blood multi-parametric flow cytometry assay inhibition of dendritic cell migration by transforming growth factor-b increases tumor-draining lymph node meta stasis effective clinical responses in metastatic melanoma patients after vaccination with primary myeloid dendritic cells classical flt l-dependent dendritic cells control immunity to protein vaccine flt ligand enhances the cancer therapeutic potency of naked rna vaccines dendritic cell-specific delivery of flt l by coronavirus vectors secures induction of therapeutic antitumor immunity pd- blockade enhances the vaccination-induced immune response in glioma silencing ido in dendritic cells: a novel approach to enhance cancer immunotherapy in a murine breast cancer model searching for the human genetic factors standing in the way of universally effective vaccines global analyses of human immune variation reveal baseline predictors of postvaccination responses key: cord- -tp o fxx authors: oliveira, cláudia c.; van hall, thorbald title: alternative antigen processing for mhc class i: multiple roads lead to rome date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: tp o fxx the well described conventional antigen-processing pathway is accountable for most peptides that end up in mhc class i molecules at the cell surface. these peptides experienced liberation by the proteasome and transport by the peptide transporter tap. however, there are multiple roads that lead to rome, illustrated by the increasing number of alternative processing pathways that have been reported during last years. interestingly, tap-deficient individuals do not succumb to viral infections, suggesting that cd t cell immunity is sufficiently supported by alternative tap-independent processing pathways. to date, a diversity of viral and endogenous tap-independent peptides have been identified in the grooves of different mhc class i alleles. some of these peptides are not displayed by normal tap-positive cells and we therefore called them teipp, for “t-cell epitopes associated with impaired peptide processing.” teipps are hidden self-antigens, are derived from normal housekeeping proteins, and are processed via unconventional processing pathways. per definition, teipps are presented via tap-independent pathways, but recent data suggest that part of this repertoire still depend on proteasome and metalloprotease activity. an exception is the c-terminal peptide of the endoplasmic reticulum (er)-membrane-spanning ceramide synthase trh that is surprisingly liberated by the signal peptide peptidase (spp), the proteolytic enzyme involved in cleaving leader sequences. the intramembrane cleaving spp is thereby an important contributor of tap-independent peptides. its family members, like the alzheimer’s related presenilins, might contribute as well, according to our preliminary data. finally, alternative peptide routing is an emerging field and includes processes like the unfolded protein response, the er-associated degradation, and autophagy-associated vesicular pathways. these data convince us that there is a world to be discovered in the field of unconventional antigen processing. defective ribosomal product (drip) proteins generates small peptides. potentially, a multitude of proteolytic systems may generate antigenic peptides, but the proteasome is responsible for the liberation of majority of them. inhibition of proteasome activity strongly decreased the pool of mhc class i-binding peptides ( ) . proteasomal cleavage typically creates a peptide's c-terminus compatible with mhc class i binding, and peptides are typically extended at their n-terminus ( , ) . peptides generated in the cytosol are translocated into the endoplasmic reticulum (er) by the tap /tap peptide transporter, where they have access to the peptide loading complex (plc), which is located within the er. tap is a heterodimeric member of the atp-binding cassette (abc) family of transporters, and peptide binding induces atp hydrolysis and transport across the er membrane. once in contact with the plc, the er-amino peptidase eraap (also known as erap ) trims n-extended peptides to a length appropriate for mhc class i binding ( ) ( ) ( ) . chaperone tapasin promotes the formation of stable mhc class i/peptide complexes and acts as an editor. additionally, calnexin facilitates the early folding of mhc class i heavy chains, whereas calreticulin and erp are involved in peptide loading ( ) ( ) ( ) . this pathway is also known as the proteasome-tap pathway and is considered as the conventional processing route because it is the mainstream pathway operating in cells under normal conditions ( ) ( ) ( ) . however, cells are equipped with alternative routes leading to liberation and loading of peptides into mhc class i molecules. these routes are independent of one or more molecules from the conventional pathway such as the proteasome, tapasin, or tap. this has become apparent from studies on cells with deficiencies in the conventional processing pathway. in this review, we will discuss what is known to date regarding alternative enzymes and routes to peptide loading compartments of endogenously generated peptides that feed the direct mhc class i pathway, especially important in cases of failure of the conventional route. we have not included interesting literature on cross-presentation pathways for mhc class i peptides. more than years ago, several papers made the important discovery that a large oligopeptidase, called tripeptidyl peptidase ii (tppii), participates in endoproteolytic activity in the cytosol and partially compensates for a deficient proteasomal activity ( ) ( ) ( ) ( ) . increased tppii activity even allowed for cell survival in lethal conditions of proteasome inhibition ( , ) . in these conditions, tppii activity also partially restored peptide presentation in mhc class i molecules and it was speculated that it could account for the generation of some epitopes independently or in cooperation with the proteasome ( ) . in fact, a paper from seifert et al. showed that tppii was involved in the generation of an epitope from the human immunodeficiency virus (hiv) protein negative factor (nef) ( ) . after that, an increasing number of proteolytic enzymes have been implicated in the generation of peptide-epitopes independently of the proteasome. insulindegrading enzyme (ide) generates an epitope from the human melanoma antigen mage-a ( ) . thimet oligopeptidase (top) and nardilysin are required for the generation of three other clinically relevant ctl epitopes: the tumor-antigen prame, an epitope from epstein-barr virus (ebv) protein ebna c, and an epitope from the melanoma protein mart- ( ) . these enzymes are part of an array of cytosolic endo-and exo-proteases that complement proteasomal activity and degrade proteasome products ultimately into amino acids. importantly, the process of peptide liberation from the protein context is inevitably coupled to the destruction pathway and all proteases mentioned above also destroy some antigenic peptides ( ) ( ) ( ) ( ) . peptides that are "rescued" from total destruction are transported by tap into the er and can potentially bind mhc class i molecules. in eukaryotic cells, secretory and membrane proteins contain a signal sequence essential for protein targeting to the er, the entrance for the secretory pathway ( , ) . these signal sequences are typically composed of three domains: a hydrophobic core (h region) of - amino acids, a polar c-terminal end (c region) with small uncharged amino acids, and a polar nterminal region (n region) with a positive net charge ( ). after insertion into the protein-conduction channel, signal peptides are usually cleaved from the preprotein by signal peptidase (sp) ( ). thereafter, signal peptides, which are small domains and trapped in the er membrane, can undergo intramembrane proteolysis by cleavage within their transmembrane region by the presenilintype aspartic protease signal peptide peptidase (spp) ( , ). peptide ligands suitable for mhc class i binding are thought to be generated after the intramembrane proteolysis by spp that promotes the release of signal peptide fragments from the er membrane ( , ). the spp-cleaved fragments in the vicinity of the cytosol can get access to the cytosol again and be further processed by the proteasome and transported by tap into the er. most hla class i molecules donate their leader sequences for binding to the non-classical hla-e, and the cleavage of these signal sequences is mediated by sp and spp ( - ). these leader peptides are even the most dominant source of peptides for hla-e. proper surface expression of hla-e prevents cytotoxic action of natural killer cells that continuously sense the presence of peptide/hla-e complexes through their cd /nkg receptors ( - ). the absence of these complexes at cell surface results in failure of interaction with cd /nkg a receptors, which activate nk cells for killing their targets. pioneering studies by peter cresswell and victor engelhard in revealed that most peptides presented at the surface of tap-deficient cells were derived from signal sequences, specific protein regions at the n-terminus of proteins ( , ). indeed, the parts of the leader peptide within the er membrane that are closest to the er lumen are released there and can get access to mhc class i grooves independent of proteasomes or tap (figure ) . after the cleavage of the transmembrane sequence by spp, the peptide fragments are released and can associate with mhc class i/β m nascent molecules. this intramembrane proteolysis by spp is thought to be important for the clearance of the er membrane by removing small protein remnants anchored at figure | classical and alternative pathways for mhc class i presentation. cells with deficiencies in components of the mhc class i processing pathway, such as tap, can present a repertoire of peptides derived from alternative processing pathways. different "housekeeping" cell functions such as signal peptide cleavage, protein maturation in the golgi, and protein/organelle disposal via autophagy can provide peptide ligands for mhc class i loading. the membrane rendering them susceptible for subsequent degradation. the intramembrane cleavage by spp is favored by spp topology that conceals the catalytic center within the plane of the membrane. the two aspartic residues required for the proteolytic activity of spp are located within conserved (y/f)d and g(l/i/f)gd amino acid motifs in two adjacent transmembrane domains ( , ). regarding a cleavage motif for spp, no consensus cleavage site has been described. however, spp demonstrates a strong preference for substrates with helix-destabilizing residues in their transmembrane domain ( - ). amino acids like asparagine, serine, and cysteine disturb a perfect alfa-helical conformation of the transmembrane domain and are therefore referred to as "helix-bending" or "helix-breaking" residues. signal peptides have been shown to contain amino acids with "helixbreaking" capacity within their h region, which critically influence their proteolytic processing by spp ( , ( ) ( ) ( ) . the disturbance of the α-helix caused by these residues is thought to facilitate intramembrane proteolysis. this and other issues would be clarified with the atomic resolution of this protease but this is still lacking due to its technical difficulties. we can have an approximation of that by looking at the crystal structure of presenilin/spp homologs recently published ( ) ( ) ( ) ( ) . jr is an spp homolog from the archaeon methanoculleus marisnigri that harbors nine transmembrane helices, similar to what is predicted for spp, with tmd and tmd containing the yd and gxgd motif, respectively. the two catalytic aspartate residues are located close to each other and approximately Å into the lipid membrane. proteolytic activity occurs in the presence of water molecules that gain access to the catalytic aspartates through a large cavity between two terminal domains ( ) . the three-dimensional structure of a human presenilin comprised into the γ-secretase complex has also been described ( ) . for the near future, we can expect more information on the catalytic activity of this family of proteases, including spp, which is definitely an important contributor of peptides for mhc class i presentation. our recent data revealed an additional role for intramembrane proteolysis by spp. regardless its name, spp also appeared to liberate a c-terminal peptide, independent of proteasome activity. the processing of c-terminal regions of a type ii protein inserted in the er membrane leads to the presentation of peptides independently of proteasome and tap ( , ) . the cterminal region from the ceramide synthase trh , which is a multiple membrane-spanning protein in the er, contains a mer peptide-epitope that is located at the very c-terminal end of the protein and protrudes into the lumen of the er ( - ). the trh protein has a housekeeping function and is ubiquitously expressed. inhibition of spp activity blocked the generation of the trh peptide. experiments with mutant forms of the trh protein indicated that the intramembrane cleavage by spp occurs at the direct upstream region of the t cell epitope within the lipid bilayer ( ) . we speculated that spp activity in the er membrane is sufficient to liberate the minimal c-terminal -mer peptide and release of this peptide into the er lumen. other proteolytic enzymes, such as amino-peptidases, were dispensable. further carboxy-terminal processing was not needed since the epitope is located at the very c-terminal end of the protein. direct release of the liberated peptide into the er lumen is very likely, due to the type ii transmembrane orientation of the trh protein tail (nterminus to c-terminus orientation). the exact peptide loading mechanism of the trh membrane peptide, however, remains to be determined. what triggers the cleavage of trh by spp is not known yet. in addition to its liberation function of small transmembrane substrates, spp has been shown to associate with misfolded membrane proteins in complexes where spp is represented as a monomer, dimer, or multimer ( ) . it was suggested that such high molecular weight complexes act as chaperones to dispose of membrane aggregates ( , ) . the role of spp in this degradation machinery might be to liberate such aggregates from the er membrane. these discoveries were based on the viral us and us proteins, which successfully labels mhc class i molecules in the er for retrograde transport to push this protein back to the cytosol for degradation by proteasomes ( , ) . interestingly, the spp family member, presenilin , seems to be associated with this membrane proteolysis as well ( ) . since spp and presenilin have opposing preference for type i and type ii transmembrane orientations, such a "degradome" machinery might be responsible to clear the er membranes. this type of machinery is called the erassociated degradation (erad) pathway. erad is an er quality control system, which monitors the integrity of nascent or erresident proteins and targets incorrectly folded or misassembled proteins to degradation. the disposal of misfolded mhc class i heavy chains also occurs through the erad pathway in the absence of viral interference ( ) . clearly, viruses take advantage of these existing pathways and hijack them in order to evade immune recognition by cytotoxic t cells ( , ) . another role for spp in erad has come from a recent paper describing the cleavage of the unfolded protein response (upr) regulator xbp u by spp ( ) . xbp u is a type ii membrane protein and undergoes intramembrane cleavage within a conserved type ii tm domain while integrated in a complex containing spp, the protease derlin- , and the e ligase trc , which prime spp for xbp u cleavage. an ectodomain shedding of spp substrates prior to spp cleavage is thought to be required and normally performed by sp in signal sequences but such cleavage was unnecessary in the case of xbp u, similarly to what we have described for trh ( , ) . in general, the upr induces a strong downregulation of mhc class i molecules at the cell surface ( , ) . thus, spp activity seems to have a direct impact on mhc class i peptide presentation by cleavage of leader sequences from nascent proteins and the liberation of some c-termini for mhc class i loading. a more indirect role that impacts on mhc class i presentation has been revealed and occurs through an erad pathway. this is also supported by a recent study using a systemslevel strategy reporting the involvement of spp in the network that coordinates the erad response ( ). the group of yewdell showed more than a decade ago that peptides located at the c-terminus of er-targeted proteins are very efficiently generated and presented on mhc class i ( ) ( ) ( ) ( ) . the location of the peptide was essential to be at the very end of the c-terminus of the protein, not requiring c-terminal trimming, in line with the fact that there is poor carboxypeptidase activity in the er ( ) . they described the presentation of tap-independent peptides from one er-resident protein, jaw , and proteins in the secretory pathway, like ovalbumin and cd . in each case, the peptides were efficiently liberated from the very c-terminus by the activity of yet unidentified endo-proteases to be generated as class i ligands. based on this pathway of peptide liberation, the authors provided the term "c-end rule" to highlight the capacity of er-resident proteases to liberate class i ligands from the cterminal ends of er-targeted proteins. the liberation of peptides without the intervention of the proteasome can also occur in the trans-golgi network ( , ) . the main proteolytic enzyme involved was shown to be furin, a known protease of the trans-golgi network normally required for the maturation of secreted proteins (e.g., growth factors and neurotransmitters) by cleaving at precise stretches of three to four basic residues ( ) . furin is part of a family of proprotein convertases that comprises nine members ( ) . three members (pc / , pace , and pc ) including furin are widely expressed and together they take part in a variety of processes occurring in the trans-golgi network, cell surface, or endosomes. this leads to the activation or inactivation of receptors, ligands, enzymes, viral glycoproteins, or growth factors ( ) . furin also has important functions during development by processing substrates like bone morphogenetic protein (bmp ), a member of the tgf-β superfamily that plays a critical role in heart development ( ) . furin processes a wide variety of precursor proteins after the c-terminal arginine residue in the preferred consensus motif -arg-x-arg/lys-arg↓-x-(x is any amino acid and "↓" indicates the cleavage position) ( ) . initially, this pathway was studied with the use of a model peptide at the cterminus of the secreted hepatitis hbe protein. furin-processed antigens targeted to the secretory route were presented by mhc class i at the cell surface and could elicit functional cd t-cell responses in vivo in a tap-independent fashion ( , ) . the ctermini of secretory or er-localized proteins thus appear to be processed for presentation to cd t cells (figure ). the generation of mhc class i ligands described above defines several alternative ways to generate peptide ligands without the intervention of the proteasome. these represent unusual pathways for peptide generation. now, we will discuss a different constraint in the conventional antigen presentation pathway related to the blockade of peptide entrance in the er due to tap deficiency. in human beings, tap-deficiency syndrome has been described in several independent families and results from mutations in either one of the subunits of the peptide transporter, tap and tap ( , ) . interestingly, these tap-deficient individuals do not succumb to viral infections, suggesting that cd t cell immunity is sufficiently supported by alternative tap-independent processing pathways. to date, a diversity of viral and endogenous tapindependent peptides have been identified in the grooves of different mch class i alleles. importantly, these tap-deficient patients harbor a polyclonal cd t-cell repertoire that is capable of recognizing peptides from the ebv virus, like protein lmp , presented on tap-deficient cells ( ) . the tap-independent processing pathway is capable of generating enough mhc class i/peptide complexes in order to keep immunosurveillance and control of viral infections. studies with tap -knockout mice have shown that surface expression levels of mhc class i are indeed lower, but the remaining complexes do induce a broad and polyclonal repertoire of cd t-cells ( ) ( ) ( ) . the tcr usage was shown to be very comparable to that of wild-type mice and tap -knockout mice were capable of mounting anti-viral cd t cell responses. together, these data show that, although crippled, the mhc class i-presented peptide repertoire in the absence of tap is sufficient to support cd t cell immunity. interestingly, peptides emerging from alternative tapindependent routes appeared to be immunogenic. following immunizations in mice with tap-deficient tumor cells, specific cd t-cells were induced that recognize tap-deficient cells, but not normal cells ( , ) . these t-cell epitopes seemed to be selectively presented by tap-deficient cells but not under normal conditions. this alternative peptide repertoire emerges due to processing defects and therefore these peptides were named "t cell epitopes associated with impaired peptide processing" (teipp). the molecular identification of some teipp peptides revealed that they can be diverse in length (from -mer to -mer), amino acid composition, and mhc class i binding, as some are presented by classical mhc class i molecules and others by the non-classical mhc molecule hla-e and the mouse homolog qa- b ( table ) ( , [ ] [ ] [ ] [ ] . they are derived from normal housekeeping proteins with ubiquitous expression, but are surprisingly not loaded on mhc class i in cells with an intact antigen-processing machinery. they constitute normal self-peptides (non-mutated, not pathogen-or tumor-specific) and can be regarded as real neo-antigens. the immunogenicity of teipp peptides exists because they are not presented by normal cells including the thymus. during thymic development, t cells are subjected to two subsequent processes called positive and negative selection ( , ) . negative selection is necessary for the maintenance of self-tolerance as it induces the deletion or inactivation of potentially autoreactive thymocytes ( ) . we recently demonstrated that teipp-specific cd t-cells indeed do not undergo negative selection and are thereby available for therapeutic exploitation. since the peptides recognized by teipp-specific ctl are derived from housekeeping proteins, we tried to understand why teipps are not presented by processing intact cells. collectively, our data show that teipp peptides are actually produced within processing proficient cells, but somehow are not or not sufficiently presented by their surface mhc class i molecules. taking the trh -derived teipp peptide as a model, we have analyzed the expression of the trh gene in several epithelial populations isolated from wild-type and tap -ko mice ( ) . this analysis revealed the same level of rna transcripts between the normal and knockout populations, suggesting comparable protein levels in both cell types. the liberation of the trh peptide is performed by spp, which is active in tap-positive as well as tap-negative targets ( ) . overexpression of the trh gene in tap-positive cells leads to surface presentation in mhc class i ( ), still in a proteasome-independent way. moreover, proteasome inhibition in tap-positive cells results in presentation of the endogenous trh peptide ( ) , indicating that, indeed, this teipp peptide is generated in all cells but loses competition with the overwhelming amount of tap-imported peptides in tappositive cells. some alternative peptides, like the ones derived from ebv proteins were shown to be presented on tap-positive cells to comparable extent, although using alternative pathways. moreover, our data suggest that the limited quantity of the trh peptide-epitope in the er is the main cause of selective presentation in tap-deficient cells. interestingly, gradual increase of overexpression correlated with the degree of recognition by the teipp-specific ctl clone, implying that tap transport actually constitutes a strong barrier for teipp peptides. the study of human teipp antigens corroborates these findings. one antigenic peptide is encoded by the human calca gene and derives from the signal sequence of preprocalcitonin (ppct) protein. this peptide is liberated in the er lumen by sequential cleavage with sp and spp, independently from proteasomes ( table ) ( ) . the presentation of the ppct peptide to specific ctl was found in human lung and medullary thyroid carcinomas that had very low expression of tap. presentation of the ppct peptide occurred also in normal non-transformed cells, such as dendritic cells (dcs), after knockdown of tap. overexpression of the calca gene in dcs and tap-positive tumor cells resulted in recognition by the specific ctl clone ( ) . identification of additional human teipp antigens at the molecular level will enable cd t cell targeting of otherwise ctl-resistance tap-negative tumor variants ( , ) . together, these findings support the model of peptide competition in the er as a factor that prevents presentation of peptides from alternative sources, and shape a picture of alternative processing pathways that emerge upon defects in the conventional one (figure ). the precise loading mechanism of tap-independent signal peptides into mhc class i molecules is not known, since processing by sp and spp is thought to take place outside of the plc. this sophisticated machinery for optimizing ligand length and quality and facilitating peptide loading onto nascent mhc class i molecules greatly facilitates peptide loading by physical bridging transporters to chaperones for loading and also "edits" the repertoire of bound peptides to maximize their affinity ( ) . the plc molecule tapasin tethers mhc class i molecules to the peptide transporter acting together with the chaperone calreticulin and the oxidoreductase erp ( ). tapasin can sense the quality of peptide bound by mhc class i complexes, and allows successive rounds of peptide binding until a certain affinity threshold is met. trimming of incoming peptides by eraap can be necessary for obtaining a good peptide length before selection by a defined mhc class i allele. a recent paper states that mhc class i molecules initially bind a variety of peptides, including some lowaffinity or n-terminally extended ones but then quickly dissociate from the molecule followed by the selection of the best fitting candidates ( ) . however, tap-independent leader peptides do not arrive in the er via tap and thus lack these editing and optimizing chaperones. in the absence of tap transporters, the loading of peptides into mhc class i occurs without a fully functional plc at hand. in that respect, inefficient loading of leader peptides in tap-positive cells can be expected, whereas in cells devoid of tap, this alternative er entrance mechanism allows emergence of these peptides. so here, the plc floats in the er membrane and is dispatched from the entry site of peptides. indeed, differential mass spectrometry analysis showed an enhanced presentation of leader peptides in cells lacking the peptide transporter ( ) . after all, mhc class i molecules get loaded with the available repertoire: from conventional or unconventional sources. but how do these "untapped" peptides find their way to empty mhc class i molecules at all? it has been shown that loading of peptides in mhc class i can occur with the minimal components of tapasin-erp -mhc class i complexes ( ), but we have to assume that the chances of a peptide to find the plc machinery in the er are scarce. on the other hand, tap-deficient cells harbor more peptide-receptive class i molecules compared to normal cells ( ) . these peptides might actually be actively chaperoned toward these open grooves. chaperones with high peptide binding capacity in the er are heat shock proteins (e.g., hsp ) and pdi, an isomerase that efficiently binds free peptide ( ) . it is possible that tap-independent peptides are captured by these molecules and chaperoned to mhc class i. recent studies suggest that the tapbpr molecule ("tapasin-like") binds those mhc class i proteins that are not bound to tapasin in the plc, so we might hypothesize that this pool of peptide-receptive grooves may function to load leader peptides ( ) . however, this still needs to be investigated. other ways to access peptide-receptive mhc has been proposed and are related to the intracellular traffic of hydrophobic peptides. studies with several epitopes from the lmp protein of the ebv showed that peptides, which possess a high hydrophobicity index, were presented in a tap-independent manner ( ) . the proposed mechanism describes the generation of these peptides outside the er, since proteasome activity is needed for presentation, and subsequently free diffusion across the er membrane possible due to their high hydrophobicity. these peptides might transgress membranes spontaneously or via alternative membrane transporters. a recent study by tey et al. showed that the processing of a peptide antigen from the human cytomegalovirus (hcmv) latency associated protein, pul , occurs entirely in the vesicular pathway and is mediated by autophagy ( ) . also other examples of autophagy enhanced mhc class i presentation of viral antigens were reported ( , ) . during autophagy, large portions of cytoplasmic content, including proteins and organelles, are encapsulated in double membrane vesicles called autophagosomes ( ) ( ) ( ) ( ) . the autophagosomal membrane has been proposed to originate from the er ( , ) and peptide-receptive mhc class i molecules might be present in autophagosomes, allowing for loading of peptides in these vesicular compartments ( ) . in addition, recirculating mhc class i molecules from the cell membrane end up in endosomes and can have contact with autophagosomes before returning to the endocytic network. transit for membrane-associated proteins between autophagosomes and endosomes has been observed by live cell imaging ( ) . moreover, peptides generated by the proteasome seem to get access to the endocytic vesicular pathway as well. we recently found at least one example for this in our teipp repertoire proportions and combinations. the mhc class i peptide repertoire can therefore be compared to a painter's palette where the different "colors" (peptides) are mixed and used to create a colorful and complex "picture." that is generated by the proteasome but is tap-independently presented ( table ) ( ) . the presentation of this peptide was surprisingly enhanced by blockade of the proton pump in endosomes, indicating that this antigen most likely crosses the vesicular pathway ( ) . finally, mass spectrometry analysis of the tapindependent peptide repertoire pointed at proteins located in the vesicular compartment as an important source ( ) ( ) ( ) . though these alternative processing and loading compartments have not fully been unraveled, these data strongly support the notion that the vesicular pathway and autophagy contributes to antigen presentation by mhc class i molecules. on summarizing, we can conclude that, even though the conventional proteasome-tap pathway represents the major source of the mhc class i peptide repertoire, alternative processing pathways clearly complement the total pool. this multitude of mhc class i processing pathways can be compared to a colorful palette where the painter combines the different colors that will end up in different proportions and combinations in the final picture (figure ) . in situations of viral infections, cellular transformation, or other initiators of stress, the contribution of these alternative pathways might gain importance. the spp and maybe their family members are convincing examples of alternative proteolytic systems that feed the alternative routes of antigen presentation. the precise molecular identification of alternative loading mechanisms unto peptide-receptive mhc class i molecules, whether it be in the er or in autophagosomes, still needs indepth investigation. in that respect, peptides can walk on multiple different paths before ending up in the grooves of mhc class i and therefore illustrate the old expression that multiple roads lead to rome. the protease inhibitor, n-acetyl--leucyl--leucyl-leucyl--norleucinal, decreases the pool of major histocompatibility complex class i-binding peptides and inhibits peptide trimming in the endoplasmic reticulum s proteasomes and immunoproteasomes produce mainly n-extended versions of an antigenic peptide the sizes of peptides generated from protein by mammalian and s proteasomes. implications for understanding the degradative mechanism and antigen presentation an ifngamma-induced aminopeptidase in the er, erap , trims precursors to mhc class i-presented peptides eraap customizes peptides for mhc class i molecules in the endoplasmic reticulum the er aminopeptidase erap enhances or limits antigen presentation by trimming epitopes to - residues mechanisms of mhc class i-restricted antigen processing and cross-presentation recent advances in antigen processing and presentation proteasome and peptidase function in mhc-class-i-mediated antigen presentation towards a systems understanding of mhc class i and mhc class ii antigen presentation proteases in mhc class i presentation and cross-presentation the known unknowns of antigen processing and presentation a giant protease with potential to substitute for some functions of the proteasome a proteolytic system that compensates for loss of proteasome function antigen processing by nardilysin and thimet oligopeptidase generates cytotoxic t cell epitopes integration of the ubiquitin-proteasome pathway with a cytosolic oligopeptidase activity an essential role for tripeptidyl peptidase in the generation of an mhc class i epitope production of an antigenic peptide by insulin-degrading enzyme a major role for tppii in trimming proteasomal degradation products for mhc class i antigen presentation pathway for degradation of peptides generated by proteasomes: a key role for thimet oligopeptidase and other metallopeptidases the efficiency of human cytomegalovirus pp ( - ) cd + t cell epitope generation is determined by the balanced activities of cytosolic and endoplasmic reticulum-resident peptidases the cytosolic endopeptidase, thimet oligopeptidase, destroys antigenic peptides and limits the extent of mhc class i antigen presentation the translocon: a dynamic gateway at the er membrane approaching the mechanism of protein transport across the er membrane requirements for signal peptide peptidasecatalyzed intramembrane proteolysis on the mechanism of spp-catalysed intramembrane proteolysis; conformational control of peptide bond hydrolysis in the plane of the membrane structural analysis of hepatitis c virus core-e signal peptide and requirements for cleavage of the genotype a signal sequence by signal peptide peptidase maturation of hepatitis c virus core protein by signal peptide peptidase is required for virus production signal peptide peptidase cleavage of gb virus b core protein is required for productive infection in vivo the crystal structure of gxgd membrane protease flak structure of a presenilin family intramembrane aspartate protease crystal structure of bacillus subtilis signal peptide peptidase a structure of signal peptide peptidase a with c-termini bound in the active sites: insights into specificity, self-processing, and regulation three-dimensional structure of human gamma-secretase new role of signal peptide peptidase to liberate c-terminal peptides for mhc class i presentation peptide transporter tap mediates between competing antigen sources generating distinct surface mhc class i peptide repertoires mammalian ceramide synthases two mammalian longevity assurance gene (lag ) family members, trh and trh , regulate dihydroceramide synthesis using different fatty acyl-coa donors selective cytotoxic t-lymphocyte targeting of tumor immune escape variants signal peptide peptidase (spp) assembles with substrates and misfolded membrane proteins into distinct oligomeric complexes signal peptide peptidase is required for dislocation from the endoplasmic reticulum a high-coverage shrna screen identifies tmem as an e ligase involved in er-associated protein degradation the human cytomegalovirus us gene product dislocates mhc class i heavy chains from the endoplasmic reticulum to the cytosol interactome analyses of mature gamma-secretase complexes reveal distinct molecular environments of presenilin (ps) paralogs and preferential binding of signal peptide peptidase to ps er quality control in the biogenesis of mhc class i molecules a membrane protein required for dislocation of misfolded proteins from the er the hcmv gene products us and us target mhc class i molecules for degradation in the cytosol signal peptide peptidase functions in erad to cleave the unfolded protein response regulator xbp u the unfolded protein response (upr)-activated transcription factor x-box-binding protein (xbp ) induces microrna- expression that targets the human antigen peptide transporter (tap ) mrna and governs immune regulatory genes stimulation of an unfolded protein response impairs mhc class i expression defining human erad networks through an integrative mapping strategy two novel routes of transporter associated with antigen processing (tap)-independent major histocompatibility complex class i antigen processing promiscuous liberation of mhc-class i-binding peptides from the c termini of membrane and soluble proteins in the secretory pathway trimming of antigenic peptides in an early secretory compartment tap-independent delivery of antigenic peptides to the endoplasmic reticulum: therapeutic potential and insights into tap-dependent antigen processing peptide diffusion, protection, and degradation in nuclear and cytoplasmic compartments before antigen presentation by mhc class i major histocompatibility complex class i viral antigen processing in the secretory pathway defined by the trans-golgi network protease furin furin-processed antigens targeted to the secretory route elicit functional tap -/-cd + t lymphocytes in vivo furin at the cutting edge: from protein traffic to embryogenesis and disease the multifaceted proprotein convertases: their unique, redundant, complementary, and opposite functions the activation and physiological functions of the proprotein convertases furin is the major processing enzyme of the cardiacspecific growth factor bone morphogenetic protein proteolytic activation of the spike protein at a novel rrrr/s motif is implicated in furin-dependent entry, syncytium formation, and infectivity of coronavirus infectious bronchitis virus in cultured cells postendoplasmic reticulum rescue of unstable mhc class i requires proprotein convertase pc description of hla class i-and cd -deficient patients: insights into the function of cytotoxic t lymphocytes and nk cells in host defense clinical and immunological aspects of hla class i deficiency human peptide transporter deficiency: importance of hla-b in the presentation of tap-independent ebv antigens positive selection of self-and alloreactive cd + t cells in tap- mutant mice tap -deficient mice select a cd + t cell repertoire that displays both diversity and peptide specificity tap mutant mice are deficient in antigen presentation, surface class i molecules, and cd - + t cells generation of cd + t cells specific for transporter associated with antigen processing deficient cells alternative peptide repertoire of hla-e reveals a binding motif that is strikingly similar to hla-a cd + t cell responses against tap-inhibited cells are readily detected in the human population the nonpolymorphic mhc qa- b mediates cd + t cell surveillance of antigen-processing defects nonclassical mhc class ib-restricted cytotoxic t cells monitor antigen processing in the endoplasmic reticulum antigen presentation in the thymus for positive selection and central tolerance induction selection of the t cell repertoire the thymic medulla: a unique microenvironment for intercellular self-antigen transfer preprocalcitonin signal peptide generates a cytotoxic t lymphocyte-defined tumor epitope processed by a proteasome-independent pathway different expression levels of the tap peptide transporter lead to recognition of different antigenic peptides by tumor-specific ctl strategies to counteract mhc-i defects in tumors a novel category of antigens enabling ctl immunity to tumor escape variants: cinderella antigens tapasin-the keystone of the loading complex optimizing peptide binding by mhc class i molecules in the endoplasmic reticulum the first step of peptide selection in antigen presentation by mhc class i molecules features of tap-independent mhc class i ligands revealed by quantitative mass spectrometry selective loading of high-affinity peptides onto major histocompatibility complex class i molecules by the tapasin-erp heterodimer peptide-receptive class i major histocompatibility complex molecules on tap-deficient and wild-type cells and their roles in the processing of exogenous antigens tap-translocated peptides specifically bind proteins in the endoplasmic reticulum, including gp , protein disulfide isomerase and calreticulin the binding of tapbpr and tapasin to mhc class i is mutually exclusive processing of a multiple membrane spanning epstein-barr virus protein for cd (+) t cell recognition reveals a proteasome-dependent, transporter associated with antigen processing-independent pathway autophagy mediates transporter associated with antigen processing-independent presentation of viral epitopes through mhc class i pathway alternative pathways for mhc class i presentation: a new function for autophagy autophagy enhances the presentation of endogenous viral antigens on mhc class i molecules during hsv- infection autophagy and adaptive immunity when autophagy meets viruses: a double-edged sword with functions in defense and offense unveiling the roles of autophagy in innate and adaptive immunity endoplasmic reticulum and golgi complex: contributions to, and turnover by eating the endoplasmic reticulum: quality control by autophagy generation of mhc class i ligands in the secretory and vesicular pathways the itinerary of autophagosomes: from peripheral formation to kiss-and-run fusion with lysosomes dominant contribution of the proteasome and metalloproteinases to tapindependent mhc-i peptide repertoire allele-dependent processing pathways generate the endogenous human leukocyte antigen (hla) class i peptide repertoire in transporters associated with antigen processing (tap)-deficient cells diversity of natural self-derived ligands presented by different hla class i molecules in transporter antigen processing-deficient cells a transporter associated with antigen-processing independent vacuolar pathway for the mhc class i-mediated presentation of endogenous transmembrane proteins financial support was received from the portuguese foundation for science and technology (grant sfrh/bd/ / to co). gamblin artists colors (portland, or, usa) is acknowledged for the free use of figure . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -p wlw d authors: olson, brian m.; sullivan, jeremy a.; burlingham, william j. title: interleukin : a key mediator of suppression and the propagation of infectious tolerance date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: p wlw d the importance of regulatory t cells (tregs) in balancing the effector arm of the immune system is well documented, playing a central role in preventing autoimmunity, facilitating graft tolerance following organ transplantation, and having a detrimental impact on the development of anti-tumor immunity. these regulatory responses use a variety of mechanisms to mediate suppression, including soluble factors. while il- and tgf-β are the most commonly studied immunosuppressive cytokines, the recently identified il- has been shown to have potent suppressive function in vitro and in vivo. furthermore, not only does il- have the ability to directly suppress effector t cell responses, it is also able to expand regulatory responses by propagating infectious tolerance and generating a potent population of il- -expressing inducible tregs. in this review, we summarize research characterizing the structure and function of il- , examine its role in disease, and discuss how it can contribute to the induction of a distinct population of inducible tregs. the immune system has evolved to establish multiple layers of defense against a variety of pathogens and diseases. however, concurrent with these effector responses are a robust network of regulatory responses that are able to keep the effector branch of the immune system in check and ensure that they do not lead to potentially harmful autoimmunity. these suppressive responses are mediated by a myriad of cell types, including myeloid-derived suppressor cells as well as macrophages with suppressive function [such as tumor-associated macrophages ( ) ], but suppressive function is most commonly associated with regulatory t cells (tregs). t cells with potential suppressive activity were identified in the seminal research by gershon and kondo as well as nishizuka and sakakura more than years ago, showing that lymphocytes can suppress t cell responses and that this tolerance could be transferred into naive mice ( ) ( ) ( ) ( ) . however, after this foundational work, research into tregs went through a period of controversy and conflicting results, with difficulty in identifying a molecular basis for their suppressive function leading some to question their existence. following more than a decade of studies aimed at elucidating the mechanisms that mediate treg activity, interest was rekindled in the mid- s with the transformational research of sakaguchi and colleagues, who specifically identified a population of cd +cd + t cells that had suppressive function, which were coined as naturally occurring thymic-derived tregs, or natural abbreviations: apc, antigen-presenting cell; dc, dendritic cell; eae, experimental autoimmune encephalitis; ebi , epstein-barr virus-induced gene ; il, interleukin; itreg, induced regulatory t cell; nima, non-inherited maternal antigen; nk, natural killer; ntreg, natural regulatory t cell; pap, prostatic acid phosphatase; tgf, transforming growth factor; treg, regulatory t cell. tregs (ntreg) ( ) . these tregs were later identified as also expressing the intracellular transcription factor foxp , and were found to mediate suppression against a wide array of effector immune responses, including cd + and cd + t cells, b cells, natural killer (nk), and nk-t cells, and even inducing dendritic cells (dcs) and macrophages into a more tolerogenic phenotype. however, while ntregs play a central role in mediating tolerance to a variety of self antigens, they are recognized as not being the primary mediator of tolerance to pathogens and other antigens encountered in the periphery. this role belongs to a broad class of cells classified as peripherally derived or inducible tregs (itregs), which are cd + or cd + t cells which enter the periphery as naive t cells but encounter their antigen under conditions which are not conducive to the generation of productive effector responses, such as environments rich in immunosuppressive cytokines such as tgf-β. when activated in these conditions, itregs gain potent suppressive functions, inhibiting t-cell proliferation and effector functions in an antigen non-specific fashion, and play a central role in mediating regulation and propagating infectious tolerance in a variety of malignancies, including infectious diseases and cancer. inducible tregs are further divided into subclasses of itregs, which are classified based largely on the mechanisms they use to mediate regulation (though the functional mechanisms of suppression utilized by these various itregs are not strictly limited to each subpopulation -for example, multiple itreg populations use surface molecules such as ctla- or pd- to mediate infectious tolerance). tr induced regulatory cells mediate suppression primarily through the secretion of the immunosuppressive cytokine il- , and are further characterized by their lack of foxp and cd expression which are expressed by ntregs ( ) . the second class of itregs are th cells, which were one of the earliest populations of tregs and were identified as playing a role in mediating tolerance in experimental autoimmune encephalitis (eae). these cells express cd and foxp and predominantly utilize tgf-β to mediate suppression, with minimal expression of il- and il- ( ). while the tr and th populations of itregs were long considered to be the only defined induced regulatory populations, research has identified another population of induced tregs that can are potent mediators of suppression as well as in the propagation of infectious tolerance: itr regulatory cells. these inducible regulatory cells were identified by dario vignali and colleagues and mediate suppression primarily through the expression of the regulatory cytokine il- ( ) . in this review, we will discuss the identification and characterization of il- , how it mediates suppression, the role it has been shown to play in disease, and the importance of il- and more specifically itr cells in propagating infectious tolerance. interleukin belongs to the il- family of cytokines, which is a group of heterodimeric cytokines that are composed of one of five subunits [p , p , p , p , and epstein-barr virus-induced gene (ebi )] that come together in various combinations to form il- , il- , il- , and il- , as illustrated in figure a ( ). despite their shared components, these cytokines run the spectrum of immunological effector functions. il- is a proinflammatory cytokine that is closely associated with the activation of th immune responses. it is predominantly expressed by monocytes and dcs, and its expression can be triggered by activated t cells. the inflammatory activity of il- is clearly seen in efforts to target its activity in a variety of diseases. in patients with malignancy, research has shown that recombinant il- can elicit anti-tumor responses in vivo ( ) . alternatively, efforts to inhibit the inflammatory effects of il- have been developed, including il- -blocking antibodies used to treat autoimmune disorders such as eae, where it has been shown to prevent uncontrolled immune responses ( , ) . similar to il- , il- also has inflammatory activity and can drive th responses, as well as promoting the activity of nk and th cells ( ) . as opposed to il- and il- , il- has a wide range of immunomodulatory activities. while it can promote th development, il- can also inhibit th responses and promote the suppression of t-cell responses depending on the microenvironment ( ). which can signal through gp or il- rβ homodimers, or through a unique gp :il- rβ heterodimer, which results in the formation of a novel stat :stat heterodimer that has distinct effects on target cells: maximal suppression, il- expression, and their conversion into itr regulatory t cells. while il- , il- , and il- can all play a role in promoting inflammatory immune responses, the youngest member of the il- family, il- , is a purely immunosuppressive cytokine. il- was identified in the mid- s, first reported by dario vignali and colleagues, and was soon after reported by his group and others to be a potent mediator of suppression ( , ) . il- is a heterodimer composed of the p and ebi subunits, which were both identified as being over-expressed by tregs and not effector cells ( ) . the potential of these two subunits coming together to form a novel heterodimer was first described in by devergne and colleagues, who found that cells transfected with p and ebi lead to the secretion of a p -ebi heterodimer ( ) . in this report, it was suggested that given the expression of ebi in many tissues replete with immune cells, it was likely that this heterodimer had immunomodulatory function -however, no functional studies were conducted for another years. recent studies into the formation of this heterodimer found that subunits from human and mouse can bind to the opposite species, indicating that the protein-protein interactions that form il- are novel to the il- family and conserved between species ( ) . furthermore, the protein binding sites were unique when compared to those used for il- and il- , and that no single mutation could disrupt this interaction ( ) . this is particularly significant, as the design of therapeutic interventions aimed at targeting the suppressive activity of il- could focus on small-molecule inhibitors of this interaction which would selectively target il- while leaving il- and il- unaffected. in addition to having a unique function when compared to the other il- family members, il- is also unique in that rather than being expressed primarily by antigen-presenting cells (apcs), il- is expressed primarily by tregs. since it was identified in , dozens of reports have been published describing il- expression in both thymus-derived and peripheral tregs. this includes a subset of cd +cd +foxp + ntregs in humans, mice, and even pigs ( , , ) , though this expression is thought to occur only in a subset of il- + ntregs and is not constitutive ( ) . research has also identified expression of il- in a population of il- -induced cd + tregs, defined as itr cells ( ) . in addition to cd + tregs, il- has also been shown to be expressed and mediate antigen-specific suppression in a population of cd + tregs in patients with prostate cancer ( ) . interestingly, other non-immune cell types have also been shown to express il- , including tumor cells ( , ) and potentially an even broader tissue expression profile in the course of inflammation ( ) . however, in all of these cell types, it has been noted that il- expression is minimal in unactivated t cells -rather, these cells need to become activated for the induction of il- , such as through engagement of their t-cell receptor or following inflammation ( , , ) . this suggests that il- may be more associated with the suppressive activity of tregs in peripheral tissues rather than a constitutive marker of tregs. the suggested expression of il- by multiple cells types, including both natural and induced treg, emphasizes the need to further characterize the mechanisms that regulate the expression of il- in these populations. after being expressed and secreted by tregs, il- then acts on its target cells following binding to the il- receptor. however, as is the case with the subunits that make up the il- family of heterodimeric cytokines, the receptors for the il- family are also composed of five different subunits: il- rβ , il- rβ , il- r, wsx, and gp (as illustrated in figure b) . the il- receptor is composed of il- rβ and gp , which are also associated with the il- and il- receptors, respectively ( , ). following binding of il- to its receptor, its signal is transduced through stat and stat , which can also form a unique heterodimer and result in the expression of target genes including p and ebi , resulting in a feedback loop promoting increased il- expression ( ). however, il- is also unique from the other members of the il- family in that it can also signal through a homodimer of its receptor subunits. however, when il- binds to one of its homodimeric receptors, only one branch of its signal transduction pathway is activated (either stat or stat for gp :gp or il- rβ :il- rβ homodimers, respectively), resulting in a partial loss of the suppressive activity of il- compared with signaling through the fully functional il- rβ -gp heterodimer receptor, as diagramed in figure b ( ). the use of these subunits sheds some light onto the target of il- activity; gp is expressed in most cell types ( ), whereas il- rβ is expressed predominantly on activated t cells, nk cells, and to a lesser extent dcs and b cells ( ). since its discovery, the predominant mechanism of suppression associated with the activity of il- is its ability to suppress t-cell proliferation and effector functions. foundational work into the activity of il- utilized il- a −/− and ebi −/− mice, finding that cd + treg lacking il- expression had a significantly reduced ability to suppress t-cell proliferation ( ) , an observation that has been repeated in numerous models by several groups ( , , ( ) ( ) ( ) . the ability of il- to suppress t-cell responses has also been clearly illustrated in studies using recombinant il- (ril- ), where it can decrease t-cell proliferation as well as t-cell cytokine expression, though these studies have been somewhat complicated by the difficulty in generating an active heterodimeric form of il- ( , , , ) . in related studies, the ectopic expression of il- by conventional cd + t cells (using a transfected il- expression construct) results in these conventional t cells gaining a regulatory phenotype, manifested by the ability to potently suppress t-cell proliferation ( , ) . the suppressive activity of il- is not limited to cd + tregs, as a population of cd +ctla- + tregs was also found to suppress the proliferation of autologous t cells in a contact-independent, il- -dependent fashion ( ) . while mechanistic studies into il- have focused on its ability to suppress cd + and cd + t-cell proliferation, il- has also been shown to have a role in suppressing th responses. tregs expressing il- have been shown to inhibit the differentiation of cd + t cells into th effector cells, and mice lacking ebi have a significant increase in the production of il- ( , [ ] [ ] [ ] . this has also been reproduced in studies using ril- , in which treatment with ril- reduces th differentiation as well as the function of th cells ( , ) . in addition to its effects on th immunity, one report has even suggested that ril- can lead to decreased antibody titers ( ). while this is the only report to our knowledge to associate il- activity with the suppression of humoral immunity, it has significant implications toward the precise mechanism of action of il- . while in vitro studies have clearly shown that il- is able to directly act on effector t cells (supported by the expression profiles of the il- receptor subunits), the ability of il- to suppress antibody responses could suggest that il- is also able to act on other cell populations, though it could also be a reflection of the inhibition of helper t cell responses that contribute to humoral immunity. given the direct suppressive activity of il- , there has been interest in evaluating the role that il- can play in the development of a variety of diseases (summarized in table ). several diseases have been shown to be associated with increased il- expression, including multiple inflammatory diseases, coronary artery disease, and cancer. in individuals with acute myeloid leukemia, the development of disease has been shown to be associated with elevated plasma levels of il- ( ) . this has been supported by results in lung cancer, where a study evaluating ebi levels in lung cancer patients found that ebi levels are elevated in patients with malignancy, predicts for poor outcome, and is an independent prognostic indicator of disease (although this study only examined ebi , and not the p subunit of il- ) ( ) . additionally, in murine models of melanoma and colorectal carcinoma, the establishment of tumors has been shown to lead to increased il- expression in cd + tumor-infiltrating lymphocytes, which are subsequently able to suppress t-cell proliferation ( ) . this likely contributes to the inhibition of the anti-tumor effects of adoptively transferred cd + and cd + t cells in this melanoma model. the loss of il- has also been shown to be associated with the development and exacerbation of disease, including many inflammatory diseases such as encephalomyelitis and inflammatory bowel disease ( table ). in multiple models of encephalomyelitis, wild-type tregs can prevent the onset and severity of disease. however, animals that lack functional il- were shown to have enhanced inflammatory immune responses and increased disease ( , , ). similar observations have been shown in inflammatory bowel disease, liver fibrosis, and models of lethal autoimmune disease ( , , , ) . conversely, given that the loss of il- is associated with increased incidence and severity of inflammatory diseases, the induction of il- expression has been shown to alleviate a variety of disease symptoms ( table ). in models of inflammatory bowel disease, il- gene therapy and the adoptive transfer of il- -expressing tregs have been shown to cure colitis symptoms ( , ) . the same holds true in collagen-induced arthritis, where ril- reduces the frequency and severity of arthritis and a decrease in inflammatory immune responses ( , ) . as opposed to these inflammatory diseases, tumor models have shown that il- contributes to tumorigenesis ( , ) . these effects are mediated through both immune-directed and tumordirected effects, as il- can act to suppress tumor-infiltrating lymphocytes that may have anti-tumor activity, as well as potentially supporting the proliferation of tumor cells by promoting angiogenesis ( , ) . while the direct suppressive activity of il- has been established in numerous reports in vitro and in vivo, research into immune tolerance has shown that the low frequency of individual regulatory populations alone are largely insufficient to control effector immunity. therefore, to expand the breadth of suppressive immunity, tregs are able to induce and mobilize additional regulatory immune cells. this concept of infectious tolerance is central to the ability of the immune system to maintain tight control of effector responses, whereby tregs can transfer suppressive function onto a nominally conventional t cell population. suppressive cytokines play a central role in the propagation of infectious tolerance, including il- , which has been shown to play an important role in the expansion of regulatory immunity. the concept of infectious tolerance was first proposed by gershon and kondo in the early s, where they showed that, "tolerance . . . can be spread from one cell to another" ( ). this was further elucidated by benjamin and waldmann, who used antibodies blocking t cell populations to induce tolerance to skin grafts ( ) , and later in elegant studies by qin and colleagues from the same group, who used congenically marked t cells to show that suppressive activity can be transferred from one cell population to another ( ) . as additional molecular data regarding the suppressive mechanisms of treg has become available, it has become clear that tregs can secrete cytokines that can induce naïve and even effector t cells to gain a regulatory phenotype. this can occur by directly targeting effector t cells and causing them to gain a suppressive phenotype, as well as targeting dc populations and causing them to promote the conversion of effector cells into regulatory cells ( ) . the most commonly thought of cytokines involved in this conversion are treg-produced il- and tgf-β, which can drive the generation of tr and th cells, respectively. however, the ability to transmit infectious tolerance is also a characteristic of il- , the production of which can cause the conversion of conventional effector t cells into induced regulatory cells that are potent mediators of suppression in vitro and in vivo. some of the earliest reports of il- began to shed light on the potential role of this cytokine in infectious tolerance. in a report by niedbala and colleagues in , they found that a ril- fusion protein induced the proliferation of a population of cd +cd +foxp + t cells, and which expressed il- and suppressed t-cell proliferation in vitro ( ) . additionally, in another report utilizing a recombinant single-chain il- , it was found that treatment of mice with ril- resulted in a significant increase in il- (but not tgf-β) production by cd + t cells in draining lymph nodes ( ). when these mice were examined further for the impact of il- on treg function, they found that administration of il- led to an increase in the frequency of cd +cd + tregs that expressed foxp and il- , and that il- promoted the proliferation of these t cells ( ). furthermore, when these cd +cd + t cells were adoptively transferred they could protect animals from collagen-induced arthritis in an il- dependent fashion ( ). these data together suggest that il- is able to promote the expansion of il- producing itregs, and that these tregs are able to mediate suppression in vitro and in vivo. while these studies provided the earliest evidence that il- could play a role in the propagation of infectious tolerance, it remained unclear whether this induced regulatory population also expressed il- , or whether il- played any role in mediating suppression in these induced regulatory cells. this was addressed frontiers in immunology | immunological tolerance ( ) in an expansive report from by collison and colleagues in late that not only specifically studied the role that il- plays in the conversion of conventional t cells into induced tregs, but also evaluated the role that il- has in mediating the suppressive function of these itreg ( ) . in this report, they show that treating either human or mouse conventional cd +foxp − t cells with il- causes these t conv to begin to express il- (but not il- nor tgf-β), and that these t conv cells can then suppress t-cell proliferation in a contact-independent fashion. further supporting the lack of a role for il- and tgf-β, blocking either of these cytokines did not affect the suppressive function of these cells whereas blocking il- significantly abrogated this suppression, suggesting that these il- -induced regulatory cells represented a novel population of itregs rather than the conventional tr or th cells, which they defined as itr cells. the conversion of conventional t cells into an il- -expressing inducible treg was also shown to occur when t conv were cultured with ntreg, which have been shown to express higher levels of il- when cultured with conventional t cells ( ) . when mouse ntreg were cultured with t conv cells, the t conv cells began to express il- and were then able to suppress t-cell proliferation ( ) . interestingly, this conversion was found to require il- and il- expression by ntregs; however, once these t conv cells had gained a regulatory phenotype, il- was not required for their suppressive activity. in a later report, the same group confirmed that this conversion of t conv into il- -expressing itregs occurred in a contact-independent fashion through the activity of il- , but did not require il- nor tgf-β ( ) . furthermore, in this report they also show that maximal treg suppression requires not only il- expression but also contact with t conv that can subsequently be converted into itr cells ( ) , further highlighting the importance of infectious tolerance in the overall suppressive activity of il- . the generation of itr cells has also been shown to occur naturally following the onset of various diseases. in one model, mice were infected with an intestinal parasite that induces an inflammatory response followed by the expansion of treg responses in the intestine. following this infection, cd + foxp + ntregs in the spleen were found to have negligible levels of il- expression, whereas cd +foxp + at the site of infection had a significant frontiers in immunology | immunological tolerance increase in il- ( ) . interestingly, when cd +foxp − conventional t cells were examined for il- , negligible levels were found in the spleen, whereas cd +foxp − t cells at the infection site had a significant increase in il- expression ( ) . similar results were observed in two different tumor models (mc colorectal and b melanoma tumor cell lines), where tumor inoculation led to an increase in il- expression in cd +foxp + and cd +foxp − t cells that infiltrated the tumor, whereas there was negligible il- expression in splenic t cells ( ) . furthermore, these tumor-infiltrating cd +foxp − t cells were able to suppress t-cell proliferation in vitro in an il- -dependent fashion, indicating that tumor establishment led to the generation of itr cells ( ) . the observation that tumor formation leads to the generation of itr cells suggests that these cells may play a role in promoting tumor development, a characteristic that is associated with other induced treg populations. in a variety of malignancies, increased frequencies of tregs has been shown to correlate with a poor prognosis for patients, though this observation is not absolute ( ) . the profoundly suppressive tumor microenvironment has been shown to promote the generation of regulatory immune responses, using factors such as tgf-β or adenosine to mediate the conversion of effector lymphocytes into itregs ( , ) . furthermore, these tumor-infiltrating itreg have been shown to have greater suppressive activity that ntreg, both in terms of the levels of suppression as well as the mechanisms used ( , ( ) ( ) ( ) . however, this does not appear to be the case with il- , as tumorinfiltrating cd +foxp + ntregs had higher levels of suppression then infiltrating foxp − itr cells ( ) . this likely reflects the multitude of suppressive mechanisms that ntreg are able to utilize to mediate suppression, as tumor-infiltrating ebi −/− ntreg were able to suppression t-cell proliferation at equal levels compared with wild-type ntreg ( ) , and even treg that lack both il- and il- expression can still mediate suppression through factors such as trail ( ). however, itr cells appear to lack this functional plasticity, as ebi −/− mice do not have tumor-infiltrating induced regulatory cells with suppressive function, and itr cells lacking ebi and/or il- p lack efficacy in preventing autoimmune responses in a variety of models in addition to these tumor models ( ) . despite this requirement for il- , itr cells, and the role of il- expression on the propagation of infectious tolerance is an important component of the suppressive tumor microenvironment. when rag −/− mice are challenged with tumors and then receive adoptively transferred wild-type cd + and cd + t cells, these t cells are able to mediate an anti-tumor response and keep tumor growth in check ( ) . when wild-type tregs are transferred as well, the tumors grow rapidly, reflecting the ability of treg to suppress the anti-tumor response associated with the transfer of the cd + and cd + t cells, both by directly suppressing t-cell proliferation as well as converting these conventional t cells into regulatory cells ( ) . however, when tumor-bearing animals receive an adoptive transfer containing wild-type cd + t cells and ebi −/− cd + t cells, the growth of these tumors was reduced by approximately % ( ) . this suggests that the conversion of t conv into itr cells is a significant contributor to the suppression of anti-tumor immunity, and that the therapeutic targeting of this regulatory population could promote anti-tumor responses. the dependence of itr cells on il- also suggests that these cells may have different characteristics regarding their long-term phenotypic and functional stability. given the nature of induced regulatory cells, which gain or lose immunosuppressive activity depending on the microenvironment in which they are activated, the stability of these induced populations is thought to be transient. however, data regarding the stability of itr cells in vivo suggests otherwise. in numerous models, the transfer of itreg was shown to mediate clinical efficacy for several weeks following a single adoptive transfer, suggesting that these cells retain their suppressive function for an extended period of time ( ) . furthermore, when congenically marked itr or th cells were injected into mice and recovery was measured over time, it was found that % of the injected itr cells were recoverable after week, % after weeks, and % after more than weeks, compared with only % of th cells that were recovered week following transfer ( ) . additionally, these cells retained their suppressive function; even days following injection, adoptively transferred itr cells were able to suppress t-cell proliferation at the same levels as freshly isolated itr cells, whereas th cells had significantly reduced suppressive function ( ) . this suggests that itr cells may represent more of a terminally differentiated regulatory population, and rely on the potent suppressive activity of il- to mediate suppression. however, further work is necessary to characterize these itr cells, and other suppressive mechanisms that they may gain or lose over time. as we look toward the future of immune regulation and infectious tolerance, it is essential to focus not only on identifying novel mediators of tolerance, but how these populations can be reliably identified. this is particularly relevant with the generation of induced tregs during the propagation of infectious tolerance, as the plastic nature of these populations makes them challenging to identify and track over time. even itr cells, which may represent more of a stable regulatory population than tr or th cells, are incredibly challenging to identify. while these cells are predominantly identified based on their robust expression of il- , the detection of il- expression can be daunting. the nature of the il- family of cytokines requires the expression of all five subunits to be interrogated to ensure that it is il- that is being expressed by a putative regulatory population (even though tregs do not express significant levels of the other il- family members). this is also manifested in difficulty detecting il- protein levels using current commercially available reagents and the lack of an antibody that jointly recognizes a conformational epitope from il- , thus requiring ebi , p , and the other il- subunits to be examined. additionally, the difficulty in generating functionally active ril- also delayed research and led to the necessity of multiple levels of evaluation in studying the suppressive effects of il- . as these reagents are developed and become commercially available, it is to be hoped that increased research into il- will better define and characterize tregs that express this cytokine. while il- expression can be analyzed on fixed or lysed cell populations, what would be ideal would be a series of cell surface www.frontiersin.org markers that can be used to identify itr cells, allowing these cells to be isolated and further characterized. currently, itr cells are characterized only by their expression of ctla- and cd , as well as a lack of intracellular foxp ; however, a gene microarray comparing itr cells with conventional treg found that there was no significant genetic signature that could be used to distinguish one regulatory population from the other ( ) . furthermore, while treatment with ril- was shown to induce a population of cd +cd +cd − tregs that express il- , these cells were not evaluated for expression of il- , causing cd to remain one of the potential markers of itr cells, though its expression is clearly not restricted to itr cells ( ). this highlights the importance of elucidating markers for itr cells that have not yet been evaluated [such as gitr, pd- , cd , or even helios, whose expression is traditionally associated with ntreg but was recently suggested to also be present on itreg ( , ) ] that can facilitate the identification of itr cells without expression analysis. as clearly illustrated in table , il- plays an important role in a variety of diseases. furthermore, research from our group has identified that il- -expressing tregs also play a central role in mediating tolerance in transplantation tolerance, immune responses to non-inherited maternal antigens (nimas), and prostate cancer [ref. ( ) ; and olson et al., unpublished results], which we also found were dependent on the expression of ctla- . interestingly, when we examined collagen type v-specific regulatory responses, we did not find a role for il- [contrary to results obtained from other groups ( , ) ] nor ctla- , though we did find that these responses relied heavily on tgf-β. this suggests that the regulatory responses we identified in the transplant, nima, and prostate cancer patients may have been relying on itr cells, whereas the responses we identified to collagen v were ntregs, and suggests that potentially ctla- and il- dependency may be a technique that can be used to identify this population of inducible regulatory cells. in our research evaluating the role of il- expression in antigen-specific tolerance in prostate cancer patients, we identified cd +ctla- + il- -expressing t cells specific for the prostate tumor-associated antigen prostatic acid phosphatase (pap), which were present in some patients with prostate cancer ( ) . immediately following immunization with a dna vaccine targeting pap, these antigen-specific cd +ctla- + t cells prevented the detection of concurrent pap-specific effector responses; however, in long-term follow ups, we found that pap-specific effector responses could be detected in these individuals. these results raise questions regarding the nature of these cd +ctla- + il- -expressing regulatory cells; in particular, whether they represent a population of cd + t cells that have been induced in the periphery to gain il- expression and suppressive activity, or alternatively if they are an antigen-specific thymus-derived population of ntregs. if these pap-specific cd + regulatory responses represent a population of ntreg cells, their presence in the periphery and tumor microenvironment pre-and immediately post-immunization would suppress and prevent the detection of pap-specific effector responses (as well as potentially induce the generation of il- -expressing tregs), whereas the long-term generation and expansion of effector responses could eventually outnumber these suppressive responses and ultimately lead to the desired goal: the generation of productive, antigen-specific anti-tumor immunity (figure a) . alternatively, if these papspecific cd + regulatory cells represent an induced population of cd + itr cells, then their presence pre-immunization would be able to convert antigen-specific effector responses into additional regulatory responses (thus furthering the propagation of infectious tolerance) until extended period following immunization when effector responses overcome these regulatory responses, either by simply outnumbering regulatory cd t cells or by preventing the conversion of effector cells to regulatory cells through the generation of a non-immunosuppressive microenvironment ( figure b) . regardless, both of these models suggest that the goal of antigen-specific immunotherapies is not simply to generate effector responses, but rather to tip the balance between antigen-specific effector and regulatory responses toward productive anti-tumor immunity. further research into these antigenspecific populations, how they are affected by antigen-specific vaccination, how they affect the generation of antigen-specific effector immune responses, and whether they have any predictive value toward the ultimate efficacy of anti-tumor vaccines, remains to be elucidated. while cd + tregs are not as well studied as their cd + treg counterparts, both cd + ntreg and itreg have been identified and characterized, including reports in individuals with cancer ( ) . in our published studies, the reliance of pap-specific cd +ctla- + t cells on il- for mediating contact-independent suppression (with no identifiable role played by il- nor tgf-β) suggests that these cells may be more akin to a population of cd + itr cells which are dependent on il- for contact-independent suppressive activity, as opposed to ntreg which are able to utilize multiple mechanisms of contactindependent suppression. furthermore, our observation that the suppressive function of il- -expressing cd +ctla- + treg is temporally regulated following antigen-specific immunization suggests that this population may be able to be modulated depending on the tumor microenvironment. however, additional research is required to determine how antigen-specific il- -expressing treg are affected by antigen-specific immunization, as well as how these il- + treg responses are generated in tumor-bearing individuals. to better characterize the generation and fate of itr cells, it will be important to shed light onto the mechanisms that regulate the expression of il- by tregs. it is clear that expression of il- by ntreg and itreg requires activation, whether through inflammatory responses, non-specific stimulation of the t-cell receptor, or through encounter of antigen by antigen-specific tregs ( , , ) . additionally, it appears that foxp does not directly play a role in the regulation of il- expression, providing further evidence that il- serves primarily as a potent mediator of suppression in induced regulatory populations rather than foxp + ntregs ( ). however, the regulation of foxp does potentially open up new avenues of potential means of il- regulation. foxp , along with other factors associated with tregs such as ctla- , are specifically hypomethylated in ntreg cells, resulting in increased access to the transcriptional complex and higher expression levels compared to t conv cells, where these sequences are preferentially hypermethylated ( , ) . additionally, the expression of various cytokines has been shown to be epigenetically regulated, including il- and frontiers in immunology | immunological tolerance if the observed pap-specific cd +ctla- + t cells represent a population of natural tregs, pre-immunization samples (left panels) have pap-specific cd + ntregs present (red cells) that utilize il- to suppress the activity of pap-specific effector cells (dark green) both in the periphery (top panels) as well as in the tumor microenvironment (bottom panels), as well as the ability to induce a population of il- -expressing tregs (light green). administration of a dna vaccine encoding pap leads to the presentation of pap-derived epitopes on the surface of apcs immediately post-immunization (center panels), leading to the expansion of antigen-specific effector cells. however, these cells are in small numbers, and when they traffic to the tumor site, they are outnumbered by pap-specific ntreg that are able to suppress their proliferation and effector functions. it is not until long-term follow up when these effector responses are able to outnumber antigen-specific ntreg, leading to the generation of productive anti-tumor immunity. (b) in a model where cd +ctla- + t cells represent a population of novel cd + itr cells (light green cells), these itregs would be able to convert effector cells (dark green) into additional itreg through their expression of il- , thus propagating infectious tolerance to prevent the generation of productive anti-tumor immunity both pre-immunization as well as immediately post-immunization. this process would be predicted to continue until long-term follow up, when antigen-specific effectors could expand to a sufficient level to outnumber these itreg responses, and potentially prevent the generation of induced antigen-specific treg by promoting tumor destruction and a non-suppressive tumor microenvironment. tgf-β, which can in turn induce epigenetic changes that can lead to the generation of itreg populations ( ) ( ) ( ) ( ) . this raises the possibility that the induction of il- expression in itr cells may be epigenetically regulated, which would permit the heritable transmission of il- expression into subsequent progeny itr cells while maintaining flexibility for altered expression levels based www.frontiersin.org on the immune profile of the microenvironment. to date, epigenetic regulation of il- expression has not been specifically evaluated -however, regions of the il- p promoter have been shown to become methylated to regulate il- expression by dcs ( ) and il- p intronic regions can become demethylated in non-activated tregs ( ) . additionally, the il- rβ receptor has also been shown to be epigenetically regulated ( ) , suggesting that il- could also be regulated using an epigenetic mechanism. many challenges also remain regarding the various populations of induced regulatory cells, and how these populations complement each other. clearly there is a role for il- in the generation of itr cells, though the converse does not appear to be true, with il- not appearing to play a central role in the generation of il- -secreting tr or tgf-β-secreting th populations. this may suggest that these populations may have distinct roles in the suppression of inflammatory immune responses. alternatively, this could be a reflection of the redundancy of the immune system, having multiple layers of regulatory populations that can mediate similar effects, but that perhaps itr cells represent more of a terminally differentiated regulatory cell that is mobilized when high levels of immunosuppression are required. while studying the differentiation pattern of this population raises significant experimental challenges, these studies will be essential to better understand the nature of t-cell functional plasticity, as well as how il- -expression fits into this process. doing so will allow for the identification of methods that can be used to control and guide these regulatory responses to design effective therapies for cancer, autoimmunity, and tissue transplantation. identification and manipulation of tumor associated macrophages in human cancers thymus and reproduction: sex-linked dysgenesia of the gonad after neonatal thymectomy in mice cell interactions in the induction of tolerance: the role of thymic lymphocytes infectious immunological tolerance immunoregulatory circuits among t-cell sets. identification of a subpopulation of t-helper cells that induces feedback inhibition immunologic self-tolerance maintained by activated t cells expressing il- receptor alpha-chains (cd ) weiner hl. induction and mechanism of action of transforming growth factor-beta-secreting th regulatory cells the inhibitory cytokine il- contributes to regulatory t-cell function il- family cytokines: immunological playmakers interleukin- : biological properties and clinical application curcumin inhibits experimental allergic encephalomyelitis by blocking il- signaling through janus kinase-stat pathway in t lymphocytes functional blocking monoclonal antibodies against il- p homodimer inhibit adoptive transfer of experimental allergic encephalomyelitis il- : one cytokine in control of autoimmunity il- is a novel cytokine with therapeutic effects against collagen-induced arthritis through the expansion of regulatory t cells and suppression of th cells epstein-barr virus-induced gene and the p subunit of interleukin form a novel heterodimeric hematopoietin distinct subunit pairing criteria within the heterodimeric il- cytokine family cutting edge: human regulatory t cells require il- to mediate suppression and infectious tolerance porcine t-helper and regulatory t cells exhibit versatile mrna expression capabilities for cytokines and co-stimulatory molecules human cd + cd + foxp + regulatory t cells do not constitutively express il- il- -mediated induction of a potent regulatory t cell population human prostate tumor antigen-specific cd + regulatory t cells are inhibited by ctla- or il- blockade il- over-expression increases apoptosis sensitivity and suppresses cell growth in human cancer cells tumor-derived il- promotes tumor growth by enhancing myeloid cell accumulation and angiogenesis il- is a novel responsive anti-inflammatory cytokine -a new system of categorizing anti-inflammatory cytokines exacerbation of delayed-type hypersensitivity responses in ebv-induced gene- (ebi- )-deficient mice il- production by inducible costimulator (icos)-positive regulatory t cells reverses established il- -dependent allergic airways disease deletion of interleukin (il)- p induces liver fibrosis in dominant-negative tgfbeta receptor type ii mice aberrant expression of treg-associated cytokine il- along with il- and tgf-beta in acute myeloid leukemia identification of epstein-barr virus-induced gene as a novel serum and tissue biomarker and a therapeutic target for lung cancer decreased plasma il- levels are related to the left ventricular ejection fraction in coronary artery diseases erythromycin enhances cd +foxp + regulatory t-cell responses in a rat model of smoke-induced lung inflammation airway inflammation and ige production induced by dust mite allergen-specific memory/effector th cell line can be effectively attenuated by il- prevention of autoimmune diabetes by ectopic pancreatic beta-cell expression of interleukin- interleukin- enhances lyme arthritis in borrelia-vaccinated and -infected mice induction of tolerance by monoclonal antibody therapy infectious" transplantation tolerance the battle against immunopathology: infectious tolerance mediated by regulatory t cells regulatory t cell suppression is potentiated by target t cells in a cell contact, il- -and il- -dependent manner regulatory t cells in cancer cutting edge: tumor-specific cd + t cells infiltrating prostatic tumors are induced to become suppressor cells induced and natural regulatory t cells in human cancer expansion of human t regulatory type cells in the microenvironment of cyclooxygenase overexpressing head and neck squamous cell carcinoma increased ectonucleotidase expression and activity in regulatory t cells of patients with head and neck cancer targeting human inducible regulatory t cells (tr ) in patients with cancer: blocking of adenosineprostaglandin e( ) cooperation the plasticity of regulatory t cell function expanded subpopulation of foxp + t regulatory cells in renal cell carcinoma coexpress helios, indicating they could be derived from natural but not induced tregs expression of helios in peripherally induced foxp + regulatory t cells inducible reprogramming of human t cells into treg cells by a conditionally active form of foxp epigenetic mechanisms of regulation of foxp expression t cell receptor stimulation-induced epigenetic changes and foxp expression are independent and complementary events required for treg cell development epigenetic regulation of cytokine gene expression in t lymphocytes the regulation of il- production by immune cells epigenetic suppression of the tgf-beta pathway revealed by transcriptome profiling in ovarian cancer development and maintenance of regulatory t cells changes in il a methylation pattern in livers from mice fed ddc aberrant methylation of il- rbeta gene in lung adenocarcinoma cells is associated with unfavorable prognosis . collison frontiers in immunology | immunological tolerance key: cord- -u gznxq authors: huang, weishan; solouki, sabrina; carter, chavez; zheng, song-guo; august, avery title: beyond type regulatory t cells: co-expression of lag and cd b in il- -producing t cell lineages date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: u gznxq type regulatory cd (+) t (tr ) cells express high levels of the immunosuppressive cytokine il- but not the master transcription factor foxp , and can suppress inflammation and promote immune tolerance. in order to identify and obtain viable tr cells for research and clinical applications, co-expression of cd b and lag has been proposed as a unique surface signature for both human and mouse tr cells. however, recent studies have revealed that this pattern of co-expression is dependent on the stimulating conditions and the differentiation stage of the cd (+) t cells. here, using an il- (gfp)/foxp (rfp) dual reporter transgenic murine model, we demonstrate that co-expression of cd b and lag is not restricted to the foxp (−) tr cells, but is also observed in foxp (+) t regulatory (treg) cells and cd (+) t cells that produce il- . our data indicate that il- -producing tr cells, treg cells and cd (+) t cells are all capable of co-expressing lag and cd b in vitro following differentiation under il- -inducing conditions, and in vivo following pathogenic insult or infection in the pulmonary mucosa. our findings urge caution in the use of lag /cd b co-expression as sole markers to identify tr cells, since it may mark il- -producing t cell lineages more broadly, including the foxp (−) tr cells, foxp (+) treg cells, and cd (+) t cells. the mammalian immune system has evolved both effector and regulatory immune axes to protect the host from invading pathogens, along with a control mechanism to tune the level of immune reactivity against self-and non-self-agents to prevent host tissue damage. interleukin- (il- ) is a regulatory cytokine with a demonstrated anti-inflammatory function and plays an essential role in preventing allergic inflammation ( ), autoimmunity ( ) , and pathogen-induced immunopathology ( , ) , but can also promote the establishment and maintenance of chronic infection ( , ) . il- has been reported as a product of activation of multiple immune cell lineages. innate immune cells including dendritic cells (dcs) ( ) , macrophages ( ) , neutrophils ( ) , and innate lymphoid cells (ilcs) ( ) have been reported to express il- in vivo and in vitro. il- is also expressed by many cell subsets of the adaptive immune system, including b cells ( ) and t cells comprising the foxp − cd + ( ), foxp + treg ( ) , and cd + t cell subsets ( ) . regulatory t cells are defined by their immunosuppressive function, and the three aforementioned subsets of il- -producing t cells have been reported as phenotypically distinct regulatory t cell subsets, playing important roles in promoting immune tolerance and/or suppressing inflammation in both mouse and human ( ) ( ) ( ) ( ) ( ) ( ) . among the il- -producing t cells, the foxp − cd + t cell subset, also known as type regulatory t cells (tr cells), are inducible in the periphery and have a pivotal role in limiting inflammation ( , ( ) ( ) ( ) . tr cells have been shown to prevent allergic asthma ( ) and atopic dermatitis ( ) in murine models. in both mouse models and humans, induction of tolerance via specific antigen immunotherapy (sit) is accompanied by induction of tr cells ( , ) . therefore, tr cells have strong promise as a potential therapeutic approach for inflammatory diseases. tr cells can be differentiated from naïve cd + t cells upon tcr engagement in the presence of il- in vitro ( ) , and in order to identify and obtain viable tr cells for clinical application, co-expression of lag and cd b has been recently proposed to be a cell surface signature of the foxp − il- high tr cells ( ) . lag is a structural homolog of the cd molecule and can bind to mhc class ii with high affinity ( , ) . lag is highly expressed by il- + cd + t cells ( ) , as well as by activated effector t cells ( ) and foxp + treg cells ( ) . cd b is the α integrin subunit, highly expressed by nk cells ( ) . cd b is up-regulated in t cells that may produce il- and/or pro-inflammatory cytokines ( ) ( ) ( ) . in addition to foxp − tr cells, il- can be highly up-regulated in activated foxp + treg and cd + t cells under inflammatory conditions and/or upon tcr activation. given the importance of being able to identify foxp − tr cells, including under clinical conditions, and to gain a better understanding of the selectivity of co-expression of lag and cd b as a cell surface signature for il- -producing cells, we sought to determine whether co-expression of lag and cd b can mark a broader range of t cell subsets that are actively producing high levels of il- . using a murine model carrying an il- gfp /foxp rfp dual reporter system, we find that co-expression of lag and cd b is a generic feature of the il- -producing foxp − cd + , foxp + cd + , and cd + t cell subsets. the capacity of co-expression of lag and cd b in marking il- high t cell subsets is dependent on the disease conditions and anatomical location of the cells. furthermore, co-expression of lag and cd b is also a shared feature of human il- -producing foxp − cd + , foxp + cd + , and cd + t cell subsets. our data reveal that co-expression of lag and cd b is a generic signature of il- -producing t cells, which is broader than previously appreciated. abbreviations: nb, nippostrongylus brasiliensis; tr cell, type regulatory t (cd + tcrβ + foxp − il- + ) cell; treg cell, foxp -expressing regulatory t cell; hdm, house dust mite; sr, saccharopolyspora rectivirgula; wsn, influenza a/wsn/ (h n ). all mice were on the c bl/ background. rag −/− (b . s -rag tm mom /j), il- gfp (b (cg)-il tm . karp /j) ( ) , and foxp rfp (c bl/ -foxp tm flv /j) ( ) reporter mice were from the jackson laboratory (bar harbor, me). single reporter strains were crossed to generate an il- gfp /foxp rfp dual reporter strain as we recently reported ( ) . human peripheral blood samples were procured from the new york blood center collected from healthy cohorts. all experiments were approved by the office of research protection's institutional animal care and use committee and institutional review board at cornell university. all fluorescent antibodies are listed in "fluorochrome-target (clone; annotation if desirable)" format below. purified anti-cd / ( ; fc block), cd ε ( - c ), cd ( . ), ifn-γ (xmg . ), and il- (c . ) antibodies were from biolegend (san diego, ca); pacific blue-cd ( - . ), fitc-tcrβ (h - ), apc-lag (c b w), pe-cy -cd b (hmα ), and pe-cy -cd l (mel- ) were from biolegend; efluor -cd (gk . ); alexa fluor -cd (gk . ) were from ebioscience; bd horizon v -cd (im ), pe-cd (im ), and apc-cy -tcrβ (h - ) were from bd biosciences; percp-cy . -cd α ( . ) was from tonbo biosciences. purified anti-cd ε (otk ) and cd ( . ), efluor -cd α (rpa-t ) fitc-cd (okt ), and apc-foxp ( a/e ) were from ebioscience; pe-il- (jes - f ), alexa fluor -lag ( c c ), and percp-cy . -lag ( c c ) were from biolegend; fitc-cd b (ak- ) and alexa fluor -cd (rpa-t ) were from bd biosciences. human trustain fcx (fc receptor blocking solution) was from biolegend; cell fixable viability dye efluor was from ebiosciences. cells from various organs were isolated as we recently described ( ) . briefly: blood cells were collected through cardiac puncture, and red blood cells were lysed before analysis; lungs were minced and digested in . mg/ml liberase tl (sigma, st. luis, mo) in • c for - min, then filtered and red blood cells were lysed before analysis; intestines were flushed, opened longitudinally, and inner contents removed with the blunt end of scissors, then cut into . -cm fragments, followed by digestion in u/ml collagenase viii (sigma) in • c for h, filtered, and lymphocytes isolated using gradient separation by % and % percoll (ge healthcare, wilkes-barre, pa) solutions; perigonadal adipose tissues were minced and digested in u/ml collagenase i (worthington biochemical corp., lakewood, nj) in • c for min, filtered and red blood cells were lysed before analysis. - u/ml dnase i (sigma) were added during digestion to reduce cell death triggered by free dna. foxp rfp il- gfp dual reporter mice were injected with µg/mouse anti-cd ε ( - c ) intraperitoneally on day and , and analyzed on day , as previously described ( ) . mice were given l nb larvae per mouse through subcutaneous injection, as we previously described ( ) . cells from the lungs were analyzed days post infection ( dpi). mice were given daily intranasal exposures of µg house dust mite (dermatophagoides pteronyssinus) protein extract (xpb d a . from greer) in pbs, for consecutive days. cells from the lungs were analyzed h post the last treatment. mice were intranasally exposed to µg saccharopolyspora rectivirgula (sr, atcc ) extract on consecutive days each week as previously described ( ) , for weeks. cells from the lungs were analyzed on the last day of the fourth week. mice were intranasally infected with ld ( pfu) wsn per mouse, as we previously described ( ) . cells from the lungs were analyzed days post infection ( dpi). mouse tcrβ + foxp rfp− cd − cd l + splenic naïve t cells were sorted on bd facs aria ii or fusion systems (bd biosciences, san jose, ca), then cultured with mitomycin-c (sigma, µg/ml) treated antigen-presenting cells (apcs; rag −/− splenocytes) at : ratio in the presence of anti-cd ε ( µg/ml), anti-cd ( µg/ml), recombinant murine (rm) il- (r&d systems, - ng/ml), anti-ifn-γ and anti-il- ( µg/ml) for days. human peripheral blood mononuclear cells (pbmcs) were isolated from blood (new york blood center, long island, ny) using gradient separation in ficoll-paque plus (ge healthcare). pbmcs were cultured in full rpmi- medium for min in • c, then non-adherent cells were used to enrich for cd + t cells using anti-human cd microbeads (miltenyl biotec, san diego, ca) or cd + t cells using a human cd isolation kit (biolegend). adherent cells were treated with mitomycin-c (sigma, µg/ml) in • c for min and used as apcs. anti-human cd ε ( µg/ml) and cd (cd . , ebioscience, - µg/ml), recombinant human (rh) il- (peprotech, u/ml), il- (peprotech, u/ml), il- (r&d system, ng/ml), and ifn-α b (r&d system, ng/ml) were added to differentiate human il- -producing t cells. three days after cultures were set up, cells were stimulated with pma ( ng/ml, sigma-aldrich), ionomycin ( . µm, sigma), brefeldin a ( µg/ml), and golgistop ( . µl/ml, bd biosciences) for h as we previously described ( ) , and subjected to surface staining and intracellular staining (see details below). surface staining of live cells were done in the presence of fc block and fixable viability dye. to detect human cd in activated t cells, anti-cd antibody was added into the intracellular staining panel. for intracellular cytokine staining, cells were fixed with % paraformaldehyde (electron microscopy sciences, hatfield, pa), permeabilized and stained with anti-cytokine antibodies in pbs/ . % saponin (sigma). staining for human transcription factor foxp was performed with a foxp staining buffer kit (ebioscience). flow cytometry data were acquired on lsrii, facs aria ii or fusion systems (bd biosciences), and analyzed in flowjo (tree star, ashland, or). all analyses were performed on fixable viability dye negative singlet population. non-parametric mann-whitney tests and one-way anova were performed using graphpad prism v . (graphpad, san diego, ca), with p ≤ . considered statistically significant. "ns" refers to "no significance." both il- -producing cd + and cd + t cells lag and cd b co-expression was previously reported to be a cell surface signature for both mouse and human il- -producing cd + t cells that lack the expression of foxp (also known as type regulatory t cells, tr cells) ( ) . we and others have previously reported that co-culturing murine naïve cd + t cells with antigen presenting cells (apcs) in the presence of anti-cd , anti-cd , anti-ifn-γ, anti-il- , and il- can efficiently induce the differentiation of tr cells ( , , ) , which express high levels of lag and cd b. our recent data also demonstrated that this protocol can induce il- production in bulk t cell populations that include both cd + and cd + t cells ( figure a) . surprisingly, the resultant il- -producing cd + t cells induced in vitro through this protocol also exhibited high levels of lag /cd b co-expression ( figure a , last plot). in fact, il- -producing cd + t cells can express higher levels of lag and cd b than their il- producing cd + counterparts induced in the same cell culture ( figure b) . these data suggest that co-expression of lag and cd b is not an exclusive cell surface signature of the tr cells, and may be a shared feature of both il- -producing cd + and cd + t cells. both il- -producing tr and treg cells il- production can be significantly elevated in the pulmonary mucosa during the late stages of parasitic infection by nippostrongylus brasiliensis (nb), predominantly by cd + t cells that are lag /cd b double positive ( , ) . we recently observed that both foxp − and foxp + t cells are capable of producing il- in the pulmonary tissues post nb infection ( ) . to determine whether co-expression of lag and cd b is exclusive to foxp − tr cell subset, we infected il- gfp /foxp rfp dual reporter mice with nb, and analyzed the il- -producing t cells. we found that, as previously described, a large majority of the il- -producing t cells in the lungs of nb-infected mice express high levels of both lag and cd b (figures a,b) , and are predominantly cd + t cells ( figure c) . however, interestingly, these il- -producing lag + cd b + cd + t cells included both foxp + and foxp − cd + t cells subsets (figure c , last plot; and figure d ). to further determine whether lag /cd b co-expression is correlated with il- and/or foxp expression in cd + t cells, we compared the percentage of lag /cd b double positive populations in foxp − il- − , foxp − il- + , foxp + il- − , and foxp + il- + cd + t cells isolated from the lungs of nb-infected mice. we found that regardless of expression of foxp , nb infection did not lead to significant up-regulation of lag /cd b co-expression on il- − cd + t cells (figures e,f) . however, the percentage of the lag + cd b + population of il- + cd + t cells is significantly higher than their il- − counterparts ( figure f) . moreover, the levels of lag and cd b expression are similar between the foxp + and foxp − counterparts of il- + cd + t cells (figure g) . therefore, il- -producing cd + t cells, regardless of foxp expression, have a high capacity of coexpressing lag and cd b. together with the data shown in figure , these data suggest that co-expression of lag and cd b is a generic feature of il- -producing t cells, including foxp − tr cells, foxp + treg cells and cd + t cells. the composition of lag + cd b + il- -producing t cells differs in different disease models il- plays an essential role in pulmonary inflammatory diseases, which has been reported in multiple murine models of lung disease, including allergic asthma ( ) , hypersensitivity pneumonitis (hp) ( ) and influenza pneumonia ( ) . we examined whether the expression of lag and cd b would differ based on the inflammatory response in three mouse models of lung inflammation. mice carrying the il- gfp /foxp rfp dual reporters were exposed intranasally to house dust mite (hdm) protein extract (as a model of allergic asthma), saccharopolyspora rectivirgula (sr) (as a model of hp/farmer's lung disease), or infected intranasally with wsn/flu virus (as a model of influenza infection). we observed significant percentages of il- -producing t cells in the lung tissue of mice exposed to hdm (figure a) , sr (figure c) , or wsn/flu virus ( figure e) . these il- -producing t cells all co-expressed high levels of lag and cd b, and include foxp + cd + , foxp − cd − , and cd + subsets in all disease models analyzed (figure ) . however, the relative proportions of il- -producing foxp + cd + , foxp − cd − , and cd + subsets differed in the various disease models (figure ) . in contrast to the composition of il- -producing lag + cd b + t cells induced by nb infection, in which the foxp − cd + subset is the majority ( % in figure d ), in hdm-induced allergic asthma model, the largest subset of the il- -producing lag + cd b + t cells in the lungs are foxp + cd + t cells ( %), followed by foxp − cd + t cells ( %), while cd + t cells are only around . % (figure b ). in the sr-triggered farmer's lung disease model, foxp + cd + t cells are the majority of the il- producing lag + cd b + t cell subset in the lungs, however, there are similar percentages of foxp − cd + and cd + t cells ( % each) (figure d) . strikingly in the murine model of influenza infection, cd + t cells are the largest subset of the il- -producing lag + cd b + t cells in the lungs ( %), followed by foxp − cd + t cells, while foxp + cd + are a minority ( figure f) . these data compared the composition of il- -producing lag + cd b + t cells in various murine models of pulmonary inflammatory diseases. along with the model of parasitic infection shown in figure , our data suggest that co-expression of lag and cd b marks all il- -producing t cell lineages in the pulmonary system, and relative abundance of the marked t cell subsets is dependent on the type of immune response as shown in the disease models. the composition of lag + cd b + as discussed above, we demonstrated that co-expression of lag and cd b is a generic feature of il- -producing t cells in vivo in pulmonary tissues under multiple inflammatory conditions (figures , ) . to determine whether this feature is applicable to il- -producing cells in other organs, we injected il- gfp /foxp rfp dual reporter mice with an anti-cd ε antibody that has been shown to stimulate pronounced il- production by t cells through tcr activation in vivo ( , ) . we analyzed il- -producing t cells in blood, lymph nodes (ln), lung, fat and small intestine, and found that coexpression of lag and cd b marked a portion of the il- producing t cells following tcr activation in vivo, which again included foxp + cd + , foxp − cd + , and cd + t cell subsets ( figure a ) in all organs analyzed. an interesting note is that the relative abundance of foxp + cd + , foxp − cd + , and cd + subsets among the il- -producing lag + cd b + t cells vary significantly in different organs of the same mice ( figure b) . upon tcr activation in vivo, foxp + cd + t cells are the major population that are il- + lag + cd b + in the blood, lymph nodes and lungs, while cd + t cells are the majority of il- + lag + cd b + t cells in the perigonadal fat and small intestine ( figure b) . these data suggest that co-expression of lag /cd b marks all three il- -producing t cell subsets in figure | composition of il- -producing lag + cd b + t cells in lung inflammatory and infectious disease models. all experiments were performed using mice carrying the il- gfp /foxp rfp dual reporter system, and cells were isolated from the lungs and gated on live singlets for analysis as shown in figure a . representative facs plots of lag /cd b co-expression by il- -producing cells, and among these cells, ratio of cd + vs. cd + t cells, and foxp − vs. foxp + cd + subpopulations are shown, followed by pie chart showing the proportions of cd + foxp + , cd + foxp − , and cd + t cells in il- -producing lag /cd b double positive t cell population. mice were exposed intranasally to: (a,b) house dust mite (hdm) protein extract for consecutive days, and analyzed h post the last treatment; (c,d) saccharopolyspora rectivirgula (sr) for consecutive days every week for weeks, and analyzed in the end of the fourth week; (e,f) influenza a (wsn) viruses, and analyzed dpi. n ≥ , combined from three independent experiments. data presented as mean ± s.e.m. multiple organs and the relative abundance of the foxp + cd + , foxp − cd + and cd + t cell subsets in il- -producing lag + cd b + t cells is dependent on the anatomical location of the cells. human il- -producing cd + and cd + t cells exhibit a lag + cd b + phenotype in murine models, we demonstrated that co-expression of lag and cd b marks il- -producing foxp + cd + , foxp − cd + , and cd + t cells under different inflammatory conditions in the lungs (figures , ) , as well as in different organs when tcr is activated in vivo (figure ) . to further determine whether different subsets of il- -producing t cells exhibit the shared feature of co-expression of lag and cd b in humans, we isolated cd + and cd + t cells from human peripheral blood and cultured them under il- -inducing conditions. we found that as in the mouse, human il- producing foxp + cd + , foxp + cd + , and cd + subsets all up-regulated both lag and cd b expression, with a significant lag /cd b double positive population (figure ). our data presented in this report demonstrated that, as previously reported ( ) , co-expression of lag and cd b identifies foxp − il- high tr cells, but that these markers are not exclusive for tr cells in human and mouse. furthermore, we find that the il- -producing lag + cd b + t cell population is composed of foxp + cd + , foxp − cd + , and cd + t cell subsets, and this composition varies depending on the disease conditions and anatomical locations of the cells. the importance of this work is emphasized by the need to specially identify foxp − tr cells, especially under clinical conditions, and our findings suggest that the use of lag and cd b should be combined with other markers to uniquely identify these cells. using tr cell clones derived from human naïve cd + t cells and purified by an il- secretion assay, gagliani, roncarolo and colleagues identified co-expression of lag , cd b, and cd as cell a surface signature of il- -producing cd + t cells, and demonstrated that co-expression of lag and cd b is sufficient to distinguish foxp − il- high tr cells from t helper and/or regulatory subsets that expressed lower levels of or no il- in both mouse and human ( ) . however, it was recently reported that il- -producing t cells derived from human cd + memory t cells exhibit low levels of surface expression of lag and cd b ( ) , suggesting that the pattern of co-expression of lag and cd b may vary in human il- -producing cd + t cells, which may be associated with whether they were derived from naïve precursors vs. memory cells. indeed, it is clear that co-expression of lag and cd b can mark il- -producing tr -like cells, and serve to help eliminate those t cells lineages that are not capable of producing il- for potential clinical interest. however, other non-tr -like il- -producing t cells can also co-express these markers. whether co-expression of lag and cd b allows efficient recovery of il- high t cells may be dependent on the proportion of il- high t cells that are co-expressing lag and cd b, which may differ depending on whether the relevant il- -producing cells had different origins and/or underwent different activation regimes. both lag and cd b can individually be up-regulated in activated t cells, regardless of their production of the anti-inflammatory il- or pro-inflammatory cytokines ( ) ( ) ( ) ( ) ( ) ( ) . co-expression of lag and cd b is more restricted to il- -producing subsets, as previously described in foxp − cd + t cells ( ) . our data here demonstrated that this is a generic feature of il- -producing cells, including foxp − cd + , foxp + cd + , and cd + subsets. the cooccurrence of il- production and lag + cd b + may be explained in two ways. the first explanation is that il- stimulation through autocrine and/or paracrine may induce the expression of lag and cd b in the stimulated cells, therefore, il- -capturing t cells [cells detected by roncarolo's group in ref ( ) ] exhibited high levels of lag /cd b coexpression. this hypothesis would place il- up-stream of lag and cd b expression, which is unlikely, as recent studies by flavell and huber's groups showed that the il- receptor is dispensable for tr cell differentiation, including the lag /cd b double positive feature; instead, il- receptor signaling is critical for maintaining the cell fate commitment and functional performance of the differentiated tr cells ( ) . therefore, it is more likely that lag and cd b signaling pathways function cooperatively to activate the expression of il- . this hypothesis is more reasonable, given our data that lag and cd b co-expression is a generic feature of il- producing cells in multiple subsets. lag is a structural homolog of the cd molecule, and can bind to mhc class ii with higher affinity than cd ( , ) . in unstimulated t cells, lag is retained in the intracellular compartment and degraded in the lysosome. upon t cell activation, lag traffics from the lysosomal compartment to the cell surface through a protein kinase c (pkc) dependent pathway ( ) . cd b is the integrin α subunit and plays a critical role in cell-cell interaction and adhesion ( ) . the cd b pathway activates multiple downstream effector signaling pathways, among which is the ras/mapk signaling cascade ( , ) . we recently reported that the ras/mapk signaling pathway functioning downstream of the tcr is indispensable for il- production by foxp − cd + cells, through activation of the expression of the transcription factor interferon regulatory factor (irf ) ( ) . pkc can regulate ras signaling to downstream effectors, and can activate mapk signaling in both ras-dependent and independent manners ( , ) . the interplay between pkc and ras may regulate signals that connect lag and cd b downstream pathways, leading to up-regulation of il- . given the complexity of these pathways, comprehensive understanding of the mechanism(s) underlying the co-expression of these markers with il- will require significantly more in-depth analyses. regardless of the mechanism, our findings indicate that lag /cd b co-expression does not uniquely identify foxp − tr cells, but is a more general indicator of il- production in t cell lineages. despite the shared feature of co-expression of lag and cd b by il- -producing foxp + cd + , foxp − cd + , and cd + t cells, we also observed interesting discrepancies in the proportional composition of these three il- high t cell subsets that are all lag /cd b double positive in the lung mucosa of different pulmonary inflammatory disease models, as well as in different anatomical locations in the same mice upon tcr activation in vivo. for example, parasite infection (nb) and mite allergen (hdm) induced predominantly il- producing lag + cd b + t cells that are cd + with very few that are cd + . in the nb infection model, the majority of il- + lag + cd b + cd + t cells are foxp − tr cells, while in hdm-exposed mice, foxp + treg cells are the majority. in the bacterial exposure (sr) and viral infection (flu) models, we observed an increased proportion of il- + lag + cd b + t cells that are cd + , which is the predominant population in the flu model ( figure f ). this discrepancy in the composition of il- -producing lag + cd b + t cells may be due to the difference of the microenvironment in which the il- producing cells are being induced. factors that may affect the different composition of the il- high lag + cd b + t cells may include the type of immune response, abundance and affinity of the tcr ligands, the cytokines induced and orchestrated by the stimuli, and the nutritional microenvironments that favor different subsets of the t cells. for example, type i ifn can facilitate the preferential induction of il- -producing effector cd + t cells through inducing and sustaining expression of the irf and blimp transcription factors ( ) . type i interferon is significantly elevated during influenza infection ( ) but much less so by hdm exposure ( ) . another possible orchestrator could be the cytokine il- , which has been reported to be directly required for il- induction in cd + t cells ( ) and cd + foxp − ( ) but not cd + foxp + t cell subsets ( ) . transcription factors and their interacting molecular networks that exhibit differential functions for il- induction in different t cell lineages might provide an answer as well. for example, blimp- is indispensable for il- induction in foxp + and foxp − cd + t cells ( , ) , as well as in cd + t cells ( ) . ahr interaction with cmaf is critical in il- induction in foxp − tr cells in response to an il- -supplemented environment, but is insufficient in inducing il- production in foxp + treg cells under the same conditions ( ); ahr expression was not critical in il- -producing cd + t cells either ( ) , suggesting that ahr signaling has a multifaceted function in regulating the level of il- expression in different t cell lineages. a more comprehensive understanding of the t cellintrinsic molecular features that are shared or distinct among the il- -producing cd + , foxp − cd + and foxp + cd + t cells awaits further investigation. our data reported here demonstrate that co-expression of lag and cd b marks il- high t cell subsets that are foxp + cd + , foxp − cd + , or cd + in both human and mouse, and thus does not uniquely identify foxp − tr cells. however, this finding does not negate the feasibility of utilizing co-expression of lag and cd b in marking a broader range of immunosuppressive il- high t cell populations that have potential therapeutic effects for clinical application. further investigations are required to determine the levels of regulatory and pro-inflammatory cytokine production in the bulk lag + cd b + t cells, and to determine and compare the ability of the foxp + cd + , foxp − cd + , and cd + subsets of the lag + cd b + t cells in suppressing effector immunity and inflammation in vivo. wh and aa conceived research, designed experiments, analyzed and interpreted data, and wrote the manuscript. wh, ss, and cc performed experiments; s-gz contributed reagents and intellectual input. potential role of interleukin- -secreting regulatory t cells in allergy and asthma interleukin- -deficient mice develop chronic enterocolitis in the absence of endogenous il- , mice acutely infected with toxoplasma gondii succumb to a lethal immune response dependent on cd + t cells and accompanied by overproduction of il- , ifn-gamma and tnf-alpha a defect in interleukin- leads to enhanced malarial disease in plasmodium chabaudi chabaudi infection 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nonpathogenic bacteria alleviating atopic dermatitis inflammation induce il- -producing dendritic cells and regulatory tr cells il- and regulatory t cells cooperate in allergen-specific immunotherapy to ameliorate allergic asthma birch pollen immunotherapy leads to differential induction of regulatory t cells and delayed helper t cell immune deviation suppression of autoimmune inflammation of the central nervous system by interleukin secreted by interleukin -stimulated t cells cd /major histocompatibility complex class ii interaction analyzed with cd -and lymphocyte activation gene- (lag- )-ig fusion proteins characterization of the major histocompatibility complex class ii binding site on lag- protein cd +cd -lag + regulatory t cells controlled by the transcription factor egr- lag- , a novel lymphocyte activation gene closely related to cd lag- expression defines a subset of cd (+)cd (high)foxp (+) regulatory t cells that are expanded at tumor sites cutting edge: the mouse nk cell-associated antigen recognized by dx monoclonal antibody is cd b (alpha integrin, very late antigen- ) immature dendritic cells suppress collagen-induced arthritis by in vivo expansion of cd b+ regulatory t cells alpha beta integrin is the major collagen-binding integrin expressed on human th cells functional specialization of memory th cells revealed by expression of integrin cd b expression of interleukin- in intestinal lymphocytes detected by an interleukin- reporter knockin tiger mouse identifying foxp -expressing suppressor t cells with a bicistronic reporter itk signalling via the ras/irf pathway regulates the development and function of tr cells th -polarized immune response in a murine model of hypersensitivity pneumonitis and lung fibrosis dendritic cell-mhc class ii and itk regulate functional development of regulatory innate memory cd + t cells in bone marrow transplantation the aryl hydrocarbon receptor interacts with c-maf to promote the differentiation of type regulatory t cells induced by il- an essential role for th -type responses in limiting acute tissue damage during experimental helminth infection regulatory t cells and il- in allergic inflammation interleukin- modulates the severity of hypersensitivity pneumonitis in mice tr -like t cells -an enigmatic regulatory t cell lineage il- receptor signaling is essential for tr cell function in vivo trafficking of lag- to the surface on activated t cells via its cytoplasmic domain and protein kinase c signaling the role of integrins alpha beta and alpha beta in cell-cell and cell-substrate adhesion of human epidermal cells integrin-mediated ras-extracellular regulated kinase (erk) signaling regulates interferon gamma production in human natural killer cells integrin signalling during tumour progression regulation of ras signaling specificity by protein kinase c protein kinase c activates the mek-erk pathway in a manner independent of ras and dependent on raf type i ifn signaling facilitates the development of il- -producing effector cd (+) t cells during murine influenza virus infection tlr stimulation in human plasmacytoid dendritic cells leads to the induction of early ifn-inducible genes in the absence of type i ifn type i interferon is required for t helper (th) induction by dendritic cells persistent loss of il- responsiveness in cd + memory t cells abrogates il- expression in a recall response cutting edge: il- induces the transcription factor c-maf, cytokine il- , and the costimulatory receptor icos that coordinately act together to promote differentiation of il- -producing tr cells tregspecific il- ralpha deletion uncovers a key role for il- in treg function to control autoimmunity the transcription factors blimp- and irf jointly control the differentiation and function of effector regulatory t cells role of blimp- in programing th effector cells into il- producers cd + t cell help and innate-derived il- induce blimp- -dependent il- production by antiviral ctls highly activated cytotoxic cd t cells express protective il- at the peak of coronavirus-induced encephalitis we thank a. redko for animal care and l. zhang the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © huang, solouki, carter, zheng and august. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - mzsaz a authors: wium, martha; jonker, hester isabella; olivier, adriaan jacobus; bellstedt, dirk uwe; botes, annelise title: dna vaccines against mycoplasma elicit humoral immune responses in ostriches date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: mzsaz a in ostriches, the population densities resulting from intensive rearing increases susceptibility to pathogens such as mycoplasmas. in addition to good management practices, vaccination offers an attractive alternative for controlling mycoplasma infections in food animals, instead of using antibiotics, which often leave unacceptable residues. the use of live attenuated vaccines, however, carry the concern of reversion to virulence or genetic recombination with field strains. currently there are no commercially available vaccines against ostrich-infecting mycoplasmas and this study therefore set out to develop and evaluate the use of a dna vaccine against mycoplasma infections in ostriches using an oppa protein as antigen. to this end, the oppa gene of “mycoplasma nasistruthionis sp. nov.” str. ms was cloned into two dna vaccine expression vectors after codon correction by site-directed mutagenesis. three-months-old ostriches were then vaccinated intramuscularly at different doses followed by a booster vaccination after weeks. the ability of the dna vaccines to elicit an anti-oppa antibody response was evaluated by elisa using the recombinant oppa protein of ms as coating antigen. a statistically significant anti-oppa antibody response could be detected after administration of a booster vaccination indicating that the oppa protein was successfully immunogenic. the responses were also both dose and vector dependent. in conclusion, the dna vaccines were able to elicit an immune response in ostriches and can therefore be viewed as an option for the development of vaccines against mycoplasma infections. the ostrich (struthio camelus var. domesticus) is the largest living, flightless bird species belonging to the ratite group ( ) . although ostriches are traditionally endemic to africa, parts of arabia and the middle east ( ), they are reared in several countries across the world. south africa, however, is currently the largest producer of ostrich products (meat, leather, and feathers) on international markets ( ) . here, the majority of ostriches destined for slaughter are reared in free-range systems, but when environmental conditions are unfavorable for grazing, they are reared in grow-out camps from about months of age ( ) . mycoplasma, a genus of bacteria that belongs to the prokaryotic class mollicutes, is known to be highly infectious in these intensive rearing systems ( , ) . characteristic features of mycoplasma include the lack of a cell wall, small at-rich genome and a minimal set of genes ( , ) . the species found to specifically infect ostriches are "mycoplasma struthionis sp. nov." str. ms , mycoplasma sp. str. ms , and "mycoplasma nasistruthionis sp. nov." str. ms ( , ) . they are typically associated with upper respiratory tract infections ( ) , reduced growth rates, downgrading of carcasses and in extreme cases, chick mortalities ( , ) . the severity of infections increases with exposure to environmentally induced stress, other bacterial and viral infections. of the three mycoplasma species, "mycoplasma nasistruthionis sp. nov." str. ms was found to have the highest prevalence ( ) amongst ostriches and therefore was the focus of this study. mycoplasma infections are currently managed using a combination of biosecurity systems and antibiotics. with antibiotics there is the risk of resistance ( ) as well as an accumulation of residues in meat with concomitant risks for consumers ( ) . development of whole organism vaccines for use in ostriches is limited by the slow-growing nature of these organisms and the cost of medium required to sustain growth. dna vaccines, on the other hand, do not require large scale cultivation of the pathogen, can be produced at relatively low cost and are more temperature stable than other vaccines ( , ) . it is therefore an attractive alternative for use in ostriches where the vaccine market is small, relative to e.g., poultry, and ostriches are usually farmed in semi-arid or arid regions where temperatures are frequently high and cold storage of vaccines can be problematic. similar to live vaccines, they are also able to stimulate humoral and cellular immunity as indicated by measuring antibody, t-helper cell and cytotoxic t-lymphocyte responses ( ) . but unlike live vaccines cannot revert back to an infectious form ( ) . el gazzar et al. ( ) has reported the reversion to virulence of a commonly used live mycoplasma vaccine in poultry, ts- , whilst another has reported possible genetic recombination between live vaccines and field strains upon long term use ( ) . this exchange or transfer of genes could have a negative impact on either the pathogenicity or transmissibility of field strains. chromosomal integration of dna vaccines is of some concern, but preclinical and clinical studies have shown the rate of integration of plasmid dna into the host genome to be lower than that of spontaneous mutations ( , ) . to date, dna vaccines have not been tested in ostriches or any other ratites. the aim of this study was to use the oppa protein of "m. nasistruthionis sp. nov." str. ms as antigen in the development and evaluation of dna vaccines in ostriches at different doses and using different vectors. a dna vaccine is a plasmid expression vector containing a gene that codes for a protein antigen. as a result of their parasitic lifestyle ( ), mycoplasmas possess, and rely on, a wide range of transmembrane transport systems for their survival. the extracellular components of these transporters are ideal targets for vaccine development ( , ) . in this study the extracellular oppa domain of an oligopeptide permease (opp) transporter was chosen as protein antigen ( , ) . using mouse models, oppa has to date been evaluated as antigen in subunit vaccines against brachyspira pilosicoli ( ) , moraxella catarrhalis ( ) , and yersinia pestis ( ) , as well as against haemophilus parasuis in pigs ( ) . oppa has, however, not been evaluated in any organism as part of a dna vaccine. in this study we report, for the first time, that a dna vaccine can elicit a humoral immune response in ostriches using oppa as antigen. this response was both dose and vector dependent. site-directed mutagenesis of the oppa gene cultures of "m. nasistruthionis sp. nov." str. ms (genbank: km . ) were obtained from mr j.j. gouws (faculty of veterinary science, onderstepoort, university of pretoria). genomic dna (gdna) was isolated from these cultures using a method described by hempstead ( ) . the type a oppa gene ( ) pci-neo (promega) and vr (vical inc.) were chosen as vaccine vectors (supplementary figure ) . the pci-neo vector is a mammalian expression vector that contains a cmv enhancer/promoter region, chimeric intron and sv late polyadenylation signal sequence. the vr vector also contains a cmv enhancer/promoter region, but in combination with a cmv intron, tissue plasminogen activator (tpa) signal peptide sequence and a bovine growth hormone polyadenylation signal sequence. the mutated oppa gene was sub-cloned into each vector using restriction digestion. for pci-neo, acci and mlui (fastdigest, thermo scientific) and for vr , bamhi (fermentas) restriction enzymes were used according to the manufacturer's instructions. cultures of the pci-neo_oppa and vr _oppa vaccine plasmid were prepared and the respective plasmids purified with an endotoxin-free plasmid dna purification kit (nucleobond r xtra midi plus ef, macherey-nagel, germany). yields were determined using a nanodrop spectrophotometer. prior to large scale plasmid isolation for vaccination, the nucleobond kit's ability to yield supercoiled pci-neo_oppa and vr _oppa plasmids was evaluated by digesting plasmids with the acci and bamhi restriction enzymes, respectively, followed by electrophoresis on a % (w/v) agarose gel. to prepare a sufficient amount of plasmid for vaccination, the plasmids were prepared in batches over several days. the purified plasmids were again evaluated as before to confirm the integrity and supercoiling of the plasmids prior to being used for vaccination. the isolated plasmids were diluted to , , and , µg/ml with sterile pbs ( mm nacl, . mm kcl, mm na hpo , and . mm kh po , ph . ), respectively, a day before use. dilutions were prepared under aseptic conditions, in sterile serum glass bottles, closed with inert silicone stoppers and sealed with a tear away center aluminum cap (sigma-aldrich). ethical approval was obtained from the stellenbosch university animal ethics committee (su-acum - ) as well as the south african department of agriculture, forestry and fisheries (reference: / / / / ) in terms of section of the animal disease act (act no. of ). a group of ostrich chicks of ± -months-old were randomly selected from a chick rearing unit in the fraserburg district (northern cape province, south africa). since fraserburg is outside the major commercial ostrich production region, this limits pathogen exposure during the critical - months-old rearing phase ( ) . as per standard practice, upon reaching a weight of about kg (± months of age), trial ostriches were transported back (± km) to a commercial ostrich farm in the oudtshoorn district (western cape province, south africa). three-month-old chicks were chosen for vaccination to allow sufficient time for a primary immune response to develop before being transported to a higher stress environment with concomitant increase in pathogen load and increased risk of exposure to mycoplasma. the chicks were quarantined for days after relocation and random testing was performed for avian influenza. this is required by avian influenza control measure before ostriches can be released and allowed to mix with other populations on any farm to which they have been transported. at the start of the trial, each ostrich was tagged with a unique number for identification under the right wing, in accordance with standard ostrich farming practices. the trial was conducted under similar conditions to which a commercial vaccine would be administered and therefore trial ostriches were at all times housed and treated in the same manner as non-trial ostriches on the farms. in fraserburg, the chick rearing units were open air camps ( , m ) with - chicks per camp. in oudtshoorn, ostriches were kept in open air grow-out camps ( , m ) with - ostriches per camp. the ostriches received food and water ad libitum and were only handled by trained and experienced farm personnel. trial birds were not kept separately but grouped with other ostriches of the same weight and age. the ostriches were randomly allocated to seven treatment groups of ostriches each. the pci-neo_oppa and vr _oppa vaccine groups were vaccinated at a dose of , , and , µg, respectively. the last group was the control group that did not receive any vaccine. the dose typically administered to poultry ranges from . to µg when injected intramuscularly, with a booster dose after - weeks ( - ). dunham ( ) indicated that larger animals may require a larger dose of - , µg, but that this needs to be optimized for individual vaccines since the dose required is influenced by the target species, its size and the efficiency of plasmid delivery. the different doses used in our study were therefore selected to compensate for the increase in weight of the ostriches during the course of the trial and the fact that no adjuvant was used in the vaccine formulation to limit possible skin reactions which can impact hide quality. vaccine doses were administered in a single ml volume at week and a booster injection administered at week by intramuscular injection in the upper thigh. blood samples ( ml) were drawn from the jugular vein using gx / ′′ needles (vacuette r ) and collected in vacuette r z serum sep clot activator tubes at week , , , and . serum was separated by centrifugation at low speed for min and stored at − • c. week and samples were collected in fraserburg, and week and samples in oudtshoorn. the ostriches were moved from fraserburg to oudtshoorn days prior to the week sampling and booster injections were therefore administered during the quarantine period. ostriches were monitored after vaccination for any adverse reactions. for analysis of anti-oppa antibody responses, recombinant oppa protein was produced for use as coating antigen in an enzyme-linked immunosorbent assay (elisa). to this end, the sdm corrected oppa gene was pcr amplified and sub-cloned into a pgex- t- vector (ge healthcare life science, uk) using restriction digestion. primer sequences with bamhi and noti restriction sites are shown in supplementary table . for protein expression, the pgex- t- _oppa plasmid was transformed into escherichia coli bl (de )plyss cells (promega) and freezer stocks prepared. an overnight culture (from a freezer stock) and expression cultures were prepared using terrific-broth (tb) medium. expression of the glutathione s-transferase (gst) oppa fusion protein was induced at an od between . and . by the addition of . mm iptg, and cells harvested at and h by centrifugation ( , × g at • c). the cells were resuspended in xten extraction buffer [ mm tris-hcl, mm edta, mm nacl, . % triton x- , . m dithiothreitol and % glycerol (v/v)] at µl extraction buffer per ml culture used for centrifugation. protease inhibitor was also added to the ten buffer ( tablet per ml extraction solution of complete ultra tablets, mini, easypack tablet, roche). the gst-oppa protein was isolated using glutathioneagarose chromatography (sigma-aldrich) under gravity flow at • c according to the manufacturer's instructions. a sample ( ml) was prepared for loading of the column by treating the resuspended pellets with three freeze-thawing cycles ( min at • c followed by min at − • c) and five cycles of s sonication followed by min on ice. the samples were then triturated three times through a gx / ′′ needle (avacare) into a ml injekt-f syringe (bbraun). this was repeated using a gx / " needle (nipro) followed by centrifugation at , × g for min ( • c) to obtain a clear supernatant which was loaded onto the column under gravity flow at • c. fractions ( ml) were collected and their protein concentration determined using a modified bradford assay ( ) . expression and isolation products were analyzed using sds-page ( ) and western blot analysis ( ) . microtiter plates (maxisorp, nunc) were coated overnight at • c with µl of recombinant gst-oppa protein diluted to µg/ml in carbonate buffer ( mm nahco , ph . ). to block non-specific binding, µl/well casein buffer ( mm tris-hcl, mm nacl, . % casein and . % thiomersal, ph . ) was added and the plate incubated for h at • c. serum samples were diluted : with casein-tween [casein buffer containing . % (v/v) tween r ] and µl/well added to the plate in triplicate before incubation for h at • c. biotinylated rabbit anti-ostrich ig polyclonal antibodies, prepared as previously described ( ), were next added at a dilution of : in casein-tween and incubated for h at • c. a streptavidin horseradish peroxidase (hrp) conjugate mixture [ ml streptavidin hrp (invitrogen), ml . % casein buffer, and ml % glycerol], diluted : with casein-tween, was then added ( µl/well) and the plate incubated for h at • c. finally, µl/well substrate solution ( . mg/ml abts, . µl/ml h o , . m citrate buffer, ph ) was added and absorbance measured at nm with a thermo scientific multiskan ex plate reader after min incubation at • c. after each incubation step the content of the plate was first decanted followed by washing five times with pbs-tween [ mm nacl, . mm kcl, mm na hpo and . mm kh po , ph . , . % (v/v) tween r ] and washing three times with milli-q r water. between the coating and blocking steps washing was not applied. a first-row column blank (containing all components except ostrich serum) was used on each plate to blank absorbance values. all plates contained controls to monitor plate to plate variation. the controls were serum samples representing the week , , , and sampling points of a single ostrich, randomly selected from the pci-neo_oppa , µg group based on high titers produced after vaccination. prior to the analysis of samples, the elisa was optimized with regard to coating concentration ( - µg/ml), serum dilution ( : - : ) as well as number of washing cycles between steps. non-specific binding of anti-oppa antibodies during elisa analysis was evaluated by coating the wells with carbonate buffer ( µl/well) containing no capture antigen and serum from a vaccinated bird that gave a high absorbance value at a dilution of : when using the gst-oppa protein as capture antigen. the absorbance values obtained are referred to as elisa titres, which are a measurement of the antibody levels produced when using a serum dilution of : in the elisa. the weight of trial birds was monitored and recorded at weeks , , and . to determine the possible influence of existing mycoplasma infections on immune response data, trachea swabs were collected at weeks , , , and and tested for the presence of mycoplasma infections by pcr. birds were, however, not excluded from the trial if they tested positive since ostriches are often naturally infected with mycoplasmas. swab samples were collected using dry sterile swabs (copan) which were then rinsed in µl sterile pbs buffer. the eluate was tested by pcr using a generic primer pair specific for the mycoplasma genus ( ). positive samples were further evaluated for the presence of the ostrich-infecting mycoplasmas ms , ms , and ms ( ). statistical analysis of the elisa titer data was performed using the general linear model (glm) procedure in the agrobase generation ii r (agronomix software inc.) software. analysis of variance (anova) and least significant difference (lsd) values were calculated at a significance level of . . the bp oppa gene was successfully amplified from the ms gdna and cloned into a pgem r -t easy vector as confirmed by sequencing. using sdm, all mycoplasma tga codons in this plasmid were successfully mutated to universal tgg codons in seven consecutive steps of sdm as confirmed by sequencing (supplementary figure ) . the mutated oppa gene was successfully sub-cloned into the pci-neo and vr vaccine vector as confirmed by sequencing (supplementary figure ) . large scale production of the dna vaccines was achieved and approximately mg of plasmid was isolated for each of pci-neo_oppa and vr _oppa. the / absorbance ratio of the isolated plasmids ranged from . to . . the integrity of the isolated plasmids was confirmed with agarose electrophoresis and most of the pdna was in the required supercoiled conformation. a supercoiled conformation has a higher transfection rate into mammalian cells, is less susceptible to intracellular degradation and is more effective at inducing an immune response than other plasmid conformations ( , ) . ostriches were vaccinated with the prepared dna vaccines and no adverse reactions were observed at the injection sites (redness, swelling, inflammation, or allergic reaction) during and after the trial. ostriches resumed normal behavior such as eating, walking around and exploring immediately after vaccination (no lameness or loss of appetite). sub-cloning of the mutated oppa gene into the pgex- t- vector was successful as confirmed by sequencing frontiers in immunology | www.frontiersin.org (supplementary figure ) . sds-page and western blot analyses confirmed that the recombinant oppa protein was expressed successfully as an n-terminal gst-fusion protein with the predicted size of kda ( kda due to the gst tag) (figures a,b) . bradford analysis indicated that the oppa protein was eluted between fraction and with the highest concentration obtained in the th fraction. ostriches with more than two missing sampling points were excluded from elisa and subsequent statistical analysis. there was no non-specific binding of the anti-oppa antibodies in ostrich serum and the plate control samples indicated limited plate to plate variation. the titer values resulting from vaccination with the pci-neo_oppa and vr _oppa vaccines are shown in figures a,b , respectively. the titer values of the control group, which received no vaccine, did not show significant variation over time. however, there was a slight increase in the average titer at week . vaccination with the pci-neo_oppa vaccine (figure a ) resulted in significant treatment x time interactions (p = . ), but only the and µg doses resulted in average elisa titers that differed significantly from the control. this was also only at week after a booster vaccination was administered. the average titers of the different doses were, however, not significantly different from one another. vaccination with the vr _oppa vaccine (figure b ) resulted in a significant treatment × time interaction (p < . ) and all three doses resulted in average elisa titers that differed significantly (lsd = . ) from the control. this was again only at week after a booster vaccination was administered and all the doses differed significantly from one another. the vaccinated groups as well as the control group gained weight from weeks to , but from weeks to there was an average weight loss of . kg ( table ) . there was, however, no statistically significant difference between the treatment groups and the control group over the weeks period (p = . for pci-neo_oppa and p = . for vr _oppa). the presence of mycoplasma infections was monitored with pcr during the trial ( table ) . mycoplasma infections could not be detected in any of the groups at weeks , , and . infections were, however, detected at week . at this point the ostriches had been in oudtshoorn for days, of which days were out of quarantine. in the groups that received the vr _oppa vaccine the highest percentage of infections at week was due to ms ( . %) followed by ms ( . %) and ms ( . %) ( table ) . compared to this, the groups that received the pci-neo_oppa vaccine had fewer mycoplasma infections. amongst these the highest percentage of infections was again due to ms ( . %) followed by ms ( . %) and ms ( . %). for both vaccine groups, some of the birds were infected by more than one of these mycoplasma species. the only group that had no pcr-detectable mycoplasma infections at week was the group that had received , µg of the vr _oppa vaccine and the control group. in this study, dna vaccines were developed for ostriches using the oppa gene of an ostrich-infecting mycoplasma (ms ) as vaccine antigen. the expression vectors used (pci-neo and vr ), were selected based on dna vaccine studies in other birds, and on their immunostimulatory characteristics ( ) ( ) ( ) . after vaccination of ± -month-old chicks a statistically significant anti-oppa antibody response could not be detected at week , although a general trend of increases in the average titer values was observed from week to for groups that received the vr _oppa vaccines. based on previous studies using inactivated vaccines in ostriches ( , ) , we expected a primary immune response about weeks after the first vaccination (i.e., between weeks and ). after administering a booster vaccination, both dna vaccines were able to elicit a statistically significant anti-oppa antibody response. as these responses against oppa were significant in comparison to the negative responses following the first vaccinations, this is evidence of a secondary immune response as a result of immune memory. if memory cells are produced after the initial contact with an antigen, subsequent exposure to the same antigen will allow the host to recognize the antigen faster, and with greater magnitude ( , ) . the responses elicited by the dna vaccines were, however, dose dependent as well as vector dependent since different results were produced by each vaccine when using the same dose. this highlights a possible role of the vaccine vector in the observed antibody responses. the vr _oppa vaccine, on average, elicited higher titer values compared to the pci-neo_oppa vaccine, which might be due to the tpa signal sequence of the vr plasmid, which is situated upstream of the oppa gene, and assists with protein expression in mammalian cells and export of the protein out of the cell. this would in turn increase the immunogenicity of the antigen ( ) . in addition to this, sequences in the plasmid backbone have intrinsic immunostimulatory activity which enhance the immune system's response to the expressed protein antigen ( , ) . over the whole vaccination trial, the health of the ostriches was monitored by recording weight gain. all groups gained weight up to week , but toward week their weight gain decreased. given that this trend was also observed for the control group ostriches, the weight loss cannot be ascribed to vaccination. the decrease in weight did, however, coincide with the movement of the birds to a new location. ostriches are known to be severely affected by stress and during this period of adapting to changes in social dynamic and housing environment, reduction in weight gain and even weight loss is normal amongst farmed ostriches ( ) . the presence of existing mycoplasma infections at the start of the trial, as well as changes in infection status during the trial, were also monitored by pcr analysis of trachea swabs. mycoplasma infections could only be detected at week , which was after they were moved and exposed to the farm environment in oudtshoorn for more than days. the absence of detectable mycoplasma infections at the earlier time points may be ascribed to fraserburg being outside of the commercial ostrich production region. the oudtshoorn district, on the other hand, has a higher incidence of mycoplasma infections especially during seasonal changes and our trial was conducted during late autumn. amongst the vaccinated groups, the number of detected ms and ms infections were low compared to ms , with only the µg vr _oppa and control group having no detectable ostrichinfecting mycoplasmas. in future trials, careful consideration should be given to the timing of the primary and booster vaccination relative to the time of introduction to growout conditions. it is possible that the mycoplasma infections had an influence on the observed antibody responses. in a study done by yang et al. ( ) using recombinant oppa protein as subunit vaccine against m. catarrhalis in mice, it was found that the elisa titer values were already raised at the start of the vaccination trial which they ascribed to the presence of existing systemic antibodies to oppa as a result of existing infections. despite the possible presence of antibodies due to mycoplasma infections, this did not mask the detection of a dose dependent antibody response to the oppa protein of ms during vaccination. in conclusion, this is the first study to show that dna vaccines are able to elicit an antibody response in ostriches against a mycoplasma antigen in spite of ostriches being prone to environmentally induced stress conditions that can suppress immune function. the mycoplasma oppa protein was sufficiently immunogenic to induce an anti-oppa antibody response and this response was firstly, dose dependent and secondly, required a booster vaccination. further trials are required to determine the extent of protection provided by this mycoplasma antigen relative to the timing of the primary and booster vaccination before introduction to grow-out conditions. this study (field trial) was carried out in accordance with the recommendations of the south african department of agriculture, forestry and fisheries (reference: / / / / ) in terms of section of the animal disease act (act no. of ) . the protocol was approved by the stellenbosch university animal ethics committee (su-acum - ). mw performed the sdm, prepared the dna vaccine and expression plasmids, optimized the expression of the recombinant oppa protein, and the initial optimization of the elisa. hj further optimized the elisa and evaluated the anti-oppa antibody responses during the vaccination trial. ao assisted with the vaccine trial. mw wrote the manuscript with support from ab. ab supervised the project with the assistance of db and ao. ab and db conceived the original idea. all authors provided critical feedback and helped shape 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expression and immunogenicity of m. tuberculosis ag . microbes infect measuring the effects of an ever-changing environment on malaria control comparison of five expression vectors for the ha gene in constructing a dna vaccine for h n influenza virus in chickens growth rate and hatching date in ostrich chicks reflect humoral but not cellmediated immune function generating memory with vaccination vaccination to gain humoral immune memory effect of plasmid backbone modification by different human cpg motifs on the immunogenicity of dna vaccine vectors cpg dna as a vaccine adjuvant the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © wium, jonker, olivier, bellstedt and botes. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -du heorx authors: ho, thuong thi; nguyen, giang thu; pham, ngoc bich; le, van phan; trinh, thi bich ngoc; vu, trang huyen; phan, hoang trong; conrad, udo; chu, ha hoang title: plant-derived trimeric co- k-equivalent epitope induced neutralizing antibodies against porcine epidemic diarrhea virus date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: du heorx porcine epidemic diarrhea virus (pedv) is a causative agent of a highly infectious disease with a high mortality rate, especially in newborn piglets in asian countries resulting in serious economic loss. the development of a rapid, safe, effective and cost-efficient vaccine is crucial to protect pigs against pedv infection. the coe antigen is regarded to be a major target for subunit vaccine development against pedv infection. the naturally assembled coe protein forms a homotrimeric structure. in the present study, we successfully produced a trimeric coe protein as a native structure by fusion with the c-terminal isoleucine zipper trimerization (gcn pii) motif in nicotiana benthamiana, with a high expression level shown via semi-quantified western blots. trimeric coe protein was purified via immobilized metal affinity chromatography (imac), and its trimeric structure was successfully demonstrated by a cross-linking reaction, and a native page gel. a crude extract containing the coe trimer was used for evaluating immunogenicity in mice. after and booster immunizations, the crude extract containing trimeric coe elicited elevated pedv-specific humoral responses, as demonstrated by elisa and western blot analyses. notably, a virus-neutralizing antibody assay indicated that the neutralization activities of sera of mice vaccinated with the crude extract containing coe-gcn pii were similar to those of mice vaccinated with a commercial vaccine. these results suggest that crude extract containing trimeric coe is a promising plant-based subunit vaccine candidate for pedv prevention. porcine epidemic diarrhea (ped) is a highly infectious disease identified by dehydration, acute watery diarrhea, and a high mortality rate, especially in newborn piglets ( ) ( ) ( ) . pedv, the disease causative agent of ped, spreads to several countries in the world, and resulting in serious economic loss to the swineproduction ( ) ( ) ( ) . the pedv genome comprises five open reading frames (orfs) encoding four structural proteins [the spike (s), envelope, membrane and nucleocapsid proteins] and three non-structural proteins [the replicases orf a and b, and orf ; ( , ) ]. among the structural proteins, the s protein locating on the surface of pedv virion, plays a key role in the attachment of pedv to host cell receptors ( ) ( ) ( ) . in addition, the s protein is a target for neutralizing antibody induction because it harbors virusneutralizing epitopes and is the principle antigenic determinant ( ) . the s protein is naturally assembled in homotrimeric form with a number of predicted glycosylation sites ( ) . the co- k-equivalent epitope (coe epitope) is one of the various neutralizing epitopes on the s protein of pedv which have been recognized ( ) . coe is the antigen epitope motif that was identified by the monoclonal antibody c at the cterminal end of the s protein ( ) and the s d domain ( ) . the coe protein is regarded as a critical target for the subunit vaccine development against pedv infection ( ) . the neutralizing epitope region of coe contains amino acids within the s domain extending from amino acid - ( , ) . coe has been expressed as monomer or pentamer structures in various plants including tobacco, rice, and lettuce ( ) ( ) ( ) ( ) ( ) . mice fed transgenic plants or immunized transgenic rice calli protein extracts containing the monomeric coe protein were found to have both systemic and mucosal immune responses against the coe antigen ( , ) . the immunogenictity tests of pentameric coe have not been tested. to date, the expression of trimeric coe as a native coe structure in plants and immunogenicity of trimeric coe in animals have not been reported. gcn , known as the gcn leucine zipper, is a yeast transcription factor that is responsible for the reductive reaction of amino acid deficiency ( ) . gcn can be switched from native dimer form to multimeric states by mutations in the a-and dpositions ( ) . gcn pii is generated by a core that was formed entirely of beta branched residues. gcn pii has been used for the successful production of trimeric ha proteins of h n viruses ( , ) , and trimeric s protein of pedv ( ) . in which, the gcn pii was used to trigger trimerization of the proteins of interest, and increase protein stability and solubility. the development of a rapid, valid, safe, and cost-effective vaccination strategy to protect swine against pedv is urgently needed, especially in developing asian countries producing pigs. plant-based subunit vaccines have been reported with several advantages including low manufacturing cost, effortlessness of scaling, high stability and long shelf life [for a review, see ( ) ]. in addition, low profit margins in the industrial vaccine development are provided from plants with beneficial and economical platforms ( ) . agro-infiltration methods can offer various advantages in production of substantial amounts of recombinant proteins in short times (a few days) after completing vector construction process, and therefore, this system generally exhibits as a very fast and efficient method to produce subunit vaccines ( ) . in this study, we generated a plant vector containing a dna sequence encoding the coe protein of the pedv dr strain fused gcn pii as a vector model to investigate the immunogenicity of a plant-based coe antigen in mice compared to that of a commercial vaccine. interestingly, the neutralization activities of sera of mice vaccinated with the crude extract containing coe trimer were similar to those of mice vaccinated with the commercial vaccine. our first successful initial results are expected to provide an alternative strategy to generate a plantbased trimeric coe vaccine against pedv infection for national rapid response. the coe nucleotide sequence encoding for amino acid - of the coe in the s protein of the attenuated pedv dr strain (ncbi accession number jq . ) was synthesized, codon optimized commercially in tobacco (genebank accenssion number bankit optimized mt ), and then inserted in a pez cloning vector (poch, life science missouri city, texas ). the coe gene was amplified from the vector, and replaced for the h sequence present in prtra-camv s-h -gcn pii-cmyc-his-kdel ( ) to via the bamhi and bsp i sites. the resulting expression cassette ( figure a ) was inserted into the expression vector pcb -kan ( ) via hindiii cleavage, then transformed into the agrobacterium tumefaciens [pgv in c c ; ( ) ] strain via electroporation at . kv, µf capacitance, and ohm resistance. to express the recombinant protein in planta, the agroinfiltration protocol was performed as described by pham et al. ( ) , with some modifications. briefly, bacteria containing the expression vector pcb -coe-gcn pii-cmyc-his-kdel and plant vector including hcpro ( ) , that was used as a gene silencing suppressor for enhancing the expression levels of targeting proteins in plants ( , ) , were mixed and diluted in an infiltration buffer [ mm -(n-morpholino) ethanesulfonic acid (mes), mm mgso , ph . ]. n. benthamiana plants ( weeks old) were completely infiltrated in an agrobacterium solution, and maintained in a greenhouse. six days after agro-infiltration, plant leaf samples were collected and stored at − • c. recombinant coe-gcn pii protein was purified via immobilized metal affinity chromatography (imac) as described by pham et al. ( ) . the oligomeric form of purified coe-gcn pii protein was determined by a cross-linking reaction that was described by weldon et al. ( ) and a native page. leaf samples were ground in liquid nitrogen, mixed with pbs buffer ( mm nacl, . mm kcl, mm na hpo , . mm kh po , ph . ). the crude extract was clarified by centrifugation twice at , rpm for min at • c. the coe-gcn pii protein in leaf crude extract and a number of known concentrations of anti-tnfα-nanobody-elp standard protein [ , , , ng, ( ) ] were separated in a - % sds-page gel and transferred to a pvdf membrane (millipore). the protein expression was determined via western blot that was performed as described by pham et al. ( ) using monoclonal anti-c-myc antibody. the coe-gcn pii protein expression level in the leaf crude extract was semi-quantified via western blotting by comparison of the western blot signal intensities and the coe sequence encoding the coe protein of pedv was fused with the c-terminal trimeric motif (gcn pii) for generating trimeric coe protein. the recombinant protein was fused to a c-myc tag and his-tag for downstream detection by western blot and protein purification by imac, respectively. the legumine b signal peptide and the kdel motif were used to target protein retention in the er. camv s pro, cauliflower mosaic virus s ubiquitous promoter; camv s term, cauliflower mosaic virus s terminator; asterisk, the molecular weight of proteins was calculated for unglycosylated monomers. (b) a total of . µg of total soluble proteins in crude extract containing coe-gcn pii or wild type crude extract and a series of known concentrations of the anti-nanobody-elp standard ( , , , and ng) were separated in - % gel polyacrylamide. an anti-cmyc tag antibody was used as a primary antibody. hrp-linked goat anti-mouse igg was used as a secondary antibody. the coe content in the plant crude extract was determined by comparing the blot signal intensities and those of the standard protein using imagej software. (c) sds-page analysis of the purification procedure of coe-gcn pii from total soluble protein extracts using imac; re, raw extract; ft, flow through; w, wash fraction and p, purified fraction in sds-page. (d) western blot analysis of the purification procedure of coe-gcn pii from total soluble protein extracts using imac. coe-gcnpii was immunologically detected via an anti-his monoclonal antibody. those of the anti-tnfα-nanobody-elp standard protein using imagej software. the study was approved by the ethical committee of the institute of biotechnology, academic of science and technology vietnam (vast), hanoi, vietnam. the crude extracts after days of the storage were mixed with the emulsigen r -d adjuvant (mvp technologies, g street, omaha, ne , usa) with a ratio of : (v/v), respectively. three groups of - -week-old female balb/c mouse (five per group) respectively numbered g , g , g were subcutaneously vaccinated at days , and with µl of emulsigen r -d adjuvant-formulated crude plant extracts of non-transgenic plants as negative control) or µl of the commercial vaccine against the pedv dr strain ( × tcid /dose, ctc vacc ped, korea) as positive control or µl of emulsigen r -d adjuvant-formulated crude plant extracts containing . µg of coe-gcn pii protein that was semiquantified by western blotting. the bloods of mice were collected at seven days after the second and the third immunization via the retro-orbital sinus. all mouse sera were collected separately by centrifugation. to inactivate the non-specific complement, all mouse sera were incubated at • c for min before being stored at − • c until used. pedv propagation and purification were carried out as described by hofmann et al. ( ) , with modifications. the vero e cell line (atcc r crl- tm) was propagated and incubated at • c in dulbecco's modified eagle medium (dmem) including % fetal bovine serum (fbs) and antibiotics ( µg/ml penicillin/streptomycin). the cells were cultured in a % co at • c. then, the pedv-dr strain was propagated in vero cells with µg/ml trypsin treated-tosyl phenylalanyl chloromethyl ketone (tpck) (worthington, lakewood, nj, usa). after h of cultivation, when all cells showed % cytopathogenic effects with morphological changes using cell morphology evaluation by inverted light microscopy, the infective culture fluid was harvested andfreeze-thawed three cycles. next, cellular debris was pelleted by centrifugation at , ×g for min. the clarified supernatant was then enriched by ultracentrifugation at , ×g. sucrose density gradient centrifugation ( , , %) was then used to purify pedv. to detect pedv-specific igg mouse antibodies, µg of purified pedv dr was loaded onto -wells of one sds-page gel. two-fold serial dilutions of serum samples after the nd immunization were prepared in α-minimum essential medium (mem) including % antibiotic-anti-mycotic solution (invitrogen, usa). then, tcid / . ml of pedv dr was added with an equal volume of diluted serum, and the virus-serum mixture was maintained at • c for h. next, µl of each virus-serum mixture was introduced onto vero cell monolayers in -well plates. the virus-serum mixture was removed after adsorption at • c for h. the plates were then washed for min with pbs. finally, µl of serum-free α-mem medium containing trypsin were placed into each well and maintained at • c for days. for controlling this assay, the virus control, positive serum control, negative serum control and blank control were used. the serum neutralization titres (sn titres) were defined as the highest serum dilution and consequent on inhibition of the cytopathic effect. statistical analyses for the elisa test and virus neutralization assay were carried out in sigma plot software using a t-test. the difference between sample data mean was compared and is showed as the x ± standard deviation (sd). p-values that were < . were determined to be significantly different. the expression of the coe-gcn pii protein in planta was successful demonstrated via separation of sds-page under reducing conditions, blotted and detected by western blot using an anti-c-myc monoclonal antibody ( figure b) . the apparent band shown in figure b with coe molecular weight was larger than the expected coe size predicted from the coe polypeptide sequence ( kda). one of the possible reasons for the increase in the molecular weight of coe protein is that the n-glycosylation sites located within the coe-s protein of pedv at amino acids and may influence the electrophoretic behavior during the page separation ( , ) . no coe protein was detected in wild type crude extract. the coe-gcn pii protein expression level in leaf crude extract was semiquantified by western blotting. the coe-gcn pii protein was quantified with a high expression level of ∼ % of the total soluble protein. the amount of plant-produced coe-gcn pii protein was found to be mg/kg wet weight. several publications have reported the accumulation of coe in transgenic plants ( , , ) ; however, the expression level was still lower than that in our report. we demonstrated that the accumulation of coe in tobacco leaves could be significantly improved by codon optimization for plant expression by using a strong expression system, such as agro-infiltration. the purification process was validated by collecting samples from each step of the purification procedure to analyse via sds-page and western blot using a monoclonal anti-his antibody (figures c,d) . these results indicate the enrichment and successful purification of coe-gcn pii protein from n. benthamiana leaves. the oligomeric state of the coe-gcn pii protein was successfully determined by a cross-linking reaction with bs and a separation under native condition by native-page. a band with a molecular weight of approximately kda corresponding to molecular weight of trimeric coe form was detected (figures a-c) . these results revealed that the trimeric coe protein was successfully generated in planta by the fusion of coe with gcn pii motif. since animal vaccine development should minimize downstream processing in pig immunizations, the crude plant extract was chosen for testing immunogenicity in mice. interestingly, after storing the crude extract containing trimeric coe at • c for six days, the coe content was still stable, as revealed by western blotting (see supplementary file) . the antibody-mediated humoral immune responses from vaccinated mice were first examined against the purified pedv dr strain by western blot (figure a) . before vaccination, pedv-specific igg antibody responses were not detected in mice. the western blots in figure a showed that there was a band with molecular weight of over kda detected in mice groups g (vaccinated with the commercial vaccine against pedv) and g (vaccinated with crude extract containing coe-gcn pii) that was larger than the expected size of s protein pedv ( . kda). the larger band size obtained in western blot might be explained that might be due to the influence on electrophoretic behavior by the potential n-glycosylation sites locating within the s protein of pedv during the page separation ( ) . the results indicate that pedv-specific igg antibody responses were induced in mice groups g and g after the nd immunization and the rd immunization, and the antibody responses were strongly increased in both mice groups g and g after the rd immunization. in contrast, no pedv-specific igg antibody response was detected in mice group g vaccinated with crude extracts of non-transgenic plants. different levels of pedv-specific igg, iga and igm antibodies in each mouse sera group were calculated as the reciprocals of the geometric mean titer of the five mice of each group. end-point titer of each mouse sera group was compared by the t-test. the results showed that after the nd immunization, crude extract containing coe-gcn pii (g ) elicited elevated levels of igg, iga and igm antibody responses against pedv (figures b-d) , figure | determination of the levels of pedv-specific igg, iga, and igm antibody responses via a western blot and elisas. (a) sera from five mice from each group immunized with a negative control (wild-type crude extract, g ) or a positive control (commercial vaccine, g ) or the crude extract containing coe-gcn pii (g ) were mixed, diluted times and used as a primary antibody for detecting µg of purified pedv antigen. hrp-linked goat anti-mouse igg was used as a secondary antibody. in total, ng of purified pedv per well was coated on a plate. each serum was measured in triplicate. the sera were serially diluted and analyzed via elisa. different levels of pedv-specific igg (b), iga (c), and igm (d) antibodies in each mouse sera group were calculated as the reciprocal of the geometric mean titer of the five mice of each group vaccinated with the negative control (wild-type crude extract, g ) or the positive control (commercial vaccine, g ) or the crude extract containing coe-gcn pii (g ). end-point titer of igg, iga, or igm antibodies against pedv of each mouse sera group was compared using the t-test (sigmaplot), and is presented. *p< . was defined as a statistically significant difference. reaching end-point antibody titres of : , : , : , respectively. the levels of igg, iga and igm antibody responses in mice group g were strongly enhanced after the rd immunization, having end-point antibody titres of : , : , : , respectively. notably, no statistically significant difference in pedv-specific igg antibody responses was obtained between mice group g and those in mice group g , with p-values of . after the third immunization (p < . ). therefore, plant crude extract containing the trimeric coe protein had the level of igg antibodies similar to that of commercial vaccines against the pedv dr strain after the third injection. however, level of pedv-specific iga and igm antibody responses in mice group g were lower than those in mice group g after the second and the third immunization. levels of igg, iga and igm antibody responses against pedv found in the sera of negative control mice group g were very poor. the cytopathic effect caused by the wild-type pedv dr virus of all serum samples was determined using a microscope and a representative cytopathic effect result observed under microscope of a single dilution of a serum from each group was presented (figure a) . the highest dilutions of sera that caused cytopathic effect inhibition were defined as the serum figure | virus neutralization assay. (a) cytopathic effect caused by wild-type pedv dr virus at the dilution of : of a single serum from a mouse from the groups vaccinated with the negative control (wild-type plant extract, g ) or the positive control (commercial vaccine, g ) or the crude extract containing coe-gcn pii (g ) was determined by using a microscope. (b) determination of serum neutralization titer. serum samples were firstly incubated to inactivate for min at • c. two-fold serial dilutions ( : , : , : , : , : , : , : , : ) were applied. a pedv dr strain ( µl tcid / . ml) was mixed with an equal volume of diluted sera. the virus control, positive serum control, blank control, negative serum control, and were used for controlling the assay. the serum neutralizing titres (sn titres) were expressed as the highest serum dilution and consequence on the inhibition of cytopathic effect. statistical analyses were performed using the t-test (sigmaplot) and are shown. a single dot indicates the sn value of a single mouse serum. the sd is included on a single dot that corresponds to the sn data variation of a single mouse serum with three replications. the bars indicate the average value of the test groups. *p < . was defined as a statistically significant difference. neutralizing antibody titres. the serum neutralizing antibody (sn) titer of every single mouse serum was presented in each dot ( figure b ). as expected, cytopathic effect inhibition of sera from the negative control group was observed in a low serum dilution, which produced neutralizing geometric mean titres of this group as low as . in contrast, sera of mice vaccinated with the crude extract containing coe-gcn pii and the commercial pedv vaccine (the positive control) had the ability to neutralize pedv with high neutralizing geometric mean titres: . and . , respectively. these titres were significantly different from those of the negative control group. notably, the serum neutralization titer in the crude extract containing coe-gcn pii (g ) was not significantly different compared to that of sera from mice vaccinated with the commercial vaccine, with a p-value of . . in this study, the immunogenicity in mice group g vaccinated with the coe trimer in crude extract was compared to those of mice positive control group g vaccinated with commercial pedv vaccine and mice negative control group g vaccinated with crude extracts from non-transgenic plants. as expected, western blot results demonstrated that pedvspecific igg antibodies were presented in mice group g and mice group g after the second and the third immunization. more interestingly, the elisa analyses illustrated that crude extract containing coe-gcn pii (g ) elicited strong levels of igg antibody responses against pedv, and especially the level of pedv-specific igg antibody responses found in mice group g was similar to that in mice group g after the third immunization. mucosal immune responses play an important role in the defense of pedv, and mucosal iga antibody response was found to correlate with the protection against pedv infection ( , ) , however igg antibody-mediated humoral response is also essential to protect the neonatal pig against pedv infection ( , ) . early publications showed the important role of igg in protection of gastrointestinaltract ( , ) . in this study, trimeric coe antigen (g ) was vaccinated in mice via subcutaneous route that is a conventional vaccination route widely used for various human and animal vaccines to elicit igg antibody-mediated humoral responses. interestingly, beside pedv-specific igg, pedv-specific iga and igm antibodies were presented in mice group g , however they were lower as compared to those found in mice group g . our results are comparable to several recent evidence studies that subcutaneous route can induce both antigenspecific igg antibody, and antigen-specific iga and igm antibodies in sera ( ) ( ) ( ) ( ) ( ) . moreover, su and his co-workers proposed that under some circumstances (antigen, adjuvant, delivery vehicle) systemic routes may induce systemic immune responses and muscosal immune responses against infectious diseases ( ) . in addition, the ability of the neutralizing antibodies induced in mouse sera to neutralize pedv via binding to coe epitopes related to pedv neutralization was further determined. after virus neutralization, there was an inhibition generated by neutralizing antibodies to the virus's infection cycle, containing surface binding, fusion, entry, endocytosis, and replication ( ) . the assessment of pedv-neutralizing activity illustrated that there was no statistically significant difference between the serum neutralization titres of mice vaccinated with the crude extract containing trimeric coe (g ) and that of mice vaccinated with the commercial vaccine (g ), with a p-value of . . therefore, we concluded that plant crude extract containing the trimeric coe protein had a strong immunogenicity and induced a neutralizing antibody titer similar to that of the commercial vaccine against the attenuated pedv dr strain. the enhancement of neutralizing antibody responses induced in animals after vaccination is an important requirement for vaccine development due to the powerful correlation of vaccine efficacy with neutralizing antibodies for numerous commercial vaccines ( ) . the crude extract containing trimeric coe (g ) showed neutralizing activity, with a geometric mean titer of : . . a high neutralizing antibody titer indicated that mice subcutaneously administered the crude extract containing trimeric coe possessed a strong ability to neutralize pedv. to increase the immune response, especially mucosal immune responses against pedv, oral mucosal vaccination with coe but not subcutaneous administration in animals has been previously presented to induce anti-pedv mucosal immune responses ( , ) . in addition, since pedv causes mainly intestinal infections, the coe antigen was fused with dendritic cell-targeting peptide (dcpep) and m celltargeting peptide (col) for targeting intestinal microfold (m) cells and dendritic cells (dcs) ( , , ) . the neutralizing activity mouse sera orally provided genetically engineered lactobacillus but not plant extracts expressing coe targeting m cells or dcs or both has been previously reported ( , ) . when compared to the publication of ma and coworkers, the neutralizing antibody titres obtained by oral administration in mice with the recombinant lactobacillus casei strains expressing the pedv coe antigen on the cell surfaces by fusion with dcpep or col or both dcpep and col were : , : , and : , respectively ( ) , which were lower than the neutralizing antibody titres observed in this study. these results showed that crude extract containing trimeric coe can be a promising vaccine candidate against pedv infections. in summary, the trimeric coe protein was successfully produced in plants with high expression levels. crude extract containing trimeric coe elicited strong humoral immune responses and elevated neutralizing antibody titres against pedv. in particular, the neutralizing activities of mice vaccinated with the crude extract containing coe-gcn pii were similar to those of mice vaccinated with the commercial vaccine. these results suggest that crude extract containing trimeric coe might be a potential subunit vaccine antigen against pedv infection. further studies will focus on investigating immune efficacy and protection against pedv in piglets. all datasets presented in this study are included in the article/supplementary material. the animal study was reviewed and approved by the principles of the basel declaration and recommendations of arrive guidelines, ethical committee of institute of biotechnology, academic of science and technology vietnam (vast), hanoi, vietnam on the use of animals for research. hc, np, and th designed the research. th and gn constructed vectors and performed transient expression. th purified protein and performed the cross-linking reaction, performed elisa and western blotting analyses, and performed the calculations, all data analysis and wrote the manuscript. vl and tt purified the pedv dr strain and carried out the virus-neutralizing antibody assay. uc, hp, np, tv, and hc revised the manuscript. hc is the corresponding author and 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neutralization of animal viruses maternal antibodies, childhood infections, and autoimmune diseases oral recombinant lactobacillus vaccine targeting the intestinal microfold cells and dendritic cells for delivering the core neutralizing epitope of porcine epidemic diarrhea virus oral delivery of probiotics expressing dendritic cell-targeting peptide fused with porcine epidemic diarrhea virus coe antigen: a promising vaccine strategy against pedv all authors contributed to the article and approved the submitted version. this study was financed by vast through the project: study on the expression of s oligomer of porcine epidemic diarrhea virus (pedv) in nicotiana benthamiana for the next generation of vaccine development, listed in scientific program: biotechnology, code: vast . / - . we would like to thank dr. song daesub for supplying the pedv dr strain. we also appreciate dr. do thi thao from ibt, hanoi, vietnam for the mouse experiments. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material key: cord- - ozhye q authors: yu, haijing; liu, yang; wang, hongwu; wan, xiaoyang; huang, jiaquan; yan, weiming; xi, dong; luo, xiaoping; shen, guanxin; ning, qin title: clara cell kda protein alleviates murine hepatitis virus strain -induced fulminant hepatitis by inhibiting fibrinogen-like protein expression date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: ozhye q background: fulminant hepatitis (fh) is a serious threat to human life, accompanied by massive and rapid necroinflammation. kupffer cells, the major immune cell population involved in innate immune responses, are considered to be central for fh. fibrinogen-like protein (fgl ) is a pro-coagulant protein that is substantially induced in macrophages upon viral infection, and fgl depletion represses murine hepatitis virus strain (mhv- ) infection. clara cell kda (cc ) protein is a secretory protein with anti-inflammatory properties in allergic rhinitis and asthma. however, its mechanisms of action and pathogenic roles in other disease are still unclear. in this study, we aimed to determine the role of cc in fh and the regulation of fgl by cc . methods: a mouse fh model was established by peritoneal injection of mhv- . the mice received cc protein through tail vein injection before viral infection. survival rate, liver function, liver histology, fibrin deposition, and necrosis were examined. the regulatory effect of cc on fgl expression was investigated using thp- cells and mouse peritoneal macrophages in vitro. results: in the mouse fh model induced by mhv- , the survival rate increased from to . % in the cc group compared to that in the saline-only control group. meanwhile, the levels of alt and ast in serum were significantly decreased and liver damage was reduced. furthermore, hepatic fgl , tnf-α, and il- β expression was obviously downregulated together with fibrin deposition, and hepatocyte apoptosis was reduced after administration of cc protein. in vitro, cc was found to significantly inhibit the expression of fgl in ifn-γ-treated thp- cells and mhv- -infected mouse peritoneal macrophages by western blot and real-time pcr. however, there was no direct interaction between cc and fgl as shown by co-immunoprecipitation. microarray investigations suggested that hmg-box transcription factor (hbp ) was significantly low in cc -treated and ifn-γ-primed thp- cells. hbp -sirna treatment abrogated the inhibitory effect of cc on fgl expression in human umbilical vein endothelial cells (huvecs). conclusion:cc protects against mhv- -induced fh via suppression of fgl expression in macrophages. such effects may be mediated by the transcription factor hbp . fulminant hepatitis (fh) is a serious life-threatening disease characterized by massive hepatocyte necrosis, severe liver damage, and high mortality. the underlying mechanisms and the pathogenesis of fh are not clear. however, accumulating evidence suggests that, regardless of the pathogenesis of fh, the host's inflammatory responses contribute to liver microcirculatory disorders and injuries. accordingly, it has been shown that immune cell activation and inflammatory cytokines play an important role in fh ( ) . in recent years, our laboratory has conducted extensive research on the pathogenesis of fh and found that immune cells play a key role in it. kupffer cells, natural killer (nk) cells ( , ) , cytotoxic t-lymphocytes (ctls), and double negative t-cells (dnt) ( ) ( ) ( ) in liver and the cytokines that are produced by these cells cause liver damage. prothrombinase fgl belongs to the fibrinogen superfamily and is produced by activated macrophages or endothelial cells, transforming prothrombin directly into thrombin, so as to quickly initiate the process of coagulation. this promotes the conversion of fibrinogen into fibrin, resulting in thrombosis ( ) ( ) ( ) ( ) ( ) ( ) . our study found that fgl was highly expressed in peripheral blood mononuclear cells (pbmcs) and in liver tissue of humans or mice with severe viral hepatitis, and was positively related to the severity of the disease ( , ) . gene therapy targeting fgl silencing showed that the survival rate of fulminant hepatitis mice increased from to . % ( ) . thus far, the discovery and related research involving fgl have provided new insights into the molecular mechanism of hepatocyte necrosis in fh. in view of the important role of fgl in severe viral hepatitis, investigations concerning the regulation of fgl will be beneficial in the search for new strategies for treatment of severe hepatitis. clara cell kda protein (cc ), also considered to be uteroglobin, clara cell secretory protein, is one of members of secretoglobin superfamily. expressed in mucosal epithelial cells of organs (including lungs and nose) that communicated with the outside world ( ) . cc has immunomodulatory and anti-inflammatory effects. compared to wild-type mice, cc -knockout mice exhibited excessive airway inflammation abbreviations: fh, fulminant hepatitis; mhv- , murine hepatitis virus strain ; fgl , fibrinogen-like protein ; cc , clara cell kda protein; alf, acute liver failure; pfu, plaque-forming units; pbs, phosphate-buffered saline; alt, alanine aminotransferase; ast, aspartate aminotransferase; pca, pro-coagulant activity; hrp, horseradish peroxidase; tunel, terminal deoxynucleotidyl transferase dutp nick end labeling. caused by allergic reaction and bacterial and viral infections ( ) . reduced levels of cc are associated with inflammatory and allergic airway diseases, including sinusitis, asthma and allergic rhinitis ( ) ( ) ( ) ( ) . previous studies and published articles show that cc protein can not only inhibit th cell responses by inhibiting expression of related molecules of dendritic cells and cytokines in mice with allergic rhinitis, but also can inhibit chitosan- like protein ( , ) . moreover, cc inhibits the expression of an important immune regulator, osteopontin (opn), in models of allergic rhinitis ( ) . in this study, we investigated the role of cc in hepatitis virus strain (mhv- )-induced fh in mice and explored whether cc protein could regulate fgl in the disease process. female balb/cj mice (shanghai shilaike animal seed center, shanghai, china), - weeks of age, with a body weight of . - . g, were kept in tongji hospital with food and water. mice were divided into two groups: cc group (experimental group) and phosphate-buffered saline (pbs) group (control group). this study was carried out in accordance with the recommendations of the guidelines of the national institutes of health and the animal experiment committee of tongji hospital. this study was reviewed and approved by the animal experiment committee of tongji hospital. the human monocyte cell line thp- was purchased from the cell institute of the chinese academy of sciences (shanghai, china). human umbilical vein endothelial cells (huvecs) were obtained from the biology treasure center of wuhan university, china. the chinese hamster ovary (cho) cell line was acquired from the typical culture preservation commission cell bank, the chinese academy of sciences (shanghai, china). human umbilical vein endothelial cells (huvecs) and cho cells were cultured in dulbecco's modified eagle's medium (dmem), and thp- cells were maintained in rpmi , containing % heat inactivated fetal bovine serum (fbs, gibco life technologies, usa), u/ml penicillin, and mg/ml streptomycin and cultured at • c, ml/l co and % humidity. peritoneal exudative macrophages (pems) were obtained from balb/cj mice. cells were resuspended in rpmi , supplemented with % fbs at - × cells/ml in a -well plate and incubated for h. they were then washed with rpmi medium and non-adherent cells discarded. the adherent cells were macrophages and were incubated for a further h. peritoneal exudative macrophages (pems) were divided into two groups. one group was supplemented with cc protein ( ng/ml) and in the other group, pbs was added. after h of stimulation, , plaque forming units (pfus) of mhv- was added to the cells, which were then cultured for h. peritoneal exudative macrophages (pems) were harvested and lysed for real-time pcr and western blotting analysis. cell apoptosis was detected by the terminal deoxynucleotidyl transferase dutp nick end labeling (tunel) method with a tunel apoptosis detection kit (roche, switzerland). briefly, µm sections were deparaffinized, dehydrated through an alcohol series and incubated with proteinase k for min at • c. after stopping the proteinase k digestion reaction with pbs, the samples were incubated with terminal deoxynucleotidyl transferase end-labeling cocktail (a mixture of terminal deoxynucleotidyl transferase and dutp at a ratio of : , respectively), for h at • c in an immunohistochemistry wet box. following washing and blocking, each section was supplemented with reagent (converter-pod) to cover the tissues and incubated for min at • c in a wet box. then, the liver tissue sections were washed with pbs, and colored with diaminobenzidine (dab) subsequently. hepatocytes with nucleus stained brownish yellow were considered to be apoptotic cells. the expression of fgl on thp- cells was measured by flow cytometry (bd facs canto ii, usa). briefly, cells ( × per tube) were incubated with human trustrain fcx (fc receptor blocking solution, biolegend, usa) for min at room temperature and then incubated in the dark with mouse anti-fgl antibody ( : , abnova,) or normal goat serum (an isotype control) at • c for min. cells were washed with pbs and incubated in the dark with pe-conjugated goat anti-mouse igg antibody ( : , biolegend, usa) at • c for min. cells were then washed with pbs and resuspended in µl pbs for study. liver slices were fixed in % paraformaldehyde and then embedded in paraffin. immunohistochemistry of liver tissues was performed using sp- splink detection kits (biotin-streptavidin hrp detection systems) (zsgb-bio, beijing, china) according to the manufacturer's instructions. for immunohistochemistry staining, the expression of fgl , fibrinogen, fas and tnf-receptor in mouse liver tissues was detected with polyclonal rabbit anti-mouse fgl antibody ( : , proteintech, usa), polyclonal rabbit anti-mouse fibrinogen antibody ( : , , abcam, england), polyclonal rabbit antimouse fas antibody ( : , abcam, england), and polyclonal rabbit anti-mouse tnf-receptor antibody ( : , abcam, england), respectively. after incubation with an horseradish peroxidase (hrp)-labeled goat igg fraction to rabbit igg fc, the target protein was detected using a dab kit (zsgb-bio, beijing, china). the slides were then counterstained with hematoxylin and visualized under a microscope (olympus, tokyo, japan). liver tissue and cells were homogenized in ripa lysis buffer with phenyl methane sulfonyl fluoride (pmsf) protease inhibitor. protein lysates were separated by sds-page, and western blotting was performed using a monoclonal mouse antihuman/mouse fgl ( : , abnova), a monoclonal mouse antihuman hbp ( : , santa cruz, usa), and a monoclonal rabbit anti-human/mouse β-actin ( : , , cell signaling technology, usa). liver tissues were collected from mhv- -infected balb/cj mice at h, and total rna was extracted using trizol reagent (invitrogen, usa) and then reverse transcribed into cdna by using revertra ace qpcr rt kit (toyobo, japan). the cdna was then amplified by rt-pcr by using dream taq green pcr master mix ( ×) (thermo scientific, usa). realtime quantitative pcr (qpcr) with sybr green real-time pcr master mix (toyobo, japan) was performed using a cfx real-time pcr detection system (bio-rad, usa) and mrna levels were normalized with reference to those of the house keeping gene gapdh. primer sequences for qpcr amplification were as follows: mtnf-α forward, ′ -ttt gag atc cat gcc gtt gg- ′ ; mtnf-α reverse, ′ -gcca cca cgc tct tct gt- ′ ; mil- β forward, ′ -tgt aat gaa aga cgg cac acc- ′ ; mil- β reverse, ′ -tct tct ttg ggt att gct tgg- ′ . mfgl forward, ′ -gcc aaa tgt gag tcc ctg gaa- ′ ; mfgl reverse, ′ -ttc cac cca aga gca cgt tta ag- ′ ; hfgl forward ′ -aca gtt cag gct ggt ggt- ′ ; hfgl reverse, ′ -ggc tta aag tgc ttg ggt- ′ ; hbp forward, ′ -tga agc aga agc tgg gagt- ′ ; hbp reverse, thp- cells were treated with ng/ml phorbol -myristate -acetate (pma) (sigma, usa) for h to induce differentiation toward adherent macrophage-like cells as reported previously ( ) . the cc group was supplemented with cc protein ( ng/ml). after h of stimulation, ifn-γ ( ng/ml) was added to these cells, which were then cultured for h before they were collected for western blotting and real-time pcr studies. the chinese hamster ovary (cho) cells were cultured in cm cell culture dishes with dmem supplemented with % fbs until - % confluence. next, µg pcdna . -hfgl (constructed in our lab) was mixed with µg pcdna . -hcc in serumfree dmem. the mixture was then combined with lipofectamine , (invitrogen, usa) and mixed gently. after incubation at • c for min, the solution was added to cho cells and incubated at • c in % co . four to six hour after transfection, the medium was removed and fresh medium containing % fbs was added. at h after transfection, the cells were collected for co-immunoprecipitation analysis to evaluate the interaction of cc with fgl . both huvec and thp- cells express fgl . however, in the transfection experiments, it is difficult to transfect the thp- cells with sirna, so we use huvec instead of thp- . human umbilical vein endothelial cells (huvecs) were cultured in figure | cc protein increased survival rate and reduced liver damage in mice. (a) the survival rate of cc group is higher than the control group comprised of mhv- -infected balb/cj mice treated with saline. cc protein ( µg) or saline were injected into mice by tail vein. balb/cj mice then received pfu of mhv- intraperitoneally h later to develop fulminant viral hepatitis. then, cc protein ( µg) or saline were injected into mice by tail vein following mhv- infection h later. the survival rate was observed for days (n = /group). representative data from three independent experiments are shown. the survival curve was analyzed by using the log-rank test. ***p < . compared with saline group. (b) histopathology of liver tissues (h&e staining; original magnification, × , n = /group) at h post-mhv- infection was evaluated in the two groups of mhv- -infected balb/cj mice. livers were collected from saline-treated (a) and cc -treated (b) balb/cj mice at h after mhv- infection. arrows point to inflammatory cell infiltration areas or necrotic regions with inflammation. (c) effect of cc on serum alt and ast levels (n = - /group). values represent means and standard error of three independent experiments performed in triplicate. **p < . compared with the saline group. six-well plates with dmem supplemented with % fbs until - % confluence. pmol hbp -sirna was mixed with µl serum-free dmem. two microliter lipofectamine , was gently mixed with serum-free dmem. after incubation at • c for min, the solution was added to huvecs and incubated at • c. four hour after transfection, the medium was removed and fresh medium containing % fbs was added. at h after transfection, cells were collected for real-time pcr and western blot analysis to evaluate the effects of hbp on fgl . at h after transfection, the cc group was supplemented with the cc protein ( ng/ml). after h of stimulation, ifn-γ ( ng/ml) was added to these cells. these cells were then cultured for h before they were harvested for real-time pcr studies to evaluate the effects of cc on fgl by hbp . negative control was used as a control. to detect whether there was a potential interaction between cc protein and fgl , cho cells were transfected with pcdna . -hcc and pcdna . -hfgl for h. cells transfected with empty plasmid pcdna . (mock) were used as negative controls for cc gene transfection. immunoprecipitation and immunoblotting were performed by using pierce co-immunoprecipitation kit (pierce, usa). total cell proteins were extracted as previously described ( ) . the proteins were immunoprecipitated by mouse anti-human fgl antibody ( : , abnova). for co-immunoprecipitation experiments, western blotting was performed using both rat anti-human uteroglobin/scgb a antibody ( : , r&d, usa) frontiers in immunology | www.frontiersin.org and mouse anti-human fgl antibody ( : , abnova). control isotype rat igg was used as a negative control for primary antibodies. the human cc coding region gene, including a bp sequence, was amplified from homogenized human turbinate tissue by rt-pcr. in this study, the sequences of pcr primers for cc were as follows: hcc -forward, ′ -ccc tcc acc atg aaa ctcg- ′ ; hcc -reverse, ′ -tga gat gct tgt ggt tta ttg aag- ′ . the pcr products were cloned into peasy-t cloning vector (transgen, beijing, china) and then subcloned into hindiii/xbai site of pcdna . vector (invitrogen, usa) to form eukaryotic expression plasmids pcdna . -hcc . microarray analysis was used to screen changes in genome-wide gene expression patterns in thp- cells with or without cc protein. the changes in over , human gene expression patterns were assessed using affymetrix gene microarrays (human genome u plus . ) (capitalbio co.,ltd., beijing, china). three replicates were used for microarrays analysis. data obtained from the experiments are expressed as means ± sem. survival curve comparisons were performed with the log rank test. multiple group analyses for data were evaluated by one-way analyses of variance. analyses of two group results were performed using student's t-test to evaluate the statistical significance of differences. values of p < . indicated significance. to establish an animal model of mouse fh, mhv- was injected intraperitoneally to balb/cj mice ( mice/group). to further study the role of cc in fh, recombinant mouse cc protein ( µg/mouse) or saline was administrated into the tail vein h prior to mhv- infection. the same dose of cc protein or saline was then administered h later. the survival rate of the cc and saline groups was observed for days. the results showed that mice in the two groups began to die at h after injection of mhv- and exhibited symptoms of horripilation, slow activity, and reduced food consumption. in the cc group mice were alive on day after infection, mice alive on day , and of ( . %) mice recovered from fulminant viral hepatitis. at the same time, in saline treated group, there were mice alive on day , mice alive on day after infection, and no mice survived to day . that is to say, the mice in the saline group died within or days. three of ( . %) mice of the cc group recovered from fulminant viral hepatitis ( figure a) . to better understand the mechanisms underlying the biological effects of the cc protein, liver function (alt and ast levels in serum) and liver histology in mice of mhv- -infected was performed. liver tissues were harvested h following mhv- infection, and liver histology was detected by h&e staining. these results showed that there was substantial inflammatory cell infiltration and widespread necrosis of hepatocytes in the liver tissue of the saline group mice (figure ba ). there were rare or no infiltrating inflammatory cells, and few or no hepatocyte necrosis in the livers of mice in the cc group h after mhv- infection (figure bb) . serum alt and ast levels in mice were observed h after mhv- infection. the results showed that serum alt and ast levels in the saline group reached a peak h after mhv- infection, but there was no significant increase in the cc group compared to the levels in the control group (p < . , figure c) . these results suggested that cc protein has a role in protection against mhv- -induced liver injury in mice. to further elucidate the mechanisms of reduced liver injury following cc protein injection, we investigated the cytokines tnf-α and il- β expression. because these two cytokines play a crucial role in the liver damage of fh. they are characterized by an increase in apoptosis. levels of tnf-α and il- β in liver tissues were markedly reduced in the cc group (as shown in figure a) . hepatic apoptosis (figure b ) was significantly reduced in the cc group. we and collaborators have a long standing interest in studying the role of fgl in viral hepatitis. fgl has been verified to play an essential role in the progression of fulminant viral hepatitis as we appreciate from previous reports. we have provided liver pathology figures and liver function for mhv- infected mice with a fgl gene knockout as shown in supplementary figure . the data was comparable with previous reports from our center and collaborators. from this current study we shown that cc plays a protective role in liver damage.to study the related molecules of cc in mhv- -induced fh mice, we evaluated whether there was crosstalk between fgl and cc . we found that the expression of fgl in the liver of mice was reduced h after mhv- infection and treatment with cc protein (figures a,b) . furthermore, fibrin deposition, an indicator of liver injury associated with fgl expression in fh, was also decreased in the livers of cc -treated mice compared to that in controls (figure c ). this indicates that cc treatment reduced liver injury after viral infection by inhibiting fgl expression. we examined the effect of increasing doses of cc protein ( , , , and ng/ml) on ifn-γ-induced fgl expression in thp- cells. cc treatment showed a . % decrease in thp- cells compared to that in control after stimulation with ng/ml ifn-γ for h. cc protein inhibited fgl expression between doses of ng/ml and ng/ml (figure a ). in particular, ng/ml cc protein had the strongest inhibitory effect on fgl expression among the doses, and we chose this dose for the following experiments. we explored the effect of different time points of stimulation with a concentration of ng/ml cc protein. after stimulation with cc protein for , , and h compared to the pbs control, the strongest inhibitory effect on fgl expression was noted at h; hence, we chose this time point for the following studies ( figure b ). an increasing number of studies suggest that macrophages are the primary source of fgl . in order to ascertain that cc has a direct effect on macrophages, we treated thp- cells with recombinant cc and assessed the expression of fgl . unlike in controls, ifn-γ induced a significant increase in fgl expression. this effect was attenuated when cells were treated with cc protein (figures c,d) , revealing that cc directly reduces the levels of fgl in macrophages. to further explore the possibility that cc protein directly acts on macrophages, we infected murine pems with mhv- in the presence of recombinant cc and determined fgl expression. compared to levels in the controls, mhv- infected macrophages exhibited a significant increase in fgl production, and this effect was abolished by using cc protein (figures a,b) , indicating that cc directly modulates fgl production in macrophages. in order to determine genes that were downregulated after stimulation by cc protein, we used dna microarray analysis to screen for differentially expressed genes. thp- cells were cultured and pma was added to induce differentiation into macrophages. the production of fgl was stimulated by ifnγ. the experimental group was treated with cc protein for microarray detection of differentially expressed genes. the results showed that the most obviously downregulated genes were ube w, hectd , mir , atrx, sox , hbp , and fgl (supplementary table ) . and then these genes were tested by qpcr. however, ube w, hectd , mir , atrx, and sox was not differentially expressed by qpcr, while hbp and fgl were still down-regulated genes. dna microarray analysis identified hbp as a down-regulated gene involved in the pathological processes of the regulation of cc . recently, very limited studies have explored the role of hbp in fh. nevertheless, the mechanistic functions of hbp in fh remain largely unexplored. therefore, we selected this gene for further study. qpcr analysis confirmed that mrna levels of hbp were significantly decreased in thp- cells after cc protein stimulation compared to that in the pbs control group (figure a ). we knocked down hbp using hbp -sirna. then, transfection of hbp -sirna into huvecs was detected by qpcr and western-blotting methods. as expected, hbp knockdown led to significantly decreased expression of hbp (figures b,c) . furthermore, hbp knockdown impaired expression of fgl (figure d ), suggesting that hbp was able to activate fgl . hbp -sirna was used to transfect huvecs. then, ifn-γ was added to induce the expression of fgl followed by stimulation with cc protein ( ng/ml) after h. finally, we explored the expression of fgl by qpcr. the results showed that hbp -sirna treatment abrogated the inhibitory effect of cc on fgl expression in huvecs (figure ) . that is to say, cc could suppress fgl expression in macrophages. such an effect may be mediated by the transcription factor hbp . it is well-known that cc protein can suppress the immune response. in animal models of allergic diseases of the respiratory tract, most of evidences confirm this inhibition ( ) . its function in fh has not been investigated yet. here, we used a murine fh model established by mhv- infection to explore the effects of cc in this disease process. to determine the role of cc in the pathogenesis of fh, cc protein was injected into a mouse fh model established by mhv- infection. mhv- -induced liver injury in cc -treated mice occurred rarely and the areas of lesions were much fewer than those in saline-treated control mice. in summary, these results suggested that cc could reduce pathological liver damage in this fh model together with lower mortality rates followed by mhv- infection. mhv- induced fulminant viral hepatitis progresses rapidly and infected mice die within - days. previous studies suggested fgl played a vital role in this process with a - % increase of survival when fgl was deleted ( , , , ) . multiple inflammatory factors or mediators including tnf-α and ifn-γ, il- β and c ar have been demonstrated to promote fh progression with significant discrepancies between liver damage and survival rate ( ) ( ) ( ) ( ) , which is accordant with our observation that cc substantially alleviated liver injury though survival rate improved mildly. the survival rate based on hours may be more accurate to examine the effect of cc on fh. it is speculated that fgl can mediate lethality in mhv- -induced fh. this is due to the fact that fgl induces the deposition of fibrinogen, which leads to activation of the coagulation cascade and induction of procoagulant activity ( ) . to determine whether the tissue necrosis was mediated by fgl in cc -treated mice following infection, fgl expression was observed. results suggested that the expression of fgl was significantly increased in mhv- -induced fh mice and cc treatment significantly reduced the production of fgl in the infected liver and serum. in addition, decreased fibrinogen deposition was also observed in the livers of cc -treated mice. therefore, our research results strongly clarify that the lower mortality of cc -treated mice after mhv- infection is due to the lower levels of fgl and decreased fibrinogen deposition. indeed, it has been reported that fgl is expressed on macrophages, and the expression of fgl is believed to be induced by ifn-γ and tnf-α ( ) . cultured thp- cells activated by ifn-γ or il- have been demonstrated, with induction of fgl expression and enhanced activation of human prothrombin ( ) . therefore, in this study, we explored this cell line to investigate the modulation of cc on fgl . surprisingly, we found that cc directly inhibited ifn-γ-induced fgl expression in thp- cells. as we know, ifn-γ has proved to be the main cytokine that leads to the development and progression of fh. also, it was shown that ifn-γ might exert its own proinflammatory biological function through enhancing fgl expression. therefore, in our study, cc might counter the effect of ifn-γ in the setting of fh, which substantiates its role in fh. these results demonstrated that cc regulates the expression of fgl in macrophages. in the current study, we used co-immunoprecipitation to analyze binding between cc and fgl . in this study, we investigated possible protein-protein interactions between cc and fgl in vitro. the chinese hamster ovary (cho) cells transfected with pcdna . -hcc and pcdna . -hfgl . cellular proteins were immunoprecipitated with anti-cc antibody or anti-fgl antibody. immunoblotting was performed with anti-fgl and anti-cc antibodies. immunoprecipitation of protein extracts from pcdna . -cc and pcdna . -fgl co-transfected cho cells with anti-fgl or anti-cc antibody followed by western blotting with fgl and cc antibodies indicated that cc did not co-immunoprecipitate with fgl , showing that there is no direct relationship between cc and fgl (data not shown). the results showed that cc has no direct interaction with fgl . from our previous study the gene of fgl contributed profoundly in mhv- induced fulminant hepatitis and is extensively expressed in macrophages and endothelium ( , ) . our microarray indicated a cc down-regulated fgl expression and this is further confirmed by qpcr and western blotting in vivo (peritoneal macrophages) and in vitro (thp- , macrophage cell line). therefore, it is reasonable to focus on macrophages to display the effect of cc on fgl expression and eventually mice survival. we entirely agree there may be other possibilities for a protective effect of cc to contribute to the disease process. this is worth further studies. the potential receptor of cc has not been revealed yet. our previous study have demonstrated that cc have effect of dendritic cells in allergic rhinitis ( ) . in this research, we evaluated the effect of cc on macrophages functions and found fgl was substantially down-regulated upon cc treatment, therefore, we speculate that potential cc receptor may be also expressed on macrophages. the potential target of cc on other immune cells cannot be excluded. dna microarray analysis is one of the most powerful approaches for the potential identification of unexpected genes involved in pathogenic processes. by using this approach, hmgbox transcription factor (hbp ) was found to be one of the most downregulated genes after cc treatment of thp- cells. hbp is a well-described transcriptional repressor that modulates expression of genes involved in cell cycle progression. in a recent study, it was found that hbp is a direct target of mir- and confirmed that hbp modulates the inhibitory function of mir- -aso in hepatosteatosis and carcinogenesis simultaneously ( ) . hbp is an endogenous inhibitor of the wnt signaling pathway in both normal and cancer cells. the tumor suppressor role of hbp has been reported in some malignancies, such as oral cancer and glioma ( ) . however, an association between hbp and fgl has not been investigated yet. the current study clearly demonstrated that cc protects against mhv- induced fh via suppression of fgl expression. such effects might be mediated by hbp . however, the functional status of hbp in the cc pathway requires further research, and such studies are conducting in our laboratory. in conclusion, we demonstrated that cc could limit the immunopathological damage in mhv- -induced fh mice. our results suggest that enhancing cc expression by an immunotherapeutic approach might be an effective treatment for fh. hy performed all the described experiments and wrote the manuscript. yl assisted with some experiments, analyzed experimental results, and edited the manuscript. hw analyzed experimental results. xw reviewed and 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hepatic failure in mice the novel cd +cd + regulatory t cell effector molecule fibrinogen-like protein contributes to the outcome of murine fulminant viral hepatitis cytokine-induced hepatic apoptosis is dependent on fgl /fibroleukin: the role of sp /sp and stat /pu. composite cis elements intact type immunity and immune-associated coagulative responses in mice lacking ifngamma-inducible fibrinogen-like protein the nlrp inflammasome and il- β accelerate immunologically mediated pathology in experimental viral fulminant hepatitis tnfα, and fgl contribute to coagulation and complement activation in virus-induced fulminant hepatitis fulminant hepatic failure in murine hepatitis virus strain infection: tissue-specific expression of a novel fgl prothrombinase clara cell -kda protein inhibits t(h) responses through modulating dendritic cells in the setting of allergic rhinitis mir- promotes the growth of osteosarcoma in a hbp -dependent mechanism the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material table | list of down-regulated genes by dna microarray. in order to determine genes that were downregulated after stimulation by cc protein, we used dna microarray analysis to screen for differentially expressed genes. briefly microarray analysis was used to screen changes in genome-wide gene expression patterns in thp- cells with or without cc protein. the changes in over , human gene expression patterns were assessed using affymetrix gene microarrays (human genome u plus . ) (capitalbio co. ltd., beijing, china). three replicates were used for microarrays analysis. thp- cells were cultured and pma was added to induce differentiation into macrophages. the production of fgl was stimulated by ifn-γ. the experimental group was treated with cc protein for microarray detection of differentially expressed genes. the results showed that the most obviously downregulated genes were ube w, hectd , mir , atrx, sox , hbp , and fgl . key: cord- - l rrlm authors: poh, chek meng; zheng, jian; channappanavar, rudragouda; chang, zi wei; nguyen, thi h. o.; rénia, laurent; kedzierska, katherine; perlman, stanley; poon, leo l. m. title: multiplex screening assay for identifying cytotoxic cd (+) t cell epitopes date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: l rrlm the cytotoxicity of epitope-specific cd (+) t cells is usually measured indirectly through ifnγ production. existing assays that directly measure this activity are limited mainly to measurements of up to two specificities in a single reaction. here, we develop a multiplex cytotoxicity assay that allows direct, simultaneous measurement of up to different specificities of cd (+) t cells in a single reaction. this can greatly reduce the amount of starting clinical materials for a systematic screening of cd (+) t cell epitopes. in addition, this greatly enhanced capacity enables the incorporation of irrelevant epitopes for determining the non-specific killing activity of cd (+) t cells, thereby allowing to measure the actual epitope-specific cytotoxicity activities. this technique is shown to be useful to study both human and mouse cd (+) t cells. besides, our results from human pbmcs and three independent infectious animal models (mers, influenza and malaria) further reveal that ifnγ expression by epitope-specific cd (+) t cells does not always correlate with their cell-killing potential, highlighting the need for using cytotoxicity assays in specific contexts (e.g., evaluating vaccine candidates). overall, our approach opens up new possibilities for comprehensive analyses of cd (+) t cell cytotoxicity in a practical manner. the cytotoxicity of epitope-specific cd + t cells is usually measured indirectly through ifnγ production. existing assays that directly measure this activity are limited mainly to measurements of up to two specificities in a single reaction. here, we develop a multiplex cytotoxicity assay that allows direct, simultaneous measurement of up to different specificities of cd + t cells in a single reaction. this can greatly reduce the amount of starting clinical materials for a systematic screening of cd + t cell epitopes. in addition, this greatly enhanced capacity enables the incorporation of irrelevant epitopes for determining the non-specific killing activity of cd + t cells, thereby allowing to measure the actual epitope-specific cytotoxicity activities. this technique is shown to be useful to study both human and mouse cd + t cells. besides, our results from human pbmcs and three independent infectious animal models (mers, influenza and malaria) further reveal that ifnγ expression by epitope-specific cd + t cells does not always correlate with their cell-killing potential, highlighting the need for using cytotoxicity assays in specific contexts (e.g., evaluating vaccine candidates). overall, our approach opens up new possibilities for comprehensive analyses of cd + t cell cytotoxicity in a practical manner. after priming and activation by antigen-presenting cells in secondary lymphoid organs, epitope-specific cd + t cells expand in numbers, migrate to sites of infection, and kill infected cells ( ) . cd + t cells also form part of the memory response. they can rapidly expand in number and eliminate pathogens after secondary challenge. also, cd + t cells can recognize tumor antigens presented by cancer cells and eradicate them before they metastasize ( ) . the cytotoxicity of cd + t cells requires direct contact with target cells, although the capability of each cd + t cell to eliminate target cells remains uncertain ( , ) . regardless, these cell-to-cell contacts enable cd + t cells to release cytotoxic granules and death ligands, thereby killing the target cells ( ) . interferon-gamma (ifnγ) is one of the critical cytokines for establishing effective immune responses. in the context of t cells, this cytokine helps to drive the polarization of cd + t cell response to t h , which directs the overall t cell response toward cell-mediated immunity. in cd + t cells, activated subsets start to produce ifnγ rapidly, with the dominant epitope-specific cd + t cells expanding preferentially over subdominant ones ( , ) . ifnγ production by activated cd + t cells also promotes t h polarization ( ) , enhances cell motility and cytotoxicity ( ) , and exerts antiviral immunity in a wide variety of cells through ifnγ receptor signaling ( ) . ifnγ also plays a vital role in regulating the contraction of effector cd + t cells after the clearance of primary infection ( , ) . despite its multiple functions, ifnγ produced by activated epitope-specific cd + t cells is widely used as an indirect marker to demonstrate the functional efficacy of epitope-specific cd + t cells. for example, as assays for measuring ifnγ produced by cd + t cells can be highly robust (e.g., intracellular cytokine staining, ics), ifnγ is often used as a surrogate marker in screening assays for identifying potential cytotoxic cd + t cell epitopes. however, one should note that the relationship between these two parameters is not necessarily correlated. the cytotoxicity of cd + t cells against their cognate targets can be measured directly, which was initially assayed through radioactive labeling of target cells, mostly using chromium- ( ) . fluorescence-based methods have since supplanted the radioactive labeling methods due to their higher sensitivity and better safety profile ( ) . however, these assays are typically used in a single target format, allowing measurements of cytotoxic potential of one or two cd + t cell attributes. this approach is not highly robust, which limits its potential use in large-scale screening studies. when there is a need to test many cd + t cells epitopes, the amount of starting materials (mice or human pbmcs) that are needed will inevitably increase substantially, which may pose difficulties for conducting comprehensive analyses. in this study, we demonstrated the feasibility of measuring cytotoxicity activities of up to twenty-four specificities using just three fluorescent dyes in a single reaction, thereby increasing the multiplexity and capability to screen multiple cd + t cell epitopes simultaneously. we used cd + t cells from human pbmcs to analyze previously known hla-a * -restricted epitopes of influenza a viruses in vitro, showing that only some of these epitopes can elicit a robust cytotoxic response. in mouse models, we demonstrated that this method allows for screening of cd + t cells harvested from different anatomical sites and that our approach allows accurate measurement of cell killing that is attributable to cognate cd + t cells. we further demonstrated that ifnγ expression by epitope-specific cd + t cells does not necessarily correlate with its cytotoxic potential. our results highlight that this multiplex assay has potential uses in identifying potent cytotoxic cd + t cell epitopes. besides, epitopes that have differential effects on the ifnγ production and cytotoxic activity of cd + t cells can be identified by this approach in a rapid manner. peptides of > % purity were purchased from genscript (piscataway, nj), reconstituted at mg/ml with dimethyl sulphoxide (dmso), and stored at− • c. all healthy donors and patients provided written, informed consents to participate in the study. ethics approvals were obtained from the human research ethics committee of the university of hong kong and monash health (hrec/ /monh/ ), royal melbourne hospital (local reference number: / ). blood samples from the subjects were screened for blood-borne pathogens and sent for human leukocyte antigen (hla) typing. patient blood samples were collected in sodium heparin tubes and pbmcs isolated as previously described ( ) . briefly, blood was diluted : (v/v) with pbs, overlaid on histopaque- (sigma-aldrich, st. louis, mo) and centrifuged at g for min without brake. isolated pbmcs were washed with pbs and resuspended in fbs+ % dmso for liquid nitrogen storage. six-to eight-week-old balb/cj, c bl/ j, and human dipeptidyl peptidase- knock-in (hdpp -ki) mice on c bl/ j background ( ) were used in these experiments. mice were bred under specific-pathogen-free conditions. all animal experiments and procedures were approved by the following: a vaccinia virus carrying h n np, ha, na, m , m genes, and human il- previously reported by us was used as an experimental vaccine ( ) . mice were anesthetized and given x pfu of this vaccine through the intranasal route twice at weeks apart. three weeks after the second vaccination, mice were challenged with mld h n (a/puerto rico/ / ) through the intranasal route. weight loss and survival of mice were followed for up to weeks. plasmodium berghei anka clone cy (pba) ( ) and plasmodium berghei nk (pbnk ) ( ) parasites were passaged in c bl/ j mice, and infected erythrocytes were resuspended in alsever's solution and stored in liquid nitrogen. to infect mice, × infected erythrocytes were injected through the intraperitoneal route. parasitaemia was monitored by flow cytometry ( ) . all mers-cov experiments were carried out in the university of iowa absl- facility. mice were infected with × pfu of human isolate of mers-cov (mers-cov-emc) and these mice were challenged after weeks with × pfu of a mouse-adapted strain of mers-cov. spleens from naïve mice were dissociated using a µm cell strainer with a syringe piston to release splenocytes in rpmi complete medium, supplemented with % fetal bovine serum (fbs) and u/ml penicillin-streptomycin (thermofisher scientific, waltham, ma). splenocytes were resuspended with ack lysis buffer ( mm nh cl, mm khco , . mm edta; all chemicals from sigma-aldrich) for at least a minute before washing with rpmi complete medium. the splenocytes were split into up to groups and pulsed with relevant peptides at a final concentration of mg/ml. treated cell were labeled with unique combinations of celltracker cmfda, cmtmr, and deep red dyes (thermofisher scientific; table s ) and were then washed with rpmi complete media. equal numbers of labeled cells from each group were combined and transferred into recipient mice at a total volume of µl and µl pbs for intranasal and retro-orbital routes, respectively. after - h, recipient mice were sacrificed to harvest donor splenocytes and bronchoalveolar lavage, which were labeled with live/dead fixable violet stain (thermofisher scientific) before acquisition by flow cytometry. thawed pbmcs were washed twice with rpmi complete medium, resuspended in ml fresh medium in ml falcon tube and left to recover at • c, % co overnight at about • horizontal tilt with loose cap ( ) . after recovery, cells were split into two groups: one group was treated with cd + t cell isolation kit and the other group treated with cd + nanobeads for depletion (biolegend, san diego, ca). the target cells obtained from the negative fraction from the latter group were split into groups for peptide pulsing and dye labeling as described earlier. equal numbers of cells from each group were then combined together and split into two groups: one group to be mixed with the isolated cd + t cells and the other group without. cells were seeded in a -well flat-bottom plate and incubated at • c, % co overnight. the next day, cells were labeled with live/dead fixable near ir stain (thermofisher scientific) before acquisition using a flow cytometer. splenocytes from mice at up to × cells were seeded together with mg/ml mouse il- (biolegend), µl bd golgiplug (becton dickinson, franklin lakes, nj) or brefeldin a (thermofisher scientific) and µg peptide in -well tissue culture plates, followed by incubation at • c, % co for h. for plasmodium studies, the mouse il- addition was omitted. they were then stained with zombie aqua fixable viability kit (biolegend) for min, washed and followed by antibody stainings for min, selected from the following panel: purified cd / (clone ); cd pe/dazzle (clone a ); cd ε brilliant violet (clone - c ); cd apc/cy (clone gk . ); cd a percp/cy . (clone - . ); cd b percp/cy . (clone yts . . , all from biolegend). after washing, cells are fixed in % formaldehyde, permeabilized with . % saponin (sigma-aldrich) or per buffer (bd bioscience), followed by staining with ifnγ fitc (clone xmg . , biolegend/ebioscience). the formula for the calculation of cytotoxicity of antigen-specific cd + t cells are as follows: ( ) mann-whitney u-test was used to analyse for statistical significance between two groups. for comparisons between more than two groups, one-way analysis of variance (anova) was used if sample data follow a normal distribution. otherwise, kruskal-wallis test and dunn's post-test were used. dunnett's or dunn's multiple comparisons were performed with the irrelevant control (csp , ova, mog, or ebo - ) designated as the control group. flow cytometric analysis was carried out with flowjo (treestar, ashland, or) and calculations were performed with prism (graphpad, la jolla, ca). in contrast to the conventional method of using a single cell labeling dye (e.g., cmfda) to create two distinct target populations during flow acquisition (figure a) , we first explored the possibility of combining three different labeling dyes to exponentially increase the number of target populations that can be distinguished ( figure b) . this strategy was inspired by the use of multicolor-coded mhc multimers to test different antigen-specific t-cell responses in a single sample ( ) . by modifying the concentrations of each dye, up to different groups of donor cells can be created (table s ). when equal numbers of cells from each group were combined and transferred into recipient mice through the intravenous route, all different groups of donor cells could be identified by flow cytometry (figure b) . the clear demarcation of each donor group raises the possibility of directly measuring the cytotoxicity of multiple cd + t cell specificities simultaneously in a single mouse (see below). this possibility is in sharp contrast to the conventional method, which requires one mouse for testing a single cd + t cell epitope. we previously developed a vaccinia-based influenza a virus (iav) vaccine (henceforth called vax a) that can express five h n iav genes and human interleukin- gene in mice ( ). this experimental vaccine is known to induce robust cd + responses in mice. mice vaccinated twice with vax a via intranasal route could be fully protected by a lethal heterologous influenza virus challenge (a/pr/ / ; mld ), whereas all unvaccinated mice succumbed to the challenge (figure a) . we used this animal model to evaluate our multiplex in vivo cytotoxicity assay. we divided mice into three groups, and they were subjected to different treatments ( figure b . naïve: no treatment; vax a + pr : vaccination followed by a virus challenge; pr : virus challenge only). at day post-challenge, we prepared donor splenocytes from unrelated naïve mice and divided them into eight groups. each group of donor cells were pulsed and labeled with a specific combination of peptide and dye ( table s ). the treated donor cells were then transferred into these mouse groups via intravenous or intranasal routes (figure c , upper and lower panel), and they were recovered from these recipient mice the following day. as shown in figure c , we could identify these labeled cells and distinguish them from each other in all experimental conditions. furthermore, donor cells pulsed with influenza epitopes np (cmfda+) and ha (cmtmr+) experienced high levels of cytolysis in vaccinated mice upon challenge with influenza ( figure c , highlighted by * in the middle panels, and figure d ), but not in unvaccinated mice. however, the csp (cmtmr++) control group experienced significant cytolysis in both the spleen and bronchoalveolar lavage of the unvaccinated mice, with this effect more pronounced in latter tissue compartment (figure d , left vs. right panels). this observation suggested that the inflammatory microenvironment contributed to substantial non-specific killing in these unprotected mice. the proinflammatory milieu is known to induce fas receptor expression in different cells ( ) ( ) ( ) , and others have shown that bystander cell lysis by activated cd + t cells occurs in a tnfα-or fasl-dependent manner ( ) ( ) ( ) . thus, we believe that the non-specific cytolysis by activated cd + t cells in our model could occur similarly. we also believe that the bystander killing of target cells due to dysregulated inflammation resulting from iav infection can also extend to cells labeled with iav epitopes. thus, the inclusion of irrelevant epitopes in this multiplex assay provides a unique opportunity to quantitate this bystander killing effect. consequently, we used csp data points from individual mice to normalize against the rest of the corresponding data points to obtain figure e . these normalized data clearly showed that cd -mediated cytolysis against influenza epitopes are also elevated in the lung mucosa of vaccinated mice. analyzing cytotoxicity of multiple epitope-specific human cd + t cells simultaneously in vitro next, we tested the potential of this multiplex approach using cd + t cells harvested from human pbmcs. we obtained frozen pbmc samples from hla-a * : -positive healthy individuals and influenza-infected patients to purify cd + t cells. we also harvested homologous non-cd + cells from the corresponding samples and use them as target cells in the assay. these target cells were divided into groups and pulsed with different hla-a * : -restricted influenza virus epitopes (table s ). these epitopes are experimentally known to induce influenza-specific cd + t cell responses in hla-a * : -positive individuals (influenza research database and immune epitope database). peptide-pulsed target groups were differentially labeled with unique combinations of fluorescent markers, pooled together and split equally into two groups, with one group incubated together with homologous cd + t cells and the other group without. the latter group serves as the naïve population, which is essential for the calculation of epitope-specific cd + t cell cytotoxicity. table s , then inoculated into recipient mice and re-harvested the next day to analyze remaining target cells. representative flow plots of mice from different experimental groups were shown, depicting the gating strategies to identify corresponding donor cell groups. corresponding positions for target cells treated with csp , ha , np , and dmso are highlighted in the top right panel for naïve mice as examples. (d-e) the analysis of cytolytic activity of specific cd + t cells, before (d) and after (e) normalization, were shown. *p < . , mann-whitney u-test. of sixteen tested iav epitopes, six (h n m - , h n pa - , h n pb - , h n pb - , h n np - , and h n np - ) could evoke significant levels of cytotoxicity from their cognate cd + t cells, both in healthy individuals ( figure a ) and in hospitalized patients with acute influenza (figure b ). for these six cytotoxic cd + t cell epitopes, infected individuals consistently elicited stronger responses than the healthy individuals did (one-way anova with holm-sidak's post-comparison; p < . ). hence, apart from identifying multiple epitope-specific cd + t cells that elicited cytotoxicity toward their targets simultaneously, the results of the multiplex in vitro cytotoxicity assay correlated with the immune status of individuals, i.e., the rapid proliferation of influenza-specific cd + t cells in infected individuals led to elevated levels of cytotoxic activities. ebola does not circulate in the region where our pbmc donors reside. we reasoned that hla-a * : -restricted ebola virus epitope, ebo - ( ) could serve as an appropriate irrelevant epitope to demonstrate the specificity of our multiplex cytotoxicity assay, as well as setting a baseline for comparison. as expected, no cytotoxic activity against ebo - could be detected, proving the specificity of this multiplex assay for testing human cd + t cells from the blood. all influenza virus epitopes used in the above human study were previously shown to stimulate cd + cell responses in various immune assays (table s ) . we were surprised to find that only a minority of these epitopes can elicit cytotoxic killing from their cognate cd + t cells. as hla-typed pbmc samples were highly limited, it was difficult for us to study this phenomenon comprehensively. therefore, we used different animal models to test whether cytotoxicity of epitope-specific cd + t cells can differ from corresponding ifnγ production. we first used the vaccinated mouse model described above to check for this phenomenon. we expanded the number of epitopes to be tested by searching through the influenza research database for h -k d -binding pr h n epitopes and their variants found in h or h subtype (if available). we selected a total of epitopes for a -color combination screening (table s ) . we also chose to perform the multiplex in vivo cytotoxicity assay and ifnγ-ics assay (figure a) in separate experiments in order to avoid issues of spillover signals from brightly labeled donor splenocytes. both results were then overlaid onto a single graph to allow for better visual comparison ( figure b ; non-h n viral epitopes are highlighted in gray and variant epitopes are in the same dotted box). in vaccinated mice subjected to a lethal pr challenge, the immunodominant epitopes np and ha consistently produced robust responses in both assays (figure b , left and ). however, the following epitopes did not give concordant results: flunp- , fluha- , fluha- , and flupb f - (figure b, arrows) . especially for flunp- , the peptide elicited a very significant positive response in cytotoxicity assays but not in assays measuring ifnγ expression. interestingly, with the exception of fluha- , iav variant epitopes that were present in h n or h n subtypes elicited similar cytotoxicity and ifnγ expression by cd + t cells as the corresponding homologs present in pr , suggesting that pr specific cd + t cells may tolerate small changes in the epitope sequence, enabling them to target cells infected by other iav subtypes. in unvaccinated mice, cytotoxicity against np and flunp- were significantly elevated upon lethal pr challenge, albeit not at levels seen in vaccinated mice ( figure c ). more importantly, intracellular ifnγ staining did not show any significant difference between any of the tested peptides. in the above mouse work, we observed apparent discrepancies between ifnγ production, a common marker of t cell activation, and actual cytotoxicity in some iav-specific cd + t cells. we wondered if we could observe this phenomenon in other disease models. to answer this, we used the pathogenic malaria model for evaluation. the malaria parasite plasmodium berghei anka (pba) causes a severe neurological complication termed experimental cerebral malaria in susceptible c bl/ j mice. at days post-infection, mice infected with pba were subjected to both assays with the eleven previously known h- b malaria epitopes (table s ). in pba-infected mice, pb -, f -, and pbt -specific cd + t cells displayed significantly elevated killing activities. interestingly, only pb and pbt -specific cd + t cells, but not those specific for the f epitope, registered significant increases in ifnγ production ( figure a) . a similar result also occurs when a different parasite strain, pbnk , was used to infect mice ( figure b) . additionally, we investigated this phenomenon in a mouse model of mers-cov infection ( ) . in this model, hdpp -ki mice were infected with mers-cov-emc. after weeks, these mice were challenged with a ld ( pfu) mouseadapted strain of mers-cov. at the peak of the t cell response, splenocytes were harvested and assayed for epitopespecific cd + t cell activity, with the epitopes shown in table s , by ifnγ expression and in vivo cytotoxicity assays. as shown in figure , cd + t cells recognizing all the tested figure | feasibility of performing multiple in vitro cytotoxicity assay in a single well. frozen pbmcs from (a) healthy and (b) influenza-infected hla-a* : individuals were rested in a • c/ % co incubator overnight. non-cd + t cells (target cells) were isolated from cd + t cells by macs, split into equal groups and pulsed with corresponding peptides (including one additional group that is incubated with dmso; peptide sequences are shown in table s ). pulsed target cells are combinatorically labeled with cell labeling dyes (celltrace violet, celltrace yellow, celltracker cmfda and celltracker deep red), mixed in equal numbers and split into two groups: one group incubated with cd + t cells and the other group without. cells are incubated at • c/ % co overnight and analyzed the next day by flow cytometry to calculate the specific lysis of target cells by cognate cd + t cells. ****p < . , one-way anova with holm-sidak's multiple comparison test against ebo - (control). cd + t cell epitopes show elevated ifnγ levels in challenged mice, but this was not corroborated in cytotoxic assays. when comparing s -and m -specific cd + t cell activities, the former showed potent cytotoxicity while the latter had no cytotoxicity, despite both of them having comparable levels of ifnγ expression (figure ). in this study, we expanded the conventional in vivo cytotoxicity assay from two parameters to twenty-four parameters. this functional assay allows the direct assessment of multiple cd + t cell specificities simultaneously in a single mouse or human pbmc sample, leading us to the finding that not all epitopespecific cd + t cells that express ifnγ exhibit concomitant direct cytotoxicity. importantly, we demonstrate that this phenomenon is not limited to three different pathogen mouse models: iav, malaria and mers-cov, but it also appears on human pbmc screening against experimentally known iav cd + epitopes. these observations raise concerns about t cell studies in applications such as validating epitope candidates for vaccine studies. currently, ifnγ-ics remains widely used for such a purpose, with those eliciting ifnγ production regarded as responding positively to a pathogen insult, leading them to be selected as potential targets of vaccine design. however, this may not mean the t cells induced by this kind of epitope-based vaccines can always control the disease, as has been shown in figure | discrepancy between direct and indirect measurements of cd + t cell effector functions in an influenza mouse model. splenocytes from vaccinated mice challenged by a lethal pr infection were used to measure epitope-specific cd t cell effector functions through direct (in vivo cytotoxicity) and indirect (ifnγ-ics) assays. twenty-three different peptides were employed for the screening (table s ) to fully utilize the capability of our multiplex assay. (a) the representative gating strategy for ifnγ-ics assay is shown. (b-c) the results for vaccinated (b) and unvaccinated (c) mice following challenge are shown. epitopes grouped within dashed boxes are homologs of each other, with those in gray originating from h n or h n . ****p < . , ***p < . , **p < . , *p < . , kruskal-wallis test with dunn's post-test against irrelevant control (csp ). red and black asterisks refer to corresponding data from multiplex in vivo and ifnγ-ics assays respectively. frontiers in immunology | www.frontiersin.org table s ) that were published in the literature were employed in the screens, with the results as shown. ***p < . , **p < . , *p < . , kruskal-wallis test with dunn's post-test against irrelevant control (ova). red and black asterisks refer to corresponding data from multiplex in vivo and ifnγ-ics assays respectively. a simian immunodeficiency virus vaccination/challenge study ( ) , and a human iav trial ( ) . although no cytotoxicity data were obtained in those studies, we argue that the possible discrepancies between ifnγ production and actual cytotoxic potential of the epitope-specific cd + t cells may be one of the causes. in this respect, we believe our multiplex cytotoxicity assay can further validate the epitope candidates in terms of the cytotoxicity potential of the cognate cd + t cells. in particular, our approach can be a robust tool for revealing a more comprehensive picture of the protective role of cd + t cells stimulated by t cell-based vaccines. furthermore, the multiplex cytotoxicity assay is also useful for evaluating cd + t cell cytotoxicity responses in t cell therapies such as those involving chimeric antigen receptor (car)-t cells or cancer vaccines. the multiplex cytotoxicity assay described here produces results that are comparable to that when the conventional single-plex cytotoxicity assay was performed instead (data not shown), which offers several advantages. firstly, the number of recipient mice needed in a given experimental setup can be reduced, resulting in lower costs, better compliance with animal ethics (the r rule), and data that are more reproducible across multiple epitopes. secondly, cd + t cell killing activities in different compartments can be analyzed simultaneously, which will provide data useful for evaluating the efficacy of a vaccine in inducing both systemic and mucosal figure | discrepancy between direct and indirect measurements of cd + t cell effector functions in a middle east respiratory syndrome (mers) mouse model. the hdpp -ki c bl/ j mice were infected with × mers-cov-emc via intranasal route. after weeks, these mice were challenged with × pfu of mouse-adapted strain of mers-cov via intranasal route. spleens from these mice were harvested and stimulated with the corresponding peptides in the presence of brefeldin a. ifnγ expression from cd t cells in the splenocytes were quantified (black). in parallel, splenocytes from healthy donor mice were used for multiplex in vivo cytotoxicity assay with the corresponding peptides and transferred into transduced/challenged recipients at day post-infection. the recipient splenocytes were harvested and analyzed by flow cytometry (red). *p < . , **p < . , ***p < . , ****p < . , kruskal-wallis test with dunn's post-test against irrelevant control (ova). red and black asterisks refer to corresponding data from multiplex in vivo and ifnγ-ics assays respectively. adaptive immunity. thirdly, the expansion of parameters available for screening allows one to include one or more irrelevant epitopes in the cytotoxicity assay. by definition, irrelevant epitopes should not elicit destruction by cd + t cells. however, it has been shown that uninfected cells also undergo apoptosis and necrosis during iav infection ( ) . hence, we argue that such non-specific killing can apply to intended target cells, and the destruction of irrelevant target cells acts as a control to measure non-specific killing activity, permitting the calculation of actual cd + t cell-mediated cytotoxicity levels. the expression of ifnγ in epitope-specific cd + t cells has been widely used to measure cell activation and hence represents a marker to evaluate the efficacy of cd + t cell epitopes. this expression and other commonly used maker staining methods (e.g., tetramer staining) are indirect methods of assessing t cell functionality. one would expect that cd + t cells that upregulate ifnγ production should be cytolytic against its cognate targets. however, in an hiv setting, single-cell analysis of individual hiv-specific cd + t cells in vitro revealed discordance between ifnγ expression and cytolytic potential ( ) . in three different mouse models of infection tested in our study, we showed that such discrepancies also occur in some epitope-specific cd + t cells. in most cases, the discrepancies exist in specific cd + t cells that were ifnγ-negative but possess vigorous cytolytic activity. however, this can also happen vice versa. continuous ifnγ signaling by activated epitope-specific cd + t cells was shown to lead to their reduction of cytolytic potential ( ) , suggesting that these cells may downregulate ifnγ production in order to maintain their cytolytic potential. on the other hand, the finding of substantial ifnγ production by cd + t cells that possess low cytotoxic potential, such as the m -and m -specific cd + t cells in our mers-covinfected mice, was also unusual. similar phenomena could also be found in humans ( ) , but the mechanism behind this is yet to be determined. the mechanisms regulating the ifnγ production and cytolytic potential of epitope-specific cd + t cells may be highly complex, and future research on this topic is needed. nevertheless, our results point out the disadvantage of relying solely on ifnγ production as a marker to evaluate cd + t cell activation, particularly in screening of cd + t cell epitopes for vaccine candidates, as one may miss out cd + epitopes that are useful in generating protective cd + t cells. besides, evaluations of pathogen-specific cd + t cell activities in diseases settings should be cautious when ifnγ is the only marker used in the assessments. we believe that our multiplex assay might able to fill these gaps. the datasets generated for this study are available on request to the corresponding author. the studies involving human participants cd (+) t cells: foot soldiers of the immune system recent advances in targeting cd t-cell immunity for more effective cancer immunotherapy in vivo killing capacity of cytotoxic t cells is limited and involves dynamic interactions and t cell cooperativity mechanisms and dynamics of t cellmediated cytotoxicity in vivo how do cytotoxic lymphocytes kill cancer cells? immunodominance in virus-induced cd (+) t-cell responses is dramatically modified by dna immunization and is regulated by gamma interferon the rapidity with which virus-specific cd + t cells initiate ifn-gamma synthesis increases markedly over the course of infection and 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clone fas ligand-mediated lysis of self bystander targets by human papillomavirus-specific cd + cytotoxic t lymphocytes perforin-independent cd (+) t-cell-mediated cytotoxicity of alveolar epithelial cells is preferentially mediated by tumor necrosis factor-alpha: relative insensitivity to fas ligand mapping hla-a , -a and -b supertype-restricted t-cell epitopes in the ebolavirus proteome attenuation of simian immunodeficiency virus sivmac infection by prophylactic immunization with dna and recombinant adenoviral vaccine vectors expressing gag a synthetic influenza virus vaccine induces a cellular immune response that correlates with reduction in symptomatology and virus shedding in a randomized phase ib live-virus challenge in humans cellular inhibitor of apoptosis protein ciap protects against pulmonary tissue necrosis during influenza virus infection to promote host survival a high-throughput single-cell analysis of human cd (+) t cell functions reveals discordance for cytokine secretion and cytolysis regulation of ifn-gamma signaling is essential for the cytotoxic activity of cd (+) t cells correlation between interferon-gamma secretion and cytotoxicity, in virusspecific memory t cells we thank mike richards, jeni mitchell, and teresa so for their technical supports. we acknowledge the assistance from faculty core facility in hku medicine and flow cytometry platform in singapore immunology network for flow cytometry analyses. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © poh, zheng, channappanavar, chang, nguyen, rénia, kedzierska, perlman and poon. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - ikzg authors: pan, xiaocheng; zhang, nianzhi; wei, xiaohui; jiang, yinan; chen, rong; li, qirun; liang, ruiying; zhang, lijie; ma, lizhen; xia, chun title: illumination of prrsv cytotoxic t lymphocyte epitopes by the three-dimensional structure and peptidome of swine lymphocyte antigen class i (sla-i) date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: ikzg to investigate ctl epitope applications in swine, sla- (*) -restricted peptide epitopes matching porcine reproductive and respiratory syndrome virus (prrsv) strains were explored by crystallography, biochemistry, and the specific pathogen-free (spf) swine experiments. first, nine predicted prrsv peptides were tested by assembly of the peptide-sla- (*) (psla- (*) ) complexes, and the crystal structure of the sla- (*) complex with one peptide (nsp -tmp ) was determined. the nsp -tmp peptide conformation presented by psla- (*) is different from that of the peptides presented by the known psla- (*) and psla- (*)hs complexes. two consecutive pro residues make the turn between p and p of nsp -tmp much sharper. the d pocket of psla- (*) is unique and is important for peptide binding. next, the potential sla- (*) -restricted peptide epitopes matching four typical genetic prrsv strains were identified based on the peptide-binding motif of sla- (*) determined by structural analysis and alanine scanning of the nsp -tmp peptide. the tetrameric complex of sla- (*) and nsp -tmp was constructed and examined. finally, taking nsp -tmp as an example, the ctl immunogenicity of the identified prrsv peptide epitope was evaluated. the spf swine expressing the sla- (*) alleles were divided into three groups: modified live vaccine (mlv), mlv+nsp -tmp , and the blank control group. nsp -tmp was determined as a prrsv ctl epitope with strong immunogenicity by flow cytometry and ifn-γ expression. our study developed an integrated approach to identify sla-i-restricted ctl epitopes from various important viruses and is helpful in designing and applying effective peptide-based vaccines for swine. the development of new viral vaccines should be increasingly focused on biosafety, especially to remove the viral genetic material and to avoid the possibility of recombinant viruses developing due to the use of vaccines. in view of this central idea, diverse viral vaccines, such as cytotoxic t lymphocyte (ctl) and b cell epitope vaccines, have been experimentally researched in animals for the ongoing control of viral diseases and immunological deficiency diseases ( ) . porcine reproductive and respiratory syndrome virus (prrsv) is one of the most important swine pathogens and has caused significant economic losses in the swine industry worldwide for two decades ( ) . prrsv is an enveloped positive-strand rna virus with a viral genome of ∼ kb in length and contains open reading frames (orfs) ( ) . orfs a and b are situated ′proximal to the polycistronic genome and encode two large non-structural replicase polyproteins, pp a, and pp b, which are processed into at least non-structural proteins (nsps). eight relatively small genes following orf in the ′ to ′ direction encode four membrane-associated glycoproteins, three membrane proteins, and a nucleocapsid protein. progress has been made in identifying nsp function related to rna synthesis (nsp and nsp ), subgenomic mrna synthesis regulation (nsp ), membrane-rearrangement (nsp and nsp ), replicative endonuclease (nsp ), major virulence factors (nsp - ) , and viral pathogenesis and host immunity (nsp , nsp , nsp , nsp , and nsp ) ( ) . the frequent mutation and recombination of the prrsv rna genome have resulted in the emergence of numerous variants ( , ) . these phenomena can cause the emergence of some virulent strains, such as the highly pathogenic prrsvs that are causing enormous economic losses in asia ( ) , and have led to the failure of vaccines against new emerging prrsvs. there are considerable challenges and specific requirements in the development of novel vaccines to prevent prrs, such as the ctl-epitope vaccine ( , ) . the greatest challenge is that prrsv can markedly suppress the swine immune defense system ( ) . the current evaluation of the prrsv vaccine is based on its induced antibody response. although high antibody titers can be produced after immunization, protection is not ideal because the key neutralizing antibodies (nabs) against prrsv appear late, typically > days post-infection (dpi), and usually at low levels ( ) . furthermore, nabs are usually specific for the homologous prrsv strain and confer little cross-protection against heterologous strains ( , ) . regarding ctl-mediated immunity, specific ctl responses have been observed in prrsv ( , , ) . the virulent type (lena) prrsv resulted in increased il- α production and a higher percentage of cd + t cells and ifnγ-producing cells compared with controls. cross-reactivity against divergent prrsv is also associated with cytotoxic cd +ifnγ and cd -ifnγ+ cells to a different extent ( ) . prrsv-specific t cells could be observed as early as weeks after infection, with the viral loads decreasing in persistent infection ( ) . modified live vaccines (mlvs) could induce ctl immune responses and confer better protection against heterologous prrsv strains than inactivated prrs vaccines ( ) . these findings indicate that specific cd + ctl immunity may play an important role in controlling prrsv infection. however, there is limited clear and direct evidence of ctls eliminating prrsv infection, and more basic immune reagents, such as the tetramer of swine major histocompatibility complex (mhc) class i with prrsv peptide epitope, are required to address these important issues ( ) . swine mhc class i has been referred to as swine lymphocyte antigen (sla-i). there are three classical sla-i loci (sla- , sla- , and sla- ) in the swine genome, and all are dominantly expressed ( ) . sla-i molecules can present viral peptide epitopes to swine cd + t cells and induce the ctl response to kill the infected cells ( ) . similar to human mhc (also known as human leukocyte antigen, hla), sla-i molecules are a highly polymorphic gene superfamily whose peptide-binding specificities are significantly influenced by highly variable sites ( ) . thus, far, more than sla-i genes have been cloned (ipd; http://www.ebi.ac.uk/ipd/index.html), and two three-dimensional ( d) structures of peptide-sla-i (p/sla-i) molecules have been determined, revealing the peptide presentation characteristics of sla-i molecules in swine ( , ) . thus, the situation is favorable for the design and development of a novel viral ctl vaccine against swine prrs based on these d structures of sla-i molecules. in an attempt to identify anti-prrsv ctl epitopes in this study, first, predicted peptide epitopes derived from prrsv were synthesized, and a trimolecular complex, the structure of the epitope from prrsv-nsp (tmppgfely, termed nsp -tmp )-bound sla- * (psla- * ), was solved. next, the potential sla- * -restricted peptide epitopes matching four typical genetic prrsv strains were identified. finally, the immunogenicity of the ctl epitope was identified. our results provide a novel strategy, i.e., the use of the mhc-restricted structural mechanism, to identify and validate ctl epitopes that could be used to develop a peptide-based vaccine against swine prrs. peptide epitopes were predicted by the netmhcpan . server (http://www.cbs.dtu.dk/services/netmhcpan/) based on the whole protein sequences of four typical prrsv strains (vr , genbank accession no. ef . ; hb- . , genbank accession no. eu . ; jxwn , genbank accession no. ef . ; and chsx , genbank accession no. kp . ). these potential non-apeptides were predicted using the sla- * allele (genbank accession no. hq ) and purified to > % purity by analytical reverse-phase high-performance liquid chromatography (hplc) (scilight biotechnology) ( table ) . these peptides were stored in lyophilized aliquots at − or − • c after synthesis and were dissolved in dimethyl sulfoxide (dmso) before use. refolding of the sla- * complex to assemble the psla- * complexes with each non-apeptide (table ) , sla- * heavy chain (hc) and swine β m(sβ m) inclusion bodies were refolded (in a : : molar ratio) via the gradual dilution method we described previously ( , ) . the sla- * hc and sβ m inclusion bodies were also refolded without peptides or with non-combined peptides as negative controls. in addition, the sla- * hc and sβ m inclusion bodies were refolded with a positive peptide (amino acid sequence nsdtvgwsw) as a positive control. after h of incubation at • c, the remaining soluble portion of the complex was concentrated and then purified via chromatography in a superdex / column, followed by resource-q anion-exchange chromatography (ge healthcare), as previously described ( ) . crystallization and data collection of psla- * the purified complex ( kda) of psla- * with the nsp -tmp peptide (amino acid sequence tmppgfely, derived from residues - of the prrsv non-structural protein) was dialyzed against crystallization buffer ( mm tris-hcl ph . , mm nacl) and concentrated to mg/ml. the sample was then mixed with reservoir buffer at a : ratio and crystallized via the hanging-drop vapor diffusion technique at and k. index kits (hampton research, riverside, ca) were employed to screen the crystals. with a protein concentration of mg/ml, crystals of psla- * were obtained in - days from index solution no. ( . m bis-tris ph . , . m ammonium acetate, % peg ) at • c. diffraction data were collected at a resolution of . Å (psla- * ) with an in-house x-ray source (rigaku micro-max desktop rotating anode x-ray generator with a cu target operated at kv and ma) and an r-axis iv ++ imaging plate detector at a wavelength of . Å. the crystals were first soaked in reservoir solution containing % glycerol as a cryoprotectant and then flash-cooled in a stream of gaseous nitrogen at − • c ( ). the collected intensities were indexed, integrated, corrected for absorption, scaled, and merged by using the hkl package ( ) . the structures of psla- * with nsp -tmp were solved via molecular replacement using the molrep program with hla-a * (pdb code, q ) as the search model. extensive model building was performed by hand with coot ( ), and restrained refinement was performed with refmac . additional rounds of refinement were conducted by using the phenix.refine program implemented in the phenix package ( ) with isotropic atomic displacement parameter (adp) refinement and bulk solvent modeling. the stereochemical quality of the final model was assessed with the procheck program ( ) . data collection and refinement statistics are listed in table . determination of the circular dichroism spectra and thermal unfolding of psla- * the thermostability of sla- * with six mutant peptides was examined via circular dichroism (cd) spectroscopy. cd spectra were measured at • c in a jasco j- spectropolarimeter equipped with a water-circulating cell holder. far-uv cd spectra ( - nm) were collected at a protein concentration of . mg/ml in mm tris (ph . ) buffer in a cuvette with a length of mm at . -nm spectral resolution. the ellipticity at nm was continuously recorded during heating. thermal denaturation curves were obtained by monitoring the cd value at nm in a cell with an optical path length of mm as the temperature was raised from to • c at a rate of • c/min. the temperature of the sample solution was directly measured with a thermistor. the fraction of unfolded protein was calculated from the mean residue ellipticity (θ ) by the standard method: the unfolded fraction (%) is expressed as (θ -θ n )/(θ u -θ n ), where θ n and θ u are the mean residue ellipticity values in the fully folded and fully unfolded states. the midpoint transition temperature (tm) was determined by fitting the data to the denaturation curves by using the origin . program (originlab), as described previously ( ) . the tetrameric psla- * complex was constructed according to a previously described method ( ) . briefly, a sequence containing a bira enzymatic biotinylation site was added to the c-terminus of the sla- * hc via pcr. the pcr primers and conditions were as described previously ( ) . then, the entire construct was cloned into the pet- a(+) vector, which was subsequently transfected into escherichia coli strain bl (de ) for protein expression. the inclusion bodies of recombinant sla- * hc containing the bira site and of sβ m were refolded with the nsp -tmp peptide as described above. the psla- * complex was then purified and biotinylated by using the bira enzyme (avidity aurora, co). finally, the complex was purified and tetramerized by mixing psla- * -bsp with pe-labeled streptavidin (biosource international, camarillo, ca) at a molar ratio of : , after which the samples were separated by using kda millipore tubes. sds-page electrophoresis was used to determine the efficiency of tetramerization. a total of nine specific pathogen-free (spf) swine ( kg, - weeks old). beijing center of spf swine breeding and management) expressing the sla- * alleles were divided into three groups: mlv, mlv+nsp -tmp , and a blank control group. for initial immunization, the mlv and mlv+nsp -tmp groups were injected with an attenuated prrsv vaccine according to the manufacturer's instructions (boehringer-ingelheim, ingelvac). after seven days, for the second immunization, the mlv + nsp -tmp group was injected with the nsp -tmp peptide mixed with complete freund's adjuvant (cfa, : emulsification). the mlv group was injected with the mlv peptide mixed with cfa. seven days later, peptide mixed with incomplete freund's adjuvant (ifa, : emulsification) was injected into the mlv+nsp -tmp group. the mlv group was injected with mlv mixed with ifa. the immune dose of the peptide was . mg/kg body weight. the control group was injected with phosphate-buffered saline (pbs), deionized water mixed with cfa ( : emulsification), and deionized water mixed with ifa ( : emulsification) at the same time as the immunization group. equivalent volumes were used in the immunization group and the control group. blood was collected from the anterior vena cava, and peripheral blood mononuclear cells (pbmcs) were isolated by the kit according to the manufacturer's instructions (solarbio). the pbmcs were incubated for min at • c in staining buffer (pbs with . % bsa and . % sodium azide) containing the pe-labeled tetrameric complex and the fitc-labeled anti-cd monoclonal antibody. the cells were then washed once with staining buffer and detected via flow cytometry. more than cell events were acquired for each sample. cells stained with pe-labeled tetramers and a fitc-labeled anti-cd monoclonal antibody were counted as ctl response cells ( ) . the results for fluorescence-activated cell sorting (facs) data are presented as the mean ± standard error of the mean (sem) for the three animals in each group. statistical analysis was performed using graphpad prism (https://www.graphpad.com) for windows. significant differences (p < . ) between means were tested by two-tailed student's t-test. immunization with nsp -tmp one week after immunization with nsp -tmp or deionized water, pbmcs were collected from immunized and control groups. these pbmcs were stimulated with nsp -tmp peptide at a concentration of µg/ml. pha was added at the same concentration to each positive control group, while an equivalent volume of pbs was added to the negative groups. swine ifn-γ in the supernatant was detected via an elisa kit according to the manufacturer's instructions (invitrogen) after the cells had been incubated at • c for h. six sla-i alleles were cloned from landrace pigs. sla- * showed better prrsv peptide-binding ability than the others (table s ) according to in silico prediction (http://www.cbs.dtu.dk/services/netmhcpan). nine prrsv peptides, all of which could be presented by sla- * , were synthesized to test this prediction ( table ) . all nine peptides could form complexes with sla- * and swine β m (psla- * ) by in vitro refolding. the stable psla- * complexes were further used to screen the crystal structures. d structure of psla- * sla- * in complex with nsp -tmp was crystallized in the p space group with a high resolution of . Å ( table ) . one asymmetric unit contains only one sla- * molecule. the psla- * complex displays a canonical p/mhc i structure, including the α , α , and α domains of the hc and the light chain sβ m. nsp -tmp is located in the peptide-binding groove (pbg) formed by the α and α domains ( figure a) . the root mean square differences (rmsds) between sla- * and two other solved p/sla i structures (sla- * , pdb code: qq ; sla- * hs , pdb code: h ) were found to be . and . , respectively, indicating similarities among the overall structures of the p/sla i molecules. the nsp -tmp peptide is fixed by hydrogen bonds with residues in the n-and ctermini of the pbg, and no hydrogen bonds were observed in the middle portion (p -p ) (figure b) . based on the surface model, the p and p residues are located outside the pbg, and their side chains are solvent accessible, especially the p residue, which is at the top position of the nsp -tmp peptide conformation ( figure c ). pocket composition of psla- * bound to nsp -tmp peptide the compositions and polarities of the six pockets of psla- * are shown in figure , and the interactions between the nsp -tmp peptide and these pockets are listed in table . the pockets of psla- * , p/sla- * , and p/sla- * hs are compared in figure . the a pocket of psla- * , composed of leu , tyr , phe , tyr , glu , tyr , leu , ser , and tyr , fixes p -thr via hydrogen bonds and strong van der waals forces (vdws) (figure a ; table ). the residues forming the a pockets of sla i molecules, including ser , are highly conserved (figure ) . in most mhc i molecules of other (figure b ; table ). the residue composition of sla- * is similar to that of sla- * , and only the residue at position (lys/val) is different (figure ) . the c, d, and e pockets usually form a large cavity in the middle portion of the pbg. the amino acid compositions of these three pockets in sla- * are shown in figures c-e. no hydrogen bonds or salt bridges were found in these structures; instead, many vdws were observed between the three pockets and the nsp -tmp peptide ( table ) . the d pocket is critical for the peptide selection of sla- * and sla- * hs because of the charged residue at position ( , ) . the non-polar met causes the d pocket of sla- * to be hydrophobic, in contrast to the charged d pocket of sla- * or sla- * hs (figure ) . the f pocket of psla- * consists of asn , ala , leu , tyr , leu , asp , tyr , ile thr , and lys and shows numerous interactions with p -tyr, reflecting a key anchoring site ( figure f ). p -tyr can form hydrogen bonds and many vdws with the residues of the f pocket ( table ). the f pockets of both psla- * and p/sla- * can accommodate p -tyr, and only two different residues (asn/gly and ala/thr ) were found between the two f pockets (figure ) . the nsp -tmp peptide conformation presented by sla- * is different from that of the peptides presented by sla- * and sla- * hs (figure ) . because of the two consecutive pro residues, the turn between p and p of the nsp -tmp peptide is much sharper than that in the other two peptides (figures a,b) . previous studies on sla- * and sla- * hs showed that the residue at position plays a key role in peptide binding by fixing the p residue with a salt bridge or hydrogen bond (figures c,d) . in contrast, no salt bridge or hydrogen bond forms between the p -pro of nsp -tmp and met of sla- * . to determine the peptide-binding motif of sla- * , the peptide nsp -tmp was mutated by alanine scanning ( ) , and cd spectra were used to test the stability of psla- * complexes with these mutant peptides ( figure ) . the in vitro refolding and cd results showed that the binding stabilities of p -ala, p -ala, and p -ala mutant peptides are significantly lower than that of the wild-type nsp -tmp peptide. although p -pro cannot form a hydrogen bond or salt bridge with the d pocket of sla- * , its ala mutant still impairs the stability of the psla- * complex. according to these results, the p , p , and p residues are the primary anchor residues of the epitope peptides presented by sla- * . the b, d and f pockets accommodate these primary anchor residues and determine the peptide-binding motif of sla- * , similar to sla- * and sla- * hs . the b and f pockets accommodate the p and p anchor residues of the binding peptide, respectively, and their preference for p and p anchor residues is determined by their amino acid composition. figure shows the pocket composition of sla- * . figure shows that the amino acid composition of the b and f pockets of sla- * is very similar to that of sla- * . differential amino acids are found only at one or two individual sites and do not form direct contacts with the side chains of the p or p residues of the binding peptide. because of the similar b and f pockets, the p and p residues of the sla- * -binding peptides should be the same as in the sla- * peptides. the sla- * binding peptide ( ) and the sla- * binding peptide have a large overlap at the p and p residues ( table ). the b pocket of sla- * accommodates multiple uncharged residues, while the f pocket mainly binds phe, tyr and trp. the in vitro refolding results for the peptides supported this reasonable speculation ( table ) . the uncharged d pocket of sla- * might accommodate various uncharged p residues, unlike those of sla- * and sla- * hs . peptides with p -ala cannot provide sufficient affinity, unlike larger amino acids, such as l, m, f, s, n, and p ( table ). in summary, the preliminary peptide-binding motif of sla- * is expected to contain the following combination: x-(s/m/f/w/t/v/i/l)-(l/p/m/f/s/n)-x-x-x-x-x-(f/y/w). the predicted peptide epitopes derived from whole protein sequences of four typical prrsv strains were screened: the first isolated strain, vr ; the low-virulence strain hb- . ; the highly pathogenic strain jxwn ; and the chsx strain, which was responsible for a recent epidemic in china (figure ) . most sla- * -restricted prrsv peptides are located in the non-structural protein and the rna-dependent rna polymerase (rdrp) encoded by orf a and b. although numerous mer sla- * -binding peptides exist in each of these four prrsv strains (∼ peptides), only peptides were found to be completely conserved in all four strains (table s ) . rdrp contains conserved peptides, which is a much greater number than in the other proteins. identification of nsp -tmp as the ctl epitope by using the tetramer technique and the detection of swine ifn-γ the tetrameric psla- * complex was constructed (figures a-c) ( ) . six landrace pigs expressing the sla- * genes were used to check the immunogenicity of nsp -tmp peptide (figure ) . a total of , events were recorded by the flow. the ratio of psla- * tetramer and cd double-positive cells was at a rate of ∼ . - % in the mlv+nsp -tmp -immunized group, which was significantly higher than in the control group (p = . ). the mlvimmunized group was significantly higher than in the control group (p = . ); however, there was no significant difference between the mlv+nsp -tmp -immunized group and the mlv-immunized group (p = . ) (figure b) . additionally, swine ifn-γ expression in the peripheral blood of each pig was detected according to the methods used by kumar and walker ( , ) . secreted ifn-γ was detectable in all of the immunized pigs but was lower than the lowest detectable limit in all control pigs ( figure c) . these data indicated that nsp -tmp , as the ctl epitope, could stimulate specific ctl immunity in swine. ctl epitopes might be a requirement of optimal prrsv immunity for the control and treatment of prrsv infection ( , ) . in our study, a novel approach was used to select prrsv ctl epitopes, i.e., starting from a computer prediction for prrsv peptides, followed by in vitro complex refolding with sla- * , the analysis of the complex crystal structure, the identification of sla- * -restricted potential epitopes from whole genomes of different prrsv strains, and finally, verification of the immunogenicity of sla- * -restricted prrsv epitope. the crystal structure of sla- * is the third to be solved for an sla-i allele. the crystal structure of sla- * exhibits the typical structural characteristics of an mhc i complex. the structure of sla- * is very similar to those of sla- * and sla- * hs , indicating that the overall combination of heavy chains, light chains, and peptides in the swine sla-i complex is highly conserved. however, in terms of peptide binding, the sla- * structure not only reflects the common features of sla-i alleles but also exhibits unique allelic-specific characteristics. similar to the previously resolved sla- * and sla- * hs , the n-terminus of the sla- * pbg is open because the amino acid at position of the a pocket is a small ser (figure a) , but in other species such as humans and mice, the amino acid at this position is a large trp ( , ) . the peptide-binding motif of sla- * , like that of sla- * and sla- * hs , is determined by the three pockets b, d and f together, while the hla-i molecule is mostly determined by the two pockets b and f. these common characteristics indicate that sla-i has its own unique species features in binding peptides. in the b and f pocket composition, sla- * and sla- * are very similar, and only the non-critical amino acids in the individual positions are different (figure ) , resulting in a large overlap of the anchoring residues accommodated in their b and f pockets ( ) . in the structures of sla- * and sla- * hs , the d pocket plays a key role in fixing the bound peptides, with a strong salt bridge between the charged residue and the p residue of the peptides ( , ) . the uncharged met of sla- * cannot form strong charge interactions with p residues similar to those observed for sla- * and sla- * hs (figure ) . nevertheless, the d pocket is still important in determining the peptide binding of sla- * and prefers uncharged residues of a certain size to form sufficient vdws. the three p/sla i structures indicate that regardless of its properties, the d pocket is critical in determining the peptide-binding motif of sla-i, and this phenomenon is expected to be a common feature among different sla-i alleles. sla- * was predicted in silico to present more prrsv peptide epitopes than other sla-i alleles cloned from landrace pigs, and the in vitro refolding results confirmed that most of the predicted prrsv peptides could be bound by sla- * . four typical prrsv strains of the north american genotype were used to screen sla- * -restricted binding peptides. according to the summarized peptide-binding motifs, approximately peptides in each prrsv strain could be presented by sla- * . these peptides are unevenly distributed in different regions, with the nsp / / proteins encoded by orf a, nsp / / encoded by orf b and gp / exhibiting most of the candidate peptide epitopes. approximately one out of three peptides are conserved among the four prrsv strains, and approximately half of these peptides are encoded by orf b of rdrp. ctl-epitope-based vaccines present advantages in terms of safety, specificity, and usability and are successfully used to control many viruses, such as hiv, hpv, and dengue virus ( ) ( ) ( ) ( ) . although studies aimed at developing an anti-prrsv epitope-based vaccine have been performed, no mature product is currently available ( ) ( ) ( ) . our data indicated that rdrp (especially nsp / / ) may be the best target for developing a prrsv vaccine to induce a ctl response to genetically heterologous strains. tetramers of p/mhc i alleles are basic reagents that are used in immunological studies ( , , ) . however, the absence of sla-i tetramers limits effective and convincing research on swine antiviral ctl responses, especially regarding accurate quantitative research. in this study, the crystallized nsp -tmp peptide was used to produce the tetramer for evaluating sla- * -restricted ctl responses. the nsp -tmp epitope could induce cd and tetramer double-positive ctls at a rate of ∼ . - % in mlv+nsp-tmp -immunized pigs, similar to the results obtained for other known efficiently protective viral ctl epitopes found in humans and mice by facs ( ) ( ) ( ) . our results also showed that the mlv used (produced by the vr strain) could induce a ctl response specific to prrs. somewhat disappointingly, we do not have live prrsv with which to challenge these swine groups and evaluate the protection of mlv and nsp-tmp epitope. however, nsp -tmp was identified as an immunogenic epitope that could stimulate the proliferation of specific cd + ctls and the expression of ifn-γ in spf landrace pigs bearing sla- * alleles. immunization enhancement with nsp -tmp produces a specific ctl response similar to that of immunization with mlv, indicating that the peptide vaccine can produce effective immunoprotection and thus that it is feasible to develop an effective prrsv polypeptide epitope vaccine. in conclusion, we solved the crystal structure of sla- * and described its prrsv peptide-binding map according to its preliminary peptide-binding motif determined via biochemical analyses. using the tetramer of sla- * , the immunogenicity of nsp -tmp was identified by facs and the expression of ifn-γ. the results increase our understanding of how to acquire a viral ctl vaccine against swine prrs disease. in addition, this study provides a complete and credible method for identifying sla-i-restricted viral epitopes, demonstrating the feasibility of peptide vaccines in antiviral immunity of swine. based on our experimental results, we encourage and promote the development of a safe peptide vaccine that can effectively activate ctl immune protection, solve the safety problems figure | identification of the functional nsp -tmp ctl epitope. (a) immunization program for spf pigs. nine pigs expressing sla- * were divided into three groups: the mlv group, mlv+peptide group and blank control group. the mlv and mlv+peptide groups were injected with an attenuated prrsv vaccine as the first immunization. seven days later, the mlv+peptide group was injected with the nsp -tmp peptide mixed with cfa, and the mlv group was injected with mlv mixed with cfa as the second immunization. at day , the mlv+peptide group was injected with the nsp -tmp peptide mixed with ifa, and the mlv group was injected with mlv mixed with ifa as the third immunization. pigs in control group were injected with pbs, deionized water mixed with cfa, and deionized water mixed with ifa. (b) nsp -tmp -specific ctls stained with pe-labeled sla- * tetramer and fitc-labeled anti-cd monoclonal antibody were detected via flow cytometry. (c) the secreted ifn-γ contents of the control group and immunized group were measured via elisa kit. the secreted ifn-γ content of the control group was less than the lowest detectable limit ( pg/ml). caused by conventional attenuated vaccines, and provide new ideas for controlling not only prrs but also the extent of african swine fever. the coordinates and structural characteristics of psla- * have been deposited in the protein data bank under accession number ylx; the sequence of sla- * is available at the national center for biotechnology information (ncbi) database under accession number hq . the animal trials in this study were performed according to the chinese regulations for laboratory animals-the guidelines for the care of laboratory animals (ministry of science and technology of people's republic of china) and laboratory animal requirements for environment and housing facilities (gb - , national laboratory animal standardization technical committee). the license number associated with this research protocol is cau - , which was approved by the laboratory animal ethics committee of china agricultural university. the protocol adhered to the recommendations in the institute for laboratory animal research's guide for the care and use of laboratory animals. full protection of swine against foot-and-mouth disease by a bivalent b-cell epitope dendrimer peptide assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states the structural biology of prrsv the prrsv replicase: exploring the multifunctionality of an intriguing set of nonstructural proteins analysis of orf and full-length genome sequences of porcine reproductive and respiratory syndrome virus isolates of genotypes and retrieved worldwide provides evidence that recombination is a common phenomenon and may produce mosaic isolates complete genome sequence of a novel variant porcine reproductive and respiratory syndrome virus (prrsv) 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responses a phase i trial of a human papillomavirus (hpv) peptide vaccine for women with high-grade cervical and vulvar intraepithelial neoplasia who are hpv positive safety and immunogenicity of a ctl multiepitope peptide vaccine for hiv with or without gm-csf in a phase i trial location and characterization of the antigenic portion of the fmdv immunizing protein assessment of the efficacy of commercial porcine reproductive and respiratory syndrome virus (prrsv) vaccines based on measurement of serologic response, frequency of gamma-ifn-producing cells and virological parameters of protection upon challenge vaccine efficacy of porcine reproductive and respiratory syndrome virus chimeras efficacy of a modified live porcine reproductive and respiratory syndrome virus (prrsv) vaccine in pigs naturally exposed to a heterologous european (italian cluster) field strain: clinical protection and cell-mediated immunity mhc-peptide tetramers to visualize antigen-specific t cells the numbers game for virus-specific cd + t cells identification of an hla-a * -restricted t-cell epitope on the mpt protein, a major secreted protein derived from mycobacterium tuberculosis, by mpt overlapping peptide screening detection, phenotyping, and quantification of antigen-specific t cells using a peptide-mhc dodecamer identification and structural definition of h -specific ctl epitopes restricted by hla-a * derived from the h n subtype of influenza a viruses we thank the shanghai synchrotron radiation facility (ssrf), shanghai, people's republic of china, for the crystal diffraction data. we thank dr. lei zhou for his help in collecting information on prrsv strains. we thank prof. george f. gao of the chinese academy of sciences. cx: design of the study. xp and nz: data collection. xp, yj, and ql: analysis and interpretation of data. cx, nz, and xw: drafting the article. cx, xp, rl, lz, and lm: critical revision of the article. xp, nz, xw, yj, rc, ql, rl, lz, lm, and cx: final approval of the version to be published. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material key: cord- -corvd cs authors: li, chen; liu, jiaxin; zhang, xin; wei, shina; huang, xiaohong; huang, youhua; wei, jingguang; qin, qiwei title: fish autophagy protein exerts negative regulation on antiviral immune response against iridovirus and nodavirus date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: corvd cs autophagy is an important biological activity that maintains homeostasis in eukaryotic cells. however, little is known about the functions of fish autophagy-related genes (atgs). in this study, we cloned and characterized atg , a key gene in the autophagy gene superfamily, from orange-spotted grouper (epinephelus coioides) (ecatg ). ecatg encoded a -amino acid protein that shared and % identity to seabass (lates calcarifer) and humans (homo sapiens), respectively. the transcription level of ecatg was significantly increased in cells infected with red-spotted grouper nervous necrosis virus (rgnnv). in cells infected with singapore grouper iridovirus (sgiv), ecatg expression declined during the early stage of infection and increased in the late stage. fluorescence microscopy revealed that ecatg mainly localized with a dot-like pattern in the cytoplasm of grouper cells. overexpression of ecatg significantly increased the replication of rgnnv and sgiv at different levels of detection, as indicated by increased severity of the cytopathic effect, transcription levels of viral genes, and levels of viral proteins. knockdown of ecatg decreased the replication of rgnnv and sgiv. further studies showed that overexpression ecatg activated autophagy, decreased expression levels of interferon related cytokines or effectors and pro-inflammatory factors, and inhibited the activation of nuclear factor κb, ifn-sensitive response element, and ifns. in addition, ectopic expression of ecatg affected cell cycle progression by hindering the g /s transition. taken together, our results demonstrated that fish atg exerted a crucial role in virus replication by promoting autophagy, down-regulating antiviral ifn responses, and affecting the cell cycle. autophagy is a conserved cell biological pathway that delivers cytoplasmic components to lysosomes for degradation and elimination of useless or harmful substrates to maintain cell homeostasis in all eukaryotic cells ( ). this fundamental process involves formation of double membrane autophagosomes, which fuse with lysosomes to degrade the sequestered cargo ( , ) . viruses depend on the host cell's machinery to replicate the genome and generate progeny virus particles. autophagy, as a cell steward, has been reported to play an important role in virus replication. many studies have reported that viruses cause accumulation of autophagosomes and exploit these membrane structures as "virus factories" ( ) . in addition, several studies have confirmed that autophagy interacts with the innate antiviral immune response ( ) ( ) ( ) . autophagy can amplify the innate immune response that is mediated by nucleic acid-sensing toll-like receptors and enhance delivery of cytosolic pathogen-associated molecular patterns ( ) . additionally, some autophagy factors can downregulate rig-i (retinoic acidinducible gene i)-like receptors and type i interferon (ifn) signaling and suppress inflammasome activation ( , ) . the process of autophagosome formation is regulated by several autophagy-related genes (atgs) ( ) . in the autophagy gene superfamily, atg is a key gene that plays an important role in early autophagosome formation. enhanced or reduced atg levels affect the occurrence and alteration of autophagy pathways. on one hand, atg protein can conjugate to atg to form a complex with the multimeric protein atg , and the atg -atg -atg complex facilitates extension of the autophagosome ( , ) . on the other hand, the combination of atg complex and autophagic vesicle membrane can promote recruitment of lc (atg ) to autophagic vesicles ( ) . atg is involved in various physiological and pathological processes. in lipid metabolism, silencing or knocking out atg can lead to lipid deposition. atg also plays a role in regulating ifn immune and inflammation responses ( ) . the grouper (epinephelus spp.) is a well-known mariculture species that is widely distributed in south china and southeast asia. in , the scale of grouper breeding in china was , t, which was . % higher than that in . however, outbreaks of viral diseases have caused heavy economic losses in the grouper aquaculture industry. two representative pathogens are singapore grouper iridovirus (sgiv) and red-spotted grouper nervous necrosis virus (rgnnv) ( , ) . current research on the prevention and control of viral diseases in grouper is mainly focused on exploration of the anti-virus immune network and key immune genes. although numerous immune regulatory molecules have been found to play vital roles in the grouper antiviral response ( ) ( ) ( ) ( ) ( ) , the roles of atgs in the replication of sgiv or rgnnv have not been reported. in other studies of aquatic viruses, proliferation of svcv was significantly reduced in beclin- (atg ) and lc (atg )-depleted endothelial progenitor cells. however, references to atg in aquatic animal viruses are limited ( ) . in this study, we cloned a key autophagy related gene (atg ) from orange-spotted grouper (e. coioides) (ecatg ) and investigated the roles of ecatg in autophagy, innate immunity, and cell cycle. our results provide new insights into the roles of fish atg in virus infection. based on several expressed sequence tag sequences of ecatg from the grouper spleen transcriptome ( ), primers (table ) were designed to amplify the full-length open reading frame (orf) of ecatg . identity analysis between ecatg and other atg sequences was performed using blastp searches of the ncbi database. amino acid alignments were conducted using mega . software and edited with the genedoc program. the phylogenetic analysis was carried out using the boot-strapped neighbor joining method in clustalx . software. orange-spotted groupers ( - g) used in this study were purchased from a local farm in hainan province and kept in a laboratory recirculating seawater system as described previously ( ) . the relative expression level of ecatg was examined using quantitative real-time pcr (qrt-pcr) in selected tissues, including liver, spleen, head kidney, kidney, heart, intestine, brain, gill, stomach, muscle, fin, and skin. all tissues were collected from three fish. a grouper spleen (gs) cell line was established in our lab ( ) , and cells were propagated and maintained at • c in leibovitz's l- medium (gibco, usa) supplemented with % fetal bovine serum (gibco, usa). sgiv and rgnnv were isolated in our laboratory and propagated in gs cells with titer of tcid /ml as described previously ( , ) . to clarify the molecular function of ecatg in vitro, ecatg was subcloned into the vectors pegfp-c and pcdna . - ×ha using the primers listed in table . all recombinant plasmids were confirmed by dna sequencing. gs cells were transfected with ecatg sirna (siecatg : ′ -gaaagagauguacccugcugcuuua- ′ ) or same volume negative control for h, and then infected with sgiv or rgnnv for , , and h. at the end of each incubation period, the total rna or protein of cells were extracted for detection. gs cells were seeded onto cover slips ( × mm) in -well plates. after allowing the cells to adhere for h, pegfp-c and pegfp-ecatg plasmids were transfected into gs cells using the transfection reagent lipofectamine (invitrogen, carlsbad, ca, usa). at h post-transfection, cells on the cover slips were washed with phosphate buffered saline (pbs), fixed with % paraformaldehyde for h at room temperature, and then stained with , -diamidino- -pheny-lindole (dapi) for min. finally, cells were observed under a fluorescence microscope (leica, wetzlar, germany). total rna isolation was performed using the sv total rna isolation system (promega, usa) according the manufacturer's instructions, and reverse transcription was carried out using revertra ace (toyobo, osaka, japan). qrt-pcr was performed in an abi quant studio device (applied biosystems, carlsbad, ca, usa). the expression levels of viral genes and host immune genes were detected. the relative expression ratio of the selected gene vs. β-actin (reference gene) was calculated using the − ct method. reactions of sybr green were performed in a µl volume containing µl of × sybr r premix ex taq tm , . µl of each forward and reverse primer ( µm), . µl of water, and µl of cdna. all experiments were performed in triplicate, and the cycling parameters were chosen according to the manufacturer's instructions. cells were collected and lysed in ripa buffer. proteins were separated by % sds-page and transferred onto immobilon-p polyvinylidene difluoride membranes (millipore, temecula, ca, usa). blots were incubated with the indicated primary antibody: anti-ha ( : , dilution), anti-β-actin ( : , dilution), anti-atg ( : dilution), anti-lc ( : , dilution), anti-sgiv major capsid protein (mcp) ( : , dilution), anti-rgnnv capsid protein (cp) ( : , dilution). subsequently they were incubated with horseradish peroxidase (hrp)-conjugated goatanti-rabbit igg ( : , dilution). mouse monoclonal anti-ha antibody was purchased from sigma (usa). mouse monoclonal anti-β-actin antibody and rabbit polyclonal atg antibody were purchased from proteintech (rosemont, il, usa). rabbit monoclonal anti-lc antibody was purchased from abcam (usa). hrp-conjugated goat anti-rabbit and anti-mouse antibodies were purchased from kpl (usa). the polyclonal anti-mcp antibody of sgiv and the polyclonal anti-cp antibody of rgnnv were prepared in our lab. immunoreactive proteins were visualized using an enhanced hrp-dab chromogenic substrate kit (tiangen, beijing, china). to evaluate the promoter activity regulated by ecatg , luciferase reporter plasmids, including interferon-sensitive response element (isre)-luc, ifn -luc, and nuclear factor (nf)-κb (clontech, usa), were used for co-transfection. briefly, gs cells were transiently transfected with the luciferase plasmids together with the indicated ecatg expression vectors using lipofectamine reagent. the prl-sv renilla luciferase vector was used as an internal control. luciferase activity of total cell lysates was measured using a luciferase reporter assay system (promega). statistical analysis was performed using spss version . oneway anova was used to evaluate the variability between treatment groups ( * p < . , * * p < . ). the full-length orf of ecatg was obtained using pcr amplification. sequence analysis indicated that ecatg encoded a -amino acid protein that shared % and % identity to seabass (lates calcarifer) and humans (homo sapiens), respectively ( figure a) . phylogenetic analysis indicated that ecatg was closely related to the fish subgroup, followed by amphibians, birds, and mammals ( figure b) . to analyze the tissue distribution, qrt-pcr was conducted in different tissues of healthy juvenile orange-spotted grouper. frontiers in immunology | www.frontiersin.org ecatg was constitutively expressed in all the analyzed tissues in healthy grouper, and it was relatively high mrna levels in the brain, liver, and fin (figure a) . to analyze the gene expression profiles in response to different viral infections, the transcription levels of ecatg were examined in rgnnv or sgiv infected cells. the transcription levels of ecatg were significantly increased in rgnnv infected cells. in sgiv infected cells, the expression levels of ecatg first decreased within h post-injection and then increased after h ( figure b ). to demonstrate the subcellular localization of ecatg , pegfp-ecatg was transfected into grouper cells, and fluorescence was observed under fluorescence microscopy. green fluorescence was observed in the cytoplasm in ecatg transfected grouper cells, and most of these cells exhibited fluorescence aggregation (figure ) . in pegfp-c transfected cells, fluorescence was distributed both the cytoplasm and nucleus. the results showed that ecatg was a cytoplasmic protein. to clarify the function of ecatg , the eukaryotic expression vector of pcdna . - ×ha-ecatg was constructed, and the recombinant plasmid successfully expressed ha-ecatg protein after being transfected into gs cells ( figure a ). on the contrary, ecatg protein level was decreased after sirna silencing ( figure b ). autophagy is characterized by the formation of autophagosomes. conjugation of the essential lc to phosphatidylethanolamine is required for autophagosome biogenesis. therefore, lc lipidation is used as a faithful marker of autophagy activation ( , ) . to assess whether ecatg overexpression affected gs autophagy, we investigated the level of lc lipidation in cells overexpressing or silencing ecatg . the lc -ii (the lipidated form) level was higher in cells transfected with ecatg compared with control cells (figure a) , and ecatg knockdown reduced the lc -ii level (figure b) , which suggests that ecatg might activate autophagy by promoting lc lipidation in gs cells. to clarify the effects of ecatg overexpression on virus infection, ecatg transfected cells were infected with sgiv or rgnnv, and then viral replication was investigated. severity of the cytopathic effect (cpe) induced by sgiv infection evoked at h (figure a) . the amount and severity of vacuoles induced by rgnnv infection also increased in ecatg overexpressing cells compared to the empty vector transfected cells. at the transcription level, the expression of sgiv mcp, icp , vp , and litaf increased in ecatg overexpressing cells after sgiv infection ( figure b) . the transcription of rgnnv cp and rdrp genes also increased compared with control cells after rgnnv infection ( figure c) . consistently, ectopic expression of ecatg also increased the protein levels of sgiv mcp and rgnnv cp (figure d) . meanwhile, the effects of silencing ecatg on sgiv and rgnnv replication were also studied. quantitative analysis results showed that the transcription level of sgiv mcp, icp , vp , and litaf decreased after ecatg knockdown ( figure a) . the transcription of rgnnv cp and rdrp genes also decreased compared with negative control cells ( figure b) . consistently, ecatg knockdown decreased the protein levels of sgiv mcp and rgnnv cp in gs cells ( figure c) . together, ecatg was speculated to promote sgiv and rgnnv replication in gs cells. to explore the potential mechanism involved in the action of ecatg in fish virus infections, the roles of ecatg on the host interferon immune and inflammation response were evaluated. the pcdna . - ×ha-ecatg and the empty vector were transfected into gs cells, and cells were harvested at , , and h. the transcription levels of host immune factors and pro-inflammatory cytokines were detected using qrt-pcr. as shown in figure , expression levels of interferon related cytokines or effectors, including irf , irf , mda , isg , lgp , mxi, ifp , myd , and traf , were all decreased in ecatg overexpressing cells compared with control vector transfected cells. in addition, we also found that the expressions of pro-inflammatory cytokines such as interleukin (il)- β, il- , il- , and tumor necrosis factor alpha, were all significantly decreased in ecatg overexpressing cells (figure ). to further explore the roles of ecatg during fish virus infection, the promoter activity of reporter genes in typical antiviral pathways, including isre, type i ifn, and nf-κb, were measured using the plasmids isre-luc, inf-luc, and nf-κb-luc. as shown in figure a , ecatg overexpression suppressed the promoter activity of these genes. in addition, siecatg was co-transfected with the plasmids isre-luc, inf-luc, and nf-κb-luc, and the promoter activity of three reporter genes were measured. the results showed that sirna-mediated atg knockdown increased the promoter activity of three reporter genes ( figure b) . thus, we proposed that ecatg negatively regulates nf-κb and the ifn immune responses. mammalian atg plays a causal role in regulating cell cycle progression ( , ) . whether ecatg has the similar effects on cell cycle remains uncertain. to explore the role of ecatg on cell cycle progression, gs cells were transfected with pcdna . - ×ha or ecatg . ectopic expression of ecatg clearly inhibited the g /s transition compared to the empty vector overexpressing cells. the percentages of g phase cells in pcdna . - ×ha and ecatg overexpressing cells were . and . %, respectively (figure ) . those results indicated that ecatg may affect cell cycle progression from the g to the s phase and arrest cells in the g phase. autophagy is a highly conserved pathway, and it plays an important role in resistance to intracellular viruses and other pathogens ( ) . autophagosome formation relies on the atg family ( ) , and atg has been studied in detail in mammals. atg is involved in autophagic membrane extension and curvature, lc (atg ) recruitment, and lysosome and late intracellular regeneration. it also has been implicated in the ifn immune response, inflammation response, and lipid metabolism ( , ) . in fish, the roles of atg gene in zebrafish neurogenesis and organogenesis has been reported, and the results showed that the formation of the atg -atg conjugate may depend on atg protein generation and its splicing ( ) . twelve autophagyrelated genes from yellow catfish pelteobagrus fulvidraco and their transcriptional responses to waterborne zinc exposure were also characterized ( ). however, little has been reported about the roles of atg in virus replication and its relationship with the innate antiviral immune response in aquatic animals by far. in the present study, an atg homolog from orange-spotted grouper (ecatg ) was cloned and its roles during fish virus infection were investigated. ecatg encoded a -amino acid protein that shared % and % identity to seabass (lates calcarifer) and humans (homo sapiens), respectively. atg functions as an e (b) knockdown of ecatg decreased rgnnv gene transcription including cp and rdrp. one-way anova was used to evaluate the variability between treatment groups (*p < . , **p < . ). (c) virus protein level after transfection with siecatg . the level of sgiv-mcp or rgnnv-cp was detected by western blot, and β-actin was used as the internal control. band intensity was calculated using quantity-one software and ratios of mcpor cp/β-actin was assessed (p < . ). ligase-like enzyme ( ) . ecatg possesses all the characteristic features of canonical ubiquitin ligase, including two ubiquitinlike domains, a helix-rich domain, and the conserved calpain cleavage sites ( , ) . as a preliminary step to unravel the physiological role of ecatg , the mrna tissue distribution was determined. the present results indicated that the mrna expression of ecatg was ubiquitous within all the tested tissues. the ubiquitous distribution suggested that autophagy was implicated in many metabolic pathways among the tissues. originally autophagy was identified as a response to nutrient deficiency, so it is thought to be a receptor of cellular energy and metabolism ( ) . however, it is now evident that autophagy can be induced by a variety of factors, including starvation, reactive oxygen figure | ecatg overexpression decreased expression of interferon related cytokines or effectors. gs cells were transfected with ecatg , then cells were harvested at the indicated time. expression levels of host ifn associated genes were determined using qrt-pcr. one-way anova was used to evaluate the variability between treatment groups (*p < . , **p < . ). species, endoplasmic reticulum stress, microbial invasion and so on ( ) . based on this, we detected the expression of ecatg under the stimulation of two viruses. transcription levels of ecatg increased from the early stage of rgnnv infection, suggesting that rgnnv infection may significantly induce autophagy activity to facilitate its proliferation. in sgiv infected cells, the expression levels of ecatg firstly decreased within h post-infection and then increased after h. this pattern might be caused by the lack of cellular nutrition. with the cell growth, metabolism and virus replication, the nutrient deficiency in cells will increase autophagy activity over time. autophagy is an important cellular process by which atg initiates the formation of double membrane vesicles (dmvs). recently, the contribution of an autophagy protein, atg , to viral replication has been demonstrated ( ) , and atg was identified as an interacting protein for the hepatitis c virus ns b ( ) . the altered expression level of ecatg in gs cells infected with sgiv and rgnnv suggested that ecatg might play an essential role in the grouper response to fish virus infection, so the impact of ecatg overexpression on virus proliferation were investigated. overexpression of ecatg promoted sgiv and rgnnv replication, evidenced by the severity of cpe, the increased transcription levels of viral genes, and the increased levels of viral proteins. knockdown of ecatg decreased sgiv and rgnnv replication by assessing transcription and protein levels of viral genes. the results suggested that atg might share conserved function to viral replication from fish to mammals. studies of mammals suggested that atg -atg promotes viral replication by negatively regulating the ifn response ( ) . the atg -atg conjugate interacts directly with the mitochondrial antiviral-signaling protein (mavs) and retinoic acid-inducible gene i (rig-i) through the n-terminal caspase recruitment domain (card), resulting in inhibition of type i ifn production ( ) . n-terminal fragments of rig-i and ifn-α possess great capacity to activate ifn-β and isre promoters in atg deficient cells ( ) . when the formation of autophagosomes was promoted, the activity of the ifnβ promoter was decreased so that autophagy contributed to sustained hepatitis c virus infection ( ) . here, overexpression of ecatg in grouper cells not only decreased the expression levels of several interferon related cytokines or effectors, but also negatively regulated the expression of pro-inflammatory factors. moreover, the ectopic expression of ecatg significantly decreased isre, ifn, and nf-κb promoter activities, and figure | overexpression of ecatg decreased the transcription of proinflammatory factors. gs cells were transfected with ecatg , then cells were harvested at the indicated time. expression levels of proinflammatory factors were determined using qrt-pcr. one-way anova was used to evaluate the variability between treatment groups (*p < . , **p < . ). knockdown of ecatg increased promoter activities of nf-κb, ifn, and isre. the promoter activity was measured using the luciferase reporter gene assay. one-way anova was used to evaluate the variability between treatment groups (*p < . , **p < . ). knockdown of ecatg raised promoter activities of these reporter genes. atg -atg inhibits the production of ifn in canonical autophagy while playing the opposite role in alternative autophagy ( ) . thus, overexpression of ecatg might activate canonical autophagy in gs cells. overexpression of ecatg up-regulated the level of lc -ii, indicating that ecatg can activate autophagy. taken together, we speculated that ecatg decreased interferon immune response and activated autophagy might contribute greatly to its promoting effect on sgiv and rgnnv replication. in mammals, atg can induce cell cycle arrest at the g /s phase by up-regulating expression of p (a cyclindependent kinase inhibitor) at the level of post-transcription in response to challenges such as nutrient deficiency ( , ) . considering that atg is a key and relatively conserved protein, we speculated that fish atg might play a similar role in cell cycle progression. in the present study, ecatg affected cell cycle progression from the g to the s phase and arrested cells in the g phase. it was also reported that the replication level and virus titer of rgnnv were greater in cells released from the g phase or s phase of the cell cycle compared to cells released from the g phase ( ) . those results suggested that overexpression of ecatg may facilitate rgnnv replication. however, whether fish atg affects the cell cycle by regulating p requires further investigation. in conclusion, a key autophagy related gene (atg ) from orange-spotted grouper (e. coioides) (ecatg ) was cloned, and the roles of ecatg in autophagy, innate immunity, and cell cycle were investigated in this study. the results showed that ecatg plays crucial roles in virus replication via promoting autophagy, down-regulating antiviral ifn responses, and affecting cell cycle. this study identified a link between the autophagic machinery and innate immune signaling against viral infection. the datasets for this manuscript are not publicly available because this data has not been published. requests to access the datasets should be directed to jingguang wei, weijg@scau.edu.cn. all animal-involving experiments of this study were approved by the animal care and use committee of college of marine sciences, south china agricultural university, and all efforts were made to minimize suffering. qq and jw designed the experiments. cl performed the majority of the experiments, analyzed data, and wrote the manuscript. jl and xz contributed experimental suggestions. sw, yh, and xh helped to design the experiments. all authors revised the manuscript. national natural science foundation of china ( , , and ) autophagosome formation: core machinery and adaptations virus-induced double-membrane vesicles viruses and the autophagy pathway contribution of autophagy to antiviral immunity autophagy is involved in regulating the immune response of dendritic cells to influenza a(h n ) pdm infection autophagy in infection, inflammation, and immunity noncanonical autophagy is required for type i interferon secretion in response to dna-immune complexes atg a controls dsdna-driven dynamic translocation of sting and the innate immune response regulation of inflammasome signaling development by self-digestion: molecular mechanisms and biological functions of autophagy structure of atg ,atg , a complex essential for autophagy the evolving functions of autophagy in ocular health: a double-edged sword loss of atg , but not atg , in pro-opiomelanocortin neurons exaerbates diet-induced obesity characterization of a novel ranavirus isolated from grouper epinephelus tauvina sin ym. characterization, pathogenicity and neutralization studies of a nervous necrosis virus isolated from grouper, epinephelus tauvina expression and functional characterization of trif in orange-spotted grouper (epinephelus coioides) traf is a critical factor in fish immune response to virus infection fish trim functions as a critical antiviral molecule against iridovirus and nodavirus molecular cloning and characterization of fadd from the orange-spotted grouper (epinephelus coioides) grouper viperin acts as a crucial antiviral molecule against iridovirus spring viraemia of carp virus induces autophagy for necessary viral replication transcriptome analysis of orange-spotted grouper (epinephelus coioides) spleen in response to singapore grouper iridovirus characterization of two grouper epinephelus akaara cell lines: application to studies of singapore grouper iridovirus (sgiv) propagation and virus-host interaction molecules and their functions in autophagy atg -mediated autophagy deficiency in proximal tubules promotes cell cycle g /m arrest and renal fibrosis atg can regulate p expression and activation autophagy and virus infection the autophagosome: origins unknown, biogenesis complex autophagy fights disease through cellular self-digestion expression pattern and functions of autophagy related gene atg in zebrafish organogenesis characterization of twelve autophagy-related genes from yellow catfish pelteobagrus fulvidraco and their transcriptional responses to waterborne zinc exposure structure of the human atg -atg conjugate required for lc lipidation in autophagy er stress negatively regulates akt/tsc/mtor pathway to enhance autophagy porcine reproductive and respiratory syndrome virus induces autophagy to promote virus replication coronavirus replication complex formation utilizes components of cellular autophagy autophagy protein atg interacts transiently with the hepatitis c virus rna polymerase (ns b) early during infection the atg -atg conjugate associates with innate antiviral immune responses autophagy: a novel guardian of hcv against innate immune response interplay between autophagy and innate immunity in antiviral responses footand-mouth disease virus infection suppresses autophagy and nf-κb antiviral responses via degradation of atg -atg by cpro autophagy regulates ros-induced cellular senescence via p in a p mapkα dependent manner effect of atg -mediated anti-nutritional crisis in hepatocellular carcinoma rgnnv-induced cell cycle arrest at g /s phase enhanced viral replication via p -dependent pathway in gs cells the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © li, liu, zhang, wei, huang, huang, wei and qin. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - i e zkd authors: hu, wan-chung title: a framework of all discovered immunological pathways and their roles for four specific types of pathogens and hypersensitivities date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: i e zkd nan follicular helper t cells (tfh) are considered as the key helper cells for b-cell germinal centers in lymph nodes and are characterized as il- -producing t cells ( ) . follicular dendritic cells (cd +) are antigen presenting cells ( ) , whereas lymphoid tissue inducer cells (lti) are the innate lymphoid cells for tfh ( ) . bcl is a key transcription factor in tfh development ( ) . tgf-β induced by a stat signal can constrain the differentiation of il- -producing helper t cells ( ) . il- production is related to stat and stat activation. il- production is also related to stat activation because immunosuppressive prolactin can cause stat a to suppress bcl expression ( ) . in contrast, stat b can upregulate bcl ( ) . stat a and stat b have distinct target genes in immune responses ( ) , and stat b is the transcription factor that induces tfh. tfh can induce b-cells to produce igm antibodies and il- produced by tfh facilitates b cell isotype switching to igg ( , ) . besides the protein antigen recognized by b cells and t cells, natural killer t (nkt) cells also recognize lipid antigens. the subtype inktfh plays a role in tfh responses ( ) . thus, t lymphocytes are the first to initiate adaptive host immunity ( ) ( ) ( ) , wherein different stat proteins regulate different immunological pathways. if the infection tends to be eradicable, then the host immunological pathways mentioned in the following sections are generated along with other cytokines. th immune responses are driven by il- and are induced against intracellular bacteria or protozoa ( ) . type myeloid dendritic cells (cd + mdc ) are the antigen presenting cells eradicable immune responses include th , th , th , and thαβ. tolerable immune responses include th -like (th l), th , th , in the th response ( ) . the main th effector cells are stimulatory macrophages (m ), ifn-γ-secreting cytotoxic cd t cells (cd + tc ), ifn-γ-secreting cd t cells, inkt cells, and igg -producing b-cells ( , ) . initiation of eradicable immunity also requires innate lymphoid cells to produce the initial cytokines that drive different immunological pathways. for th immune responses, the key innate lymphoid cells are ilc ( ) . stat is the key transcription factor for th immunity but t-bet also plays a vital role. th responses against self-antigens present as type delayed-type hypersensitivity, such as type diabetes mellitus or crohn's disease ( ) . th immune responses are driven by il- and is induced against extracellular parasites (helminths) ( ) . the antigen presenting cells in th immune responses are langerhans cells (cd a+) ( , ) . the main th effector cells are eosinophils (ieos), basophils/pro-inflammatory mast cells (mct, mast cell tryptase), il- -/il- -secreting cd t cells, inkt cells, ilc , and igg /ige-producing b-cells ( ) . igg activates eosinophils, and ige activates mast cells, as in acute anaphylaxis ( ) . igg eosinophils function to activate eosinophil-mediated cellular immunity against parasites or insects, whereas ige-mast cells act to expel helminths or insects through a physiological mechanism. mast cells activated by ige can release histamine, which causes bronchoconstriction, vomiting/nausea, rhinorrhea, skin itching, gastric acidification, increased local vascular permeability, or increased bowel movement. these actions can all help to physiologically expel helminths or insects. the key transcription factor in th response is stat and gata also plays a vital role in the th immunological pathway. th responses against selfantigens present as type immediate allergy, such as food/drug allergy, anaphylaxis, or urticarial ( ) . thαβ is distinct from traditional th immune responses ( ) . thαβ cells are induced against viruses and were previously called as tr cells ( , ) . thαβ immune responses are driven by ifnα/β or il- . the antigen presenting cells for thαβ responses are plasmacytoid dendritic cells (pdc) ( ) . the main effector cells of thαβ immune responses are il- -producing stimulatory nk cells (cd -cd + nk cells), il- /il- -secreting cd t cells, il- -secreting cytotoxic cd t cells (cd + tc ), inkt cells, ilc , and igg -producing bcells ( , ( ) ( ) ( ) . the cd molecule is important for antiviral immunity. the key transcription factors for thαβ response are stat and stat ( ). thαβ immune responses against self-antigens present as type antibody-dependent cytotoxic hypersensitivity, such as the acute stage of myasthenia gravis or graves' disease ( ) . il- is not merely an immunosuppressive cytokine; it can also have potent stimulatory effects on nk cells, cytotoxic t lymphocytes (ctls), and b-cells. th responses are part of the host innate immunity against extracellular bacteria and fungi ( ) . th response is driven by il- or tnfα ( ) . the antigen presenting cells in th immune responses are type myeloid dendritic cells (cd c+ mdc ) ( ) . the main th effector cells are neutrophils (n ), il- -secreting cd t cells, inkt cells, ilc (ncr+), and igg -producing b-cells ( , ) . the key th transcription factor is stat ; ap and cebp are also important transcription factors. tgf-β can suppress il- to skew the th immune response toward th ( ) . th responses against selfantigens present as type immune-complex and complementmediated hypersensitivity, such as the arthus reaction or rheumatoid arthritis ( ) . the host immunological pathways induced are mainly decided by the extracellular or intracellular location of protozoa or fungi. four igg subtypes fit the four types of acute immunological pathways. murine igg antibodies also have four subclasses and are correlated with human igg subtypes as follows: human igg <->murine igg a; human igg <->murine igg ; human igg <->murine igg b; and human igg <->murine igg ( ) . higg /migg a function against viral antigens; higg /migg function against bacterial antigen, especially polysaccharides; higg /migg b act against intracellular bacteria; and higg /migg act against parasite antigens ( ) ( ) ( ) . notably, the immune response against fungi or protozoa is mainly based on their intracellular or extracellular location. extracellular fungi such as candida spp or aspergillus spp usually trigger th immune responses, whereas intracellular fungi such as histoplasma spp. trigger th responses. tregs are the host immune inhibitory cells ( ) driven by il- and tgf-β. regulatory dendritic cells (dcreg) are the antigen presenting cells for tregs ( ) . regulatory innate lymphoid cells (ilcreg) are the initial helpers for treg production ( ) . the main effector cells for tregs are the tgf-β-producing cd t cells, foxp regulatory inkt cells, and iga-producing b-cells ( ) . stat , especially stat a is the key transcription factor for the treg pathway. however, both stat a and stat b play non-redundant roles in treg generation ( ) . they may first act sequentially with stat b activation in tfh signaling. combined signaling stat b and stat a induces treg generation. the combination of tregs and the aforementioned four immunological pathways are important to shift adaptive immunity to tolerable immunity. during initial infection, acutestage fierce cytokines can rapidly kill pathogens and infected cells or tissues. however, if the pathogen infects numerous cells in a tissue such as the liver, killing the infected cells will completely destroy the organ ( ) . thus, a regulatory t cell stat signal combined with th /th /th /thαβ will allow the generation of cd t cells with less fierce inflammatory cytokines ( ) . th like/th /th /th immunological pathways are generated during chronic infection. iga and iga are the two types of iga antibodies, with iga being dominant in the serum, whereas iga is dominant in the mucosa. tgf-β can induce iga or iga depending on the lymphoid follicle location ( ) . in the gutassociated lymphoid tissues (galts) or the peyer's patches, iga is the dominant iga antibody produced in the gastrointestinal mucosa. in the lymph nodes at other body locations, iga is the dominant iga antibody produced. however, iga is specifically related to viral protein antigens, whereas iga is especially related to bacterial antigens such as lps. the heavy-chain locus sequence of b-cell antibodies on the human chromosome is igm, igd, igg , igg , iga , igg , igg , ige, and iga . b-cells co-express igm and igd. igg , igg , and iga comprise the first group for cellular immunity, whereas igg , igg , ige, and iga can be considered as the second group for humoral immunity. the gene sequence order is important as it affects the time sequence of the isotype switch. th -like cells (non-classic th ) are initiated by tgf-β (stat signaling) and ifn-γ (stat signaling). th -like cells with foxp + regulatory characteristics have been identified ( ) . th helper cells and th -like cells are closely related ( ) . th -like cells are induced in chronic th immune responses. thus, these cells may be related to chronic inflammation such as long-term tuberculosis or leishmania infection ( ) . the effector cells of th -like immune responses include suppressive macrophages (m ), ilc , suppressive cd t cells (cd -cd +treg), iga producing b-cells, inkt cells, and ifn-γ-/tgf-β-producing cd t cells ( , , ) . the th -like response induces type delayed-type hypersensitivity, such as crohn's disease ( ) . th cells are driven by il- (stat signaling) combined with tgf-β (stat signaling) ( ) ( ) ( ) . thus, th cells are closely related to the th immunological pathway in parasite immunity ( ) . the cells are characterized as il- -secreting cd t cell. th cells are important under a chronic allergic condition such as asthma. thus, th helper cells are chronic t helper cells related to th immune response. the effector cells of th immunity include regulatory eosinophils, basophils/profibrotic mast cells (mcct, mast cell chymase, and tryptase), ilc , il- -producing cd t cells, inkt cells, and iga -producing b-cells ( , ) . th immune responses present as type allergy, including asthma ( ) . th cells are driven by il- /il- combined with tgf-β ( ). thus, th cells are closely related to the th immunological pathway against extracellular bacteria and fungi ( ) . th cells are characterized as il- -secreting cd t cells. th cells are important in chronic immune-complex-mediated diseases such as rheumatic arthritis. the th helper cell is the chronic t helper cell related to th immunity. tgf-β with stat can suppress the acute il- -producing cells and enhance the chronic il- -producing cells ( ) . owing to the role of tgfβ in th immunity, regulatory il- -producing cells have been noted. the effector cells of th immunity include regulatory neutrophils (n ), ilc (ncr-), il- -producing cd t cells, inkt cells, and iga -producing b-cells ( , ) . th immunity presents as type immune-complex hypersensitivity, including ulcerative colitis or rheumatoid arthritis ( ) . th cells are driven by il- and tgf-β ( , ) . thus, th cells are closely related to the thαβ immunological pathway against viruses ( ) . these cells also produce il- and tgf-β. thus, th helper cells are important for chronic antibody-dependent cellular cytotoxic hypersensitivity. th cells are the chronic helper t cells corresponding to thαβ helper cells. the th immune effector cells include il- -producing regulatory nk cells (cd + cd -nk cells), ilc , il- -and tgf-β-secreting cd t cells, suppressive cd t cells (cd -cd + treg), inkt cells, and iga -producing b-cells ( , , , , ) . iga of th immune responses is produced in the serum and acts against viral protein antigens. th immune responses induce type antibody-dependent cytotoxic hypersensitivity, including the chronic stage of systemic lupus erythematosus (sle) ( ) . the summary diagram includes complete picture of the × + immunological pathways ( table ). the th , th , th , and thαβ eradicable immune responses correspond with the th -like, th , th , and th tolerable the author is thankful for the instructions provided by professors alan scott, louis august bourgeois, and pien-chien huang during his ph.d. study in the department of international health, johns hopkins university, bloomberg school of public health. the author is also grateful for the guidance from professors chi-huey wong and alice lin-tsing yu in his postdoctoral research at the genomics research center of academia sinica, taiwan. this manuscript has been released as a pre-print at biorxiv ( ) . two types of murine helper t cell clone. i. definition according to profiles of lymphokine activities and secreted proteins human immune responses to plasmodium falciparum infection: molecular evidence for a suboptimal thalphabeta and th bias over ideal and effective traditional th immune response th immune responses in the progression of leprosy via molecular cross-talks of tgf-beta, ctla- and cbl-b ifn-alpha and il- induce the differentiation of human type t regulatory cells interleukin -producing cd + effector t cells develop via a lineage distinct from the t helper type and lineages th cells represent a distinct human t cell subset involved in epidermal immunity and remodeling identification of t helper type -like, foxp + regulatory t cells in human autoimmune disease the development and fate of follicular helper t cells defined by an il- reporter mouse human dendritic cell subsets lymphoid tissue inducer cells: bridges between the ancient innate and the modern adaptive immune systems bcl mediates the development of t follicular helper cells tgf-beta prevents t follicular helper cell accumulation and b cell autoreactivity prolactin inhibits bcl expression in breast cancer through a stat a-dependent mechanism stat regulates the self-renewal capacity and differentiation of human memory b cells and controls bcl- expression a sequence of the cis gene promoter interacts preferentially with two associated stat a dimers: a distinct biochemical difference between stat a and stat b il- induces differentiation of human naive and memory b cells into antibody-secreting plasma cells cutting edge: il- is a switch factor for the production of igg and igg by human b cells development of invariant natural killer t cells cutting edge: stat is required for il- -mediated bcl induction for early follicular helper cell differentiation cutting edge: induction of follicular homing precedes effector th cell development early th cell differentiation is marked by a tfh cell-like transition active tuberculosis is characterized by highly differentiated effector memory th cells type cytotoxic t lymphocytes modulate the activity of dendritic cells toward type immune responses tgfbeta signaling plays a critical role in promoting alternative macrophage activation innate lymphoid cells. innate lymphoid cells: a new paradigm in immunology th -type responses mediate spontaneous ileitis in a novel murine model of crohn's disease host protective roles of type immunity: parasite killing and tissue repair, flip sides of the same coin functional specializations of human epidermal langerhans cells and cd + dermal dendritic cells lungresident eosinophils represent a distinct regulatory eosinophil subset interleukin- promotes the development of tryptase and chymase doublepositive human mast cells accompanied by cell maturation lps promotes th dependent sensitisation leading to anaphylaxis in a pru p mouse model. sci rep il- regulates viral lung immunopathology during acute respiratory syncytial virus infection in mice cd /cd -costimulated t and t subsets: differential in vivo allosensitization generates distinct gvt and gvhd effects differentiation of human nk cells into nk and nk subsets a novel il- -producing innate lymphoid cells (ilc ) in a contact hypersensitivity mouse model interferon alpha/beta-mediated inhibition and promotion of interferon gamma: stat resolves a paradox interleukin deficiency attenuates induction of anti-tsh receptor antibodies and hyperthyroidism in a mouse graves' model th cells are an important source of il- for host protection against enteropathogenic bacteria identification of a human helper t cell population that has abundant production of interleukin and is distinct from t(h)- , t(h) and t(h) cells innate lymphoid cells-how did we miss them? transcription factor c-maf mediates the tgf-beta-dependent suppression of il- production in t(h) cells elevated th cells correlated with th cells in patients with rheumatoid arthritis selective increases in antibody isotypes and immunoglobulin g subclass responses to secreted antigens in tuberculosis patients and healthy household contacts of the patients virally induced modulation of murine igg antibody subclasses correlation between serum igg- concentrations and the antibody response to bacterial polysaccharide antigens allergen-specific ige and igg are markers of resistance and susceptibility in a human intestinal nematode infection control of regulatory t cell development by the transcription factor foxp regulatory dendritic cells: there is more than just immune activation regulatory innate lymphoid cells control innate intestinal inflammation identification of regulatory foxp + invariant nkt cells induced by tgf-beta nonredundant roles for stat a/b in directly regulating foxp cd + cd + foxp + regulatory t cells protect against t cell-mediated fulminant hepatitis in a tgf-beta-dependent manner in mice cd engagement triggers switching to iga and iga in human b cells through induction of endogenous tgf-beta: evidence for tgf-beta but not il- -dependent direct s mu->s alpha and sequential s mu->s gamma, s gamma->s alpha dna recombination il- inhibits the tgf-betadependent t cell developmental programs and skews the tgf-beta-induced differentiation into a th -like direction mononuclear cells from patients recovered from cutaneous leishmaniasis respond to leishmania major amastigote class i nuclease with a predominant th -like response four functionally distinct populations of human effector-memory cd + t lymphocytes th cells that express the transcription factor pu. drive t cell-mediated colitis via il- receptor signaling in intestinal epithelial cells il- inhibits tgf-beta-induced foxp + t cells and, together with tgfbeta, generates il- + il- + foxp (-) effector t cells stat -dependent regulation of th development il- and th in parasite immunity human mast cell subsets-distinct functions in inflammation? lung-infiltrating t helper cells as the major source of interleukin- a production during pulmonary cryptococcus neoformans infection polarization of tumor-associated neutrophil phenotype by tgf-beta: "n " versus "n il- and tgf-beta induce alloreactive cd +cd -t cells to acquire regulatory cell function antigen-specific cellular hyporesponsiveness in a chronic human helminth infection is mediated by t(h) /t(r) -type cytokines il- and transforming growth factor-beta but not by a t(h) to t(h) shift airway memory cd (+) t cells mediate protective immunity against emerging respiratory coronaviruses cd +cd -, suppressive t cells in systemic lupus erythematosus tgfbeta promotes conversion of cd + peripheral blood nk cells into cd -nk cells with similarities to decidual nk cells continuous administration of anti-interleukin antibodies delays onset of autoimmunity in nzb/w f mice a framework of all discovered immunological pathways and their roles for four specific types of pathogens and hypersensitivities the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © hu. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -f f utzx authors: lutterberg, karina; kleinwort, kristina j. h.; hobmaier, bernhard f.; hauck, stefanie m.; nüske, stefan; scholz, armin m.; deeg, cornelia a. title: a functionally different immune phenotype in cattle is associated with higher mastitis incidence date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: f f utzx a novel vaccine against bovine viral diarrhea (bvd) induced pathogenic antibody production in – % of bvd-vaccinated cows. transfer of these antibodies via colostrum caused bovine neonatal pancytopenia (bnp) in calves, with a lethality rate of %. the exact immunological mechanisms behind the onset of bnp are not fully understood to date. to gain further insight into these mechanisms, we analyzed the immune proteome from alloreactive antibody producers (bnp cows) and non-responders. after in vitro stimulation of peripheral blood derived lymphocytes (pbl), we detected distinctly deviant expression levels of several master regulators of immune responses in bnp cells, pointing to a changed immune phenotype with severe dysregulation of immune response in bnp cows. interestingly, we also found this response pattern in % of non-bvd-vaccinated cows, indicating a genetic predisposition of this immune deviant (id) phenotype in cattle. we additionally analyzed the functional correlation of the id phenotype with health parameters and diseases in a retrospective study over months. the significantly increased prevalence of mastitis among id cows emphasizes the clinical relevance of this deviant immune response and its potential impact on the ability to fight infections. vaccines are the most effective and also affordable disease-prevention tools ( ) and maternal antibodies protect the offspring from infections in man ( ) and animals ( ) . in cattle, pre-partum vaccination with various virulence factors of bacterial or viral infectious diseases is a standard procedure which effectively stimulates the production of specific antibodies ( , ) . a novel production technology using an allogeneic cell line ( ) and the addition of a highly potent adjuvant to pregsure bvd, a new vaccine against bovine viral diarrhea (bvd), induced fatal immune reactions with production of alloreactive antibodies in - % of vaccinated cows ( ) ( ) ( ) . these alloantibodies were transmitted to calves, regardless of kin, via the colostrum of these dams ( , ) and caused bovine neonatal pancytopenia (bnp) ( , ( ) ( ) ( ) , even years after the vaccine has been taken off the market ( ) . bnp calves suffered from hemorrhagic diathesis, thrombo and leukocytopenia and bone marrow depletion, which resulted in a deadly bleeding disorder ( , , ) . several authors hypothesized alloimmune reactions against the major histocompatibility complex class i (mhc i) as causal for bnp ( ) ( ) ( ) ( ) . this hypothesis is still controversial since mhc i alloantibodies would be expected to bind all nucleated cells, not only to blood-derived and hematopoietic progenitor cells ( , ) . also transcriptome analyses provided evidence that mhc i can be excluded as single causal agent for bnp-associated alloantibodies ( ) . as other authors claimed the possibility of a genetic predisposition of bnp dams for production of bnp inducing alloantibodies ( ) ( ) ( ) , our studies focused on investigating a general difference in immune responses between pregsure bvd vaccinated control cows and bnp donors. we wanted to analyze if there is evidence for differential expression of master transcription factors of cellular immune response in lymphocyte proteome of controls and bnp dams. interestingly, we found a markedly different immune phenotype in bnp dams, characterized by significant hyperproliferation of lymphocytes and a deviant immune signaling pathway compared to controls. we were next interested to see if we could find evidence for this immune phenotype in cows that were never vaccinated with pregsure bvd. further testing showed that this altered immune phenotype was also detectable in % of cows from a pregsure bvd unvaccinated cohort. this confirmed its natural, not vaccineinduced, appearance in cattle and gave more evidence for immune deviant (id) phenotype as a genetic predisposition. in further in-depth experiments we found that interleukin- (il- ) plays an important role in diverging immune response of id lymphocytes. in addition to these immune response studies, we analyzed the functional correlation of the id phenotype with overall health parameters and diseases in a retrospective study over months. here, a significantly increased mastitis incidence in cows with an id phenotype became apparent, highlighting the clinical impact of altered immune response. in this study, peripheral blood derived lymphocytes (pbl) of a total of cows were used. eleven of these cows had previously been vaccinated with pregsure bvd, of which six did not produce bnp-inducing antibodies (bvd vaccinated control cows) and five showed alloreactive antibody production (bnp dams) resulting in their calves dying from hemorrhagic diathesis with confirmed thrombocytopenia, leukocytopenia and bone marrow depletion. the remaining cows were healthy controls (female, age: - years, first to eight lactation period) from one dairy farm never vaccinated with pregsure bvd. no experimental animals were killed through this study. withdrawal of blood was permitted by the local authority regierung von oberbayern, munich, permit no. . - - - . - - . bovine venous blood samples were collected in tubes with heparin sodium . i.u.. blood was diluted with equal parts staining of × cells per well was performed with anti-bovine cluster of differentiation (cd) (mouse igg monoclonal, bio-rad abd serotec, puchheim, germany; : ), anti-bovine cd (mouse igg a monoclonal, bio-rad abd serotec; : ) and fitc-conjugated anti-bovine igm (bio-rad abd serotec, puchheim, germany; : ) antibodies, diluted in staining buffer ( % bsa + , % nan in pbs). respective secondary antibodies anti-mouse igg fitc and anti-mouse igg a fitc (both santa cruz biotechnology; heidelberg, germany : ) were added. all antibodies were incubated for min on ice. cells were washed with staining buffer between primary and secondary antibody staining steps ( × g, • c, min). for all stainings with secondary antibodies respective isotype controls were used. cells were fixed in % pfa diluted in staining buffer and stored at • c until analysis. on facs canto ii, measurement of cells was performed with facs diva software (bd biosciences). lymphocytes were gated according to forward scatter (cell size) and side scatter (intercellular granularity) properties of cells. per staining, between × and × cells were measured. pbl ( . × cells) of two pregsure bvd vaccinated control cows and two bnp dams were stimulated with pwm and cona ( µg/ml) for h. for lc-ms/ms analysis, peptides were separated on a reversed chromatography column ( µm id × cm, acclaim pepmap c . Å/size, lc packings, thermo fisher scientific, bremen, germany) and the analysis was conducted with an ultimate nano-hplc system (dionex, idstein, germany). nano-hplc was connected in a linear quadruple ion trap-orbitrap (ltq orbitrap xl) mass spectrometer (thermo fisher scientific, bremen, germany). the mass spectrometer was operated in the data-dependent mode to automatically switch between orbitrap-ms and ltq-ms/ms acquisition. up to most intense ions were selected for fragmentation on the linear ion trap using collision induced dissociation at a target value of ions and subsequently dynamically excluded for s. elevated spectra were imported into progenesis software (version . ). after comparison and normalization, spectra were exported as mascot generic files and searched against the ensembl bovine database (version ) with mascot (matrix science, version . . ). peptide assignment was reimported to progenesis software. all unique peptides allocated to a protein were considered for quantification. only proteins quantified with at least two peptides were included for further analysis. for hierarchical cluster analyses of normalized protein abundances, arithmetic means of two respective vaccinated control and bnp samples were generated and proteins with similar expression patterns were clustered with perseus software algorithm (version . . . ; computational systems biochemistry, martinsried, germany) ( ) . proteins with a ratio of at least fold in normalized abundance between control and bnp samples were defined as differentially. for protein expression analyses with western blots, pbl were first lysed in lysis buffer ( m urea, m thiourea, mm dithioerythritol, % chaps). proteins were then separated by sds-page on % gels ( µg protein/slot) and blotted semidry onto . × cm pvdf membranes (ge healthcare, freiburg, germany) and blocked with % bsa ( h). blots were incubated overnight with respective primary antibodies: rabbit anti-pstat tyr , rabbit anti-pstat tyr (cell signaling, darmstadt, germany, : ) or mouse anti-beta actin (sigma, taufkirchen, germany, : ). as secondary antibodies, either hrp-coupled goat anti-rabbit igg (h+l) antibody (cell signaling, darmstadt, germany, : ) or goat anti-mouse igg (h+l) antibody (sigma-aldrich, taufkirchen, germany, : ) were used ( h). signals were detected by enhanced chemiluminescence on xray film (super- g ortho, fuji; christiansen, planegg, germany). films were scanned on a transmission scanner and densitometric quantification of western blot signals was performed using imagej software (open source: http:// imagej.nih.gov/ij/). abundances of pstat and pstat were subsequently normalized to beta actin. milk production and health parameters (observed by veterinarians) of non-pregsure bvd vaccinated cattle from one dairy farm were analyzed for months retrospectively. results were then correlated to immune response data of in vitro assays. seventy-three control cows with low proliferation rate after cona stimulation and id cows, which showed a bnp-like hyperproliferation to cona, were included in this study. ten health parameters [daily milk yield, average lactation performance ( days), milk structure (lactose, fat, urea, somatic cell count), fertility parameters (amounts of inseminations, calving-to-conception intervals, medicinal induction of oestrus, ovarian cysts)] and diseases [diseases of musculoskeletal system, claws, digestive tract, respiratory system, and metabolic disorders (ketosis, hypocalcemia)] were analyzed. all parameters were recorded and listed by the same veterinarians and the statistical analysis was performed using the odds ratio and chi-square distribution. data were analyzed in prism software (graphpad, version . ) with kolmogorow-smirnow (ks) test. if ks test was significant (p < . ; normal distribution), student's t-test was used for statistical analysis, if ks test was not significant (p > . ; no normal distribution) statistics were performed using mann-whitney test. for statistical analysis of health parameters, we used odds ratio and chi-square distribution. differences were considered statistically significant with the following p-values: * p < . , * * p < . , * * * p < . , and * * * * p < . . after in vitro stimulation ( h) with t and b cell mitogen pwm ( ) and t cell mitogen cona ( ), a clearly divergent reaction of lymphocytes from pregsure bvd vaccinated cows became evident. cells from cows known to produce alloreactive bnp antibodies after vaccination (bnp lymphocytes) proliferated significantly stronger ( . -fold) than cells from vaccinated control dams after pwm stimulation ( figure a , bnp to ctr, * * * * p < . ) and cona stimulation ( -fold stronger; figure b , bnp to ctr, * * * * p < . ). thus, in vitro proliferation assays revealed a highly significant hyperproliferation of bnp lymphocytes demonstrating an increased reaction to polyclonal immune stimulation in these cows. to exclude differences in lymphocyte subset percentages between control and bnp dams as a possible cause for the differential responses toward polyclonal immune stimulation, we analyzed the distribution of cd + , cd + , and b cells in control and bnp pbl. there were no significant differences in proportional composition of subpopulations between pregsure bvd vaccinated control and bnp animals (figure ). to investigate if observed deviant reactions to in vitro stimulation originated from differences in immune cell regulation, we executed a differential proteomics experiment with pwm and cona treated lymphocytes as well as unstimulated cells. overall, we detected , proteins. hierarchical cluster analysis of complete proteomes already visualized fundamental differences in protein abundances between pregsure bvd vaccinated control and bnp cows in constitutive protein tyr signals were normalized to beta-actin and quantified using image-j software. protein expression is shown in mean ± sd. for statistical analyses student's t test was performed. levels (ce, figures a,b) . differences in protein expression were also confirmed after immune stimulation (pwm and cona, figures a,b , hierarchical clustering of all identified proteins). these proteome analyses therefore revealed substantial quantitative differences on protein level. next, we used our dataset to perform more in-depth analyses on part of the proteome involved in transcription pathways of immune cell regulation. to identify possible differences in master transcription factor expression, we compared lymphocytes from vaccinated control and bnp cows after polyclonal in vitro stimulation. signal transducers and activators of transcription (stat) induce different t helper (th) responses in mice and man ( ) as master transcription factors. therefor we analyzed all identified stats from both proteomic approaches and characterized usage of different stat pathways as a response to immune stimulation in both cow groups. after polyclonal activation of lymphocytes with pwm, stat and stat a were selectively upregulated at least -fold in bnp lymphocytes ( figure c) . cona stimulation of control cells, however, caused upregulation of stat and stat expression levels (figure d) , whereas bnp pbl showed enhanced stat in immune response ( figure d) . thus, control and bnp pbl upregulated different master transcription factors as response to stimulation pointing to usage of different immune pathways. since we decided to further analyze the differential activation of stats through t cell mitogen cona, we determined figure | stat inhibitor wp selectively inhibits hyperproliferation of lymphocytes from bnp cows. stat inhibitor did not inhibit proliferation of control pbl (white bar, n = ), but significantly inhibited cona stimulated bnp cells (black bars, n = , ***p < . ). percentage of inhibition rate shown in mean ± sd. student's t-test was used for statistical analyses. phosphorylation of stat and stat in response to immune stimulation. in lymphocytes of vaccinated controls, phosphorylation of stat tyr significantly increased after in vitro stimulation with cona compared to bnp pbl, where stat tyr phosphorylation decreased after cona stimulation (figures a,b , ctr to bnp after cona stimulation, * * p < . ). in contrast, lymphocytes of bnp dams phosphorylated stat tyr comparatively stronger than pbl of vaccinated control cows after cona (figures c,d , bnp to ctr after cona stimulation, * p < . ). thus, these experiments ascertained a qualitative difference in immune reactions between the cow groups to a t cell stimulus. since bnp lymphocytes responded to polyclonal stimulation with hyperproliferation and activation of stat , we next tested if inhibition of stat reversed respective hyperproliferation. wp (stat inhibitor) significantly reduced proliferation of bnp lymphocytes after cona stimulation (figure , inhibition of bnp compared to ctr, * * * p < . ), but not of control lymphocytes. this verified the importance of stat -pathway for the hyperproliferation in bnp pbl after polyclonal t cell stimulation. since we first detected the deviant immune phenotype in bnp cows, we next tested cows that were never vaccinated with pregsure bvd in order to clarify whether the deviant phenotype was induced through vaccination or if it occurs naturally in a certain subset of cows. therefore, we examined response of lymphocytes to different polyclonal stimuli in a large group of pregsure bvd unvaccinated cows. to exclude differences caused by environmental factors, cows all came from the same farm. in in vitro proliferation assays with t cell mitogen cona we observed that % of the unvaccinated cows reacted similar to bnp dams by showing a hyperproliferative reaction to cona (immune deviant (id) phenotype; figure a , reaction difference id to controls or bnp to controls, * * * * p < . ). lymphocytes from id cows with a hyperproliferative immune phenotype did not show significant differences to lymphocytes of bnp cows in these assays (figure a , reaction difference bnp to id). these data confirmed the natural occurrence of this id phenotype in cattle and gave stronger evidence for genetic predisposition instead of vaccine-induced appearance. in order to further characterize the pathway targeted by the polyclonal activators pwm and cona we tested additional mitogens. the t cell mitogen banlec ( ) , which activates human t cells through the il- pathway ( , ), led to similar hyperproliferation of pbl from bnp dams as observed with cona ( figure a , reaction difference factor: . , bnp to controls, * * * * p < . ). pbl of id cows responded to banlec stimulation with a similar immune response intensity as bnp cows (figure b , reaction difference factor: . , id compared to controls, * * * * p < . ). the reaction of lymphocytes showed no significant differences between id and bnp cows. since the results with cona and banlec pointed to a response via il- pathway, we next tested stimulation with purified bovine il- only. after il- stimulation, lymphocytes of id cows also reacted excessively (figure c , reaction difference factor: . , id to controls, * * * * p < . ) and did not show significant differences to bnp lymphocytes ( figure c , reaction difference factor: . , bnp to controls, * * * * p < . ). finally, we tested further signature cytokines for different th immune responses, bovine interferon gamma (ifnγ; th ) and interleukin (il- ; th ), to analyze differentiation of t helper subsets. we detected no significant differences between lymphocytes of vaccinated and unvaccinated controls and bnp cases after ifnγ or il- stimulation in vitro (figures d,e) . this proves a crucial role for il- in the deviant immune responses, but not for ifnγ or il- . in this study, the proliferative phenotype of controls and id cows could be repeatedly tested during an overall observation period of months. all of the sampled immune deviant cows showed hyperproliferative reaction to cona at least three figure | immune deviant phenotype also detectable in unvaccinated cows. (a) in in vitro proliferation assays, % of unvaccinated cows showed a bnp-like hyperproliferative reaction to cona [****p < . vs. ctr (white bar, n = )]. lymphocytes from id cows (gray bars, n = ) with a hyperproliferative immune phenotype showed no significant (ns) differences to lymphocytes of bnp cows (black bars, n = , technical replicates n = ). (b) after banlec stimulation, id lymphocytes reacted hyperproliferative in comparison to control lymphocytes (****p < . ) and the proliferation rates of these id lymphocytes did not show significant differences (ns) compared to bnp lymphocytes. (c) after il- stimulation, lymphocytes of id cows also reacted excessively (****p < . vs. ctr) just like lymphocytes from bnp donors (ns). (d/e) after bovine ifnγ (d) and il- (e) stimulation, no significant differences between lymphocytes of unvaccinated and bnp cows were determined (ns). proliferation rate shown in mean ± sd. student's t-test was used. times. overall, two-thirds of the id animals consistently showed significant differences in their immune response compared to the unvaccinated control cows (figure a ). since we wanted to know if this newly discovered immune deviant phenotype related to impaired health parameters in respective cows, we retrospectively analyzed milk production, health parameters and different diseases of control and id cows from the same farm never vaccinated with pregsure bvd. from a total of parameters tested, there was no difference in parameters. however, mastitis incidence was significantly different between both cow groups. we detected that id cows suffered from mastitis twice as often as control cows (figure b , * p < . ). at the moment of blood sampling, all cows were clinically healthy and showed no signs of mastitis which could potentially influence adverse effects to immune stimulation. thus, this clearly indicates that the hyperproliferative phenotype of id cows has clinical relevance apart from the response to the bvd vaccine. the hyperproliferative phenotype of bnp cows (black) and id cows (gray) as well as the low proliferation of control cows (white) were consistently detectable over the entire observation period ( months). three representative control cows and id cows are shown and two bnp cows, white symbols = controls, gray symbols = id, black symbols = bnp. (b) id cows (gray, n = ) suffered twice as often (difference in incidence *p < . ) from mastitis than the control cows (white, n = ). incidence of mastitis shown in percent. odds ratio and chi-square distribution was used for statistical analyses. bnp was the unwanted result of a fatal immune response to vaccination with pregsure bvd ( , ) . five to ten percentage of vaccinated cattle produced disease inducing antibodies which were transferred via colostrum, killing % of receiving calves, regardless of kin ( , ) . it is still unclear, why a certain subgroup of cows responded differently to the vaccination. we hypothesize that the origin of this altered immune response lies in the existence of a naturally occurring deviant immune phenotype. in this study, we could effectively confirm a quantitative difference in immune cell activation of bnp pbl through hyperproliferative responses in vitro (figures a,b) . a shift in lymphocyte subpopulations (cd + , cd + , b cells) as a potential reason for these differential responses toward polyclonal immune stimulation could be excluded (figure ) . the hierarchical clustering of identified lymphocyte proteins from our shotgun proteomics experiment substantiated a general quantitative difference in constitutive expression of proteins in pbl of controls and bnp dams (ce, figures a,b) . after immune stimulation, these differences increased even further (pwm/ cona, figures a,b) . in order to perform more indepth investigations of potential functional differences, we subsequently focused on the analysis of master transcription factors of immune cell regulation from our dataset. as a result, we detected several differentially expressed stats. stats initiate the differentiation of various th cell subsets ( ) as firstresponse master regulators and provide lineage specificity by promoting the differentiation of a given th cell subset while opposing the differentiation to alternative th cell subsets ( ) . this makes them especially interesting to us, since expression of different stats in controls and bnp specimen indicates divergent immune response pathways in these groups. in control lymphocytes we identified stat with increased expression ( figure d ) and significantly higher activation (figures a,b) after t cell stimulation with cona. in mice and man, stat is important for the differentiation of naive cd + t cells to th cells ( ) or to type regulatory (tr) t cells ( ) . from these findings, we conclude that after immune stimulation, bovine control lymphocytes use the stat pathway and we hypothesize that control cows react with a th or tr immune response in polyclonal stimulation assays. the effect of stat inhibition could not be tested, since we found no stat specific inhibitor that was commercially available. in contrast to controls, pbl proteome of bnp dams showed higher expression levels of stat ( figure d) , with increased phosphorylation (figures c,d) in bnp lymphocytes after immune stimulation. stat could therefore be a possible master regulator for the deviant immune response in bnp dams. in mouse and man, stat induces the development of follicular th (tfh), th , and th cells ( , , ) . our findings proved a stat pathway dependent immune response of bnp lymphocytes after polyclonal stimulation, but so far it is unknown which th subsets exactly are regulated by stat in cows. the dependence of the hyperproliferative phenotype of immune deviant pbl on stat was shown by inhibition of respective factor using wp , , a cell permeable tyrphostin analog which blocks the stat pathway through inhibition of janus kinase (jak ) protein tyrosine kinase ( ) . as a result, we observed a significantly reduced proliferation to % in bnp pbl, but no effect in control pbl (figure ). our findings therefore show, that the deviant immune response of bnp donors is associated with stat /jak pathway. further studies will be needed to clarify whether immune cells from control cows differentiate to th or tr subsets and if bnp donor cows develop tfh, th , or th cells after immune stimulation. hyper-reactivity of bnp pbl and the stat driven differentiation of tfh cells after immune stimulation are supported by findings of other research groups, describing higher alloantibody levels in bnp cows compared to pregsure bvd vaccinated control dams ( ) . this group also hypothesized, that differences between bnp and control dams are likely due to genes controlling the quantitative alloantibody response after vaccination ( ) . to find evidence for a genetic predisposition of this immune deviant bnp phenotype in cows ( , ) , we additionally analyzed the immune phenotypes of non-pregsure bvd vaccinated cows. in our study, % of cows (id cows) showed a bnp-like hyperproliferative response to t cell stimulation in vitro (id, figures a,b) . this percentage fits to the findings of benedictus et al., who hypothesized a heritability of % in dams for the development of bnp in the calf ( ) . this proves the existence of an immune deviant phenotype among a certain subpopulation of cattle, which is not triggered by vaccination with pregsure bvd but occurs naturally. the immune deviant response described here in these id cows clearly depends on il- ( figure c) , which we could prove through hyperproliferative response of bnp and id lymphocytes to stimulation with banlec ( figure b) , a mitogen that activates human t cells ( , ) via the il- pathway ( ). for bovine t cells, the specific impact of il- itself was not described so far. in man and mice, however, il- has pleiotropic autocrine or paracrine functions that regulate proliferation of t cells ( ) . it also plays a key role in promoting development, homeostasis and function of regulatory t cells though il- /stat signals ( ) and balances expression of different stats in th cells of mice ( ) . therefore, the association of il- with the immune deviant response in cows is a very interesting finding which merits further in-depth investigations in future studies. to determine whether the newly detected deviant immune phenotype is of clinical relevance, we analyzed clinical parameters and diseases in cows from the sampled dairy farm and compared incidence between controls (n = ) and id animals (n = ). interestingly, we found a significantly increased mastitis incidence in id cows compared to controls ( figure b ). this highly important correlation points to an altered immune reaction of id cows to classical mastitis pathogens, facilitating mastitis onset in these animals. in conclusion, we could prove the existence of an immune deviant phenotype in % of cattle, regardless of the vaccination status. the id phenotype shows altered reactions to immune stimulation identical to bnp dams, such as hyperproliferation of lymphocytes, stat /jak regulated pathway and association with il- . these features potentially triggered alloantigenproduction after maternal pregsure bvd vaccination, causing bnp. the correlation of the id phenotype with high mastitis incidence ( figure b ) underlines its clinical relevance. simply put: vaccination saves lives vaccines and pregnancy: past, present, and future pros and cons of vp -specific maternal igg for the protection of enterovirus infection vaccination of pregnant cows with espa, espb, gamma-intimin, and shiga toxin proteins from escherichia coli o :h induces high levels of specific colostral antibodies that are transferred to newborn calves serological, colostral and milk responses of cows vaccinated with a single dose of a combined vaccine against rotavirus, coronavirus and escherichia coli f (k ) the risks of using allogeneic cell lines for vaccine production: the example of bovine neonatal pancytopenia case control study to investigate risk factors for bovine neonatal pancytopenia (bnp) in young calves in southern germany bovine neonatal pancytopenia: is this alloimmune syndrome caused by vaccine-induced alloreactive antibodies? incidence of bovine neonatal pancytopenia in farms in germany pancytopenia and haemorrhage in young beef calves calf-level factors associated with bovine neonatal pancytopeniaa multi-country case-control study effect of the vaccination scheme on pregsure(r) bvd induced alloreactivity and the incidence of bovine neonatal pancytopenia bovine neonatal pancytopeniacomparative proteomic characterization of two bvd vaccines and the producer cell surface proteome (mdbk) pregnancy boosts vaccine-induced bovine neonatal pancytopenia-associated alloantibodies ingestion of colostrum from specific cows induces bovine neonatal pancytopenia (bnp) in some calves demonstration of early functional compromise of bone marrow derived hematopoietic progenitor cells during bovine neonatal pancytopenia through in vitro culture of bone marrow biopsies vaccineinduced antibodies linked to bovine neonatal pancytopenia (bnp) recognize cattle major histocompatibility complex class i (mhc i) alloantibodies against mhc class i: a novel mechanism of neonatal pancytopenia linked to vaccination haematopoietic depletion in vaccine-induced neonatal pancytopenia depends on both the titre and specificity of alloantibody and levels of mhc i expression pathogenicity of bovine neonatal pancytopenia-associated vaccineinduced alloantibodies correlates with major histocompatibility complex class i expression a novel rnaseqassisted method for mhc class i genotyping in a non-model species applied to a lethal vaccination-induced alloimmune disease bovine neonatal pancytopenia is a heritable trait of the dam rather than the calf and correlates with the magnitude of vaccine induced maternal alloantibodies not the mhc haplotype bovine neonatal pancytopenia (bnp): novel insights into the incidence, vaccination-associated epidemiological factors and a potential genetic predisposition for clinical and subclinical cases a genetic predisposition for bovine neonatal pancytopenia is not due to mutations in coagulation factor xi the perseus computational platform for comprehensive analysis of (prote)omics data in vitro activation of human t and b lymphocytes by pokeweed mitogen mitogenic stimulation of t cells reveals differing contributions for b - (cd ) and b - (cd ) costimulation banana lectin: a brief review musa acuminata (del monte banana) lectin is a fructose-binding lectin with cytokine-inducing activity isolation and characterization of banlec-i, a mannoside-binding lectin from musa paradisiac (banana) sera from dams of calves with bovine neonatal pancytopenia contain alloimmune antibodies directed against calf leukocytes transcriptional and epigenetic regulation of t-helper lineage specification t-bet and gata orchestrate th and th differentiation through lineagespecific targeting of distal regulatory elements the competitive nature of signal transducer and activator of transcription complex formation drives phenotype switching of t cells genes associated with t helper cell differentiation and function stat regulates cytotoxicity of human cd + cd + t cells in blood and lymphoid follicles wp disrupts janus kinase- and induces caspase-dependent apoptosis in acute myelogenous leukemia cells the role of interleukin- during homeostasis and activation of the immune system interleukin- and stat in regulatory t cell development and function opposing regulation of the locus encoding il- through direct, reciprocal actions of stat and stat a functional different immune capacity in cattle is associated with higher mastitis incidence the authors thank sven reese for statistical analyses and roxane degroote for critical discussions. the manuscript was released as a pre-print at https://www.biorxiv.org/content/early/ / / / ( ) . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord- -e rpryrh authors: tomasello, elena; pollet, emeline; vu manh, thien-phong; uzé, gilles; dalod, marc title: harnessing mechanistic knowledge on beneficial versus deleterious ifn-i effects to design innovative immunotherapies targeting cytokine activity to specific cell types date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: e rpryrh type i interferons (ifn-i) were identified over years ago as cytokines critical for host defense against viral infections. ifn-i promote anti-viral defense through two main mechanisms. first, ifn-i directly reinforce or induce de novo in potentially all cells the expression of effector molecules of intrinsic anti-viral immunity. second, ifn-i orchestrate innate and adaptive anti-viral immunity. however, ifn-i responses can be deleterious for the host in a number of circumstances, including secondary bacterial or fungal infections, several autoimmune diseases, and, paradoxically, certain chronic viral infections. we will review the proposed nature of protective versus deleterious ifn-i responses in selected diseases. emphasis will be put on the potentially deleterious functions of ifn-i in human immunodeficiency virus type (hiv- ) infection, and on the respective roles of ifn-i and ifn-iii in promoting resolution of hepatitis c virus (hcv) infection. we will then discuss how the balance between beneficial versus deleterious ifn-i responses is modulated by several key parameters including (i) the subtypes and dose of ifn-i produced, (ii) the cell types affected by ifn-i, and (iii) the source and timing of ifn-i production. finally, we will speculate how integration of this knowledge combined with advanced biochemical manipulation of the activity of the cytokines should allow designing innovative immunotherapeutic treatments in patients. specifically, we will discuss how induction or blockade of specific ifn-i responses in targeted cell types could promote the beneficial functions of ifn-i and/or dampen their deleterious effects, in a manner adapted to each disease. type i interferons (ifn-i) were the first cytokines discovered, over years ago, based on their potent anti-viral effects ( , ) . ifn-i play a crucial, non-redundant role in vertebrate anti-viral defenses ( ) ( ) ( ) . ifn-i also mediate protective effects in other physiopathological contexts, including cancer ( ) and multiple sclerosis (ms) ( ) . on the contrary, ifn-i responses can be deleterious in a number of circumstances, including bacterial or fungal infections ( - ), many autoimmune diseases ( ) , and, paradoxically, certain chronic viral infections ( ) ( ) ( ) . it is only recently that an integrated picture has emerged of the cellular and molecular mechanisms regulating the production of ifn-i and underlying their functions. much knowledge was gained recently on another class of potent innate anti-viral interferons, the lambda, or type iii ifns (ifn-iii). we will review knowledge on ifn-i/iii (ifns) and discuss how it could be harnessed to develop innovative therapeutic strategies aimed at surgically tuning ifn activity toward protective responses in a manner adapted to each disease. we will focus on ifn-α/β/λ because they are the best characterized ifns and already used therapeutically. recent reviews are covering information on other ifn-i subsets including ifn-ε, which is produced at mucosal sites and promotes local anti-viral defenses ( , ) . dendritic cells (dcs) are rare heterogeneous mononuclear phagocytes functionally characterized by their unique efficacy for antigen-specific activation of naïve t lymphocytes. dcs are sentinel cells of the immune system, able to sense and integrate a variety of danger input signals for delivery of output signals instructing the activation and functional polarization of effector immune cells. in mammals, five major dc subsets exist, which differ in their expression of innate immune recognition receptors (i r s) and in their functional specialization: monocyte-derived dcs (modcs), langerhans cells, cd b + dcs, xcr + dcs, and plasmacytoid dcs (pdcs) ( ) . a recurrent theme of this review will be the intricate relationships between ifns and dcs, since these cells can be a major source and/or target of these cytokines under various conditions. www.frontiersin.org the first section will synthesize current knowledge on ifn production and protective anti-viral functions. the i r s and downstream signaling pathways responsible for ifn-i production during viral infection will be listed. the roles of different cell types for this function will be discussed. the two main mechanisms through which ifn-i promote anti-viral defense will be reviewed, succinctly for direct anti-viral effects and in greater details for immunoregulatory functions. the second section will focus on the detrimental functions of ifn-i. selected diseases will be discussed to illustrate how different, and sometimes opposite, processes underlie deleterious ifn-i responses depending on the physiopathological contexts. ifn-i induction of unbridled inflammatory responses causing lethal tissue damage will be discussed as a major pathological mechanism during bacterial encounters secondary to influenza infection or in some autoimmune diseases. inappropriate functional polarization of immune responses by ifn-i will be discussed as one potential cause for enhanced susceptibility to bacterial or fungal infections. the complex and disputed role of ifn-i in chronic viral infections will be reviewed, with emphasis on the physiopathology of the infections by human immunodeficiency virus type (hiv- ) and human hepatitis c virus (hcv), with an outlook for the development of novel immunotherapeutic strategies to combine with anti-viral drugs. the third section will recapitulate how the balance between beneficial versus deleterious ifn-i responses is modulated by several key parameters including (i) the source and timing of ifn-i production, (ii) the cell types affected by ifn-i, and (iii) the signaling pathways activated by ifn-i. in the last section, we will speculate how integration of all the knowledge discussed before combined with advanced biochemical manipulation of the activity of the cytokines should allow designing innovative immunotherapeutic treatments, based on induction or blockade of specific ifn-i responses in targeted cell types. this"activity-by-targeting"concept is based on the design of novel "immuno-ifns" consisting in covalent association between a mutated ifn-i with decreased affinity for its receptor and an antibody with high avidity for a molecule specifically expressed on target cell types ( ) . this design ensures lack of activity of the immuno-ifns on all cell types but those targeted, contrary to previous strategies using ifns with close to maximal potency that were still able to mediate strong off-target effects despite their coupling to cell-type specific antibodies and/or their local delivery. type i interferons expression is not detectable under steady state conditions in vivo using classical methods such as gene expression analysis by rt-pcr or protein titration by elisa or bioassays. however, mice deficient for the expression of the alpha chain of the ifn-i receptor (ifnar ) harbor alteration in the ontogeny or functions of various cell types ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . hence, extremely small or localized but functionally relevant quantities of ifn-i must be produced under steady state conditions ( ) . indeed, the existence of steady state responses to ifn-i in various organs in vivo was demonstrated by using reporter mice expressing the firefly luciferase under the control of the promoter of ifnb ( ) or of mx ( ) , a canonical ifn-i-stimulated gene (isg). steady state ifn-i responses are promoted by gut commensals ( ) . early and transiently after many viral infections, large amounts of ifns can be detected, in blood and spleen in the case of systemic infections or locally in the case of confined infections. ifn induction during viral infections results from the detection of specific danger signals by specialized i r s. this includes the detection of pathogen-associated molecular patterns as well as the sensing of stress signals or damage-associated molecular patterns ( , ) . based on the nature and intracellular location of the danger signals that induce the production of the cytokines, the cellular sources of ifns during viral infection can be classified in two main groups. infected cells often contribute to ifn production as a response to their sensing of endogenous viral replication, or consecutive to the metabolic stress induced during massive translation of viral structural proteins, or as a result of plasma membrane perturbations upon viral entry. specific subsets of uninfected cells can also significantly contribute to ifn production upon engulfment of material containing viral-derived nucleotide sequences and sensing of these molecules in endosomes by specific i r s. all sensing pathways leading to ifn induction converge on the activation of interferon response factors or (irf / ), which are the master transcription factors inducing ifn genes. most cell types constitutively express irf but not irf or only at low levels. irf expression requires ifn-i stimulation. ifn-β can directly be induced by irf . all but one of the ifn-α subtypes require irf for their induction. hence, ifn-β secretion promotes its own production and that of ifn-α in an autocrine manner ( , ) . this positive feedback loop strongly amplifies ifn production during viral infections, promoting fast and widespread induction of cell-intrinsic anti-viral defenses in uninfected cells to prevent virus dissemination. other feedback loops tightly regulate ifn-i production positively or negatively. this section reviews different mechanisms controlling ifn production and how they could play different roles in host/virus interactions. different innate immune recognition receptors are involved in sensing various types of viral nucleic acids in distinct categories of cells during viral infections, which may promote different types of anti-viral defenses. for each selected sensor shown, the types of viral nucleic acids recognized and the downstream signaling cascade induced are represented in a simplified, schematic manner. the potential specific role of each cell type in anti-viral defenses is also indicated at the bottom of each panel. (a) potentially all types of infected cells can detect endogenous viral replication through cytosolic sensors triggering their local production of ifn-β/λ to control viral replication in an autocrine and paracrine fashion in infected tissues. (b) uninfected xcr + dcs selectively produce high levels of ifn-λ and ifn-β upon engulfment of materials containing dsrna and the consecutive triggering of tlr in endosomes. the receptor of ifn-λ is mostly expressed by epithelial cells. hence, xcr + dcs might be involved in inducing local ifn responses in virally infected epithelial tissues. since xcr + dcs are especially efficient at producing ifn-iii upon hcv stimulation, they might contribute to local or systemic ifn production during infection with this virus, to promote ifn-λ-mediated protection of hepatocytes. uninfected xcr + dcs and other uninfected cells may produce some ifn-β upon engulfment of materials containing ssrna and the consecutive triggering of tlr in endosomes. the contribution of this pathway to anti-viral defense is not well understood yet, in part because mouse tlr is deficient for this function. (c) uninfected pdcs selectively produce high levels of all subsets of ifns upon engulfment of materials containing ssrna or cpg dna and the consecutive triggering of tlr / in endosomes. however, pdcs seem to be activated for this function only in lymphoid tissues. hence, pdc might contribute to systemic ifn production during blood-borne viral infections or as a failsafe mechanism activated upon abnormal widespread dissemination of a viral infection once it has escaped local confinement at its portal of entry. cm, cell membrane; nm, nuclear membrane. particular nucleotide sequences or tertiary structures, their signaling pathways and their physiological significance have been recently reviewed ( , ) . cytosolic rna sensors encompass dexd/h helicases among which the retinoic-acid-inducible gene (rig)-i-like receptors (rlrs) have been the most studied, namely rig-i and melanoma differentiation associated gene (mda ). rig-i recognizes rna with a -ppp or -pp ( ) (uncapped) moiety, or double-stranded rna (dsrna), both structures being present in viral, but not in cytosolic eukaryotic, rna molecules. mda might specifically recognize long dsrna fragments. both rig-i and mda contain a dexd/h box-containing rna helicase domain, and caspase recruitment domains (card / ), which bind to mitochondrial anti-viral signaling protein (mavs). rna/rlr/sting molecular complexes initiate a signaling cascade leading to irf / -dependent induction of ifns (figure ). other dexd/h helicases can promote ifn-i production in dcs, www.frontiersin.org although their physiological roles for in vivo immune defenses against viral infections remain to be established ( ) . cytosolic dna sensors able to induce ifn-i (mostly ifn-β) and ifn-iii encompass molecules belonging to different protein families, including dexd/h helicases, the inflammasome component ifn-γ-inducible protein (ifi ) , the z-dna binding protein (zbp ), and the cyclic gmp-amp (cgamp) synthase (cgas) ( , ) . most of the cytosolic dna sensors activate sting and lead to irf / -and nfκb-dependent induction of ifn-β and ifn-iii. many cell types express zbp and are able to produce ifn-i upon triggering of this molecule, including macrophages, dcs, and fibroblasts following an hsv- infection ( , ) . upon dna binding, cgas catalyzes the production of cgamp. cgas is critical for the detection of lentiviruses including hiv- / ( , ) and can contribute to sensing of, and protection against, other rna viruses, including in vivo in mice ( ) . cgamp also acts as a secreted second messenger signal alerting uninfected cells to directly induce their expression of intrinsic immune anti-viral defenses. the cgas/sting/irf signaling cascade and the irf transcription factor are "master" inducers of cell-intrinsic immunity able to control the replication of most dna and some rna viruses at least in part independently of ifns ( ) . infected cells become a factory for production of viral particles. hijacking of the translation apparatus of the host cell for massive production of viral structural proteins leads to an overload of the capacity of the er for correct folding of newly synthesized proteins. er overload induces a homeostatic response of the cell, the unfolded protein response (upr). upr aims at restoring normal er functions by inhibiting translation. upr activation in infected cells contributes to prevent viral replication, including through inhibition of the production of viral proteins, promotion of ifn-i production, and induction of cell suicide ( ) . toll-like receptors (tlrs) are among the first and best characterized i r s. tlrs are transmembrane proteins with a leucine-rich repeat extracellular domain involved in ligand recognition and an intracellular toll/interleukin- receptor domain essential for signaling ( ) . among the nine tlrs conserved between mouse and human, tlr , tlr , tlr , and tlr are located in endosomes where they can detect the abnormal presence of nucleic acids such as occurs upon endocytosis of virions or of virally infected cell material. tlr recognizes dsrna, tlr / ssrna, and tlr dna sequences containing unmethylated cytidinephosphate-guanosine (cpg) motifs. tlr fine specificity and signaling pathways have been reviewed recently ( ) and are summarized in figure . we will discuss the expression patterns and functions of endosomal tlrs with regards to ifn production in uninfected specialized immune cell types, pdcs and xcr + dcs. plasmacytoid dcs uniquely produce very large amounts of ifns in response to in vitro stimulation with many viruses, without being infected ( ) . ifn-i mrnas represent up to % of all mrnas in pdcs at the peak of their activation ( ) . in vitro, upon exposure to influenza virus, herpes virus type , cytomegaloviruses, or vesicular stomatitis virus, individual pdcs produce - times more ifns than total pbmcs, monocytes, modcs, cdcs, neutrophils, and fibroblasts ( ) ( ) ( ) ( ) ( ) ( ) . however, in vitro, high molarity infection of cdcs with certain viruses unable to inhibit ifn-i production in their target cells can also induce massive ifn-β secretion ( ) . pdcs produce high levels of all subtypes of ifns, contrary to many other cell types including infected cells, which often preferentially produce ifn-β ( , ) . in vivo, pdc depletion during systemic viral infections leads to over % decrease of ifn-i production, while the total number of pdcs producing ifn-i (< , in one mouse) is much lower than the total number of infected cells ( ) ( ) ( ) ( ) ( ) ( ) . this shows that in vivo also individual activated pdcs produce much more ifn-i/iii than most other cell types, including virus-infected cells. the professional ifn-producing function of pdcs largely results from their high constitutive and selective expression of irf , tlr , and tlr (figure ) . these molecules are pre-associated in readyto-signal complexes located in specialized endosomes specific to pdcs ( , ) . pdcs must also be equipped for efficient sensing and up-take of virions and virus-infected cells. the corresponding cell surface i r s remain to be identified. selective expression of tlr in xcr + dc endows them with a unique ability to produce very high amounts of ifn-β and ifn-iii upon stimulation with dsrna or hcv irrespective of their own infection. xcr + dcs are very potent for antigenspecific activation of cd + t cells, in particular through crosspresentation of exogenous antigens that they have captured from other cells and processed for association with class i major complex histocompatibility (mhc-i) molecules ( ) . in mice, xcr + dcs are crucial for the initiation of protective adaptive immune responses against tumors and a variety of viruses ( ) . mouse and human xcr + dcs constitutively and selectively express high levels of tlr (figure ) . they produce large amounts of ifn-iii and ifn-β upon stimulation with a synthetic mimetic of dsrna, polyinosinic:polycytidylic acid (polyi:c) ( , ) . human xcr + dcs uniquely respond to stimulation with hcv by producing large amounts of ifn-iii in a tlr -dependent manner ( , ) , irrespective of their own infection. positive feedback loops. in addition to irf induction, other positive feedback mechanisms exist to amplify the production of ifns rapidly after initiation of a viral infection as illustrated by the following selected examples. ifns induce the expression of many cytosolic rna/dna sensors and of tlr . this broadens the spectrum of host's cell types able to detect endogenous viral replication for ifn induction. induction of oasl by ifns in human cells enforces rig-i signaling, counteracting viral immune frontiers in immunology | microbial immunology evasion genes interfering with this sensing pathway ( ) . the ifninducible ribonuclease l (rnasel) generates viral and cellular rna degradation products, which engage rlrs for amplification of ifn production ( , ) . the ifn-inducible protein kinase r (pkr) stabilizes ifn-i mrna ( ) . to prevent unbridled responses deleterious for the host, ifn activity must be tightly controlled including during viral infections. several negative feedback loops exist to terminate ifn production, after anti-viral defenses have been activated. the isg ubiquitin specific peptidase (usp ) binds to ifnar , preventing it from recruiting signal transducer and activator of transcription (stat ). ifns induce the expression of tam receptor tyrosine kinases in dcs, monocytes, and macrophages. tam receptors associate and signal in part through ifnar . they activate the suppressors of cytokine signaling- / (socs- / ). socs inhibit tlr and rlr signaling, thereby terminating ifn production ( ) . tam receptor ligands, gas and pros, bind phosphatidylserine on dying cells and are produced by activated dcs and monocytes/macrophages. thus, ifn induction of tam inhibitory receptors on uninfected phagocytic immune cells could limit their propensity to produce the cytokines upon engulfment of dying virally infected cells. ifns induce tetherin on most cell types. pdcs express a receptor for tetherin, leukocyte immunoglobulin-like receptor, subfamily a (with tm domain), member (lilra ). lilra triggering on pdcs inhibits their production of ifn-i. hence, through lilra engagement by tetherin, pdcs can monitor their efficacy at inducing an antiviral gene expression program in neighboring cells through ifns, and timely terminate their ifn production. how positive and negative feedback loops integrate in time and space to promote optimal kinetics and intensity of ifn production in order to efficiently control viral infection without causing severe immunopathology is not completely understood. positive feedback loops may occur very rapidly after initiation of viral infection to allow rapid secretion of high levels of the cytokines for fast and strong induction of anti-viral cell-intrinsic immunity. negative feedback loops occur likely later to terminate the response and thus avoid chronicity of cytokine production and its ensuing deleterious effects. pdcs do not constitute the major source of ifn production upon local infections by several viruses in the lung or in the female reproductive tract. pdcs are dispensable for resistance against these infections ( , , ) . during pulmonary infection by newcastle disease virus (ndv), ifn-i are produced locally in the lungs mainly by infected alveolar macrophages. lung pdcs do not express the cytokines ( ) . selective depletion of lung alveolar macrophages leads to systemic dissemination of ndv, and to a strong activation of pdcs for ifn-i production specifically in the spleen. even in the case of systemic viral infections such as caused by intravenous injection of ndv or intraperitoneal injection of mouse cytomegalovirus (mcmv), pdc ifn production is confined to the spleen. it is not detected in other organs even those with high viral replication ( , ) . hence, splenic pdcs are especially prone to high level ifn production upon systemic acute viral infections. pdcs located in non-lymphoid organs, in particular mucosal barrier tissues, appear to be inhibited for ifn production. thus, ifn production by infected cells serves as first line of defense to block virus replication at its portal of entry in the body. ifn production by uninfected pdcs might constitute a failsafe mechanism mainly activated in the spleen when viral infection gets systemic ( ) . under these conditions, to promote health over disease, the benefits for the host of producing high circulating levels of ifns in order to induce widespread cell-intrinsic anti-viral defenses might prevail over the deleterious effects that this could cause on certain cell types or tissues. indeed, pdcs are required for protection against hsv- and hsv- in mice only in systemic but not local infections ( ) . this observation is consistent with the crucial role of pdcs for protection of mice against systemic infection by mouse hepatitis virus (mhv), a fast replicating coronavirus ( ) . conflicting results have been obtained on the role of pdcs during intranasal influenza infection ( , ( ) ( ) ( ) . a possible explanation is that pdc ifn production contributes to resistance to highly pathogenic influenza strains that might systemically spread from the lung early after infection, even if at low levels. another intriguing observation is that ifns are critical for host resistance to mcmv and that pdcs are the major source of ifns in this infection model but are dispensable for virus control ( ) . studies are ongoing to understand this apparent paradox. patients bearing genetic mutations disrupting endosomal tlr signaling do not appear to suffer from life-threatening viral infections ( , ) , contrary to patients impaired in ifnar signaling ( , ) . a notable exception is the specific susceptibility to severe herpes virus encephalitis in individuals' deficient for tlr signaling ( , ) . however, contrary to extracellular tlr, endosomal tlr have evolved under strong purifying selection in human beings ( ) . hence, while pdcs and endosomal tlr might have been required for protection of our species against viral infections in the past, this appears not to be the case anymore perhaps due to improved social, hygiene, and health care in modern society ( ) . attesting to the importance of ifns for anti-viral defense in vertebrates, many mammalian viruses encode immune evasion genes specifically inhibiting the production of ifns in infected cells ( , ) . pdcs or xcr + dcs might be essential for ifndependent host protection against these viruses, because they are spared from the intracellular functions of viral immune evasion genes ( ) . to the best of our knowledge, mcmv does not encode for immune evasion genes inhibiting ifn production. however, mcmv manipulates ifn-i responses through specific inhibition of stat functions in infected cells. thus, pdcs might be dispensable for resistance against systemic mcmv infection due to sufficient levels of ifn production by infected cells locally in all infected tissues. hepatocyte responses to ifn-iii appear to play a www.frontiersin.org critical role in human resistance to hcv. in infected hepatocytes, hcv induces the expression of cellular micrornas binding to ifn-iii mrna and leading to its degradation. uninfected xcr + dcs produce high levels of ifn-iii in vitro upon hcv stimulation ( , ) . hence, during acute hcv infection in vivo, xcr + dc may be a strong and early source of ifn-iii not subjected to virus immune evasion strategies, therefore, contributing to protect naturally resistant individuals. in secondary lymphoid organs, a subset of macrophages is critical for the clearance of viruses from the lymph ( ) . these macrophages are located on viral entry routes, near to subcapsular sinuses where the afferent lymph drained from non-lymphoid tissues flows. contrary to other subsets of macrophages, subcapsular sinus macrophages are highly susceptible to viral infection, because they constitutively express only low levels of effector molecules of cell-intrinsic anti-viral immunity and because their responses to ifns are inhibited by their high constitutive expression of usp . subcapsular sinus macrophages rapidly become infected by viruses incoming from the lymph and produce large amounts of ifns. this altruistic suicide prevents virus dissemination to other adjacent cell types and promotes the induction of innate and adaptive anti-viral immunity ( ) . upon instruction by ifns, cells express a wide variety of viral restriction factors, whose combined action blocks pathogen invasion by interfering with the different stages of viral life cycle (figure a ). this has been extensively reviewed recently ( ) and will only be described succinctly here. virus fusion with host cell membrane can be blocked by cholesterol- hydrolase (ch h) that inhibits sterol biosynthesis. some viruses enter cells by escaping from endosomes/lysosomes, which can be blocked by interferon inducible transmembrane (ifitm) proteins. virus uncoating can be blocked by tripartite motif (trim) proteins, such as trim α, which bind to hiv- capsid thus promoting its degradation, and by myxoma resistance gtpases, mx , and mx , which efficiently trap viral structural proteins at an early stage following virus entry into the cell. mx inhibits a number of viruses, including influenza virus through sequestration of its nucleocapsid. mx associates with host cyclophilin a and hiv- capsid protein. virion assembly can be blocked at transcriptional, translational, and posttranslational levels. the adenosine deaminase acting on rna (adar ) and the apolipoprotein b mrna editing enzyme, catalytic polypeptide-like (apobec) deaminases induce viral rna destabilization and hypermutation ( , ) . the sterile alpha motif and histidine-aspartic domain (hd) containing protein (samhd ) blocks reverse transcription by hydrolyzing dntps ( ) . adar , apobec, and samhd functions have been mainly studied in infections by hiv- and other retroviruses. the , -oligoadenylate synthase (oas) proteins, the ifn-induced proteins with tetratricopeptide repeats (ifit), and pkr inhibit viral and host protein translation by using complementary mechanisms ( ) . the major post-translation modification induced by ifns is the binding of the ubiquitinlike modifier isg to several viral and host proteins, a process called isgylation. most of the known isgylated proteins are targeted for degradation, with few exceptions that are on the contrary stabilized like irf ( ) . finally, the egress and budding of virions of many enveloped viruses can be inhibited by tetherin or by viperin ( ) . many anti-viral isgs have been functionally characterized only recently, largely thanks to large-scale screening approaches. they display a variable degree of viral specificity ( , ) that might inversely relate to the extent of their side effects on host cells ( figure b ). anti-viral effectors acting on a broad spectrum of viruses often target key metabolic pathways that are also crucial for host cell functions. this is the case for the control of cholesterol metabolism by ch h ( ) or of protein translation by pkr, oas, or ifits ( ) . other anti-viral restriction factors such as mx may specifically target one molecule of a very restricted set of viruses with no apparent side effects on host cells. some anti-viral isgs target specific functions critical for only a restricted array of viruses and might similarly exert side effects only on a subset of host cell types. for example, samhd inhibits retrovirus replication through dntp depletion, which might more specifically affect proliferating host cells. hence, the infected host must balance the intensity, breadth, and location of isg induction to circumvent viral replication while preventing life-threatening damages to vital cell types or tissues. one of the mechanisms contributing to this balance is translational control of the expression of isgs, especially those with pro-apoptotic or anti-proliferative functions ( ) . while many anti-viral isgs are transcriptionally activated in most ifn-stimulated cells, their translation can be specifically blocked in uninfected cells by cellular microrna. this inhibition is relieved upon cell infection through negative regulation of the function of the rna-induced silencing complex. hence, ifn stimulation of uninfected cells prepares them for rapid and strong induction of cell-intrinsic anti-viral defenses upon viral infection while avoiding their unnecessary exposure to the toxic effects of certain isgs. further knowledge on the functions and the dynamic regulation of isgs is essential to develop novel therapeutic strategies against viral infections aiming at modulating ifn responses to promote their protective anti-viral cell-intrinsic functions over their deleterious toxic effects. a better understanding of the immunoregulatory effects of ifns will also help. type i interferon can modulate the functions of a broad spectrum of immune cells ( figure a ). we will review this knowledge, focusing on the functions of dcs, nk cells, t cells, and b cells, since they are involved in the control of most viral infections. we will discuss the hypothesis that dcs play a central role in ifn-i orchestration of innate and adaptive immunity for the induction of optimal anti-viral defenses (figure ) . during viral infections and cancer immunosurveillance, ifn-i constitute one of the most important input signal acting on dcs to promote their delivery of appropriate output signals to t cells, b cells, and nk cells for protective immunity ( figure a ). dcs deliver three types of signals to activate and functionally polarize t cells. signal is the triggering of the t cell receptor by viral peptide-mhc complexes. signal is the triggering of activating t cell co-stimulation receptors such as cd or cd by the cd / and cd co-stimulation molecules expressed on dcs. signal corresponds to cytokines, which can promote t cell proliferation and acquisition of specific effector functions. under steady state conditions, most dcs are in an immature state characterized by low level expression of mhc-ii (signal ) and co-stimulation molecules (signal ) and by the lack of production of t cell-activating www.frontiersin.org cytokines (signal ). upon activation, including early after viral infections in vivo, dcs up-regulate their expression of signal and activating signal and secrete t cell-activating cytokines. this process is called dc maturation. gene expression profiling of dcs stimulated by microbial stimuli identified a core set of genes upregulated in mature dcs irrespective of the stimulus they receive, irrespective of the subset they belong to, and conserved across evolution ( ) . most of these genes are induced during dc maturation in part through cell-intrinsic ifn-i signaling ( ) . consistently, cell-intrinsic ifnar signaling in dcs is required in many circumstances for the induction of protective immunity, including efficient cd t cell responses during viral infection or tumor development ( ) ( ) ( ) , th responses upon polyi:c injection independently of il- or ifn-γ effects ( , ) , as well as follicular helper t cell and humoral responses ( , ) . mechanistically, ifn-i promote dc immunogenicity for efficient t cell activation through a variety of effects ( figure b) . it drives dc up-regulation of signal in vivo during viral infections ( ) and boosts their capacity to cross-present antigens for increased delivery of signal to cd t cells ( ) ( ) ( ) . it shapes their delivery of activating signal , in particular inducing il- and promoting or inhibiting il- depending on experimental conditions ( , ) . finally, it is necessary to induce their metabolic shift from mitochondrial oxidative phosphorylation to aerobic glycolysis, which fuels the increased needs in energy and the expansion of the intracellular organelles required for the production and proper intracellular routing of the signal and proteins ( , ). selective inactivation of ifnar on cdcs compromises mouse resistance to mcmv and mhv infections ( , ) . in contrast, ifnar expression is not required on nk cells for protection against mcmv and on pdcs, t cells, and b cells for early control of mhv replication ( , ) . although cell-intrinsic ifn-i signaling in nk cells can promote their activation ( ) (figure a) , ifn-i-induced il- trans-presentation by dcs plays a more prominent role for this function in many conditions including in vivo during mcmv infection ( , ) ( figure c) . cell-intrinsic ifn-i signaling in cd t cells ( ), cd t cells ( , ) , and b cells ( ) can also contribute to their efficient activation and functional polarization (figure ). this depends on experimental settings. cd t cell-intrinsic ifn-i responses are crucial for mounting efficient cytotoxic cd t cell responses against lcmv but are less critical against vaccinia virus and vesicular stomatitis virus ( , , ) . mechanistically, cell-intrinsic ifn-i signaling in cd t cells can promote their survival during their antigen-induced proliferation ( ) . cell-intrinsic signaling in dcs and cd t cells may act in a synergistic manner. indeed, conditional inactivation of ifn-i responsiveness was required to occur simultaneously in both of these two cell types to dramatically affect cd t cell expansion upon vaccination with a modified ankara vaccinia virus ( ) . in summary, ifn-i generally play a crucial, beneficial, role in immune defenses against viral infections, both through the induction of cell-intrinsic anti-viral defenses and through the orchestration of innate and adaptive immunity. however, if these responses are not properly regulated, they can contribute to diseases as we will now discuss. a frequent side effect of ifn-i treatment against cancer or chronic viral infections is the induction of autoimmune reactions. consistently, isg expression is a hallmark of many spontaneous systemic or tissue-specific autoimmune diseases, including systemic lupus erythematosous (sle), sjogren's syndrome, psoriasis, and other skin disorders ( ) . the dysregulation of ifn-i responses observed in patients with these autoimmune diseases likely results from both genetic and environmental factors. genome-wide association studies show that polymorphisms in genes involved in ifn-i responses strongly correlate with increased susceptibility to many autoimmune diseases ( ) . diverse environmental factors can also contribute to the onset of autoimmune diseases. microbial infections often precede first clinical manifestations of autoimmune diseases. whether infections ( ) and/or alterations in the commensal microbiota of the affected barrier tissues ( , ) are the cause or rather the consequence of autoimmunity is still matter of debate. infection-or dysbiosis-induced tissue damages and unbridled ifn-i responses can contribute to initiate autoimmune reactions. gender is another prominent factor affecting susceptibility to autoimmune diseases. women are more prone to autoimmunity, which may result from endocrine regulation of ifn-i responses. pdc ifn-i production is enhanced in human and mouse females, due at least in part to cell-intrinsic enhancement of tlr / responses by the female hormone estradiol ( ). in autoimmune diseases, different mechanisms could operate to initiate the dysregulation of immune responses leading to a vicious circle of reciprocal activation between innate ifn-i responses and adaptive self-reactive lymphocyte responses (figure ) . adaptive immune cells are educated to spare "self." this occurs through negative selection of potentially autoimmune b and t cells during their development in the bone marrow or thymus, respectively, a process called central tolerance. self-reactive b or t cells that have escaped this pruning can be either deleted or functionally inactivated once they have egressed in secondary lymphoid organs or non-lymphoid tissues, a process called peripheral tolerance. in some individuals, polymorphisms in genes involved in the promotion of central or peripheral tolerance lead to a higher number, diversity, and/or responsiveness of self-reactive lymphocytes in the periphery, in particular of b cells secreting anti-dna or anti-rnp antibodies ( , ) . mammalian dna or rna are poor inducers of pdc ifn-i induction under normal conditions. however, pre-existing anti-dna or anti-rnp autoantibodies can break this innate tolerance of pdc. indeed, antibodies binding to self nucleic acids can protect them from degradation and compact them into nanoparticles that are very effective for the induction of ifn-i in pdc (figure ) . dna-containing immune complexes (ics) are frequently found in the serum of sle patients (sle-ics) and can activate pdc ifn-i production ( ) . in turn, pdc ifn-i activate cdcs, monocytes ( ) , and b cells, leading to a vicious circle of reciprocal activation between dcs and frontiers in immunology | microbial immunology figure | a simplified model of the deleterious role of ifn-i in several autoimmune diseases. when exposed to different kinds of injuries (microbial infection, commensal microbiota dysbiosis, chemical or physical insults), healthy tissues can undergo cell damage and death. these events induce the release of apoptotic bodies encompassing self rna or dna. neutrophil recruitment and activation in inflamed tissues can also constitute a potent source of self nucleic acids, through the release of neutrophil extracellular traps (net). self rna or dna can associate with cationic peptides (e.g., ll ) as shown in psoriatic patients or with inflammatory molecules (e.g., high mobility group box , hmgb ) to generate nanoparticles that are extremely efficient for ifn-i production by pdc and eventually other cell types. pdc can also be efficiently activated for ifn-i production by immune complexes (ics) generated by the association between self nucleic acids and auto-antibodies as frequently found in the serum of systemic lupus erythematosus patients. ifn-i promote the differentiation and/or the maturation of antigen-presenting cells, in particular different subsets of dc. activated dc can then present self-antigens for activation of auto-reactive t cd + cells, including follicular helper lymphocytes, which in turn activate auto-reactive b cells for auto-antibody secretion, leading to a vicious circle of reciprocal activation between innate and auto-reactive adaptive immune cells. idc, immature dc; mdc, mature dc; mo-dc, monocyte-derived dc. see main text for further details. self-reactive lymphocytes and to the exacerbation of autoimmune responses (figure ) . certain infections or dysbiosis of the commensal microbiota of the affected barrier tissues could promote chronic production of host amphiphatic peptides able to combine with eukaryotic dna or rna, likely released from dying cells, thus forming pdc-activating nanoparticles. indeed, in psoriatic skin, both a high expression of ll and a massive infiltration of pdcs is observed ( ) (figure ) . hence, to treat many autoimmune diseases, novel therapeutic strategies could be designed to target dysregulated pdc ifn-i production or b cell activation by ifn-i. one of the most common complications of primary infections by many respiratory viruses, in particular influenza virus, is a lifethreatening pneumonia due to secondary pulmonary infections by bacteria, such as streptococcus pneumoniae, staphylococcus aureus, or haemophilus influenza ( , ) . these pathologies affect especially infants, elderly, and immunocompromised patients. retrospective studies indicate that secondary bacterial pneumonia was highly recurrent in lung tissues isolated from patients who died during last century influenza pandemics, independently of antibiotic availability ( , ) . influenza virus induces high ifn-i responses in human beings and mice. in both hosts, secondary bacterial infections are lethal only when they occur in a limited time window following primary viral infection ( - days), around the peak of ifn-i responses, before complete virus clearance. mouse models of viral/bacterial coinfections are being used to dissect disease mechanisms ( ) . ifnar -deficient mice appear more resistant to secondary pulmonary bacterial infections, showing that ifn-i responsiveness contributes to disease ( ) . similarly, after lymphochoriomeningitis virus (lcmv) infection, wild-type but not ifnar -deficient mice are more susceptible to lpsinduced septic shock ( ) . several mechanisms may contribute to the detrimental role of ifn-i in secondary bacterial infections ( figure ) . early during viral infection, ifn-i decrease the host ability to control bacterial replication, by dominantly polarizing immune responses toward anti-viral functions, simultaneously inhibiting the development of the types of immune responses required for protection against most bacterial infections. ifn-i can inhibit the production of chemokines required for the recruitment to the respiratory tract of antibacterial effector innate immune cells, in particular neutrophils or monocytes/macrophages ( , ) (figure ) . depending on the experimental models used, ifn-i can on the contrary induce a ccr -dependant recruitment of classical monocytes ( ) . in infected tissue, ifn-i might skew the functional polarization of resident or infiltrating monocytic cells toward immunosuppression, because it does limit their antibacterial functions by inhibiting their il- production ( ) ( ) ( ) while it might promote their production of il- and nitric oxygen intermediates. the exact nature of infiltrating monocytic cells is not clear and could correspond to activated classical monocytes, modcs, monocyte-derived macrophages, or myeloidderived suppressor cells (mdscs). the boundaries between these putatively different cell types are currently ill-defined ( ) . these cells could fuel local replication of monocyte/macrophage-tropic bacteria ( ) , be immunosuppressive ( ) or contribute to local immunopathology ( ) . the role of ifn-i on monocytes/macrophages is complex and will require further investigations to determine when it is protective versus deleterious and what the underlying mechanisms are. depending on the context, ifn-i can either promote or inhibit the induction of th cytokines such as il- and ifn-γ, and myeloid cell responses to ifn-γ ( , ( ) ( ) ( ) . ifn-i can also polarize cd t cell responses toward th at the expense of th , while the th -type cytokines il- a and il- are required for host defense against pulmonary www.frontiersin.org bacteria by inducing the production of anti-microbial peptides and of tissue repair molecules ( figure ) ( ) ( ) ( ) . ifn-i may not only affect host resistance to bacterial infection, but also host tolerance, i.e., the ability of the host to tolerate a given burden of pathogen without undergoing excessive tissue damages ( , ) . hence, to counter ifn-i deleterious effects during secondary bacterial infections, it will be important to better delineate the respective contribution of lung tissue tolerance modulation and of immune-mediated resistance weakening. another well documented example of deleterious effects of ifn-i due to their inappropriate functional polarization of immune responses is the enhanced susceptibility to fungal infections of patients with genetically determined hyperactive ifn-i responses, as exemplified in the hereditary disease chronic mucocutaneous candidiadis (cmc) (figure ) ( ) . patients with cmc have a significant deficit in th cd t cells, at least in part as a consequence of altered responsiveness to il- or il- . several stat mutations were identified in patients with autosomal dominant cmc. gain-of-function stat mutations were found to hard wire cd t cell responses to cytokines toward stat signaling, compromising their stat -dependent ability to produce il- upon il- or il- stimulation. this was associated to induction of a global ifn-i transcriptomic signature in blood ( ) . deleterious ifn-i effects on immunity to candida might not only occur in cmc patients but also in other types of individuals upon secondary fungal infections occurring shortly after a primary viral infection, likewise to the situation discussed above for secondary bacterial infections. indeed, polyic induced ifn-i abrogate innate immunity to systemic candidiasis in mice ( ) , and ifnar-deficient mice can be more resistant to candida infection under certain experimental settings ( ) . however, the role of ifn-i in the modulation of the ability of immunocompetent hosts to control fungal infection is disputed ( , ) . the inhibition of th responses by ifn-i could be protective in at least one important human pathology, ms (figure ) . ms represents a striking exception to the previously discussed detrimental role of ifn-i in autoimmune diseases. indeed, a large proportion of ms patients have low serum ifn-i activity and low isg levels. these ms patients present a significant reduction of ms relapse upon ifn-β administration ( ) . the underlying mechanisms are not yet completely unraveled. however, in the experimental autoimmune encephalitis mouse model of ms, th responses bear a major contribution to nervous system damages and are inhibited by the il- and il- induced upon ifn-i administration ( ) . in summary, ifn-i responses can be deleterious in autoimmunity by promoting a vicious circle of reciprocal activation between innate immune cells and auto-reactive cd t or b lymphocytes. ifn-i responses can also be deleterious upon secondary bacterial or fungal infections in the lung or the kidneys occurring shortly after a primary viral infection, by compromising the recruitment of anti-microbial innate effector cells and/or by preventing the proper functional polarization of immune responses. we will now discuss how ifn-i responses can also compromise host immune defenses against certain viruses and promote chronic infections. different lcmv strains such as armstrong and clone- (cl ), respectively, lead to acute versus chronic infections in mice. a hallmark of chronic lcmv infection is the loss of the proliferative potential and effector functions of anti-viral cd t cells, a process called exhaustion. exhausted cd t cells are characterized by a high expression of the inhibitory receptors pd- , ctla , and lag- ( ) . in vivo blockade of these inhibitory receptors can reverse t cell exhaustion and allow resolution of the chronic infection ( ) . ifn-i and isgs are induced early after infection with all strains of lcmv, albeit to lower levels with those leading to chronic infection. this early ifn-i production is critical to limit viral replication ( ). in models of acute infection, ifn-i responses rapidly return to normal, undetectable, levels, before viral replication is completely controlled. in contrast, isg induction is maintained in chronic infection, including the expression of pd- ligands on apcs and of the immunosuppressive il- cytokine, consistent with a prolonged expression of ifn-i albeit at low levels ( , ) . in vivo neutralization of ifn-i by antibody administration promoted resolution of chronic lcmv cl infection, allowing the frontiers in immunology | microbial immunology restoration of functional anti-viral cd t cell responses at least in part through cd t cell-and ifn-γ-dependent mechanisms ( , ) . during persistent lcmv cl infection, chronic low level ifn-i production polarizes cd t cell responses toward t follicular helper (tfh) rather than th functions. thus, chronic ifn-i responses promote enhanced anti-viral b cell responses but facilitate cd t cell exhaustion due to deficient cd t cell help, therefore contributing to host failure to prevent chronic infection ( ) . strikingly, establishment of chronic infection by lcmv cl could also be prevented by early administration of two shots of a high dose of exogenous ifn-i, at days and post-lcmv inoculation. this treatment allowed viral clearance by rescuing anti-viral cd t cell from exhaustion ( ) . altogether, these studies show that the timing and duration of ifn-i production during viral infections is critical in determining how this response will impact the balance between the virus and the host. an early and robust but transient production of ifn-i promotes strong induction of cell-intrinsic viral restriction mechanisms as well as adequate polarization of adaptive anti-viral immune responses, which combined effects lead to viral clearance. in contrast, if the production of ifn-i is too low and/or too late, both viral replication and low ifn-i responses become chronic, their combined action leading to induction of immunosuppressive effects and to inadequate functional polarization of cd t cells. this results in cd t cell exhaustion and maintenance of chronic infection. chronic viral replication and cd t cell exhaustion is also a hallmark of hiv- infection. we will now discuss the complex and disputed role of ifn-i in this disease. both in hiv- infection and in its most relevant animal model, infection of non-human primates with simian immunodeficiency virus (siv), disease progression after the acute phase of the infection is associated with high and chronic expression of isgs while ifn-i production is inconsistently detected ( ) ( ) ( ) . in contrast, the individuals that do not progress toward disease despite persistent high viral loads show much lower immune activation, in particular low isg expression, after the acute phase of the infection ( ) ( ) ( ) ( ) . hence, chronic low levels of ifn-i are associated to disease progression independently of the level of viral replication. therefore, an outstanding question still open for a better understanding of the physiopathology of hiv- infection is whether chronic ifn-i responses are merely a marker of progression, or whether they are implicated in driving disease development. in addition to mechanisms similar to those uncovered in the mouse model of chronic lcmv infection, during hiv- infection other effects of ifn-i could promote a vicious circle of reciprocal activation between chronic viral replication and sustained, deleterious immune responses (figure ). very early after hiv- infection, in most individuals, ifn-i production might be too weak or too late to induce a combination of cell-intrinsic defense mechanisms and of immune responses efficient enough to prevent later establishment of chronic infection. on the contrary, as demonstrated in the case of the mouse model of lcmv infection, ifn-i responses could favor cd t cell exhaustion, either by direct cell-intrinsic effects on cd t cells (figure , ) or by contributing to deprive them from cd t cell help (figure , ) . several effects of ifn-i might compromise anti-hiv- th responses or more generally contribute to the global depletion of cd t cells. these mechanisms include functional polarization of anti-hiv- cd t cells toward tfh rather than th responses, cxcl production leading to enhance recruitment of memory cd t cells to the sites of viral replication where they fuel chronic viral replication with new hiv- target cells (figure , to ) , direct pro-apoptotic and anti-proliferative effects on cd t cells (figure , ), as well as trail induction on pdcs licensing them for killing cd t cells irrespective of their infection (figure , ) ( , ) . altogether, these mechanisms entertain chronic viral replication and continuous depletion of cd t cells, leading to the dramatically enhanced susceptibility to opportunistic infections (figure , ) characteristic of the acquired immunodeficiency syndrome (aids) (figure , ) . other lines of evidences have been reported to support a deleterious role of pdc activation during hiv- infection. women undergo faster hiv- disease progression than men with similar viral loads, which may result in part from the highest ifn-i production of women's pdcs including in response to hiv- stimulation ( ) . pdc recruitment and activation in the vaginal mucosa of female macaques early after local siv inoculation contribute to attract and activate cd t cells, which can then be infected and promote virus dissemination from its portal of entry ( ) . however, in vivo blockade of pdc ifn-α production by administration of tlr / -antagonistic oligonucleotides early after siv infection of macaques did not decrease t lymphocyte activation, which suggests that additional sources of ifn-i likely contribute to the immune dysfunction observed in siv/hiv- infections. targeting dysregulated ifn-i responses during hiv- infection might represent an interesting adjuvant therapeutic strategy to highly active antiretroviral treatments. administration of ifn-i in the non-pathogenic siv infection model of sooty mangabeys was not sufficient to switch it into a pathogenic model. no cd t cell depletion ensued, no hyperactivation of immune responses were observed. viral loads were even significantly decreased. however, this could be consistent with the positive impact of early and high dose ifn-i administration in chronic lcmv infection ( ) . indeed, during the review process of this manuscript, it was reported that, early during primary siv infection in the pathogenic rhesus macaque model, a high dose injection of ifn-i was protective while neutralization of endogenous ifn-i was deleterious. in contrast, in the same animal model, prolonged ifn-i administration accelerated disease development in the chronic stage of the infection ( ) . in mice with a humanized immune system, pdc depletion strongly decreased isg induction and enhanced viral replication both in the acute and chronic phases of hiv- infection. however, pdc depletion during chronic infection decreased infection-induced t cell apoptosis and increased t cell numbers in lymphoid organs ( ) . these results further emphasize the dual role of ifn-i and pdcs in the physiopathology of hiv- infection. a strong and transient production of ifn-i early after infection benefits the host by lowering the set-point of viral replication during chronic infection. sustained production of low levels of ifn-i during chronic infection contributes to immune dysregulation and cd t cell depletion. further studies will be necessary to examine whether complementing standard-of-use antiretroviral drugs with pdc www.frontiersin.org depletion, ifn-i neutralization, or selective inhibition of t cell responses to ifn-i could yield additional benefits to chemotherapy in non-human primates during chronic siv infection. ifn-i administration has been used for many years to treat another human chronic viral disease, hcv infection. roughly, half of the patients do not show sustained virological responses (svr). the treatment causes severe side effects in many individuals. new chemotherapeutic drugs very potent at blocking hcv replication in vivo have recently become available. hence, whether ifn-i administration still constitutes a viable treatment against chronic hcv infection is being questioned ( , ) . we will now discuss this issue. chronic hcv infection is the main cause of liver cirrhosis and hepatocellular carcinoma. there is currently no vaccine against hcv. the most common therapy for chronic hcv patients is the administration of recombinant pegylated ifn-α (peg-ifn-α) combined with the anti-viral drug ribavirin. however, because of ifnar pleiotropic expression, ifn-α administration induces severe side effects including flu-like syndrome, fever, fatigue, myalgia, and nervous depression ( figure a ) ( ) . moreover, only about half of treated patients harbor svr ( ) . prior-totreatment high hepatic isg expression is a negative predictor of svr upon peg-ifn-α therapy. high isg expression in untreated patients likely results from chronic but low ifn-i production triggered by persistent hcv replication. indeed, hepatocytes from non-responder patients were found to be infected at a greater frequency and to exhibit dampened antiviral and cell death responses ( ) . what the cellular sources of ifn-i production are and why they persist only in non-responder patients still remain to be established. in chronic hcv infection, cytotoxic effector lymphocytes may contribute to the development of hepatocarcinoma by causing low level but sustained hepatocyte death and renewal. in contrast, local production of ifn-γ in the liver by nk and t lymphocytes could promote resistance to disease through non-cytolytic control of viral replication. as discussed previously for lcmv and hiv- , low chronic production of endogenous ifn-i in hcv patients could compromise both innate and adaptive anti-viral immune responses. chronic exposure to ifn-i could dampen the ability of frontiers in immunology | microbial immunology utr of ifnl mrna to promote their degradation. the favorable ifnl allele associated with responsiveness to peg-ifn-α treatment may allow endogenous expression of sufficient levels of ifnl for efficient induction of cell-intrinsic anti-viral defenses in hepatocytes. this process is, however, hampered by the limited expression of the receptor for this cytokine (ifnλr ) in these patients. peg-ifn-α treatment might promote resolution of the infection by inducing ifnλr in these patients, potentiating their response to their endogenous production of ifnl . in the patients that do not respond to peg-ifn-α treatment, endogenous levels of ifnl are insufficient for efficient induction of cell-intrinsic anti-viral defenses in hepatocytes, due to the degradation of the corresponding mrna in infected hepatocytes. in these patient's hepatocytes, however, ifnλr is already expressed to high levels prior to treatment due to their high endogenous ifn-i responses. administration of exogenous ifn-λ might cure these patients. see main text for further details. nk and cd t cells to produce ifn-γ ( , ) and promote cd t cell exhaustion ( ) . it could also induce an antagonist form of cxcl , a chemokine required for recruitment to the liver of anti-viral nk and cd t cell effectors ( ) . it may also polarize monocytes toward immunosuppressive functions ( ) . therefore, better understanding ifn-i effects in hcv infection is critical to improve care of both responders and non-responder patients to peg-ifn-α. for responder patients, the issue is to modify the treatment to favor beneficial antiviral and immunoactivating effects over side effects strongly affecting patient's quality of life (figure a ). this might be achieved by specific delivery of ifn-i to targeted cell types as discussed later. for non-responder www.frontiersin.org patients, the issue is to understand the mechanisms underlying treatment failure to determine whether alternative therapies could be designed (figure a) . genome-wide association studies identified various single nucleotide polymorphisms (snp) in the gene encoding il- b/ifn-λ , one of the ifn-iii, as well in its and non-coding regions ( ) ( ) ( ) ( ) . one snp, called rs , is located kb upstream of the ifnl gene. patients harboring the cc genotype have a favorable prognosis to ifn-i treatment. patients with the tt genotype are at high risk of treatment failure ( , ) . in europeans, the favorable cc genotype is the major, most common, ifnl allele. the unfavorable tt snp is the minor allele. the frequency of these alleles is reversed in africans. the favorable allele allows escape of ifnl mrna from degradation by cellular microrna induced upon hcv infection ( ) . until recently, ifnl genotypes and hepatic isg expression were considered as independent predictors of response to peg-ifn-α treatment in hcv patients ( ) . here, we propose a potential explanation, which integrates both factors in a relatively simple model ( figure b ). our main hypothesis is that efficient control of hcv infection depends on hepatocyte response to ifn-λ rather than ifn-α. this is supported by reports that ifn-λ induces a stronger and more sustained isg expression in hepatocyte cell lines in vitro ( ) , and that polyi:c-induced control of hcv replication in humanized liver in chimeric mice is correlated to the induction of ifn-λ but not ifn-i in human hepatocytes ( ) . responder patients harboring the favorable ifnl allele preventing the degradation of the corresponding rna in infected cells might express significant levels of endogenous ifn-λ , although this is disputed. however, they express only low levels of ifn-λr , which limits ifnλ efficiency ( figure b ) ( ) . how these patients benefit from peg-ifn-α treatment could be that it induces ifn-λr expression on hepatocytes thus boosting endogenous ifn-λ effects ( ) . in contrast, high isg-expressing non-responder patients harboring the unfavorable ifnl allele might not express enough ifn-λ for virus control. however, they do express ifn-λr as a result of their endogenous production of ifn-i. hence, peg-ifn-α might be ineffective in these patients because they already express ifn-λr but fail to produce endogenous ifn-λ due to the degradation of its mrna in infected hepatocytes (figure b) . these patients may be good candidates for peg-ifn-λ therapy, currently undergoing clinical development. since the expression of ifn-λr is mainly restricted to epithelial cells, melanocytes, and hepatocytes, some of the side effects related to ifn-i treatment might be strongly attenuated in peg-ifn-λ therapy. however, as ifn-i are key to induce anti-viral immune responses, it will be critical to determine whether, beside viral clearance, peg-ifn-λ therapy can also induce long-term immune protection against hcv. ifn-i transduce intracellular signals through a single receptor, ifnar, but via a multitude of downstream signaling pathways. the janus activated kinase (jak)/stat pathway was the first to be identified ( ) . ifnar is composed of two distinct subunits, ifnar and ifnar , which are constitutively associated with members of the jak family, tyrosine kinase (tyk ) and jak , respectively ( ) . the binding of ifn-i to their receptor leads to the phosphorylation of jak and tyk , which in turn induce the phosphorylation and activation of the stat proteins ( ) . different stat complexes can form upon triggering of ifnar (figure ) . a transcriptional complex that forms in most conditions of ifn-i stimulation and induces the expression of many molecules of cell-intrinsic anti-viral immunity is interferonstimulated gene factor (isgf ), a heterotrimer composed of pstat , pstat , and irf ( ) (figure ) . following its translocation into the nucleus, isgf binds to isre regulatory sequences in target genes. many molecules playing a key role in the function of innate or adaptive immune leukocytes are also induced by isgf , including cd , cd , or il- in dc, and granzyme b in nk cells. isgf is generally composed of stat phosphorylated on tyr and ser and of stat phosphorylated on tyr . however, alternative isgf complexes have been described in various contexts which could participate to the diversity of ifn-i effects ( ) . the pstat homodimer also plays a prominent role in cell-intrinsic ifn-i-dependent gene induction. it binds ifnγactivated sequences (gas) and controls the expression of many pro-inflammatory molecules ( ) . pstat homodimers can form upon stimulation with either ifn-i or ifn-γ. many gasregulated genes can be induced by either cytokines. depending on cell types, jak signaling downstream of ifnar can lead to the activation of virtually all stat proteins and to their combinatorial association into a variety of complexes with different affinities for specific gas elements ( ) ( ) ( ) (figure ) . this diversity contributes to ifn-i induction of different transcriptional programs in distinct cell types ( ) . stat complex formation depends in part on the relative abundance of stat molecules in the cell ( ) . while stat , stat , stat , and stat can be activated in most cell populations, stat and stat are mainly activated in lymphocytes ( ) . for example, quiescent nk cells express more stat than stat , leading to constitutive association of ifnar to stat in these cells. hence, quiescent nk cells mount pstat homodimer-dependent responses to ifn-i stimulation, including ifn-γ production and t-bet-driven proliferation (figure ) ( , ) . changes in stat levels can also occur upon the differentiation/activation of a given cell type and lead to a shift in its functional response to the cytokines ( ) . upon activation, nk cells decrease their expression of stat and increase that of stat , shifting their ifn-i response from stat -dependent in a quiescent state to stat dependent in pre-activated cells. this translates into opposite ifn-i effects on ifn-γ production and proliferation for quiescent versus pre-activated nk cells ( ) . however, this outcome can be modulated by simultaneous exposure to other cytokines such as il- or il- / . a reverse stat -to-stat shift occurs in dc during their maturation, shifting their functional responses from inhibition to activation of il- production in response to combined stimulations with ifn-i and cd l ( ) . this frontiers in immunology | microbial immunology ifn-i binding to ifnar triggers the phosphorylation of tyk and jak , which in turn phosphorylate a variety of stat proteins. activated stats are able to form complexes, as homo-or hetero-dimers. the heterodimer stat -stat binds to a third partner, ifn-regulatory factor (irf ), in order to form the isgf complex. this complex translocates into the nucleus and binds to specific regulatory sequences, ifn-stimulated response elements (isre), to activate the expression of many interferon-stimulated genes (isgs). in particular, isgf induces most, if not all, of the isgs encoding effector molecules of cell-intrinsic anti-viral defenses such as oas or mx . alternative jak/stat pathways include the formation of stat or stat homodimers, which may drive different functional responses to ifn-i. stat homodimers bind to ifnγ-activated sequences (gas) in the promoter of certain isgs, which may promote inflammatory, anti-proliferative, and pro-apoptotic responses. stat homodimers also bind to gas but promote ifn-γ production and pro-proliferative responses. enables mature dc to efficiently activate cd t cells. other yet unknown mechanisms control the formation of different stat complexes in distinct cell types. the nature and dynamics of the signaling pathways triggered by ifn-α or -β were evaluated in bulk cultures of human blood leukocytes using flow cytometry ( ) or high throughput mass cytometry ( ) . a diversity of phosphorylation patterns of stat / / was observed upon ifn-i stimulation. ifn-α activation induced phosphorylation of stat , stat , and stat in most cell types, peaking at min ( ) . ifn-β-induced stat phosphorylation was found to be poor in b cells as compared to monocytes and t cells ( ) . however, the underlying mechanism remains to be identified since b cells did not express lower amount of ifnar or stat or enhanced levels of the inhibitory socs molecule. the high stat activation in monocytes led to their induction of ifn-i-dependent pro-apoptotic genes while this was not the case in b cells. these results strikingly differ from those obtained in the other study upon ifn-α stimulation, where stat phosphorylation was on the contrary lower in cd + monocytes and was prolonged in b cells and nk cells ( ) . the differences between these two studies might have resulted from the use of different subsets and doses of ifn-i. in any case, both studies consistently reported that cd t cells showed the highest activation of stat . all cd t cells but not all cd t cells activated stat and for a longer time ( ) . ifn-β activation of stat was delayed in cd t cells and b cells as compared to cd t cells and monocytes ( ) . different stat complexes may lead to distinct transcriptional programs linked to different biological functions (figure ) . more systematic studies are needed to understand this complexity. besides changing stat levels between cell types or www.frontiersin.org activation states, the processes controlling differential formation of stat complexes downstream of ifnar triggering remain to be identified. in addition to jak/stat signaling, other pathways can be activated downstream of ifnar, including those involving the phosphatidylinositol -kinase (pi k), mitogen-activated protein kinases (mapk), and the crk adaptor molecules ( , ) . this leads to the activation of other transcription factors such as irf, nf-κb, or pu. , which contribute to orchestrate cell responses to the cytokines by regulating both distinct and overlapping sets of genes as compared to stat ( , ) . in summary, ifnar signals through a remarkable diversity of pathways, including but not limited to diverse combinations and kinetics of stat phosphorylations. this explains at least in part the diversity of ifn-i effects, including their induction of opposite responses depending on the physiopathological contexts and/or the nature of the principal responding cell types ( , ) . ifn-iii induce the same signaling pathways as ifn-i, although they engage a different heterodimeric receptor, composed of the il- ra and il- rb chains and preferentially expressed on epithelial cells including hepatocytes. in mice and human beings, numerous ifn-i subtypes exist. functional and population genetic analyses showed that these ifn-i subtypes significantly differ in their functions ( ) ( ) ( ) ( ) ( ) ( ) . hence, one of most extraordinary feature of ifn-i biology is how ifn-i subtypes can elicit so many pleiotropic and diverse functions by interacting with the same receptor complex ( ) . both ifnar and ifnar are required for the initiation of ifn-i-dependent signals, as mice deficient in either one are highly susceptible to viral infections ( , ) . the assembling of the ifnreceptor ternary complex is a two-step process. first, a binary complex is formed by the binding of one side of the ifn molecule to ifnar . then, a single binary complex interacts with ifnar via the other side of the ifn molecule. the stability of the ternary complex will be determined in part by the association and dissociation kinetics between the cytokine and the two receptor chains, as well as by ifnar expression levels since the cell surface concentrations of the receptor subunits are relatively low. hence, both the affinity of ifn-i subsets for ifnar and the amounts of ifn-i, ifnar , and ifnar will regulate their biological effects ( figure a ) ( , ) . cell membrane density of ifnar and ifnar is also involved in differential ifn-β-versus ifn-α-induced functional activities, such as anti-proliferative function ( ) . a variety of cell-intrinsic parameters can also impact the lifetime of the ifn-receptor ternary complex, such as the rate of endocytosis/degradation/recycling of signaling complexes, and negative isg regulators such as usp that decrease the affinity of ifn-ifnar binding ( , ) . based on a definition of a prototypic cytokine-receptor binding module and by analogy with the epo receptor system, ifn-i subtypes were originally postulated to form ternary complexes of differing architectures, resulting in distinct geometry and assembling of intracellular signaling components ( ) . experimental evidence rejected this hypothesis. rather, the differential activities of ifn-i subtypes are determined by the stability of the ligand/receptor ternary complex ( , ) . differential affinities of the ifn-i subtypes for ifnar and ifnar extracellular domains generate subtype-specific signaling cascades and biological outcomes ( figure a ) ( , ) . crystal structure of ternary ifn-i/ifnar /ifnar complex illuminated the biochemical complexity of ifn-i interaction with their cognate receptors ( ) . the main conformational features of ifn-i/ifnar /ifnar ternary complexes are conserved among the different ifn-i, but are quite different from the other cytokine receptors ( , ) . in the formation of the binary ifn-i/ifnar complex, ifn-i ligand discrimination resides on differential energetics during the interaction of anchor points with ifnar , shared by all ifn-i, as well as on key amino acid substitution among ifn-i subtypes ( ) . ifnar then performs major conformational changes to interact with ifn-i associated in the binary complex, thus displaying an optimized functional plasticity ( ) . these differences in the chemistry of ifn-i subtype interaction with ifnar and ifnar thus explain the different affinities of ifn-α versus ifn-β within ternary complex and their differential activities ( ) . the functions regulated by ifn-i strongly depend on the main responding cell types ( figure b ). this has been studied in vitro by examining the functional consequences of the stimulation of different cell types with ifn-i, and in vivo by determining the contribution of cell-intrinsic ifn-i responses of different cell types to resistance or susceptibility to various diseases. an emerging concept is the central role of dc responses to ifn-i for induction of protective immunity against viral infections or tumors (figure ) . the development of mutant mice allowing conditional genetic inactivation of ifnar in a cell-type specific manner using the cre-lox system ( ) has been instrumental in accelerating our understanding of how different cell types respond to ifn-i in vivo and what their respective contribution is to protective or deleterious ifn-i responses. this has been investigated most extensively in viral infections ( , , , , ) but also in cancer ( , ) , bacterial infections ( ), autoimmunity ( , ) , sepsis ( ), or inflammatory diseases ( ) . efforts are being pursued to better understand which cell types respond to ifn-i in a manner promoting protective versus deleterious effects in different physiopathological settings. that knowledge will considerably help to develop novel strategies to modulate ifn-i functions for promoting health over disease. the development of mutant mice allowing conditional genetic inactivation of stat , stat , and stat ( - ) will help better understanding how different signaling pathways in different cell types determine the outcome of ifn-i response in vivo in various conditions. this knowledge might lead to the development of strategies aiming at targeting a given cell type with a specific subset of ifn-i, or in the presence of antagonists of certain signaling pathways, to surgically tune ifn-i responses in vivo toward the most desirable outcome. frontiers in immunology | microbial immunology for example, the affinity of ifn-β for ifnar is -times higher than that of ifn-α , and ifnβ is much more potent in inhibiting cellular proliferation or (continued ) ifn-i can also determine distinct functional outcomes. for example, during viral infections, early and transient high levels of ifn-i promote protective dc and t cell responses, while delayed, chronic and low level ifn-i production compromises host immune defenses and promotes chronic viral infections. within a given cell type, the outcome of ifn-i stimulation also depends on time of exposure to these cytokines relative to other modulatory signals (timing relative to other stimuli). for example, in naïve cd t cells, tcr signaling prior to ifn-i stimulation leads to increased expression of stat and promotes ifn-γ production and proliferation, while ifn-i stimulation prior to tcr triggering leads to stat -dependent anti-proliferative and pro-apoptotic effects. the formation of specific stat complexes is a highly dynamic process. it depends not only on the cell type but also on its specific state at the time it sees ifn-i. hence, major parameters controlling the effects of ifn-i in a given cell type also include its microenvironment ( figure c ) and the timing of its exposure to the cytokines both in terms of duration of the stimulation and of previous activation history ( figure d) . the tam receptor ligand gas is expressed within tumor cells in various solid cancers ( , ) . elevated gas expression is of bad prognosis in different cancers ( , ) . in a mouse model of ovarian cancer, early during tumorigenesis tumor-infiltrating dcs were found to be immunogenic and promote antitumor immunity, but they were later altered in the course of tumor development to acquire immunosuppressive properties beneficial to the tumor ( ) . one may thus hypothesize that expression of tam soluble ligands in certain tumors and of tam receptors on tumor-infiltrating dcs might contribute to dampen dc response to ifn-i and therefore facilitate their polarization by the tumor microenvironment into immunosuppressive cells (figure c) . acute versus chronic exposure to ifn-i can lead to strikingly opposite effects on a given cell type ( , , ) . in addition to duration, the time when a cell is exposed to ifn-i can also dramatically impact its functional response, depending on its previous activation history ( figure d) . in vitro stimulation of dcs with ifn-β can lead to opposite outcomes depending whether it occurs simultaneously to, or after, tnfα-induced maturation. ifn-β polarizes dcs toward th induction in the former case, and toward il- -secreting t cells in the latter case. these opposite effects result at least in part from the differential expression of il- / by dcs ( ) . similarly, ifn-i effect on the functional polarization of cd t cells is strongly modulated by the other cytokines present in the lymphocyte microenvironment at the same time ( ) . ifn-i can also mediate opposite effects on cd t cells depending whether it occurs before or after cognate engagement of the t cell receptor. indeed, while cd t cells have the potential to respond to ifn-i by inducing both stat -and stat -dependent genes, this depends upon their activation history. naïve cd t cells respond mostly to ifn-i through stat signaling, leading to the inhibition of their proliferation and eventually to the induction of their apoptosis. however, cognate triggering of the t cell receptor causes a decrease in stat and an increase in stat expression in cd t cells. this leads to a shift of their ifn-i response from stat -to-stat signaling, resulting in the promotion of their proliferation and ifn-γ production. during lcmv infection, this mechanism promotes stat -dependant expansion of anti-viral cd t cells, but stat -dependant inhibition of naïve cd t cell proliferation ( ) . since the late s the clinical potential of ifn-i for the treatment of patients suffering of viral infection or cancer diseases has been widely acknowledged ( ) . today, this expectation is tempered because ifn-i treatment can induce severe side effects and sufficient doses cannot be administered in patients. therefore, there is a strong need to create tuned ifn molecules devoid of side effects. based on our current understanding of ifn-i responses as reviewed above, many parameters could be tuned individually or in a combined manner to modulate ifn-i activity to promote their beneficial effects over the deleterious ones in a number of diseases. these parameters include modifying the affinity of ifn-i for its receptor, playing with the local quantity/concentration of ifn-i and with the duration of its delivery, and modulating the nature of the cells that are responding to ifn-i. we will discuss here novel strategies being developed to deliver ifn-i to, or block ifn-i responsiveness of, a specific target cell type in vivo (figure ). if ifn-i-induced side effects are a consequence of the pleiotropic nature of ifn-i, and if the bioactivities mediating deleterious effects have some degree of independence from those mediating beneficial effects, one could mutate the ifn-i molecules in order to skew their activity toward a desired bioactivity. indeed, introducing key mutation in ifn-α allowed increasing its affinity to ifnar by a factor of . accordingly, this ifn-α mutant is times more potent in inhibiting cell proliferation, but as potent as wt ifn-α in inducing an anti-viral state ( ) ( ) ( ) . hence, it is possible to tune ifn activity by modifying its binding to ifnar. however, translating such an approach for the design of molecules for clinical application is severely hampered by the poor understanding we have on the ifn-i bioactivities mediating the side effects. furthermore, we are far from having established the list of bioactivities that could be differentially modulated by changing the stability of the ifn-i/ifnar complex. we know more about the frontiers in immunology | microbial immunology cell types that mediate beneficial versus deleterious ifn responses in various diseases. hence, we will now discuss strategies aimed at focusing ifn activity to specific cell types to promote health over disease. several strategies have been developed to specifically target ifns on tumor cells, tumor-infiltrated immune cells or infected tissues. these strategies include intra-lesional injection ( , ) , adenoviral-mediated gene transfer ( ) ( ) ( ) , engineered tumorinfiltrating monocytes ( ) , and fusion of ifns with a cleavable protecting shell ( ) . another strategy to increase cytokine accumulation within the tumor or infected tissue is antibody-mediated targeting of cytokine delivery, where a cytokine moiety is fused to an antibody directed against a specific cell surface marker (figure ) . the fusion molecule retains both antigen-binding and ifn-i bioactivities, and is enriched at the targeted site upon in vivo injection ( ) ( ) ( ) ( ) . when targeted to human cd , ifn-i inhibited the proliferation of lymphoma cells engrafted in immunodeficient mice ( ) . an ifn-i targeted to a tumor antigen can also amplify the therapeutic effect of the antibody by acting on tumor-infiltrated dcs, thus increasing antigen cross-presentation and antitumor cytotoxic t cell responses ( ) . on non-targeted cells, the antibody conjugation negatively impacts ifn-i potency, but only modestly ( , , ) (figure a) . fusion molecules generally retain full ifn-i biological activity on the cells expressing the antibody target ( figure b) . hence, this difference only leads to a modest ratio between the ifn-i specific activity measured on target and non-target cells (figure b) . such a targeting efficiency is definitely too low to reduce the toxic effect of ifn-i administration, because it will not specifically focus ifn-i activities on "beneficial cells" without stimulating "deleterious cells." the engineering of immuno-ifn-i must be improved to reach the very high targeting efficacy required to significantly diminish the treatment side effects. we recently reported an innovative strategy reaching this goal ( ) . it is based on the postulate that the antibody moiety of an immuno-ifn-i stabilizes the ifn-i/receptor-complex by avidity. it also takes into account the fact that the biological potency of an ifn-i is proportional to the stability of the ifn-i/receptor complex up to a certain threshold beyond which increasing the stability does not increase its potency ( , ) . ifn-α and ifn-β are used in most immuno-ifn-i studies. they have evolved to retain close to maximal potency. hence, their targeting by an antibody that only provides a modest gain in terms of biological potency. however, it is expected that decreasing the affinity of the ifn-i for its receptor, by introducing a mutation, would increase the targeting effect of the antibody (figures c,d) . this is indeed the case. using an ifn-i with a single point mutation that dramatically decreases its affinity for ifnar ( figure c ) allows engineering immuno-ifns that are up to -fold more potent on cells expressing the antibody target ( figure d) . the three log targeting efficiency of these novel types of immuno-ifns is found for various activities measured in vitro or in vivo when delivered in mice. if the toxic side effect experienced by the patients treated with ifn-i is due to systemic ifn-i activity, this targeting technology may find considerable clinical applications since such engineered immuno-ifns are virtually inactive while "en route" and are activated only after binding of the fused antibody to the desired target. it remains to define the useful targets according to pathologies, for example, tumor cells themselves and professional cross-presenting xcr + dcs for cancer ( , , ) , or hepatocytes for chronic hcv infection. to treat autoimmune diseases, novel therapeutics targeting ifn-i have been developed, including two ifn-α-neutralizing monoclonal antibodies currently in clinical trials (sifalimumab and rontalizumab) ( , ) . however, long-term systemic neutralization of ifn-i activity may increase susceptibility to viral infection and tumor development. alternative strategies are needed to specifically inhibit ifn-i deleterious effects in these diseases without globally compromising ifn-i anti-viral and www.frontiersin.org anti-tumoral functions. the sequential nature of the assembling of the ifn-i/receptor complex opens the possibility to design ifn-i antagonists specifically targeting the cell subsets responsible for ifn-i deleterious effects. an ifn-α carrying a single amino acid substitution that blocks the ifn-i/ifnar interaction engages ifnar in a complex, which cannot bind ifnar ( ) . since the binary ifn-i/ifnar complex is devoid of any ifn-i activity, such mutant behaves as a potent ifn-i antagonist. when linked to an antibody specific for a cell surface marker, the antagonistic activity of the mutant ifn-i should be significantly reinforced specifically on the cells expressing the target. hence, it should be possible to design and construct targeted antagonists that inhibit responsiveness to endogenous ifn-i specifically on the cell subsets on which the cytokines act to promote autoimmunity or severe side effects, leaving the other cells fully responsive. for example, in chronic hcv patients treated with peg-ifn-α, one of the most deleterious side effects is nervous depression, which might be prevented by co-administration of an ifn-i antagonist specifically targeting neurons or other cells of the central nervous system. in the last decade, several major technological breakthroughs and the generation of novel animal models have remarkably advanced our understanding of the mode of action of ifns. in vitro high throughput screening allowed systematically studying the functions of isgs by ectopic expression or knock-down. advance biophysical investigation of the interactions between ifn-i and the ifn-i receptor allowed to rigorously investigate the mechanistic basis for the differential bioactivities of ifn-i subtypes. the analyses of the responses of different cell types to ifns or to viral infection, in vitro but also in vivo in various pathologies, demonstrated that ifn-i often mediate beneficial versus deleterious roles by acting on different cell types. from integrative analysis of these data, a picture is now emerging suggesting that it will be possible to segregate protective from deleterious ifn-i effects, based (i) on their differential induction depending on ifn-i subsets or on the magnitude/timing of ifn-i production, (ii) on their conditioning in different tissues, (iii) or on their occurrence in different cell types. hence, innovative immunotherapeutic treatments are being designed to tune ifn-i activity toward desired effects in order to promote health over disease in a manner adapted to each physiopathological condition. in particular, a proof-of-concept has been made in vitro that it will be possible to target ifn-i activity on given cell types or tissues to administer to patients sufficiently high doses of the cytokine at the site of interest while limiting unwanted effects in other tissues or cell types. the next steps will be to demonstrate efficacy of this strategy in vivo in preclinical animal models. importantly, to foster the development of these innovative immunotherapies, major efforts are still warranted to continue delineating which cell types are mainly responsible for the protective versus deleterious effects of ifn-i in different diseases. combining high throughput technologies and systems biology approaches will also advance our understanding of the molecular mechanisms dynamically controlling ifn-i responses in health and diseases, which should reveal potentially novel therapeutic targets. virus interference. i. the interferon virus interference. ii. some properties of interferon functional role of type i and type ii interferons in antiviral defense inborn errors of interferon (ifn)-mediated immunity in humans: insights into the respective roles of ifn-alpha/beta, ifn-gamma, and ifn-lambda in host defense suppressor of cytokine signaling regulates the immune response to infection by a unique inhibition of type i interferon activity new insights into cancer immunoediting and its three component phases 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cell antigen (bdca )(+) dendritic cells are a potent producer of interferon-lambda in response to hepatitis c virus antiviral activity of human oasl protein is mediated by enhancing signaling of the rig-i rna sensor activation of ifn-&# ; expression by a viral mrna through rnase l and mda rnase l releases a small rna from hcv rna that refolds into a potent pamp protein kinase r contributes to immunity against specific viruses by regulating interferon mrna integrity tam receptors are pleiotropic inhibitors of the innate immune response alveolar macrophages are the primary interferon-alpha producer in pulmonary infection with rna viruses differential type i interferon induction by respiratory syncytial virus and influenza a virus in vivo crosstalk between components of the innate immune system: promoting anti-microbial defenses and avoiding immunopathologies plasmacytoid dendritic cells and toll-like receptor -dependent signalling promote efficient protection of mice against highly virulent influenza a virus plasmacytoid dendritic cells are dispensable during primary influenza virus infection clearance of influenza virus from the lung depends on migratory langerin+cd b− but not plasmacytoid dendritic cells human tlr- -, - -, and - -mediated induction of ifn-alpha/beta and -lambda is irak- dependent and redundant for protective immunity to viruses pyogenic bacterial infections in humans with myd deficiency impaired response to interferon-alpha/beta and lethal viral disease in human stat deficiency herpes simplex virus encephalitis in a patient with complete tlr deficiency: tlr is otherwise redundant in protective immunity herpes simplex encephalitis in children with autosomal recessive and dominant trif deficiency evolutionary dynamics of human toll-like receptors and their different contributions to host defense type interferons and the virus-host relationship: a lesson in detente subcapsular sinus macrophages prevent cns invasion on peripheral infection with a neurotropic virus enforced viral replication activates adaptive immunity and is essential for the control of a cytopathic virus interferon-stimulated genes: a complex web of host defenses protein kinase pkr and rna adenosine deaminase adar : new roles for old players as modulators of the interferon response dna deamination mediates innate immunity to retroviral infection samhd host restriction factor: a link with innate immune sensing of retrovirus infection a diverse range of gene products are effectors of the type i interferon antiviral response the transcription factor stat- couples macrophage synthesis of -hydroxycholesterol to the interferon antiviral response reciprocal inhibition between intracellular antiviral signaling and the rnai machinery in mammalian cells plasmacytoid, conventional, and monocyte-derived dendritic cells undergo a profound and convergent genetic reprogramming during their maturation crosspriming of cd + t cells stimulated by virus-induced type i interferon type i interferon is selectively required by dendritic cells for immune rejection of tumors host type i ifn signals are required for antitumor cd + t cell responses through cd {alpha}+ dendritic cells dendritic cells require a systemic type i interferon response to mature and induce cd + th immunity with poly ic as adjuvant direct type i ifn but not mda /tlr activation of dendritic cells is required for maturation and metabolic shift to glycolysis after poly ic stimulation type i interferons potently enhance humoral immunity and can promote isotype switching by stimulating dendritic cells in vivo johansson-lindbom b. type i interferon signaling in dendritic cells stimulates the development of lymph-noderesident t follicular helper cells differential responses of immune cells to type i interferon contribute to host resistance to viral infection a type i interferon autocrine-paracrine loop is involved in toll-like receptorinduced interleukin- p secretion by dendritic cells tlrdriven early glycolytic reprogramming via the kinases tbk -ikkvarepsilon supports the anabolic demands of dendritic cell activation type i ifn-mediated protection of macrophages and dendritic cells secures control of murine coronavirus infection direct action of type i ifn on nk cells is required for their activation in response to vaccinia viral infection in vivo dendritic cells prime natural killer cells by trans-presenting interleukin cutting edge: the direct action of type i ifn on cd t cells is critical for sustaining clonal expansion in response to a viral but not a bacterial infection type i interferons act directly on cd t cells to allow clonal expansion and memory formation in response to viral infection direct stimulation of t cells by type i ifn enhances the cd + t cell response during cross-priming cutting edge: enhancement of antibody responses through direct stimulation of b and t cells by type i ifn cd t cells specific for lymphocytic choriomeningitis virus require type i ifn receptor for clonal expansion innate inflammatory signals induced by various pathogens differentially dictate the ifn-i dependence of cd t cells for clonal expansion and memory formation concomitant type i ifn receptor-triggering of t cells and of dc is required to promote maximal modified vaccinia virus ankara-induced t-cell expansion antiviral immune responses: triggers of or triggered by autoimmunity? is chronic plaque psoriasis triggered by microbiota in the skin? genetic dysbiosis: the role of microbial insults in chronic inflammatory diseases the tlr-mediated response of plasmacytoid dendritic cells is positively regulated by estradiol in vivo through cell-intrinsic estrogen receptor alpha signaling accumulation of peripheral autoreactive b cells in the absence of functional human regulatory t cells cvid-associated taci mutations affect autoreactive b cell selection and activation human lupus autoantibody-dna complexes activate dcs through cooperation of cd and tlr ifn priming is necessary but not sufficient to turn on a migratory dendritic cell program in lupus monocytes plasmacytoid dendritic cells sense self-dna coupled with antimicrobial peptide the pathology of influenza virus infections how do viral infections predispose patients to bacterial infections? predominant role of bacterial pneumonia as a cause of death in pandemic influenza: implications for pandemic influenza preparedness pathology of human influenza revisited immune dysfunction and bacterial coinfections following influenza type i interferon: friend or foe? a role for ifn-alpha beta in virus infection-induced sensitization to endotoxin type i ifns mediate development of postinfluenza bacterial pneumonia in mice synergistic stimulation of type i interferons during influenza virus coinfection promotes streptococcus pneumoniae colonization in mice intranasal poly-ic treatment exacerbates tuberculosis in mice through the pulmonary recruitment of a pathogen-permissive monocyte/macrophage population innate and adaptive interferons suppress il- alpha and il- beta production by distinct pulmonary myeloid subsets during mycobacterium tuberculosis infection host-directed therapy of tuberculosis based on interleukin- and type i interferon crosstalk inflammation. -hydroxycholesterol suppresses interleukin- -driven inflammation downstream of type i interferon dendritic cells, monocytes and macrophages: a unified nomenclature based on ontogeny cytomegalovirus impairs antiviral cd + t cell immunity by recruiting inflammatory monocytes tnf/inos-producing dendritic cells are the necessary evil of lethal influenza virus infection influenza a inhibits th -mediated host defense against bacterial pneumonia in mice influenza a virus exacerbates staphylococcus aureus pneumonia in mice by attenuating antimicrobial peptide production role of tissue protection in lethal respiratory viral-bacterial coinfection disease tolerance as a defense strategy gainof-function human stat mutations impair il- immunity and underlie chronic mucocutaneous candidiasis type i interferon inhibits interleukin- production and inflammasome activation type i interferons promote fatal immunopathology by regulating inflammatory monocytes and neutrophils during candida infections ifnalpha/beta signaling is required for polarization of cytokine responses toward a protective type pattern during experimental cryptococcosis interferonbeta production via dectin- -syk-irf signaling in dendritic cells is crucial for immunity to c. albicans aberrant type i interferon regulation in autoimmunity: opposite directions in ms and sle, shaped by evolution and body ecology janus-like effects of type i interferon in autoimmune diseases coregulation of cd + t cell exhaustion by multiple inhibitory receptors during chronic viral infection type i interferon suppresses de novo virus-specific cd th immunity during an established persistent viral infection timing and magnitude of type i interferon responses by distinct sensors impact cd t cell exhaustion and chronic viral infection distinct transcriptional profiles in ex vivo cd + and cd + t cells are established early in human immunodeficiency virus type infection and are characterized by a chronic interferon response as well as extensive transcriptional changes in cd + t cells chronic cd + t-cell activation and depletion in human immunodeficiency virus type infection: type i interferon-mediated disruption of t-cell dynamics host genes associated with hiv- replication in lymphatic tissue global genomic analysis reveals rapid control of a robust innate response in siv-infected sooty mangabeys nonpathogenic siv infection of african green monkeys induces a strong but rapidly controlled type i ifn response downregulation of robust acute type i interferon responses distinguishes nonpathogenic simian immunodeficiency virus (siv) infection of natural hosts from pathogenic siv infection of rhesus macaques comparative transcriptomics of extreme phenotypes of human hiv- infection and siv infection in sooty mangabey and rhesus macaque hiv turns plasmacytoid dendritic cells (pdc) into trail-expressing killer pdc and down-regulates hiv coreceptors by toll-like receptor -induced ifn-alpha plasmacytoid dendritic cells express trail and induce cd + t-cell apoptosis in hiv- viremic patients sex differences in the toll-like receptor-mediated response of plasmacytoid dendritic cells to hiv- glycerol monolaurate prevents mucosal siv transmission type i interferon responses in rhesus macaques prevent siv infection and slow disease progression plasmacytoid dendritic cells suppress hiv- replication but contribute to hiv- induced immunopathogenesis in humanized mice chronic hepatitis c: future treatment host-targeting agents in the treatment of hepatitis c: a beginning and an end? the interferons: years after their discovery, there is much more to learn immune responses to hcv and other hepatitis viruses interferon lambda alleles predict innate antiviral immune responses and hepatitis c virus permissiveness altered interferon-alpha-signaling in natural killer cells from patients with chronic hepatitis c virus infection natural killer cells are polarized toward cytotoxicity in chronic hepatitis c in an interferon-alfa-dependent manner coexpression of pd- , b , cd and klrg on exhausted hcv-specific cd + t cells is linked to antigen recognition and t cell differentiation evidence for an antagonist form of the chemokine cxcl in patients chronically infected with hcv monocyte activation by interferon alpha is associated with failure to achieve a sustained virologic response after treatment for hepatitis c virus infection genetic variation in il b and spontaneous clearance of hepatitis c virus genetic variation in il b predicts hepatitis c treatment-induced viral clearance a variant upstream of ifnl (il b) creating a new interferon gene ifnl is associated with impaired clearance of hepatitis c virus the favorable ifnl genotype escapes mrna decay mediated by aurich elements and hepatitis c virus-induced micrornas distinct and overlapping genomic profiles and antiviral effects of interferon-lambda and -alpha on hcv-infected and noninfected hepatoma cells targeted induction of interferon-lambda in humanized chimeric mouse liver abrogates hepatotropic virus infection ifn-lambda receptor expression is induced in chronic hepatitis c and correlates with the ifn-lambda genotype and with nonresponsiveness to ifn-alpha therapies how cells respond to interferons jak-stat pathways and transcriptional activation in response to ifns and other extracellular signaling proteins regulation of type i interferon responses stat and irf : beyond isgf . jakstat ( ) single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators major differences in the responses of primary human leukocyte subsets to ifn-beta critical role for stat activation by type interferons in the interferongamma response to viral infection immunomodulatory functions of type i interferons high basal stat balanced by stat induction to control type interferon effects in natural killer cells type interferon induction of natural killer cell gamma interferon production for defense during lymphocytic choriomeningitis virus infection changing partners at the dance: variations in stat concentrations for shaping cytokine function and immune responses to viral infections dendritic-cell maturation alters intracellular signaling networks, enabling differential effects of ifn-alpha/beta on antigen cross-presentation mechanisms of type-i-and type-ii-interferon-mediated signalling type i interferon [corrected] gene induction by the interferon regulatory factor family of transcription factors complex modulation of cell type-specific signaling in response to type i interferons alternate interferon signaling pathways ifn-beta induces serine phosphorylation of stat- in ewing's sarcoma cells and mediates apoptosis via induction of irf- and activation of caspase- usp -based negative feedback control is induced by type i and type iii interferons and specifically inactivates interferon alpha response interferon-alpha and -beta differentially regulate osteoclastogenesis: role of differential induction of chemokine cxcl expression differential activity of type i interferon subtypes for dendritic cell differentiation evolutionary genetic dissection of human interferons the receptor of the type i interferon family interferons as biomarkers and effectors: lessons learned from animal models protection against progressive leishmaniasis by ifn-beta multifaceted activities of type i interferon are revealed by a receptor antagonist receptor density is key to the alpha /beta interferon differential activities structural and dynamic determinants of type i interferon receptor assembly and their functional interpretation the type i interferon receptor: structure, function, and evolution of a family business differential receptor subunit affinities of type i interferons govern differential signal activation structural linkage between ligand discrimination and receptor activation by type i interferons distinct and nonredundant in vivo functions of ifnar on myeloid cells limit autoimmunity in the central nervous system type i interferons protect t cells against nk cell attack mediated by the activating receptor ncr lymphadenopathy in a novel mouse model of bartonella-induced cat scratch disease results from lymphocyte immigration and proliferation and is regulated by interferon-alpha/ beta cytosolic rig-i-like helicases act as negative regulators of sterile inflammation in the cns expression of type i interferon by splenic macrophages suppresses adaptive immunity during sepsis myeloid type i interferon signaling promotes atherosclerosis by stimulating macrophage recruitment to lesions stat activation is responsible for il- -dependent t cell proliferation through preventing apoptosis: generation and characterization of t cell-specific stat -deficient mice generation of mice with a conditional stat null allele conditional stat ablation reveals the importance of interferon signaling for immunity to listeria monocytogenes infection loss of stat from mouse mammary epithelium results in an increased neu-induced tumor burden inactivation of stat in mouse mammary epithelium during pregnancy reveals distinct functions in cell proliferation, survival, and differentiation meta-analysis of microarray data identifies gas expression as an independent predictor of poor survival in ovarian cancer axl and growth arrest-specific gene are frequently overexpressed in human gliomas and predict poor prognosis in patients with glioblastoma multiforme gas expression identifies high-risk adult aml patients: potential implications for therapy ovarian cancer progression is controlled by phenotypic changes in dendritic cells ifnalpha activates dormant haematopoietic stem cells in vivo timing of ifn-beta exposure during human dendritic cell maturation and naive th cell stimulation has contrasting effects on th subset generation: a role for ifn-beta-mediated regulation of il- family cytokines and il- in naive th cell differentiation combinatorial flexibility of cytokine function during human t helper cell differentiation regulating type ifn effects in cd t cells during viral infections: changing stat and stat expression for function interferons at age : past, current and future impact on biomedicine inquiring into the differential action of interferons (ifns): an ifn-alpha mutant with enhanced affinity to ifnar is functionally similar to ifn-beta an interferon alpha mutant optimized by phage display for ifnar binding confers specifically enhanced antitumor activities the stability of the ternary interferon-receptor complex rather than the affinity to the individual subunits dictates differential biological activities intra-lesional low-dose interferon alpha a therapy for primary cutaneous marginal zone b-cell lymphoma intratumoral injection of interferon-alpha and systemic delivery of agonist anti-cd monoclonal antibodies synergize for immunotherapy delivery of interferon alpha using a novel cox -controlled adenovirus for pancreatic cancer therapy the efficacy of radiotherapy relies upon induction of type i interferondependent innate and adaptive immunity a trial of intrapleural adenoviral-mediated interferon-alpha b gene transfer for malignant pleural mesothelioma tumortargeted interferon-alpha delivery by tie -expressing monocytes inhibits tumor growth and metastasis targeting cytokines to inflammation sites livertargeting of interferon-alpha with tissue-specific domain antibodies antibody-based targeting of interferon-alpha to the tumor neovasculature: a critical evaluation targeting ifn-alpha to b cell lymphoma by a tumor-specific antibody elicits potent antitumor activities targeting the tumor microenvironment with interferon-beta bridges innate and adaptive immune responses targeted delivery of interferon-alpha via fusion to anti-cd results in potent antitumor activity against b-cell lymphoma targeted delivery of interferon-alpha to hepatitis b virus-infected cells using t-cell receptor-like antibodies variations in the unstructured c-terminal tail of interferons contribute to differential receptor binding and biological activity safety and pharmacodynamics of rontalizumab in patients with systemic lupus erythematosus: results of a phase i, placebo-controlled, double-blind, dose-escalation study safety profile and clinical activity of sifalimumab, a fully human anti-interferon alpha monoclonal antibody, in systemic lupus erythematosus: a phase i, multicentre, double-blind randomised study mutation of the ifnar- receptor binding site of human ifn-alpha generates type i ifn competitive antagonists the studies performed in the laboratories are supported by funding from inserm, cnrs, aix-marseille university, the labex mabimprove, and the european community's seventh framework programme fp / - (grant agreement for gilles uzé, european research council starting grant agreement number for marc dalod including salary support to emeline pollet and thien-phong vu manh). we thank past and present laboratory members for their contribution to studies on dcs or ifns. we apologize for not quoting certain studies because of space limitations. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord- -viy y authors: burrack, kristina s.; morrison, thomas e. title: the role of myeloid cell activation and arginine metabolism in the pathogenesis of virus-induced diseases date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: viy y when an antiviral immune response is generated, a balance must be reached between two opposing pathways: the production of proinflammatory and cytotoxic effectors that drive a robust antiviral immune response to control the infection and regulators that function to limit or blunt an excessive immune response to minimize immune-mediated pathology and repair tissue damage. myeloid cells, including monocytes and macrophages, play an important role in this balance, particularly through the activities of the arginine-hydrolyzing enzymes nitric oxide synthase (nos ; inos) and arginase (arg ). nitric oxide (no) production by inos is an important proinflammatory mediator, whereas arg -expressing macrophages contribute to the resolution of inflammation and wound repair. in the context of viral infections, expression of these enzymes can result in a variety of outcomes for the host. no has direct antiviral properties against some viruses, whereas during other virus infections no can mediate immunopathology and/or inhibit the antiviral immune response to promote chronic infection. arg activity not only has important wound healing functions but can also inhibit the antiviral immune response during some viral infections. thus, depending on the specific virus and the tissue(s) involved, the activity of both of these arginine-hydrolyzing enzymes can either exacerbate or limit the severity of virus-induced disease. in this review, we will discuss a variety of viral infections, including hiv, sars-cov, lcmv, hcv, rsv, and others, where myeloid cells influence the control and clearance of the virus from the host, as well as the severity and resolution of tissue damage, via the activities of inos and/or arg . clearly, monocyte/macrophage activation and arginine metabolism will continue to be important areas of investigation in the context of viral infections. tissue -resident and monocyte-derived macrophages are innate immune cells that play a key role in normal tissue homeostasis, presentation of foreign and self antigens following infection or injury, pathogen clearance, and resolution of inflammation and wound healing. depending on the microenvironment, macrophages can be programed to adopt a variety of proinflammatory, regulatory, resolving, and immunosuppressive activation phenotypes, particularly in vivo. these activation states exist as a complex continuum of overlapping phenotypes; however, macrophage subsets with distinct functions have been defined ( ) . macrophages are considered m -polarized when stimulated by ifn-γ or toll-like receptor (tlr) ligands, such as lipopolysaccharide (lps), to express inducible nitric oxide synthase (inos; nos ) and produce nitric oxide (no). nos enzymes metabolize l-arginine to citrulline and no. no is a short-lived gaseous messenger with physiological and pathological effects. nanomolar concentrations of no, generated by endothelial nos and neuronal nos, are important for maintaining homeostasis, regulating vasodilation, and for the aggregation, recruitment, and adhesion of platelets to the vascular endothelium. inos generates micromolar levels of no that modulates various pathophysiological processes and is important for killing intracellular pathogens ( ) . in contrast, m -polarized macrophages result following stimulation of cells with a variety of stimuli, including type cytokines such as interleukin (il)- or il- . m -polarized macrophages express a distinct l-arginine-metabolizing enzyme, arginase (arg ), which hydrolyzes l-arginine to l-ornithine and urea. l-ornithine can be further metabolized to polyamines, which participate in a variety of fundamental cellular functions (e.g., proliferation, cell membrane transport), and l-proline, which is an essential component of collagen. in addition to playing important roles in defense against extracellular parasites and tissue repair, arg expression and activity in myeloid cells have emerged as a major regulator of innate and adaptive immune responses ( ) . other m -like suppressive or anti-inflammatory macrophages include myeloid-derived suppressor cells (mdscs) and tumor-associated macrophages (tams). mdscs are considered to be an immature population of myeloid cells, including both monocyte-like (gr- /ly- c + ) and neutrophil-like (gr- /ly- g + ) populations, associated with tumors or infections that suppress proinflammatory responses ( , ) . depending on the context, mdscs have been shown to mediate their suppressive activity via no-and/or arg -dependent mechanisms. importantly, macrophages are not permanently programed, but are considered "plastic" -that is, macrophages have been shown to change activation phenotypes depending on the local environment. although the m /inos and m /arg division is generally appropriate, arg can be induced in m -like macrophages under certain conditions. thus, due to the spectrum of activation states for macrophages, a framework for macrophage-activation nomenclature was recently suggested ( ) . in an attempt to avoid confusion in this review, we focused on the specific effects of the l-arginine metabolizing enzymes inos or arg on the pathogenesis of viral infections, noting other activation markers where appropriate. increasing evidence suggests that myeloid cell programing, inos, and arg contribute to the pathogenesis of numerous virus infections, suggesting that therapies that target these cells and pathways may be beneficial for the treatment of some virus diseases. in this review, we highlight recent studies of viral infections where myeloid cell polarization -resulting in expression of inos or arg -contribute to viral control or the development of chronic virus infection and mediate the resolution of tissue damage or cause immunopathology. no has antimicrobial activity against a number of bacteria, parasites, and fungi ( , ) . additionally, no has been shown to have direct antiviral effects in vitro and/or in vivo against several viruses, including dna viruses such as herpes simplex virus type- (hsv- ), ectromelia virus (ev), and vaccinia virus (vv) ( , ), as well as some rna viruses such as vesicular stomatitis virus (vsv) ( ), japanese encephalitis virus (jev) ( ) , dengue virus (denv) ( ) , and coxsackievirus ( table ) ( ) ( ) ( ) ( ) . there are several advantages of using no as an antiviral agent. for instance, unlike complement and antibody, no can readily pass through cellular membranes into neighboring cells as well as some viruses. additionally, no is likely to act on a variety of both viral and virally exploited cellular targets, inhibiting viral replication as well as limiting the capacity of viruses to develop resistance. lastly, the effect of no is independent of immune recognition of the infected cell, in contrast to that of antiviral lymphocytes, which could be important in virusinfected cells where expression of mhc class i molecules may be downregulated and in some virally infected tissues such as the brain where expression of mhc class i and ii molecules is limited. in initial studies in vitro, inhibition of ev, vv, and hsv- replication in mouse raw . macrophages and in primary mouse macrophages following ifn-γ treatment was shown to be largely dependent on no production ( , ) . additionally, pharmacologic inhibition of nos or genetic deletion of nos resulted in increased viral titers and mortality following ev infection in mice ( , ) . moreover, no affects several events in the late stages of the life cycle of vv, including viral dna replication, viral protein synthesis, and virion maturation in vitro ( ) . these studies provided some of the first evidence that macrophage-produced no has direct antiviral effects. in addition to inhibiting hsv- replication in vitro, macrophage-derived no has been shown to have anti-hsv properties in vivo. in a mouse model of hsv- -mediated corneal disease, inos was highly induced in the trigeminal ganglion (tg) of hsv- -infected mice, and its expression was markedly reduced in mice depleted of macrophages ( ) . depletion of macrophages prior to hsv- infection resulted in markedly reduced inos expression and higher viral loads in the tg of infected mice ( , ) , suggesting that macrophages were the main source of inos expression in the affected tissues following hsv- infection and that no had important anti-hsv- properties in vivo. consistent with these data, inhibition of nos activity resulted in increased viral loads in the tg ( ) . additional studies showed that f / + gr- + inflammatory monocytes were recruited to the eye via an ifn-α-driven ccl gradient and restricted hsv- replication in that tissue via no production ( ) . it was further shown that no production by f / + macrophages in the brains of hsv- -infected mice blocked viral replication in a partially tlr -and tlr -dependent mechanism ( ) . finally, following footpad inoculation, hsv- -infected nos −/− mice displayed a delayed clearance of virus from the dorsal root ganglia (drg) and exhibited an increase in the frequency of virus reactivation in drg ( ) . the reactivity of no and its higher oxides and nitrosothiol products ( ) makes it likely that a variety of molecular targets are involved in its antiviral action. it has been shown that no can inhibit ribonucleotide reductase ( , ) , a rate-limiting enzyme in dna synthesis, and no can lead to the deamination of mammalian and bacterial dna ( , ) , which may be important antiviral mechanisms. indeed, hsv- encodes its own ribonucleotide reductase and although it is not required for hsv- replication in vitro, it is necessary under conditions where the intracellular pool of deoxynucleotides is limited ( , ) . thus, by inactivating this cellular and/or viral enzyme, no may halt virus replication by directly inhibiting viral dna synthesis. in addition to hsv- , treatment of primary human cells with an no donor following infection with human cytomegalovirus (hcmv), a beta-herpesvirus, resulted in a significant reduction of early and late viral protein expression ( ) . consistent with these in vitro data, nos −/− mice ( /sv/ev x c bl/ f ) exhibited increased viral titers and mortality following infection with murine cmv (mcmv; smith vr strain) ( ) . nitric oxide has also been shown to have antiviral properties on a chicken herpesvirus, marek's disease virus (mdv), which can cause t cell lymphomas in chickens: addition of no-generating compounds inhibited viral replication in chicken fibroblasts ( ) . additionally, the treatment of chickens with an inhibitor of inos increased the level of mdv replication in vivo ( ) . further studies demonstrated that no production was limited to chickens that were genetically resistant to tumor development following mdv infection or to chickens that were vaccinated before being inoculated with mdv ( ) . thus, no appeared to be produced in both types of resistance to tumor development in marek's disease, either acquired after vaccination or genetic. together, these findings suggest a role of no in the protective immune mechanisms against marek's disease, possibly through its activity on viral replication. finally, studies with hbv, a hepadnavirus associated with acute and chronic hepatitis, demonstrated that hbv replicated to higher levels in the livers of hbv-transgenic nos −/− mice than control transgenic mice, and transgenic nos −/− mice had increased frontiers in immunology | antigen presenting cell biology liver disease ( ) . it was further demonstrated that no production by mononuclear cells, most likely macrophages, in the liver mediated most of the antiviral activity resulting from ifn-γ production by virus-specific t cells ( ) , suggesting an antiviral role for macrophage-derived no following hbv infection in mice. in addition to dna viruses, macrophage-derived no also exerts antiviral effects against a number of rna viruses. inhibition of jev, a mosquito-transmitted flavivirus that causes encephalitis in humans, in ifn-γ-activated raw . macrophages in vitro correlated with no production, and ifn-γ-activated raw . macrophage-mediated inhibition of jev replication in murine neuroblastoma n cells was no-dependent ( ) . moreover, inhibition of nos activity led to increased mortality in jev-infected mice ( ) . in terms of its mechanism of action, no was found to inhibit jev rna synthesis, viral protein accumulation, and virus release from infected cells in vitro ( ) . these data suggest that no may be directly or indirectly inhibiting viral enzymes and/or other www.frontiersin.org cellular components required for viral replication, and this may subsequently block viral protein synthesis. additionally, no may interfere with the release and/or maturation of virions. monocyte/macrophage-derived no may also block replication of denv, another mosquito-transmitted flavivirus. infection with denv resulted in increased levels of no in patients with dengue fever, the classic form of the disease ( ) . additionally, inos expression was induced in cd + monocytes from a subset of acutely infected individuals ( ) . it was further shown that ex vivo infection of human monocytes with denv- resulted in increased inos expression, and inhibition of inos activity led to increased denv antigen detection in these cells ( ) . moreover, treatment of c / mosquito cells with an no donor resulted in reduced denv-positive cells ( ) . these data suggest that denv replication is susceptible to no-mediated inhibition. consistent with this, nos −/− mice were shown to be more susceptible to denv infection, resulting in more severe disease and increased lethality in mouse models of denv- and denv- infection ( , ) . it was further demonstrated that, following denv infection in vivo, il- and il- induced ifn-γ production, resulting in inos expression and no production, which contributed to viral control ( , ) . in addition to monocyte/macrophage-derived no, a recent study demonstrated that platelets isolated from patients with dengue fever had increased l-arginine transport and increased no production compared to platelets from healthy controls ( ) . however, no has anti-aggregatory properties, and mendes-ribeiro et al. ( ) found that dengue patients exhibited decreased collagen-induced platelet aggregation, consistent with the vascular leak and hemorrhagic manifestations of dengue hemorrhagic fever/dengue shock syndrome (dhf/dss), thus establishing an association between reduced platelet aggregation, enhancement of the l-arginine-no pathway, and dhf/dss ( ) . in contrast, getts and colleagues showed that experimentally abrogating no activity during west nile virus (wnv) encephalitis, a related flavivirus, in no-competent mice at a specific, relatively late time point prolonged survival of infected mice, while pharmacological inactivation throughout disease did not ( ) . combined, these data suggest that although during denv infection ifn-γ-induced no production has a role in antiviral defense, it is likely that dysregulation of the il- / -ifn-γ-no axis leads to immune-mediated damage in certain flavivirus infections. along these lines, it has also been shown that treatment of mice with a nos inhibitor increased mortality rates following sindbis virus (sinv) infection ( ) , suggesting a protective role for no during this particular cns infection. however, sinv replication in the brain was unaffected. furthermore, treatment of neuroblastoma cells with no donors had little effect on sinv replication but increased cell viability ( ) . these data suggest that no protects mice from fatal sinv-induced encephalitis by a distinct mechanism that does not directly involve the inhibition of virus growth but rather may enhance survival of the infected neuron until the immune response can control virus replication. nitric oxide also plays an antiviral role during cns infection with reovirus. infection of neonatal mice with the prototypic neurotropic reovirus strain (t a) induced inos expression in brain areas demonstrating reovirus antigen expression and associated virus-induced injury ( ) . reovirus also induced inos expression following in vitro infection of primary neuronal and glial cultures. reovirus was shown to infect a subpopulation of microglial cells in vitro ( ) , suggesting that direct virus interaction may induce inos in this specialized population of macrophages. treatment of neuronal cultures with an no donor inhibited viral replication whereas a nos inhibitor increased viral growth ( ) , suggesting inos has the potential to exert antiviral activity in vivo. finally, coxsackievirus infection has been shown to induce expression of inos in macrophages infiltrating the hearts of infected mice ( ) . treatment of wt mice with a nos inhibitor and infection of nos −/− mice resulted in more severe coxsackievirus-induced pancreatitis and myocarditis, elevated viral loads in tissues, and decreased survival compared to wt mice following coxsackievirus b (cvb ) infection ( , , ) . similarly, nos −/− mice infected with coxsackievirus b exhibited decreased survival and delayed viral clearance compared to wt mice ( ) . these data suggest an antiviral effect of no against coxsackievirus infection. consistent with this, it was demonstrated that no inhibits the a and c proteinases of cvb in vitro ( ) . additionally, cvb -infected outbred mice showed significantly reduced signs of myocarditis after treatment with no donors ( ) . despite its protective capacity during some viral infections, no can also contribute to immunopathology. the pathological effects of no are likely due, at least in part, to oxidative damage caused by the interaction of no with oxygen radicals such as the superoxide anion radical o − and hydrogen peroxide (h o ). for example, although addition of an no donor to virusinfected mdck cells reduced influenza a and b viral burden in vitro ( ) , treatment of mice with inhaled no (ino) did not decrease the viral load of influenza a (mouse-adapted h n strain)-infected mice; in fact, prophylactic treatment with ino resulted in enhanced weight loss and decreased survival following infection ( ) , suggesting a pathogenic role for no. consistent with this, chickens, which show a high level of mortality and associated pathology following avian influenza infection, had higher levels of inos expression in the lungs compared with h n influenza-infected ducks, which show relatively minor symptoms following influenza infection ( ) . additionally, akaike and colleagues ( ) found evidence of the production of peroxynitrite, which is generated through the reaction of no and o -, in the lungs of influenza a (mouse-adapted h n strain)-infected mice. moreover, inhibition of nos resulted in enhanced survival and decreased pneumonia, but not decreased viral loads, in influenzainfected mice ( , ) , suggesting that no was contributing to pathogenesis rather than having direct antiviral effects. nos −/− mice also survived a lethal dose of influenza a virus (pr/ / strain) infection with little histopathologic evidence of pneumonitis; however, in these studies no infectious virus was detected in nos −/− mice at day after infection ( ) . the enhanced viral control in nos −/− mice was shown to require the activity of ifn-γ ( ), with nos −/− mice also producing increased virusspecific igg a antibody titers ( ) . additionally, genetic deletion frontiers in immunology | antigen presenting cell biology of nos or pharmacologic inhibition of nos enhanced survival of mice inoculated with the highly pathogenic (non-mouse-adapted) influenza virus strain, although mice exhibited similar viral loads to control mice in lung tissue at the peak of viral replication ( ) . influenza infection in vitro was shown to induce apoptosis, and a reduction in influenza-mediated apoptosis was noted in cells treated with a nos inhibitor ( ) . similarly, fewer apoptotic cells were found in the lungs of influenza-infected nos −/− mice, suggesting that no mediates cell death following influenza infection ( ) . the cellular source of inos/no following influenza infection in mice was shown to be ccr + inflammatory monocytes that accumulate in the lungs: ccr −/− mice survived a lethal challenge of influenza infection (pr/ / strain) and had significantly reduced accumulation of inos-expressing macrophages in the lung, with no associated increase in viral titers or dissemination ( ) . it was also recently shown that a subset of monocyte-derived dendritic cells (dcs), described as tnf-α/inos-producing dcs (tipdcs), accumulate in greater numbers during the course of lethal versus sublethal influenza infections, suggesting a pathogenic role for this subpopulation of myeloid cells ( ) . interestingly, though, aldridge et al. ( ) found that the tipdcs also stimulated a local, protective cd + t cell response in the virusinfected respiratory tract, indicating both protective and pathogenic roles for these cells in influenza infection. it was further shown that partially compromising tipdc recruitment via treatment with pioglitazone, a synthetic agonist of the peroxisome proliferator-activated receptor-γ (ppar-γ), was protective against lethal influenza challenge ( ). pioglitazone treatment led to a reduction in the levels of ccl (mcp- ) and mcp- in the bal fluid of influenza-infected mice ( ) . pioglitazone has also been shown to reduce the production of a wide range of proinflammatory molecules, including inos ( ), providing further evidence for the importance of no production by monocyte-derived cells in the pathogenesis of influenza infection. pharmacologic inhibition of nos using l-nmma also decreased pneumonitis and increased survival following intranasal infection of cba/j mice with hsv- , despite a -fold increase in viral titers in the lung at day after inoculation ( ). in contrast, treatment of balb/c mice with a different nos inhibitor [aminoguanidine (ag), administered intranasally] resulted in enhanced pneumonitis, viral titers, and mortality following infection with a different strain of hsv- ( ) . thus, the precise role of no in hsv- pneumonitis remains to be determined. no and other ros/rns were also shown to be pathogenic in the brains of mice with herpes encephalitis: inos was induced in cd b + resident microglia following intranasal infection with hsv- , and oxidative and nitrative damage was found in the brains of infected animals ( ) . a common neurological complication of hiv infection in the developed world is sensory neuronal injury accompanied by inflammation, which is clinically manifested as disabling pain and gait instability. feline immunodeficiency virus (fiv) infection of cats, which causes similar neuroinflammation together with immunosuppression in cats, resulted in induction of inos and stat- , which were predominantly produced by macrophages, in drg ( ) . additionally, inhibition of nos resulted in reduced nitrotyrosine and prevented neuronal injury in fiv-infected drg cultures in vitro ( ) . these data suggest that lentivirus infection contributes to axonal and neuronal injury through a mechanism involving m macrophage immune activation mediated by stat- and inos activation. in addition to these studies, infection of mice with the retrovirus lp-bm , which causes profound immunodeficiency, induces cd b + gr- + ly- c + mdsc-like cells that inhibit both t-and b-cell responses in an inos/no-dependent but arginase-independent fashion ( ) . this study identified an important -and only recently appreciated -role for inosexpressing myeloid cell-mediated suppression of b cell responses in retrovirus infection. the oxidative effects of no have also been shown to inhibit immune cells, particularly t cells. this phenomenon has been appreciated for a number of years in the context of tumors ( ), where myeloid suppressor cells can inhibit the anti-tumor t cell response via the effects of no in addition to other mechanisms ( , ) . in a similar manner, it has been shown that no can inhibit the antiviral immune response. mcmv clearance from balb/c mice is predominantly cd + t cell-mediated. a recent report showed that mcmv infection in balb/c mice induced cd b + ly- c hi inflammatory monocyte recruitment from the bone marrow to infected tissues that was dependent on ccr signaling ( ) . this recruitment was shown to inhibit antigen-specific cd + t cell activation, expansion, and cytotoxic activity via no production, thus facilitating viral persistence ( ) . in a similar fashion, no may contribute to a defective immune response following infection of mice with an attenuated neurotropic coronavirus (rj . strain of mouse hepatitis virus). rj . infected wt mice exhibited mild acute encephalitis, followed by a non-lethal, chronic demyelinating disease ( ) . in marked contrast, rj . infection of mice that transgenically express ccl in the brain (ccl tg) ineffectively cleared virus and rapidly succumbed to the infection ( ). ccl tg mice mounted a dysregulated immune response, characterized by increased accumulation of inos-expressing macrophages and microglia as well as regulatory t cells, but decreased arg expression ( ) . these data suggest that persistent ccl overexpression establishes and sustains an immunological milieu that may predispose mice to a defective immune response to a typically minimally virulent virus. arginase activity is important for wound healing and tissue regeneration through the production of polyamines and proline ( ) . in the context of some viral infections, arginase activity and m macrophage activation have been shown to be beneficial for tissue repair following virus-induced damage. for instance, resolution of severe respiratory syncytial virus (rsv)-induced bronchiolitis in mice is mediated by m macrophages that counteract cyclooxygenase (cox)- -induced lung pathology ( , ) . arg was induced in the lungs of rsv-infected mice, and its induction was shown to be il- rα-dependent ( ) . additionally, wt macrophages adoptively transferred into rsv-infected il- rα −/− mice restored the www.frontiersin.org m phenotype in the lungs and decreased lung pathology ( ) . it was further shown that the lipoxogenase pathway was important for m macrophage activation and lung resolution following rsv infection ( ) . most recently it was demonstrated that treating mice with agents that sustain arg expression (e.g., il- /anti-il- immune complexes) limited rsv-induced lung pathology ( ) . consistent with a pathogenic role for inos/no following influenza infection (described above), it was recently shown that the presence of airway bacteria polarize alveolar macrophages into a m phenotype, thus limiting influenza-mediated lethal lung inflammation. wang and colleagues ( ) demonstrated that priming with staphylococcus aureus, which commonly colonizes the upper respiratory mucosa, attenuated influenzamediated lung injury via tlr signaling that recruited peripheral ccr + cd b + monocytes into the alveoli ( ) . these monocytes polarized alveolar macrophages into a m phenotype characterized by high arg as well as ym , fizz , and il- expression ( ) . it was further shown that s. aureus-primed m alveolar macrophages inhibited inflammatory cell recruitment to the lung, including neutrophils, nk cells, and cd t cells ( ) . s. aureus-primed m alveolar macrophages also expressed higher levels of the inhibitory ligand pd-l ( ) , suggesting that expression of a combination of anti-inflammatory cytokines and inhibitory ligands could be the mechanisms by which s. aureusprimed m alveolar macrophages limit influenza-mediated lung inflammation. as discussed above, coxsackievirus b (cvb ) infection causes myocarditis in human beings as well as in male balb/c mice. although female mice do not develop severe myocarditis, both male and female mice have comparable numbers of infiltrating macrophages and viral titers in the heart following cvb infection ( ) . the macrophages infiltrating the heart in male mice were skewed toward a m phenotype characterized by high expression of inos ( ) as well as m -associated cytokines such as ifn-γ and il- ( ) . additionally, inhibition of nos resulted in increased viral titers and higher mortality in cvb -infected mice ( ), consistent with an antiviral role for no during cvb infection (see above). however, in contrast to male mice, the heart-infiltrating macrophages in female mice were skewed toward a m phenotype characterized by high expression of arg as well as il- and il- ( ). moreover, adoptive transfer of ex vivo-programed m macrophages significantly increased myocarditis in both male and female mice. strikingly, transfer of m -programed macrophages into susceptible male mice alleviated myocardial inflammation by modulating the local cytokine profile from a m to m phenotype and promoting peripheral regulatory t cell (treg) differentiation ( ) . using different variants of cvb , one that caused myocarditis in c bl/ mice and one that did not, it was additionally shown that the myocarditic variant induced a m macrophage phenotype ( ) . in contrast, the amyocarditic variant induced a m macrophage phenotype, which was also associated with the activation of nkt cells that promoted a treg response ( ) . the ability of nkt cells to suppress myocarditis was shown by adoptive transfer of purified nkt cells into nkt knockout (jα knockout) mice infected with the myocarditic cvb variant, which inhibited cardiac inflammation and increased treg response ( ) . cardiac virus titers were equivalent in all mouse strains indicating that nkt cells did not participate in control of virus infection ( ) . thus, although no appears to have antiviral properties against cvb , these data indicate an important role for arg -expressing m macrophages in controlling cvb -induced myocarditis. as a consequence of their co-evolution with their hosts, viruses have developed numerous strategies to evade the host immune system and ensure their own replication and survival. recent studies have identified a new evasion strategy for viruses: exploitation of the host's anti-inflammatory, wound repair response to promote chronic infection. two strains of lcmv -armstrong (arm) and clone (c )have been studied for decades as models for acute and chronic infections ( ) . infection of mice with the arm strain leads to a robust cd + t cell response that rapidly clears the virus ( ) , whereas infection with c results in t cells with impaired functionality, enabling the virus to persist ( ) . it was recently demonstrated that c infection led to an enhanced and sustained expansion of cells that resembled mdscs ( ) . these suppressive myeloid cells inhibited t cell proliferation ex vivo via an inos/no-dependent but arg -independent mechanism. another study, however, found that arg -expressing immunoregulatory antigen presenting cells induced during c infection suppressed t cell responses ( ) . most recently, it was demonstrated that t cell responses were improved -resulting in clearance of the normally chronic c infection -when either myeloid cells or t cells lacked il- production ( ) . overall, these data demonstrate the importance of inos/arg -expressing myeloid cells in viral persistence. similar to lcmv c infection, it was recently demonstrated that infection of mice with the arthritogenic alphaviruses ross river virus (rrv) and chikungunya virus (chikv) resulted in the induction of arg in macrophages in the infected and inflamed musculoskeletal tissues ( ) . it was further shown that genetic deletion of myeloid cell arg resulted in enhanced viral control in inflamed muscle tissue and reduced tissue pathology following rrv infection in mice ( ) , suggesting an important role for arg -expressing macrophages in the persistence of these chronic viruses. infection of mice with theiler's murine encephalomyelitis virus (tmev) results in persistent virus infection in the cns, which contributes to the development of a demyelinating disease that has similarities with multiple sclerosis. bowen and olson ( ) showed that cd b + ly- c + cells infiltrated the cns following infection and were the dominant cell type during the innate immune response. depletion of the cd b + ly- c + cells via administration of an anti-gr- ab resulted in reduced development of demyelinating disease and enhanced virus-specific cd + and cd + t cell responses ( ) . additionally, tmev-infected, anti-gr- ab-treated mice had decreased myelin-specific cd + t cell responses compared to control ab-treated mice during the demyelinating disease at a later time post-infection ( ) . although the expression of arg was not investigated in this study, tmevinfected mice had elevated expression of il- in the brain and spinal cord ( ), suggesting a role for this cytokine in the suppression of antiviral t cell responses, potentially through the effects of arg . interestingly, a role for the modulation of arginine metabolism in viral control versus persistence along with associated disease has recently been demonstrated for the tumor-inducing, chickenspecific herpesvirus mdv. we mentioned above that mdv was vulnerable to the antiviral properties of no, with inos being induced in genetically resistant chickens and in vaccinated chickens ( ) . in contrast, mdv induced strong macrophage arginase activity in cell extracts from adherent monocytes from genetically susceptible chickens, but not in chickens that were resistant to marek's disease, either genetically or acquired after vaccination ( ) . together, these data suggest that in the case of marek's disease, the state of resistance versus sensitivity to disease was correlated with a reciprocal balance of nos versus arginase activities in macrophages. this phenomenon of arg -mediated t cell suppression has also been recognized in human viral infections. arg mrna and protein levels were elevated in hcv-infected liver cell lines in vitro and in hcv-infected liver samples compared with paired hepatocellular carcinoma samples from the same patients or with uninfected liver tissues ( ) . additionally, the number of mdscs in chronic hcv patients correlated with levels of plasma hcv-rna ( ). cai et al. ( ) also found that mdscs from patients with chronic hcv infection suppressed t cell function via an arg -dependent mechanism. an additional study found that more pbmcs from chronic hcv patients expressed the phenotypic markers of mdscs than pbmcs from healthy controls, and these cells expressed increased levels of p phox , a component of the nadph oxidase complex ( ) , suggesting a role for ros in mdsc-mediated suppression. consistent with this, cd + mononuclear cells co-cultured with hcv-infected hepatocytes or hcv core protein suppressed t cell proliferation in a ros-dependent manner ( ) . overall, these data suggest that multiple mechanisms -including arginine metabolism and ros -may be at play in myeloid cell-mediated suppression of anti-hcv t cell responses. it has been suggested that prolonged immune activation during chronic virus infections, such as hcv and hiv, provides an environment that drives viral replication and disease progression ( , ) . moreover, immune activation can drive an anti-inflammatory response to limit immunopathology, which can be characterized by the presence of m -like macrophages. indeed, similar to hcv infection, a role for arginase and m polarized mdsc-like cells has been identified in the suppression of antiviral t cell responses following hiv infection. individuals with detectable hiv- infection showed an increase in the frequency of cd + cd + cd + monocytes, which are thought to be precursors of m macrophages, when compared to seronegative or hiv- -infected persons with undetectable viral loads, and monocyte frequency correlated positively with hiv- viremia and negatively with cd + t cell counts (in patients with counts < cells/µl) ( ) . furthermore, qin and colleagues ( ) observed elevated levels of mdscs, defined as hla-dr −/low cd b + cd +/high cd + cd − cells, in the peripheral blood of hiv- -seropositive subjects compared with healthy controls, and these mdscs suppressed t cell responses in an arg -dependent manner. moreover, pbmcs from hivseropositive patients exhibited increased levels of arginase activity ( ) . cloke and colleagues ( ) found that increased arginase activity correlated with lower cd + t cell counts, and this association was abrogated following antiretroviral treatment ( ) . additionally, exposure of pbmcs to hiv gp expanded t cellsuppressive mdscs in vitro ( ) . these data point to a direct role for arginase-expressing mdsc-like cells in the suppression of anti-hiv t cell responses. consistent with that, individuals co-infected with hiv and leishmania parasites had increased arginase activity in pbmcs and plasma compared with leishmania-only infected individuals, even though leishmania infection alone results in increased arginase activity ( ) . in addition, the parasite load in the spleen was significantly higher in co-infected patients ( ) . the arginase-expressing cells were identified as low-density granulocytes ( ) . these results suggest that increased arginase might contribute to the poor immune responses and disease outcome characteristic of patients with leishmania and hiv co-infection. hepatitis b virus (hbv) infection is another common chronic viral infection, with estimates as high as million chronically infected humans ( ) . bility and colleagues ( ) recently developed a humanized mouse model with both a human immune system and human liver cells, named the a /nsg-hu hsc/hep humanized mouse model, to study the pathogenesis of hbv infection. following hbv infection, the mice developed persistent hbv infection as well as chronic hepatitis and liver fibrosis ( ) . the liver disease was associated with a high level of infiltrating human macrophages with a m -like activation phenotype ( ) . similarly, m -like macrophage accumulation was seen in chronic hbv-infected patients, and m -like macrophage induction in the liver was associated with accelerated liver fibrosis and necrosis in patients with acute hbv-induced liver failure ( ), suggesting a role for m macrophages in persistent hbv infection. additionally, patients with acute hbv infection had increased serum levels of arginase, and this serum inhibited ifn-γ production by cd + t cells ( ) . das et al. ( ) also found decreased l-arginine levels in the circulation of chronic hbv patients with marked liver inflammation (> alt) and increased arginase activity in liver extracts taken directly ex vivo from patients with chronic hbv compared with those from patients with other types of liver pathology ( ) . they further showed that cd + t cells from chronic hbv patients, regardless of their antigen specificity, exhibited less il- but not ifn-γ or tnf-α production and impaired proliferation following tcr-dependent stimulation, indicating an aberrant antiviral t cell response in chronic hbv infection ( ) . in the a /nsg-hu hsc/hep humanized mouse model, hbv-infected mice had impaired liver t cell responses, and m macrophages were associated with t cells in the liver ( ) . expression of the tcr signaling molecule cd ζ was reduced in both peripheral and intrahepatic cd + t cells from chronic hbv patients; similarly, cd was also downregulated on cd + t cells from high viral load hbv patients ( ) . downregulation of the cd ζ molecule has previously been shown to occur in the arginine-depleted tumor microenvironment. consistent with this, in vitro transfection of cd ζ and cd restored il- production and supplementation of l-arginine partially restored cd ζ expression and t cell proliferation ( ) . these data suggest a role www.frontiersin.org for arginase activity and arginine depletion in the impairment of anti-hbv t cells functions. in the absence of inkt cells, influenza a (pr/ strain) infection was shown to induce the expansion of cd b + gr- + mdscs in the lungs of mice, which suppressed influenza-specific t cell and antibody responses through the activity of both arginase and nos, resulting in higher viral titers and increased mortality ( ) . adoptive transfer of inkt cells reversed this phenotype; mice had an increased survival rate, reduced viral titers, and increased virusspecific immune responses, suggesting a novel immunomodulatory role for inkt cells during influenza virus infection ( ) . moreover, these authors identified that influenza infection in humans induced the expansion of cd b + myeloid cells with suppressive activity that could be reduced by inkt cell activation or the inhibition of arginase and nos activity. similarly, it was recently shown that highly pathogenic h n and h n influenza virus infection induced the accumulation of cd b + gr- + cells and the expression of arg in the lungs ( ) , further supporting a role for m -polarized mdsc-like cells in promoting viral persistence and immunopathology. helminth infection induces the expression of type cytokines and is associated with m macrophage activation, as determined by arg , fizz , and ym expression. indeed, osborne and colleagues ( ) found that arg , fizz , and ym were highly induced in the ileum of mice infected with the helminth trichinella spiralis (ts). interestingly, they further showed that co-infection of mice with ts and murine norovirus (mnv) resulted in decreased frequencies and numbers of mnv-specific cd + and cd + t cells within the small intestine and spleen as well as decreased polyfunctionality of these t cells, compared to ts-only infected mice ( ) . additionally, the defective t cell responses were associated with increased viral loads in the double-infected mice compared to the mono-infected controls ( ) , suggesting that ts-elicited m -activated macrophages inhibited the antiviral t cell response to mnv. lastly, neutralization of ym , a chitinase-like molecule, in co-infected mice partially restored antiviral immunity and was associated with enhanced control of viral replication ( ) . these data point to a new mechanism by which arg -expressing macrophages inhibit antiviral responses. cumulatively, these data are reminiscent of macrophages found in tumors (e.g., mdscs, tams) that have been shown to suppress anti-tumor t cell responses via a variety of no-and/or arg -dependent mechanisms ( , ) . indeed, in a mouse model of human papillomavirus (hpv)-induced cancer, arg -expressing cd b + f / + macrophages infiltrated the tumors and inhibited t cell responses, including virus-specific t cells, by suppressing t cell proliferation and promoting a regulatory phenotype ( ) . moreover, depletion of the tumor-infiltrating macrophages resulted in reduced tumor growth and increased tumor infiltration by virus-specific cd + t cells ( ) . thus, increasing evidence points to a direct role for arginase-expressing m -polarized cells in the suppression of antiviral t cell responses and the persistence of a variety of important pathogenic viruses. in addition to the actions of inos and arg , mdsc-like cells can employ other mechanisms to promote chronic viral infections, which were recently reviewed by goh and colleagues ( ) . in contrast to some parasitic infections where m macrophages limit th cell-mediated immunopathology, m -polarized macrophages have been shown to promote immunopathology in some viral infections. for example, it was recently demonstrated that sars-cov infection of mice induced suppressive alveolar macrophages that inhibited the induction of antiviral t cell responses, a phenotype that was reversed by the adoptive transfer of activated bone marrow-derived dcs into mice prior to virus infection ( ) . additionally, sars-cov-infected mice lacking hematopoietic stat- expression were shown to have greater weight loss and lung pathology, and this was associated with the activation of m macrophages ( ) . to further test the role of m macrophages in enhanced pathogenesis following sars-cov infection, the authors generated stat- /stat- double knockout mice due to the established role for stat- in driving m macrophage activation in response to il- /il- stimulation. stat- /stat- double knockout mice, which reversed the upregulation of m macrophages observed in stat- -deficient mice, had reduced lung disease and prefibrotic lesions ( ) . these data support the notion that m macrophages contribute to sars-cov pathogenesis. in another example, mice deficient in the ifn-γr exhibit more severe disease following infection with murine gammaherpesvirus- (mhv- ), including interstitial and intra-alveolar fibrosis that is reminiscent of idiopathic pulmonary fibrosis (ipf) in human beings. in this model, alveolar macrophages were recruited to the lungs of mhv- -infected ifn-γr −/− mice, were associated with areas of fibrosis, and exhibited a m -polarized phenotype characterized by the expression of fizz , ym , and arg ( ) . additionally, lung tissue from patients with ipf showed increased expression of arg in alveolar macrophages compared with normal lung ( ) . these results suggest that virus-induced upregulation of arg could be mediating lung fibrogenesis. mhv- infection in ifn-γr −/− mice also resulted in fibrosis in lymphoid tissues such as the spleen, which is a site of latent mhv- infection, and the liver ( , ) . similar to the lung, mhv- infection in the absence of ifn-γr signaling induced a m macrophage response in the spleen, characterized by high arg expression along with fizz and m /th cytokines such as il- , resulting in fibrotic disease in the spleen ( ) . moreover, depletion of t cells prevented mhv- -mediated fibrosis in ifn-γr −/− mice ( ) , suggesting that m macrophages were further driving th activation to possibly create a m /th cytokine-induced cycle, resulting in the exaggerated pathology. in contrast to ifn-γr −/− mice, inos was induced in the spleen of mhv- -infected wt mice ( ) , indicating an important role for ifn-γ in inducing a m -associated immune response to control gamma-herpesvirus infection and limiting arg -mediated immunopathology. macrophages and other myeloid cells have marked phenotypic heterogeneity, as a result of distinct cellular differentiation programs, distribution in tissues, and responsiveness to various endogenous and exogenous stimuli. indeed, macrophages have well-established roles in development, tissue homeostasis, coordinating the adaptive immune response and inflammation, as well as directing tissue resolution and repair following damage -processes that are often modulated via the actions of the arginine-hydrolyzing enzymes nos and arg . we have highlighted a number of viral infections in which these enzymes have a beneficial effect: no has antiviral properties against a variety of viruses, and arginase activity can mediate tissue repair and regeneration following a viral insult (table ) . however, no production can also result in immunopathology in some virus infections, and the suppressive functions of arg -expressing macrophages can promote immunopathology. additionally, some viruses have exploited the immune-suppressive properties of inos-and/or arg -expressing macrophages to evade the immune response, particularly the antiviral t cell response, resulting in chronic viral infections. clearly, inos-and/or arg -mediated responses are important in many viral infections. thus, there is the potential to develop the means to selectively stimulate or inhibit either m or m responses to mediate viral clearance or repair tissue damage. due to the overlap in immunosuppressive mechanisms of inos-and/or arg -expressing suppressor cells, therapeutic strategies under development to limit the immunosuppressive effects of myeloid cells in cancer may be beneficial in treating persistent/chronic virus infections. however, as described above, inos and arg activity can be both beneficial and detrimental during certain viral infections. therefore, further research is needed to define the molecular and tissue-specific mechanism(s) by which inos and arg influence the clearance of viral pathogens as well as the injury and repair of tissues. in addition, a better understanding of the pathways regulating macrophage polarization (specifically inos and/or arg induction and activity), macrophage trafficking, and the precise effects of inos and arg activity on other immune cells 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nitric oxide donors inhibit the coxsackievirus b proteinases a and c in vitro, virus production in cells, and signs of myocarditis in virus-infected mice molecular basis of "suppressor" macrophages. arginine metabolism via the nitric oxide synthetase pathway selection of genetic variants of lymphocytic choriomeningitis virus in spleens of persistently infected mice massive expansion of antigen-specific cd + t cells during an acute virus infection viral persistence alters cd t-cell immunodominance and tissue distribution and results in distinct stages of functional impairment macrophage and t cell produced il- promotes viral chronicity innate immune cd b+gr- + cells, suppressor cells, affect the immune response during theiler's virus-induced demyelinating disease clinical significance and functional studies of myeloid-derived suppressor cells in chronic hepatitis c patients myeloid suppressor cells induced by hepatitis c virus suppress t-cell responses through the production of reactive oxygen species macrophage polarization and hiv- infection contribution of immune activation to the pathogenesis and transmission of human immunodeficiency virus type infection hepatitis b virus infection hpv tumor associated macrophages suppress antitumor t cell responses myeloid-derived suppressor cells: the dark knight or the joker in viral infections? murine gammaherpesvirus-induced fibrosis is associated with the development of alternatively activated macrophages work in dr. morrison's laboratory is supported by nih-niaid grants u ai and r ai . kristina s. burrack was supported by nih-niaid training grant t ai . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord- -zf vl authors: mayor-ibarguren, ander; busca-arenzana, carmen; robles-marhuenda, Ángel title: a hypothesis for the possible role of zinc in the immunological pathways related to covid- infection date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: zf vl nan • zinc deficiency may be common and associated with severe infection. • zinc helps to enhance the interferon type response to the virus and participates in many regulatory pathways. • low levels of zinc have been associated with higher il- responses. • il- plays an important role in severe lung injury due to covid- infection. • zinc inhibits sars-cov rna polymerase, and thus its replication capacity. • zinc may increase the efficacy of antimalarial agents, since they are zinc ionophores. • differences in mortality due to covid- infection may be explained to some degree by− il- gene polymorphism. zinc (zn) is the second most abundant trace metal in the human body after iron. however, unlike iron, there is no specialized zinc store ( ) . zinc's functions can be classified as catalytic, structural, and regulatory ( ) . for example, important zinc metalloenzymes include alkaline phosphatase, rna polymerases, and alcohol dehydrogenase ( ) . zinc deficiency can precipitate an immune system imbalance, exemplified in severe deficiency by high susceptibility to infections, skin disorders, gastrointestinal disorders, weight loss, growth retardation and male hypogonadism, amongst other symptoms ( ) . while severe zinc deficiency is rare, mild to moderate deficiency is more common worldwide ( ) . there are very low levels of free zinc in plasma, since it is mostly bound to proteins such as albumin, alpha- -macroglobulin (a m), and transferrin. plasma zinc levels are therefore only around µg/ml, equal to . % of total body zinc, but are still the most important reservoir for zinc homeostasis, which requires "free" or "labile" zinc mobilization ( , ) . kinetic studies suggest that only a small proportion of total body zinc ( %) represents the "functional pool" of zinc, located within the liver and other tissues, that exchanges rapidly with that found in the plasma ( , ) . when this functional pool is depleted, zinc deficiency ensues ( ) . intracellular zinc is distributed in zinc-storing vesicles called zincosomes, the nucleus and other organelles. in cytoplasm, zinc mostly binds zinc-chelating proteins called metallothioneins (mts). zinc homeostasis is understood to be the correct balance of zinc distribution. internal zinc homeostasis is regulated by the cooperative activities of two metal transporter protein families. one family consists of solute-linked carrier (slc or znt) exporters, and the other family consists of solute-linked carrier (slc or zip) importers ( , ) . for instance, most labile zinc in the body is absorbed by intestinal epithelial cells via slc a protein, and excessive zinc is excreted through the kidneys, and the intestine via slc a ( ) . we are currently experiencing an unprecedented covid- pandemic caused by a novel rna coronavirus called sars-cov- , which can produce a severe acute respiratory distress syndrome (ards) ( ) . it was first detected in wuhan province in china at the end of ( ) , and on march , who characterized covid- as a pandemic ( ) . the reported mortality rate for those infected varies between countries ( . - . %) with the most important focus previously in italy and spain and currently in the usa, uk, and brazil ( ) ( ) ( ) ( ) . age, male sex, and pre-existing chronic metabolic diseases including diabetes, cardiovascular disease, and obesity are associated with greater severity of infection ( ) . there is no specific treatment yet. many agents are being used with variable success, but none have had their efficacy demonstrated in clinical trials. examples include: antimalarial agents such as chloroquine and hydroxychloroquine, antivirals such as lopinavir/ritonavir and remdesivir, and tocilizumab as an anti-interleukin (il- ) receptor antibody ( ) ( ) ( ) ( ) . remdesivir has shown good initial clinical outcomes in a clinical trial, when treatment started within days of symptom onset ( ). in contrast, in a double-blind randomized trial of patients with severe covid- (hypoxia and radiographically confirmed pneumonia) in china, time to clinical improvement was not statistically different with remdesivir compared with placebo taken for days (median time to improvement vs. days; hazard ratio for improvement . [ % ci . - . ]) ( ) . mixed and controversial results have also been published regarding antimalarial agents ( ) . thus, more robust data are needed before conclusions can be drawn regarding treatment. il- and il- may play an important role in severe lung inflammation, leading to acute respiratory distress syndrome, which can result in patient death ( , ) . this pathway appeared relevant in sars-cov, producing severe acute respiratory syndrome (sars), and in mers-cov, producing middle east respiratory syndrome (mers) ( ) . high serum levels of proinflammatory cytokines [il- , il- , il- , interferon γ (ifnγ), and transforming growth factor-β] and chemokines (ccl , cxcl , cxcl , and il- ) were found in patients with sars with severe disease compared with individuals with uncomplicated sars ( ) . mers-cov infections of dendritic cells and macrophages result in robust and sustained production of pro-inflammatory cytokines and chemokines such as tnfα, il- , cxcl- , ccl- , ccl- , ccl- , and il- ( ) . the purpose of this article is to highlight the key roles that zinc can play in covid- infection (summarized in figure ), based on pre-existing evidence of its role in immune system function and viral infections, as well as its estimated possible deficiency in at-risk populations. zinc deficiency may be present in up to % of the population worldwide. the elderly especially are at higher risk of zinc deficiency and its adverse effects ( ) . impairment of zinc homeostasis has also been demonstrated in metabolic diseases including diabetes, obesity, and cardiovascular disease ( ) . many antihypertensive drugs such as ace inhibitors, angiotensin receptor antagonists, and thiazide diuretics are zinc chelators ( ) . iron and calcium may interfere with zinc absorption too ( ) . the us food and nutrition board recommends intake of and mg/day for adult men and women, respectively ( ) . apart from calcium and iron, non-digestible plant ligands such as phytate, some dietary fibers, and lignin chelate zinc and inhibit its absorption. measurement of plasma zinc levels is the most useful clinical test for zinc deficiency, despite limited sensitivity, and specificity ( ) . also, plasma zinc levels remain stable even with low dietary intake, due to homeostasis in the body, decreasing in blood only when deficiency is very prolonged ( ) . the absence of a dedicated store for zinc repletion results in impairment of function when zinc status is compromised. homeostasis maintains a constant intracellular zinc concentration and a plasma concentration within the reference range of - µm ( . - . mg/l) ( ) . low plasma zinc has been defined as < mcg/dl (< . µm) ( ). when zinc intake decreases, homeostatic mechanisms initially maintain the plasma concentration within the reference range, but when deficiency is severe or prolonged, the concentration decreases. however, although plasma zinc concentration moderately correlates to habitual intake, the test also has limited specificity because zinc levels are depressed during inflammatory disease states or pregnancy and increase with acute catabolic states ( ) . in mild diseases, with c-reactive protein (crp) levels of mg/l, a % decrease in zinc is observed. in severe infectious diseases, crp levels can reach - mg/l, with a much greater decrease in zinc levels ( - %) ( ) . if crp levels are normal, plasma zinc measurements are more reliable. moreover, the test has limited sensitivity since patients with mild zinc deficiency may have normal plasma levels ( ) . the copper:zinc ratio may be an interesting marker for the diagnosis of zinc deficiency, since the latter leads to an increase in copper absorption ( ) . to be reliable, this ratio must be higher than . . however, critical patients may have high levels of copper, reflecting the effects of the systemic inflammatory response, thus not reliably representing their actual levels ( ) . a marker that might be more sensitive to the nutritional status of zinc is the ratio of apo/holo activities of angiotensin converting enzyme ( ) . zinc levels may also be measured in neutrophils, lymphocytes, or erythrocytes, but these assays generally have poor sensitivity ( , ) . ruz m et al. reported that zinc levels in neutrophils do not change, even in the event of changes in plasma concentrations during experimentally-controlled zinc depletion ( ) . metfah et al. found that zinc levels in lymphocytes, granulocytes, and platelets decreased significantly only during the late zinc depletion phase ( ) . interestingly, plasma zinc levels did not change even during the late zinc depletion phase in this study. in contrast, they found that activity of ecto- ′ -nucleotidase (an integral zinc-dependent plasma enzyme located on most mammalian cells) was significantly decreased during mild zinc deficiency. when measured in neutrophils, zinc deficiency is defined as < mcg/ cells ( ) . when measured in lymphocytes, zinc deficiency is defined as < mcg/ cells ( ) . taking all this into account, we highlight that there is no good reliable definition for zinc deficiency, besides a low plasma concentration with respect to normal reference levels, which may not be representative, especially in acute states or mild grades. zinc homeostasis in immune system pathways is complex, since it participates both in pro-inflammatory and regulatory pathways, and much of the data comes from preclinical in figure | legend: a schematic view of the involvement of zinc in various signaling pathways. green arrows: zinc-mediated activation. red t bar arrows: zinc-mediated inhibition. blue arrows: flow of activation pathway. obesity, cardiovascular disease, diabetes, and aging are associated with zinc deficiency. − gg polymorphism on the il- promoter gene is associated with zinc homeostasis impairment and elevated il- levels which contribute to lung damage. zinc deficiency may increase ace- receptor activity on type pneumocytes and other cells that are infected by sars-cov- , mainly in the lower respiratory tract. zinc inhibits rdrp, blocking viral rna replication. zinc-finger protein zcchc senses viral rna and activates through rig- -like receptor a cascade that results in an increase in the interferon type response. ifn type stimulates synthesis of antiviral proteins such as rnasel and pkr. zinc helps to regulate the same kind of responses by activating the a protein that inhibits traf downstream activation, and by inhibiting pde, which results in increased levels of cgmp that will activate pka that will inhibit nf-κb. zinc also inhibits stat- dimerization, blocking active stat signaling from the il- receptor. acronyms: zcchc : zinc finger cchc domain-containing protein . rig- , retinoic acid-inducible gene i; mavs, mitochondrial antiviral-signaling protein; tank, traf family member-associated nf-κb activator; iκkε, i kappa b kinase epsilon; tbk , tank binding kinase ; irf , interferon regulatory factor ; tlr, toll-like receptor; myd , myeloid differentiation primary-response protein ; irak, interleukin- receptor-associated kinase; traf- , tumor necrosis factor receptor-associated factor ; tak : ikk, i kappa b kinase; nfκb, nuclear factor kappa b; a , zinc protein; pde, phosphodiesterase; cgmp, cyclic guanosine-monophosphate; gmp, guanosine-monophosphate; pka, protein kinase a; inf- , interferon type ; jak, janus kinase; stat, signal transducer and activator of transcription; rnase l, ribonuclease l; pkr, rna-activated protein kinase; ace- , angiotensin-converting enzyme ; rdrp, rna-dependent rna polymerase. vitro studies. despite this, it seems clear that deficient or excessive zinc levels can lead to malfunction of the adaptive and innate immune systems. zinc regulates the proliferation, differentiation, maturation and functioning of lymphocytes, and other leukocytes ( ) . it also regulates the immune response, and its deficiency increases susceptibility to inflammatory and infectious diseases, including pneumonia ( ) . zinc sulfate supplementation at mg/day for months reduced acute lower respiratory tract infection morbidity vs. placebo in a clinical trial ( ) . zinc is essential in both the adaptive and innate immune systems ( ) . for instance, the functionality of natural killer (nk) cells, which are essential for maintaining the immune response against viruses and tumors, is affected by low levels of zinc ( ) . furthermore, zinc supplementation significantly increased nk cell numbers in whole blood cultures and nk cell activity in vivo ( , ) . in this latter study, zinc supplementation in subjects with low or borderline-normal circulating zinc increased the concentration of this ion and improved nk lytic activity, as well as modulating plasma il- . zinc homeostasis directly influences the formation of lymphocytes and the secretion of cytokines and indirectly alters their stimulation by the innate immune system ( ) . there is also evidence that unregulated zinc homeostasis in macrophages impairs phagocytosis and results in an abnormal inflammatory response ( ) . in a study performed in mice, a diet deficient in zinc was associated with more pronounced airway inflammation after agricultural organic dust exposure, compared with normal dietary zinc intake ( ) . this was partially explained by the fact that macrophages maintained in a zinc-deficient environment exhibited increased cxcl and il- production, as a result of increased nf-kb activation. also, pulmonary zinc deficiency may be one of the mechanisms by which hiv- infection impairs alveolar macrophage immune function and facilitates severe pulmonary infection in these individuals ( ) . zinc also has a role in viral recognition. the zinc-finger protein zcchc binds rna and facilitates the detection of intracellular rna viruses by activating retinoic acid-inducible gene-i (rig- )-like receptors (rlrs), including rig-i and mda ( ) . this action triggers the activation of the antiviral response mediated by downstream activation of antiviral genes ( ) . in this process, kinases such as tbk and iκk further phosphorylate the interferon regulatory transcription factor (irf ) and iκb-alpha, the nk-κb inhibitor, leading to activation of irf and nf-κb, which results in interferon type upregulation ( , ) (see figure ) . interferon alpha-induced signaling results in upregulation of antiviral proteins (rnase l and pkr), known to degrade viral rna and inhibit its translation ( ) . zinc also exerts an inhibitory effect on the activation of nf-κb, through the expression of the a protein. a is a zinc-finger protein that negatively regulates tumor necrosis factor receptor (tnfr) and toll-like receptor (tlr)-initiated nf-κb pathways ( ) . furthermore, zinc acts as an inhibitor of cyclic nucleotide phosphodiesterase (pde). when pde is inhibited, cyclic nucleotide cgmp (cyclic guanosine monophosphate) is elevated, leading to the activation of pka (protein kinase a), and subsequent inhibition of nf-κb ( ) . additionally, zinc supplementation has been shown to downregulate inflammatory cytokines by decreasing gene expression of il- β, tnf-alpha, and by inhibiting nf-κb activation ( ) . nutritional immunity is a process by which the host organism sequesters trace minerals during an infection so that their availability to pathogens is limited ( ) . during infection and inflammation, there is a transient transfer of zinc from serum to the organs, causing temporarily low serum zinc levels, which normalize during resolution of the inflammatory response ( , ). thus, a sufficient level of zinc is essential during responses to infection. zinc signals act in an anti-inflammatory manner during sepsis by regulating the pro-inflammatory response, due to cellular uptake of zinc by zip as shown in a polymicrobial model of sepsis in mice ( ) . zinc deficiency was strongly associated with an elevated risk of exaggerated inflammation and mortality due to sepsis in a murine model ( ) . in this study, mice with a zinc-deficient diet had a % reduction in plasma zinc levels compared with those with a normal diet, and had a significantly lower survival rate of % in the context of sepsis. based on the studies mentioned above, one could hypothesize that an initial chelation of zinc would trigger an antiviral response mediated by interferon type (ifn-i). however, ensuring an adequate level of zinc would be necessary to regulate this response, since zinc participates as an inhibitory agent at many points in this pathway (see figure ) . indeed, an early ifn-i response was shown to be optimal, while a delayed ifn-i response was associated with ards in a study with sars-covinfected mice ( ) . ifn- subtypes were studied alone and in combination with other antiviral drugs for the treatment of sars and mers, in vitro and in vivo, with some beneficial reports, but later failed to improve outcomes in humans ( ) ( ) ( ) . despite this, sars-cov- appears to be more sensitive than mers or sars-cov to ifn type , and its use as prophylaxis or treatment is also being studied ( ) . although it is also hypothesized that it should be tested on the early phase of infection, late phase anti-ifn type treatment could be beneficial for treating severe disease ( ) . there is some evidence that sars-cov- infection triggers expression of numerous ifn-stimulated genes, which is thought to induce inadequate ifn responses ( ) . although there are no specific data regarding zinc in this pathway for sars-cov- , zinc may limit infection through upregulation of ifn-alpha production and an increase in its antiviral activity ( , ) . in this latter in vitro study, when cultures of white blood cells from elderly subjects were supplemented with µm zinc (the physiological concentration), they produced ifn in amounts comparable to those from the younger subjects. we hypothesize that transient zinc deficiency during infection could result in a hyperinflammatory state in those with prior zinc deficiency. also, zinc deficiency has been linked to a loss of taste and smell, symptoms recently attributed to infection by this virus ( , ) . in our opinion, this could be a consequence of a transient acute zinc deficiency produced during infection. zinc deficiency may diminish protein synthesis in taste bud cells, reduce alkaline phosphatase activity in taste buds, alter a zinc-containing salivary protein, block the taste pore region of the taste bud or lead to central nervous system dysfunction ( ) . il- appears to be important in triggering severe lung damage during sars-cov- infection. sustained elevation of il- is postulated as being responsible for severe immune-mediated lung damage as well as for macrophage activation syndrome (mas) that might overlap in patients with severe covid- ( ) . there is much evidence for how this cytokine storm may be related to zinc levels. firstly, il- induces expression of metallothioneins (mt) and alpha- -macroglobulin (a m) (both zinc-binding proteins), which can reduce zinc bioavailability. il- , mt, and a m increase with age and impaired zinc availability contributes to immunosenescence ( ) . secondly, zinc acts as an anti-inflammatory element, downregulating many pro-inflammatory signaling pathways, such as il- -mediated activation of stat- ( ) . thirdly, il- production seems to be increased in zinc-deficient elderly subjects. furthermore, obese patients with lower dietary intake of zinc present with lower plasma and intracellular zinc levels, along with upregulated gene expression of il- alpha, il- beta, and il- , compared with patients with higher zinc intake ( ) . in this in vivo study, mg of pure zinc supplementation resulted in a significant . % decrease in il- release from white blood cells in healthy elderly subjects. fourthly, a polymorphism has been described in the il- gene that is related to impaired zinc homeostasis. an il- promoter gene single nucleotide polymorphism (snp) at position − has been studied in several age-related diseases, such as cardiovascular disease, alzheimer's disease, diabetes, and cancer ( ) ( ) ( ) . zinc deficiency induces a progressive demethylation of the il- promoter in thp cells, which correlated to increased il- expression ( ) . genetic variation at the il- - g/c locus is involved in determining il- production and the immune response. elderly subjects with gg genotypes (called c-) have more risk of developing atherosclerosis due to higher il- production, impaired k cell cytotoxicity, increased mt gene expression, and low zinc ion availability compared with c+ carriers ( ) . for instance, in elderly individuals aged - years, c+ polymorphism was associated with il- levels of . pg/ml and zinc levels of . µg/dl, whereas c-polymorphism was associated with il- levels of . pg/ml and plasma zinc of . µg/dl, these differences being statistically significant. in another study, c+ carriers had significantly higher plasma zinc levels, lower mt production, higher red blood cell zinc levels, and good nk cell cytotoxicity, as shown in an in vivo study performed in elderly subjects ( ) . thus, patients with il- - gg polymorphism (c-carriers) may be susceptible to developing a severe infection due to sars-cov- , leading to an increase in il- levels that produce a cytokine storm related to impaired zinc homeostasis. interestingly, this polymorphism seems to be twice as common in people from italy ( . %) and other mediterranean countries, compared with northern european countries such as germany ( . %) ( ) . this might explain, to some degree, the difference in mortality rates observed between these countries; as of the march, italy recorded , confirmed cases and , deaths, while germany had , cases and deaths ( ) . to date, germany has one of the lowest case fatality rates at . % as of the beginning of may, compared with italy ( . %). it is probable that other factors, such as differences in early identification of cases and correct isolation, and differences in the proportion of the population that is elderly, may also have been important. nevertheless, studies on genetic susceptibility for developing covid- pneumonia and severe illness are underway ( , ). there are no data regarding the prevalence of this polymorphism in other countries such as the uk or usa, which are known foci of the pandemic. the usa has almost , , infected cases with more than , deaths, which translates to a fatality rate of . % ( ) . zinc has shown its ability to inhibit sar-cov rna polymerase ( ) . zn + cations, especially in combination with zn ionophore pyrithione, inhibited sars-cov rna-dependent rna polymerase, rdrp. a more than % reduction in overall rna synthesis was observed at zinc levels of µm, while < % activity remained at zinc levels of µm. this finding would make zinc a potential antiviral agent for coronavirus diseases. additionally, chloroquine and hydroxychloroquine, among their other specific mechanisms, act as zinc ionophores and promote cellular uptake of zinc-a mechanism which may increase the effectiveness of these compounds in inhibiting the replicative capacity of the virus ( , ) . sars-cov- and sars-cov require angiotensin-converting enzyme (ace ) for entry into target cells. zinc exposure reduced recombinant human ace- activity in rat lung ( ) . ace- is a zinc metallopeptidase that contains a hexxh motif that functions as the zincbinding domain at its active site. in this in vitro study, in the presence of µm zinc, activity was significantly (p < . ) decreased in rat lung and rhace- compared with or µm zinc. in the presence of , µm zinc, activity was further reduced (p < . ) in all three preparations compared with , , and µm zinc. thus, hypothetically, zinc deficiency could facilitate sars-cov- infection of target cells due to an increase in ace- activity that could facilitate binding with sars-cov- . in conclusion, the world is facing a pandemic caused by a novel coronavirus, with some countries suffering a higher burden of disease. the infection is known to more severely affect older people with various chronic comorbidities such as obesity, hypertension, and diabetes. zinc has a known role in the regulation of immunity. a plausible biological mechanism for the involvement of zinc in this condition exists, which we summarize in figure . its supplementation, alone or as an adjuvant to medicines that are currently being used to treat active infection, could be beneficial due to its effect on many key factors in the regulation of a severe immune response during infection. zinc supplementation could be a novel treatment for people at high risk of zinc deficiency who develop severe pneumonia due to covid- . we believe there is enough evidence to further investigate how zinc status or homeostasis is involved in the pathogenesis of severe illness produced by sars-cov- infection, and its potential role as an active treatment should be assessed in clinical trials. zinc in infection and inflammation metal elements and gene 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nutrition in health and disease effect of the inflammatory response on trace element and vitamin status assessment of marginal zinc status in humans zinc deficiency and clinical practice assessment of plasma and red cell trace element concentrations, disease severity, and outcome in patients with critical illness zinc deficiency in patients with idiopathic taste impairment with regard to angiotensin converting enzyme activity erythrocytes, erythrocyte membranes, neutrophils and platelets as biopsy materials for the assessment of zinc status in humans ecto ' nucleotidase ( 'nt) as a sensitive indicator of human zinc deficiency multiple impacts of zinc on immune function zinc supplementation for prevention of acute respiratory infections in infants: a randomized controlled trial zinc and the immune system alterations in human natural killer cell activity and monocyte cytotoxicity induced by zinc deficiency t-helper type cytokine release is enhanced by in vitro zinc supplementation due to increased natural killer cells effect of zinc supplementation on plasma il- and mcp- production and nk cell function in healthy elderly: interactive influence of + mt a and− il- polymorphic alleles zinc and immunity: an essential interrelation the role of zinc and zinc homeostasis in macrophage function insufficient zinc intake enhances lung inflammation in response to agricultural organic dust exposure hiv- -transgene expression in rats decreases alveolar macrophage zinc levels and phagocytosis the zincfinger protein zcchc binds rna and facilitates viral rna sensing and activation of the rig-i-like receptors pathogen recognition and innate immunity glycogen synthase kinase β regulates irf transcription factor-mediated antiviral response via activation of the kinase tbk virus-triggered ubiquitination of traf / by ciap / is essential for induction of interferon-beta (ifn-beta) and cellular antiviral response zinc potentiates the antiviral action of human ifn-alpha tenfold functional significance of zinc-related signaling pathways in immune cells zinc-dependent suppression of tnf-alpha production is mediated by protein kinase a-induced inhibition of raf- , i kappa b kinase beta, and nf-kappa b antioxidant effect of zinc in humans zinc dyshomeostasis during polymicrobial sepsis in mice involves zinc transporter zip and can be overcome by zinc supplementation zinc deficiency increases organ damage and mortality in a murine model of polymicrobial sepsis dysregulated type i interferon and inflammatory monocytemacrophage responses cause lethal pneumonia in sars-cov-infected mice. version sars: systematic review of treatment effects treatment with lopinavir/ritonavir or interferon-β b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset treatment of middle east respiratory syndrome with a combination of lopinavir-ritonavir and interferon-β b (miracle trial): study protocol for a randomized controlled trial type interferons as a potential treatment against covid- the use of antiinflammatory drugs in the treatment of people with severe coronavirus disease ( ). (covid- ): the perspectives of clinical immunologists from china heightened innate immune responses in the respiratory tract of covid- patients zinc ions inhibit replicaction of rhinoviruses zinc supplementation reconstitutes the production of interferon-alpha by leukocytes from elderly persons zinc and taste disturbances in older adults: a review of the literature anosmia and ageusia: common findings in covid- patients the effects of feeding a zinc-deficient diet on taste acuity and tongue epithelium in rats the role of cytokines including interleukin- in covid- induced pneumonia and macrophage activation syndrome-like disease zinc-binding proteins (metallothionein and alpha- macroglobulin) and immunosenescence zinc regulates the acute phase response and serum amyloid a production in response to sepsis through jak-stat signaling zinc supplementation in the elderly reduces spontaneous inflammatory cytokine release and restores t cell functions interleukin- gene alleles affect the risk of alzheimer's disease and levels of the cytokine in blood and brain interleukin- - g > c polymorphism affects the association between il- plasma levels and insulin resistance in type diabetic patients the interleukin- − g>c promoter polymorphism is associated with a higher risk of death after an acute coronary syndrome in male elderly patients zinc deficiency enhanced inflammatory response by increasing immune cell activation and inducing il promoter demethylation the− g/c polymorphism of il- is useful to screen old subjects at risk for atherosclerosis or to reach successful ageing zinc deficiency and il- − g/c polymorphism in old people from different european countries: effect of zinc supplementation. zincage study thüsen j, van der eerden m. histopathology and genetic susceptibility in covid- pneumonia human leukocyte antigen susceptibility map for severe acute respiratory syndrome coronavirus zn( +) inhibits coronavirus and arterivirus rna polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture chloroquine is a zinc ionophore new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid- ? concentrationdependent effects of zinc on angiotensin-converting enzyme- activity ( . ) all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © mayor-ibarguren, busca-arenzana and robles-marhuenda. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -a f l iu authors: dou, dan; revol, rebecca; Östbye, henrik; wang, hao; daniels, robert title: influenza a virus cell entry, replication, virion assembly and movement date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: a f l iu influenza viruses replicate within the nucleus of the host cell. this uncommon rna virus trait provides influenza with the advantage of access to the nuclear machinery during replication. however, it also increases the complexity of the intracellular trafficking that is required for the viral components to establish a productive infection. the segmentation of the influenza genome makes these additional trafficking requirements especially challenging, as each viral rna (vrna) gene segment must navigate the network of cellular membrane barriers during the processes of entry and assembly. to accomplish this goal, influenza a viruses (iavs) utilize a combination of viral and cellular mechanisms to coordinate the transport of their proteins and the eight vrna gene segments in and out of the cell. the aim of this review is to present the current mechanistic understanding for how iavs facilitate cell entry, replication, virion assembly, and intercellular movement, in an effort to highlight some of the unanswered questions regarding the coordination of the iav infection process. influenza viruses belong to the orthomyxoviridae family and are classified as either type a, b, c, or the recently identified type d ( , ) . influenza a viruses (iavs) and type b viruses (ibvs) contain , negative-sense, single-stranded viral rna (vrna) gene segments ( figure a ) ( , ) , which encode transcripts for essential viral proteins, as well as several strain-dependent accessory proteins ( figure b) . in comparison, influenza type c and d viruses only possess seven vrna gene segments, as the hemagglutinin-esterase fusion protein vrna replaces the hemagglutinin (ha or h) and the neuraminidase (na or n) vrnas ( , ) . iavs will be the main focus of this review since they are the primary agents responsible for influenza pandemics, and a major contributor to the annual influenza epidemics in the human population ( ) . the natural reservoir for iavs is wild aquatic birds, but they commonly infect other species, including humans, and have even been isolated from penguins in antarctica ( ) ( ) ( ) ( ) . the ability to adapt to multiple species is a major reason why iavs are more diverse than ibvs, which are essentially exclusive to humans. despite the host-range differences, many similarities do exist between these two viruses. both possess a host-derived lipid membrane, referred to as an envelope, which is decorated on the surface with the viral membrane proteins ha, na, and to a lesser extent the matrix (m ) protein ( figure c ) ( ) ( ) ( ) . the envelope is supported underneath by the matrix (m ) protein, and inside, the eight vrnas are found as individual viral ribonucleoprotein (vrnp) complexes ( figure c , bottom). each vrnp is comprised of a vrna that is wrapped around numerous red circles represent the ′ m pppg cap, black lines denote the - nucleotide, host-derived primers that are obtained by the cap-snatching mechanism of the viral polymerase. a(n) corresponds to the ′ poly-a tail produced by reiterative stuttering of the viral polymerase. the smaller mrnas (empty boxes) represent transcripts that encode nonessential accessory proteins found in many strains, whereas those that are less prevalent (pb -s , m , and ns ) are not illustrated ( ) ( ) ( ) ( ) ( ) ( ) . (c) diagram of an influenza a or b virus. the viral membrane proteins ha, na, and m are shown, along with the eight viral ribonucleoproteins (vrnps), and the matrix protein m that supports the viral envelope. to highlight the vrnp components, the illustration beneath the virus is not to scale. a single vrna gene segment is shown wrapped around multiple nucleoprotein (np) copies with the conserved promoter regions in the ′ and ′ utrs forming a helical hairpin, which is bound by a single heterotrimeric viral rna-dependent rna polymerase (pb , pb , and pa). (d) top view of an influenza virus cross-section showing the vrnp " + " configuration. vrnps are depicted with black circles as it is not known if the positioning of a particular vrnp is conserved or interchangeable. frontiers in immunology | www.frontiersin.org july | volume | article copies of the viral nucleoprotein (np) and bound by a single copy of the heterotrimeric viral polymerase, consisting of pb , pb , and pa ( ) ( ) ( ) . the polymerase binds the vrnas at a helical hairpin that results from the base pairing between the conserved semi-complimentary ′ and ′ ends ( ) ( ) ( ) . morphologically, iavs can either form spheres with a diameter of ~ nm or filaments that can reach up to µm in length [reviewed in ref. ( ) ]. however, upon passaging in eggs, or mdck cells, the filamentous form is generally lost ( , ) . several studies have attributed the morphology change to m , presumably through its function in supporting the envelope ( ) ( ) ( ) . regardless of the virion shape, ha is the most abundant viral envelope protein, followed by na, and m ( ) . recent work has shown that the viral envelope also contains host membrane proteins ( , ) . these proteins are likely recruited based on the lipid composition at the plasma membrane budding site, which can differ between cell types ( , ) . through possible interactions with each other and m , the eight vrnps typicallly form a + configuration inside the virus ( figure d ) ( , ) . the + configuration may have a mechanistic function, as it is also conserved in type c and d viruses that only possess vrnas ( ) . further supporting the mechanistic concept, it was recently shown that iavs can package cellular ribosomal rna (as a vrnp) when one of the vrnas is made unavailable ( ) , possibly explaining how type c and d viruses acquire their "eighth" vrna. the classification of iavs into subtypes is based on the genetic and antigenic properties of the surface antigens ha and na, which mediate viral entry and release, respectively ( , ) . to date, ha (h - ) and na subtypes (n - ) have been found in iavs isolated from aquatic birds ( ) . two additional subtypes for ha (h and h ) and na (n and n ) have recently been identified in bats ( , ) , but in contrast to the ha and na subtypes from the more traditional avian iavs, these do not appear to recognize sialic acid (sa) ( ) ( ) ( ) . despite the numerous possible subtype combinations, only three have consistently persisted in the human population, causing the following pandemics in the process: and (h n ), (h n ), and (h n ) ( ) . currently, only the h n and h n subtypes, as well as the two antigenically distinct ibv lineages (victoria and yamagata), are endemic in the human population ( ) , which is why many iav vaccines include two representative iav and ibv strains ( ) . a significant challenge in battling iavs is the constant evolution of the surface antigens (ha and na) in response to pressure from the host immune system, which is referred to as antigenic drift and antigenic shift. antigenic drift is most evident in circulating seasonal iavs, where substitutions by the polymerase that cause mutations in the surface antigen epitopes have continuously been selected to enable reinfection of the same host ( ) . antigenic shift is responsible for the development of the iav pandemics, and it relies on the less the terminal sialic acid residues are displayed with an α- , linkage, as well as an α- , linkage, to illustrate the "linear" and "bent" presentations. (b) illustration of iav cell entry. (i) iavs initiate cell entry by using the ha receptor-binding domain (located in the ha region) to associate with sialylated glycoconjugates on a host "receptor." binding to the "receptor" triggers endocytosis. (ii) the virus then traffics to the endosome where the lower ph facilitates a conformational change in ha, exposing the fusion peptide (located in the ha region) for insertion into the endosomal membrane. (iii) the ha pre-hairpin conformation begins to collapse, forming a six-helix bundle that promotes hemifusion of the viral envelop with the endosomal membrane. at some point, the m channel opens to release the viral ribonucleoproteins (vrnps) from m by acidifying the viral interior. (iv) ha further collapses into a trimer of hairpins to promote the formation of the fusion pore, which (v) releases the vrnps into the cytosol. (vi) the exposed nuclear localization signals (nls) on the vrnps are recognized by the adaptor protein importin-α, leading to the recruitment of importin-β that (vii) facilitates the transport through the nuclear pore complex (npc) and into the nucleus. frontiers in immunology | www.frontiersin.org july | volume | article frequent process of reassortment, which involves the exchange of vrnas between two iavs during co-infection of a cell ( , , ) . while reassortment can happen between two related iavs, antigenic shift occurs when the reassortment process yields a new iav subtype. iavs are also under constant negative selection due to the functional requirements of the viral proteins, and the constraints of the limited genome. several roles have been reported for most of the iav proteins. these include the function of ha in receptor binding, as well as membrane fusion, and viral release by the sialidase activity of na. to perform these functions, the proteins need to correctly fold, oligomerize, and as for the genome itself, they have to be properly trafficked and packaged into new virions. thus mutations that benefit one property may hinder another. the goal of this review is to highlight these functional requirements by providing a summary of the mechanisms iavs have evolved to facilitate cell entry, replication, virion assembly and movement, with particular attention to how iavs coordinate the infection process. iavs initiate the infection process by using the ha molecules on the viral envelope. upon reaching a potential host cell, the ha receptor-binding site attaches the virus to surface glycoconjugates that contain terminal sa residues (figure a ) ( , , ) . iavs then scan the cell surface for the proper sialylated "receptor" by using the sialidase function of na to remove local sas and liberate nonproductive ha associations ( ) . currently, the "receptor's" identity remains unknown, but it is generally thought that has from avian iavs have higher specificity for receptors with α- , -linked sas that have a "linear" presentation ( , ) , whereas has from human iavs prefer an α- , linkage, which results in a more "bent" presentation ( figure a ) ( , ) . while these preferences correlate with sa linkages in the respective hosts ( ) , several studies have shown that matching ha receptor binding preferences with the sa linkages in a particular host is not essential for infection, but is more critical for transmission ( ) ( ) ( ) ( ) . this implies that the iav "receptor" either displays significant cell tropism in the airways or that iavs can potentially use more than one receptor. despite the unknown identity of the receptor, it is clear that ha-mediated binding to the receptor triggers endocytosis of the virion ( figure b , step i). the endocytosis can either occur in a clathrin-dependent manner, involving dynamin and the adaptor protein epsin- ( - ), or by macropinocytosis ( , , ) . once inside the cell, the virus is trafficked to the endosome, where the low ph activates the m ion channel ( , , ) , and causes a large conformational change in ha that exposes the fusion peptide ( figure b , step ii) ( ) ( ) ( ) . opening of the m ion channel acidifies the inside of the viral particle, releasing the packaged vrnps from m ( figure b , step iii), which enables the transfer of the vrnps to the host cytoplasm following ha-mediated fusion ( , fusion of the viral-endosomal membranes by ha occurs through multiple steps [reviewed in refs. ( , ) , and requires cleavage of ha by host cell proteases into two subunits, ha and ha ( , , ) ]. the cleavage (see ha proteolytic activation at the golgi or plasma membrane) is required to enable the exposure of the fusion peptide on the n-terminus of the ha upon the ph change in the endosome ( ). once exposed, the fusion peptide inserts into the endosomal membrane, while the c-terminal transmembrane domain (tmd) anchors ha in the viral membrane, creating a pre-hairpin conformation (see figure b , step ii "box"). the ha trimers then fold back on themselves creating a hairpin that begins to position the two membranes in close proximity to each other (see figure b , step iii "box"). the hairpin bundles then further collapse into a six-helix bundle, and in doing so, the two membranes come closer together enabling the formation of the lipid stalk, and the subsequent fusion of the two membranes ( figure b , step iv). to date, not all of these stages have been observed with ha and some have been inferred based on observations of related fusogens from other viruses. in contrast to the early steps in iav entry, vrnp trafficking to the nucleus following the fusion event is highly dependent on the host cell machinery and transport pathways [reviewed in ref. ( ) ]. supported by numerous studies, the current model is that the newly released cytoplasmic vrnps use the importin-αimportin-β nuclear import pathway to gain entry to the host cell nucleoplasm ( figure b , steps vi and vii) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . to initially engage this pathway, it is thought that the vrnps use the surface exposed nuclear localization sequences from the numerous np molecules to recruit the adapter protein importin-α ( ) ( ) ( ) . upon binding to the vrnp, importin-α is recognized by the importin-β transport receptor, which directs the vrnp to the nuclear pore complex, where it is transported into the nucleoplasm. recent improvements in imaging and rna labeling techniques have made it possible to monitor the entire entry process in single cells ( , , ( ) ( ) ( ) . the cumulative results from these studies show that iavs can deliver their vrnps from the cell surface to polyadenylation "reiterative stuttering" the nucleus in approximately h, with entry and fusion occurring rather quickly (~ min), and nuclear import requiring the bulk of the time ( ) . a striking observation from these studies is the efficiency with which the eight vrnas reach the nucleus, indicating how effectively vrnps recruit the host nuclear import factors. supporting this observation, it was shown that np adaptation to the importin-α isoforms of a particular species is crucial for productive iav infections ( ) . while the bulk of the vrnp trafficking work has been carried out using various immortalized cell lines, the potential species related differences, and the essential role of vrnp trafficking in reassortment, emphasize the need for further methodology development to examine the details of iav entry in primary cells and tissue explants. inside the nucleus, the heterotrimeric viral rna-dependent rna polymerase carries out the transcription and replication of the vrnas [reviewed in refs. ( , ) ]. the replication of the influenza genome involves two steps: transcription of complimentary rna (crna), followed by transcription of new vrna copies using the crnas as templates. the crnas are produced by an unprimed process that relies on the correct complementation of free rntps (generally gtp and atp) with the ′ end of the vrna template ( figure a ) ( , ) . the nucleotide complementation locks the vrna template into the polymerase active site within the pb subunit and results in the formation of an a-g dinucleotide from which the crna is elongated ( ) . upon exiting the polymerase, the crna associates with newly synthesized np molecules and a single copy of the viral polymerase to assemble into a crnp ( ) . currently, it is thought that the newly produced viral polymerases, which are incorporated into the crnps, generate multiple vrna copies in a manner similar to crna transcription ( figure b) . however, there is one distinction related to the difference in the positioning of the longer ′ end of the positivesense crna. due to the increased length, the crna is positioned in the polymerase such that the rntp annealing and dinucleotide formation is likely to occur at the nucleotides located and bases from the crna ′ end (figure b , pathway i) ( , ( ) ( ) ( ) . the dinucleotide primer then has to dissociate and reanneal to the nucleotides at the ′ end prior to elongation ( figure b) . alternatively, the crna ′ end could reposition within the polymerase due to rntp bin ding, resulting in the generation of full-length vrna transcripts directly ( figure b , pathway ii). the transient nature of the rntp annealing and dinucleotide formation makes it technically challenging to exclude either possibility. the remaining task of assembling a vrnp is analogous to crnp formation. viral mrna transcription from the vrna templates is primed, making it significantly more efficient than crna and vrna transcription ( ) . the viral polymerase obtains the primers through a mechanism termed cap snatching ( ) , which is aided by the association with the cellular rna polymerase ii c-terminal domain (figure ) ( ) ( ) ( ) . for cap snatching, the viral polymerase uses the pb subunit to bind to ′ caps of nascent host transcripts ( ) and the pa subunit endonuclease domain to cleave - nucleotides downstream of the ′ cap ( ) ( ) ( ) . the pb cap-binding domain then rotates to position the newly acquired capped primer into the pb catalytic center where it is extended using the vrna as a template ( ) . finally, each transcript is polyadenylated through a reiterative stuttering′ process, which occurs when the polymerase encounters the short poly-u sequence at the vrna ′ end (figure "box") ( , ) . this process likely involves multiple cycles of dissociation, repositioning, and reannealing of the mrna to this template region of the vrna to achieve polyadenylation. during the course of infection, mrna synthesis occurs before crna and vrna transcription, and mrna transcription is much more abundant because the use of primers significantly increases the initiation efficiency ( ) . the initial mrnas are transcribed by the vrnp-associated polymerases and exported from the nucleus for translation by cytoplasmic ribosomes ( ) . however, the m and ns transcripts also possess donor and acceptor splice sites that match well with those in human transcripts ( ) . these sites recruit the cell spliceosome, which produces the spliced transcripts that encode for the m and ns proteins, respectively ( ) ( ) ( ) ( ) ( ) . the ns transcript has been reported to maintain a similar ratio of non-spliced and spliced transcripts throughout infection ( ) , whereas the ratio of the spliced m transcripts (encoding m ) have been shown to increase during infection ( ) . these observations imply that ns and ns are always equally expressed, while m expression is more biased toward the later stages of infection. however, it is likely that the splicing efficiency of the ns and m transcripts differs between iav strains ( , ) . iav protein synthesis is entirely dependent on the translation machinery of the host cell. following nuclear export [reviewed in ref. ( ) ], the translation of the viral mrnas is divided between cytosolic ribosomes (for pb , pb , pa, np, ns , ns , and m ) and endoplasmic reticulum (er)-associated ribosomes for the membrane proteins ha, na, and m (figure , steps i and ii). nuclear localization sequences on the newly synthesized np proteins and polymerase subunits (pb , pb , and pa) target these proteins into the nucleus by recruiting the importin-α-importin-β pathway that is utilized for vrnp nuclear import (figure , step iii). the np and pb proteins are imported individually, whereas the pb and pa proteins are imported as a heterodimer ( , ) . in the nucleus, these newly synthesized proteins assist in viral mrna transcription and vrna replication. np monomers bind to nucleotide stretches with a partial g bias in vrnas, and presumably crnas, to assemble vrnps and crnps through a process that may be regulated by the np phosphorylation (figure , steps v and vii) ( ) ( ) ( ) . the heterotrimeric polymerase assembles and binds to the newly formed crnps to transcribe vrnas ( figure , step vi) that upon formation into vrnps can generate additional viral mrna (figure , step viii) , or crna transcripts (figure , step ix) ( , ) . the viral rna-binding protein ns is synthesized early and also imported into the nucleus, where it can act as an inhibitor of interferon signaling [reviewed in ref. ( ) ]. in addition, ns may contribute to viral mrna export from the nucleus by linking the viral transcripts to the cellular nuclear export components tap/nxf , p , rae , e b-ap , and the nucleoporin nup ( ) . ns (alternatively known as the nuclear export protein) and m are imported into the nucleus as well. multiple studies have implicated these two proteins in the nuclear export of vrnps ( , , ( ) ( ) ( ) ( ) . while the mechanism remains unclear, current data support a model where m acts as an adaptor protein linking ns to vrnps (figure , step x) ( , ) . through established interactions with crm , ns is then able to target the vrnp complex to the crm nuclear export pathway for transport to the cytoplasm ( ), where m potentially prevents the re-import of vrnps by blocking access to the np nuclear localization sequences (figure , step xi) ( ) . within the cytoplasm the vrnps are trafficked toward the plasma membrane for viral assembly by rab . rab facilitates the interaction by associating with the viral polymerase pb subunit ( ) , potentially providing a quality control mechanism that ensures new virions incorporate vrnps carrying a polymerase. earlier studies proposed that vrnps specifically associate with rab on recycling endosomes, which use microtubules for transport toward the cell surface ( figure , step xiia) ( ) ( ) ( ) ). an alternative model has recently been proposed where infection causes tubulation of the er membrane network and the vrnps bind to rab molecules that have localized to this network for trafficking toward the plasma membrane ( figure , step xiib) ( ) . currently, it is not known how vrnps are transferred to the plasma membrane in either model, or how iavs incorporate all eight of the different vrnps in a " + " configuration. while several studies have indicated that specific vrnp associations likely contribute to the packaging of the eight vrnps ( , , ) , the underlying mechanisms remain to be established. the iav membrane proteins, which are ultimately destined for the viral envelope, are synthesized by ribosomes associated with the er membrane. similar to cellular secretory proteins, ribosome-nascent chain complexes containing na, ha, or m are co-translationally directed to the er by interactions of their hydrophobic targeting sequences with the signal recognition particle (srp) (figure , step ii) ( ) ( ) ( ) ( ) . the cleavable signal sequence on ha facilitates the interaction with srp, whereas na and m use their respective tmd as an er targeting sequence. once bound, srp targets the ribosome-nascent chain complexes to the srp receptor in the er membrane (figure , step iii) , which transfers the ribosome to a sec protein-conducting channel known as the translocon ( - ) . linked to the dependence on srp, mutations that alter the targeting sequence hydrophobicity of cellular secretory proteins have been shown to decrease their er targeting and subsequent synthesis ( , ) . although this aspect has not been examined for the iav membrane proteins, there is evidence that the hydrophobicity of their er-targeting sequences change ( , ) , which suggests iavs potentially use this mechanism to titrate na and ha expression. the translocon enables passage of the elongating na, ha, and m polypeptides into the er lumen and facilitates the membrane partitioning of their respective tmd segments through a lateral gate ( , ) . to activate the membrane integration, the tmd segments have to be of the appropriate length and hydrophobicity ( , ) . in human h n and h n viruses, these criteria are conserved in the tmds of ha and m , but not in the tmd of na, as it has become progressively less hydrophobic in the h n viruses ( ) . the uncharacteristic hydrophobicity loss was shown to be possible because of the na tmd being positioned at the n-terminus ( ) . the positioning (~ amino acids from the c-terminus), combined with the slow rate of ribosomal translation (~ amino acids per second), likely provides these nontypical tmds with significant time to properly orientate and facilitate membrane insertion during the co-translational translocation process. during translocation, the n-terminus of ha and m is directly translocated into the er lumen, whereas na inverts, positioning the c-terminus in the er lumen ( , ) . in addition, ha and na receive multiple n-linked glycans. the glycans are transferred by the oligosaccharyltransferase to asn-x-ser/thr sequences, and vary in number as well as positioning based on the strain, or subtype ( ) . one function of the glycans is to increase the folding efficiency of na and ha by recruiting the lectin chaperones (calnexin and calreticulin) and the associated oxidoreductase erp , which aids in disulfide bond formation ( , ( ) ( ) ( ) . this is especially crucial for the ha and na proteins that possess a significant number of intramolecular disulfide bonds (e.g., six in has, eight in n , and nine in n ) ( ) ( ) ( ) . by contrast, m possesses two intermolecular disulfide bonds in its tetrameric conformation ( ) . depending on the subtype, na tetramers also possess or more intermolecular disulfide bonds. oligomerization of ha involves the trimerization of independently folded monomers, whereas na tetramerization has been proposed to result from the pairing of two co-translationally formed dimers, which assemble through a process involving the n-terminal tmd of na ( , ) . in line with this model, it has been shown that the tmd is essential for proper na folding, and that the decreasing hydrophobicity in the n tmds functions to frontiers in immunology | www.frontiersin.org july | volume | article support the folding and oligomerization of the enzymatic head domain ( , ) . iavs easily achieve the protein concentrationdependent requirement for oligomerization due to the abundance of ha and na that is synthesized during an infection. however, these high synthesis levels at the er can also be deleterious by activating the er-stress response. indeed, several studies have shown that iav replication does activate the er-stress induced unfolded protein response ( , ) , but this response is also mitigated by the inhibition of the eif α-kinase and stress granule formation through the functions of other viral proteins ( ) . despite everything that is known about the synthesis and assembly of the iav membrane proteins, several aspects have yet to be addressed. these include obtaining atomic structures of fulllength ha and na in a membrane, something that should become easier to address with the advances in cryo-electron microscopy structure determination. identifying if the na protein removes sa residues directly from substrates within the golgi, as this could decrease the effectivity of nonmembrane permeable na inhibitors. it is also unclear how iavs regulate the timing and expression levels of the viral proteins as viral mrna transcription shows little temporal variation ( , ) . while it is likely that m is regulated in part by splicing ( , ) , this does not apply to ha and na. recent work has linked na and ha regulation to the nucleotide composition of the ′coding regions for their er-targeting sequences, which dramatically differ from the profile of corresponding regions in human secretory protein mrnas ( , ) . an obvious candidate for post-transcriptional regulation is the viral rna-binding protein ns . indeed, many studies have shown that ns can increase translation of particular mrnas, possibly by enhancing the translation initiation rate through the recruitment of eif- g to the ′region of viral mrnas ( , ( ) ( ) ( ) ( ) ( ) . however, a clear mechanistic picture for influenza protein regulation is lacking. ha traffics from the er as a fusion incompetent precursor termed ha . to gain its fusion function, ha must be cleaved into the subunits ha and ha ( , , ) . the cleavage occurs in either a monobasic, or a multibasic, cleavage site ( ) . multibasic sites are commonly found in highly pathogenic avian iavs and are cleaved by furin, a calcium-dependent serine endoprotease that is located within the trans-golgi network ( ) . furin is also ubiquitously expressed ( ) , which is one of the major reasons why avian iavs with a multibasic cleavage site are generally more pathogenic. by contrast, human (and low pathogenic avian) iavs encode for has with a monobasic cleavage site, which have been shown to be processed by different proteases in human respiratory epithelial cells. these include the transmembrane protease serine s- member (tmprss ), human airway trypsin-like protease (hat), and possibly tmprss ( , ) . hat localizes at the plasma membrane where it can either cleave newly synthesized ha or the ha found in cell-associated virions ( , ) . similar to furin, tmprss resides in the trans-golgi network, where it cleaves ha en route to the plasma membrane. the m ion channel is thought to prevent the premature activation of ha following cleavage by equilibrating the slightly acidic ph of the golgi ( , ) . distinct from furin, tmprss expression has been found to be more restricted to the upper and lower respiratory tract, whereas hat was mainly shown to be expressed in the upper respiratory tract ( ) . these cell tropisms suggest that lower respiratory infections are likely mediated by tmprss , and could be one of the primary reasons human iavs are confined to the epithelial layer of the respiratory tract. compared with the bulk lipid profile of the plasma membrane, iav envelopes are enriched in cholesterol and sphingolipids ( ) , indicating that they bud from distinct apical plasma membrane regions often referred to as "rafts" ( ) . however, infectious iavs must possess mechanisms to target the eight vrnps, m , ha, na, and m to these sites in the membrane ( , ) . ha is believed to localize to these distinct regions based on fatty acid modifications of the c-terminal cysteine that occur in the golgi ( ) ( ) ( ) ( ) , whereas na enrichment has previously been attributed to a property in the c-terminus of the tmd ( ) . in contrast, m has been shown to accumulate at the boundaries of these budding domains ( ) , and the cytosolic protein m has been proposed to localize to the budding region by associating with the short cytoplasmic tails of ha and na ( ) . however, it is equally plausible that na and ha create membrane domains with a unique lipid profile that have a high affinity for m . finally, the vrnps, delivered to the cell periphery by rab , are thought to localize to the budding site by binding to m ( , ) . in addition to orchestrating the assembly of the correct viral components at the apical budding site, iavs also have to remodel the membrane to induce bud formation, and ultimately scission of the viral envelope from the plasma membrane. to promote bud formation, the virus must first induce significant curvature in the membrane and then constrict the two opposing membranes of the viral envelope to help to facilitate membrane scission. curvature can be induced by (i) protein or "molecular" crowding on one leaflet of a bilayer, (ii) association of curved or "bending" proteins with the bilayer, (iii) biased accumulation of cone shaped lipids in one leaflet of the bilayer, or (iv) the cytoskeleton ( ) . based on cumulative data regarding budding, iavs appear to induce membrane curvature through a combination of these mechanisms. indicative of using molecular crowding and bending proteins, several studies have demonstrated that ha and na expression is sufficient to induce budding, and that the efficiency and shape uniformity benefit from the presence of m ( ) ( ) ( ) ( ) . these results indicate that the abundance of ha and na on one side of the membrane can contribute to curvature. it also is intriguing to speculate that the asymmetric ( ) shape of na plays a role in this process as it is often seen clustering in the viral membrane ( , ) . by contrast, m appears to be analogous to a membrane-bending protein as it recruited to the cytosolic side of the membrane budding site, oligomerizes upon reaching the membrane, and these oligomers have been modeled to form curved structures ( ) ( ) ( ) . based on these properties, it is plausible that m significantly influences the membrane curvature at the budding site, potentially explaining its role in discerning whether iavs form spheres or filaments ( , ) . the ion channel m localizes to the budding site boundary and has also been shown to contribute to iav scission by functioning as a membrane-bending protein ( , ) . the membrane-bending property of m is localized in an amphiphilic α-helix that can incorporate the amino acid side chains from its hydrophobic face into a leaflet of the bilayer. with this domain positioned in the cytosol, the intercalation results in negative membrane curvature, which has been proposed to facilitate viral bud neck formation and scission, presumably by decreasing the distance between the two opposing membranes of the viral envelope ( ) . while much of the framework concerning iav budding has been established, it has been difficult to identify the details of the budding process, in part due to the mobility and heterogeneity of the plasma membrane. the lack of strong phenotypes from domains proposed to contribute to budding could also imply that iavs have built redundancy into the budding process ( ) ( ) ( ) . the possibility of redundancy is certainly plausible, as iavs contain the necessary components to allow for a combination of lipid recruitment, molecular crowding, and a membrane-bending protein. iav cell release and movement once the newly assembled iavs bud, their release is highly dependent on the sialidase activity of na. na is a homotetramer, and each subunit is comprised of a short n-terminal cytoplasmic tail (six amino acids), followed by a tmd, a length variable stalk, and a globular enzymatic head domain ( ) . the globular head domain forms a -bladed propeller structure, where each blade is comprised of four antiparallel β-sheets that are stabilized by disulfide bonds ( , , ) . the catalytic tyr residue is found in a highly conserved active site that forms a deep pocket in the center of each monomer ( ) . all of the residues necessary for catalysis exist within each monomer ( ) , which has made it difficult to reconcile why na evolved to function as a tetramer ( , , ) . structures of the enzymatic head domain indicate that na tetramers bind up to five calcium ions and calcium has been shown to contribute to na activity ( , , ) . however, it remains unclear why influenza na has evolved to position a calcium ion at the tetrameric interface. na facilitates viral release by catalyzing the hydrolysis of the glycosidic linkage that attaches sa to underlying sugar molecules ( ) ( ) ( ) . by removing local sa residues, na prevents ha binding at the cell surface, which facilitates the release of the virus during budding (figure a and step vi) ( , ). na has also been shown to promote the separation of iavs by removing sa residues from the n-linked glycans located on the ha and na molecules in the viral envelope ( figure b and step vii) ( ) . in contrast to ha, nas from human iavs show a general preference for α , -linked sa with variable abilities to cleave α , -linked sa residues ( , , ) . however, a thorough analysis of na sa preference is lacking. more recent studies have found that some strains possess nas that are inefficient enzymes, but still capable of sa binding, raising the question of whether a poor na enzyme could contribute to, or replace, the ha receptor-binding function ( , ) . the movement of iavs from cell to cell in the respiratory epithelium is significantly different from that in immortalized cell lines grown in liquid culture due to the presence of different cell types and a mucus layer. the mucus layer provides a protective barrier for the epithelium and is rich in heavily glycosylated mucins that can interact with iavs and limit cell binding ( , ) . studies measuring viral movement through mucus and respiratory epithelial cells have shown that na-mediated cleavage of sas from mucins enhances iav movement through the mucus layer and infectivity ( figure c and step viii) ( , , ) . recent work showed that this function may also apply to transmission, as iavs that possess low na activity, and are inhibited by mucus, are deficient in aerosol and contact transmission ( ) . iavs are constantly exposed to negative and positive selection pressure, which shapes how the virus evolves. the functional requirements of each iav protein, such as enzyme catalysis, substrate binding, oligomerization, and domains that perform essential interactions with host proteins all combine to create substantial negative selection pressure that often manifests in the form of sequence conservation. negative pressure can also come from functions within the vrna sequences. these include promoters and "packaging signals, " but are also likely to involve aspects such as the formation of structural elements, or possibly mediating vrnp interactions that generate the + assembly in viral particles. in addition, the exposure of iavs to the immune response and constantly changing environments such as host, temperature, ph, cell type, and antivirals result in positive selection pressure. experimentally, addressing each type of selection has its caveats, but clearly a holistic picture of both iav and host functions are required to begin predictions of evolutionary constraints on the virus. most studies on the influenza evolutionary process focus primarily on antigenic drift and antigenic shift. however, all the viral transcribed rnas are subject to replication errors by the viral polymerase, which are estimated at per , - , nucleotides ( ) ( ) ( ) . consequently, both the viruses and the viral proteins are likely to exist as large heterogeneous populations during an infection. as many iav proteins are homo-oligomers this can potentially generate heterogeneity within individual protein complexes that could have functional advantages. by applying single particle and single cell analysis, these types of aspects are beginning to be investigated ( ) . another interesting approach is deep mutational scanning, which has been used to examine the site-specific amino acid tolerance of iav proteins in general, and in the context of different selection pressure ( ) ( ) ( ) ( ) . currently, the best characterized protein in iavs is ha, which has two primary functions, (i) to initiate binding to the host cell and (ii) to deliver the vrnps to the host cell cytosol by fusing the viral and endosomal membranes. these functions are efficiently divided between the two domains of ha (ha and ha ), created by proteolysis. the receptor-binding site responsible for entry is located in the considerably larger ha subunit that is known to be immunodominant, explaining the high sequence variability in this region ( ) . by contrast, the smaller ha subunit, containing the fusion peptide that is necessary to deliver the viral genome to the host cell, shows considerably higher sequence conservation. this organization is logical from the viral perspective as the large ha subunit likely blocks antibody recognition of ha . the viral downside is the need to escape antibodies that inhibit the receptor-binding pocket without losing specificity and the binding function. based on this knowledge, several exciting new strategies are being developed to elicit the production of antibodies that target the more conserved region of ha ( ) ( ) ( ) . the hope is that these strategies will generate broadly neutralizing antibodies that recognize multiple ha subtypes from iavs and the distinct lineages in ibvs, providing longer lasting immunity and alleviating the threat of potential pandemics. a similar approach using na would likely provide additional benefits. however, our knowledge of na lags behind ha. currently, it is still not known why na has evolved to function as a tetramer, which is relevant because this property presumably restricts the potential antigenic drift (mutations) it can accommodate and still function. iav replication and spreading frontiers in immunology | www.frontiersin.org july | volume | article a relatively overlooked feature in the replication process is the contributions of host rna-binding proteins (rbps). human cells are predicted to encode over , rbps, of which are predicted to interact with mrnas ( ) . as a rna virus, it is highly likely that iavs have evolved to utilize this enormous network of rbps, which is supported by observations that some rbps inhibit iav replication, whereas others contribute ( ) ( ) ( ) . it should also be considered that changes in rbps have been associated with various cancers, which could possibly influence the susceptibility to influenza infections ( , ) . with the growing interest in rna biology, this aspect of iav infections is likely to receive considerable attention in the future. in terms of iav antivirals, the recent progress in determining the structures and mechanisms of the viral polymerase should significantly aid in the current development of drugs aimed at inhibiting different aspects of iav transcription ( ) . through continued progress in defining the fundamental mechanisms that are necessary for iav infections, replication and intercellular movement, it should become possible to minimize the annual burden caused by iavs. rd wrote the review with input from dd, rr, hÖ, and hw. dd, rr, hÖ, and hw put together the figures and wrote the figure legends. characterization of a novel influenza virus in cattle and swine: proposal for a new genus in the orthomyxoviridae family mapping of the influenza virus genome: identification of the hemagglutinin and the neuraminidase genes influenza virus genome consists of eight distinct rna species influenza vaccines: challenges and solutions an 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nanoparticles generate heterosubtypic influenza protection structure and function analysis of an antibody recognizing all influenza a subtypes a census of human rna-binding proteins cellular rna binding proteins ns -bp and hnrnp k regulate influenza a virus rna splicing cellular ddx rna helicase inhibits influenza a virus replication but is counteracted by the viral ns protein multiple nuclear-replicating viruses require the stress-induced protein zc h a for efficient growth dissecting the expression landscape of rna-binding proteins in human cancers rna-binding proteins in cancer: old players and new actors inhibitors of influenza a virus polymerase the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord- -n x z authors: zelaya, hortensia; alvarez, susana; kitazawa, haruki; villena, julio title: respiratory antiviral immunity and immunobiotics: beneficial effects on inflammation-coagulation interaction during influenza virus infection date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: n x z influenza virus (ifv) is a major respiratory pathogen of global importance, and the cause of a high degree of morbidity and mortality, especially in high-risk populations such as infants, elderly, and immunocompromised hosts. given its high capacity to change antigenically, acquired immunity is often not effective to limit ifv infection and therefore vaccination must be constantly redesigned to achieve effective protection. improvement of respiratory and systemic innate immune mechanisms has been proposed to reduce the incidence and severity of ifv disease. in the last decade, several research works have demonstrated that microbes with the capacity to modulate the mucosal immune system (immunobiotics) are a potential alternative to beneficially modulate the outcome of ifv infection. this review provides an update of the current status on the modulation of respiratory immunity by orally and nasally administered immunobiotics, and their beneficial impact on ifv clearance and inflammatory-mediated lung tissue damage. in particular, we describe the research of our group that investigated the influence of immunobiotics on inflammation–coagulation interactions during ifv infection. studies have clearly demonstrated that hostile inflammation is accompanied by dysfunctional coagulation in respiratory ifv disease, and our investigations have proved that some immunobiotic strains are able to reduce viral disease severity through their capacity to modulate the immune-coagulative responses in the respiratory tract. influenza virus (ifv) is a major respiratory pathogen of global importance, and the cause of a high degree of morbidity and mortality, especially in high-risk populations such as infants, elderly, and immunocompromised hosts. given its high capacity to change antigenically, acquired immunity is often not effective to limit ifv infection and therefore vaccination must be constantly redesigned to achieve effective protection. improvement of respiratory and systemic innate immune mechanisms has been proposed to reduce the incidence and severity of ifv disease. in the last decade, several research works have demonstrated that microbes with the capacity to modulate the mucosal immune system (immunobiotics) are a potential alternative to beneficially modulate the outcome of ifv infection. this review provides an update of the current status on the modulation of respiratory immunity by orally and nasally administered immunobiotics, and their beneficial impact on ifv clearance and inflammatory-mediated lung tissue damage. in particular, we describe the research of our group that investigated the influence of immunobiotics on inflammation-coagulation interactions during ifv infection. studies have clearly demonstrated that hostile inflammation is accompanied by dysfunctional coagulation in respiratory ifv disease, and our investigations have proved that some immunobiotic strains are able to reduce viral disease severity through their capacity to modulate the immune-coagulative responses in the respiratory tract. keywords: immunobiotics, influenza virus, inflammation, coagulation, respiratory immunity introduction influenza virus (ifv) is a member of the orthomyxoviridae family that contains a negative-sense, single-stranded, segmented rna genome protected by a capsid of viral ribonucleoproteins. this virus is categorized into subtypes based on the expression of hemagglutinin (ha) and neuraminidase on the surface of the viral envelope. influenza is a highly contagious viral infection that has a substantial impact on global health and ifv is a major respiratory pathogen that causes a high degree of morbidity and mortality, especially in high-risk populations such as infants, elderly, and immunocompromised hosts. given the high capacity of ifv to change antigenically, acquired immunity is often not effective to limit infection and therefore vaccination must be constantly redesigned to achieve protection. improvement of respiratory and systemic innate immune mechanisms has been proposed to reduce the incidence and severity of ifv disease. in the last decade, several research works have demonstrated that microbes with the capacity to modulate the mucosal immune system (immunobiotics) are a potential alternative to beneficially modulate the outcome of ifv infection. this review provides an update of the current status on the modulation of respiratory immunity by orally and nasally administered immunobiotics, and their beneficial impact on ifv clearance and inflammatorymediated lung tissue damage. in particular, we describe the research of our group that investigated the influence of immunobiotics on inflammation-coagulation interactions during ifv infection. studies have clearly demonstrated that hostile inflammation is accompanied by dysfunctional coagulation in respiratory ifv disease, and our investigations have proved that some immunobiotic strains are able to reduce viral disease severity through their capacity to modulate the immune-coagulative responses in the respiratory tract. the first barrier that protects the host against ifv infection is the respiratory epithelium through its capacity to recognize the viral attack. when ifv successfully overcomes the respiratory barrier constituted by the mucus layer and the ciliar movement, it mediates its attachment and internalization into respiratory epithelial cells to start its replication ( ) . during the viral attack, several pathogen-associated molecular patterns (pamps) are exposed and recognized by pattern-recognition receptors (prrs) expressed in respiratory cells (figure ). it is now well established that the most important prrs involved in the recognition of ifv are the toll-like receptor (tlr)- and tlr and the rna recognition protein rig- ( ) . tlr is expressed in endosomes and is able to recognize viral double-stranded rna (dsrna) that is produced during viral replication; while endosomal tlr and cytoplasmic rig-i recognize single-stranded rna (ssrna). rig-i signals through mitochondrial antiviral signaling protein. the pamps-pprs interaction leads to the activation of several signaling pathways that induce the activation of nuclear factor κb (nf-κb) and interferon (ifn) regulatory factor (irf ) and the production of type i and iii ifns and inflammatory cytokines ( ) . type i ifns, especially ifn-β, produced and released during the earlier stages of ifv infection are key to develop an antiviral state in the respiratory tract. it was reported that human bronchial epithelial cells release preformed ifn-β in response to ifv challenge inducing a protective role ( ) . ifns produced by infected cells are able to act in a paracrine or autocrine manner activating their receptors (ifnar) and increasing the expression of hundreds of genes that counteract viral replication. functional genomic studies have identified several of the ifn-induced factors that have important roles in controlling ifv replication ( ) including the ifn-inducible transmembrane proteins , , and ( ), mx proteins ( ) , and ′, ′-oligoadenylate synthetase (oas)-rnaasel system ( ) . proinflammatory cytokines and chemokines produced as a result of tlr and rig-i activation during ifv infection are also important for the generation of the respiratory antiviral innate immune response. infection of epithelial cells by ifv increases the expression of tnf-α, il- , il- , ccl (mip- ), ccl (rantes), ccl (mip- α), and cxcl (ip- ) ( ). the production of these cytokines is complemented by activity of inflammasomes that induce the activation of caspase- and promote the generation of the active forms of il- β and il- (figure ) . ifv has been shown to activate mainly the nlrp inflammasome which is essential for the protection against the virus since several studies demonstrated that mice lacking nlrp or caspase- have decreased il- β and il- secretion and increased mortality after ifv challenge ( ) ( ) ( ) . the proinflammatory cytokines and chemokines are responsible for the activation of resident immune cells such as innate lymphoid cells, alveolar macrophages, and dendritic cells (dcs) as well as for the recruitment of neutrophils, macrophages, and lymphocytes into the respiratory tract ( , ) (figure ) . respiratory cells infected with ifv express ha on their surface that is important for its recognition by nk cells ( ) . it was established that ha expressed by the infected cells is recognized by nkp and pkp receptors of nk cells that then mediated the lysis of ifv-infected cells ( ) . macrophages activated during ifv infection produce ifns, il- , tnf-α, and nitric oxide synthase that amplify the inflammatory response. in addition, macrophages limit the viral spread by the elimination of apoptotic-infected cells and through phagocyte-mediated opsonophagocytosis of ifv ( ) . the production of proinflammatory cytokines during the generation of the respiratory innate immune response against ifv also conditions the adaptive immune response, which includes the production of virusspecific systemic and mucosal antibodies as well as the induction of specific t cell responses ( ) . after exposure to ifv there is an activation of antibody responses in the respiratory tract. upper airway exposure results primarily in an iga response while the contact of ifv with the deep lung induces an increased production of pathogen-specific igg ( ) . following exposure to ifv in the airways there is an antigen uptake and processing by dcs, activation of cd + th cells, and generation of iga-producing plasma cells that populate airway lamina propria. secretory iga has a noninflammatory protective function since these antibodies can bind to virus without activating complement or stimulating the release of inflammatory mediators by innate immune cells ( , ) . iga prevents ifv from adhering to the epithelial surface by inducing viral agglutination, and masking adhesion epitopes. in the deep lung, when ifv reach the alveolar space, there is a differentiation and expansion of antibody-secreting plasma cells that are committed to the production of igg. induction of neutralizing respiratory and serum igg antibodies is a key event in the defense against influenza infection since igg prevents systemic spread ( ) . influenza infection in the lungs also activates the cellular adaptive immune response by stimulating the production of ifn-γ by th cells that effectively activate cd + t cells and macrophages, which clear virus and infected cells from the lungs ( ) . therefore, during uncomplicated influenza, adaptive immune response ultimately results in clearance of ifv from the lungs through the activity of virus-specific antibodies and cd + and cd + t cells. the gut microbiome, which is defined as the collective group of microorganisms and their associated genes within the intestinal tract, is considered as a key player in the modulation of host intestinal immune responses ( , ) . in fact, the impact of gut commensal bacteria on the innate and adaptive immune responses to enteric pathogens has been recognized conclusively ( ) ( ) ( ) . however, the effect of gut microbiome on the immune responses in distal mucosal sites and its impact in the outcome of respiratory infections has recently been exposed. in this regard, some studies have demonstrated an important role for intestinal microbiota in maintaining respiratory antiviral immunity against ifv ( , ) . iwasaki and colleagues observed that commensal bacteria within the gut, especially gram-positive bacterial populations, had an important role in supporting an appropriate immune response to ifv infection in the respiratory tract ( ) . the work demonstrated that oral antibiotic treatments impaired the resistance of mice to the intranasal infection with ifv as noted by the elevated lung viral titers when compared to non-antibiotictreated animals. results indicated that gut gram-positive bacteria provided protection by triggering an adequate inflammatory response through inflammasomes activation. in antibiotictreated mice, synthesis of pro-il- β, pro-il- , and nlrp was impaired even at the steady state. in addition, depletion of grampositive bacterial populations in the gut resulted in an alteration of the distribution and activation of respiratory dcs at steady state as well as in a diminished dcs migration from the lung to the draining lymph nodes, resulting in reduced activation of cd + and cd + t cells after influenza challenge ( ) . alteration of respiratory dcs activities also correlated with impaired expansion of influenza-specific b cells and reduced influenza-specific antibodies. by using germ-free and antibiotic-treated mice challenged with ifv, abt et al. ( ) showed that the absence or the alteration of intestinal microbiota induced an exacerbated weight loss, a greater drop in blood oxygen saturation, increased mortality, and elevated lung viral titers indicating a weaker ability to resist influenza. even more, germ-free and antibiotic-treated mice infected with ifv experienced higher epithelial cell necrosis, peribronchiolar inflammation, severe bronchiole epithelial degeneration, and epithelial hyperplasia when compared to conventional animals ( ) . interestingly, those effects were observed when both the pr strain and the x -gp virus, a less pathogenic strain of ifv that causes minimal mortality and morbidity in conventional mice, were used. consistent with the work by ichinohe et al. ( ) , germ-free and antibiotic-treated mice challenged with ifv had an impaired adaptive immune response as shown by the lower influenza-specific antibodies (serum igm and igg), fewer number of ifv-specific t cells present in lungs, as well as a reduced capacity of specific t cells to produce effector cytokines such as tnf-α, mip- α, il- , and ifn-γ ( ) . moreover, authors demonstrated that the alterations of adaptive immune responses were related to defects in the early innate immune response mediated by macrophages. in fact, transcriptional profiling and computational analyses of macrophages from antibiotic-treated mice indicated a reduced expression of antiviral genes including ifnb, tnfa, il b, irf , mx , and oas a when compared to immunobiotics for influenza virus infection frontiers in immunology | www.frontiersin.org december | volume | article conventional mice. in addition, functional assays of macrophages from antibiotic-treated mice demonstrated that those cells had a defective response to type i ifns and an impaired capacity to limit ifv replication ( ) . the cellular and molecular mechanisms through which the gut microbiome and their derived signals maintain and modulate immune responses in distal mucosal sites are poorly understood. two possible mechanisms that are not mutually exclusive have been proposed to explain this beneficial effect of the gut microbiome. one possibility is that distal mucosal and peripheral immune cells are directly exposed to bacterial products that activate prrs in the steady state and help to maintain the normal immune tone. there is evidence that bacterial products from gut commensals such as peptidoglycan can be absorbed and circulate throughout the host and help to modulate the normal development of immune cells ( ) . in line with this hypothesis, iwasaki and colleagues speculated that bacterial products from gut commensals trigger prrs to stimulate immune cells systemically and that factors released by those cells supported steady-state production of pro-il- β, pro-il- , and nlr proteins. this idea was sustained by their observation that intestinal injection of tlr ligands restored immune responses to ifv in antibiotic-treated mice ( ) . another possibility is that commensal bacteria may indirectly influence systemic and distal mucosal immune responses through immune factors released from the intestinal mucosa including cytokines, chemokines, and grow factors. these research works demonstrated that the gut microbiome provides signals to sustain antiviral innate immune defense mechanisms in the respiratory tract allowing robust and efficient effector responses upon challenge by viral pathogens such as ifv. therefore, the role of the gut microbiome in regulating respiratory antiviral immunity represents an exciting area of research that could help to provide the scientific basis for the development of novel prevention strategies for lung infectious diseases. however, several questions need to be answered to identify new alternatives to improve antiviral respiratory defenses by modulating the microbiota. how the different microbial species from the gut microbiota influence the common mucosal immune system? which prrs are activated by the gut microbiota to functionally modulate antiviral immunity locally and in distal mucosal sites? which cellular functions are modulated by the microbiota after prr activation? has the microbiota the ability to influence immune responses to other respiratory viruses? are similar immune mechanisms activated by the microbiota in high-risk populations (infants, elderly, immunocompromised hosts) in which respiratory viral infections are more frequent and severe? is it possible to beneficially modulate antiviral respiratory defenses by orally administering selected microorganisms with immunomodulatory capacities? research from the last years has provided some answers for the last question. the first studies that assessed the capacity of immunobiotics to favorably modulate the immune response against ifv focused on the humoral immunity ( table ) . yasui et al. ( ) reported that the oral administration of bifidobacterium breve yit improved the production of anti-ifv igg antibodies in serum of ifv-infected mice. the yit strain reduced viral titers, improved the survival rate, and decreased the severity of the symptoms associated to the influenza infection. similarly, it was shown that orally administered non-viable lactobacillus pentosus b ( ) or viable lactobacillus brevis kb ( ) were able to improve the levels of respiratory specific iga and igg antibodies of mice challenged with ifv. moreover, the improved humoral response induced by these strains correlated with significant reduction of viral titers, body weight loss, and a decrease of the alterations of physical conditions induced by ifv. more recently, kikuchi et al. ( ) demonstrated a beneficial effect on the outcome to ifv infection related to an improved respiratory humoral response in lactobacillus plantarum ayatreated mice. in addition, the work proposed a mechanism for the distal immunomodulatory activity induced by orally administered immunobiotics. authors showed that l. plantarum aya fed to mice impacted in peyer's patches (pps) inducing an activation of antigen presenting cells (mainly cd b + dcs) and increasing the production of il- . those changes promoted an igm-to-iga class switch recombination, the differentiation of iga + b cells into plasma cells, and improved the production of mucosal iga in both the intestine and the respiratory tract. those studies show that immunobiotics are capable to modulate the production of systemic and mucosal antibodies against influenza and therefore, to enhance the humoral immune response (figure ) . however, the precise mechanism by which orally administered immunobiotics induce iga production in distant mucosal sites remains unclear. it was also demonstrated that immunobiotics are able to improve cellular immune response against ifv (figure ) . in this regard, it was reported that orally administered lactobacillus casei shirota improved the outcomes of ifv infection of aged ( ) and infant mice ( ) by increasing systemic and respiratory nk cell activity and improving the production of ifn-γ and tnf-α by respiratory lymphocytes. both studies also demonstrated that ifv titers were significantly reduced in aged and infant mice treated with the shirota strain ( , ) . similar to the mechanism proposed to explain the improvement of humoral response, it was postulated that immunobiotic l. casei shirota stimulated th cells and nk cells in pps and induced a mobilization of those cells to lungs and respiratory-associated lymphoid tissues where they produced ifn-γ and enhanced the antiviral defenses. several other studies corroborated these findings by showing similar effects for orally administered lactobacilli ( , ) . immunobiotic lactobacillus strains (l. gasseri tmc , l. rhamnosus gg, or l. plantarum cc ) beneficially modulated nk cells activity and th response against ifv, diminished virus titers and reduced lung pathological changes ( , ) ( table ) . more recently, kawahara et al. ( ) described the improvement of respiratory antiviral response by an orally administered bifidobacteria strain. it was shown that bifidobacterium longum mm- increased respiratory nk cell activity and ifn-γ production resulting in improved clinical symptoms, reduced mortality, and decreased virus titers after ifv challenge. research work has also demonstrated that nasal administration of immunobiotics is an interesting alternative to improve cellular response against influenza infection ( - ) ( table ) . hori et al. ( ) observed that balb/c mice nasally treated with non-viable l. casei shirota had increased levels of il- , ifnγ, and tnf-α in mediastinal lymphoid nodes and lungs. this improved cellular respiratory immunity correlated with a beneficial clinical outcome to ifv challenge. similar observations were performed with nasally administered l. pentosus s-pt ( ) and l. rhamnosus gg ( ). other recent studies have also demonstrated the ability of immunobiotics to improve respiratory innate antiviral defenses in the respiratory tract (table ; figure ) . it was reported that orally administered non-viable l. plantarum l- improved protection against ifv by increasing type i ifn production ( ). the work clearly demonstrated that the increased production of ifn-β induced by the immunobiotic strain correlated with the reduction of viral loads in lungs as well as the improved survival of infected mice. more recently, it was shown that l. gasseri sbt enhanced survival rates and reduced lung viral titers in mice infected with ifv ( ). interestingly, authors observed that the lung expression of the antiviral genes mx and oas a was enhanced in l. gasseri sbt -treated mice and that the inflammatory response triggered by ifv was differentially regulated inducing a lower inflammatory damage ( ). our group has also reported a beneficial regulation of the ifv-triggered inflammatory response by immunobiotics. lung damage induced by ifv is known to be produced by virus replication as well as the uncontrolled inflammatory response that is characterized by a hypersecretion of proinflammatory cytokines, especially tnf-α, il- β, and il- ( ). the adequate production of inflammatory factors is necessary to protect against ifv infection together with an appropriate regulation with anti-inflammatory cytokines to prevent the damage of lung tissue. thus, the proper balance of cytokines is a key factor in determining the outcome of ifv infection. in this regard, we observed that orally ( ) or nasally ( ) administered immunobiotic l. rhamnosus crl differentially regulated the levels and kinetics of inflammatory cells and cytokines in mice after ifv challenge. in our experimental model, we observed increased levels of respiratory tnf-α, il- , neutrophils, and macrophages in crl -treated mice early after the challenge with ifv. later, proinflammatory cytokines and infiltrated cells started to decrease in immunobiotic-treated animals in contrast to control mice, in which those parameters continued increasing. the trend toward lower inflammatory factors and cells registered later during ifv infection in l. rhamnosus crl -treated mice correlated with a reduced severity of pulmonary damage when compared to control mice ( , ). chen et al. ( ) also investigated the ability of orally administered enterococcus faecalis kh to beneficially modulate the innate immune response to influenza infection. authors observed that kh strain protected c bl/ mice against ifv as observed by the reduced mortality, weight loss, and lung viral titers. as expected, ifv enhanced the levels of proinflammatory mediators in the respiratory tract including il- , tnf-α, ifn-γ, il- β, il- , and mcp- while the treatment with e. it is not clear how immunobiotics initiates the cross-talk with the immune system in order to modulate the respiratory antiviral immunity. it is not known exactly which prrs are activated by immunobiotics in the intestinal or respiratory mucosa to functionally modulate antiviral immunity locally and in distal mucosal sites, respectively. neither it has been determined with exactitude which cellular functions are modulated by immunobiotics immediately after prr activation. research from the last decade has demonstrated that the immunomodulatory effects of probiotic bacteria are the consequence of complex interactions between several bacterial molecules and host receptors located in different immune and non-immune cells ( , ). it has also been shown that the immunomodulatory properties of immunobiotics are dependent on the strains. therefore, studies carried out with certain strains cannot be easily extrapolated to other bacteria, even those of the same genus and species ( , ). consequently, it is still necessary to carry out deeper studies to find out the molecular mechanisms by which immunobiotics beneficially influence the respiratory antiviral immunity. the studies mentioned before showed the potential of immunobiotics to be used for the reduction of the incidence and severity of ifv infections. however, in addition to deepening the knowledge of their mechanisms of action, several other points should be considered for the efficient application of immunobiotics in humans. for example, it is necessary to determine the best time as well as the most appropriate route for their administration. immunobiotics used as components of functional foods can be included in diets on a regular basis and thus help to improve respiratory defenses, especially in high-risk populations and during the seasons with the highest incidence of respiratory infections occurs. in this sense, in a randomized controlled trial we demonstrated that l. rhamnosus crl (administered in a yogurt formulation) improved mucosal immunity and reduced the incidence and severity of intestinal and respiratory infection in children ( ). hence, the incidence of infectious events was reduced from % in the placebo group to % in the group that received the probiotic yogurt. furthermore, there was also a significant reduction in the occurrence of indicators of disease severity such as fever and the need for antibiotic treatment in children receiving the probiotic yogurt. this immunobiotic yogurt (yogurito ® ) has been included into official national nutritional programs in argentina and is given daily to children at schools in several provinces thanks to the government actions. epidemiological studies in the schools receiving the immunobiotic product have shown a reduction in the incidence of infections and in the associated school absenteeism (alvarez et al., unpublished results). on the other hand, as mentioned earlier the nasal administration of immunobiotics is more efficient than the oral administration to enhance respiratory immunity. this route of administration poses a practical disadvantage considering that the treatments with immunobiotics showed favorable results when they were used before the infectious challenges. in this way, it would be necessary to predict the exact moment in which the viral pathogen will be in contact with the host in order to carry out the prophylactic immunobiotic treatment. this option could be used for example during a school or work outbreak in which cases of respiratory infections occur and it is desired to prevent or reduce the severity of infections in asymptomatic individuals. for an intervention of these characteristics, it would be also important to determine the exact time after the contact with the virus in which it is possible to administer immunobiotics to achieve the beneficial effect. in a recent study, percopo et al. ( ) have defined this as "the window of opportunity. " the work evaluated the effect of the nasal administration of live or inactivated l. plantarum ncimb in a mice model of severe respiratory infection with the pneumonia virus of mice (pvm) and found that immunobiotic treatment promoted full survival from acute pvm infection when administered within day after virus challenge ( ) . similar studies would be of value in ifv infection models. another point of interest is related to the duration of the improvement of respiratory defenses after the last immunobiotic administration. our studies have showed that the immunomodulatory effect of some nasally administered immunobiotics persisted for at least days (villena et al., unpublished results) . other studies have also reported short-term protection after nasal treatment with different immunobiotic strains ( ). interestingly, garcia-crespo et al. ( ) found that adult mice primed nasally with l. plantarum ncimb or lactobacillus reuteri f were completely protected against lethal pvm infection and that protection persisted for at least months after the initial priming. these findings open an interesting challenge in the study of immunobiotics to improve the defenses against ifv, since it would be very useful to establish the duration of the protective effect for each strain and treatment, since in the majority of cases these long-term studies were not taken into account. ifv infections often result in mild to moderate lung infection; however, life-threatening disease can occur. it has been demonstrated that the most severe disease outcomes are associated with secondary bacterial pneumonia caused primarily by staphylococcus aureus or streptococcus pneumoniae ( ) . taking into account the high incidence of viral infections and the frequency of associated secondary bacterial infections which contribute to aggravate the health status of the host and reduce its chance of recovery, various approaches for preventing and treating influenza and secondary bacterial pneumonia are been investigated. a wide range of antibiotics and anti-inflammatory drugs has been tested in mice [reviewed in ref. ( ) ]. it would be of interest to evaluate the potential beneficial effect of immunobiotics on these circumstances. in this regard, preliminary studies from our laboratory showed that nasally administered l. rhamnosus crl is able to improve survival, reduce bacterial cell counts in lung and blood, and limit lung inflammatory damage caused by s. pneumoniae infection in mice produced after the infection with ifv or respiratory syncytial virus (rsv) (villena et al., unpublished results) . these results opened an interesting topic for future investigations. finally, it would be also of interest to investigate whether immunobiotic treatments may influence other physiological systems involved in the defenses against viral respiratory infections such as the coagulation system. our group has made some progress in this regard, as mentioned below. coagulation is an extremely ordered process that involves the interaction of three key components: endothelial cells (ecs), platelets, and coagulation factors. tissue injury that activates ecs typically initiates coagulation that is characterized by the binding of platelets to activated ecs and the formation of the platelet plug. almost simultaneously, tissue factor (tf) released by ecs result in factor x activation, which induces thrombin and the generation of fibrin strands to strengthen the platelet plug leading to a stable platelet-fibrin clot. all these processes are tightly regulated by anticoagulant and fibrinolytic mechanisms to avoid thrombotic and/or haemorrhagic complications. a key role has been attributed to ecs in the temporal and special regulation of coagulation activation. resting ecs avoid the inappropriate plug formation by controlling platelet adhesion and activation and generating several anticoagulant factors providing a non-thrombogenic barrier ( , ) . once activated or injured, ecs expose collagen to blood, increase platelet binding and aggregation, reduce the expression physiological anticoagulant factors, increase the expression of tf and von willebrand factor, and suppress the fibrinolytic activity ( , ) . all these changes in the hemostatic system facilitate thrombosis in the infected or inflammated tissue. both hemorrhagic and thrombotic complications have been described during ifv infection. influenza is able to cause pulmonary hemorrhage and edema related to coagulopathy or induce uncontrolled thrombosis through an over-activated coagulation (figure ) ( , ) . animal models have helped to explain the mechanisms by which ifv infection activates coagulation and key role has been attributed to tf. it was described that ifv activates coagulation by enhancing tf production, thrombin generation and fibrin deposition in c bl/ mice ( ) . in a mice model of ifv infection, it was recently shown that wild-type animals increased lung tf expression and activation of coagulation but presented alveolar hemorrhage ( ) . moreover, selective deletion of tf in epithelial cells from lung significantly reduced tf expression after ifv infection and had higher alveolar hemorrhage and immunobiotics for influenza virus infection frontiers in immunology | www.frontiersin.org december | volume | article reduced survival than controls. on the contrary, deficiency of tf in either respiratory myeloid cells or ecs did not enhanced alveolar hemorrhage or modified survival of ifv-infected mice ( ) . these results indicate that an appropriate modulation in the production of tf in the lung during ifv infection is necessary to maintain tissue hemostasis avoiding hemorrhage and excessive fibrin deposition. production of tf by lung epithelial cells will be required to maintain alveolar hemostasis during ifv infection, while excessive release of tf by macrophages and ecs would contribute to pathology and lung tissue injury ( , ) . it is considered that ecs may play an important role in the pathogenesis of ifv. influenza infection is able to induce alveolar edema and pulmonary hemorrhage through the alteration of ecs via several mechanisms, including direct damage and loss of tight junctions and apoptosis ( ) . in addition, recognition of damageassociated molecular patterns such as hmgb or oxidized phospholipids through tlr activates ecs to drive lung injury ( ) . direct stimulation of tlr by viral rna also results in the upregulation of tf and the downregulation of thrombomodulin (tm) in ecs ( ) . at the same time, the inflammatory activation of ecs leads to the activation of the coagulation cascade. inflammation caused by ifv infection increases various proinflammatory cytokines such as tnf-α, il- β, and il- that induce the secretion of tf by ecs and monocytes ( ) . in addition to their roles in coagulation, activated proteins such as thrombin, fxa, and fviia also enhance the inflammatory response. the inflammatory potentiating abilities of coagulation factors are mediated through their activation of protease-activated receptors (pars) that are expressed in platelets, ecs, macrophages, and respiratory epithelial cells ( ) . the tf/thrombin/par- pathway has been associated to the promotion of a deleterious innate inflammatory response to ifv infection in mice ( , ) . therefore, both the hyper-inflammatory response and the aberrant activation of coagulation, which are potentiated with each other, are involved in severe influenza pneumonia and are key events that have to be controlled in order to reach a favorable resolution of the infectious process. considering the importance of the coagulative response in the outcome of influenza infection and the ability of immunobiotics to beneficially influence the immune response to this respiratory pathogen, we wonder whether some immunobiotic strains would be able to beneficially modulate the immuno-coagulative response triggered by ifv. for this purpose, we performed challenge-infection experiments in mice and evaluated the influence of viable and non-viable immunobiotic l. rhamnosus crl strain on the respiratory immuno-coagulative response induced by ifv ( , ) . our data demonstrated that oral administration of l. rham nosus crl to mice significantly reduced lung viral titers and tissue damage after the challenge with ifv ( ). we later explored the capacity of nasally administered l. rhamnosus crl , alive or heat killed, to reduce the influenza burden of disease ( ). those treatments induced a significant decrease in ifv titers in lungs, lessened pulmonary damage, and increased survival. interestingly, a similar effect was achieved with the nasal administration of viable and non-viable crl strain. moreover, the nasal route was more efficient than the oral administration to protect mice against ifv infection ( , ). the protective effect achieved by the immunobiotic strain was related to its ability to modulate the respiratory antiviral immune response, particularly to its capacity to improve the levels of ifn-γ and ifn-β in the respiratory tract (figure ) . type i ifns trigger the activation of the jak-stat pathway and increase the expression of antiviral genes. in addition, ifn-γ is produced by immune cells, especially th cells, and it further improves antiviral immune response by inducing activation of nk cells and macrophages. therefore, the modulation of type i ifns and ifn-γ would be responsible of the reduction of viral loads in ifv-infected mice previously treated with the crl strain, similarly to other immunobiotic strains as mentioned before ( table ) . we demonstrated that the crl strain increased the levels of gut cd + cd + ifn-γ + t cells, induce a mobilization of these lymphocytes into the lung and enhanced the respiratory production of ifn-γ and the activity of local antigen presenting cells ( , , ) . it was also noted that nasal administration was more effective than the oral route to increase pulmonary cd + cd + ifn-γ + t cells ( , ). the mechanism by which nasally administered viable or heat-killed l. rhamnosus crl improves ifn-γ + t cells population is not clear. however, our studies support the possibility that the immunobiotic strain l. rhamnosus crl impact in the nasalassociated lymphoid tissue or bronchial-associated lymphoid tissue producing an innate imprinting in antigen presenting cells that contribute to the enhanced number and activity of cd + cd + ifn-γ + t cells. our studies also showed that immunobiotic treatments were able to beneficially modulate the activation of coagulation during respiratory viral infection, an effect that was not reported before ( , ). then, our studies were the first in demonstrating a beneficial modulation of the immune-coagulative response during respiratory trl activation and ifv infection induced by immunobiotic microorganisms (figure ) . although ifv is an ssrna virus, it generates dsrna replication intermediates that activate tlr and contribute to the initiation of the antiviral respiratory immune response. in fact, ifv triggers type i ifn secretion through tlr recognition in immune (myeloid dcs or macrophages) and non-immune (fibroblasts or pneumocytes) cells ( ) . challenge-infection experiments with respiratory viruses in tlr −/− mice showed that tlr does not modify the clearance of viral pathogens but it is relevant for the modulation of the lung inflammatory response ( , ) . it was showed that wild-type mice mount a robust inflammatory response in the lung after ifv infection and that this process is significantly diminished in tlr −/− animals ( ) . tlr −/− mice showed a longer survival when compared wild-type animals and this effect was associated with a reduction of inflammatory cells recruitment and lower levels of inflammatory factors in the respiratory tract. other in vivo studies also demonstrated that tlr activation by poly(i:c) enhanced proinflammatory cytokines and immunobiotics for influenza virus infection frontiers in immunology | www.frontiersin.org december | volume | article antiviral factors expression ( ), altered vascular permeability ( ) , and incremented the levels of d-dimers indicating that coagulation and fibrinolysis were triggered. in line with these findings, it was observed that the levels of d-dimers in tlr −/− mice were significantly lower than in wild-type animals after poly(i:c) administration ( (neutrophils and macrophages) and proinflammatory mediators (il- β, tnf-α, il- , and il- ) in the respiratory tract. moreover, tlr activation also induced an increase in tf expression and thrombin-antithrombin complex (tatc) levels in the lung while it reduced tm expression. these inflammatory-coagulative modifications were accompanied by respiratory tissue alterations and impairment of lung function ( , ) . of interest, we demonstrated that orally ( ) or nasally ( ) administered immunobiotics before the challenge with poly(i:c) differentially modulated the inflammatory-coagulative response. l. rhamnosus crl was able to reduce and increase the expression of tf and tm, respectively, after the respiratory activation of tlr . thus, the crl strain significantly diminished coagulation activation in blood and in the respiratory tract after the nasal stimulation with poly(i:c). we also evaluated pulmonary coagulation during ifv infection ( , ). the respiratory virus induced activation of coagulation in the lungs of infected mice as demonstrated by the increased levels of respiratory tatc. these procoagulant changes were related to alterations in the expression of tm and tf in lungs. our findings are in line with previous studies in humans and animal models of influenza infection demonstrating increased lung fibrin deposition and enhanced numbers of intravascular thrombi in the respiratory tract ( , , ) . we demonstrated that immunobiotic treatment is able to significantly diminish the activation of coagulation in ifv-challenged mice. in fact, lower levels of respiratory tatc and a reduced expression of tf was observed in l. rhamnosus crl -treated mice infected with ifv when compared to controls ( , ). as mentioned before, ifv promote a procoagulant state directly through its capacity to infect ecs and monocytes stimulating the expression of tf ( , ) . in addition, ifv induce activation of coagulation indirectly by the enhancement of proinflammatory factors such as il- ( , ) . therefore, the ability of immunobiotics to modulate the ifv-triggered immune-coagulative response could be explained by their direct influence on viral replication related to the enhancement of the antiviral state in the respiratory mucosa, and indirectly through the modulation of the inflammatory response. considering this last point, we performed experiments using anti-il- r blocking antibodies in order to evaluate the role of the regulation of the inflammatory response in the reduction of coagulation activation. results showed that il- is important for the regulation of coagulation induced by the immunobiotic l. rhamnosus crl ( ). blocking of il- r abolished the capacity of the crl strain to change the expression of tm and tf in the lungs. this was in line with our previous studies evaluating the ability of l. rhamnosus crl to confer protection against inflammatory damage induced by tlr activation or rsv infection, which showed that il- is a key factor for the reduction of lung injury ( ) . additionally, it was demonstrated that lethal disease caused by ifv infection is prevented by il- administration through the reduction of lung immunopathology ( ) . moreover, tf expression and procoagulant activity of macrophages and ecs are reduced by il- ( , ) . therefore, we demonstrated that immunobiotic administration induce an early increase in the levels of tnf and il- in the respiratory tract after poly(i:c), rsv, or ifv challenge, while the levels of those proinflammatory factors are significantly reduced later during infection ( , , ) . the early increase of proinflammatory mediators and the augmented levels of ifn-γ explain the ability of l. rhamnosus crl to diminish viral replication while the improved production of il- would lead to a beneficial modulation of the immune-coagulative response which results in a reduced severity of lung damage. it has been suggested that respiratory viral infections increase the risk of venous thromboembolism and ischemic heart disease through ecs perturbation, coagulation activation, reduction of anticoagulant factors, and inhibition of fibrinolysis ( ) ( ) ( ) . then, our studies suggest that immunobiotics could be an interesting alternative not only to reduce the incidence and/or severity of respiratory viral infections, but in addition to reduce the risk of atherothrombotic alterations associated to respiratory viral infections. research from the last decade has clearly demonstrated that beneficial microorganisms are able to modulate respiratory tract immunity and promote the resolution and lessen the severity of respiratory infections caused by pathogens such as ifv. studies in animal models have demonstrated that orally or nasally administered immunobiotics are able to improve protection against ifv by three main mechanisms. first, immunobiotics increase the respiratory antiviral state by their capacity to improve levels of type i ifns, the number and activity of antigen presenting cells, nk cells, cd + ifn-γ + t, and iga + b lymphocytes, as well as the levels of systemic and mucosal specific antibodies. second, immunobiotics beneficially modulate the ifv-triggered respiratory inflammatory response by inducing changes in the levels and kinetics of proinflammatory factors and immunoregulatory cytokines such as il- that allow the clearance of virus with a minimal inflammatory lung tissue damage. finally, as demonstrated by our recent research works, immunobiotics modulate lung immune-coagulative response triggered by tlr activation or ifv infection, mainly by downregulating lung tf and restoring tm levels. studies in animal models suggest that immunobiotics would influence principally the innate immune response, modulating in that way the early antiviral inflammatory response and the subsequent cellular and humoral immune responses. therefore, immunobiotics would have mainly an adjuvant effect. however, the exact molecular mechanisms by which immunobiotics differentially modulate the innate antiviral immune response against ifv remain to be elucidated. additionally, a growing number of studies in humans have examined the effect of immunobiotics on the incidence and severity of ifv infection. considering the impact of immunobiotics in the innate immune response clinical studies have evaluated principally their potential adjuvant effects on ifv vaccination ( table ) . although mechanistic studies have not been addressed in depth, there is promising evidence for beneficial effects of immunobiotics on human respiratory health and resistance against ifv. these observations might be helpful to propose new influenza a penetrates host mucus by cleaving sialic acids with neuraminidase the 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and release of active tissue factor-bound microparticles anticoagulant properties of the anti-inflammatory cytokine il- in a factor xa-activated human monocyte model risk of myocardial infarction and stroke after acute infection or vaccination risk of deep vein thrombosis and pulmonary embolism after acute infection in a community setting crosstalk between inflammation and thrombosis oral intake of lactobacillus fermentum cect enhances the effects of influenza vaccination a probiotic fermented dairy drink improves antibody response to influenza vaccination in the elderly in two randomised controlled trials lactobacillus gg as an immune adjuvant for live-attenuated influenza vaccine in healthy adults: a randomized double-blind placebo-controlled trial daily intake of heat-killed lactobacillus plantarum l- enhances type i interferon production in healthy humans and pigs evaluation of the immune benefits of two probiotic strains bifidobacterium animalis ssp. lactis, bb- (r) and 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influenza-like illness and immunological response to influenza virus key: cord- -x dq sy authors: wan, dongshan; jiang, wei; hao, junwei title: research advances in how the cgas-sting pathway controls the cellular inflammatory response date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: x dq sy double-stranded dna (dsdna) sensor cyclic-gmp-amp synthase (cgas) along with the downstream stimulator of interferon genes (sting) acting as essential immune-surveillance mediators have become hot topics of research. the intrinsic function of the cgas-sting pathway facilitates type-i interferon (ifn) inflammatory signaling responses and other cellular processes such as autophagy, cell survival, senescence. cgas-sting pathway interplays with other innate immune pathways, by which it participates in regulating infection, inflammatory disease, and cancer. the therapeutic approaches targeting this pathway show promise for future translation into clinical applications. here, we present a review of the important previous works and recent advances regarding the cgas-sting pathway, and provide a comprehensive understanding of the modulatory pattern of the cgas-sting pathway under multifarious pathologic states. pattern-recognition receptors (prrs) serve as innate cellular sensors of danger signals, such as pathogen-associated molecular patterns (pamps) or danger-associated molecular patterns (damps), and yield cellular-stress response. dna molecules are vital genetic components within cells, which are compartmentalized restrictively into specific regions. the occasionally misplaced dna is degraded rapidly by scavenger cells and extracellular or intracellular ribonucleases. aberrant accumulation of dna is relevant to tissue damage ( ) . in , several research teams discovered a new protein on the endoplasmic reticulum (er) which can be activated by immune-stimulatory dna (isd) and initiate type-i interferon (ifn) responses, which was named "stimulator of interferon genes" (sting, also known as mita, eris) ( ) ( ) ( ) . sting does not bind to dna directly, and bacteria-derived cyclic di-guanylate monophosphate (c-dgmp) or cyclic di-adenosine monophosphate (c-damp) were confirmed to be ligands for sting ( , ) . subsequently, it was found that some dna sensors can facilitate sting activation, such as interferon gamma inducible protein (ifi ) ( ) . however, sting activation could not be fully explained by the upstream factors/ligands that had been found. it was postulated that an unknown upstream regulator might be responsible for sting activation. in , wu and sun found that cyclic guanosine monophosphate-adenosine monophosphate (cgamp) was a novel secondary messenger serving as a ligand of sting ( ) . beside it, they purified a new protein named "cyclic-gmp-amp synthase" (cgas) that had cytosolic dna-sensing ability and can synthesize cgamp ( ) . also, they found that the cgas-cgamp-sting pathway was indispensable for host anti-viral immunity ( ) . their work filled in the gaps missing from upstream of sting. stimulator of interferon genes or cyclic-gmp-amp synthase is expressed widely in a broad spectrum of cells including immune, non-immune, cancer cells ( ) . mounting evidence has demonstrated that the cgas-sting pathway is important for mediating cellular immune sensing, and shows particular responses pattern to the isd distinguished from other nucleotide-sensing pathways. it is also regulated delicately by several molecules or feedback loops to maintain cellular homeostasis. nevertheless, cgas-cgamp-sting pathway itself has distinctive or even opposing effects under different conditions. in this review, we cover the roles of cgas-sting pathway in cellular type-i ifn immune response, and several cellular processes including autophagy, survival and senescence. we also summarize the literature on intrinsic cellular mechanisms modulating cgas-sting pathway as well as its cross-regulations with other dna-sensing pathways. moreover, the inflammationmodulation capacities of this pathway in infectious disease, inflammation and cancers have been elucidated too, and a pervasive pattern of this pathway has been described, which could provide a plausible explanation of the contradictory findings of studies. finally, current or prospective therapeutic strategies targeting the pathway, and issues that need to be addressed in the future, are discussed. cyclic-gmp-amp synthase belongs to the structurally conserved cgas/dncv-like nucleotidyltransferase (cd-ntases) superfamily. the latter is expressed universally in prokaryotes and eukaryotes, and can use purines or pyrimidines selectively as substrates for the production of linear or cyclic di-or even tri-nucleotide compounds, which act as secondary intracellular messengers ( ) . cyclic-gmp-amp synthase is distributed mainly in the cytosol (also nucleus in some specific conditions) ( ) . generally speaking, cgas is activated upon the recognition of b-type double-stranded dna (dsdna) without sequence-specificity but not a-type dsdna or rna ( , ) . hybrid dna:rna or stemlike single-stranded dna (ssdna) are also low-affinity ligands for cgas ( , ) . after binding with ligands, cgas undergoes an allosteric structural change, and subsequently catalyzes its substrates guanosine triphosphate (gtp) and adenosine triphosphate (atp) to produce a mixed phosphodiester-linked cyclic dinucleotide: g( - )pa ( - ) p cgamp (abbreviated as , -cgamp or cgamp) ( ) . cgas also catalyze the synthesis of linear dinucleotides such as amp- -atp, gmp- -gtp, and amp- -gtp as intermediate products ( ) . there are two major dsdna-binding sites on opposite sides of the catalytic pocket: a and b site. site a is the primary contact surface for dsdna, whereas site b is complementary, binding another dsdna. it allows for cgas to the formation of a : cgas:dsdna complex structure directed into two orientations with dsdna at least bp ( ) ( ) ( ) (figure a) . increased numbers of back-to-back dimers of cgas hold the two dsdna molecules together and permit successive recruitment of cgas which, consequently, forms a n: cgas:dsdna higherordered "ladder-like" oligomerization, with cgas arrayed "head to head/tail to tail" ( , ) . the dna-binding protein hu, mitochondrial transcription factor a (tfam), or bacterial high mobility group box protein (hmgb ) can bend the dsdna into a u-shaped structure and, thus, facilitate binding of cgas dimers to the same strand as it travels in opposite directions ( ) (figure b) . human cgas, unlike mouse cgas, is prone to formation of this ladder-like network with long dsdna, because of the human-specific residues k and l . these two dsdna-interfacing residues of site a loosen the interaction of dsdna with cgas, leading to dsdna curving and allowing more convenient binding for the next adjacent cgas ( , ) ( figure c) . finally, accumulated cgas-dsdna complexes can go through a liquid-phase separation and condense into gellike droplets as a reaction unit ( figure d) . this conformation requires a sufficiently long dsdna strand to form multivalent interaction positions, also requires the function of the n-terminal tail of cgas and a recently discovered dsdna-binding site in the catalytic domain of cgas (site c) ( , ) . meanwhile, the n-terminal tail of cgas mediates cgas localization onto the membrane by binding to phosphatidylinositol , bisphosphate (pi ( , ) p ) and prevents liberation of cgas and oligomerization, but can release cgas during cell stress ( ). the structure of cgas determines long strand dsdna (> - , bp) could potentially stimulate the enzyme activity and cgamp production of cgas ( ). the ability of human cgas to discriminate long dsdna strands from shorter dsdna may contribute to the specific sensing and recognition of the "danger dna" of pathogens, necrotic cells or cancer cells rather than irrelevant shorter dsdna, thereby enhancing the immunity against them specifically. double-stranded dna is restricted into the nucleus or mitochondria and is rarely present in the cytoplasm. extrinsic dsdna from pathogens such as viruses, bacteria, transcellular vesicles or rupture of dying cells can be internalized into the cytosol in several diverse ways ( - ). these extrinsic dsdna sources are engulfed by endosome through phagocytosis and digested immediately by dnaseii when fusing with lysosomes ( , ). however, some escaping mechanisms under certain conditions could help protect them from being degraded. for example, antimicrobial peptide ll could efficiently transports self-dna from endosome into cytosol of monocytes ( ). cell oxidative stress can lead to phagosomal acidification delay and probably release endosome context including dsdna owing to increased membrane permeability ( , ). the intrinsic self-dsdna can also be segregated inaccurately and released into the cytosol ( , ). for example, genomic dna (gdna) injury as a result of genotoxic stress and dna self-instability or replication errors leads to double-strand breaks (dsbs) and can be repaired by several ways ( ). impaired mediators of dna-damage repair response mediators, such as ataxia telangiectasia mutated (atm)-rad , poly adpribose polymerase (parp) and breast cancer / (brca / ) multiple cgas molecules can bind two double-stranded dnas (dsdna) to form a n: cgas:dsdna higher-ordered "ladder-like" oligomerization. mitochondrial transcription factor a (tfam) can bend the dsdna into a u-shaped structure and promote polymerization. (c) cgas can recognize b-type dsdna. in humans, the cgas dna-interfacing residue of site a loosens the interaction of dsdna to curve dsdna away for more convenient binding with next adjacent cgas. cgas can catalyze gtp and atp to synthesize cyclic guanosine monophosphate-adenosine monophosphate (cgamp). the n-terminal tail binds to the cell membrane, associating with phosphatidylinositol , -bisphosphate [pi ( , )p ]. (d) accumulation of cgas-dna complex goes through a liquid-phase separation and condenses into gel-like droplets. are associated with persisting dsbs and accumulation of cytosolic dna ( - ). extra-nuclear micronuclei formation during mitosis is a source of cytosolic dsdna caused by dsbs ( , ). followed by homologous recombination repair of collapsed replication forks, dna cleavage by methyl methanesulphonate (mms) and ultraviolet-sensitive (mus ) also lead to cytosolic dsdna presenting ( ). furthermore, manually cre/loxp recombination technology can induce dsdna damage during dna cleavage, which results in the accumulation of cytoplasmic dsdna ( ). in normal cellular mitotic processes, chromosomal dna can be exposed to the cytoplasm, while it is hard to bind and trigger cgas ( ). in addition, mitochondrial dna (mtdna) is also a considerable ligand of cgas and can be released into the cytosol under mitochondrial stress or dysfunction of proteins which participates in maintaining mitochondrial operations ( , , ) (figure a) . cells have several types of nucleases to restrict cytosolic dna to avoid cgas activation. for example, three-prime repair exonuclease (trex also known as dnaseiii) is a cytosolic dna exonuclease which removes unprotected dsdna from the cytosol ( ) . rnaseh locates to the nucleus and specifically degrades the rna in rna:dna hybrids participating in dna replication ( ) . dnaseii is a lysosomal dnase which degrades undigested dna in endosomes or autophagosomes to prevent their entry into the cytoplasm ( ). sam domain and hd domain-containing protein (samhd ) is characterized as a dntpase and restricts reverse transcription of the rna virus ( , ) . samhd can also stimulate the exonuclease (but not the endonuclease) activity of mre to degrade nascent cdns (including cgamp) might be exported by some ways. (c) inflammatory signaling mediated by the cgas-sting pathway. after sensing dna, cgas produces cgamp and extracellular cdns, promoting stimulator of interferon genes (sting) to undergo dimerization. sting can exit from the endoplasmic reticulum (er), and be translocated from the er to the er-golgi vesicle, and arrives at the golgi. sting and tank binding kinase (tbk ) can be oligomerized and cluster at the golgi. the sting-tbk /iκb kinase (ikk) signalosome forms a scaffold to phosphorylate interferon regulatory factor (irf ) and inhibitor of nf-κbα (iκbα). then, dimerized irf and the activated canonical nf-κb p /p complex can be translocated into the nucleus as transcription factors to promote transcription of type-i ifn. (d) autophagy initiation and degradation. sting activation on er triggers er stress and mechanistic target of rapamycin complex (mtorc ) dysfunction. er stress and mtorc dysfunction can stimulate the unc- like autophagy activating kinase (ulk ) complex and beclin -phosphatidylinositol -kinase catalytic subunit type (pi kc ) complex. autophagy-related protein (atg ) and light chain (lc ) are associated with genesis and elongation of the autophagosome. after autophagy initiation, cgas-sting is ubiquitinated and binds with p . then, they are packaged into autophagosomes and terminally sorted to lysosomes (bold arrows represent main signaling pathways, thinner arrows represent regulatory signaling pathways, and dashed arrows represent bypass or suspicious pathways). ssdna, and start dna-repair responses at stalled replication forks ( ) . depletion of samhd leads to the cleaving of nascent ssdna by the activity of mre endonuclease and cytosolic translocation of gdna ( ) . deficiency of any of these nucleases can lead to accumulation of self-dna in the cytoplasm, thereby activating the cgas-sting pathway against dna molecules ( ) (figure a ). the production of asymmetrically linked , -cgamp catalyzed by cgas has the highest affinity for sting to promotes sting dimerization ( , ) . cgamp as a second messenger can be also transferred among cells in several ways to pass danger signaling of frontiers in immunology | www.frontiersin.org cytosolic dna. intercellular gap junction consists of two docking hexamer channels formed by different connexins, which allows many small molecules, including cgamp, to pass bi-directionally through cells. and intercellular transfer of cgamp through gap junction is largely dependent on connexin ( ) ( ) ( ) . additionally, cgamp can be packaged into virons and pre-notify newly infected cells ( , ) . cell fusion is a distinct manner for intracellular transmission of the human immunodeficiency virus (hiv); cgamp also enter membrane-fused bystander cells in this way ( ) . extracellular vesicles such as exosomes can contain cgamp along with viral dna, host gdna or mtdna, and mediate cells communication ( , ) . there were no evidences that cgamp could be pumped out to extracellular space by a channel/transporter. however, it was found that slc a can transmit cyclic dinucleotides (cdns) into cell plasma ( , ) . notably, ectonucleotide pyrophosphatase/phosphodiesterase family member (enpp- ) can degrade extracellular cgamp ( ) (figure b ). besides triggering sting, these exogenous cgamp can directly bind to cgas and prompt its activation as well ( ) . after binding to cgamp, the "lid" region of the sting dimer undergoes a conformational change that converts sting from an inactive "open" formation to an active "closed" formation. following that, the sting dimer translocates from the er to perinuclear er-golgi intermediate compartment (ergic) vesicles, finally arriving at the golgi to form punctuate structures with downstream molecules ( , ) . er-retention of sting caused by mutations results in reduced ifn signaling ( , ) . the translocon-associated protein β (trapβ) recruited by inactive rhomboid protein (irhom ) initially forms the trap translocon complex that mediates sting exit from the er ( , ). they both assist cytoplasmic coat protein complex-ii (copii) to drive er-vesicle formation and carry the sting complex to the golgi ( , ) . trafficking sting can bind directly to and be phosphorylated by tank binding kinase (tbk ) dimer or iκb kinase (ikk) complex ( , , ) . the c-terminal tail (ctt) of sting is a linear unfolded segment, which determines the optimization of combination specificity. sting ctt in mammals tends to bind tbk , whereas in fish it tends to activate nuclear factor-kappab (nf-κb) signal ( ) . the sting phosphorylation site ser in the ctt cannot reach the kinase-domain active site of its directly bound tbk , instead can reach the kinase-domain active site of the next adjacent tbk binding with another sting and be phosphorylated, while tbk phosphorylate each other ( , ) . hence, sting and tbk can aggregate on the golgi to form the sting signalosome. clustering sting undergoes palmitoylation and full activation ( ) . it is also possible for sting-ikk to cluster and form the sting signalosome in this manner. the sting-tbk /ikk signalosome produces a scaffold to phosphorylate interferon regulatory factor (irf ) or inhibitor of nf-κbα (iκbα). activated irf undergo dimerization ( ) . the activation of iκbα leads to its polyubiquitination and degradation by the proteosome, thereby eliminating its inhibition of nf-κb. there is also evidence suggesting that nf-κb activation might not require sting trafficking from the er ( ) . then, the dimerized irf or activated nf-κb p /p (p is also known as rela) complex are translocated into the nucleus as transcription factors and bind to the promoter of type-i ifn to aid the transcription of type-i ifn ( , , ) . meanwhile, activation of nf-κb p /relb can prevent recruitment of p and inhibit the p /p signal ( ) (figure c ). expressed type-i ifn can propagate among cells in paracrine or autocrine manners. the binding of ifnα/β with its receptor triggers janus kinase (jak) and signal transducer and activator of transcription (stat) pathways, then induce transcription of type-i ifn-stimulated genes (isgs), which have ifn-sensitive response elements (isres) in their -untranslated regions (utrs) ( ) . irf can also bind partially to several isres alone ( ) . herein, the expression of some isgs including interferon-induced protein with tetratricopeptide repeats (ifit) and pro-inflammatory cytokines such as tumor necrosis factor α (tnfα), interleukin (il)- , c-x-c motif chemokine ligand (cxcl ) and c-c motif chemokine ligand (ccl ) is increased remarkably by the cgas-sting pathway ( ) . furthermore, cgas and sting are both isgs, suggesting a positive feedback loop in spreading of the ifn signal ( , ) . stimulator of interferon genes activation on the er also triggers an er stress response with an "unfolded protein response (upr) motif " on the c-terminus of sting, which leads to and er stress-mediated autophagy ( , ) . sting-tbk activation and er stress also induce mechanistic target of rapamycin complex (mtorc ) dysfunction ( ) . er stress or reduced mtorc signaling activates unc- -likeautophagy activating kinase (ulk ) complex and the beclin- -class iii phosphatylinositol -kinase (pi kc also known as vps ) complex, which promotes initiation of the classical autophagy path ( ) . cgas can also interact directly with the autophagy protein beclin- -pi kc complex and trigger autophagy ( ) . furthermore, cgas-dsdna polymer can form a liquid-phase condensate (as mentioned above), which could theoretically be an initiator of autophagy ( ) . after autophagy initiation, autophagy-related protein (atg ) undertakes the genesis of the autophagosome along with light chain (lc ) undergoing lipidation, thereby resulting in elongation of the autophagosome ( ) . lc can also be recruited directly by ergic-loading sting and bypass the classical autophagy pathway ( , ) . cgas-sting-mediated autophagy can spread to the whole cell and help the elimination of intracellular microorganisms, subcellular organelles or misfolded proteins, as well as the er itself that loads the sting signalosome ( - ) ( figure d ). cgas-sting-mediated autophagy is also indispensable for removing cytosolic dna and inflammatory signaling factors to restrict the inflammatory response raised by the pathway itself ( ) . excessive signaling of the autophagy cascade can lead to irreversible apoptosis termed "autophagic cell death" ( ) . consequently, oligomerized cgas or sting undergoes ubiquitination and is packaged into autophagosomes with the help of p , to be terminally sorted into lysosomes ( , , , ) . cgas or sting is digested immediately in the autophagolysosome after transient activation of downstream signaling ( , , , ) . autophagy functions as a negative feedback loop which ensures transient cgas-sting signaling and avoids consistent over-reaction of the pathway. thus, impairment of autophagy may give rise to destructive inflammatory diseases ( ). we cataloged factors in the literature that could potentially up-or down-regulate expression of cgas/cgamp/sting in pre-translational or post-translational stages (tables , ) . the regulatory mechanisms of tbk , irf, and nf-κb in signaling pathways associated with expression of type-i ifn are outside the scope of this review. pyhin family member absent in melanoma (aim ) is a cytoplasmic dsdna sensor. it can recruit apoptosis-associated speck-like protein containing a card (asc) by its pyhin domain and form the aim inflammasome. the inflammasome activates caspase- , which activates il- and trigger pyroptosis ( ) . the aim pathway could counteract the cgas-sting pathway ( ) . first, cgas is a target for caspase- cleavage ( ) . second, gasdermin d activated by caspase- can lead to potassium ion (k + ) efflux which inhibits cgas ( ) . conversely, the cgas-sting pathway can trigger the aim inflammasome or nlr family pyrin domain containing (nlrp ) by several means, and the process lags behind canonical ifn signaling ( , ) . in this way, the inhibitory nucleic-acid sensor nlr family card domain containing (nlrc ) can counteract sting by binding and occupying it, but viral dna as a possible nlrc ligand can reverse its occupation of sting ( ) (figure a) . another pyhin family member, ifi , is a dna sensor located in the nucleus. ifi can bind to viral dna sequences or damaged chromatin dna and be translocated to the cytoplasm to recruit sting cooperatively with tnf receptor associated factor (traf ) and p ( , ) . several studies have shown that ifi (which can stimulate the phosphorylation and recruitment of sting and tbk ) is required for the full response of sting ( , ) ( figure b) . conversely, cgas can partially enter the nucleus and interact with ifi to promote its stability ( ) . therefore, it is inferred that during viral infection, ifi can facilitate recognition of decapsidated viral dna in the nucleus, while cgas in the cytoplasm engages with viral gene transcription products ( , ) . however, sting signaling can trigger ifi degradation by tripartite motif-containing (trim ) ubiquitination ( ) . tlr is also an important prr for multiple pamps ( ) . tir domain-containing adaptor-inducing ifnβ (trif) is downstream of several subtypes of tlrs (including tlr ). trif may be responsible for interacting with sting and helping the dimerization of sting ( ) . during viral infection, the tlr -myeloid differentiation primary response (myd )-irf / pathway is necessary for mouse monocytes recruitment to lymph nodes, whereas the sting pathway is necessary for local production of type-i ifn ( ) . however, sting signaling can induce suppressor of cytokine signaling (socs ) expression, which can negatively regulate myd activity ( ) (figure c ). oxidized mtdna can be released into the cytoplasm during cell stress elicited by hypoxia, viral infection and mitochondrial damage, etc.; oxidized mtdna is resistant to degradation by the cytosolic nuclease trex ( ) . in addition, mtdna accompanied with tfam (a mtdna-binding protein that can bend mtdna) is also a reasonable target for recognition by cgas ( , ) . however, during regulated cell death (as represented by apoptosis), it undergoes mtdna release but has certain mechanisms to ensure a minimal cgas-stingmediated immune response. mitochondrial outer membrane permeabilization (momp) activation, which is executed by bcl- -associated x protein (bax) and bcl- antagonist or killer (bak), is a highly controlled conserved process in regulated cell figure | interaction of the cgas-sting pathway with other dna-sensing pathways and its role in cell survival. (a) absent in melanoma (aim ) pathway and pyroptosis and necroptosis. aim can be triggered by cgamp and form an inflammasome, consequently triggering interleukin (il)- production and pyroptosis. stimulator of interferon genes (sting) trafficking to the lysosome ruptures the lysosome membrane, resulting in k + efflux and activation of the nlrp inflammasome, leading to pyroptosis. cyclic-gmp-amp synthase (cgas) and interferon regulatory factor (irf ) can be a target for caspase- cleaving. gasdermin d can lead to k + efflux and inhibition of cgas. (b) interferon gamma inducible protein (ifi ). ifi can be transported to the cytoplasm to help to recruit sting and tank binding kinase (tbk ). ifi as a pyhin family protein may form the inflammasome only in theory. (c) toll-like receptor (tlr) pathway. tir domain-containing adaptor-inducing ifnβ (trif) may be responsible for helping the dimerization of sting. sting signaling can induce suppressor of cytokine signaling (socs ) expression, which negatively regulates myd activity. (d) apoptosis. mitochondrial outer membrane permeabilization (momp) formed by bax/bak induced by mitochondrial stress can release oxidized mitochondrial dna (mtdna) and cytochrome c into the cytosol. oxidized mtdna is a suitable ligand for cgas recognition and is resistant to dnaseiii (trex ) degradation. cytochrome c binds to apoptotic protease-activating factor (apaf ) and initiates the formation of an apoptosome cooperatively with caspase- to activate caspase- , which can induce apoptosis. caspase- can cleave cgas. death. bak and bax activated by apoptosis signals cooperatively form a pore-like conformation on the mitochondrial outer membrane, leading to a permeability change of outer and also inner membranes ( , ) . consequently, the mitochondrial matrix, including cytochrome c and oxidized mtdna-tfam, is released into the cytoplasm ( , ) . cytochrome c binds to apoptotic protease-activating factor (apaf ) and initiates the formation of the apoptosome cooperatively with caspase- , which further triggers the intrinsic apoptosis program ( ) . in vivo and in vitro studies have shown that an absence of caspase- is associated with greater release of type-i ifn ( , ) . this occurs because caspase- and its downstream caspase- can cleave cgas and irf to restrain deleterious inflammation ( ) (figure d) . the cgas-sting pathway can also initiate programmed cell death. activation of sting enhances phosphorylation and activation of receptor interacting serine/threonine kinase (rip ) and mixed lineage kinase domain-like pseudokinase (mlkl). proapoptotic bcl binding component (puma), a member of bh -only family, is subsequently activated in a rip /mlkl-dependent manner, which promotes leakage of mtdna by momp ( , ) . activated irf can bind directly to bax to form irf /bax complex and induce apoptosis ( ) . excessive cgas-sting-mediated autophagy signaling can cause "autophagic cell death" and prevent malignant transformation of cells through dna damage ( , ) . sting trafficking to the lysosome can broaden permeabilization of the lysosome membrane, thereby rupturing the lysosome and releasing its contents, resulting in "lysosomal cell death (lcd)". lcd further triggers k + efflux and nlrp activation, ultimately resulting in pyroptosis ( , ) (figure d) . moreover, stimulating sting-dependent type-i ifn and tnfα signals simultaneously can lead to necroptosis of tumor cells ( , ) . cell senescence is recognized as a permanent arrest of the cell cycle, and is common in aging, immunity, ontogenesis and infectious defense ( ) . it lacks a specific biomarker but can be identified by the expression of several antiproliferative molecules (representatively rb-p andp -p pathway) ( ) . during senescence, changes in the nuclear structure and loss of the nuclear lamina protein disrupt the integrity of the nuclear envelope, leading ultimately to dna damage and cytoplasmic chromatin fragments ( ) . cellular senescence can be accelerated by accumulation of cytoplasmic chromatin in turn ( ) . these senescent cells produce the senescence-associated secretory phenotype (sasp), which shapes an inflammatory microenvironment ( ) . the cgas-sting pathway has been reported to be involved in the recognition of cytoplasmic chromatin fragments from senescence-related dna damage, and mediate the expression of sasp genes ( ) ( ) ( ) ( ) . along with these actions, the expression of trex and dnaseii is inhibited by dna damage through the inhibition of e f/dp (a potential transcription factor of trex and dnaseii) ( ) . for hematopoietic stem cells (hscs), dna damage can promote excessive secretion of type-i ifn in the hsc niche and activate p pathway, both of which can lead to long-term senescence and exhaustion of hscs ( , ) . hscs expressing a circular rna named "cia-cgas" in the nucleus, however, are protected from this exhaustion as a result of cia-cgas having stronger affinity than that of self-dna, which prevents it from being sensed ( ) . it implied a novel target to manipulate the immune environment in bone marrow and help for finding treatment approaches for hematopoiesis-based diseases, such as aplastic anemia. utilizing cellular senescence to restrain tumor growth is discussed below. cgas-sting signaling has an essential role in defense against a broad spectrum of intracellular dna and rna viruses ( , , ) . hiv is a typical rna retrovirus: there is neither dsdna in its genome, nor production of nucleic acids ( ) . nevertheless, cgas can detect the presence of hiv. rna:dna hybrids synthesized during reverse transcription that can be sensed by cgas explain (at least in part) this phenomenon ( ) . cgas may be triggered by endogenous dna broken and released during hiv infection as well theoretically. however, some studies found the new mechanisms. the early reverse-transcription production of hiv- can flank short stem loops with paired base, which lead to the production of y-type dna containing unpaired guanosines that can activate cgas well ( ) . moreover, nucleolus protein non-pou domain-containing octamer-binding protein (nono), as a sensor of capsid components of hiv, can help cgas to be translocated to the nucleus and assist cgas to sense hiv dna accompanied by polyglutamine-binding protein (pqbp ) ( , ) . the assistance proffered by nono in assisting cgas to sense dna is also associated with its role in constructing a ribonuclear complex with dna-dependent protein kinase (dna-pk) subunits around hexamethylene bisacetamide-inducible protein (hexim ), termed as "hexim -dna-pk -paraspeckle components-ribonuclear protein complex (hdp-rnp), " which also has a role in repair of dna damage and transduction of genotoxic signals ( ) . this complex is also required to accompany cgas-pqbp in sensing dna virus, such as kaposi's sarcoma-associated herpes virus ( ) . in addition, during virus infection, sting activation can lead to global suppression of translation in cells, which restricts viral replication ( ) . compared with hiv- , hiv- is less infective because it can infect dendritic cells (dcs) and elicit an anti-virus immune response. as a result, hiv- can cross-protect against hiv- ( ). this phenomenon has been attributed to the fact that hiv- (instead of hiv- ) can encode protein vpx, which overcomes the samhd restriction of dntp in dcs ( , ) . hiv- can infect dcs via vpx presentation, nevertheless, hiv- still cannot be fully sensed and induce an efficient immune response owing to certain escape mechanisms. whether it is hiv- or hiv- , a completely robust ifn response is required at pre-and post-integration sensing stages ( ) . cgas in dcs can detect reverse-transcribed cdna of hiv- before and after integration, whereas hiv- sensing is after genome integration owing to its capsid protection ( , ) . it was suggested that during initial infection by hiv- , nucleotides are recruited into the intact capsid through the hexamer pores on the hiv- capsid. therefore, the capsid-coated hiv- virus prevents the encapsidated reverse-transcription production from being sensed by the cytosolic nucleic-acid sensors ( ) . hiv- capsids can be ubiquitinated and then degraded by the host e ubiquitin ligase function of trim , which leads to detection of viral dna, meanwhile hiv- could use some host protein like cyclophilins to evade the sensing ( , ) (figure a) . similarly, other viruses also have evasion mechanisms to escape cgas-sting pathway surveillance (table ) . therefore, identifying and preventing such viral-evasion factors could be a viable means to design novel anti-viral drugs. cgas-sting pathway is responsible to protect against intracellular or extracellular bacterial infection (especially hsv, herpes simplex virus; cmv, cytomegalovirus; irhom , inactive rhomboid protein ; trapβ, translocon-associated protein β; kshv, kaposi's sarcoma-associated herpes virus; hdp-rnp, hexim -dna-pk-paraspecklecomponents-ribonuclear protein complex; lana, latency-associated nuclear antigen; virf , viral interferon regulatory factor ; plpro, papain-like protease; lc , light chain ; mtor, mechanistic target of rapamycin; traf , tnf receptor associated factor ; hcv, hepatitis c virus; hpv, human papilloma virus; hbv, hepatitis b virus; hiv, human immunodeficiency virus; nf-κb, nuclear factor-κ b. intracellular infections). cdns (e.g., c-dgmp, c-damp, and cgamp) produced by bacteria are essential for the regulation of bacterial function, such as biofilm formation, colonization, and reproduction ( , ) . as ligands for sting, cdns can bind directly to and activate sting independently of cgas, which contributes to several immune responses from bacteria ( ) . usually, bacteria can enter or be engulfed by the cell through the endophagosome and be sequestered from the cytosolic sense receptor. some bacteria, such as mycobacterium tuberculosis (mtb), can survive in vacuoles, resulting in an insufficient cellular immune response to defend against it ( ) . in contrast, the esx- secretion system of the mycobacterium can translocate the phagosomal vacuolar matrix including bacterial genome molecules into the cytoplasm and trigger the cgas-sensing pathway ( ) . for other bacteria, such as legionella pneumophila or and brucella abortus, the host guanylate binding proteins (gbps) facilitate rupture of phagosome vacuoles and are indispensable for controlling their infection ( , ) . autophagy signaling mediated by cgas/sting is also involved in microorganism clearance mentioned above ( , ) . bacteria have evolved strategies to confront this pathway too. bacterial phosphodiesterase cdnp produced by mtb or group-b streptococci can degrade cdns ( , ) . cpsa (a type of mtb lytr-cpsa-psr domain-containing protein) can prevent autophagy responses for eliminating pathogens ( ) . chlamydia trachomatis inclusion membrane proteins can maintain the stability of the inclusion membrane and avoid inclusion lysis (leading to pathogen antigens leaking out and being detected by the host cell) ( , ) . yersinia outer protein j (yopj) deubiquitinates sting and impedes the formation of the sting signalosome ( ) . the cgas-sting pathway activation even impedes the elimination of listeria monocytogenes because bacterial dna can be packaged into evs and transferred into t cells, where it induces apoptosis of t cells ( , ) . several protozoans, such as toxoplasma gondii and malaria parasites, have an intracellular period in their lifecycle. t. gondii could engage cgas-sting exclusively ( ) . however, irf activation inducing isg expression promotes t. gondii development independently of ifn expression ( ) . p. falciparum can target erythrocytes, lacking a nucleus and unable produce ifn, but infected erythrocytes can secrete evs containing parasitic gdna to monocytes and trigger cgas ( ) . the immune system is regulated by a complicated network. disorder of immune signaling can elicit non-infectious inflammatory or autoimmune diseases. excessive, uncontrolled production of type-i ifn can lead to a spectrum of inflammation diseases termed "type-i interferonopathies, " which have some common manifestations ( ) . cgas-sting is the one of main sources of type-i ifn, acts as a cellular immune-sensing signaling axis, and is involved in type-i interferonopathies. stimulator of interferon genes -associated vasculopathy with onset in infancy (savi) is a typical sting-related hereditary inflammatory type-i interferonopathy, and is manifested by interstitial lung disease, dermatomyositis and arthritis. its pathology is featured by leukocytoclastic vasculitis and microthrombotic angiopathy of small dermal vessels ( , ) and patients can also suffer from lymphopenia ( ) ( ) ( ) ( ) . the etiology of savi is a gain of function (gof) mutant in sting which leads to constitutive sting activation without cdns stimulation ( ) . currently, several mutant amino acids residues have been found in or close to the dimerization domain (v m, n s, g e, v l, and v m) ( , , , ) , as well as r g, r s, r q, and c y in the cgamp-binding domain ( ) . other types of type-i interferonopathies, such as systemic lupus erythematosus (sle) and aicardi-goutières syndrome (ags), have relationships with defective clearance of cytosolic nucleic acids caused by congenital dysfunction of trex , rnaseh , and samhd . sle is a heterogeneous autoimmune disease which has prominent type-i and also -ii ifn signatures ( ) . ags comprises some systemic autoimmune syndromes overlapping with sle, and can be classified as a "lupuslike disease" ( ) . additionally, ags also causes severe developmental neurological disorders, including cerebral calcifications, encephalopathy and cerebral atrophy. systemic lupus erythematosus is a representative model for elucidating the mechanism of type-i interferonopathies. in sle, the level of self-dna which is packaged into apoptosis-derived membrane vesicles along with the level of anti-dsdna antibody is increased in the serum of patients ( ) . a study revealed increasing levels of isgs (including cgas/sting) as well as the cgamp-detected ratio in peripheral-blood mononuclear cells of sle patients ( ) . as innate immune cells, dcs have essential roles in antigen presentation, cytokine secretion, and priming the adaptive response of immune cells ( ) . plasmacytoid dcs (pdcs) can internalize and recognize self-dna and they are the main source of type-i ifn in serum during sle ( ) . ifnα/β is essential for complete function of immature pdcs ( ) . ifnα/β and il- can induce mitochondrial oxidative stress in dcs and decrease atp production, which blocks proton-pump function and increases ph of lysosomes. this process inhibits mitochondrial degradation and blocks mtdna clearance, which engages the cgas-sting pathway ( , ). moreover, monocytes may sense mtdna through cgas-sting and differentiate into dcs ( , ). neutrophil extracellular traps (nets) are complexes released by neutrophils exposed to stimuli or autoantibody immune complexes. nets comprise extracellularly released chromatin, myeloperoxidase enzymes, and also oxidized mtdna. in lupus-like diseases, nets can be induced by ifnα/β and may play a major part in priming pdcs ( , ) . all mechanisms stated above contribute to a more aggravated type-iifn response and exacerbate disease. a similar phenomenon can be observed in savi, ataxia telangiectasia (at) and artemis deficiency ( ) . however, compared with savi, dcs in sle can prime t-cell maturation significantly and increasing secretion of pro-inflammatory cytokines, such as il- and tnfα can also lead to activation of adaptive immunity ( figure a) . the cgas-sting pathway can mediate systemic inflammation as well as autoimmune activation. however, it is also involved in the local inflammation of multiple tissues. with regard to ischemic myocardial infarction (mi), cardiac macrophages can sense dying ruptured cells and lead to fatal post-mi cardiac inflammation, which is reversed by ablation of cgas/sting/irf ( , ) . in a non-alcoholic steatohepatitis (nash) model induced by a methionine-and choline-deficient or high-fat diet, lipotoxicity can cause mitochondrial damage and up-regulate sting/irf expression in hepatocytes, which in turn promotes lipid accumulation and inhibits glycogen synthesis. all above bring out hepatic inflammation and hepatocytes apoptosis ( ) . in this model, mice with deficiency of sting presents alleviated insulin resistance and lower levels of low-density lipoprotein in serum, and also decreased hepatic inflammation and fibrosis/steatosis, in which hepatic macrophages/kupffer cells may take a big part ( , ) . lipotoxicity can induce p to be phosphorylated through the cgas-sting-tbk pathway, which causes aggravated protein inclusions in hepatocytes and it indicates that p could be a biomarker for nash prognosis ( ) . mtdna-dependent inflammation induced by lipotoxicity also occurs in adipose tissue and endothelial cells of blood vessels, which contributes to tissue inflammation, insulin resistance, and cardiovascular diseases ( , ) . in traumatic brain injury (tbi), local injury initiates breakdown of the blood-brain barrier and global neuroinflammation ( ) . sting expression is up-regulated in tbi and can lead to increased expression of pro-inflammatory cytokines and enlargement of secondary injury ( ) . reduced autophagy-associated protein expression induced by sting may contribute to the dysfunction of autophagy and dampen the elimination of necrotic tissue, thereby intensifying inflammation ( ) . during silicosis, silica can yield cytosolic dsdna release and engage cgas-sting, which activates dcs and macrophages to cause severe lung inflammation. it also leads to death of epithelial cells through the nlrp pathway and pulmonary fibrosis ( ) . similarly, mtdna release in renal tubule cells has been found to be associated with acute kidney injury by cytotoxic drugs and chronic renal fibrosis ( , ) . neurodegenerative diseases are correlated with local inflammation ( ) . in the central nervous system (cns), microglia is considered to be the main source of cgas-sting-dependent ifn expression ( ) . in neurodegenerative diseases, levels of the marker of microglia activation-cluster of differentiation (cd ), and pro-inflammatory cytokines are increased ( ) . a significant feature of parkinson's disease (pd) is the neuronal loss of cerebral nuclei (especially dopaminergic neurons in the substantianigra). serine/threonine-protein kinase pink and e ubiquitin-protein ligase parkin are ubiquitinrelated factors that take part in removing damaged mitochondria by autophagy, and their dysfunction lead to the early onset of pd ( ) . parkin −/− and pink −/− mice, following exhaustive exercise, show inflammation and loss of dopaminergic neurons, which can be rescued by loss of sting ( ) . similarly, at is a genetic disease caused by missense mutation of a dna-repair protein: atm. at patients usually show neurodegenerative defects (especially ataxia) complicated with telangiectasia on their eyes or body, deficiency of adaptive immune cells, and predisposition to cancer ( ) . nevertheless, up-regulation of expression of type-i ifn can also be found in at patients and mice, causing them to be prone to autoimmune diseases ( , , ) . this syndrome is related to p -mediated senescence but also the chronic inflammation mediated by the cgas-sting pathway which engages cytosolic uncombined broken gdna caused by atm dysfunction ( ) . in addition, accumulation of cellular mtdna occurs in age-related macular degeneration characterized by retinal pigmented epithelium (rpe) degeneration. this can trigger chronic inflammation by cgas-sting pathway, in which nlrp inflammasomes, inflammatory/apoptotic caspases are also involved ( , ) . with regard to other diseases in which adaptive immune cells prime, cgas-sting has a different role. multiple sclerosis (ms) is a local inflammatory disease of cns. ms is characterized by over-reactive microglia, infiltration of self-reactive t cells, demyelization of nerve fibers and hyperplasia of gliocytes. autoantibodies against proteins expressed in immune-privileged regions of cns also contribute to its pathogenesis ( ) . in ms, ifnα/β can attenuate disease severity effectively. this implies a protective role for type-i ifn in cns, which is considered to counteract the pro-inflammatory ifnγ ( ) . using experimental autoimmune encephalitis (eae) as a ms model, sting was found to be indispensable for amelioration of type-i ifnmediated neuroinflammation, and it could be induced by a conventional anti-viral drug ganciclovir ( ) . ultraviolet (uv) radiation is a factor inversely related to the morbidity of ms ( ) . it was found that uv-b irradiation can recruit inflammatory monocytes and produce type-i ifn in a sting-dependent manner ( ) . all above indicate that cgas-sting-ifnα/β pathway may have a beneficial effect on some cns inflammatory diseases such as ms. a possible reason for the observed effect above is due to indoleamine , -dioxygenase (ido), which can catabolize tryptophan (trp) oxidatively. trp withdrawal or trpoxidative catabolites can interact with general control non-derepressible (gcn ) and mtor, of which both can control cellular aminoacid metabolism and suppress t helper (t h ) cells immunity ( ) . ifnα/β is a potent ido inducer to suppress proliferation of cd + t h cells and promote differentiation of foxp + regulatory t (t reg ) cells, which are believed to suppress cnsspecific autoimmunity ( , ) . in addition, dna released from dying cells can be internalized directly by t cells and sensed by cgas-sting pathway, which leads to enhancement of the t h transcription factor gata but suppression of the t h transcription factor t-bet. consequently, this process polarizes naive t cells toward t h differentiation ( ). studies mentioned above may (at least in part) explain why the cgas-sting signal is a negative regulator of ms. the inhibitory role of cgas-sting in inflammation is also attributed to its apoptosis-triggering role. in some subtypes of savi and mouse models, apoptosis of blood-vessel endothelial cells or bronchial epithelial cells and leucopenia can be observed (especially t-cell lymphopenia) ( , , ) . when the sting signal is stimulated, apoptosis occurs more frequently in normal or cancerous t cells ( ) . also, bone-marrow chimeras and gene-knockout studies have shown that t cells defect in savi are not associated with type-i ifn signaling or cgas ( , ) . localization of sting at the golgi can cause delay of t-cell mitosis and reduced proliferation independently of irf and tbk ( ) . furthermore, a "upr motif " on the c-terminus of sting can cause er stress and upr, resulting in ca + overloading and t-cell death ( ) . a controversial view is that b cells express sting variously and may undergo apoptosis through this way ( , , ) . however, simultaneous signaling by sting and the b-cell antigen receptor can promote b-cell activation and antibody production independently of type-i ifn ( ) (figure b) . as for some diseases with inflammatory responses involved, the acute phase of pancreatitis causes dying acinar cells to produce free dsdna, which activates cgas-sting signaling in macrophages, and exacerbates inflammation severity ( ) . however, in the chronic phase of pancreatitis, cgas-sting activation decreases pancreatic inflammation, which may be mediated by limiting t h response ( ). for gut mucosal immunity, transient stimulation of sting could strengthen the function of antigen-presenting cells (apcs) and promote t h and t h immune responses against microbes ( ) . chronic sting signaling, however, elicits an il- response to control the inflammation and avoids inflammatory enterocolitis such as bowel disease ( ) . sting knockout mice present reduced numbers of goblet cells, a decreased ratio of commensal versus harmful bacteria and compromised t reg cells in the gut, making it prone to enterocolitis ( ) . chromosomal instability (cin) is an intrinsic feature of cancer, and results spontaneously from errors in chromosome segregation during the mitosis of cancer cells. cin can also be induced manually by radiotherapy or chemotherapy, which causes dsbs. it results in micronuclei formation outside the nucleus, of which rupture brings out irrepressible accumulation of cytosolic self-dna and engages cgas ( , , ). however, normal mitotic processes involve exposure of chromosomal dna to the cytoplasm, but this cannot initiate a substantial inflammatory reaction or apoptosis because nucleosomes can suppress dsdna-cgas binding in a competitive manner ( ). an appropriate immune response against tumors via a type-i ifn plays an indispensable part in limiting tumors and prolonging host survival ( ) . it was found sting-deficient mice are prone to developing several types of cancer and have poor survival under a tumor burden, whereas stimulation of sting can elicit robust immunity to tumors ( ) ( ) ( ) . a mechanism is many cancer cells expressing cgas can recognize cytosolic dna and produce cgamp to stimulate secretion of type-i ifn through sting ( , ) . excessive expression of trex in cancer cells, which can be induced by radiotherapy, attenuates this progression ( ) . cgas-sting can also promote senescence of cancer cells through the p -p pathway ( ) . cgas-sting-mediated autophagy contributes to autophagic cell death if mitotic crisis occurs to avoid transformation of cancer cells ( ) . melanoma cells can also transfer cgamp produced by them to proximal non-cancerous host cells through gap-junction channels and activate sting in these cells, which contributes to the recruitment of tumor-infiltrating immune cells such as natural killer (nk) cells ( , ) . expression of the nk cellspecific ligand nkg d retinoic acid early transcript (rae ) on cancer cells is highly up-regulated by sting once nk cells permeate into tumor tissue ( ) . the activation of sting in the endothelium within the tumor microenvironment (tme) could contribute to the remodeling of tumor vasculatures, and may have positive effects on tumor regression ( ) . dendritic cells are the main source of type-i ifn in several types of tmes and are dependent on sting signaling ( ) . more preferentially than macrophages, infiltrating dcs take up tumor-derived dna or cgamp from dying cell fragments by phagocytosis ( , , , , ) . moreover, cancer cells can package dna into exosomes and transfer dna to dcs ( ) . produced cgamp by cancer cells can also be transferred to dcs through forming gap junction ( ) . by activating cgas-sting signal in dcs, cd α + subtype dcs secret chemokines such as ccl and cxcl and crossprime infiltrating anti-tumor cd + t cells ( , , ( ) ( ) ( ) . in contrast, numbers of immune-suppressing cells such as t reg cells, myeloid-derived suppressor monocytes and m macrophages have been reported to be decreased ( , , ) . expression of il- /il- rα complex is up-regulated in myeloid cells with the help of sting/type-i ifn and promotes tumor regression ( ) . tumor cells can evade intrinsic cellular surveillance in different ways. in various cancer cell lines, cgas, sting, tbk , and irf are mutated frequently and their decreased expressions are also related to the high level of methylation ( ) . sting expression has been shown to be suppressed by the alternative lengthening of telomeres (alt) pathway, which is responsible for prolonging the telomere length and maintaining the proliferation of tumor cells ( ) . a hypoxic environment in tumor cells can lead to accumulation of lactic acid and is associated with the inhibition of tumor-conditional dcs and reduced expression of ifn signaling molecules ( ) . in breast cancer, functional up-regulation of expression of human epidermal growth factor receptor (her ), a ligand-independent receptor tyrosine kinase (rtk), can arrest the expression of rac-alpha serine/threonineprotein kinase (akt ) (a key factor in the mtor pathway), which is reported to inhibit the activation of cgas and tbk ( , ) . patients with lung adenocarcinoma have a low probability of survival if they have reduced expression of cgas ( ) . thus, expression of the cgas-sting and dna-damage marker histone γh ax in tumor cells could be considered as independent prognostic factors to predict therapy response and clinical outcome, and could be superior to that of traditional markers like immunogenic cell death and t cells number ( ) . however, some scholars have arrived at opposite conclusions. when dsbs occur in cancer cells, cgas can be relocated to the nucleus and obstruct the formation of the parp -timeless complex, thereby inhibiting homologous recombination repair and maintaining cin, which potentiates tumor evolution ( , ) . it has also been reported that cgas recognizing cin activates non-canonical nf-κb signaling and potentiates cellular metastasis programs ( ) . furthermore, sting −/− mice are resistant to skin carcinogenesis in a , -dimethylbenz(a)anthracene (dmba)-treatment model. it has been demonstrated that when dmba-induced nuclear dna leaks into the cytoplasm, sting can induce chronic inflammatory stimulation that contributes to cancer development ( ) . during brain metastasis, cgamp transferred to bystander cells (e.g., astrocytes) can also produce ifnα and tnfα in the tme but, in this context, it will support tumor development and chemoresistance ( ) . coordinating with myeloid cells penetrating into the tumor, myeloidderived suppressor cells can also be recruited through the c-c chemokine receptor type (ccr ) ( ) . another study found that microparticles yielded by tumor cells can turn macrophages into the m type through cgas-sting-tbk , contrary to previous findings ( ) . immune-system interactions with tumor cells are complicated. the effect of cgas-sting on cancer is dependent on the type of tumor, host immune state, activated cell types, therapeutic intervention, and the magnitude of cgas-sting activation. like inflammation generated by cgas-sting, a time-dependent inflammatory anti-tumor response mediated by cgas-sting may be present. temporary activation of cgas-sting in innate immune cells could enhance the anti-tumor effect, whereas sustained activation of cgas-sting might induce immune tolerance of the tumor. more investigations are necessary to ascertain the exact role of cgas-sting in oncology, and elucidate the specific advantages and adverse effects of targeting the cgas-sting pathway in cancer therapy ( figure ). considering the pivotal role of the cgas-sting pathway in infection, inflammation and cancer, positive modulation of the pathway signaling is a promising way to enhance the immune state and restrict microorganisms or heterogeneous cells, whereas negative modulation can control aberrant inflammation. radiotherapy or chemotherapy drugs such as cisplatin or cyclophosphamide can induce dsbs and micronuclei, then trigger the cgas-sting pathway to enhance tumor immunogenicity ( ) ( ) ( ) . in addition, parp inhibitors such as olaparib have promising effects on cancer cells lacking brca because of their cooperative dna-repair functions ( ) . although cgas activation is inhibited by nucleosomes, taxol can induce mitotic cell-cycle arrest and sustain divided chromatin in the cytosol to activate the cgas-sting pathway slowly, and accumulation of signaling could stimulate apoptosis of cancer cells ( ). inhibitors of topoisomerase or used conventionally as chemotherapy drugs trigger minor damage to dna and accumulation of cytosolic dna, which can engage cgas and enhance the anti-tumor or anti-infection responses of cells ( ) ( ) ( ) . cgas-sting is essential on anti-tumor immune checkpoints therapies. for example, blockade of cd -signal regulatory protein α (sirpα) signaling on dcs can activate nadph oxidase (nox ) and increase the ph in phagosomes along with incomplete degradation of mtdna, which can trigger cgas-sting ( ) . sting deficiency in mice abrogates the anti-tumor effect of cd blockade ( ) . a similar phenomenon also can be seen in anti-programmed cell death- (pd- ) therapy ( ) . there is greater infiltration of ifnγ + cells and cd + t cells and pd- /pd- ligand (pd-l ) expression in tme treated by sting-ligand derivatives ( ) . therefore, in several types of tumors, combined administration of a sting agonist and immune-checkpoint antibody could elicit a more curative outcome compared with one therapy alone ( , ) . viruses can infect cells lacking cgas-sting more effectively, and have higher oncolytic activity compared with virus therapy alone. hence, the use of oncolytic viruses such as talimogene laherparepvec is beneficial for treating tumors with low expression of cgas/sting. sting expression can be regarded as a prognostic measurement for such therapy ( ) . some artificial analog molecules of cdns, such as , dimethylxanthenone- -acetic acid (dmxaa) and -carboxymethyl- ( h) acridone (cma) can bind the cdn pocket of mouse-specific sting dimers and promote conformational transition of sting from inactive "open" to an active "closed" state ( , ) . dmxaa showed convincing efficacy in restraining tumors in mice ( ) . however, dmxaa is restricted in activating mouse-specific sting but not humanspecific sting, which could be an explanation for the failure of dmxaa in treating non-small-cell lung cancer patients in a phase-iii clinical trial (nct ) ( ) . nonetheless, with three substitutions (g i, q i, and s a), human sting can also be induced by dmxaa to undergo conformational transition ( ) . another new compound, amidobenzimidazole, has been found to be an agonist of sting without lid closure and has potential therapeutic value ( ) . cyclic dinucleotides and their derivates that can stimulate sting directly are candidate adjuvants to restrain tumors. intratumoral administration of c-damp, c-dgmp, or cgamp analogs alone or combined with other adjuvants or tumor antigens have shown anti-tumor effects ( , ) ; phase-i or ii clinical trials (nct , nct , and nct ) of dithio-(rp,rp)-c-damp (known as adu-s ) are ongoing ( ) . to avoid the degradation and ensure maximal phagocyte internalization of cdns, endosomolytic nanoparticles have been designed to package and deliver cdns. for example, ph-sensitive nanoparticles (e.g., stingnanoparticles) can release their contents if located in acidic endosomal environments ( ) . for treatment of type-i interferonopathies, lessons can be taken from the treatment of canonical autoimmune disease such as sle, but there are several differences. for example, it was found that corticosteroid pulse therapy, γ-immunoglobulins, disease-modifying anti-rheumatic drugs, anti-cd , and some immunosuppressants (e.g., methotrexate) have limited efficacy against savi ( , ) . jak inhibitors such as ruxolitinib, tofacitinib and baricitinib that reduce type-i ifn downstream signaling have shown therapeutic value against savi, but further verification of their efficacy is needed ( ) . moreover, novel immune therapies, such as anti-ifnα and anti-ifnar immunoglobin, are in clinical trials for sle. these could also be tested against savi in the future ( ) . pharmaceutically screening has revealed that some antimalaria drugs, such as suramin, have an inhibitory effect on cgas by blockade of interaction between dna and cgas ( ) . in addition, novel small molecules such as ru or g can occupy the enzymatic pocket of species-specific cgas to abrogate cgamp synthesis ( , ) . recently, a study found that aspirin can acetylate cgas at three lysine residues and block cgas activity ( ) . with regard to sting, the cyclopeptide astin c can block irf recruitment onto the sting signalosome ( ) . the molecule h- can block the palmitoylation of human-sting ( ) . all of these agents are potential candidates for alleviating type-i interferonopathies. the cgas-sting pathway is primarily responsible for the modulation of immune response in cells when facing cytosolic dsdna challenge. moreover, it is complicatedly cross-regulated by other cellular processes or cellular signaling networks. the exact fundamental mechanism of the pathway in cells and the effect on the whole organism in specific states is not completely clear and requires further investigation. in conclusion, the cgas-sting pathway has dichotomous roles in the immune system. in general, cgas-sting-type-i ifn signaling can promote the innate immune response in myeloid cells but alleviate the adaptive immune response exerted by t cells and b cells. cgas shows high expression in apcs such as macrophages and dcs, but sting is expressed in most cells ( ) . cgas-sting signaling corresponding to cytoplasmic dsdna in apcs can boost innate and adaptive immunity transiently. in this situation, the dna challenge signal is limited to only macrophages and dcs. their pro-inflammatory and antigenpresenting functions to adaptive immune cells are promoted in the short-term. if the signal spreads to other bystander cells, such as t cells, b cells, local resident cells by means of cgamp transfer, or just aberrant sting activation by gof, it causes apoptosis in bystander cells or adaptive immune cells and immune tolerance in the long-term. therefore, it is reasonable to conclude that the intrinsic function of the cgas-sting pathway is essential for the innate immune system responses of the host immediately after pathogen invasion or abnormal cell appearing. once the challenge persists, the cgas-sting pathway controls the adaptive immune system to avoid chronic, detrimental inflammatory reactions or autoimmune diseases. the inflammatory response exists universally in almost all physiologic and pathologic progressions. cgas-sting is pivotal in modulating cellular inflammation, so it is promising to extend our conception of the cgas-sting pathway onto more diseases with inflammatory responses involved, especially cns-based diseases such as stroke, in which the inflammatory reaction exists but was recognized less. moreover, for targeting the cgas-sting pathway for therapeutic purposes, drugs should be optimized to augment the desirable effect and prevent its unwanted effects. for example, to eliminate tumor cells or infectious agents, agonists of cgas-sting would be a rational option if designed to target apcs exclusively but not t cells or b cells. in this scheme, the antitumor immune response is enhanced while avoiding apoptosis of adaptive immune cells and infiltration of immune suppressor cells. also, most research on the cgas-sting pathway has focused on its ifn-expressing role but overlooked autophagy and cell-death roles, which are also main downstream signaling of the pathway. therefore, some drugs, such as emricasan, are potential apoptosis inhibitors that may have a complementary effect on ameliorating apoptosis of blood-vessel endothelial cells or bronchial epithelial cells, and lymphopenia, in savi. until now, studies of the cgas-sting pathway have been done mainly in the laboratory but it has large space to be explored in clinical or translational fields. additionally more prrs and cellular immune-surveillance pathways may remain to be discovered to piece together the molecular puzzles of the cell. dw drafted the manuscript and drew the figures. wj and jh conceived and revised the review. frontiers in immunology | www.frontiersin.org recognition of endogenous nucleic acids by the innate immune system sting is an endoplasmic reticulum adaptor that facilitates innate immune signalling the adaptor protein mita links virus-sensing receptors to irf transcription factor activation an endoplasmic reticulum ifn stimulator, activates innate immune signaling through dimerization c-di-amp secreted by intracellular listeria monocytogenes activates a host type i interferon response sting is a direct innate immune sensor of cyclic di-gmp ifi is an innate immune sensor for intracellular dna cyclic gmp-amp synthase is a cytosolic dna sensor that activates the type i interferon pathway pivotal roles of cgas-cgamp signaling in antiviral defense and immune adjuvant effects an expression atlas of human primary cells: inference of gene function from coexpression networks bacterial cgas-like enzymes synthesize diverse nucleotide signals structural mechanism of cytosolic dna sensing by cgas structure of human cgas reveals a conserved family of second-messenger enzymes in innate immunity cytosolic rna:dna hybrids activate the cgas-sting axis sequence-specific activation of the dna sensor cgas by y-form dna structures as found in primary hiv- cdna cgas produces a - -linked cyclic dinucleotide second messenger that activates sting the catalytic mechanism of cyclic gmp-amp synthase (cgas) and implications for innate immunity and inhibition the cytosolic dna sensor cgas forms an oligomeric complex with dna and undergoes switch-like conformational changes in the activation loop cyclic gmp-amp synthase is activated by double-stranded dna-induced oligomerization structure of the human cgas-dna complex reveals enhanced control of immune surveillance cgas senses long and hmgb/tfam-bound u-turn dna by forming protein-dna ladders dna-induced liquid phase condensation of cgas activates innate immune signaling human cgas catalytic domain has an additional dna-binding interface that apoptotic caspases suppress mtdna-induced sting-mediated type i ifn production obsessive-compulsive disorder is associated with broad impairments in executive function: a meta-analysis ribonuclease h mutations induce a cgas/sting-dependent innate immune response restriction by samhd limits cgas/sting-dependent innate and adaptive immune responses to hiv- host restriction factor samhd limits human t cell leukemia virus type infection of monocytes via sting-mediated apoptosis samhd acts at stalled replication forks to prevent interferon induction cyclic gmp-amp containing mixed phosphodiester linkages is an endogenous high-affinity ligand for sting cyclic gmp-amp synthase is an innate immune sensor of hiv and other retroviruses cell intrinsic immunity spreads to bystander cells via the intercellular transfer of cgamp connexin-dependent transfer of cgamp to phagocytes modulates antiviral responses cancer-cell-intrinsic cgas expression mediates tumor immunogenicity viruses transfer the antiviral second messenger cgamp between cells transmission of innate immune signaling by packaging of cgamp in viral particles cgas-mediated innate immunity spreads intercellularly through hiv- envinduced membrane fusion sites priming of dendritic cells by dna-containing extracellular vesicles from activated t cells through antigen-driven contacts cells infected with herpes simplex virus export to uninfected cells exosomes containing sting, viral mrnas, and micrornas slc a is an importer of the immunotransmitter cgamp slc a transports immunoreactive cyclic dinucleotides hydrolysis of -cgamp by enpp and design of nonhydrolyzable analogs cgas facilitates sensing of extracellular cyclic dinucleotides to activate innate immunity sting regulates intracellular dna-mediated, type i interferon-dependent innate immunity activation by translocation from the er is associated with infection and autoinflammatory disease atg a controls dsdna-driven dynamic translocation of sting and the innate immune response irhom is essential for innate immunity to dna viruses by mediating trafficking and stability of the adaptor sting tmed potentiates cellular ifn responses to dna viruses by reinforcing mita dimerization and facilitating its trafficking autophagy induction via sting trafficking is a primordial function of the cgas pathway phosphorylation of innate immune adaptor proteins mavs, sting, and trif induces irf activation sting specifies irf phosphorylation by tbk in the cytosolic dna signaling pathway modular architecture of the sting c-terminal tail allows interferon and nf-kappab signaling adaptation structural basis of sting binding with and phosphorylation by tbk a conserved plplrt/sd motif of sting mediates the recruitment and activation of tbk activation of sting requires palmitoylation at the golgi ubiquitination of sting at lysine controls irf activation non-canonical nf-kappab antagonizes sting sensor-mediated dna sensing in radiotherapy immunomodulatory functions of type i interferons dna-binding landscape of irf , irf and irf dimers: implications for dimer-specific gene regulation attenuation of cgas-sting signaling is mediated by a p /sqstm -dependent autophagy pathway activated by tbk positive feedback regulation of type i ifn production by the ifn-inducible dna sensor cgas positive feedback regulation of type i interferon by the interferon-stimulated gene sting sting-mediated disruption of calcium homeostasis chronically activates er stress and primes t cell death sting senses microbial viability to orchestrate stress-mediated autophagy of the endoplasmic reticulum chronic innate immune activation of tbk suppresses mtorc activity and dysregulates cellular metabolism mtor signaling in growth, metabolism, and disease crosstalk between the cgas dna sensor and beclin- autophagy protein shapes innate antimicrobial immune responses a brief history of autophagy from cell biology to physiology and disease autophagy in the renewal, differentiation and homeostasis of immune cells sting directly activates autophagy to tune the innate immune response mycobacterium tuberculosis is protected from nadph oxidase and lc -associated phagocytosis by the lcp protein cpsa extracellular m. tuberculosis dna targets bacteria for autophagy by activating the host dna-sensing pathway upr, autophagy, and mitochondria crosstalk underlies the er stress response dnase a deficiency uncovers lysosomal clearance of damaged nuclear dna via autophagy self-consumption: the interplay of autophagy and apoptosis trafficking-mediated sting degradation requires sorting to acidified endolysosomes and can be targeted to enhance anti-tumor response the dna inflammasome in human myeloid cells is initiated by a sting-cell death program upstream of nlrp aim recognizes cytosolic dsdna and forms a caspase- -activating inflammasome with asc antagonism of the sting pathway via activation of the aim inflammasome by intracellular dna inflammasome activation triggers caspase- -mediated cleavage of cgas to regulate responses to dna virus infection gasdermin d restrains type i interferon response to cytosolic dna by disrupting ionic homeostasis a noncanonical function of cgamp in inflammasome priming and activation viral dna binding to nlrc , an inhibitory nucleic acid sensor, unleashes sting, a cyclic dinucleotide receptor that activates type i interferon non-canonical activation of the dna sensing adaptor sting by atm and ifi mediates nf-kappab signaling after nuclear dna damage nuclear ifi induction of irf- signaling during herpesviral infection and degradation of ifi by the viral icp protein ifi is required for dna sensing in human macrophages by promoting production and function of cgamp ifi and cgas cooperate in the activation of sting during dna sensing in human keratinocytes cgas-mediated stabilization of ifi promotes innate signaling during herpes simplex virus infection viral dna sensors ifi and cyclic gmp-amp synthase possess distinct functions in regulating viral gene expression, immune defenses, and apoptotic responses during herpesvirus infection sting-mediated ifi degradation negatively controls type i interferon production assembly and localization of toll-like receptor signalling complexes sting requires the adaptor trif to trigger innate immune responses to microbial infection sequential activation of two pathogen-sensing pathways required for type i interferon expression and resistance to an acute dna virus infection cross-regulation of two type i interferon signaling pathways in plasmacytoid dendritic cells controls anti-malaria immunity and host mortality oxidative damage of dna confers resistance to cytosolic nuclease trex degradation and potentiates sting-dependent immune sensing mitochondria and cell death: outer membrane permeabilization and beyond mitochondrial inner membrane permeabilisation enables mtdna release during apoptosis apoptotic caspases prevent the induction of type i interferons by mitochondrial dna apoptotic caspases suppress type i interferon production via the cleavage of cgas, mavs, and irf signalling strength determines proapoptotic functions of sting puma amplifies necroptosis signaling by activating cytosolic dna sensors autophagic cell death restricts chromosomal instability during replicative crisis induction of necroptotic cell death by viral activation of the rig-i or sting pathway tnfalpha and radioresistant stromal cells are essential for therapeutic efficacy of cyclic dinucleotide sting agonists in nonimmunogenic tumors cellular senescence: aging p ink a induces an age-dependent decline in islet regenerative potential autophagy mediates degradation of nuclear lamina extranuclear dna accumulates in aged cells and contributes to senescence and inflammation senescenceassociated secretory phenotypes reveal cell-nonautonomous functions of oncogenic ras and the p tumor suppressor cytoplasmic chromatin triggers inflammation in senescence and cancer downregulation of cytoplasmic dnases is implicated in cytoplasmic dna accumulation and sasp in senescent cells innate immune sensing of cytosolic chromatin fragments through cgas promotes senescence cgas is essential for cellular senescence dna-damage-induced type i interferon promotes senescence and inhibits stem cell function bacterial c-di-gmp affects hematopoietic stem/progenitors and their niches through sting a circular rna protects dormant hematopoietic stem cells from dna sensor cgas-mediated exhaustion pqbp is a proximal sensor of the cgas-dependent innate response to hiv- nono detects the nuclear hiv capsid to promote cgas-mediated innate immune activation p induces formation of neat lncrna-containing paraspeckles that modulate replication stress response and chemosensitivity hexim and neat long non-coding rna form a multi-subunit complex that regulates dna-mediated innate immune response stingdependent translation inhibition restricts rna virus replication inhibition of hiv- disease progression by contemporaneous hiv- infection samhd is the dendritic-and myeloid-cell-specific hiv- restriction factor counteracted by vpx reshaping of the dendritic cell chromatin landscape and interferon pathways during hiv infection the capsids of hiv- and hiv- determine immune detection of the viral cdna by the innate sensor cgas in dendritic cells hiv- activation of innate immunity depends strongly on the intracellular level of trex and sensing of incomplete reverse transcription products hiv- uses dynamic capsid pores to import nucleotides and fuel encapsidated dna synthesis restriction of hiv- and other retroviruses by trim hiv- evades innate immune recognition through specific cofactor recruitment cyclic di-gmp: second messenger extraordinaire cyclic gmp-amp signalling protects bacteria against viral infection a bacterial cyclic dinucleotide activates the cytosolic surveillance pathway and mediates innate resistance to tuberculosis tuberculosis pathogenesis and immunity mycobacterium tuberculosis differentially activates cgas-and inflammasome-dependent intracellular immune responses through esx- brucella abortus triggers a cgas-independent sting pathway to induce host protection that involves guanylate-binding proteins and inflammasome activation beneficial effects of yogasanas and pranayama in limiting the cognitive decline in type diabetes group b streptococcus degrades cyclic-di-amp to modulate stingdependent type i interferon production inhibition of innate immune cytosolic surveillance by an m. tuberculosis phosphodiesterase absence of specific chlamydia trachomatis inclusion membrane proteins triggers premature inclusion membrane lysis and host cell death the chlamydia trachomatis inclusion membrane protein cpos counteracts sting-mediated cellular surveillance and suicide programs yersinia yopj negatively regulates irf -mediated antibacterial response through disruption of sting-mediated cytosolic dna signaling intracellular bacteria engage a sting-tbk -mvb b pathway to enable paracrine cgas-sting signalling sting-dependent type i ifn production inhibits cell-mediated immunity to listeria monocytogenes induction of interferon-stimulated genes by irf promotes replication of toxoplasma gondii malaria parasite dna-harbouring vesicles activate cytosolic immune sensors type i interferon in rheumatic diseases activated sting in a vascular and pulmonary syndrome inherited sting-activating mutation underlies a familial inflammatory syndrome with lupus-like manifestations stimulator of interferon genes-associated vasculopathy with onset in infancy: a mimic of childhood granulomatosis with polyangiitis severe combined immunodeficiency in stimulator of interferon genes (sting) v m/wild-type mice sting-associated vasculopathy develops independently of irf in mice disease-associated mutations identify a novel region in human sting necessary for the control of type i interferon signaling personalized immunomonitoring uncovers molecular networks that stratify lupus patients aicardi-goutieres syndrome and the type i interferonopathies apoptosisderived membrane vesicles drive the cgas-sting pathway and enhance type i ifn production in systemic lupus erythematosus expression of cyclic gmp-amp synthase in patients with systemic lupus erythematosus. arthritis rheumatol the role of dendritic cells in autoimmunity human plasmacytoid dentritic cells elicit a type i interferon response by sensing dna via the cgas-sting signaling pathway interferon priming is essential for human cd + cell-derived plasmacytoid dendritic cell maturation and function interleukin- beta induces mtdna release to activate innate immune signaling via cgas-sting induction of dendritic cell differentiation by ifn-alpha in systemic lupus erythematosus neutrophil extracellular traps enriched in oxidized mitochondrial dna are interferogenic and contribute to lupus-like disease netting neutrophils are major inducers of type i ifn production in pediatric systemic lupus erythematosus type i ifnrelated netosis in ataxia telangiectasia and artemis deficiency cytosolic dna sensing promotes macrophage transformation and governs myocardial ischemic injury irf and type i interferons fuel a fatal response to myocardial infarction activation of the sting-irf pathway promotes hepatocyte inflammation, apoptosis and induces metabolic disorders in nonalcoholic fatty liver disease expression of sting is increased in liver tissues from patients with nafld and promotes macrophage-mediated hepatic inflammation and fibrosis in mice sting-mediated inflammation in kupffer cells contributes to progression of nonalcoholic steatohepatitis lipotoxicity induces hepatic protein inclusions through tank binding kinase -mediated p /sequestosome phosphorylation sting-irf triggers endothelial inflammation in response to free fatty acid-induced mitochondrial damage in diet-induced obesity priming the inflammatory pump of the cns after traumatic brain injury stingmediated type-i interferons contribute to the neuroinflammatory process and detrimental effects following traumatic brain injury sting-dependent sensing of self-dna drives silica-induced lung inflammation mitochondrial damage and activation of the sting pathway lead to renal inflammation and fibrosis mitochondrial damage causes inflammation via cgas-sting signaling in acute kidney injury how neuroinflammation contributes to neurodegeneration sensing of hsv- by the cgas-sting pathway in microglia orchestrates antiviral defence in the cns chronic neurodegeneration induces type i interferon synthesis via sting, shaping microglial phenotype and accelerating disease progression the roles of pink , parkin, and mitochondrial fidelity in parkinson's disease parkin and pink mitigate sting-induced inflammation atm and the molecular pathogenesis of ataxia telangiectasia autoimmune phenomena in ataxia telangiectasia mitochondrial dna has a pro-inflammatory role in amd cgas drives noncanonical-inflammasome activation in age-related macular degeneration multiple sclerosis type i/ii interferon balance in the regulation of brain physiology and pathology activation of the sting-dependent type i interferon response reduces microglial reactivity and neuroinflammation modulation of the immune system by uv radiation: more than just the effects of vitamin d? ultraviolet b irradiation causes stimulator of interferon genes-dependent production of protective type i interferon in mouse skin by recruited inflammatory monocytes. arthritis rheumatol engineering dna nanoparticles as immunomodulatory reagents that activate regulatory t cells activation of the sting adaptor attenuates experimental autoimmune encephalitis nucleic acid sensing by t cells initiates th cell differentiation sting-associated lung disease in mice relies on t cells but not type i interferon hierarchy of clinical manifestations in savi n s and v m mouse models intrinsic antiproliferative activity of the innate sensor sting in t lymphocytes agonist-mediated activation of sting induces apoptosis in malignant b cells b cell-intrinsic sting signaling triggers cell activation, synergizes with b cell receptor signals, and promotes antibody responses sting signaling promotes inflammation in experimental acute pancreatitis habtezion a. sting signalling protects against chronic pancreatitis by modulating th response stingactivating adjuvants elicit a th immune response and protect against mycobacterium tuberculosis infection sting-dependent signaling underlies il- controlled inflammatory colitis the cytosolic sensor sting is required for intestinal homeostasis and control of inflammation the multifaceted role of chromosomal instability in cancer and its microenvironment immunogenic cell death in cancer and infectious disease sting contributes to antiglioma immunity via triggering type i ifn signals in the tumor microenvironment sting-dependent cytosolic dna sensing mediates innate immune recognition of immunogenic tumors suppression of sting associated with lkb loss in kras-driven lung cancer dna exonuclease trex regulates radiotherapy-induced tumour immunogenicity growing tumors induce a local sting dependent type i ifn response in dendritic cells tumorderived cgamp triggers a sting-mediated interferon response in nontumor cells to activate the nk cell response rae ligands for the nkg d receptor are regulated by sting-dependent dna sensor pathways in lymphoma sting activation reprograms tumor vasculatures and synergizes with vegfr blockade dendritic cells but not macrophages sense tumor mitochondrial dna for cross-priming through signal regulatory protein alpha signaling exosomes shuttle trex -sensitive ifn-stimulatory dsdna from irradiated cancer cells to dcs host type i ifn signals are required for antitumor cd + t cell responses through cd {alpha}+ dendritic cells sting-dependent cytosolic dna sensing promotes radiation-induced type i interferondependent antitumor immunity in immunogenic tumors targeting dna damage response promotes antitumor immunity through sting-mediated t-cell activation in small cell lung cancer intratumoral sting activation with t-cell checkpoint modulation generates systemic antitumor immunity sting signaling remodels the tumor microenvironment by antagonizing myeloid-derived suppressor cell expansion il- is a component of the inflammatory milieu in the tumor microenvironment promoting antitumor responses suppression of sting signaling through epigenetic silencing and missense mutation impedes dna damage mediated cytokine production extrachromosomal telomere repeat dna is linked to alt development via cgas-sting dna sensing pathway downregulation of membrane trafficking proteins and lactate conditioning determine loss of dendritic cell function in lung cancer her recruits akt to disrupt sting signalling and suppress antiviral defence and antitumour immunity akt kinase-mediated checkpoint of cgas dna sensing pathway dna damage predicts prognosis and treatment response in colorectal liver metastases superior to immunogenic cell death and t cells chromosomal instability drives metastasis through a cytosolic dna response inflammation-driven carcinogenesis is mediated through sting carcinomaastrocyte gap junctions promote brain metastasis by cgamp transfer host stingdependent mdsc mobilization drives extrinsic radiation resistance tumor cellderived microparticles polarize m tumor-associated macrophages for tumor progression parp inhibition enhances tumor cell-intrinsic immunity in ercc -deficient non-small cell lung cancer parp inhibitor efficacy depends on cd (+) t-cell recruitment via intratumoral sting pathway activation in brca-deficient models of triple-negative breast cancer radiotherapy combined with novel sting-targeting oligonucleotides results in regression of established tumors topoisomerase inhibition promotes cyclic gmp-amp synthasedependent antiviral responses topoisomerase ii inhibitors induce dna damage-dependent interferon responses circumventing ebola virus immune evasion cgas/sting axis mediates a topoisomerase ii inhibitor-induced tumor immunogenicity cd blockade triggers t cell-mediated destruction of immunogenic tumors sting activation of tumor endothelial cells initiates spontaneous and therapeutic antitumor immunity a sting agonist given with ox receptor and pd-l modulators primes immunity and reduces tumor growth in tolerized mice magnitude of therapeutic sting activation determines cd (+) t cellmediated anti-tumor immunity recurrent loss of sting signaling in melanoma correlates with susceptibility to viral oncolysis species-specific detection of the antiviral small-molecule compound cma by sting bindingpocket and lid-region substitutions render human sting sensitive to the species-specific drug dmxaa the sting agonist dmxaa triggers a cooperation between t lymphocytes and myeloid cells that leads to tumor regression randomized phase iii placebo-controlled trial of carboplatin and paclitaxel with or without the vascular disrupting agent vadimezan (asa ) in advanced non-small-cell lung cancer design of amidobenzimidazole sting receptor agonists with systemic activity sting ligand c-di-gmp improves cancer vaccination against metastatic breast cancer endosomolytic polymersomes increase the activity of cyclic dinucleotide sting agonists to enhance cancer immunotherapy efficacy of the janus kinase / inhibitor ruxolitinib in the treatment of vasculopathy associated with tmem -activating mutations in children suramin potently inhibits cgamp synthase, cgas, in thp cells to modulate ifnbeta levels small molecule inhibition of cgas reduces interferon expression in primary macrophages from autoimmune mice development of human cgas-specific small-molecule inhibitors for repression of dsdnatriggered interferon expression the cyclopeptide astin c specifically inhibits the innate immune cdn sensor sting targeting sting with covalent small-molecule inhibitors trim -mediated monoubiquitination of cgas for cytosolic dna sensing the ubiquitin ligase trim regulates innate immune responses to intracellular doublestranded dna the e ubiquitin ligase rnf facilitates the cgas-mediated innate immune response rnf temporally regulates virus-triggered type i interferon induction by two distinct mechanisms the e ubiquitin ligase amfr and insig bridge the activation of tbk kinase by modifying the adaptor sting trim inhibits cgas degradation mediated by selective autophagy receptor p to promote innate immune responses p inhibition provides anti-dna virus immunity by regulation of usp phosphorylation and sting activation usp recruits usp to promote innate antiviral response through deubiquitinating sting/mita the deubiquitinase cyld is a specific checkpoint of the sting antiviral signaling pathway sumoylation promotes the stability of the dna sensor cgas and the adaptor sting to regulate the kinetics of response to dna virus senp potentiates cgas activation by relieving sumo-mediated inhibition of cytosolic dna sensing g bp promotes dna binding and activation of cgas zcchc is a cosensor of cgas for dsdna recognition in innate immune response manganese increases the sensitivity of the cgas-sting pathway for double-stranded dna and is required for the host defense against dna viruses tmem is a binding partner and regulator of sting-mediated inflammatory signaling in macrophages the erassociated protein zdhhc is a positive regulator of dna virus-triggered, mita/sting-dependent innate immune signaling association of abnormal elevations in ifit with overactive cyclic gmp-amp synthase/stimulator of interferon genes signaling in human systemic lupus erythematosus monocytes s k-sting interaction regulates cytosolic dna-mediated activation of the transcription factor irf glycogen synthase kinase beta regulates irf transcription factor-mediated antiviral response via activation of the kinase tbk trim short isoform preferentially promotes dna and rna virus-induced production of type i interferon by recruiting gsk beta to tbk lsm a plays a critical role in antiviral immune responses by regulating mita level in a cell-specific manner trim promotes dna virus infections by inhibiting innate immune response trim alpha is a negative-feedback regulator of the intracellular dna and dna virustriggered response by targeting sting the ubiquitin ligase rnf regulates antiviral responses by mediating degradation of the adaptor protein mita usp negatively regulates antiviral responses by deubiquitinating sting oligoadenylatesynthetase-family protein oasl inhibits activity of the dna sensor cgas during dna virus infection to limit interferon production interactome and proteome dynamics uncover immune modulatory associations of the pathogen sensing factor cgas sensing of bacterial cyclic dinucleotides by the oxidoreductase recon promotes nf-kappab activation and shapes a proinflammatory antibacterial state the ca( +) sensor stim regulates the type i interferon response by retaining the signaling adaptor sting at the endoplasmic reticulum nitro-fatty acids are formed in response to virus infection and are potent inhibitors of sting palmitoylation and signaling nlrc , a member of the nlr family of proteins, is a negative regulator of innate immune signaling induced by the dna sensor sting ppm a regulates antiviral signaling by antagonizing tbk -mediated sting phosphorylation and aggregation ptpn / -mediated dephosphorylation of mita/sting promotes its s proteasomal degradation and attenuates innate antiviral response nlrx sequesters sting to negatively regulate the interferon response, thereby facilitating the replication of hiv- and dna viruses nlrx promotes immediate irf -directed antiviral responses by limiting dsrnaactivated translational inhibition mediated by pkr mitigating sox -potentiated immune escape of head and neck squamous cell carcinoma with a sting-inducing nanosatellite vaccine mediates hypoxia-induced immunosuppression by repressing cgas nrf negatively regulates sting indicating a link between antiviral sensing and metabolic reprogramming kdm histone demethylases repress immune response via suppression of sting herpes simplex virus abrogates the cgas/sting-mediated cytosolic dna-sensing pathway via its virion host shutoff protein, ul herpes simplex virus tegument protein vp abrogates cgas/sting-mediated antiviral innate immunity species-specific deamidation of cgas by herpes simplex virus ul protein facilitates viral replication evasion of the sting dna-sensing pathway by vp / of herpes simplex virus herpes simplex virus gamma . protein inhibits sting activation that restricts viral replication human cytomegalovirus protein ul inhibits dna sensing of cgas to mediate immune evasion type i interferon production by inactivating the dna sensor cgas without affecting sting essential role of hcmv deubiquitinase in promoting oncogenesis by targeting anti-viral innate immune signaling pathways human cytomegalovirus tegument protein ul inhibits sting-mediated signaling to evade antiviral immunity the herpesviral antagonist m reveals differential activation of sting-dependent irf and nf-kappab signaling and sting's dual role during mcmv infection human cytomegalovirus-encoded us targets mavs and sting signaling to evade type i interferon immune responses inhibition of cgas dna sensing by a herpesvirus virion protein cytoplasmic isoforms of kaposi sarcoma herpesvirus lana recruit and antagonize the innate immune dna sensor cgas modulation of the cgas-sting dna sensing pathway by gammaherpesviruses sars coronavirus papain-like protease inhibits the type i interferon signaling pathway through interaction with the sting-traf -tbk complex poxviruses evade cytosolic sensing through disruption of an mtorc -mtorc regulatory circuit viral and metazoan poxins are cgamp-specific nucleases that restrict cgas-sting signalling zika virus elicits inflammation to evade antiviral response by cleaving cgas via ns -caspase- axis denv inhibits type i ifn production in infected cells by cleaving human sting dengue virus ns b protein targets cgas for degradation and prevents mitochondrial dna sensing during infection dna tumor virus oncogenes antagonize the cgas-sting dna-sensing pathway influenza a virus targets a cgas-independent sting pathway that controls enveloped rna viruses hepatitis c virus ns b blocks the interaction of sting and tbk to evade host innate immunity hepatitis c virus ns b protein targets sting and abrogates rig-i-mediated type i interferon-dependent innate immunity hepatitis b virus polymerase disrupts k -linked ubiquitination of sting to block innate cytosolic dnasensing pathways hiv- /siv vpx targets a novel functional domain of sting to selectively inhibit cgas-stingmediated nf-kappab signalling we thank k. wood from barrow neurological institute for discussions and editing. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © wan, jiang and hao. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -tkqxb ql authors: toman, miroslav; celer, vladimir; kavanová, lenka; levá, lenka; frolichova, jitka; ondráčková, petra; kudláčková, hana; nechvátalová, kateřina; salat, jiri; faldyna, martin title: dynamics and differences in systemic and local immune responses after vaccination with inactivated and live commercial vaccines and subsequent subclinical infection with prrs virus date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: tkqxb ql the goals of our study were to compare the immune response to different killed and modified live vaccines against prrs virus and to monitor the antibody production and the cell mediated immunity both at the systemic and local level. in the experiment, we immunized four groups of piglets with two commercial inactivated (a —progressis, a —suivac) and two modified live vaccines (b —amervac, b —porcilis). twenty-one days after the final vaccination, all piglets, including the control non-immunized group (c ), were i.n., infected with the lelystad strain of prrs virus. the serum antibody response (igm and igg) was the strongest in group a followed by two mlv (b and b ) groups. locally, we demonstrated the highest level of igg antibodies in bronchoalveolar lavages (balf), and saliva in group a , whereas low iga antibody responses in balf and feces were detected in all groups. we have found virus neutralization antibody at dpv (days post vaccination) and higher levels in all groups including the control at dpi (days post infection). positive antigen specific cell-mediated response in lymphocyte transformation test (ltt) was observed in groups b and b at dpv and in group b at dpv and in all intervals after infection. the ifn-γ producing lymphocytes after antigen stimulation were found in cd (−)cd (+) and cd (+)cd (+) subsets of all immunized groups days after infection. after infection, there were obvious differences in virus excretion. the virus was detected in all groups of piglets in serum, saliva, and occasionally in feces at dpi . significantly lower virus load was found in groups a and b at dpi . negative samples appeared at dpi in b group in saliva. it can be concluded that antibodies after immunization and infection, and the virus after infection can be detected in all the compartments monitored. immunization with inactivated vaccine a —progressis induces high levels of antibodies produced both systemically and locally. immunization with mlv-vaccines (amervac and porcilis) produces sufficient antibody levels and also cell-mediated immunity. after infection virus secretion gradually decreases in group b , indicating tendency to induce sterile immunity. the goals of our study were to compare the immune response to different killed and modified live vaccines against prrs virus and to monitor the antibody production and the cell mediated immunity both at the systemic and local level. in the experiment, we immunized four groups of piglets with two commercial inactivated (a -progressis, a -suivac) and two modified live vaccines (b -amervac, b -porcilis). twenty-one days after the final vaccination, all piglets, including the control non-immunized group (c ), were i.n., infected with the lelystad strain of prrs virus. the serum antibody response (igm and igg) was the strongest in group a followed by two mlv (b and b ) groups. locally, we demonstrated the highest level of igg antibodies in bronchoalveolar lavages (balf), and saliva in group a , whereas low iga antibody responses in balf and feces were detected in all groups. we have found virus neutralization antibody at dpv (days post vaccination) and higher levels in all groups including the control at dpi (days post infection). positive antigen specific cell-mediated response in lymphocyte transformation test (ltt) was observed in groups b and b at dpv and in group b at dpv and in all intervals after infection. the ifn-γ producing lymphocytes after antigen stimulation were found in cd − cd + and cd + cd + subsets of all immunized groups days after infection. after infection, there were obvious differences in virus excretion. the virus was detected in all groups of piglets in serum, saliva, and occasionally in feces at dpi . significantly lower virus load was found in groups a and b at dpi . negative samples appeared at dpi in b group in saliva. it can be concluded that antibodies after immunization and infection, and the virus after infection can be detected in all the compartments monitored. immunization with inactivated vaccine a -progressis porcine reproductive and respiratory syndrome (prrs) is the most economically significant infectious disease currently affecting swine worldwide. typical clinical symptoms of prrs are mild to severe respiratory disease in infected newborn and growing pigs, and reproductive failure in pregnant sows. two genotypes of the prrs virus (prrsv) have been identified: european (type ) and north american (type ). there are considerable genetic and virulence differences between and within prrsv genotypes ( - ) correlated with a lack of cross-protection by vaccines ( ) ( ) ( ) ( ) ( ) . highly pathogenic strains of prrsv (hp-prrsv) have been identified within both genotypes ( ) ( ) ( ) . depending on viral strain and immune status of the host, some swine farms may have pigs subclinically infected, whereas others experience severe reproductive, and/or respiratory disease. infection with both "classical" and highly pathogenic strains is associated with aberrant host immune response ( , ) . swine are the only known natural host of prrsv and the primary target cells for replication of prrsv are porcine alveolar macrophages (pams) ( ) . the first stage is represented by acute infection, resulting in viremia - h post-infection (pi), and lasting for several weeks despite the presence of circulating antibodies. in the second, persistent stage of infection, the virus is no longer detected in blood and lungs, and pigs no longer exhibit signs of clinical disease. in this stage, viral replication is primarily localized in lymphoid organs, including tonsils, and lymph nodes ( ) . infection with prrsv elicit poor innate and adaptive immune responses associated with immune modulation and incomplete viral clearance in most of the pigs, depending on their age, and immune status ( , ( ) ( ) ( ) . infection with certain prrsv strains induced significant suppression of nk cell cytotoxic activity ( ) . the quantity of pro-inflammatory cytokines is significantly lower than in other viral infections and is strain dependent ( ) . prrsv is also a poor inducer of ifn-α. infection with prrsv induces an antibody response (production) by - dpi but with no evidence of protection against prrsv infection; serum neutralizing antibodies appear only later, typically ≥ days pi ( ) . the virus also evades host cell-mediated immunity most likely by the promotion of immunosuppressive cytokines il- and tgf-β resulting in delayed onset of th immune response ( ) . similarly, an immunosuppressive function of prrsv was shown to probably be mediated by the cytokines il- and tgf-β and action of treg ( ) ( ) ( ) . immunosuppression induced by prrsv facilitates other viral and bacterial infections ( , , ) . vaccination is the principal means used to control and treat prrsv infection. several comprehensive review articles have been published recently. they critically evaluate different vaccination approaches against the prrs virus and indicate the main weaknesses of current vaccines and vaccination strategies ( ) ( ) ( ) ( ) . among others the problem are caused by high heterogeneity and occurrence of highly pathogenic strains and therefore efforts have been made to develop vaccines with a broad spectrum of effects ( , , , ( ) ( ) ( ) ( ) . however, the opinion still prevails that vaccination is more cost-beneficial over other health interventions ( ) ( ) ( ) . our study had the following three aims: ) to establish complex immune response characteristics using several methodological approaches; ) to monitor the dynamics in different compartments and in a time-dependent manner after vaccination and the challenging infection; ) to compare the types of immune responses after vaccination with inactivated or live attenuated vaccines and subsequent challenge using a homologous strain. twenty-five weaned piglets aged weeks and weighing - kg of the large white breed from a prrsv negative herd were used. the negative status of the animals was confirmed by serology using commercial elisa kit (idexx labs). the use of animals was approved by the branch commission for animal welfare of ministry of agriculture of the czech republic (approval protocol no. mze- ) as a part of project as a part of project respig (qj ). four commercial vaccines were used. their characteristics are in table . the virus was clarified by centrifugation, and its concentration was determined by plaque assay. the concentration of stock virus used in experiments was × plaque forming units per ml. twenty-five piglets were used in the experiment. the piglets were assigned to five groups of five animals each according to weight and gender. the animals were housed in bsl isolation rooms, keeping animals from only one experimental group in each room. the animals were left to acclimate for days after stocking. all piglets were clinically healthy at the time the experiment started. on day (d ) two groups of piglets (a and a ) were immunized. each animal was administered ml of inactivated vaccine by an intramuscular (i.m.) injection. after days (d ), piglets in these groups were revaccinated with the same dose, and piglets from the other two groups (b and b ) were immunized with ml of a mlv vaccine. the health status of piglets was monitored on a regular basis, including temperature measurements, and samples of blood and other body fluids were taken for respective examinations at preset time intervals. after an additional days (d ), all pigs, including control group (c ), were infected with ml of the live prrs virus. the piglets were monitored for another days and then slaughtered (d ). euthanasia was performed by exsanguination after combined anesthesia with a tkx (telazol-ketamin-xylazin) mixture containing . mg/ml tiletamine and . mg/ml zolazepam (telazol, virbac, carros, france), . mg/ml ketamine (vetoquinol, lure, france), and . mg/ml xylazine (bioveta, ivanovice na hane, czech republic), administered intramuscularly in a final volume of . ml/kg body weight. as well as collection of blood and other body fluids (intestinal contents, bronchoalveolar lavage), an autopsy was performed and organs (lung parenchyma, spleen, lymph nodes,...) were collected for virological examination. blood samples for serum and heparin-treated blood samples were taken from the jugular vein. group saline samples were collected using ropes which were left in the hutch for h. individual fecal samples were collected when handling the animals. bronchoalveolar lavage fluid (balf) sampling was performed for the first time on live animals and for the second time after slaughter. the intravital lavage was performed with the animals under general anesthesia (a mixture of xylazine and ketamin) without the use of an endoscope by a method described earlier ( ) . pigs were positioned in the sternal recumbency. an endotracheal tube was inserted into the trachea and ml of sterile pbs (ph . ) was injected into the distal parts of the airways, toward the bronchus. about % of the infused saline was recovered as balf aspirate and was filtered and centrifuged for min at g. supernatant was stored at − • c prior to serological analyses. total rna from experimental samples of sera, oral fluids, and bal ( µl) was extracted using a nucleospin r rna ii kit (macherey-nagel), in accordance with the manufacturer's instructions (protocol for total rna preparation from biological fluids). the rna obtained was eluted in µl rnase-free water and immediately used for qrt-pcr amplification. remaining rna was frozen at − • c for subsequent use. isolated rna was used for qrt-pcr amplification by ez-prrsv tm mpx . real time rt-pcr kit (tetracore), in accordance with the manufacturer's instructions. quantification of the virus genome copies was based on quantification standards included in the kit. for the evaluation of systemic and local antibody production two elisa methods were used. all swine sera tested were examined by commercially available elisa test ingezim prrs universal (ingenasa), in accordance with the manufacturer's instructions, to examine for the presence of n protein specific igg antibodies. all swine sera, oral fluids, and bal tested were examined by home-made indirect elisa test based on recombinant nucleocapsid protein n of prrs virus (developed previously in our department) for detection of specific igm, igg, and iga antibodies. optimal antigen, serum, and antibodies concentrations were determined by checkerboard titration of positive and negative porcine sera. the cut-off value was determined by defining the upper prediction limit based on the upper tail of the t-distribution of negative control od readings, at a confidence level of . %. positive serum with an absorbance corresponding to the calculated cut-off was included in all test plates. the recombinant n protein diluted in mm bicarbonate-carbonate buffer ph . to a final concentration of . µg/ml was coated on -well-microtiter plates (maxisorp ii r immunoplates, nunc, denmark) overnight at • c. the wells were then blocked with % skimmed milk in pbs for min at • c and then washed with pbs. positive and negative controls were included in each test plate. each sample diluted in % skimmed milk in t-pbs (pbs with . % tween and . m nacl) was added in duplicates on antigen-coated wells with some differences among different types of samples. one hundred microliters of serum samples diluted : (for detection of igm antibodies) or µl of non-diluted samples of bal (for detection of igg and iga antibodies) were incubated for min at • c. two hundred and fifty microliters of oral fluids diluted : were incubated h at • c (for detection of igg antibodies). subsequently the plates were washed three times with t-pbs and antibody binding was detected by incubation for min at • c with µl of anti-pig igm peroxidase conjugate ( : , , bethyl), anti-pig igg peroxidase conjugate ( : , , sigma), or with anti-pig iga peroxidase conjugate ( : , , bethyl) separately (diluted in t-pbs with % skimmed milk). after washing the plates as described above, µl per well of the tmb-complete (test-line) substrate was added. the optical density (od) was measured at nm after an incubation time of - min at room temperature. the virus neutralization test for detection of prrsv neutralization antibodies was performed as follows. samples of sera were diluted : in dmem medium (sigma-aldrich) supplemented with % fbs. then, heat inactivated sera ( • c for min) were diluted -fold serially in flat-bottom -well-microplate (nunc). next, equal volume ( µl) of media containing prrsv pfu (lelystad-capm v- ) was added to each well. following incubation ( min at • c) marc- cells were added to each well ( × per well) in µl media per well. after days of cultivation ( • c, % co ), the cytopathic effect (cpe) of prrsv on marc- was evaluated by optical microscopy. the reciprocal value of the last sera dilution causing % reduction of cpe was defined as virus neutralization antibody titer. the lymphocyte transformation test was performed according to the method published earlier ( ) . peripheral blood mononuclear cells (pbmc) were obtained by gradient centrifugation post infection d ++ ++ + + + + + + -+ + + + + + ++ + + + + + + ++ the end of experiment d + ++ ++ + ++ + + + ± -+ + ++ + frontiers in immunology | www.frontiersin.org (histopaque- , sigma-aldrich). concentration of the cells was adjusted to , cells in µl of rpmi- medium supplemented with % of autologous serum, iu/ml penicillin, µg/ml streptomycin, and µg/ml gentamicin). they were incubated with the µg of optimal concentration of the specific antigen (moi . ) for days at • c in % co . negative controls were incubated with rpmi- medium only. all samples were evaluated in triplicate. h-thymidine was added on the last day of cultivation. subsequently, the cells were harvested (filtermate harvestor, packard bioscience company, usa), and h-thymidine incorporation was measured by a microplate scintillation and luminescence counter (topcount nxt tm , packard bioscience company) in counts per minute (cpm). the results were expressed in terms of stimulation indexes (si), which were calculated as the ratio of cpm in stimulated samples vs. cpm in non-stimulated controls. the number of ifn-γ producing cells was calculated by elispot techniques. commercially available porcine ifn-γ elispot kit ( - hpw- , mabtech) was used in accordance with the manufacturer's instructions. the number of cells used in the test was × /well. prrsv was used for stimulation of the antigen-specific response in multiplicity of infection (moi) . . mitogen cona at a concentration µg/ml was used as a positive control. cells without stimulation were used as a negative control. incubation lasted for h. spots were detected using elispot reader system elro tl (aid, germany). the results were recalculated to the number of cd + lymphocytes. the × of pbmc per well was stimulated with prrs virus in moi . for h. the × of cultured pbmc were pelleted and µl of primary monoclonal antibody cocktail containing anti-cd (igg b, clone - - , wsu, monoclonal antibody center, usa), anti-cd (igg a, clone - - , wsu, usa), and anti-γδ tcr (igg , clone pgbl a, wsu, usa) and µl of heat-inactivated goat serum was added. the cells were incubated for min at • c and then rinsed twice with cell washing solution. then, µl of goat anti-mouse secondary antibody cocktail (anti-igg b: dylight , anti-igg a: alexa fluor , and anti-igg : pe-cy ) was added and the cells were incubated for another min at • c. the cells were rinsed and then µl of anti-cd antibody (igg , clone ppt , southern biotech, pre-stained with alexa fluor dye using zenon antibody labeling kit, invitrogen) was added and the cells were incubated, rinsed twice, and fixation and permeabilization for subsequent intracellular staining was performed by solutions a and b of intra stain kit (dako cytomation, usa) ( ) . finally, µl of rpe-conjugated anti-ifn-γ antibody (clone cc , abd serotec uk) was added and the cells were incubated for min. the cells were measured as soon as possible using bd lsr fortessa flow cytometer (becton-dickinson, usa). at least , events were acquired. the post-acquisition analysis of data was performed using the facs diva software (becton-dickinson, usa). the following lymphocyte subpopulations were identified: (cd + ) γδ + cd + , (cd + ) γδ + cd − , (cd + γδ − ) cd + cd + , (cd + γδ − ) cd − cd + , (cd + γδ − ) cd + cd − , (cd + γδ − ) cd − cd − , and cd − cd + . the percentage of ifn-γ-positive cells was established for each subpopulation. the normality of data distribution were confirmed. experimental groups were compared using non-parametric man-whitney test. data from different dates were compared using non-parametric wilcoxon test for paired samples. after vaccination with inactivated vaccines (a and a ) the first igm in the serum started to appear days after the first dose in some piglets, and days after the second dose in all animals of the a group ( figure a and table ). igg antibodies appeared in all animals of both groups days after the second dose ( figure b) . the level of antibodies in the a group was significantly higher than in the group given the a vaccine. in groups of piglets vaccinated with mlv vaccines (b and b ), both igm and igg antibodies appeared days after vaccination. on day after immunization, their antibody responses were comparable to that of the a group. after infection, we identified a further increase in antibodies in the vaccinated groups. for the a group, a further increase in serum igg antibodies was observed after week and especially at and days after infection, when this antibody level significantly exceeded the values in the mlv immunized groups (b and b ) and the control one (c ). the a group showed a sharp increase on post infection days and , and the level of serum igg antibodies at these intervals was comparable to a . in groups immunized with mlv (b and b ), serum igg levels increased after , , and days post infection, but did not reach the a group values. in the control, non-immunized group, the first igm antibodies appeared days after infection, with a significant increase on day and . igg antibody levels appeared and days after infection but were lower than in the immunized groups. the virus neutralization antibody was detected in sera of animals days after vaccination (figure c) . these antibodies were detected in some animals in the groups a and a only. at the end of experiment (d , days post-infection) the high level of virus neutralization antibodies were detected in all groups except b group, in which significantly lower level was found ( figure c) . local antibodies in the balf performed days post vaccination we detected low levels of iga in all immunized groups (figure a) and igg in a , a , and b (none in b ) with the level in the a group being significantly higher (figure b) . days after infection we detected low levels of iga antibodies in a , b , b , and control group (c ) and low levels of igg antibodies in all immunized groups (none in c ). local antibodies were detected in the saliva of the a group in low concentrations and days after the second vaccination and in all groups after infection with variability in individual intervals and with no statistically significant between-group difference ( figure c) . in the feces, local antibodies (iga) were detected from week after the second immunization dose in groups a and a , and from week after mlv immunization in groups b and b ( figure d ) increased levels of antibody were detected in all groups including control after infection with no statistically significant betweengroup difference. a positive cell-mediated response after lymphocyte stimulation with specific antigen in vitro (stimulation index in ltt above ) was observed in the b group after and days post vaccination and and days post-infection (figure ) . a positive stimulation index was detected in the b group at days post vaccination only as a non-specific basal stimulation occurred from days post vaccination in this group (figure a) . groups immunized with inactivated vaccines a and a showed a marked non-specific stimulation of cells even without using antigen ( figure a ) and, therefore, it was impossible to demonstrate the effect of antigen addition and thus cell-mediated immune response. cell-mediated immune response after challenge infection was positive in all vaccinated groups and in the control group after days post infection, using elispot in pbmc from bronchoalveolar lavages. the results were recalculated to the number of cd + lymphocytes. the differences between individual animals, but no significant differences between groups were detected ( figure d) . we detected ifn-γ producing lymphocytes after prrs antigen stimulation. the most marked differences from control were found in cd − cd + and cd + cd + (and partly also in cd − + and γδ + + ) subsets of all immunized groups days after infection. in the groups vaccinated with live vaccines (b and b ) , the virus load was demonstrated in serum and saliva from day after immunization, in balf days after immunization (the only time point when the lavage was taken), and in feces occasionally days after vaccination, then in all piglets days after immunization (data not shown). no clinical signs were observed in piglets after infection. elevated body temperature was occasionally found in the first days, independent of the experimental group. however, viral shedding was noted, with between-group differences. the virus appeared in serum, saliva, and feces in all groups including the control group days after infection (figures a,b,d) . the virus was detected in balf days after infection in a , a , and c groups (figure c) . virus shedding was decreased in immunized groups and days after infection with the level in the a and b group being significantly lower compared to control group days after infection. negative samples appeared days after infection in saliva (in b group) and in feces (b and b groups). the goals of our study were ( ) to establish comprehensive immune response characteristics using several methodological approaches and monitor the dynamics in different compartments and in a time-dependent manner after vaccination and the challenging infection and ( ) to compare the immune response to different killed and modified live vaccines against prrs using these methodological tools. in order to compare the immune response after vaccination with different vaccines, we used a model of vaccinations of young piglets (beginning at weeks of age) and given vaccination intervals and subsequent infections, regardless of the fact that manufacturers' recommendations were different (especially in progressis). there are only a few papers published providing a comprehensive picture of immune response after vaccination against prrsv ( ) ( ) ( ) ( ) because the majority of the existing studies are based mainly on the evaluation of the vaccination effectiveness by monitoring the immune responses found in the blood ( , , , ( ) ( ) ( ) . our results show that antibodies after immunization and infection, and the virus after infection, can be detected in all the monitored compartments (blood, respiratory tract, intestine). by repeated sampling and simultaneous monitoring of the antibody and cell-mediated immunity and virus shedding systematically and locally, we have managed to get comprehensive information about the dynamics of the immune response after vaccination or prrs virus infection. in practical diagnostics of field samples is an effort to seek simple approaches to obtain tentative information on the epidemiological situation of the herd. one current trend is the monitoring of antibody levels and shedding of the virus in the oral fluid ( ) ( ) ( ) . in our experiment, the antibody detection rate in the oral fluid collected with ropes in pens was sufficient. the levels of antibodies detected after vaccination were low, but they increased after challenge infection. these findings confirms the possibility of using this approach for preliminary characteristics in the herd. it was interesting to observe the dynamics of antibody levels and viral shedding in feces too. this is an approach which is not often used for prrsv infection monitoring but is used in other situations where feces samples are more readily available than samples from other sources ( , ). we were wondering, among other things, to what extent infections occurring systemically, or locally in the respiratory tract occur in this remote compartment. our findings show that fecal samples can also be used for prrsv infection monitoring. detection of both the viruses and antibodies is not entirely consistent, because they appear in individual animals, and cease at later intervals, therefore, it is necessary to consider these findings as approximate. they can be used for herd-or pen-level testing, but not for establishing a diagnosis in individual animals. it appeared to be technically difficult to demonstrate specific cell-mediated immunity. the partial results were provided by each of the three methods used and a comprehensive picture could be obtained by compiling this information. therefore, it was not possible to use only data of ifn-γ production in elispot, although it is currently the most commonly used method for cmi ( , , , , , ) . a positive cell-mediated response after lymphocyte stimulation with specific antigen in vitro (in lymphocyte transformation test) was observed in mlv groups and especially in the b group as a non-specific basal stimulation occurred from days post-vaccination in b group. the strong non-specific stimulation of pbmc without specific antigen were detected in groups a and a immunized with inactive types of vaccine. this non-specific stimulation of cells in vivo masks the overall picture, and thus specific cell-mediated immunity cannot be demonstrated. this effect is attributed to the use of strong adjuvants in inactivated types of vaccines. in the test of ifn-γ production and detection with elispot, which is very often used to identify cmi both in experimental studies ( , ) , and in the field ( , ) , we have shown an increase in both blood and cells acquired by lavage, but the individual variability among the animals was too high and, consequently, there were no differences found between the groups under study. we detected also ifn-γ producing lymphocytes after prrs antigen stimulation in all immunized groups days after infection. the most marked differences from control were found in cd − cd + and cd + cd + (and partly also in cd − cd + and γδ + cd + ) subsets of lymphocytes. the cd − cd + subpopulation belongs to cytotoxic groups of cells, cd + cd + is considered a group of th memory cells ( ) . in another study the expression of cytotoxic cd + cd + and cd + cd − was described which help to recover from prrs infection ( ) . there were qualitative and quantitative differences in the immune responses to the inactivated vaccines and to mlv ones. after immunization with the inactivated vaccine (especially a -progressis), high levels of antibodies were produced generally (serum), which were mostly of the igm, and igg isotypes, and also locally (saliva, balf), both igg and iga. nevertheless it should be noted that we have applied progressis to piglets in our study, while the manufacturers declare the use of this vaccine for gilts and sows. cell-mediated immunity was detected only after infection, high non-specific cell stimulation was detected after vaccination and therefore any specific response could not be demonstrated in these intervals. the antigen specific cell mediated immunity after inactivated vaccine is rarely described ( ) . most work describes low or no cmi after vaccination with inactivated vaccine. after immunization with mlv vaccines, sufficient levels of antibodies in serum and balf (igg) were also produced, but lower than after the inactivated vaccine administration. the levels of iga antibodies in balf were comparable but low. low levels of virus neutralization antibodies after vaccination can be explained by a short interval between vaccination and infection, since neutralizing antibodies after vaccination or prrs infection occur within days ( ) . the dynamics of virus shedding after vaccination and infection is often used for monitoring vaccine efficacy ( , , ) . the decrease in virus secretion was observed days after mlv immunization and disappearance in days ( ) . in another study the excretion of virus was described still for days after vaccination with for porcilis or amervac vaccine ( ) . demonstration of cell-mediated immunity and reduction in viral load correlate with studies by other authors and support the preferred use of mlv vaccines in the control of prrs infection ( , ) . the question is to what extent these results are influenced by the composition of vaccines from different manufacturers and to what extent different types of vaccines (inactivated vs. live attenuated). there was an obvious difference in the quality between the inactivated vaccines, whereas the character of the immune response to both mlv vaccines was similar with only partial differences in the time-related response dynamics. the vaccine b (amervac) showed a more pronounced decrease in virus secretion and a tendency to induce sterile immunity, while b (porcilis) vaccine had a more pronounced of cmi in lymphocyte transformation test. it should be noted that the strain used in porcilis had a higher genetic link with the lelystad strain compared to the strain of amervac ( , ) which, however, probably did not significantly affect the above characteristics. despite the fact that many studies focused on prrs immunoprophylaxis have already been published and many procedures are implemented in the agricultural industry, a universal model does not yet exist ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the use of live attenuated vaccines is generally preferred as was also confirmed in our field study ( ) . in this study, we controlled the infection by a repeated blanket immunization with mlv vaccine (porcilis), followed by targeted immunization of gilts, and sows. the success of the strategy selected and evidence of virus eradication from the given herd were demonstrated by introducing sentinel animals into a fattening herd. based on this result, we believe that control programs can be adopted even in herds with continual throughput housing without interrupting production. however, in this case, vaccination is only one of the necessary preconditions and the introduction of very strict principles of good biosecurity is of no less importance. the use of animals was approved by the branch commission for animal welfare of ministry of agriculture of the czech republic (approval protocol no. mze ) as a part of project as a part of project respig (qj ). mt, vc, js, and mf designed the study. mt, ll, js, and kn performed the experiments. ll, hk, lk, po, and jf performed the lab work and analyzed the data. lk produced the figures and statistical analysis. mt wrote the manuscript. js and mf participated in manuscript preparation. all authors read and approved the final manuscript. the study was supported by projects no. qj , qj , and ro of the 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(prrs) modifiedlive virus vaccine on sow reproductive performance in endemic prrs farm successful elimination of prrs virus from an infected farrow-to-finish herd by vaccination key: cord- -i n x uc authors: hwang, ji young; silva-sanchez, aaron; carragher, damian m.; garcia-hernandez, maria de la luz; rangel–moreno, javier; randall, troy d. title: inducible bronchus–associated lymphoid tissue (ibalt) attenuates pulmonary pathology in a mouse model of allergic airway disease date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: i n x uc inducible bronchus associated lymphoid tissue (ibalt) is an ectopic lymphoid tissue associated with severe forms of chronic lung diseases, including chronic obstructive pulmonary disease, rheumatoid lung disease, hypersensitivity pneumonitis and asthma, suggesting that ibalt may exacerbate these clinical conditions. however, despite the link between pulmonary pathology and ibalt formation, the role of ibalt in pathogenesis remains unknown. here we tested whether the presence of ibalt in the lung prior to sensitization and challenge with a pulmonary allergen altered the biological outcome of disease. we found that the presence of ibalt did not exacerbate th responses to pulmonary sensitization with ovalbumin. instead, we found that mice with ibalt exhibited delayed th accumulation in the lung, reduced eosinophil recruitment, reduced goblet cell hyperplasia and reduced mucus production. the presence of ibalt did not alter th priming, but instead delayed the accumulation of th cells in the lung following challenge and altered the spatial distribution of t cells in the lung. these results suggest that the formation of ibalt and sequestration of effector t cells in the context of chronic pulmonary inflammation may be a mechanism by which the immune system attenuates pulmonary inflammation and prevents excessive pathology. inducible bronchus associated lymphoid tissue (ibalt) is an ectopic lymphoid tissue that forms in the lung following inflammation or infection ( , ) . large b cell follicles that often contain germinal centers (gcs) and a dense network of follicular dendritic cells (fdcs) are the most prominent features of ibalt ( ) . unlike conventional lymph nodes, ibalt is not encapsulated, but is instead embedded in the lung tissue, most often along large bronchi or filling the peri-vascular space of pulmonary artieries ( , ) . despite being in a mucosal tissue, ibalt often lacks a welldefined m cell-containing dome epithelium ( ) and its high endothelial venules (hevs) express peripheral lymph node addressin (pnad) ( ) rather than mucosal addressin cell adhesion molecule (madcam). thus, although ibalt clearly participates in pulmonary mucosal immune responses, it lacks some of the features often associated with classic mucosal lymphoid organs. unlike conventional lymphoid organs, which develop independently of antigen during embryogenesis ( , ) , ibalt forms following pulmonary infection or inflammation ( , ) . although ibalt may develop at any time after birth given the proper pulmonary insult, it most easily forms in neonatal mice due to the relative paucity of tregs and the relative abundance of il- -producing γδt cells ( ) ( ) ( ) . similarly, infant humans also seem to develop (or maintain) ibalt more easily than adults, as the frequency of ibalt areas in the lungs of healthy subjects progressively declines with age ( , ) . interestingly, the neonatal period is the same developmental window when microbial exposure, or lack thereof, imprints the immune system to be more or less susceptible to developing atopic responses to foreign antigens ( , ) or developing autoreactive responses to self antgens-the so-called hygiene effect. given that it functions as a lymphoid tissue ( ) , it is not too surprising that ibalt plays an important role in immunity to pulmonary infections. for example, mice lacking conventional lymphoid organs develop ibalt following infection with influenza and are able to resist doses of virus that would normally be lethal to mice lacking ibalt ( ) . conversely, mice lacking the chemokines that promote ibalt formation succumb to much lower doses of influenza virus ( ) . those same chemokines are essential for the proper formation of pulmonary granulomas, the recruitment of antigen-specific t cells to the lung and the development of protective immunity to mycobacterium tuberculosis ( , ) . moreover, the pulmonary administration of inflammatory agents that trigger ibalt formation also leads to enhanced resistance to influenza, sars corona virus, pneumovirus, francicella tulerensis and coxiella burnetti ( , , ( ) ( ) ( ) . thus, the presence of ibalt is clearly beneficial in the context of pulmonary infections. in contrast, ibalt is a feature of pulmonary pathology that is associated with a variety of chronic lung diseases, including chronic obstructive pulmonary disease (copd) ( , ) , rheumatoid lung disease ( ), hypersensitivity pneumonitis ( ) , and asthma ( , ) , suggesting that ibalt may exacerbate these clinical conditions. for example, copd patients make autoantibodies that react with pulmonary antigens ( ) and patients with advanced disease have reactive ibalt areas with well-developed gcs that show evidence of antigendriven selection ( ) . similarly, patients with rheumatoid lung disease develop large gc-containing areas of ibalt that are surrounded by plasma cells that produce autoantibodies ( ) . thus, many investigators have concluded that the presence of ibalt contributes to pathology in the context of chronic inflammatory diseases. however, it is not clear whether ibalt is a cause or consequence of pulmonary inflammation or whether it contributes in any way to pulmonary pathology ( ) . given the link between ibalt and pulmonary pathology, we tested whether pre-existing ibalt would affect acute allergic responses to a pulmonary allergen. surprisingly, we found that the presence of ibalt did not exacerbate allergic responses to repeated pulmonary sensitization with ovalbumin (ova). instead, mice with ibalt had reduced th -associated mrna expression, less eosinophil recruitment to the lungs and airways, attenuated goblet cell hyperplasia and reduced mucus production following pulmonary sensitization and challenge with ova. interestingly, the presence of ibalt did not alter the initial priming of th cells, but instead delayed their recruitment to the lung and altered their spatial distribution following challenge-th cells were preferentially sequestered in ibalt, which reduced the numbers of th cells in the lung parenchyma. these results suggest that the formation of ibalt and sequestration of effector t cells in the context of pulmonary inflammation may be a mechanism by which the immune system attenuates pulmonary inflammation and prevents excessive pathology. to test the role of ibalt in a mouse model of allergic lung inflammation, we intranasally administered µg lps times over the course of days starting on day after birth and analyzed the lungs by histology on day ( figure a) . as expected ( ) , we observed ibalt structures with separated b and t cell zones and distinct fdc networks in the lpstreated lungs, but not in the control lungs ( figure b) . we also quantified the number of gc b cells by flow cytometry and found that gc b cells were present in lps-treated lungs, but not in control lungs (figures c,d) . given that gcs only form in organized lymphoid tissues, we conclude that pulmonary exposure of neonates to lps promotes the formation of functional ibalt areas. to test the effect of ibalt on the immune response to a pulmonary allergen, we administered lps (or pbs) to neonatal mice as described above, allowed the mice to rest until they were weeks old, then intranasally sensitized the ibalt and control groups with µg ova in combination with low dose ( . µg) lps on days , , and and challenged them on days , , , and with µg ova ( figure e ). we first examined the gc b cell response in the lungs of sensitized and challenged mice and found that gc b cells were abundant in the lungs of mice with ibalt, but were rare in control mice and almost undetectable in completely naïve mice (figures f,g) . we also examined the inflammatory response in the lung and airways by cytospin and found that the proportion (figures h,i) and number (figures j,k) of eosinophils were elevated in mice that had been sensitized and challenged with ova compared to those in naïve mice, however, the proportion (figures h,i) and number (figures j,k) of eosinophils in mice with ibalt were reduced compared to those in control mice. reductions in eosinophils were observed in both the lungs (figures h,j) and the airways (figures i,k) of mice with ibalt compared to those in control mice. the proportions of macrophage/monocytes, lymphocytes, and neutrophils are also shown in figure h and the numbers of those cells are shown in supplementary figure . we also found that total serum ige concentration was elevated in control mice, but less ige was detected in mice with ibalt ( figure l) . upon a histological examination of lung sections, we found that ovaexposed mice with pre-existing ibalt had densely-packed areas of lymphocytes to one side of the bronchi, but relatively clear airspaces, whereas ova-exposed control mice had extensive areas of diffuse inflammation that surrounded the bronchi and extended into the alveolar airspaces ( figure m) . thus, contrary to our expectations, the presence of ibalt did not exacerbate ova-induced pulmonary inflammation, but rather reduced the inflammatory response. the presence of ibalt could have altered t cell priming so that fewer th cells were initially generated after antigen exposure. to test this possibility, we next used il- reporter ( get) mice to enumerate th cells. the get mice have the enhanced green fluorescent protein (egfp) targeted into the il- locus with an internal ribosome entry site (ires) separating the il- coding sequence from the egfp coding sequence ( ) . thus, any cells that express il- mrna will also express egfp and can be easily identified using flow cytometry ( ) . therefore, we treated neonatal get mice with lps to initiate ibalt formation, waited until they were adults and then intranasally sensitized ibalt and control mice with µg ova and . µg lps on days , , and (figure a) . two days after the last sensitization, we enumerated il- reporter (egfp)-expressing cd + t cells. we found a significant expansion of egfp + cd + t cells in both ibalt and control mice relative to naïve mice, but did not observe any difference in the frequencies ( figure b ) or numbers ( figure c ) of egfp + cd + t cells between control and ibalt groups. these data suggested that ibalt did not dramatically affect th priming in the lung. although the previous assay evaluated th priming, we had no way of enumerating antigen-specific cells. therefore, to test this possibility in another way, we transferred x naïve ovaspecific cd + t cells (ot-ii cells) into control and ibalt mice on day after birth, sensitized the recipient mice with ova on days , , and , challenged them on days , , , and and enumerated the responding cells days later in the lung ( figure d ). we found that the frequency ( figure e ) and number ( figure f ) of ot-ii cells that accumulated in the lungs of sensitized and challenged mice were the same in control and ibalt groups. these data suggest that the presence of ibalt does not affect ova-specific t cell accumulation in the lung after sensitization and challenge. although th priming and expansion appeared normal in mice with ibalt, it was not clear whether the final effector cells could actually make th cytokines. therefore, to determine whether cd t cells primed by ova sensitization and challenge could actually produce th cytokines, we sensitized and challenged mice with and without pre-existing ibalt and measured the mrna expression of th cytokines in the lungs h after the last ova challenge ( figure a) . we found that mrnas encoding il- , il- , and il- were increased in ova-exposed and challenged lungs relative to their expression in naïve lungs, but we did not observe a significant difference between the control and ibalt groups ( figure b) . we next tested whether cd + t cells from ova sensitized and challenged control and ibalt mice were equally capable of making th cytokines. to test this possibility, we collected cells from the lung tissue, restimulated them with pma and calcimycin for h and assessed the production of il- , il- , and il- by intracellular staining. we found that the total number of activated cd hi cd + t cells was increased in control and ibalt mice relative to that in naïve mice (figures c,d) , but there was little difference between the control and ibalt groups. we also found that the numbers of cd hi cd + t cells that made il- , il- , or il- were increased in control and ibalt mice relative to those in naïve mice ( figure e ), but there was little difference between the control and ibalt groups. these data suggest that the presence of ibalt does not significantly alter the number of t cells capable of producing th cytokines following sensitization and challenge. th cytokines act on other cells in the lung, such as epithelial cells and macrophages, and promote their activation and differentiation, which changes gene expression. for example, genes like chitinase- -like- (chi l ) and mucin ac (muc ac) are expressed in in the lung following il- signaling ( ) . thus, we next examined the expression of mrnas encoding these genes in the lungs of ibalt and control mice after ova sensitization and challenge as in figure a . we found that mrnas encoding chi l and muc ac were increased in ova-exposed and challenged lungs relative to their expression in naïve lungs, but their expression in the ibalt group was significantly less than that in the control group ( figure f) . thus, the expression of genes that are responsive to th cytokines were reduced in the lungs of ibalt mice relative to those in control mice. these data are more consistent with the reduced histopathology that we see in the lungs of ibalt mice compared to those in the lungs of control mice, suggesting that the difference between the groups is not in th priming and expansion, but in the response to th cytokines. in the experiments above, we were unable to independently control the initial steps of t cell priming and expansion. to rigorously show that these steps were unaffected by the presence or absence of ibalt, we next generated ova-specific th effector t cells in vitro and showed that, upon restimulation, a high frequency of these cells made il- , whereas less than % made ifnγ and almost none of them made il- ( figure a ). we next adoptively transferred x of these th cells into -day-old control or ibalt mice and challenged them with ova on days , , , and ( figure b ). one day after the final challenge, we enumerated donor t cells in the lungs and found that a similar frequency ( figure c ) and number ( figure d ) of donor t cells had accumulated in the lungs of both groups. we also enumerated gc b cells in the lungs and found that a high frequency ( figure e ) and number ( figure f ) of gc b cells accumulated in the lungs of mice in the ibalt group, but not in the lungs of control or naïve mice. despite having similar numbers of donor th cells in the lungs of each group, the histology of the lungs was dramatically different, with the control group exhibiting extensive goblet cell hyperplasia and diffuse inflammatory infiltrate throughout the lungs, whereas the ibalt mice exhibited minimal goblet cell hyperplasia and dense lymphoid accumulations (ibalt) adjacent to, but not surrounding, the airways and in the perivascular space ( figure g) . we also quantified the amount of th cytokines in the bal fluid h after the last challenge and found that, although the amount of il- , il- , and il- was slightly less than in the bal fluid of ibalt mice than in control mice, the differences were not significant ( figure h ). together, these data demonstrate that, despite starting with the same number of th cells in the lung, the biological outcome of inflammation was dramatically different in control and ibalt mice. given that the biological outcome of disease was so different despite starting with similar numbers of fully differentiated th cells, we reasoned that ibalt must engage some sort of regulatory mechanism. one important cell type that has the ability to powerfully attenuate inflammatory responses is the treg population ( ) . tregs are immunosuppressive cd + t cells that have differentiated in a way that promotes the expression of the transcription factor, foxp ( ) . cd + foxp + tregs can reduce eosinophilia ( ) , impair humoral responses and secret an inhibitory cytokines, such as il- and tgfβ ( ) , which suppress the effector functions of activated t cells and reduce inflammation. thus, changes in treg number or activity can have dramatic consequences on immune responses regardless of the number of antigen-specific effector t cells. given the immunosuppressive capacity of tregs, we enumerated cd + foxp + t cells in the lungs of ibalt and control mice following ova sensitization and challenge. we found that although the frequency ( figure a ) and number (figure b ) of tregs increased in the lungs following ova sensitization and challenge, there was no difference in tregs between ibalt and control groups. thus, alterations in treg numbers do not explain why mice with ibalt exhibit attenuated allergic responses in their lungs. previous reports show that pulmonary administration of lps to the lung conditions epithelial cells in a way that promotes the over-expression of a ( ) , an ubiquitinmodifying enzyme that blunts nf-kb signaling thereby reducing expression of pro-th cytokines like il- ( ) . thus, we were concerned that the exposure of neonatal mice to lps may permanently suppress pulmonary inflammatory responses. to test this possibility, we treated neonatal mice with lps or pbs to trigger ibalt formation and, when the mice were -weeks old, transferred ova-specific th effector cells and challenged the recipients times with either ova or pbs ( figure c) . we then enzymatically digested the lung tissue, sorted epcam + cd − sca − cd − bronchial epithelial cells and performed qpcr for tnfaip , the gene encoding the a enzyme. we found that tnfaip expression was similar in all groups of bronchial epithelial cells (figure d) , regardless of whether they came from mice that received lps as neonates or whether they came from mice that were challenged with ova as adults. we also performed qpcr to quantify the expression of the inflammatory cytokines, il- and tslp. we found that although il- expression was strongly increased following ova challenge, it was not altered by previous exposure to lps (figure e ). we also found that tslp expression was not changed by either ova challenge or previous exposure to lps ( figure e) . thus, the pulmonary exposure of neonatal mice to lps did not permanently alter the ability of bronchial epithelial cells to express inflammatory cytokines. despite the overall reduction in th -driven pathology in the mice with ibalt, the number of th effector cells seemed to be similar at the endpoint of the experiment. however, we didn't know whether they accumulated in the lung at the same rate. to determine whether the kinetics of t cell recruitment was the same in mice with and without ibalt, we generated th effectors from naïve otii cells in vitro and then adoptively transferred x th effectors h prior to a series of ova challenges ( figure a) . we then enumerated the transferred th cells and the recruited eosinophils in the lungs and bal h after each ova challenge. not surprisingly, the numbers of donor t cells and eosinophils increased over time following each challenge (figures b-e) . unexpectedly however, we observed significant reductions in the numbers of th effectors (figures b,d) and eosinophils (figures c,e) in both the lungs (figures b,c) and airways (figures d,e) of ibalt mice relative to controls after the first challenges. however, by the fourth challenge, there was no difference in the numbers of t cells. these data suggest that the presence of ibalt slows the accumulation of th effectors as well as eosinophils following pulmonary allergen exposure. these data may also explain why we saw little difference in t cell numbers at the endpoints of earlier experiments, even though we observed significant differences in lung pathology. the most unique attribute of ibalt is its dense accumulations of spatially ordered lymphocytes in a tissue (the lung) that normally has relatively few lymphocytes and rarely has them so densely organized. therefore, we next asked whether the specialized environment of ibalt alters the spatial organization of t cells in the lung following ag exposure. to test this possibility, we generated ova-specific th effector cells in vitro using otii cells. these cells were subsequently transferred into ibalt and control mice and the recipients were challenged with ova times before we examined the lungs by histology (figure a) . we performed immunofluorescence looking for the donor t cell marker (cd . ), b cells (b ), and fdcs (cd ). we found that the donor effector th cells preferentially accumulated in the ibalt areas of ibalt mice and were relatively dilute in the rest of the lung (figure b ). in contrast, the donor th effector cells were distributed more evenly in the control mice. these data were quantified in figure c . importantly, we found that the total number of donor th effector cells in the lungs of mice with and without ibalt were indistinguishable at this time ( figure d) . we also examined cd +foxp + tregs in tissue sections from mice with and without ibalt. we found that tregs were densely clustered in areas of ibalt, but were relatively dilute in the rest of the lung (figures e,f) , despite the similar numbers of tregs in the lungs of mice with and without ibalt (figures a,b) . together, these data suggest that the spatial distribution of effector th cells and tregs is affected by the presence of ibalt (they cluster together), which may explain how ibalt and control mice can have similar numbers of th cells in their lungs, but have so profoundly different outcomes in terms of eosinophil accumulation and histopathology. the above experiments used mice that had been administered lps as neonates in order to form ibalt. however, one concern with these experiments is that neonatal exposure to lps might have altered systemic immunity in some way that ameliorated subsequent inflammatory responses independently of ibalt formation. to rule out this possibility, we generated in vitrodifferentiated otii th cells and transferred x th effector cells into control mice, mice that received pulmonary lps as neonates (ibalt mice) and mice that received peritoneal lps as neonates (i.p. lps mice). the following days, we administered µg ova intranasally to all groups and measured germinal center b cells and eosinophils in the lungs h later ( figure a) . when we enumerated th effectors ( figure b ) and eosinophils ( figure c ) in the lung tissue, the numbers were reduced in the presence of ibalt compared to control mice, but in mice treated with lps intraperitoneally, the number of effector t cells and eosinophils were the same as in the control mice (figures b,c) . thus, the exposure of neonates to pulmonary lps dramatically decreased the inflammatory response in adults, whereas the exposure of neonates to peritoneal lps did not. we also observed that mice that receive pulmonary lps as neonates (ibalt mice) had a significantly higher frequency ( figure d ) and number (figure e ) of gc b cells in the lungs, whereas mice that received lps intraperitoneally as neonates (i.p. lps mice) did not accumulate gc b cells and were comparable to control mice (figures d,e) . these data indicate that systemic exposure to lps does not lead to ibalt formation (as monitored by the germinal center response in the lungs). these data are consistent with the conclusion that lps-mediated ibalt formation alleviates th -dependent inflammatory responses in the lung, independently of any systemic effect of lps on immunity. our data show that the presence of ibalt in the lungs prior to pulmonary sensitization and challenge with ova does not exacerbate th responses, but rather attenuates th -driven pulmonary pathology. in fact, mice with ibalt exhibit delayed th accumulation, reduced eosinophil recruitment, reduced goblet cell hyperplasia and reduced mucus production compared to their control counterparts. although the initial priming of th cells is not affected by the presence of ibalt, the accumulation of th cells in the lung is delayed following pulmonary challenge. more importantly, the spatial distribution of effector t cells is altered by the presence of ibalt, such that effector cd t cells as well as tregs become concentrated in ibalt areas and relatively diluted in the rest of the lung. these results suggest that ibalt functionally sequesters effector t cells, thereby limiting the exposure of the lung parenchyma to t cellproduced inflammatory cytokines, which attenuates pulmonary inflammation and prevents excessive pathology. ibalt is associated with a wide variety of inflammatory lung diseases, including copd ( ), hypersensitivity pneumonitis ( ) and rheumatoid lung disease ( ), all of which are the result of chronic exposure to antigens or inflammatory agents. although asthma is also a chronic lung disease, the mouse model of repeated sensitization and challenge with ova is more like an acute allergic response. however, our data demonstrate that the mere presence of ibalt does not necessarily lead to an increased inflammatory response or pulmonary pathology in the context of pulmonary allergen exposure as one might expect if ibalt was facilitating antigen presentation and t cell activation. therefore, despite participating in local immune responses, ibalt may be beneficial in the context of inflammatory diseases by sequestering activated lymphocytes. the idea of reducing inflammation by sequestering t cells in lymphoid tissue is consistent with the activities of the immunosuppressant drug, fty , which prevents t cells from exiting lymphoid organs-effectively sequestering them and preventing their recirculation through peripheral tissues ( , ) . in fact, treatment with fty reduces airway remodeling and pulmonary inflammation in a rat model of ova-induced asthma ( ) . interestingly, ibalt areas are expanded in the lungs of fty -treated animals, suggesting that the fty -mediated sequestration of lymphocytes can occur in either conventional lymphoid organs or in areas of ibalt in the lung ( ) . another physical change that occurs in the lung concomitantly with the development of ibalt is the formation of additional lymphatic vessels surrounding the ibalt follicles ( ) . live imaging of ibalt suggests that these lymphatic vessels may facilitate the collection of dcs from the airways ( ) . static imaging also shows that ibalt areas collect inhaled antigens and particulates ( ) , suggesting that ibalt may sequester antigen as well as t cells. in this context, it is interesting to note that mice that spontaneously develop ibalt in the context of rheumatoid lung disease are highly resistant to developing fibrosis following pulmonary challenge with bleomycin ( ) . if ibalt areas efficiently collect and sequester intratracheally administered bleomycin or remove it from the lung via lymphatics, then one would expect to observe reduced fibrosis. similarly, if ibalt areas collect and sequester pulmonary antigens like ova and remove them from the lung parenchyma, one would expect reduced pulmonary inflammation. thus, ibalt may protect the lung by collecting antigens and inflammatory substances as well as t cells. our data are also consistent with observations that ibalt attenuates inflammatory responses and improves clinical outcomes following infection with a variety of pulmonary pathogens. for example, the presence of ibalt promotes the survival of mice that are challenged with normally lethal doses of influenza virus, pneumovirus and sars corona virus ( ) and attenuates the pulmonary pathology associated with these infections. much of the clinical pathology, such as weight loss, associated with pulmonary viral infections is linked to the production of inflammatory cytokines by t cells. thus, if ibalt sequesters virus-specific effector t cells, it may limit the exposure of the rest of the lung to inflammatory cytokines and chemokines and reduce the recruitment of additional inflammatory cells-thereby attenuating pathology. however, ibalt-mediated resistance to pneumovirus is associated with impressive increases, rather than decreases, in the mrna expression of ifnγ, cxcl , and ccl in the lung ( ) . these responses were measured by pcr using mrna extracted from whole lungs, so it is difficult to know whether the increases were predominantly confined to ibalt areas or not. in addition, it is not clear whether the reported increase is due to an overall increased magnitude of the response or altered kinetics, such that more t cells are responding earlier. this last possibility seems plausible, given that accelerated antibody responses are also observed in mice with ibalt ( ) . interestingly, we also observe altered kinetics of effector t cell recruitment to the lungs following pulmonary challenge with allergen. however, we find that fewer effector th cells accumulate in the lungs at early times after allergen exposure. the differences between our results and those reported for pneumovirus may be due to the types of antigens (replicating virus vs. inert allergen) or to the types of t cells (virus-specific cd and th cells vs. allergen-specific th cells). in any case, the presence of ibalt clearly has an impact on the kinetics of pulmonary immune responses. t cells are often imprinted with particular effector functions that differ depending on the secondary lymphoid organ in which they were primed ( , ) . thus, one could envision that ibalt skews cd t cell differentiation away from a th pathway and thereby attenuates the symptoms of allergic disease. however, we found equivalent numbers and frequencies of th cells primed in mice with or without ibalt, suggesting that cd t cell differentiation is not deviated toward an alternative effector function. moreover, we observed reduced pathology in mice with ibalt following the adoptive transfer of in vitro differentiated th effector cells, demonstrating that even when t cell priming occurs in vitro, the functional outcome of the effector response is altered beneficially in mice with ibalt. interestingly, naïve t cells primed in aortaassociated lymphoid tissues in apoe −−/−− mice preferentially differentiate into tregs that suppress atherosclerosis ( ) . although the preferential formation of tregs in ibalt areas would help to explain the reduced inflammation and pulmonary pathology in mice with ibalt following ova sensitization and challenge, we did not observe changes in either the number and frequency of tregs in the lung, regardless of whether ibalt was present. nevertheless, we did find that tregs were preferentially concentrated in ibalt areas and co-localized with effector t cells, suggesting that tregs in ibalt may more effectively suppress the inflammatory functions of effector t cells in this location. thus, in the context of pulmonary sensitization with allergens such as ova, ibalt seems to alter the placement of t cells rather than changing their differentiation pathways. considering that many of the adoptively transferred th cells are being sequestered in the ibalt areas and seem to be located in the b cell follicle, it is possible that they are converting to il- -expressing tfh cells ( ) . this possibility would be consistent with the large gcs and high frequencies of gc b cells that we observe in the lungs following adoptive transfer of th cells and sensitization with antigen. in addition, ibalt-induced resistence to influenza and sars corona virus is associated with accelerated antibody responses ( ) , suggesting that local tfh cells are promoting b cell differentiation. although th -tfh cells in b cell follicles are potent producers of il- ( ), they are directing cytokine secretion toward b cells rather than pulmonary epithelial cells, which would likely lead to accelerated antibody secretion, but reduced pulmonary pathology. thus, ibalt may be sequestering t cells and directing cytokine secretion toward alternative cell types, both of which should attenuate pulmonary pathology. in summary, our data show that the presence of ibalt does not exacerbate pulmonary pathology following sensitization and challenge with an allegen, but rather attenuates th induced inflammatory responses. this same mechanism may also explain the ability of ibalt to ameliorate pulmonary pathology in the context of infection and may be a general mechanism by which ectopic follicles in a variety of tissues attempt to cope with chronic inflammatory responses. thus, we may want to develop treatments that promote, rather than prevent, ectopic follicle formation in the context of chronic inflammatory conditions. to induce ibalt, pups were intranasally administered µg lps from escherichia coli ( :b ; sigma) in µl pbs every other day starting on day after birth for a total of times. in control experiments, pups were intraperitoneally injected with µg lps in µl pbs every other day starting on day after birth for a total of times. control mice received µl pbs or nothing according to the same schedule. to induce allergic airway inflammation, adult mice were sensitized intranasally with µg ova (sigma) plus . µg lps in µl pbs and subsequently challenged intranasally with µg ova in µl pbs according to the schedule indicated in each experiment. in the dc labeling experiments, we intratracheally administered µg ova labeled with alexa flour r (invitrogen). cd + t cells were purified from the spleens of naive cd . + b congenics using ls columns and anti-cd macs beads (miltenyi biotec) according to the manufacturer's instructions. single t cell preparations were > % pure as determined by flow cytometry. th effectors were generated by activating naïve t cells for - h with plate-bound anti-cd ( µg/ml; - c ; bioxcell) and anti-cd ( . µg/ml; . ; ebioscience) in the presence of il− ( u/ml; dnax) and anti-ifnγ ( µg/ml; xmg . ; ebioscience) followed by feeding with il− ( u/ml; peprotech) for additional h. naïve and effector cd + (cd . + ) t cells ( x /mouse) were injected intravenously into no ibalt and ibalt recipients that were subsequently immunized with ova. lungs were cut into small fragments and digested with . mg/ml collagenase a (sigma) and µg/ml dnase i (sigma) in rpmi medium (gibco) at • c for min to obtain single-cell suspensions. digested tissues were mechanically disrupted by passage through a metal strainer. cell suspensions from lungs and mediastinal lns were centrifuged and resuspended in mm nh cl, mm khco , and . mm ethylenediaminetetraacetic acid (edta; lonza) for min to lyse red cells. cell suspensions were then filtered through a µm nylon cell strainer (bd biosciences), washed, and resuspended in phosphate-buffered saline (pbs) with % donor calf serum and µg/ml fc block ( . g ; trudeau institute) on ice for min before staining with fluorochromeconjugated antibodies against the following antigens: cd (rm - ), cd b (m / ), cd (pc ), cd r (b ; ra - b ), cd (fas; jo ), cd ( - ), siglec-f (e - ), and i-a b (af - . ; all from bd biosciences); cd c (n ), cd (im ), cd . (a ), cd ( e ), and foxp ; (fjk− s); all from ebioscience; cd ( d ); from biolegend; cd (nimr− ); from southernbiotech; and goat anti-rabbit-alexa flour r (invitrogen). for intracellular staining, single-cell suspensions from the lungs were stimulated on plates coated with anti-cd ( µg/ml; - c ; bioxcell) in the presence of brefeldin a ( . µg/ml; sigma) for h. the restimulated cells were surface stained, then fixed in % paraformaldehyde, made permeable with . % triton tm x− (sigma), and stained with anti-il− ( b ), anti-il− (trfk ; all from bd biosciences) or anti-il− ( a; ebioscience). cells were analyzed with a lsr ii (bd biosciences) or facscanto ii (bd biosciences) located at the university of rochester and university of alabama at birmingham flow cytometry core facility. to isolate lung epithelial cells, we perfused the lungs with pbs containing . mm edta (lonza), instilled with ml dispase ( µg/ml; corning), and incubated them in a shaker at • c for min. using forceps, lungs were torn into small pieces, and put back in the shaker at • c for an additional min in dispase ( µg/ml; corning) and dnasei ( µg/ml; worthington biochemical). digested tissues were red cell lysed, filtered through a µm nylon cell strainer (bd biosciences), resuspended in pbs with % donor calf serum, µg/ml fc block ( . g ; trudeau institute) and anti-cd macs beads (miltenyi biotec) on ice for min. cell suspensions were depleted of cd -expressing cells using ls columns (miltenyi biotec) according to the manufacturer's instructions. the flow through cells were stained with anti-cd ( ; invitrogen); anti-cd . ( ) and anti-cd (epcam; g . ; all from biolegend) and anti-sca- (ly a/e; d ; invitrogen). lung epithelial cells were sorted with facsaria ii (bd biosciences) located at university of alabama at birmingham flow cytometry core facility. lungs were instilled with ml hank's balanced salt solution (hbss; corning) containing . mm edta (lonza) and cell suspensions (≈ , cells in µl) were centrifuged at rpm for min in a shandon cytospin cytocentrifuge (cell preparation system). the cytospin pellets were air dried on glass slides, stained with shandon kwik-diff tm stain kit (thermo scientific) and were mounted with richard-allan scientific tm cytoseal tm (thermo scientific). total ige levels were determined by coating plates with µg/ml purified rat anti-mouse ige (r - ; bd biosciences). standard curve was prepared with purified mouse ige κ (c - ; bd biosciences) and bound abs in serum samples were detected with biotin rat anti-mouse ige (r - ; bd biosciences) and streptavidin-alkaline phosphatase (invitrogen). alkaline phosphate substrate (moss, inc.) was added, and color development was detected with spectramax r m (molecular devices) at nm. paraffin-embedded sections ( µm in thickness) were stained with hematoxylin and eosion (h&e) for standard histology and periodic acid-schiff (pas) for airway mucus production. frozen sections ( µm in thickness) were prepared from lungs perfused with a mixture of optimum cutting temperature (oct) compound (sakura finetek) in pbs at a : ratio. sections were fixed in cold acetone for min and blocked with fc block ( µg/ml) and % (vol/vol) normal donkey serum in pbs at • c for min. sections were then stained with the following primary abs in pbs at rt for min: goat anti-cd ε (m− ; santa cruz biotechnology), anti-cd c (hl ), and anti-cd r (b ; ra - b ; all from bd biosciences); anti-cd /cd ( e ; biolegend); and peanut agglutinin (pna; sigma). primary abs were detected with donkey antigoat-alexa flour r , rabbit fluorescein-alexa fluor r , streptavidin-alexa fluor r , and streptavidin-alex flour r (all from invitrogen) at rt for min. slides were mounted with slowfade r gold antifade mountant with ′ , diamidino− -phenylindole (dapi; invitrogen). images were collected with a zeiss axiocam digital camera (zeiss) or nikon andor clara camera (nikon). the images were obtained with a x objective for a final magnification of x . t cells in immunofluorescent images were quantified by counting cd . + cells in ibalt areas (defined manually using the outline tool) and by counting cd . + cells in non-ibalt areas (excluding large empty airways). the mean number of t cells per µm was determined using a mosaic of images obtained from control and ibalt-containing lungs that were sectioned at a similar depth and orientation. data were obtained from multiple sections from each mouse and mice per group. total rna was extracted from lungs with trizol according to the manufacturer's specifications (invitrogen) and was repurified with an rneasy mini kit (qiagen). ribonucleic acid (rna; . µg) was reverse-transcribed with superscript ii and random primers (invitrogen) and complementary deoxyribonucleic acid (cdna; ng) was amplified with following primers and probes: glyceraldehyde− -phosphate dehydrogenase (gapdh; trudeau institute), il , il , il , chi l , muc ac. tnfaip , and tslp (all from applied biosystems), with taqman r gene expression master mix (applied biosystems) and all reactions were run on a lightcycler realtime pcr system (roche). the relative level of messenger rna (mrna) expression for each gene was first normalized to the expression of gapdh and then compared to the average level of mrna expression in lungs from naïve b mice. data is expressed as logarithmic fold changes in mrna expression. lungs were instilled with ml hbss (corning) containing . mm edta (lonza) and sigmafast tm protease inhibitor cocktail tablets (sigma). total protein levels of cytokines il− , il− , and il− in lavage were quantified by mousespecific milliplex r multi-analyte kits (emd millipore) using a magpix r instrument platform and related xponent r software (luminex corporation). the readouts were analyzed with the standard version of the milliplex analyst software (emd millipore). the difference in mean values between two groups was analyzed with a two-tailed student's t-test. if three or more groups were compared, tukey's multiple comparison test was used (graphpad prism version . ). p-values of less than . were considered statistically significant. the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. the animal study was reviewed and approved by institutional animal care and use committee, university of alabama at birmingham. bronchus-associated lymphoid tissue bronchus-associated lymphoid tissue (balt) structure and function inducible bronchus-associated lymphoid tissue (ibalt) in patients with pulmonary complications of 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allergy through a induction in lung epithelial cells house dust mite allergen induces asthma via toll-like receptor triggering of airway structural cells fty : sphingosine -phosphate receptor- in the control of lymphocyte egress and endothelial barrier function fty blocks egress of t cells in part by abrogation of their adhesion on the lymph node sinus treatment with a sphingosine- -phosphate analog inhibits airway remodeling following repeated allergen exposure preferential lymphatic growth in bronchus-associated lymphoid tissue in sustained lung inflammation induced bronchus-associated lymphoid tissue serves as a general priming site for t cells and is maintained by dendritic cells inhalation of diesel exhaust for three months affects major cytokine expression and induces bronchus-associated lymphoid tissue formation in murine lungs autoreactive t and b cells induce the development of bronchus-associated lymphoid tissue in the lung a functionally specialized population of mucosal cd + dcs induces foxp + regulatory t cells via a tgf-beta and retinoic aciddependent mechanism selective imprinting of gut-homing t cells by peyer's patch dendritic cells artery tertiary lymphoid organs control aorta immunity and protect against atherosclerosis via vascular smooth muscle cell lymphotoxin beta receptors t follicular helper cells differentiate from th cells in response to helminth antigens il- -producing cd + t cells in reactive lymph nodes during helminth infection are t follicular helper cells the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © hwang, silva-sanchez, carragher, garcia-hernandez, rangel-moreno and randall. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - gz dotn authors: sallenave, jean-michel; guillot, loïc title: innate immune signaling and proteolytic pathways in the resolution or exacerbation of sars-cov- in covid- : key therapeutic targets? date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: gz dotn covid- is caused by the severe acute respiratory syndrome (sars) coronavirus (cov)- , an enveloped virus with a positive-polarity, single-stranded rna genome. the initial outbreak of the pandemic began in december , and it is affecting the human health of the global community. in common with previous pandemics (influenza h n and sars-cov) and the epidemics of middle east respiratory syndrome (mers)-cov, covs target bronchial and alveolar epithelial cells. virus protein ligands (e.g., haemagglutinin or trimeric spike glycoprotein for influenza and cov, respectively) interact with cellular receptors, such as (depending on the virus) either sialic acids, dipeptidyl peptidase (dpp ), or angiotensin-converting enzyme (ace ). host proteases, e.g., cathepsins, furin, or members of the type ii transmembrane serine proteases (ttsp) family, such as transmembrane protease serine (tmprss ), are involved in virus entry by proteolytically activating virus ligands. also involved are toll like receptor (tlr) family members, which upregulate anti-viral and pro-inflammatory mediators [interleukin (il)- and il- and type i and type iii interferons among others], through the activation of nuclear factor (nf)-kb. when these events (virus cellular entry and innate immune responses) are uncontrolled, a deleterious systemic response is sometimes encountered in infected patients, leading to the well-described “cytokine storm” and an ensuing multiple organ failure promoted by a downregulation of dendritic cell, macrophage, and t-cell function. we aim to describe how the lung and systemic host innate immune responses affect survival either positively, through downregulating initial viral load, or negatively, by triggering uncontrolled inflammation. an emphasis will be put on host cellular signaling pathways and proteases involved with a view on tackling these therapeutically. covid- is a respiratory disease whose aetiologic agent is a novel beta coronavirus (cov) called severe acute respiratory syndrome (sars)-cov- / -ncov. the initial outbreak of the pandemic began in december , and it is currently affecting the health and safety of the global community. indeed, on may , , . million worldwide cases were confirmed (probably a significant under-estimation given the number of untested asymptomatic subjects), with a death toll exceeding , . before the sars-cov- outbreak, two related highly pathogenic covs viruses, middle east respiratory syndrome (mers)-cov ( ) and sars-cov ( ) , provoked catastrophic epidemics and pandemics, respectively. unfortunately, no drugs nor vaccines have currently been approved to prevent or treat these viral episodes. the first anatomical/histological reports from the lungs of severely sars-cov- -affected patients experiencing acute respiratory disease syndrome (ards) revealed excessive inflammatory activation and destruction of the bronchial and alveolar epithelium, features already observed during the first sars pandemics in ( , ). indeed, in the latter pandemic, lung alveolar epithelial cells were identified as the most likely site of virus replication, and it was suggested that alveolar macrophages may be responsible for the dissemination of viruses within the lungs ( ). in accordance, initial histological analyses of lung biopsies from patients positive for sars-cov- have shown exfoliation of the bronchial epithelium, which may induce altered mucociliary clearance and affect host immune responses ( ). indeed, there is no doubt that the latter are involved in modulating disease onset and progression. for example, early studies report that, similarly with what was observed with sars-cov, lymphopenia [sometimes equivalent or more severe than that observed in human immunodeficiency virus (hiv) infection] is often observed in severely affected patients progressing to ards. despite, or maybe correlated with this, aberrant non-effective innate immune host responses seem associated with severe lung disease during sars ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the following sections will give an overview of the molecular and cellular mechanisms underpinning sars-cov virus infections and how lung and systemic host innate immune responses affect survival either positively, through downregulating the initial viral load, or negatively, by triggering uncontrolled inflammation. a particular emphasis will be put on the description of the host cellular signaling pathways and proteases involved with a view on tackling these therapeutically. covs are enveloped viruses with a positive-polarity, singlestranded rna genome encoding four structural proteins: the transmembrane trimeric spike glycoprotein (s, composed of two subunits s and s ), envelop (e), matrix (m), and nucleocapsid (n) ( ) . the entry of cov viruses into host epithelial cells is mediated by the interaction between the viral envelope s protein homotrimers and the cell surface receptors. following proteolytic cleavage of the cov s protein ("priming"), the s ecto-domain recognizes a membrane receptor [angiotensinconverting enzyme (ace ) for sars-cov and sars-cov- as well as dipeptidyl peptidase (dpp ) for mers-cov], whereas the s c-terminal domain is involved in cell fusion and viral entry ( ) ( ) ( ) . this mechanism of action is very similar to that used by influenza, except that the latter use sialic acids as the cognate receptor for its hemagglutinin (ha) ligand. importantly, many viruses (influenza, mers, cov, and paramyxoviruses such as hendra and nipah viruses) use similar host proteolytic enzymes for cleaving their ligands (ha and s), namely, mostly lysosomal (cathepsins b, l), furin, or trypsin-like proteases ( , ) . indeed, it is believed that it is the cellular source of these proteases that may determine the infectivity spectrum of these viruses, with the lung and the gastro-intestinal tract being high producers ( , ) . although a variety of these proteases have been studied and shown to be involved to varying degrees in virus activation, including neutrophil elastase ( ) , proteases of the type ii transmembrane serine proteases (ttsp) family [hat, transmembrane protease, serine (tmprss) , and tmprss ] have recently been demonstrated to be particularly important, albeit probably at different stages of the virus cell cycle ( , , , ) . in particular, recent research on sars-cov- has focused on tmprss and has shown it to be important (although mostly using cell lines infected with pseudotyped virus particles bearing sars-cov- s protein) for virus entry ( , ) . in that context, it has also been demonstrated that the serine protease inhibitor camostat (see also below section on therapeutic targets and conclusion) was protective ( , ) . in contrast, dpp which is necessary for the entry of mers-cov ( , ) is not involved in sars-cov- entry ( ) . unlike other sars-covs, the s protein of sars-cov- has a furin cleavage site at the boundary between the s and s subunits, which is processed during biogenesis and which may explain cov- high infectivity ( ) . although mechanistic studies are obviously still in their infancy, it is very likely that sars-cov and sars-cov- target mainly respiratory epithelial cells with similar mechanisms. indeed, as indicated above, initial work has shown that ace is the s receptor for both sars-cov ( ) and sars-cov- viruses ( , , ) , and structural studies using cryo-electron microscopy suggest a binding of two s protein trimer to an ace dimer ( , ) . whether this is strictly dependent on ace /protease expression is debatable since ace is present in other tissues in humans [such as the intestine, kidney, and testis ( ) ]. indeed, "seasonal" low pathogenic covs (e.g., cov- e, cov-oc ) infect mostly upper airways, whereas pathogenic covs (sars-cov/sars-cov- and mers) have a tropism for the distal lung and can cause severe pneumonia and ards ( ) , as currently demonstrated again in the present pandemic. indeed, potentially explaining this is the fact that seasonal coronaviruses do not use ace as a receptor. in vitro, primary nasal and tracheobronchial epithelial cells as well as the calu- bronchial cell line were shown to express ace (the latter not colocalizing with cilia), and their infection with sars-cov was shown to be highly cytotoxic ( , ) . in the distal lung, as hinted above, primary alveolar type ii epithelial (atii) cells are also permissive to sars-cov infection ( , ) . sars-cov- has also been shown to infect various respiratory epithelial cell lines including a (alveolar origin), beas -b (bronchial origin), calu- cells, as well as primary human bronchial epithelial cells ( ) . besides the lung, ace is also highly expressed in the intestine ( ) , and gastrointestinal symptoms have been recorded with covid- ( ) . it was shown that sars-cov is able to infect enterocytes as well as intestinal organoids and induces a viral response characterized by the expression of mediators related to type i and iii ifn ( ) . even if sars-cov is thought to originate from bats, the intermediate host between bats and humans is still unknown. sars-cov was previously shown to infect various wild and domestic animals, including cats, ferrets and pigs ( ) ( ) ( ) . similarly, recent work reveals that domestic animals, including ferrets and cats, are permissive to sars-cov- infection. in contrast, the virus replicates poorly in pigs, ducks, chickens, and dogs ( ) . given the described importance of host proteases in mediating infectivity of a number of viruses, it is no surprise that, upon virus infection, murine knock-out (ko) for some of these molecules has shown some protection. for example, tmprss -ko mice were protected from pulmonary disease and death following h n and h n influenza infection, but not from that of the influenza h n subtype, demonstrating some specificity and showing also that other ttsp proteases [such as desc (tmprss e) and mspl (tmprss )] or other factors may be important ( ) ( ) ( ) ( ) . similarly, tmprss ko mice showed reduced body weight and viral loads compared to wt mice in animals infected with sars-cov ( ) . also, it was demonstrated that over-expression of the human dpp in mice promoted mers-cov infection, causing lethal disease ( ) , and that tmprss was instrumental in that context ( ) . the control of viral infection requires an optimal and innate coordinated host antiviral immunity. this response is activated by various sensors, including pattern recognition receptors (prr), which recognize pathogen-associated molecular patterns (pamps). although for many viruses, viral rna is a pamp classically detected by different sensors, including toll-like receptors (tlr) (which senses double stranded (ds)rna), tlr and tlr [which sense single stranded (ss)rna], rig-i (which senses short dsrna and ssrna specific motifs), and mda- (which senses long dsrna) ( ), the sensors potentially recognizing sars-cov genomic material are still elusive. in addition, although, as mentioned above, distal peripheral lung alveolar epithelial cells seem to harbor sars-cov infection in vivo, and although respiratory epithelial cells are known to express tlr , tlr , and tlr ( , ) and initiate innate immunity in the lung ( ), the study of these cells in anti-cov responses has been hampered by their general poor permissibility to the virus in vitro (except for intestinal caco- and hek kidney epithelial cells) ( ) . in that respect, although the specific prr involved was not identified, the m protein of sars-cov was indeed shown to induce interferon (ifn)-β in a tlrrelated-traf -independent mechanism in hek cells ( ) . regarding the lung, the differentiated calu- cell line [when cultured at the air-liquid interface (ali)] is the model of choice: in that set-up, sars-cov infection triggered an inflammatory response characterized by increased production of interleukin (il)- , il- , gamma interferon (ifn-γ), inducible protein (ip- ), and activation of the transcription factor nf-κb ( ) . however, the kinetics of this response was extremely slow, and importantly, type i ifn, an important mediator of anti-viral responses, was undetected. also, another study involving a cells demonstrated that the trimeric spike s glyprotein and virus-like particles were able to modestly upregulate ccl , an important monocytic chemokine ( ) . in addition to lung epithelial cells cultured at ali, precisioncut lung slices could also be an interesting tool to study sars-cov -cells interactions ( ) , as demonstrated in influenza infections with human ( ) or animal-derived material ( ). as mentioned above, ttsps can activate virus-ligands (ha and s protein), but they are also able to modulate cell signaling pathways. for example, recombinant hat is able to activate mucin gene expression in nci-h lung epithelial cells ( ) . relatedly, we have shown both in vitro in epithelial cells and in a murine model that influenza h n is able to upregulate mucin expression and that this is dependent on human (or mouse) hat upregulation and tace activity ( ) . interestingly, haga et al. have shown that inhibiting tace prevents sars-cov cellular entry ( ) . strengthening the signaling potential of the receptors, iwata-yoshikawa et al. demonstrated in vivo that poly ic (tlr ligand) induces the expression of a variety of pro-inflammatory mediators (ccl , kc, and il- ) through the expression of tmprss ( ) . in addition, although unclear as whether it is beneficial or detrimental to the host cell, sars-cov have been shown to activate host stress response, apoptosis, and autophagy ( ) . these are also various pathways that may also need to be evaluated therapeutically in the context of the current pandemic. relatedly, we have shown that chloroquine, which also inhibits the autophagic cellular flux by decreasing autophagosomelysosome fusion, can inhibit influenza-mediated ccl production ( ) . importantly, after having established a foothold in the epithelial compartment, sars-cov can disrupt the epithelial polarity, thereby getting access to the parenchyma tissue: for example, it has been shown that the virus membrane protein e binds to pals (protein associated with lin seven ), a junction protein involved in epithelial polarity, and modifies its cellular distribution at the surface of hek- cells ( ) . myeloid cells, e.g., alveolar and interstitial macrophages or dendritic cells (dcs), elicit different immune responses toward influenza viruses, according to their subtypes ( ) . it is thus predictable that specificities may also exist with respect to sars-cov- infections. indeed, although studies are scant, these cells have generally been shown to be poorly permissive to sars-cov replication ( , , ) . however, a few studies have shown that myeloid cells can respond to sars-cov infection. indeed, dosch et al. showed that the s protein could, through tlr , trigger nf-κb activation and inflammatory responses in peripheral blood mononuclear cells (pbmc) ( ) . also, in common with epithelial cells, it was shown that pbmcs and dcs infected with sars-cov produced cytokines and chemokines such as and c-c motif chemokine ligand (ccl)- and/or c-x-c motif chemokine ligand (cxcl)- /rantes/tumor necrosis factor (tnf)/il- /il- , but, importantly, not ifn-β ( , ) . by contrast, a study performed mostly on thp- macrophages suggest that mers s protein suppresses macrophages pro-inflammatory responses through dpp -induction of irak-m and pparγ ( ) . furthermore, in an interesting " -way" system involving differentiated sars-permissive lung calu- cells and monocytederived macs and dcs, it was shown that mediators produced by calu- cells activate cytokine production by macrophages (il- β, g-csf, mip- , and tnf-α) and dcs (il- p , mip- , ifn-γ, il- , il- , and mcp- ) but that some of these calu- derived mediators (in particular il- and il- ) compromised the ability of dcs and macs to activate naïve t cells and phagocytosis ( , ). this echoes data obtained from patients suggesting that sars may in fact be partly caused by a "paralysis" of the adaptive immune system, characterized by a diminished number of immune cell types including t lymphocytes, dcs and macs ( ). demonstrating that sars-cov can induce tlr-dependent host responses in vivo, tlr , tlr , and tram ko mice were shown to be more susceptible to mouse-adapted sars-cov, albeit without exhibiting extra mortality ( ) . in comparison, mice deficient for the signaling molecule trif were highly susceptible to cov infections, exhibited diminished lung function, aberrant inflammatory responses, and importantly, higher mortality ( ) . in addition, a mouse genetic study revealed that the tlr adaptor protein ticam was a susceptibility gene to sars-cov ( ); mice ko for ticam ( ), but also myd ( ), another tlr adaptor protein, were highly susceptible to a mouse-adapted sars-cov lung infection. since polymorphisms of tlrs and myd have been associated in humans with heightened sensitivity to a variety of pathogens ( ) , these studies, in addition to demonstrating the role of tlr pathways in the sars-cov infection, suggested a human genetic predisposition to sars-cov, and this could explain the variability of severity in patients with covid- disease. forthcoming human genetic studies from international collaborative efforts (https://www. covid hg.org) could reveal genetic variants associated with sars-cov susceptibility, as in the gene encoding ace as recently suggested ( ) . indeed, ace genetic variants may be associated with a modulated ace protein expression, the sars-cov- receptor, which may explain in part patients' susceptibility to infection. genes associated with tlr pathways also represent good candidates, as demonstrated in other respiratory viral infection (e.g., influenza) where tlr variants ( ) were shown to modulate its virulence. maladaptive activation of innate immune responses (see figure b) as already mentioned above, aberrant maladaptive innate immune host responses, including "cytokine storm" events, have been associated with severe lung disease and the development of ards during sars and the covid- current episode. mechanistically, these events usually occur at a late stage of the disease, and several mechanisms have been proposed. in particular, a murine study has shown that a prolonged (albeit delayed, as demonstrated also in vitro, see above) type i ifn signaling was instrumental in triggering over-exuberant innate inflammatory monocytes-macrophages immune responses and an impaired virus-specific t-cell response ( ) . in complement to the mechanism proposed above, increased lung inflammatory protease (neutrophil elastase and metalloprotease) activity has been demonstrated in ards ( , ) , with a concomitant imbalance between protease and protease inhibitors activity ( ) . in addition, although not yet measured, to our knowledge, in sars murine models, we and others have shown increased protease-mediated lung damage in mice infected with influenza ( - ). additionally, in a mers-cov murine model, it was shown that excessive complement activation was partly responsible for exacerbated lung inflammation ( ) . lastly, "cytokines storm" may also results from socs (suppressors of cytokine signaling) inhibition ( ) . indeed, upon influenza infection, socs and socs were shown to reduce type i ifn antiviral responses in human bronchial epithelial cells ( ) . also, socs -deficient mice exhibited heightened sensitivity to influenza infection ( ) . studies about socs involvement during coronavirus infections are currently lacking and should therefore bring new interesting information. on may , , using the term "covid, " an unbiased search of already registered trials on https://clinicaltrials.gov/ retrieved , hits, and, when refined with "double blind/placebo, " hits were found. although the number of trials that are ongoing or "under recruitment" is expectedly very high, the range of molecules tested is relatively narrow and aimed at targeting mainly antivirals. these include remdesivir ( hits), lopinavir/ritonavir (also used in aids), as well as interferons ( hits) . also falling in that category are trials testing molecules aiming to block viral entry at the cellular surface by targeting ace-inhibitors ( hits) or the membrane proteases of the ttsp family (see above) using camostat mesilate ( hits). repurposing of non-antiviral drugs may offer new promising options, such as with ivermectin-an fda-approved anti-parasitic drug widely available and recently shown to inhibit sars-cov- in vitro ( ) . because the virus load is not necessarily correlated with symptoms deterioration in sars (the latter being often caused by worsening of inflammation at day - post onset of clinical signs), it follows that anti-inflammatory drugs could/should be prescribed during that stage of the disease ( ) . in that context, "classical" anti-inflammatory drugs are indeed currently being tested against covid- [e.g., methylprednisolone, budesonide, hydrocortisone, azithromycin, and non-steroïdal anti-inflammatory drugs (nsaids)]. in addition, more specific agents are also being investigated, targeting either il-β (anakinra, hits), il- signaling (siltuximab/ hits, tocilizumab/ hits, sarilumab/ hits), or cd (cd fc) with the main objective to modulate the "cytokine storm." however, chloroquine/hydroxychloroquine has, so far, undoubtedly taken the lion's share ( hits), and it has attracted a lot of media attention. in that respect, the results from an initial pan-european endeavor ("discovery"), now conducted largely in france because of enrollment difficulties, are eagerly awaited. this drug has a "mixed" mode of action. indeed, it acts as an anti-viral (presumably through inhibition of lysosomal enzymes requiring an acidic ph and of activation of endolysosomes, see above section "mechanisms of entry") and as an anti-inflammatory molecule, and it has notably been used in inflammatory rheumatic diseases ( ) . despite a relative safe profile, having been administered to millions of people over the years, worries have nevertheless arisen about cardiac issues in many individuals with severe covid- , and this will have to be properly assessed ( ) . regardless, the ultimate prize in the fight against covid- (or further sars-cov infections) undoubtedly lies with the future generation of effective vaccines and the development of neutralizing antibodies ( , ) . unfortunately, coronavirus vaccines in general have attracted less attention compared to the effort dedicated to vaccines against other potential pandemic viruses such as influenza. for example, from onwards, few sars-cov vaccines reached phase clinical trials for lack of interest from the pharmaceutical industry when it became evident that the virus was not making a "comeback" after its initial appearance. however, although probably too late for affecting the current "first wave" of sars-cov- pandemic, many pharmaceutical companies and research laboratories are now working on a plethora of vaccine formulations [for a review, see ( ) and https://clinicaltrials.gov, the latter reporting so far clinical trials on vaccines]. indeed, in pre-clinical studies, the determination of cryo-em structures of the sars-cov- s ectodomain trimer is providing a blueprint for the design of vaccines and inhibitors of viral entry ( ) . in this context, promising results show that murine polyclonal antibodies against s protein of sars-cov are able to elicit polyclonal antibody responses, preventing sars-cov- entry into cells, and thus indicating that cross-neutralizing antibodies targeting conserved s epitopes can be elicited upon vaccination ( ) . in addition to testing the best sars-cov- specific epitopes from the most suitable proteins (s, n, etc.) and way of administration (best vectors, etc.), it is important to select the best animal models. although convincing murine studies are still pending, as indicated above in the section "mechanisms of entry. . . ", studies in other animals investigated the virus susceptibility of chickens, ducks, dogs, pigs, cats, and ferrets, with the latter two being the most permissive ( ) . further up in the phylogenetic scale, a recent study reported that an inactivated vaccine candidate for sars-cov- was protective in macaques ( ) . finally, large epidemiological studies have demonstrated that bacille calmette-guerin (bcg) can heterologously protect against virus infections [e.g., yellow fever virus ( ) , probably by tapping on trained immunity mechanisms ( , ) ]. using such adjuvant-mediated strategies against sars-cov viruses may therefore be an exciting avenue worthwhile pursuing ( ) ( ) ( ) . all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. lg and j-ms received grants from the faculté de médecine sorbonne université (aap covid and université de paris (fonds d'urgence sars-cov- et covid- ) , respectively. this manuscript has 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inactivated vaccine candidate for sars-cov- bcg vaccination protects against experimental viral infection in humans through the induction of cytokines associated with trained immunity trained immunity and local innate immune memory in the lung induction of autonomous memory alveolar macrophages requires t cell help and is critical to trained immunity effects of toll-like receptor stimulation on eosinophilic infiltration in lungs of balb/c mice immunized with uv-inactivated severe acute respiratory syndrome-related coronavirus vaccine could bcg vaccination induce protective trained immunity for sars-cov- ? front immunol innate immune memory of tissue-resident macrophages and trained innate immunity: re-vamping vaccine concept and strategies host signaling and proteolytic pathways in the resolution or the exacerbation of coronavirus (cov- ) infection in covid- disease: what therapeutic targets? the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © sallenave and guillot. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -p jgyusp authors: schöler, lara; le-trilling, vu thuy khanh; eilbrecht, mareike; mennerich, denise; anastasiou, olympia e.; krawczyk, adalbert; herrmann, anke; dittmer, ulf; trilling, mirko title: a novel in-cell elisa assay allows rapid and automated quantification of sars-cov- to analyze neutralizing antibodies and antiviral compounds date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: p jgyusp the coronavirus disease (covid- ) caused by the severe acute respiratory syndrome coronavirus (sars-cov- ) is currently the most pressing medical and socioeconomic challenge. constituting important correlates of protection, the determination of virus-neutralizing antibodies (nabs) is indispensable for convalescent plasma selection, vaccine candidate evaluation, and immunity certificates. in contrast to standard serological elisas, plaque reduction neutralization tests (prnts) are laborious, time-consuming, expensive, and restricted to specialized laboratories. to replace microscopic counting-based sars-cov- prnts by a novel assay exempt from genetically modified viruses, which are inapplicable in most diagnostics departments, we established a simple, rapid, and automated sars-cov- neutralization assay employing an in-cell elisa (icelisa) approach. after optimization of various parameters such as virus-specific antibodies, cell lines, virus doses, and duration of infection, sars-cov- -infected cells became amenable as direct antigen source for quantitative icelisa. antiviral agents such as human sera containing nabs or antiviral interferons dose dependently reduced the sars-cov- -specific signal. applying increased infectious doses, the icelisa-based neutralization test (icnt) was superior to prnt in discriminating convalescent sera with high from those with intermediate neutralizing capacities. in addition, the icnt was found to be specific, discriminating between sars-cov- -specific nabs and those raised against other coronaviruses. altogether, the sars-cov- icelisa test allows rapid (< h in total, read-out in seconds) and automated quantification of virus infection in cell culture to evaluate the efficacy of nabs and antiviral drugs using reagents and equipment present in most routine diagnostics departments. by the time of writing, more than million people experienced a laboratory confirmed infection with the severe acute respiratory syndrome coronavirus (sars-cov)- and more than , people died while having coronavirus infectious disease . first surveillance studies and calculations of excess mortality rates indicate that the precise number of infections and the true number of fatalities exceed above-mentioned numbers by far. coronaviruses (covs) are positive strand rna viruses widespread among various vertebrate hosts including bats and rodents ( ) . together with four seasonal human covs (hcovs) and the two other emerging hcovs sars-cov- and mers-cov, sars-cov- is the seventh hcov causing widespread human diseases ( ) . in december , sars-cov- was first recognized in the hubei province in china ( ) from where it rapidly spread throughout the world. in addition to its genetic similarity, sars-cov- not only shares some clinical characteristics with sars-cov- ( ) but also exhibits some highly relevant particularities such as an increased spreading efficacy and the length of the course of disease ( ) . on january , the who declared the sars-cov- outbreak a public health emergency of international concern. on march , , who started to denote the outbreak a global pandemic. the center of the pandemic shifts between regions, posing danger of repetitive local and temporal reintroduction circles. thus, even countries that managed the first wave must prepare in terms of diagnostics capacities for potential future re-emergences. most sars-cov- infections lead to mild or moderate illnesses. however, a considerable fraction of cases proceeds to severe pneumonia or life-threatening acute respiratory distress syndrome. elderly individuals and people with pre-existing comorbidities such as impaired immunity, chronic respiratory diseases, cardiovascular diseases, and cancer are more prone to suffer from severe covid- . the case fatality rate (cfr) is difficult to calculate in the midst of the pandemic. depending on the age, cfr estimates of up to . % for individuals older than years and . % (range . - . ) for the general population have been reported ( ) . given the extent, pace, and severity of the covid- pandemic, diagnostics departments even in countries with highly developed medical systems struggle to provide sufficient and timely test capacities. since nucleic acid-based pathogen detection has a very short window of opportunity, assays detecting long-lasting immune responses such as antibodies are required to monitor virus spread and to estimate potential herd immunity. with some delay, most infected individuals raise a detectable humoral immune response including specific immunoglobulins (ig) m, iga, and igg ( ) ( ) ( ) . neutralizing antibodies (nabs) bind and abrogate the function of viral proteins such as the sars-cov- spike (s) protein that are essential for virus entry into host cells, e.g., through recognition of the cognate receptor ace ( ) . accordingly, monoclonal nabs exhibit strong therapeutic and prophylactic efficacies in sars-cov- -infected human ace -transgenic mice ( ) . a recent vaccination study conducted in non-human primates identified nabs as correlate of protection ( ) , indicating that a human sars-cov- vaccine should also be capable to elicit potent nab responses. accordingly, first human vaccine studies focussed on the question if nabs were induced ( ) . additionally, monoclonal nabs and nab-containing hyper-immunoglobulin preparations may be applicable as treatment against covid- ( ) . nabs are also the backbone of convalescent plasma (cp) therapies ( ) ( ) ( ) that are one of seven clinical recommendations of the idsa ( ) . based on the havoc covid- causes to the global economy, immunity passports and vaccination certificates, documenting protection through nabs, have been discussed [e.g., ( ) ]. taken together, sars-cov- -specific nabs and their quantification appear to be of central importance for the medical and socio-economic management of the pandemic. different types of neutralization tests (nt) have been developed for sars-cov- . however, to our knowledge, these assays rely on laborious microscopic counting of virus plaques or antibody-stained foci by trained personnel ( ) ( ) ( ) or on genetically modified viruses such as transgenic sars-cov- mutants ( ) or pseudo-typed viruses [e.g., vesicular stomatitis virus (vsv) or human immunodeficiency virus (hiv)] expressing the s protein of sars-cov- ( , ) . genetically modified viruses are generally prohibited in routine diagnostics laboratories and usually not applicable in less developed regions. due to the central importance of nabs and the limitations of the currently available methodology, we established a cheap, simple, fast, reliable, and automatable in-cell elisa (icelisa)-based icnt applicable in routine diagnostics departments with access to bsl laboratories. caco- (atcc htb- ) and vero e (atcc crl- ) were cultivated in roswell park memorial institute [rpmi- (gibco - )] and high glucose dulbecco`s minimal essential medium [dmem (gibco - )], respectively, supplemented with % (v/v) fcs, penicillin, and streptomycin at °c in an atmosphere of % co . sars-cov- was isolated from a patient sample using vero e and confirmed by sars-cov- diagnostic qrt-pcr. viral titers were determined by tcid titration. human ifna and ifnb were purchased from pbl assay science (# ) and peprotech (# - bc), respectively. the collection of serum samples and the virus isolation have been approved by the ethics committee of the medical faculty of the university of duisburg-essen ( - -bo, - -bo, and - -bo). anti-sars-cov- igg antibodies were detected using the elisa detecting antibodies recognizing the sars-cov- spike protein (euroimmun medizinische labordiagnostika, germany) according to manufacturer's instructions. for the analysis of neutralizing antibodies, serum samples were inactivated at °c for min. a detailed icelisa/icnt protocol is provided in supplementary file . briefly, defined doses of sars-cov- were incubated with different serum dilutions for h at °c prior to vero e infection. at - h p. i., cells (~ × /well of a -well plate) were fixed with % (w/v) paraformaldehyde/pbs. cells were permeabilized with % (v/v) triton-x- /pbs and blocked with % (v/v) fcs/pbs. the primary antibody was added and incubated for h at room temperature or overnight at °c. peroxidase-labelled secondary antibody was incubated for - h. washing steps were performed with . % (v/v) tween- /pbs. tetramethylbenzidin (tmb) substrate was added to visualize the enzyme reaction. the reaction was stopped with . m hcl. subsequently, the absorbance was measured using a microplate multireader (mithras lb ; berthold technologies). the a-n mab (abin ), a-n mab (abin ), a-e ab (abin ), and pod-coupled secondary antibodies (dianova) were used. in the case that an absolute quantification is necessary, the relative quantification of the icelisa can be transformed into an absolute quantification in terms of the virus dose using virus calibration curves (like in figure ) or using commercially available recombinant n preparations. immunoblotting was performed as described previously ( ) using antibodies recognizing the sars-cov- n protein (abin ) or gapdh (sc- , santa cruz). lysates were inactivated by sequential heat incubation ( min at °c and min at °c) before they were discharged from the bsl laboratory. proteins were visualized using peroxidase-coupled secondary antibodies (dianova) and the ecl chemiluminescence system (cell signaling technology). we hypothesized that virus-encoded proteins expressed by infected cells should be amenable as source of viral antigens for the detection and quantification by elisa. we optimized the experimental conditions such as cell type, virus dose, infection period, cell fixation method, blocking reagent, amd type and concentrations of primary and secondary antibodies ( figure and data not shown). as described in the materials and methods section and provided as detailed laboratory protocol in the supplementary information (supplementary file ), we compared different commercially available antibodies for the icelisa-based quantification of sars-cov- proteins. based on existing data on virus entry ( ), we infected human caco- cells with graded virus doses (ranging from . to pfu/cell). at days post infection (d p.i.), we fixed and stained the cells with different antibodies either recognizing the e or the n protein of sars-cov- . in accordance with high expression level of the n protein ( , ) , certain n-specific mouse iggs exhibited a signalto-noise ratio favourable for icelisa ( figure a) . a comparison of vero e and human caco- cells revealed that the icelisa is applicable to different cells ( figure b) . since the icelisa signal directly correlated with viral replication and viral antigen expression, we tested its ability to determine antiviral effects. human caco- cells were treated with graded concentrations of human interferon (ifn) a or ifnb and infected h later with sars-cov- . at d p.i., viral antigen amounts were quantified by icelisa. in accordance with a recent clinical phase trial ( ), ifnb exhibited strong and dosedependent antiviral activity against sars-cov- in human cells ( figure c) , indicating that the icelisa is applicable for future experiments addressing the efficacy of potential antiviral drugs in human cells. despite different start mois, similar icelisa signals were observed at h p.i. in vero e ( figure b) , indicating multiple rounds of virus amplification and extraordinary fast replication kinetics of sars-cov- in vero e cells, consistent with previous studies ( ) . to test if shorter infection periods might result in virus dose-dependent signals, we analyzed infected vero e cells after , , and h. sars-cov- was readily detectable in vero e cells by icelisa already at h p.i. (figures d-f) . to evaluate if the icelisa faithfully reports on the amounts of sars-cov- -expressed antigens, we infected cells with graded infectious doses and performed immunoblots and icelisas in parallel. as expected, icelisa and immunoblot signals correlated very well ( figure a ). since we observed icelisa signals already at h p.i. (figures a, d) and the n protein is an abundant component of sars-cov- particles ( ) ( ) ( ) , the n protein derived from input viruses could have contributed to icelisa signals. to evaluate this, we infected cells in the presence and absence of the translation inhibitor cycloheximide ('chx'). consistent with the notion that the icelisa signal is dominated by de novo n protein expression, the signal was significantly diminished by chx ( figure b) . taken together, these data indicate that the icelisa allowed rapid identification and relative quantification of sars-cov- replication in vero e and human cells and its inhibition by antiviral compounds. since the icelisa allowed simple and automated quantification of sars-cov- -dependent antigen expression, we tested if an icelisa-based neutralization test (icnt) can be established. we infected vero e cells for , , and h with graded sars-cov- infection doses ( , , , or , pfu/well) in the absence or presence of two convalescent sera in three different concentrations ( / , / , and / dilution). using the high infectious dose, viral antigens became dose dependently detectable by icelisa as early as h p.i. ( figure a ). both infection. even the strong icelisa signal at h p.i. was dose dependently neutralized by both sera ( figure f ). please note that the neutralizing capacity of given sera dilutions was less pronounced at higher virus doses as compared to lower virus doses, as indicated by residual icelisa signals. taken together, the icelisa resulted in a time-and virus dose-dependent signal constituting a surrogate for sars-cov- infection and replication. the fact that the infection and the resulting icelisa signal were neutralized by nabs present in immune sera indicated that the fast and automated icelisa format is applicable for icnts. although most sars-cov- nts have not been formally validated and certified, classic plaque reduction neutralization tests (prnt) are currently considered to represent the gold standard for the detection of sars-cov- -specific nabs. various commercially available igm, iga, and igg elisas have been compared to prnts [e.g., ( ) ]. to validate the novel icnt, sera- positive for sars-cov- -specific igg as determined using the euroimmun elisa (elisa ratio: . - . ) and elisa-negative sera (elisa ratio < . )-were compared side-by-side by icnt and standard prnt using pfu/well ( figure a ). one set was processed by icnt, while for the other set virus foci were stained using an antibodybased aec staining method and manually counted by microscopy. standard sars-cov- nts base on microscopic counting of plaques or antibody-stained virus foci. to enable plaque/foci recognition and individual counting by trained personnel, a countable number of pfu must be applied in prnts. depending on the prnt protocol, ( ) to pfu ( ) are used to infect each well. based on previous experiences with virus neutralization experiments ( , ) , we suspected that lower infectious doses might be more susceptible to nabs than higher virus doses, e.g., through altered ratios of nabs and antigenic regions determining neutralization, such as the receptor binding domain (rbd) of the s protein of sars-cov- . accordingly, graded infectious doses ( , - , pfu per well) showed virus dose-dependent susceptibilities to the same serum sample (supplementary figure s ). before we assessed clinical specimens, we compared icnt signals upon a high moi infection with viral progeny as determined by parallel tcid titration. as expected, we observed that icnt signals and residual virus numbers correlated very well (supplementary figure s ) . figures a-c) , the neutralizing capacity of the same sera considerably differed at the more restrictive high virus dose ( figures d-f infections of cell cultures with well-defined virus preparations in the presence or absence of compounds that may impair the infection. thus, the nt specificity results from the choice of virus applied to the cells. however, antiviral compounds can either be specific for one particular virus species (e.g., most monoclonal neutralizing antibodies), broadly active against virus families (e.g., receptor blocking agents) or even bigger genera (e.g., hyperimmunoglobulin preparations). in addition to sars-cov- , other hcovs such as the alphacoronaviruses hcov- e and hcov-nl and the betacoronaviruses hcov-oc and hcov-hku circulate autochthonously in the human population, resulting in high sero-prevalence rates ( ) . to address if the experimental sars-cov- infection in the icnt might be diminished by cross-reactive antibodies, we tested sera of individuals who have had infections with the alphacoronaviruses hcov- e or hcov-nl or betacoronaviruses such as hcov-hku ( figure a ) or a combination of hcov- e and hcov-oc ( figure b) . in contrast to the positive control containing sars-cov- -specific nabs, all tested serum samples recognizing other coronaviruses did not neutralize sars-cov- . although the limited sample size prevents definitive conclusions regarding crossneutralizing capacities of antibodies raised by other hcovs, in conjunction with consistent publications ( , ) it indicates the specificity of the icnt assay. we established a novel icelisa-based test principle for detection and relative quantification of sars-cov- . given the excellent signal-to-noise ratio between infected and uninfected cells, the test was applicable to quantify the efficacy of antiviral compounds, here shown for ifnb, and sars-cov- -specific nabs present in immune sera. compared to icelisa and icnt, standard virus titrations and prnts are far more laborious, time consuming, and expensivenot to speak from subjective and expectation biases upon usage for research. the entire icnt can be processed in less than days including the infection period. given that the protocol includes an early fixation step using % (w/v) paraformaldehyde which inactivates sars-cov- , all subsequent steps can be processed outside the biosafety level laboratory. the actual data acquisition is conducted within seconds, using standard elisa plate readers present in most routine diagnostics departments. we believe that this is advantageous compared to other nt assays that rely on more sophisticated and more expensive devises, e.g., for imaging cytometry ( ) . the icelisa and icnt provide increased data quality and precision by generating continuous data sets. since the detection antibodies can be applied in icelisa and icnts in relatively high dilutions ( / , to / , and / , of primary and secondary antibody, respectively), the assay is relatively cheap (in our case, around . € per well for both antibodies and the tmb peroxidase elisa substrate). the specificity of the icelisa and icnt is provided by the defined sars-cov- added to the cell cultures on purpose. the primary and secondary detection antibodies just serve to visualize and quantify viral antigens. thus, sars-cov- -specific antibodies can be applied for icelisa detection notwithstanding potential cross-recognition of other covs such as hcov-hku or hcov-oc -simply because these viruses are not present in the culture. obviously, such antibodies recognizing conserved residues cannot be used for classic antigen-recognizing elisas due to their inability to discriminate coronaviruses. more virus (> pfu/cell) can be applied to icnts. such high pfu icnts scrutinize the virusneutralizing capacity of sera more strictly, enabling a higher resolution compared to prnt assays that all rely on low virus numbers. it is tempting to speculate that sera exhibiting superior neutralization in high pfu icnt might be more beneficial in cp therapies. nts based on pseudo-typed viruses using heterologous expression of the sars-cov- -encoded s by non-related viruses may have certain advantages. however, genetically modified viruses are inapplicable by law in various routine diagnostics departments and usually unavailable in less developed countries. recently, an elegant surrogate assay has been designed that determines if antibodies are capable to prevent the interaction of immobilized hace with a reporter enzyme-coupled receptor binding domain (rbd) of s ( ) . this assay showed a good correlation with conventional nts (r = . ; p value < . ) and nts based on pseudo-particles (r = . ). however, without the use of genuine infectious viruses, assays relying on pseudo-typed viruses do not fully interrogate the full spectrum of antiviral effects for example if other viral proteins influence the system, e.g., by complement activation ( ) . accordingly, the icnt showed a superior correlation compared to prnt (r = . ; p value . e- ). taken together, we propose the icelisa and icnt for the quantification of sars-cov- replication and its inhibition by nabs and antiviral compounds. by changing the detection antibody and, if necessary, the cells according to the viral infection system, the test principle is transferable to all other viruses. all datasets presented in this study are included in the article/ supplementary material. the studies involving human participants were reviewed and approved by ethics committee of the medical faculty of the university of duisburg-essen ( - -bo, - -bo, and - -bo). the patients/participants provided their written informed consent to participate in this study. ls, vtkl-t, me, and dm performed research. oa, ak, and ah provided essential reagents. ls, vtkl-t, and mt analyzed the data. ls, vtkl-t, ud, and mt interpreted the data. vtkl-t and mt supervised the project. ls, vtkl-t, and mt wrote the manuscript. all authors contributed to the article and approved the submitted version. the authors received support by the stiftung universitätsmedizin essen, kulturstiftung essen, the else-kröner promotionskolleg elan, and the deutsche forschungsgemeinschaft (dfg) through grants rtg / , tr / - , and tr / - . the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. vtkl-t and ls contributed equally to this work. we thank benjamin katschinski and kerstin wohlgemuth for excellent technical assistance. mt was supported by the stiftung universitätsmedizin essen, kulturstiftung essen, the else-kröner promotionskolleg elan, and the deutsche forschungsgemeinschaft (dfg) through grants rtg / , tr / - , and tr / - . this manuscript has been released as a pre-print at biorxiv, ( ) . the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . / full#supplementary-material supplementary figure | the infectious virus dose strongly influences the neutralization capacity of serum samples. graded amounts of sars-cov- were incubated with indicated dilutions of a serum sample (chosen to exhibit a low neutralizing capacity) for h before vero e cells were infected. neutralization was evaluated by icelisa. the results of the high moi icnt correlate with residual virus after neutralization. sars-cov- was incubated with indicated dilutions of serum samples for h before vero e cells were infected. (a) neutralizing capacity was evaluated by icelisa. 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cryoelectron tomography sars-cov- (covid- ) by the numbers highly individual patterns of virus-immune igg effector responses in humans exploitation of herpesviral transactivation allows quantitative reporter gene-based assessment of virus entry and neutralization a systematic review of antibody mediated immunity to coronaviruses: antibody kinetics, correlates of protection, and association of antibody responses with severity of disease no sars-cov- cross-neutralization by intravenous immunoglobulins produced from plasma collected before the pandemic lack of cross-neutralization by sars patient sera towards sars-cov- virus reduction neutralization test: a single-cell imaging high-throughput virus neutralization assay for dengue a sars-cov- surrogate virus neutralization test based on antibody-mediated blockage of ace -spike protein-protein interaction highly pathogenic coronavirus n protein aggravates lung injury by masp- -mediated complement over-activation. medrxiv ( ) a novel in-cell elisa assay allows rapid and automated quantification of sars-cov- to analyse neutralizing antibodies and antiviral compounds the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © schöler, le-trilling, eilbrecht, mennerich, anastasiou, krawczyk, herrmann, dittmer and trilling. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -izpt p authors: chernomordik, fernando; cercek, bojan; lio, wai man; mihailovic, peter m.; yano, juliana; herscovici, romana; zhao, xiaoning; zhou, jianchang; chyu, kuang-yuh; shah, prediman k.; dimayuga, paul c. title: the role of t cells reactive to the cathelicidin antimicrobial peptide ll- in acute coronary syndrome and plaque calcification date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: izpt p the human cationic anti-microbial peptide ll- is a t cell self-antigen in patients with psoriasis, who have increased risk of cardiovascular events. however, the role of ll- as a t cell self-antigen in the context of atherosclerosis remains unclear. the objective of this study was to test for the presence of t cells reactive to ll- in patients with acute coronary syndrome (acs). furthermore, the role of t cells reactive to ll- in atherosclerosis was assessed using apoe−/− mice immunized with the ll- mouse ortholog, mcramp. peripheral blood mononuclear cells (pbmcs) from patients with acs were stimulated with ll- . pbmcs from stable coronary artery disease (cad) patients or self-reported subjects served as controls. t cell memory responses were analyzed with flow cytometry. stimulation of pbmcs with ll- reduced cd + effector t cell responses in controls and patients with stable cad but not in acs and was associated with reduced programmed cell death protein (pdcd ) mrna expression. for the mouse studies, donor apoe−/− mice were immunized with mcramp or adjuvant as controls, then t cells were isolated and adoptively transferred into recipient apoe−/− mice fed a western diet. recipient mice were euthanized after weeks. whole aortas and hearts were collected for analysis of atherosclerotic plaques. spleens were collected for flow cytometric and mrna expression analysis. adoptive transfer experiments in apoe−/− mice showed a % reduction in aortic plaque area in mcramp t cell recipient mice (p < . ). fifty six percent of adjuvant t cell recipient mice showed calcification in atherosclerotic plaques, compared to none in the mcramp t cell recipient mice (fisher’s exact test p = . ). recipients of t cells from mice immunized with mcramp had increased il- and ifn-γ expression in cd + t cells compared to controls. in conclusion, the persistence of cd + effector t cell response in pbmcs from patients with acs stimulated with ll- suggests that ll- -reactive t cells may be involved in the acute event. furthermore, studies in apoe−/− mice suggest that t cells reactive to mcramp are functionally active in atherosclerosis and may be involved in modulating plaque calcification. adaptive immunity has a major role in atherosclerosis ( ), the underlying cause of coronary artery disease (cad), associated with a variety of antigens that have been described ( ) . however, other potential self-antigens remain unknown ( ) . it has been proposed that in the chronic inflammatory state present in atherosclerosis, tolerance to self-antigens could be broken through several potential mechanisms, implicating an autoimmune component in atherosclerosis ( ) . this is supported by the reported correlation between atherosclerosis and t effector memory cells ( ) . furthermore, t effector memory cell density is associated with atherosclerotic plaque stage in humans ( ) . rupture of the atherosclerotic plaque is the most common cause of acute coronary syndromes (acs). antimicrobial peptides are an important part of the innate immune system and they are active against pathogens directly through their antimicrobial properties, or through their immunomodulatory effects ( ) . the human antimicrobial peptide ll- , a cleavage product of the cathelicidin proprotein hcap- , is present in human atherosclerotic plaques ( ) , and is associated with platelet activation and induction of thrombosis ( ) , with higher serum levels in coronary circulation compared to systemic levels in patients with st elevation myocardial infarction (stemi) ( ) . moreover, ll- is present in neutrophil extracellular traps (nets), which have been implicated in atherogenesis ( ) . they are associated with self-immunity as self-dna/ll- complexes that aggravate atherogenesis ( ) . interestingly, net burden was associated with infarct size and negatively associated with st segment resolution in patients with stemi ( ) . furthermore, ll- induced differentiation of human mononuclear cells into bone-forming cells ( ) , suggesting a potential role in calcific mineralization. ll- is a t cell self-antigen in patients with psoriasis ( ) , who have increased risk of cardiovascular morbidity and mortality ( , ) . it is not known if there is a self-reactive t cell response to ll- as a self-antigen in the context of atherosclerosis and if so, whether it is beneficial or pathogenic. cramp is the mouse homolog of the human cathelicidin proprotein hcap- , which like cramp, is proteolytically cleaved to generate a cathelin-like domain and the cationic anti-microbial domain ll- , or mcramp in mice (figure ). similar to ll- in humans, the proprotein cramp has been linked to atherogenesis in apoe−/− mice as suggested by experiments showing that cramp−/− apoe−/− mice have less atherosclerosis compared to apoe−/− mice ( ) . our group has previously shown that immunization with a low dose of the murine proprotein cramp was associated with decreased atherosclerosis in apoe−/− mice ( ) . the objective of this study was to test the role of ll- as a potential t cell self-antigen in patients with acs. furthermore, the role of t cells reactive to ll- in atherosclerosis was assessed using apoe−/− mice immunized with the ll- mouse ortholog, mcramp. the protocols were approved by the cedars-sinai institutional review board (irb). peripheral blood mononuclear cells (pbmcs) were isolated from blood collected from patients with acs within h of admission to the cedars-sinai cardiac intensive care unit. patients were consented under the approved irb protocol pro . exclusions were inability to give informed consent, age less than years old, active cancer treated with chemotherapy or radiation, patients taking immune-suppressive drugs, and pregnant women. pbmcs from stable cad patients were isolated from blood collected on the day of coronary angiography, consented under the approved irb protocol pro with the same exclusions. consented data use was limited to age, sex, ldl levels, and use/non-use of cholesterol-lowering medication. pbmcs were isolated using ficoll density gradient centrifugation and cryo-preserved in commercially available cryogenic solution (immunospot) in liquid nitrogen. cryo-preserved pbmcs from self-reported controls (n = ) were purchased from a commercial source (immunospot). cryo-preserved pbmcs were thawed, rinsed in anti-aggregation solution (immunospot), and seeded in culture plates at a density of × cells per ml of rpmi medium supplemented with % heat-inactivated pooled human serum and × antibiotic/antimycotic. peptides (lifetein) used for stimulation corresponded to ll- and the truncated cathelin domain of hcap- [cat-hcap- (aa - )]. cells were stimulated with one of the following: µg/ml ll- or cat-hcap- peptide, . × t cell stimulation cocktail containing pma and ionomycin (thermo fisher). culture medium was added at / of the starting volume h later to replenish the nutrients in the medium. cells were harvested h after seeding, stained for viability (live/dead fixable aqua dead stain kit, thermo fisher), and subjected to cell surface staining for flow cytometry using the following antibodies: cd , cd , cd , cd ra, cd ro, cd l, and cd (ccr ). isotypes were used as staining control. cd + or cd + t effector cells were gated on cd ro+cd l(−)cd (−). t effector memory cells were cd ro+cd ra(−) cd l(−)cd (−), t effector memory ra+ cells were cd ro+cd ra(+)cd l(−)cd (−). results were tabulated as response index using the following calculation ( ) : the results are expressed as response index to account for inherent variations introduced by culturing cells in vitro over time, controlled for by assessing response relative to baseline cell phenotype (% no stimulation) and maximal stimulation (% cocktail stimulation) for each subject pbmc. each data point represents one subject. in samples with sufficient cell numbers, stimulated pbmcs cultured for h were collected for rna extraction using trizol (thermo fisher) and subjected to qrt-pcr with sybr green and a primer pair for human pdcd . cyclophilin a served as the reference gene. results were expressed as fold-change relative to non-treated cells of each sample using the ct method. conditioned medium was collected after h of ll- stimulation of pbmcs and ifn-γ was measured using a commercially available elisa kit (abcam) according to manufacturer's instructions. pbmcs from control samples were stimulated with ll- alone or in the presence of either anti-human hla-a, b, c (clone w ) or anti-human hla-dr (clone l ) monoclonal antibody (biolegend; µg/ml) for h and stained for flow cytometry as described above. the studies were approved by the cedars-sinai institutional animal care and use committee. male apoe−/−mice were purchased from jackson laboratory at weeks of age and housed in a specific pathogen-free facility, kept on a -h day/night cycle, and had unrestricted access to water and food. the mcramp peptide was purchased (anaspec) with > % purity according to the manufacturer. donor mice were subcutaneously immunized with either µg mcramp in adjuvant [adju-phos (brenntag, . µl of a % solution) and monophosphoryl lipid a (mpla-sm vaccigrade, invivogen, µg)] or adjuvant alone in a final volume of µl using pbs as vehicle. one group of mice was fed normal chow and immunized at , , and weeks of age. mice were then euthanized and splenocytes isolated to assess t cell response at weeks of age using flow cytometry. another group of mice was fed normal chow and immunized at , , and weeks of age. the mice were then euthanized as t cell donors at weeks of age. recipient mice were fed high fat diet consisting of % fat, . % cholesterol (td. , envigo) starting at weeks of age until euthanasia at weeks of age. after collection of donor mouse splenocytes, red blood cells were lysed using rbc lysis buffer (biolegend), washed with pbs, and cells from the same group were pooled. splenocytes were enriched for t cells using a commercially available mouse t cell magnetic isolation kit (thermofisher) according to the manufacturer's protocol. t cells were enriched to ∼ % assessed by flow cytometry. after counting the isolated t cells, a suspension of x t cells in pbs was injected by tail vein injection into week-old recipient mice fed with high fat diet for weeks. high fat diet feeding in recipient mice continued for an additional weeks and mice were euthanized at weeks of age. at weeks of age, recipient mice were euthanized and spleens, aortas and hearts were collected. serum was collected for cholesterol level measurement. spleens were collected for flow cytometric staining and analysis. a small portion of the spleen was used for rna extraction. the aortas were dissected free of connective tissue and fat, and stained with oil red o for en face lipid staining ( ) . aortic size and plaque content were quantified by a blinded observer using image analysis software (imagepro plus version . , media cybernetics inc., rockville, maryland). heart bases were imbedded in oct (optimum cutting temperature, tissue-tek) and frozen for cryo-sectioning. ten-micron cryosections of the aortic sinus were collected. lipid was stained using oil-red-o. staining for macrophage was performed using cd antibody. collagen area was assessed using masson trichrome stain. three slides of approximately . -millimeter intervals for each animal were used for each stain and averaged. image analysis was performed using imagepro. tissue calcification in aortic sinus plaques was confirmed by alizarin red-s stain. splenocytes were stained for viability and surface markers cd , cd , cd , cd , and cd l. intracellular staining for foxp was performed after surface marker staining followed by fixation and permeabilization (ebioscience). for intracellular cytokine staining, cells were resuspended in % heat inactivated fbs-rpmi medium containing × monensin (invitrogen) and cultured at • c and % co for h. after viability and surface marker staining, cell fixation and permeabilization was performed followed by intracellular staining for il- and ifn-γ for flow cytometry. for degranulation assay to measure cd + t cell cytolytic activity, splenocytes were incubated with . µg/ml fluorescent conjugated cd a in rpmi for h followed by the addition of monensin and incubation for another h. cells were collected and stained for surface markers for flow cytometry. splenic rna was isolated using trizol and subjected to qrt-pcr using primer pairs for mouse il- β, pdcd , ctla , wnt b, runx , rankl, and osteocalcin. gapdh was used as reference gene. data were analyzed using the ct method with one adjuvant sample as calibrator. results are expressed as fold change relative to adjuvant. mouse serum levels of soluble rankl and undercarboxylated osteocalcin, the active form of osteocalcin, were measured using commercially available elisa kits (thermofisher and mybiosource, respectively) according to manufacturers' instructions. statistical analysis was performed using r software version . (r core team, ) and graphpad prism version (graphpad software, la jolla, california). data are presented as mean ± standard deviation. for multiple group analysis, significance for normally distributed samples was tested using anova followed by holm-sidak's multiple comparisons test. significance for non-normally distributed samples was tested using kruskal-wallis test followed by dunn's multiple comparisons test. correlation was analyzed using the spearman test with a two-tailed p-value. for two-group analysis, following normality testing, differences between groups were performed using t-test or wilcoxon test, accordingly. a p-value < . was considered significant, but data trends were also noted. given the role of anti-microbial peptides as potential selfantigens in atherosclerosis, and the possible association with acute events, we tested if the cleaved fragment of hcap- , the cationic antimicrobial peptide ll- , would induce differential t cell immune responses in patients with acs. peripheral blood mononuclear cells (pbmcs) from self-reported healthy controls (controls), patients with stable cad (stable) or acs were stimulated with ll- for h. pbmcs stimulated with the cathelin domain of hcap- (cat-hcap- ) served as control. baseline characteristics of the subjects are detailed in table . stimulation with ll- resulted in reduced cd + effector t cell response, in both t effector memory (tem) and t effector memory ra+ (temra) cells in pbmcs from controls and patients with stable cad, while pbmcs from patients with acs were resistant to this reduction (supplementary figure and figures a-c) . cd + effector t cell responses were trending similar to cd + effector t cells (figures d-f) . there was no significant difference in cd + effector t cell response when cells were stimulated with cat-hcap- (figure ) . there was reduced expression of programmed cell death protein (pdcd ) mrna in pbmcs from patients with acs stimulated with ll- compared to pbmcs from controls ( figure a ), but no difference was noted between groups in pdcd mrna expression when cells were stimulated with cat-hcap- ( figure b ). the nature of the self-reactive responses to cat-hcap- and ll- were investigated further by testing the relationship between the t cell response to both antigens in each subject. there was significant correlation in cd + effector t cell response ( figure d ) to cat-hcap- and ll- , but not in cd + effector response ( figure c) . the results suggest that there is potentially shared antigenic reactivity to cat-hcap- and ll- in cd + t cell responses. ifn-γ measured in conditioned medium from pbmcs stimulated with ll- showed qualitative difference in acs compared to control and stable cad patients (supplementary figure ) . the results suggest that ll- reactive t cells may be involved in the acute event. additionally, effector t cell responses in control pbmcs stimulated with ll- was partially reversed by blocking with hla class-i antibody (supplementary figure ) suggesting hla class-i mediated response. the t cell response in acs patients was further subdivided into patients with their first acs event and those who had a recurrent event. the cd + t effector response to ll- was consistent between first and recurrent acs patients, compared to patients with stable cad (supplementary figures a-c) . on the other hand, cd + t effector response was significantly higher only in the recurrent acs patients but not in the first acs event compared to stable patients (supplementary figure d ). this suggests that cd + t cell response to ll- persists in immunologic memory and is a common response in both first event acs and recurrent acs. cd + t cell effector response on the other hand seems to have evolved and is more prominent in the recurrent acs. no significant differences were observed in cat-hcap- response between first acs event and recurrent acs patients in cd + t effector (supplementary figures a-c) and cd + t effector (supplementary figures d-f) responses. to investigate the potential role of t cells that are self-reactive to the cationic antimicrobial peptide in atherosclerosis, we used the apoe−/− mouse model of atherosclerosis and immunization with the mouse ortholog of ll- called mcramp. immunization provokes a self-reactive t cell response to mcramp in apoe−/− mice to investigate the potential role of t cells reactive to the mouse cationic antimicrobial peptide mcramp in the male apoe−/− mouse model of atherosclerosis, we first tested if t cells were reactive to mcramp. apoe−/− mice fed with normal chow were immunized with mcramp at , , and weeks of age and euthanized week later for assessment of t cell response. mice injected with adjuvant alone served as control. there was no difference in cd + effector memory t cells (supplementary figure and figure a ) but a significant increase in cd + central memory (cm) t cells (figure b ) in splenocytes from apoe−/− mice immunized with mcramp. additionally, there was decreased cd +foxp + cells (supplementary figure and figure c ) and increased cytolytic activity, assessed by cd +cd a+ t cells (supplementary figure and figure d ), in splenocytes of mice immunized with mcramp compared to adjuvant. no differences were observed in cd + memory t cell subsets or in cd +foxp + treg cells (figures e-g) . to assess whether mcramp-primed t cells are functionally involved in modifying atherosclerosis, adoptive transfer of donor t cells from apoe−/− mice immunized with mcramp or adjuvant alone was performed on apoe−/− recipient mice that had been fed high fat diet for weeks prior to transfer. the week feeding with high fat diet assured that the recipient mice were already primed for atherosclerosis. recipient mice were euthanized weeks after cell transfer. recipients of t cells from mcramp immunized mice had significantly reduced aortic plaque area compared to recipients of t cells from adjuvant mice ( % reduction, p < . , figures a,b) . there was no significant difference between groups in mean body weight (mcramp = ± gr; adjuvant = ± gr) or mean serum cholesterol (mcramp = ± mg/dl; adjuvant = ± mg/dl). thus, atherosclerosis progression was reduced in the mcramp t cell recipient mice without differences in weight or serum cholesterol compared to adjuvant t cell recipient mice. aortic sinus plaque size, lipid content (oil red o staining; figures a-c) , macrophage (cd staining; figures d,e) , and collagen area (masson's trichrome staining; figures f,g) were not different between the t cell recipient groups. there were also no differences in il- β (figure a) , pdcd (figure b ) or cytotoxic t-lymphocyte-associated protein (ctla , figure c ) splenic mrna expression between the t cell recipient groups. focal staining of hematoxylin was observed in several aortic sinus sections from recipient mice, suggesting calcification in the plaque. the presence of plaque calcification was assessed with the calcium-specific alizarin red s staining. calcification in atherosclerotic plaques occurred in % of adjuvant t cell recipient control mice, compared to none in the mcramp t cell recipient mice (figures a,b and table ; fisher's exact test p = . ). immune pathways associated with t cell mediated tissue calcification were then investigated further. recipients of t cells from mcramp immunized mice had increased il- and ifnγ expression in splenic cd + t cells (supplementary figure and figures c,d) , but not in cd + t cells (figures e,f) , compared to adjuvant t cell recipient control mice. there was increased expression of wnt b mrna in splenocytes of mcramp t cell recipients, when compared to adjuvant t cell recipient control mice ( figure a) . however, there was no difference in the expression of runx mrna ( figure b) . furthermore, splenocytes from mcramp t cell recipients had increased rankl and osteocalcin mrna expression when compared to adjuvant (figures c,d) . however, no difference between groups was noted in serum rankl and undercarboxylated osteocalcin concentration (figures e,f) , which is the known active state of osteocalcin. in this study, we showed that: (a) ll- stimulation of pbmcs from patients with acs induced the persistence of cd + tem cell response compared to patients with stable cad or selfreported controls; (b) immunization of apoe−/− mice with mcramp, the cationic fragment of cramp, increased cd cm t cell activation and cytolytic activity; and (c) adoptive transfer of t cells from mice immunized with mcramp was associated with smaller atherosclerotic aortic plaque area, and absence of aortic sinus plaque calcification. our findings suggest that ll- self-reactive t cells may be important in an acute coronary event in the context of atherosclerotic disease. ll- is a t cell self-antigen in patients with psoriasis ( ), who have increased risk of early cardiovascular disease ( , ) . it has been proposed that psoriasis and atherosclerosis, both chronic inflammatory conditions, share a common inflammatory pathogenic basis ( , ) that can potentially explain the increased risk of atherosclerosis and its complications associated with this condition. tem cells have rapid effector function upon reexposure to the antigen, but a limited proliferative potential ( ) . memory t cells correlate well with atherosclerosis in both mice and humans ( , ) , and with vulnerable and ruptured plaques in humans ( ) . ll- was reported to decrease t cell proliferation in resting human pbmcs with increased cell viability without changes in cd +foxp + t reg cell percentage suggesting that in the steady-state, ll- treatment results in a degree of immunemodulation apparently independent of increased t regs ( ) . in the same report, t cells increased proliferation when co-activated with phytohaemagglutinin without affecting cell viability and with increased t reg cells. increased t cell proliferation coupled with increased t reg cells in their report suggests that t regs may be compensating for the increased proliferation, or that t regs may not have a significant role in ll- reactive t cells. our findings extend their report by demonstrating that t cell memory response to ll- was reduced in the control subjects and stable cad but persisted in samples from acs patients. this is supported by the qualitative difference in the ifn-γ secretion among the groups. these differences were associated with reduced immune checkpoint pdcd mrna expression in pbmcs from acs patients. the reduction in t cell response to ll- in the control pbmc was blocked by hla class-i antibody suggesting that the intrinsic response is at least partially mhc-i dependent. however, our results cannot rule out the possibility that adjuvant effects attributed to ll- are also involved ( ) . the observed blocking of complementary cd + t cell memory response by anti-hla class-i antibody is consistent with the report by lande et al. ( ) suggesting complementarity of t cell subset responses to ll- . whether the complementary t cell response to ll- in the controls also extend to acs as was reported for some psoriasis patients ( ) remains to be determined. thus, combined with other reports, our results suggest that the intrinsic t cell response to ll- is down-modulation but in the presence of co-activating factors such as those in the acs patients the t cell memory response persists that may be due in part to reduced checkpoint pdcd expression. these findings have potentially important clinical implications with the reported cases of acs associated with immune checkpoint inhibitor treatment ( ) . the results in our report may be a response to the acute event but adaptive immune memory to neoantigens develop over a longer time frame than that in our study (within h of admission for the acute event). it is notable that cd + tem response to ll- is consistent in patients whether it was their first acs event or a recurrent event suggesting the continued presence of memory t cells. it cannot be determined at this time whether the persistent t cell response in acs is pathogenic or a compensatory response in the acute stage of the disease. on the other hand, the cd + t effector response was altered in patients that had a recurrent event suggesting that the t cell response in acs evolved as the recurrent patients remained at risk. these observations are consistent with the notion of underlying inflammation in patients who remain at risk for a recurrent event. our results further show a correlation between the cd + t cell response to ll- and cat-hcap- . this finding may be a manifestation of expanding antigenic determinants reported in antigen spreading, wherein the original reactive antigen determinant spreads to other regions of the same protein ( ) , in this case hcap- . although speculative, it is interesting that this was not observed in cd + t cell response. it remains to be determined what the nature of the involvement of the tem response to ll- is in the acute event. studies were performed in apoe−/− mice to investigate the role of t cells reactive to the cationic antimicrobial peptide in atherosclerosis. although the cathelin domain of the proprotein hcap- /cramp is reported to be active ( ) , functional activity is mostly attributed to the cationic antimicrobial peptide domain. our results show increased cd + cm t cells and cd + t cell cytotoxic activity in mice immunized with the self-peptide mcramp coupled with decreased cd +foxp + t cells. these extend our previous report where immunization with the proprotein cramp resulted in increased cd + t cells and cytolytic activity, and reduced atherosclerosis ( ) . cd + t regs have an important role in self-tolerance, and lower levels cd + t regs have been associated with increased immune activity ( ) ( ) ( ) . we confirmed that t cells reactive to mcramp are functionally involved in atherosclerosis by the adoptive transfer of enriched t cells from mcramp-primed apoe−/− mice into recipient apoe−/− mice. to the best of our knowledge, this is the first report of reduced atherosclerosis attributed to t cells primed with mcramp. however, no differences were found in aortic sinus plaques consistent with the report of site-specificity for atherosclerosis in murine models of atherosclerosis ( ) . some key signaling pathways involved in tissue calcification potentially regulated by t cells in the recipient mice were investigated. wnt b, a member of the wnt family signaling pathway, is secreted by t lymphocytes ( , ) and activates signal transduction cascades that regulate runx , a transcription factor needed for osteoblast differentiation ( ) . rankl activates osteoclasts and bone remodeling in adult mice ( ) . t cell expression of rankl ( ) is involved in the regulation of bone metabolism ( ) . osteocalcin is secreted by osteoblasts associated with bone formation. splenocytes expressing osteocalcin induce atherosclerosis and vascular calcification in apoe−/− mice ( ). although the dysregulation of calcification pathways is manifested in differential mrna expression of specific genes involved, the systemic levels detected in serum seemed to remain in equilibrium. this is further observed in the increase in both il- and ifn-γ expressing cd + t cells. the results suggest the involvement of factors that remain to be characterized. nevertheless, these observations suggest dysregulated tissue calcification pathways in atherosclerosis potentially mediated by memory t cells. interestingly, ll- is a t cell antigen in psoriatic disease ( ) and altered bone remodeling through osteoblastosteoclast uncoupling has been proposed as an explanation for the concomitant dysregulated processes of both pathological bone formation and resorption usually found in patients with psoriatic arthritis ( ) . in our study, plaque calcification was absent in mice that were recipient of t cells primed with mcramp suggesting a role in regulating the process. coronary artery calcification is a marker of atherosclerosis and its role in cad is nuanced ( ) . on one hand, the widely used coronary artery calcium score is a strong independent risk factor of major adverse cardiovascular and cerebrovascular events ( , ) . on the other hand, calcified atherosclerotic plaques may be more stable than non-calcified plaques ( ) . the use of statins has been associated with progression of plaque calcification, in spite of their protective role in atherosclerosis ( ) . the association found in our animal experiments between recipients of mcramp-primed t cells and the lack of plaque calcification is intriguing, but its significance remains to be determined. the role of t cells reactive to ll- in humans, and their potential influence in the pathways of atherosclerotic plaque calcification need to be investigated further. limitations of the study include the potential of ll- to be a "promiscuous" hla-binding peptide or to have intrinsic adjuvant properties ( ) which coupled with the reduced pdcd expression in acs may explain the observed persistence of cd + effector cells. although anti-hla class-i antibody blocked the response to ll- in controls, our study cannot completely exclude these possibilities. the mouse studies demonstrating the involvement of t cells reactive to mcramp as a self-antigen in atherosclerosis is consistent with the observed tem cell response to ll- in acs patients, supporting the presence of a self-reactive tem cell population involved in atherosclerosis. however, the persistence of tem response to ll- in patients who suffered an acute event is not in complete alignment with the results of adoptive transfer of mcramp-primed t cells in our mouse studies. one might speculate that the controlled nature of immune priming in the mice skewed the response to be protective against atherosclerosis, even as the physiologic role of reduced calcification of mouse plaques remains to be clarified. it is also possible that the persistence of ll- reactive tem response in acs patients is a compensatory response to the inflammatory milieu that subsequently proved inadequate. these remain speculative handicapped by several limitations, including the lack of a reliable mouse model of spontaneous coronary artery plaque rupture which is the major cause of acs in humans. the original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author. the studies involving human participants were reviewed and approved by cedars-sinai irb. the patients/participants provided their written informed consent to participate in this study. the animal study was reviewed and approved by cedars-sinai institutional animal care and use committee. fc, bc, wl, pm, and pd contributed to conception and design of the study. fc, wl, jy, pm, xz, jz, and pd contributed to the data acquisition and analysis. bc and rh contributed to patient recruitment. fc, bc, wl, k-yc, ps, and pd contributed to the interpretation of the data, drafting, and revising the manuscript. all authors contributed to the article and approved the submitted version. innate and adaptive immunity in the pathogenesis of atherosclerosis antigen-induced immunomodulation in the pathogenesis of atherosclerosis t cell subsets and functions in atherosclerosis autoimmunity in atherosclerosis: a protective response losing control? effector memory t cells are associated with atherosclerosis in humans and animal models a change in inflammatory footprint precedes plaque instability: a systematic evaluation of cellular aspects of the adaptive immune response in human atherosclerosis a comprehensive summary of ll- , the factotum human cathelicidin peptide involvement of the antimicrobial peptide ll- in human atherosclerosis the endogenous antimicrobial cathelicidin ll induces platelet activation and augments thrombus 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regulatory t lymphocytes and their specific intervention in graft-versus-host and infectious diseases, autoimmunity, and cancer regulatory t cell (treg) subsets return in patients with refractory lupus following stem cell transplantation, and tgf-beta-producing cd + treg cells are associated with immunological remission of lupus site specificity of atherosclerosis: siteselective responses to atherosclerotic modulators the mouse wnt- b gene isolated from helper t cells is widely expressed and a possible oncogene in br mouse mammary tumorigenesis treatment with intermittent pth increases wnt b production by t cells in osteoporotic patients targeted disruption of cbfa results in a complete lack of bone formation owing to maturational arrest of osteoblasts soluble rankl contributes to osteoclast formation in adult mice but not ovariectomy-induced bone loss trance is a novel ligand of the tumor necrosis factor receptor family that activates c-jun n-terminal kinase in t cells tcell-mediated regulation of osteoclastogenesis by signalling cross-talk between rankl and ifn-gamma myeloid calcifying cells promote atherosclerotic calcification via paracrine activity and allograft inflammatory factor- overexpression altered bone remodeling in psoriatic disease: new insights and future directions coronary artery calcification: from mechanism to molecular imaging coronary artery calcium and long-term risk of death, myocardial infarction, and stroke: the walter reed cohort study nla/pcna guideline on the management of blood cholesterol: a report of the american college of cardiology/american heart association task force on clinical practice guidelines calcified carotid atherosclerotic plaque is associated less with ischemic symptoms than is noncalcified plaque on mdct impact of statins on serial coronary calcification during atheroma progression and regression the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © chernomordik, cercek, lio, mihailovic, yano, herscovici, zhao, zhou, chyu, shah and dimayuga. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -w f z authors: pombo, joao palma; sanyal, sumana title: perturbation of intracellular cholesterol and fatty acid homeostasis during flavivirus infections date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: w f z cellular lipid homeostasis is maintained through an intricately linked array of anabolic and catabolic pathways. upon flavivirus infections, these are significantly altered: on the one hand, these viruses can co-opt lipid metabolic pathways to generate atp to facilitate replication, or to synthesize membrane components to generate replication sites; on the other hand, more recent evidence suggests counter strategies employed by host cells, which actively modulate several of these networks in response to infection, enhancing interferon signaling by doing so, and thus creating an antiviral environment. in this review, we discuss recent data on mechanisms of alteration of lipid metabolic pathways during infection by flaviviruses, with a focus on cholesterol and fatty acid biosynthesis, which can be manipulated by the invading viruses to support replication, but can also be modulated by the host immune system itself, as a means to fight infection. metabolic reprogramming in immune cells is a recurrent phenomenon when exposed to proinflammatory stimulants in the form of pathogens or cytokines. macrophages and dendritic cells in particular are well-equipped to sense and respond to impending danger by pathogens, thus establishing the frontline of host defenses. recent studies have highlighted the extraordinary contribution that multiple host metabolic pathways confer toward the ability of innate immune cells to respond to infections ( ) . not surprisingly, some of the very same pathways that function to eradicate infection are often rewired by the invading pathogen. most viruses are known to induce aerobic glycolysis akin to the warburg effect ( , ) . more recently, perturbation in lipid metabolic pathways has also been reported for several classes of pathogens ( , ) . intracellular lipid homeostasis is achieved through a balance in biosynthetic, transport, and degradation processes. current evidence increasingly points toward an intricate relationship between host lipid metabolism and intracellular pathogens, including bacteria, viruses, and parasites. while the mechanistic details are yet to be unraveled, it is hypothesized that these pathogens, on account of their limited genome sizes, co-opt the host metabolic network to meet the energy demands and procure precursors for their anabolic processes including replication and intracellular transport. in addition, viruses alter lipid metabolism to facilitate amplification and evade the host immune response. this has been decidedly observed in cases of positive strand rna virus infections, such as dengue, west nile, hepatitis c, and several coronaviruses ( ) ( ) ( ) ( ) ( ) . marked alterations in cholesterol and fatty acid biosynthesis occur upon infection, accompanied by the appearance of distinctive compartments, believed to be their replication sites ( ) ( ) ( ) ( ) . despite diversity in their genome organization, many viruses share certain salient features, primary of which is their dependence on host factors to undergo replication, assembly, intracellular transport, and release ( ) ( ) ( ) ( ) ( ) ( ) . the intracellular life cycle of positive strand rna viruses is largely confined to the cytosol, within or on the surface of virus-induced organelle-like structures regarded as replication compartments ( ) ( ) ( ) ( ) ( ) ( ) . notwithstanding differences in transmission, host cell tropism, and pathogenesis, these viruses employ similar strategies for replication and assembly, often accompanied by reorganization of the host secretory pathway ( , , , , ) . the replication sites serve multiple purposes that function in a concerted fashion to facilitate efficient virus propagation. primarily, they offer spatial segregation of the different steps in the intracellular life cycle, such as rna translation, replication, and packaging of the viral genome into virions during assembly. viral replication compartments also enable a high local concentration of the necessary components-both viral and host-in a physically constrained space, ensuring efficient rna amplification. an equally important feature of these replication sites is to limit exposure of viral rna to the hostile cytoplasmic environment that contains cellular nucleases and sensors of the innate immune surveillance. degradation of dsrna replication intermediates is minimized by protection in membrane-delimited compartments. although lipid metabolism has received particular attention with gram-negative bacterial infection, several recent reports highlight their function in viral infections ( ) . analogous to lipopolysaccharide (lps)-mediated downregulation of sterol synthesis in case of viral infections, limiting cholesterol biosynthesis in human macrophages and fibroblasts via genetic knockdown of sterol regulatory element-binding proteins [sterol-regulatory element-binding proteins (srebps), discussed in a later section], was reported to spontaneously engage type i ifn signaling and restrict infection ( ) ( ) ( ) ( ) . initiation of anti-viral immunity thus displays a clear link with intracellular cholesterol biosynthesis, in a way that the induction of cholesterol synthesis would allow subversion of host immune responses and facilitate viral multiplication. with the advent of omics-based studies, it is increasingly becoming obvious that viruses induce large-scale alterations in host cellular metabolism ( , ( ) ( ) ( ) ( ) . among other examples are the induction of fatty acid synthesis by hepatitis c virus (hcv) in human hepatocytes, and the utilization of cellular lipid stores of hepatocytes by dengue virus. the effects of these events have been experimentally demonstrated by genetic and pharmacological inhibition of lipid biosynthetic pathways that attenuate viral pathogenesis ( , ) . these viral adaptation strategies can effectively increase available energy for virus replication and assembly, provide specific components for progeny particles, and for creating replication sites while suppressing antiviral signaling cascades. these reports highlight the intricate link between viruses and lipid metabolism. in the following sections, we discuss emerging data on fatty acid and cholesterol biosynthetic pathways that are upregulated by certain viruses to facilitate infection. fatty acid synthase (fasn) intracellular contents of fatty acids and cholesterol contribute to fuel storage as well as a source of components necessary for increased membrane production. the core reaction of fatty acid synthesis is catalyzed by fasn starting from acetyl coa and malonyl coa. once synthesized, palmitate can have several different fates, including further elongation to long chain fatty acids, which can be used for membrane production or storage in lipid droplets (lds) in the form of triacylglycerols and esterified cholesterol. lds are storage organelles consisting of triacylglycerols and steryl esters, and function as inert storage depots of excess cellular lipids. abundance and size of lds could be indicative of increased fatty acid synthesis, which might poise the cell for rapid membrane generation if needed and also maintains energy reserves ( ) . according to cellular states and their corresponding energy demands, fatty acids undergo β-oxidation to generate acetyl coa and nadh and fadh molecules in the mitochondrial matrix, for atp production via oxidative phosphorylation. viruses induce and require availability of fatty acids at several stages of their lifecycle-either to supplement energy requirements for their anabolic processes or to generate viral replication compartments, most notably observed during infection by positive strand rna viruses ( , ) . this is primarily due to the process of replication-confined to the cytoplasm-where such viruses alter the host intracellular lipid composition to create a beneficial environment. this phenomenon is exemplified by hcv, where all aspects of the viral lifecycle, including entry, replication, assembly, and release are host lipid associated ( ) . hcv requires low density lipoprotein receptor as a co-factor for entry into target cells ( ) . its replication occurs in membranous web-like compartments referred to as double membrane vesicles ( , ) and they assemble using lds as platforms ( , ) . to generate replication sites, hcv triggers synthesis of fatty acids, cholesterol, and lds ( ) ( ) ( ) ( ) . another member of the flaviviridae family, dengue, has also been reported to induce production of fatty acids ( , ) . fasn and acc were identified through a targeted sirna screen as necessary factors for efficient dengue virus replication ( , ) . drugs that inhibited fasn activity resulted in a significant attenuation in virus replication ( ) . infection with dengue virus does not affect fasn expression levels, but rather its redistribution to virus-triggered structures referred to as convoluted membranes ( ) . this phenomenon appears to be rab -mediated, a member of the gtpase family that typically resides in the er and lds. upon infection dengue ns was found to interact with rab , which allowed recruitment of fasn to viral replication sites, thus promoting fatty acid biosynthesis to increase their local concentration ( , ) . inhibiting fasn activity has a similar effect in mosquito cells with loss of infectious progeny virion production ( ). -hydroxy- -methylglutaryl-coa reductase is the rate-limiting enzyme for cholesterol biosynthesis and is regulated via a negative feedback mechanism mediated by products of the mevalonate pathway. in mammalian cells, hmgcr activity is suppressed by cholesterol imported through receptor-mediated endocytosis of low density lipoproteins ( ) . dengue infection inhibits phosphorylation of hmgcr at an inactivating site, generating a cholesterol-rich environment in the process ( , ) . this was further corroborated through inactivation of ampk and a subsequent increase in hmgcr activity, respectively ( ) . in comparison, west nile virus infection has a more direct impact on intracellular cholesterol distribution. infection was accompanied by redirecting cholesterol from the plasma membrane to virus replication sites ( ) . in mammalian cells cholesterol homeostasis is tightly regulated in a feedback mechanism via transcription factors that sense intracellular cholesterol levels ( , ) . these transcription factors are termed srebps that associate tightly with the sterol-sensing srebp cleavage-activating protein (scap) within the er membrane, via an additional interaction with the er-resident protein insig, which functions as an inhibitor of srebp ( , ) . scap has an additional role as a chaperone that mediates transport of the srebp-scap complex to the golgi network, where srebp is proteolytically cleaved by two resident golgi proteases (s p and s p) to release the transcriptionally active fragment of srebp from the membrane. the released forms of srebps are transported to the nucleus and activate transcription of target genes required for cholesterol and fatty acid biosynthesis, including hmgcr and fasn, respectively. when cholesterol levels are high, scap binds to cholesterol in the er, promoting an association with insig, and retains the complex within the er, thus reducing the synthesis of cholesterol. conversely, when cholesterol levels are low, binding of scap to insig is disrupted, and cholesterol synthesis is initiated ( ) ( ) ( ) . the authors of these studies postulated that de-enrichment of cholesterol from sites harboring sensory molecules, such as the scap-srebp-insig complex, results in activation of this signaling pathway, enabling the host cell to increase cholesterol levels to accommodate proliferation of intracellular membranes. lipid droplets are multifunctional organelles present in most organisms from bacteria to eukaryotes ( ) ( ) ( ) . these structures are particularly abundant in mammalian adipocytes and insect fat body cells. lds are mainly composed of a phospholipid monolayer and structural proteins, such as perilipins, which are involved in ld biogenesis and degradation. despite previous notions on a rather static role of lds in the maintenance of lipid homeostasis, more recently, it has become evident that lds are also present in immune cells, such as neutrophils and macrophages, where they regulate inflammatory or infectious processes ( , ) . upon stimulation with different challenges, they display an increase in abundance and thereby serve as reliable markers of immune cell activation. autophagy dependent degradation of lds has been reported for dengue virus infection in human hepatocytes ( ) . a similar activation of the autophagy pathway was recently described for zika virus infection as well ( ) . our own data (accepted, queued for publication) support a drastic upregulation of ld consumption through induction of autophagy upon both dengue and zika virus infections. this pathway appears to operate in an ancient ubiquitous protein (aup )-dependent manner, and is dictated by its ubiquitylation status. unmodified aup enabled dispersion of lds, which underwent lipophagy upon infection. this virus-triggered pathway is essential for assembly and production of newly synthesized progeny virions (in press). current consensus, therefore, supports a model where mobilization of lds in combination with increased synthesis of fatty acids and cholesterol provides a proviral environment for production of progeny virions ( ) (figure ). the interdependence of innate immune signaling processes and the regulation of sterols and fatty acid metabolism is increasingly being consolidated through emerging data ( ) . their role in production of inflammatory mediators has been reported by several groups ( ) ( ) ( ) . interferons (ifns) modulate the expression of a multitude of ifn-stimulated genes including viperin, which has been observed to be highly upregulated in response to bacterial lps, double-stranded dna, and rna analogs, and also possesses antiviral activity against a range of viruses including hcv and dengue virus ( ) . in a similar vein, inhibition of cholesterol biosynthesis also exerts an antiviral effect ( , , ) . srebps are involved in coordinating the regulation of the sterol and fatty acid biosynthesis pathways; ifns effectively inhibit srebp at both mrna and protein levels. interestingly, wnv-induced redistribution of cellular cholesterol was found to downregulate ifn-stimulated jak-stat antiviral signaling response to infection, potentially by removing cholesterol from their usual microenvironment. recent evidence suggests that alterations to cellular lipid metabolism have a more direct role in host defense, through positive regulation of the type i ifn-mediated antiviral response: for example, activation of type i ifn signaling can induce upregulation of β-oxidation and inhibition of cholesterol synthesis, in order to create a hostile cellular environment for viruses ( , ) . intracellular pathogens are known to stimulate de novo lipid and cholesterol biosynthesis to ensure their own survival. accordingly, repressing these anabolic pathways can inhibit the evolution of intracellular infections. activation of type i interferon receptors has been correlated to inhibition of cholesterol biosynthesis; however, repression of lipid metabolism in this manner is accompanied by an increase in the influx of environmental lipids, which maintain intracellular lipids and cholesterol at normal levels. thus, type i ifn signals reprogram cellular lipid metabolism, but this does not function to limit lipid availability to pathogens. it, therefore, remains unclear whether ifn-i linked repression of cholesterol biosynthesis, in the context of intracellular infection, is meant to limit nutrient availability to pathogens, or if it serves a different purpose. the scap protein acts as a sterol-sensing element, as well as a chaperone, which associates with immature srebp transcription factors in the er membrane. by knocking out or knocking down scap in macrophages, srebp activity is lowered, as well as expression of genes involved in lipid metabolism. as anticipated, de novo synthesis of cholesterol and fatty acids went down in the absence of scap, but total intracellular lipid levels remained unchanged. loss of scap also correlated with heightened resistance to viral infection in in vitro and in vivo models, confirming the functional equivalence between activation of type i interferon pathway and inhibition of lipid metabolism. culture medium supernatants from scap −/− macrophage cultures were enough to markedly increase resistance to viral challenge, when supplied to wild-type bmdms, suggesting that the higher type i interferon-mediated viral resistance was a causal effect of a secreted effector molecule, such as interferonbeta (ifnβ). in light of this, qpcr analysis revealed that both scap −/− bmdms and alveolar macrophages extracted from scap −/− mice constitutively express higher levels of ifnβ and interferon-stimulated genes (isgs), compared to wild-type macrophages. finally, blocking the interferon alpha/beta receptor (ifnar) was enough to restore interferon and isgs expression back to normal levels, as well as losing resistance to viral infection. these data strongly suggested that the absence of scap activity spontaneously triggers type i interferon production, which translates into a constitutive state of higher resistance to viral infection in macrophages ( ) . this suggests that a higher type i interferon response is specifically caused by an inhibition of cholesterol metabolism ( , ) . in support of this hypothesis, cells (immune and non-immune) with deficiency in the mevalonate pathway showed a constitutively exacerbated type i interferon response. also, addition of free cholesterol to srebp -deficient cells, or to cells with genetically impaired cholesterol metabolism, causes the exaggerated type i interferon response to decrease to basal levels ( , ) . stimulator of interferon genes (sting) is an er resident kinase, which activates interferon regulatory factor (irf ) through phosphorylation of tank binding kinase- (tbk ) ( ) . sting kinase activity is stimulated by cyclic dinucleotides, which are synthesized by cyclic gmp-amp synthase (cgas). cgas, sting, and phosphorylated tbk (ptbk ) exist in higher basal levels in srebp −/− cells compared to wild-type cells; in addition, knocking down either cgas or sting in enough to drastically lower ptbk presence in srebp -deficient cells. also, knockdown of cgas, sting, or tbk in srebp −/− cells caused the expression of ifnb and isgs to decrease to levels similar to those in wild-type cells ( ) . addition of free cholesterol to srebp -knockout cells significantly decreased ptbk , while blocking ifnar had no effect on ptbk levels, reinforcing the idea that cholesterol directly influences sting-mediated activation of tbk . these data support a model in which a lack of cholesterol in the cell makes sting more sensitive to cyclic dinucleotides, upregulating the sting-ptbk -irf signaling axis, and ultimately increasing expression of ifnb and isgs, conferring an intrinsic proinflammatory phenotype to cholesterol-deficient cells ( ) . admittedly, most of these experiments used mhv ; however, these conclusions may very well be relevant in other virus infections. repressing the cholesterol biosynthetic pathway through inhibitors of hmgcr is a common treatment for cardiovascular diseases ( ) . the clinical success of these inhibitors for human disorders provides strong support that targeting lipid metabolism can effective for human therapy. elucidating the specific alterations incurred upon virus infections would allow novel therapeutic approaches to emerge through targeted inhibition of such metabolic pathways. ifns or viral infections often result in induction of -hydroxycholesterol in macrophages-an antiviral effector, which broadly 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oztgi authors: palatnik-de-sousa, clarisa b. title: what would jenner and pasteur have done about covid- coronavirus? the urges of a vaccinologist date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: z oztgi nan vaccines are the best cost-benefit tools to control and eradicate infectious diseases. the live smallpox vaccination, called variolation, was the injection of the homologous virus and this promoted self-healing local lesions that guaranteed strong and long-lasting protection. however, since % of these variolations caused cases of smallpox in the vaccinated individuals, it was considered unsafe and was discontinued ( ) ( ) ( ) ( ) ( ) . in , edward jenner, who had been variolated, discovered the vaccination principle when he used the cowpox virus live-vaccine (vaccinia virus) to induce cross-immunity and prevent human smallpox in a child. his strong merit was to initiate the campaign that turned vaccination against smallpox obligatory and universal, and to discover that cross-protection promoted by a heterologous, although related, organism was sufficient to guarantee efficacy and reduce the safety issues of the homologous live-vaccine ( ) . in , due to the world health campaigns, smallpox was considered the first and only human viral infection ever eradicated ( , ) . ironically, jenner never knew that smallpox was induced by a virus ( , ) which suggests that, what is needed for the eradication of a disease is the systematic worldwide use of a potent and efficient vaccine. after the sabin anti-polio vaccine, which was launched in the 's, many other vaccines have been developed based on whole attenuated viruses. however, poliomyelitis was also induced by the sabin vaccine poliovirus in healthy subjects ( ) ( ) ( ) . this means that, nowadays, live-vaccination with whole wild or attenuated virus is no longer ethically possible, mainly because of the large population of immunocompromised subjects, in which a live-vaccine could cause the disease. due to these safety issues, whole virus and bacterial dead or inactivated vaccines have progressively substituted live-vaccines. we could guess that this is precisely what louis pasteur, the father of microbiology, would have done to control and eradicate covid . although he worked initially with the attenuation of viruses and bacteria, after his successful work with rabies, fowl cholera and anthrax ( ) , it became clear three steps were needed to develop a protective vaccine against infection. first, the organism should be isolated, then inactivated, and finally injected ( ) . in , pasteur's rabies vaccine employed an air dried fixed virus. semple improved the fixation by adding phenol ( ) . currently β-propiolactone is considered to be better than phenol or formaldehyde. however, it is carcinogenic ( ) . therefore, other methods like ultraviolet or gamma-irradiation, high pressure ( ) , visible ultrashort pulsed laser, and low-energy electron irradiation have been suggested ( ) . the most important advantage of whole inactivated vaccines is that, unlike the live or attenuated vaccines, they do not cause the disease ( table ). in fact, inactivated vaccines preserve the intact structure of the antigens and their b-cell epitopes that enable them to interact with the antibody paratopes, and promote the synthesis of neutralizing antibodies. they can not only stimulate the humoral, but the cellular immune responses as well, in a manner similar to live viruses, since they preserve the virus structures during inactivation ( ) . cross-presentation of conserved epitopes to the cytotoxic t cells (ctl), through the major hla class histocompatibility system, in addition to the viral pathogen associated patterns (pamps), which use innate immune receptors such as toll like receptor , can induce t cell mediated responses. in fact, in the late endosome of the infected apc three different events can occur: ( ) viral degradation following the exogenous pattern and presentation to cd t cells via the mhc class ii molecules, ( ) cross presentation pathway to cd t cells via the mhc class i molecules, and ( ) viral membrane fusion following the endogenous pathway. the above mentioned pathways along with the recognition of viral pamps, using prrs such as tlr , as well as production of cytokines such as ifn- can promote potent cellular mediated immune responses ( ) . however, in the case of influenza vaccines for instance, the inactivated formulations may not always induce t cell responses as potent as the live-vaccines. in fact, the inactivated vaccine may even prevent or suppress the induction of cross-reactive cd + t-cells ( ) . pamps are constitutive components of the virus and bacteria (viral or bacterial nucleic acids, polysaccharides, lipopolysaccharides, lipid a, monophosphoryl lipid a, bacterial peptidoglycans, etc.). they are compounds universally recognized by the innate immune system of healthy subjects, who build a natural protective response against them. in contrast, modern purified, fractionated, recombinant, or synthetic vaccines gained in safety but lost potency because they lack the pamps (table ) . while, vaccines using inactivated organisms with pamps have shown great success against polio, whooping cough, and tetanus ( , ) . if we use fixation of the structures of some of the isolates that cause the disease, then inactivate them and preserve their whole structures, the possible deleterious effect of the high mutagenicity detected in a few of the proteins of the virus ( ) would be overcome by the strong immune response generated against the whole virus structure. therefore, the mutagenicity should not be critical for the generation of protection, and would not damage the efficacy of the whole vaccine. furthermore, it might be that the whole virus inactivated vaccine could even induce some cross protection against other coronovirus agents of sars, which hold conservative structures ( ) . furthermore, whole inactivated vaccines are considered good candidates for designing universal vaccines capable of giving protection against multiple strains of influenza virus ( ) . it is true that an impressive amount of data about the dna sequencing of the virus has been gathered in a relatively short period of time and with that, the knowledge of its biological properties increased enormously ( , sequences in pubmed) ( , ( ) ( ) ( ) ( ) ( ) ( ) . however, for an urgent strategy, we could also take advantage of the lessons taught by the history of vaccinology in order to prevent the disease and save lives. furthermore, if we want to enhance the efficacy of the vaccine we should combine the inactivated virus with a good adjuvant. the adjuvant might contain saponins of quillaja saponaria molina, which induce antibodies of desired subtypes, promote both the cytotoxic antiviral cd + and cd + th cell responses against the infection and that been used with success in vaccines against leishmaniasis ( , , ) , cancer ( ) , malaria ( ), herpes zoster ( ) , and hiv ( ) . in spite of the valuable guidelines from the work of jenner and pasteur, who with much fewer resources, developed vaccines that controlled and eradicated smallpox that showed a % mortality rate ( ) and rabies with % mortality rate ( ) ; and even with all the knowledge acquired since then, there is no urgent combined international effort to produce one unique vaccine. instead, months after the description of the first covid cases in china, vaccines are reported to be in development all over the world and, only two of them contain the inactivated virus ( , ) . all the other formulations include live attenuated, non-replicating or replicating viral vector, recombinant protein, peptide base, virus-like particle, and virus dna and rna. most of these vaccines are focused on only one antigen of the coronavirus, therefore, these formulations will certainly be less potent than a vaccine made up of multiple antigens contained in the whole pathogen. furthermore, many of these formulations do not use the technology involved in any previously licensed vaccines ( ) . cepi (coalition for epidemic preparedness innovation) estimated the development of phase i clinical trials of vaccines, phase and trials for up to vaccines and progression to regulatory approval and production of up to candidates ( ) . in fact, by may th, seven vaccines had already entered phase i clinical trials: ( ) encapsulated mrna encoding protein s (moderna and niaid, usa); ( ) adenovirus expressing protein s (cansino biologics, china); ( ) dcs modified with lentivirus expressing several proteins and ctls (shenzen geno-immune medical, china); ( ) an apc modified with lentivirus expressing several viral proteins ( ); ( ) inno , sars cov dna injection (innovio, usa); ( ) chadox vaccine from the jenner institute, oxford university, (uk) which is a genetically modified adenovirus expressing coronavirus proteins ( ) , and is also being tested in a phase ii trial; and finally ( ) the whole inactivated coronavirus with alum by sinovac, china ( ) . furthermore, on july nd, the who communicates that there are covid candidate vaccines in clinical evaluation and more under pre-clinical assays ( ) . only one of the vaccines under clinical trials is currently supported by a peer reviewed scientific publication in science ( ). the results of its pre-clinical assays in the mouse, rat and non-human primate model were published before, without peer review on april th in the biorxiv. later on, on may th, the results of the chadox adenovirus vaccine of the jenner institute of oxford university were published with no peer review in the biorxiv ( ) . until june th, there has been no peer reviewed publication of this vaccine. regarding the formulations, the inactivated whole virus sinovac vaccine is composed of one isolate of sars-cov (cn ) obtained from a patient of china and alum adjuvant ( ) , while the chadox ncov vaccine of oxford is composed of a chimpanzee recombinant adenovirus, which expresses the s protein of sars-cov ( ) . sinovac biotech (china) in collaboration with several universities, public health institutions and the medical academy of the army of china have been able to produce a whole virus inactivated vaccine adjuvanted by alum that was stable and showed . to % sequence identity to other isolates also obtained from broncheoalveolar fluid (balf) of hospitalized patients (five in intensive care), from china, italy, united kingdom, switzerland and spain ( ) . the virus was propagated in cultures of vero cells in vitro and inactivated with β-propiolactone ( ) . the use of alum adjuvant is approved for human vaccines because it induces strong antibody responses, mainly of the igg and ige types that show efficacy against virus or bacterial diseases, which need neutralizing antibodies to be controlled. however, alum is a poor promoter of the cellular immune responses against pathogens ( ) . in contrast, the chadox ncov- vaccine developed by oxford university and astrazeneca is composed of a recombinant non-replicant chimpanzee adenovirus, which expresses the s protein of sars-cov- ( ) . different from the technology used for inactivated vaccines since the 's, this adenovirus platform was developed in ( ) . the authors aimed to include an adenovirus in the vaccine that would not infect humans, in order to avoid its potential rejection by human antibodies. the chosen chimpanzee adenovirus was phylogenetically related to the human adenovirus. the inventors deleted the region e of the chimpanzee adenovirus genome in order to render the virus defective and non-replicant, while the e region was excluded to increase the insert capacity. in addition, a bacterial artificial chromosome (bac) containing a codon-optimized full-length spike protein of sars-cov- with a human tpa leader sequence ( ) was added between the deleted e region and e to facilitate the genetic modifications. this approach has been reported to improve genetic stability ( ) . however, additional modifications were needed to guarantee that the e region of the virus would express a human, instead of a simian protein, that would enable the virus recognition and propagation inside human cells in in vitro culture, for large-scale virus production ( ) . regarding the number of samples, the sinovac inactivated vaccine was tested in groups of mice and rats and in cohorts of monkeys (macaca mulatta) ( ), while the chadox ncov- vaccine was only tested in groups of - mice and in rhesus monkeys using only monkeys as controls ( table ) ( ) . regarding the antibody response in mice and rats, the sinovac vaccine promoted high igg antibody titers against protein s, against its receptor binding domain, and to a lower extent against protein n ( table ) and also high titers of virus neutralizing antibodies. the cytokine expression induced by the inactivated vaccine in mice was not analyzed ( ) . in contrast, the chadox vaccine induced anti-s and s protein igg antibody titers (igg a, igg b, and igg ) and neutralizing antibodies in only three of the five balb/c mice, but showed ifn-γ, tnf-α, and il- expressed by cd + and cd + t cells and ifn-γ, tnfα, il- , and remarkably il- secreted to the supernatants ( ) ( table ) . furthermore, in vaccinated monkeys, seven days after infection, the sinovac inactivated vaccine at µg/dose induced high titers of igg antibodies directed against the s, rbd and lower levels of anti-n protein antibodies, high titers of virus neutralizing antibodies with no detected antibodydependent enhancement of disease (ade) ( ) . in contrast, anti-s protein igg and neutralizing antibodies were detected in the rhesus monkeys vaccinated with chadox ( table ) ( ) . moreover, regarding the concern of the increased proinflammatory events and cytokine storm related to the severity of covid , the sinovac vaccine was safe and did not promote any alteration in the frequencies of cd + , cd + , or cd t cells nor of secretion of ifn-γ, tnf-α, il- , il- , il- , or il- ( table ) ( ) . in contrast, increased levels of ifn-γ, tnf-α, il- , and il- were observed in monkeys vaccinated with the chadox vaccine ( ) ( table ) in which the frequencies of cytokine secreting t lymphocytes was not studied ( table ) . regarding cross-protection to other sars cov isolates, the sinovac inactivated vaccine protected mice and rats against the challenge with different virus isolates ( table ) suggesting its potential use all over the world ( ) . there is no available data concerning cross-protection for the chadox vaccine ( ) . besides, for a fair comparison of vaccine efficacies, the two sars-cov vaccines should be assayed in the same field trial, and the efficacy end-points should be determined prior to the assay. due to the urgency in saving lives, this might be not be feasible during the pandemic. however, for comparative purposes, early infection, disease, severe disease, and death due to covid or other causes should be recorded as vaccine efficacy end-points. for instance, reduction of the virus load in the nasal and pharynx mucosa indicates not only protection against early infection, but also the blockade of the transmission of infection by respiratory droplets. this means that this end point is particularly important when seeking a vaccine to interrupt the epidemic. in addition, clinical symptoms indicate disease, while pulmonary distress, cytokine storm, need of intensive care, intubation indicate severe disease. the number of deaths due to covid or other causes should also be recorded and compared in order to evaluate the reduction of mortality. notable, the sinovac inactivated vaccine reduced to zero the viral load in throat swabs (pharynx and crissum), anal swabs and all regions of both lungs of vaccinated and challenged monkeys ( ) indicating that the inactivated vaccine prevents not only the early infection but also blocks the transmission of the disease by droplets curtailing the epidemics. in contrast, no viral sgrna indicative of viral replication, could be detected in bal fluids, and in the lungs of two of six monkeys vaccinated with chadox and challenged ( ) ( table ). in fact, the lung viral load decreased by ∼ % in monkeys vaccinated with chadox . however, viral grna was detected in nose swabs of all vaccinated and challenged animals ( table ) ( ) , indicating that the chadox vaccine would not prevent the sars cov human infection nor block its transmission and interrupt the epidemics. vaccinated and infected subjects will continue to be infectious and spread sars cov- . however, the vaccine will probably, in most cases, reduce the pulmonary symptoms, and make the disease less severe. accordingly, the inventors of the chadox vaccine seem to be aware of the limitations of its efficacy when they describe it as a vaccine that prevents pneumonia in monkeys ( ) . unfortunately, neither the investigations of the sinovac nor the chadox vaccine have disclosed if any of their formulations prevent or reduces mortality. furthermore, in a report that analyzes the first results of the vaccine trials, published in nature, peter hotez considered that the oxford vaccine induced very modest titers of neutralizing antibodies and that considerable higher titers would be needed to afford protection ( ) . at the same time, hotez also says that the sinovac vaccine elicited a more promising antibody response in macaques monkeys ( ) . in spite of that, who disclosed that this vaccine is in fact being tested in uk in phase i, ii and iii trials ( ) and will be tested in a phase iii trial in brazil on , volunteers. consequently, contracts for large-scale fabrication have already been signed with the public laboratory of the brazilian ministry of health bio-manguinhos. in brazil, million doses are intended to be produced by bio-manguinhos and another million after the proven efficacy of the vaccine. at this point it is important to know which end-points of vaccine efficacy will be taken into consideration for such an important decision. on the other hand, the sinovac whole virus inactivated vaccine was also reported to have been successful in phase i and ii trials in - year olds (n = ) and in healthy elderly adults > years old (n = ) in china ( ) although these results have not yet been published in detail. more than % of the volunteers showed neutralizing antibodies ( ) . a recent contract has been signed between sinovac and the public laboratory instituto butantan of são paulo, brazil, in order to produce doses of the vaccine to immunize , healthcare professionals for a double-blind randomized phase iii trial in brazil, where the incidence of cases and deaths due to covid is still high ( ) . the results of the efficacy are expected in october . in return, the instituto butantan will gain the transfer of the technology and the license to manufacture , , doses for brazil. testing anti-covid vaccines in brazil became interesting because of the high morbidity and mortality and active expansion of the epidemics. an important warning is given by ewen callaway in his article published in nature ( ) , in which he asks for caution about the potential success of vaccines that arise from small animal or human studies. this might be the case of the moderna-niaid vaccine composed of lipid nano-particle encapsulated synthetic mrna, which encodes the spike s protein and already underwent phase i and ii clinical trials in usa ( ) . moderna company announced that phase iii trials are predicted to start in july and that studies in monkeys are underway in parallel. none of these mrna based vaccine has ever been licensed before ( ) . we conclude that the first results of anti-covid vaccine candidates confirm that the whole virus inactivated vaccine, which preserves the immunogenicity of all the antigens of the virus and contains pamps ( ) is more potent than the recombinant vaccines that have only the important s spicula protein, either expressed by an engineered adenovirus ( ) or by lnp encapsulated mrna ( ) . furthermore, the inactivated vaccine also contains the alum adjuvant. there are other examples support the superiority of whole inactivated vaccines above those expressing recombinants single antigens. for instance, . µg/dose of the trivalent inactivated influenza vaccine is as safe as, but more immunogenic than the . µg/dose of the recombinant baculovirus-expressed hemagglutinin flubok vaccine, in young children ( ) . this is a fast moving scenario and several phase clinical trials of covid vaccines have been published, either with or without peer reviews. two recombinant adenovirus vaccines expressing the s spike protein of sars-cov- , the chadox and the cansino vaccines ( , ) , and two other vaccines composed of mrna codifying for the s-protein (mrna , moderna vaccine) ( ) or its rdb domain (mrna bnt b pfizer-biontech vaccine) ( , ) have been assayed for safety and immunogenicity in phase i-ii clinical trials in humans. there were no serious adverse events related to any of the four vaccines ( ) ( ) ( ) ( ) ( ) . local and systemic reactions commonly including pain, feeling feverish, chills, muscle ache, headache, and malaise were recorded for all formulations ( ) ( ) ( ) ( ) ( ) and were reduced, in the case of the chadox vaccine, with use of prophylactic paracetamol ( ) . only the cansino vaccine was given as a single dose ( ) while chadox , moderna, and pfizer biontech vaccines were assayed in two-dose protocols ( , ( ) ( ) ( ) . anti-s protein igg responses rose by day ( ) and peaked or increased by day - , after the first ( ) or second immunization dose, respectively ( , ( ) ( ) ( ) . in addition, spike-specific t-cell responses detected by an ex-vivo interferon-γ enzyme-linked immunospot assay, peaked on day for the chadox ( ) and on day for the cansino adenovirus vaccine ( ) . moreover, the moderna mrna- , vaccine induced a th response against the s-protein peptide pools (tnf-α >il- >ifn-γ), with a minimal th cytokine expression (il- and il- ) and with cd t-cell responses, only detected at low levels, after the second vaccination ( ) . in agreement, most participants vaccinated with the pfizer-biontech mrna-rbd vaccine (bnt b ) also had th skewed t cell immune responses with rbd-specific cd + and cd + t cell expansion and ifn-γ produced by a high fraction of rbdspecific cd + and cd + t cells ( ) . furthermore, the levels of neutralizing antibodies raised by each one of the vaccines could be considered as correlates of their potential efficacy. while the mrna- of moderna disclosed % ec values ranging between and ( ), the maximal titer for the mrna rbd vaccine of pfizer-biontech was ( ) and for the chadox vaccine, from to ( ) . the cansino vaccine expresses its results as gmt ( - , ) impeding an accurate comparison ( ) . unfortunately, the results of phase i-ii clinical trial of the whole virus inactivated vaccine of sinovac have not yet been published in detail, therefore although the vaccine was tested in the largest number of individuals (n = , ) ( ), a fair comparison of the safety and immunogenicity results is not yet possible. ultimately, only the results of the phase iii trials will disclose the potential impact of the vaccines on reduction of deaths, clinical cases, and virus particles or viral rna in nasopharynx and will allow their efficacy and capability to interrupt the epidemic to be evaluated. in the imminence of a pandemic involving high mortality and economic distress, several factors could speed up the development of vaccines. one of them would be the use of an already standardized methodology. it is worth noting that most of the molecularly defined vaccines now in development would meet severe restrictions for large-scale production and this could led to an enormous delay to deliver vaccines for mass vaccination of the public. in contrast to this, nowadays large industries and public laboratories are authorized to produce inactivated vaccines against influenza. it is also reasonable to hypothesize that generation of protection and immunological memory against a group of antigens will be more efficient than that generated against a single antigen, no matter, how important it is. two concerns could be considered as the downside of the inactivated vaccines for sars diseases. the first would be the fear of an incomplete inactivation of the virus that could cause outbreaks among the vaccine production workers or in vaccinated populations ( ) . this concern is common to all vaccines produced with native antigens, which demand the production of large mass of pathogens. however, to guarantee safety, each batch of vaccine is submitted to validation of inactivation controls that include sequential passages assays of residual virus infectivity in embryonated eggs or tissue culture, and detection of live virus by tcid assays ( ) . the whole virus sars-cov- inactivated vaccine of sinovac includes validation of inactivation controls ( ) . the second concern would be the promotion of an antibody disease enhancement syndrome (ade) by the vaccine. this is usually related to non-neutralizing antibodies, which determine an increased lung pathology and it was observed before in vaccines against rsv and measles in the 's ( ) . since sars-cov- , mers-cov, and sars-cov- are phylogenetically related viruses that have caused epidemics over the last years and ade pathology was present for some sarscov- and mers vaccine candidates in animal models, there is also a concern about the induction of ade syndrome in humans vaccinated with sars-cov- vaccine candidates ( ) . however, ade pathology is not exclusive for inactivated vaccines and has been also demonstrated for the vectored vaccine expressing n protein, a replicon particle platform expressing s protein ( ), the recombinant protein s with or without gold nanoparticles ( ) and a mva vectored vaccine expressing s proteins ( , ) . with the aim of preventing these safety issues in sars-cov- vaccines cepi and the brighton collaboration safety platform for emergency vaccines (speac) convoked an expert scientific meeting on march and , in order to establish the assessment of the risk of ade during sars-cov- vaccine development ( ) . in murine models, ade was observed for an inactivated whole virus vaccine against mers ( ) and against sars-cov- ( , , ) . in fact an inactivated mers-cov vaccine, with and without adjuvant, induced in mice neutralizing antibody, reduced the viral load in lungs but showed mononuclear infiltrates containing eosinophils and eosinophils secreting il- and il- cytokines ( ) . a formalin double-inactivated sars-cov- (div) vaccine, adjuvanted or not, induced also immunopathology involving eosinophils in aged mice ( , ) . in addition, a formalininactivated sars-cov- vaccine promoted ade in nhp with macrophage and lymphocyte infiltration in the lungs and fibrin and protein-rich edema in the alveolar cavity ( ) . on the other hand, other inactivated vaccines against sars were reported as non-inducers of ade ( ) . fortunately, other studies disclosed the absence of ade in hamsters and monkeys immunized with whole inactivated vaccine against sars-cov- . these studies differed from the previous one in the use of β-propiolactone instead of formalin to inactivate the virus ( , ) . the conclusion was that, nhps could be used to evaluate the anti-covid vaccines with or without adjuvants to select the formulations with desired efficacy and reduced risk of ade ( ) . in addition, transgenic mice expressing the human ace receptor will be needed to evaluate the vaccine induced ade. the immunopathology was a consequence to a th type of response to the antigen and it was avoided in vaccines that drive the response to a th immunity, with or without adjuvants. also, it is known that the presence of fetal calf serum in the preclinical vaccine preparation may induce eosinophil influx to lungs ( ) . for instance, the passive transfer in nhps of human antibodies generated during phase trials, followed by viral challenge could be considered to assess the risk of disease enhancement ( ) . it was recommended to challenge the vaccinated animals with close related species in order to evaluate cross protection for future epidemic ( , ) . this has been done for the whole inactivated sinovac vaccine with other isolates of the sars-cov- ( ) . experts also recommended including animals vaccinated with formalin inactivated, alum-adjuvanted whole virus sars-cov- or sars-cov- , for immunopathology studies, as positive controls. this will help in establish accepted end-points to allow comparison ( ) . the group of experts considers that continuous monitoring of this risk will be needed during clinical trials. each effect observed should be discussed by the developers with their regulators who will ultimately define the actual requirements for clinical studies ( ) . if we consider that a whole virus inactivated vaccine with a potent qs saponin adjuvant is the ideal formulation for an anti-covid urgent first vaccine, the sinovac vaccine is not only the closest formulation to the ideal, only differing in the adjuvant, but also the one that can be developed the fastest. the potential use of alum, mf , as , as , or as , which contain qs saponin has been discussed ( , ) . it was concluded that, the immunopathology of sars vaccines was a consequence to a th type of response to the antigen and it was avoided in vaccines that drive the response to a th immunity, with or without adjuvants ( ) . time matters and is an extremely important factor considering the high daily rate of deaths worldwide. in fact, the assays of the sinovac vaccine in the mouse, rat, and macaque models seems to have been performed simultaneously from january to march, . in addition, this is the only vaccine with results already published in a peer reviewed journal ( ) . on the other hand, eradication of the pandemic or at least its control, as jenner knew in , will only be possible by a universal and simultaneous use of the same vaccine. this is what took place with smallpox, rabies, yellow fever, influenza, h n , etc. in spite of that, we do not see the united international effort to gather together resources for the production of enough doses of one vaccine to vaccinate the world. the support given to different research projects ( ) and the deposit of hundreds of patents confirms that. again, the urgent formulation might already be known and waiting to be rediscovered from the history of vaccinology. if different vaccines with diverse degrees of efficacy values are used, even the countries that have low incidence of covid will not be safe and will not be able to open their frontiers. to support the production of one ideal vaccine should be the common focus worldwide. maybe the observed multiple individualistic efforts that have arisen are due to the lack of leadership from the developed nations, which have the highest capacity to produce vaccines. in the usa, for instance, the economic interest of the large vaccine industries in preventive vaccination has recently decreased. conversely, they have started to invest in immunotherapies or drug treatments. in addition, in the usa there are not large public laboratories for production of the vaccines under a governmental request. consequently, the governmental public health decisions are restricted by the interests of the private vaccine companies. in contrast, in some developing countries, where infectious diseases are often the most important causes of mortality, public laboratories can produce large amounts of vaccine doses without the need to make a profit, under the auspices of their ministries of health. this is the case of instituto butantan and bio-manguinhos in brazil, instituto biológico de la plata and the administración nacional de laboratorios e institutos de la salud (anlis-malbrán) in buenos aires, argentina, and of the serum institute of india. fortunately, instituto butantan will produce the sinovac inactivated vaccine and bio-manguinhos the adenovirus chadox vaccine of oxford in brazil. the serum institute of india will also produce the chadox vaccine of oxford. finally, the modern policies for vaccine regulations should be taken into consideration. these regulations demand that phase i, phase ii, and phase iii trials should be developed with success before a government licenses a vaccine and uses it on phase iv trials and for industrialization. this usually takes at least a decade. although these tests enhance the confidence of a product, one might think that if we are dealing with a vaccine produced by a technology that is already well-established in many licensed vaccines, such as a whole inactivated virus, more rapid or simultaneous tests would be accepted as proofs of concepts ( ) . this would be another increased cost-benefit value of the vaccine, which 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, meeting: assessment of risk of disease enhancement with covid- vaccines gold nanoparticle-adjuvanted s protein induces a strong antigenspecific igg response against severe acute respiratory syndrome-related coronavirus infection, but fails to induce protective antibodies and limit eosinophilic infiltration in lungs antispike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection immunization with inactivated middle east respiratory syndrome coronavirus vaccine leads to lung immunopathology on challenge with live virus a double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge immunodominant sars coronavirus epitopes in humans elicited both enhancing and neutralizing effects on infection in non-human primates a double-inactivated whole virus candidate sars coronavirus vaccine stimulates neutralising and protective antibody responses immunogenicity and protective efficacy in monkeys of purified inactivated vero-cell sars vaccine immunogenicity and protective efficacy in mice and hamsters of a βpropiolactone inactivated whole virus sars-cov vaccine updated insights into the mechanism of action and clinical profile of the immunoadjuvant qs- : a review thanks to prof. charles greenblatt of the hebrew university of jerusalem, for helpful comments and discussion. david straker was acknowledged for the english review of this manuscript. the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © palatnik-de-sousa. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -uofygmeu authors: stäger, simona; rafati, sima title: cd (+) t cells in leishmania infections: friends or foes? date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: uofygmeu host protection against several intracellular pathogens requires the induction of cd (+) t cell responses. cd (+) t cells are potent effector cells that can produce high amounts of pro-inflammatory cytokines and kill infected target cells efficiently. however, a protective role for cd (+) t cells during leishmania infections is still controversial and largely depends on the infection model. in this review, we discuss the role of cd (+) t cells during various types of leishmania infections, following vaccination, and as potential immunotherapeutic targets. cd + t cells play a major role in protective immunity to a wide variety of pathogens, including viruses, bacteria, and protozoan parasites. however, the protective role of cd + t cells during leishmania infections has been controversial, mainly because of the discrepancy among infections with different leishmania species. different leishmania species have different tropisms and their diversity is reflected in the various clinical manifestations they induce. hence, it is not surprising that the contribution of cd + t cells to the immune response against the parasite depends on the clinical form and the species that is causing it. here, we discuss the literature on the contribution of cd + t cells to the immune response against leishmania, taking into account the various clinical forms and experimental models. cd + t cells recognize peptides that are presented in the context of major histocompatibility complex (mhc) class i molecules via the t cell receptor (tcr). although peptides presented via mhci mainly derive from endogenous antigens, various exogenous cellassociated antigens have also been shown to be uploaded onto the mhci pathway, by a process referred to as cross-presentation. leishmania antigens were also shown to be cross-presented (bertholet et al., ) . during in vivo infections, cross-presentation of leishmania antigens may result from several internalization pathways, such as direct infection, receptor-mediated uptake (woelbing et al., ) , or internalization of apoptotic vesicles (winau et al., ) . thus far, two different processing pathways have been proposed. an early work demonstrated that a surface antigen of l. amazonensis was processed in a proteasome-dependent manner within the cytosol (kima et al., ) . in contrast, a more recent study showed that cross-presentation of secreted leishmanial antigens is confined to an intraphagosomal processing pathway that is tap-and proteasome-independent (bertholet et al., ) . after activation, antigen-specific cd + t cells differentiate into effector cells and acquire the capacity to kill target cells, and produce several cytokines and chemokines (kaech et al., ; harty and badovinac, ) . among the various cd + t cell subsets, tc were shown to play a major role in the fight against several protozoan parasites (jordan and hunter, ) . the hallmark of this subset is the production of ifn-γ and tnf, and cytotoxic capacity (woodland and dutton, ) . the precise mechanism underlying cytotoxic t lymphocyte (ctl) killing of microbes is still poorly understood. ctls can exert cytotoxicity through various mechanisms: via exocytosis of lytic granula containing perforin, granzyme a/b, and/or granulysin; through the interaction between fasl and fas expressed on targets cells; via tnf; or via trail (trapani and smyth, ) . a study has also shown that reactivated memory cd + t cells efficiently killed listeria monocytogenes via a mechanism mediated by ccl and involving the induction of radical oxygen intermediates (narni-mancinelli et al., ) . direct killing of extracellular pathogen by ctls has also been described. for example, ctls can mediate killing of mycobacterium tuberculosis through the release of anti-bacterial products (stenger et al., ; canaday et al., ) . however, direct killing of schistosoma mansoni (ellner et al., ) and entamoeba histolytica (salata et al., ) is thought to be contact-dependent. ctls have also been reported to directly kill extracellular toxoplasma gondii (khan et al., ) . interestingly, killing in this case appeared to be antigen-specific. to date there is no evidence that cd + t cells can mediate protection against leishmania parasites through their cytotoxic activity. however, since ctls have been www.frontiersin.org observed in various mouse models and also in human patients, a possible protective role for leishmania-specific cytotoxic t cells should not be excluded. in addition to killing and releasing cytokines and chemokines, recent studies have ascribed a novel regulatory role for cd + t cells (sun et al., ; palmer et al., ; trandem et al., ) . regulatory cd + t cells represent a transient state of effector cd + t cells (trandem et al., ) , which is possibly induced by potent tcr stimulation, which promotes the production of the immunosuppressive cytokine il- (zhang and bevan, ) . not only do these cells produce il- , but they are also excellent killers and produce normal to higher amounts of ifn-γ and tnf (sun et al., ; palmer et al., ; trandem et al., ) . the main function of these cells is thought to lie in the prevention of immunopathology during infection without affecting the kinetics of pathogen clearance. il- -producing cd + t cells have also been observed in human patients infected with l. guyanensis (bourreau et al., ) and in patients suffering from post-kalaazar dermal leishmaniasis (pkdl; ganguly et al., ) . the role of regulatory cd + t cells in the immune response against parasitic infections is still unknown. the role of cd + t cells in the immunity to l. major has always been controversial. early studies in balb/c mice reported that cd + t cells were the main mediators of protection following cd + t cell depletion in mice infected with l. major (titus et al., ; hill et al., ; muller et al., ) . interestingly, depletion of cd + t cells was rendering susceptible balb/c mice resistant to l. major infection. the results obtained using the cd + t cell depletion model suggested that cd + t cells could potentially control l. major infection in mice. a few years later, experiments in β -microgobulin-deficient mice contradicted these findings and revealed that cd + t cells were not essential in mediating protection in l. major-infected balb/c mice (wang et al., ) . moreover, a study in cd + t cell-deficient mice demonstrated that cd −/− mice were able to control l. major infection for at least year, suggesting that cd + t cells were not required for long-lasting immunity (huber et al., ) . the contribution of cd + t cells in the control of primary l. major infection became less important also because of the strong evidence that th cells were the primary cells involved in mediating protection against cutaneous leishmaniasis (reiner and locksley, ; louis et al., ; sacks and noben-trauth, ) . several studies have demonstrated that th cells producing ifn-γ were essential in controlling l. major infection, and that failure to develop a th response resulted in susceptibility to the diseases. hence, a consensus was reached in that if a mouse generates th responses, this will lead to susceptibility; in contrast, th responses were successfully controlling infection without the help of cd + t cells. this paradigm was later challenged when new findings arose from a more natural model of infection, where metacyclic promastigotes were inoculated intradermally in the ears of c bl/ mice. in this model, cd −/− and cd + t cell-depleted mice fail to control l. major infection, and cd + t cells were thought to be necessary for supporting th responses (belkaid et al., ) . the discrepancy between the findings in the low-and the highparasite dose model was clarified by another work that compared the requirements of cd + t cells in both systems (uzonna et al., ) . interestingly, in the low infection model cd + t cells producing ifn-γ were essential for modulating cd + t cell responses toward a th response. in contrast, c bl/ mice inoculated with a high l. major dose did not require cd + t cell help to generate protective th responses. the cd + t cell requirement for optimal ifn-γ production by th cells was also proposed in a high-dose l. major infection model in balb/c mice (herath et al., ) . moreover, cd + t cell-derived ifn-γ was reported to contribute to the induction of nitric oxide production in macrophages during experimental cutaneous leishmaniasis (stefani et al., ) . although the role of cd + t cells-derived ifn-γ has been clarified, little is known about the involvement of cytotoxic cd + t cells in cutaneous leishmaniasis. in a low-dose model of l. major infection, cd + t cell responses were shown not only to be protective, but also to mediate pathology (belkaid et al., ) . hence, it is possible that ctls may be involved in the ulceration of skin lesions through tissue disruption. this suggests that perhaps two types of cd + t cell effectors are generated during l. major infection: antigen-specific cd + t cells that produce ifn-γ but lack cytotoxic activity; and ctls that are potent killers but produce little to no ifn-γ and promote pathology. although the role of cd + t cells during primary immune responses is controversial, these cells appear to play a prominent role in protecting mice from a secondary challenge (muller et al., . indeed, antigen-specific cd + t cells were expanding up to -fold in the spleen and lymph nodes of reinfected balb/c mice . this expansion correlated with a substantial production of ifn-γ, which is thought to be essential for controlling leishmania infections. these observations have major implications for vaccine design. in summary, during experimental cutaneous leishmaniasis, cd + t cells are necessary to support protective th responses through ifn-γ production, but they are also involved in the development of immunopathology. further investigations are needed to better identify various subtypes of cd + t cells that arise during cutaneous leishmaniasis. in contrast to the cutaneous models, cd + t cells have always been thought to play a major role in experimental visceral leishmaniasis (vl). over years ago, stern et al. ( ) demonstrated for the first time that cd + t cells significantly contribute to the formation of granulomas in the liver of l. donovani-infected mice. indeed, cd + t cell depletion resulted in impaired granuloma formation and exacerbation of liver disease. in agreement with these results, kaye et al. ( ) also reported a delayed onset and a decrease of the liver granulomatous response in non-obese, diabetic mice expressing transgenic i-e molecules, suggesting that antigen-specific cd + t cells are required for proper granuloma formation. cd + t cells appear to participate in controlling parasite growth in the spleen as well, since cd + t cell depletion during chronic vl significantly increased splenic parasite burden (stäger, unpublished) . this observation was underscored by the fact that adoptive transfer of antigen-specific cd + t cells during frontiers in immunology | microbial immunology chronic l. donovani infection resulted in % reduction in the splenic parasite burden (polley et al., ) . moreover, therapeutic vaccination aimed at reactivating cd + t cells during chronic vl ensued in the control of parasite growth in the spleen (joshi et al., ) . interestingly, cd + t cells do not only participate in primary responses to l. donovani, but are also the major mediators of resistance upon reinfection (stern et al., ) . indeed, protection was abrogated following cd + t cell but not cd + t cell depletion. a prominent function for cd + t cells was also described in l. infantum-infected mice. using an intradermal infection model, ahmed et al. ( ) demonstrated that cd + t cells contribute to parasite clearance in the skin of l. infantum-infected mice. another study also showed that cd + t cells purified from l. infantum-infected mice expressed ifn-γ and tnf, and displayed considerable cytotoxic activity against cells expressing leishmania antigens (tsagozis et al., ) . interestingly, killing of infected target cells was mediated by both the perforin and fas/fasl pathways (tsagozis et al., ) . the fas/fasl pathway has also been implicated in the defense against l. donovani (alexander et al., ) . indeed, gld and lpr mice, which lack a functional fas/fasl pathway, were shown to be more susceptible to l. donovani. additionally to the classical cytotoxic pathways, a novel counterregulatory function for a subset of cytotoxic cd + t cells has recently been proposed in the l. donovani infection model (martin et al., ) . in this study, cd + cd + cd + t cells are shown to suppress regulatory t cells via cd /cd l interaction during the early stages of infection in balb/c mice. cd signals through ras, pi k, and protein kinase c, leading to the induction of granzyme and perforin, and ultimately to the killing of tregs. cd + t cells may not be merely participating in the primary immune response by secreting ifn-γ and possibly killing infected target cells and/or tregs, but they could also be involved in the recruitment of inflammatory cells and in the maintenance of granulomas. indeed, a study using l. infantum demonstrated that cd + t cells expressed rantes and mip- α (tsagozis et al., ) , two chemokines that are involved in the recruitment of t cells at the inflammatory site and in the formation and maintenance of granulomas (mackay, ) . the authors proposed that cd + t cells may thus be involved in granuloma formation. this hypothesis is in agreement with the depletion data (stern et al., ) , showing that depletion of cd + t cells results in impaired granuloma formation and ultimately in disease exacerbation. despite the documented evidence that cd + t cells strongly participate in the immune response to l. donovani and l. infantum, our recent findings suggest that l. donovani induces defective antigen-specific cd + t cell responses (joshi et al., ). interestingly, mice infected with l. donovani generate cd + t cell responses with limited clonal expansion. the extension of the clonal expansion is thought to be correlated with the effectiveness in eliminating pathogens. it was calculated that a naïve cd + t cell may go through cell divisions in the first week after pathogen inoculation (badovinac et al., ) . massive clonal expansions have not only been observed during viral infections, but also following the injection of irradiated plasmodium berghei sporozoites (sano et al., ) . during l. donovani infection, cd + t cells underwent at least - rounds of division, but failed to accumulate in the spleen (joshi et al., ) . moreover, only % of cd + t cells during clonal expansion expressed markers typically associated with end-differentiated effector cells, such as klrg , unpublished) . the cause of this limited expansion is yet unknown and may depend on several factors. one of the possible explanations is limited antigen availability that may result from poor antigen-processing and presentation. processing of leishmania antigens is thought to be confined to a tap-independent, intraphagosomal pathway that is less efficient and requires higher amounts of antigen than the endoplasmic reticulum-based, tap-dependent cross-presentation pathway (bertholet et al., ) . furthermore, the major surface protease of leishmania, gp , was shown to cleave epitopes within the parasitophorous vacuole, further reducing antigen availability (garcia et al., ) . hence, leishmania antigens may be poorly presented and this poor presentation may not be enough to induce and sustain a massive clonal expansion of antigen-specific cd + t cells. nonetheless, antigen may be suddenly available in large amounts later on during l. donovani infection, since cd + t cells undergo a second round of activation, become dysfunctional, and ultimately die by "exhaustion" (joshi et al., ) ; high antigen levels have been described as a cause of cd + t cell "exhaustion" during chronic viral infections (mueller and ahmed, ). further research is needed to clarify the mechanisms involved in cd + t cell exhaustion during chronic vl. in conclusion, cd + t cells are required to control parasite growth during experimental vl and reactivation of these responses results in a dramatic reduction in parasite burden. therefore, immune interventions that target cd + t cell responses may have great therapeutic potential against vl. the role of cd + t cells in human leishmaniasis patients is still unclear and seems to depend on the various species of parasites and the disease they cause. few studies have been conducted with human vl patients. however, most of the studies ascribe a protective role for cd + t cells, in agreement with results obtained from experimental models. indeed, the control of l. infantum infection was shown not only to be associated with ifn-γ-producing cd + t cells, but also with cd + t cells (mary et al., ) . interestingly, during active vl, cd + t cells are less responsive to stimulation and a greater percentage stains positive for annexin v compared to healthy controls (clarencio et al., ). these observations correlate very well with what we observed in mice experimentally infected with l. donovani, where cd + t cells became increasingly dysfunctional during chronic infection and died by exhaustion (joshi et al., ) . whether human cd + t cells also display signs of exhaustion during active vl still remains to be tested. another study investigating cd + t cell responses in patients infected with l. chagasi has revealed that the frequency of cd + cd ro + cd + t cells is significantly decreased in the spleen of patients with active vl (clarencio et al., ). in contrast, cd + cd + t cells seem to be retained in the bone marrow of vl patients. cd , or integrin β- , is the β subunit of lfa- (cd a), cd b, cd c, and cd d. in humans, lack of cd www.frontiersin.org causes leukocyte adhesion deficiency, a disorder characterized by lack of leukocyte extravasation from blood into the tissue (bunting et al., ) . with exception of the fact that cd + cells appear in the granulomas of dogs with asymptomatic vl (sanchez et al., ) , very little is known about the function of cd + cd + t cells during vl and whether cells lacking cd expression have similar migratory capacity and effector functions to their cd + counterparts. not only is the frequency of cd + cd + t cells reduced in l. chagasi patients, but also, the level of circulating memory t cells is significantly decreased during active vl (hailu et al., ; clarencio et al., ). this observation is in agreement with our findings in the experimental model of vl, where the majority of the cd + t cells displayed an effector phenotype during chronic infection (joshi et al., ) . although cd + t cells positively correlate with cure of vl patients, one report suggested that these cells may contribute to the immunopathogenesis of pkdl (ganguly et al., ) . indeed patients suffering from pkdl showed a significant increase in the percentage of cd + t cells producing il- , which disappeared after cure (ganguly et al., ) . il- -secreting cd + t cells are thought to play a regulatory role in different viral infection models. this cd + t cell subset was shown to display great cytotoxicity and produce granzyme b, ifn-γ, and tnf (sun et al., ; palmer et al., ; trandem et al., ) . il- + cd t cells seem to represent a transient and reversible state of cd + effector t cell differentiation. its primary function is to balance pathogen clearance with bystander tissue damage (zhang and bevan, ) . interestingly, in viral model, this subset disappears after the infection is cleared. hence, it is possible that the il- -producing cd + t cells in pkdl patients are actually killing parasites and protecting patients from tissue damage, rather than suppressing protective responses. further studies are needed in order to define the nature of these cells. cd + t cells also actively participate in the immune response to cutaneous infections in human. as observed in the low-dose model in mice (belkaid et al., ; uzonna et al., ) , l. major also induces th and cd + t cells in human patients and both responses are associated with disease resolution (nateghi rostami et al., ) . cd + t cells were not only observed in large numbers in the lesions of l. major patients during the acute phase, but also during the healing process (da-cruz et al., gaafar et al., ) . the exact role of cd + t cells in l. major infections in humans is not yet known. a major correlate of protection appears to be the high amounts of ifn-γ produced by cd + t cells after restimulation (nateghi rostami et al., ) . in vitro studies have also demonstrated that leishmania-specific ctls are generated upon co-culturing human naïve t cells with antigens from l. amazonensis promastigotes and il- (russo et al., ) , or with l. major parasites (da . moreover, increased granzyme b activity was also found in patients with an active infection and was associated with a good prognosis (bousoffara et al., ) . in this study, in vitro cytotoxicity by peripheral blood lymphocytes on l. major-infected macrophages appeared to be mediated by granzyme b, suggesting that ctl activity may be involved in controlling parasite growth. it is possible, though, that the cytotoxic activity not only contributes to disease clearance, but also to the development of skin ulceration, as observed in l. major-infected mice (belkaid et al., ) . a strong cd + t cell expansion has also been observed in l. mexicana patients during the healing process (salaiza-suazo et al., ) . interestingly, lesions of patients with localized cutaneous leishmaniasis (lcl) harbor a higher number of cd + t cells compared to patients with diffuse cutaneous leishmaniasis (dcl; hernandez-ruiz et al., ) . as already observed in vl patients, cd + t cells in dcl patients, unlike lcl patients, show a reduced capacity to respond to antigen-specific stimulation during active infection. in fact, these cells displayed low cytotoxicity and only produced little ifn-γ upon stimulation, therefore showing typical signs of functional exhaustion (hernandez-ruiz et al., ) . strikingly, effector functions could be restored in vitro after stimulation with tlr agonists, highlighting the potential therapeutic benefit of the revival of cd + t cell functions in dcl patients. in contrast to the cutaneous and visceral forms of leishmaniasis -where cd + t cells seem to correlate with cure and contribute to the immune response -in mucocutaneous infections (ml) cd + t cells seem to be implicated in the pathogenesis of the disease. indeed, high numbers of cytotoxic cd + t cells were observed in ml patients (barral-netto et al., ; brodskyn et al., ) . moreover, the recruitment of granzyme a + cd + t cells is associated with lesion progression (faria et al., ) , suggesting that ctls may contribute to immunopathology. the development of ml is not only associated with the presence of ctl, but also with a high frequency of activated cd + t cells, an extreme ifnγ and tnf production, and a reduced control of inflammation due to low expression of the il- receptor (gaze et al., ; faria et al., ) . furthermore, il- -secreting cd + and cd + t cells were also found in ml patients (boaventura et al., ) . consequently, neutrophils, which are typically recruited during a th -mediated inflammatory response, were also detected in necrotic and perinecrotic areas (boaventura et al., ) . this suggests that neutrophils, together with ctls, may be involved in tissue injury and in the development of immunopathology. taken together, the literature shows that cd + t cells actively participate in the fight against most leishmania infections in humans and their presence correlates with cure. in contrast, cd + t cells in ml patients contribute to disease exacerbation. vaccination of humans with heat-killed leishmania or recombinant parasite proteins has so far failed to induce long-term immunity and only recovery from natural or experimental infection has provided proper protection. several trials have analyzed the protective effect of autoclaved l. major plus bacillus-calmette-guérin (bcg) versus bcg alone assessing the cumulative incidence of cutaneous leishmaniasis caused by l. tropica (sharifi et al., ) or l. major (momeni et al., ) , or of vl (khalil et al., ) caused by l. donovani. although no trial showed a significant effect on disease incidence, the vaccination induced skin test conversion and provided limited protection. additionally, a study showed that immunization of colombian soldiers with three doses of l. amazonensis alone was non-protective (velez et al., ) . in the human disease, there is evidence that mixed t helper cytokine profiles are present, while healing and protection against reinfection are associated with dominant th and cd + t cells. these findings suggest that it is the cytokine balance that activates or suppresses activation of macrophages harboring leishmania parasites. this, in turn, determines the outcome of the infection. thus treatments or antigen/adjuvant formulations that can alter the type of t helper response may change the course of disease (da-cruz et al., ; rogers and titus, ; mohajery, ) . for this purpose, different vaccination strategies have been examined in animal models including leishmanization (modabber, ) , killed parasite (grimaldi, ) , live attenuated parasite (titus et al., ) , and subsequently, subunit vaccines composed of recombinant or native proteins from different stages of the parasite's life cycle, and dna vaccines (skeiky et al., ; webb et al., ; stager et al., ; bottrel et al., ; campos-neto et al., ; rafati et al., ; coler et al., ) . the latter two strategies encompass candidates such as gp , gp , lack, cpb, cpa, kmp , lmsti , tsa, leif, haspb , and lpg, and have shown promising results in murine models. nonetheless, only leish f (a recombinant fusion protein of lmsti , tsa, and leif) progressed through phase i and ii clinical trials (llanos-cuentas et al., ; chakravarty et al., ) . nowadays, it is clear that cd + t cells play an important role in the mechanisms for cure of and resistance to leishmania infection, either by production of ifn-γ and activation of macrophages, or by direct killing of parasitized macrophages, or a combination of both effects. cd + t cells have been associated with protection against leishmania reinfection in murine models; however, the induction of these t cell subsets in humans seems to be also related to the healing process. today, there are several reports about different leishmanial antigens eliciting ctl responses such as p , gp , haspb (stager et al., ) , kmp (basu et al., ) , cpb (rafati et al., ) , nucleosomal histones (iborra et al., ) , lmacin (farajnia et al., ) , lmsti , and tsa (coler et al., ) . the essential point to be considered in vaccine design for a heterogeneous population, such as that of humans, is the hla polymorphism. effective vaccination against a complex parasitic infection such as leishmania would require a multivalent vaccine composed of several antigens to enhance the possibility of covering a good number of mhc types. this is possible either through recombinant fusion proteins encompassing the whole antigen or through vaccines composed of peptides from different antigens (campos-neto et al., ; rafati et al., ; mendez et al., ) . the latter strategy, called polytope vaccine, is finding its way in vaccinology because of its extraordinary properties, especially the ability to direct the immune response toward the induction of ctls (sbai et al., ; schirmbeck et al., ; robinson and amara, ) . as ctl responses play a pivotal role in defense against viruses and tumor cells, polytope vaccines have found their way in these fields but there is still no report on leishmaniasis even it has been shown that ctls could be very important in protection and long-lasting resistance to infection. recently, we took advantage of the potential of immunoinformatics tools to screen for l. major epitopes that could be presented in hla a , which is the most prevalent hla supertype in the iranian population. in vitro stimulation to recall memory cd + t cells from leishmania-infected individuals and intracellular cytokine assays for ifn-γ-producing cells confirmed that hla a positive individuals that recovered from an l. major infection successfully generated cd + t cell responses against peptides derived from lmsti and lpg- (seyed et al., ) . furthermore, walden and co-workers have mapped the t cell epitopes from kinetoplastid membrane protein of l. major (kmp ) via classical mapping for different human hla class i alleles (basu et al., ) . gazzinelli and co-workers have studied cd + t cell responses against the leishmania a antigen and mapped the cd + t cell epitopes in balb/c mice (resende et al., ) . laouini and co-workers (guerfali et al., ) and dumonteil and co-workers (dumonteil, ) started genomewide screenings for novel epitopes. using a combination of t cell epitope prediction tools, they successfully validated such epitopes in both balb/c and c bl/ mice. although understudied, cd + t cells appear to play an important role in the immune response to most leishmania infections. pilot studies in the murine model of vl have also demonstrated that adoptive transfer of antigen-specific cd + t cells (polley et al., ) or reactivation of cd + t cell responses through a therapeutic vaccine (joshi et al., ) results in the control of parasite growth. a better understanding of the mode of activation, the specificity, and effector functions of the various cd + t cell subsets generated during leishmania infections could ameliorate the design of vaccines and of novel therapeutic interventions. for the early control of parasite burden in the liver of leishmania donovani-infected mice. eur. j. immunol. , - . badovinac, v. p., haring, j. s., and harty, j. t. 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cells is mediated by fcgamma receptors and facilitates acquisition of protective immunity heterogeneity of cd (+) and cd (+) t cells cd (+) t cells: foot soldiers of the immune system this work was funded by the start up funds from the inrs-iaf to ss, and the national science foundation of iran (grant no. ) to sima rafati. we would like to thank guillermo arango duque for editing the manuscript. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord- -xtox rxs authors: li, hao-ling; huang, yan; zhou, ya-lan; teng, run-hua; zhou, shu-zhuan; lin, jia-piao; yang, yan; zhu, sheng-mei; xu, hua; yao, yong-xing title: c-x-c motif chemokine contributes to the development of neuropathic pain by increasing the permeability of the blood–spinal cord barrier date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: xtox rxs neuropathic pain is among the most debilitating forms of chronic pain. studies have suggested that chronic pain pathogenesis involves neuroimmune interactions and blood–spinal cord barrier (bscb) disruption. however, the underlying mechanisms are poorly understood. we modeled neuropathic pain in rats by inducing chronic constriction injury (cci) of the sciatic nerve and analyzed the effects on c-x-c motif chemokine (cxcl )/cxcr activation, bscb permeability, and immune cell migration from the circulation into the spinal cord. we detected cxcr expression in spinal neurons and observed that cci induced cxcl /cxcr activation, bscb disruption, and mechanical hyperalgesia. cci-induced bscb disruption enabled circulating t cells to migrate into the spinal parenchyma. intrathecal administration of an anti-cxcl antibody not only attenuated cci-induced hyperalgesia, but also reduced bscb permeability, suggesting that cxcl acts as a key regulator of bscb integrity. moreover, t cell migration may play a critical role in the neuroimmune interactions involved in the pathogenesis of cci-induced neuropathic pain. our results highlight cxcl as a new potential drug target for the treatment of nerve injury–induced neuropathic pain. neuropathic pain is caused by primary lesions or dysfunction in the nervous system, and it is among the most debilitating forms of chronic pain ( ) . its etiology is poorly understood, and this hinders the development of therapeutic and preventative strategies ( ) ( ) ( ) . one thing that is clear is that peripheral nerve injury leads to neuropathic pain by triggering radical changes that affect multiple components of the pain signaling pathway ( , ) . over the past decade, inflammatory responses after nerve injury have become an important topic in neuropathic pain research, and recent evidence suggests that neuroimmune interactions are involved in the pathogenesis of chronic pain states ( ) ( ) ( ) . accumulating evidence indicates that multiple proinflammatory mediators are released from injured nerve fibers and adjacent immune cells after nerve injury and that these mediators in turn promote central sensitization and behavioral hyperalgesia ( , ) . furthermore, animal studies have shown that peripheral nerve injury induces circulating immune cells to enter the spinal cord parenchyma, a phenomenon that may contribute to pain-related behaviors during the development of neuropathic pain ( ) ( ) ( ) . because the blood-spinal cord barrier (bscb) is the main structure regulating interactions between the immune system and the central nervous system (cns), it is reasonable to speculate that bscb dysfunction may play a critical role in the migration of circulating immune cells into the spinal cord ( ) . however, little is known about the functional states of the bscb in the context of peripheral nerve injury-induced neuropathic pain. researchers do not know how, or even whether, a remote injury can affect bscb integrity. the potential consequences of compromised bscb integrity in terms of spinal cord homeostasis and the development of pathological pain are also unclear. this matter may be illuminated through research into chemokines, a family of small cytokines (i.e., signaling proteins) that are upregulated by primary proinflammatory mediators and tumor necrosis factors ( , ) . in the cns, chemokines regulate myriad functions including neuronal development, synaptic transmission, and neuroinflammation ( ) ( ) ( ) ( ) . recent studies have shown that the c-x-c motif chemokine receptor (cxcr ) and its ligand c-x-c motif chemokine (cxcl ) are involved in the pathophysiology of allergic itches and neuropathic pain ( ) ( ) ( ) . however, the mechanism by which cxcl /cxcr signaling mediates neuropathic pain remains poorly understood. past studies have reported that cxcl promotes the entry of peripheral immune cells into the spinal cord ( , ) . on the other hand, some studies and our recent report have shown that t cell infiltration of the dorsal horn may contribute to the onset of neuropathic and inflammatory hyperalgesia ( , , , ) . however, it is unknown whether these processes contribute to hyperalgesia following peripheral nerve injury. in this study, we examined the integrity of the bscb and the migration of circulating immune cells into the spinal cord after chronic constriction injury (cci) of the sciatic nerve, which induces neuropathic pain. we also examined the activation of the cxcl /cxcr signaling pathway after cci. we aimed to elucidate the pathophysiology underlying nerve injury-induced neuropathic pain and to identify potential drug targets for the treatment of neuropathic pain. all animal experiments were conducted in accordance with the arrive guidelines ( ) and all relevant chinese laws. the experimental protocol was approved by the research ethics committee of the first affiliated hospital at zhejiang university. all measures were taken to minimize the animals' suffering and to reduce the number of animals used. adult male sprague-dawley rats ( rats in total, weeks at arrival) weighing - g were obtained from the animal center of the chinese academy of sciences. they were housed in groups ( rats/cage) in a temperature-controlled room ( ± • c) with a -/ -h light/dark cycle and ad libitum access to food and water. the rats were randomly divided into the sham surgery and cci groups (n = - per group for the behavioral test; n = - for the others). after the baseline was determined, the rats underwent the corresponding procedures on experimental day . cci was surgically induced as described in our previous publication ( ) and another study ( ) . in brief, the rats were anesthetized with intraperitoneal pentobarbital injections ( mg/kg), and the left sciatic nerve was exposed and isolated. three ligations were placed around the nerve with - chromic gut sutures (pudong jinghuan co. ltd., shanghai, china). a hindpaw twitch indicated successful nerve constriction. the muscles and skin overlying the sciatic nerve were then closed with sutures. the sham surgery was identical except for the omission of sciatic nerve ligation. all animals received hypodermic penicillin injections ( . ml/rat; mg/ml) to reduce the risk of infection. to reduce variability, all surgeries were performed by a single proficient investigator. the rats were anesthetized with an intraperitoneal injection of pentobarbital ( mg/kg) and perfused with normal saline (ns), followed by % ice-cold paraformaldehyde in phosphate buffer. the lumbar - segments were removed, post-fixed, and dehydrated in % sucrose at • c. next, -µm freefloating transverse cutting was performed using a freezing microtome at − • c. after blocking with % goat serum for h at room temperature to reduce non-specific binding, the sections were incubated for - h with the following primary antibodies: mouse anti-cxcr ( : dilution, santa cruz biotechnology, dallas, tx, usa), rabbit anti-iba ( : ; abcam, cambridge, uk), rabbit anti-gfap ( : ; proteintech, rosemont, il, usa), and rabbit anti-neun ( : , cell signaling technology, danvers, ma, usa). subsequently, the sections were incubated with an appropriate secondary antibody (fitcconjugated goat anti-rabbit or cy -conjugated goat anti-mouse, both : dilution; beyotime, shanghai, china) for h at room temperature in the dark. fluorescence signals were observed using a fluorescence microscope with appropriate filters. after the intraperitoneal injection of an overdose of pentobarbital, the spinal dorsal horn segments (lumbar - ) were dissected rapidly and stored in liquid nitrogen. frozen samples were homogenized in lysis buffer containing pmsf (beyotime). after centrifugation at , rpm and • c for min, the supernatants were collected as protein samples. sample aliquots containing equal amounts of protein were separated via sds-page and transferred onto polyvinylidene difluoride membranes. the membranes were blocked in % nonfat milk for h at room temperature, and incubated overnight at • c with rabbit anti-cxcr ( : dilution; abcam), rabbit anti-cxcl ( : , ; genetex, irvine, ca, usa), or mouse anti-gapdh ( : , ; proteintech). the membranes were then washed with tbst buffer and incubated with an appropriate secondary antibody (horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit, : , ; beyotime) for h at room temperature. after extensive washing, the densities of labeled protein bands on the blots were detected using an enhanced chemiluminescence reagent (thermo fisher, waltham, ma, usa) and captured using a chemidoc mp system (bio-rad, hercules, ca, usa). to investigate the role of cxcl /cxcr signaling in cciinduced neuropathic pain, rats were randomly divided into the sham surgery (n = - ) and cci groups. subgroups of rats that underwent cci were selected to receive intrathecal antibody or saline injections (n = ). intrathecal administration was performed by lumbar puncture, as described in a previous study ( ) . the rats were anesthetized with isoflurane ( . ml/rat, baxter international inc.; shanghai, china) in a home-made anesthesia box and placed on a plexiglass tube to broaden the intervertebral spaces. a -µl volume of normal saline or a solution containing an anti-cxcl antibody ( ng/rat, proteintech) was injected into the subarachnoid space between the l and l vertebrae with a -gauge needle. an instantaneous and rapid tail-flick indicated a successful puncture. to determine the effect of cxcl /cxcr signaling on the development of hyperalgesia, the first injection of anti-cxcl antibody was administered on experimental day after cci. daily follow-up injections were performed until day (for the behavioral experiment), unless the rats were sacrificed earlier for a bscb permeability evaluation or flow cytometry assay. to determine the effect on established hyperalgesia, the injections were administered on experimental days - after cci. rats that received normal saline injections are herein referred to as the cci + ns group, while those that received anti-cxcl antibody injections are herein referred to as the cci + anti-cxcl antibody group. hyperalgesia was assessed based on paw withdrawal responses to a calibrated series of von frey filaments (stoelting; wood dale, il, usa) as described in one of our earlier publications ( ) . in brief, the rats were individually placed in a chamber ( cm × cm × cm) in which the floor was a customized platform consisting of a grid of iron wires with -mm spacings between wires. the rats were allowed to acclimate to the chamber for ≥ min before the experiment began. a series of von frey filaments with ascending buckling forces were applied to the midplantar surface of the hindpaw ipsilateral to the site of the cci or sham surgery (herein referred to as the ipsilateral hindpaw) and the hindpaw contralateral to the surgical site (herein referred to as the contralateral hindpaw). each von frey filament was held for s, and the interval between filament applications was s. a brisk withdrawal or flinching of the hindpaw upon filament application was regarded as a positive response, and the filament applications continued until a filament produced positive responses in at least three out of five consecutive applications. the paw withdrawal threshold (pwt) was defined as the buckling force (in grams) of that particular filament. pwt testing was performed by an investigator who was blinded to the rats' group assignments. daily pwt testing began on experimental day (baseline) and continued until day after cci or sham surgery. bscb permeability was assessed with the micromolecular tracer dye sodium fluorescein (naflu; molecular weight, g/mol; sigma-aldrich; st. louis, mo, usa) according to a modified version of a published procedure ( ) . in brief, subgroups of rats that underwent sham or cci surgeries were selected to receive intravenous injections of a % naflu solution ( µl per gram of bodyweight) on experimental day . after an intraperitoneal injection of pentobarbital ( mg/kg), naflu was allowed to circulate for min, and the rats' bodies were then intracardially perfused with cold saline to remove intravascular naflu. the l and l spinal cord segments were removed and used for subsequent analyses aimed at quantifying the amount of naflu extravasated from circulation. after being weighed, the spinal cord samples were homogenized in ml of phosphate-buffered saline (pbs), and a volume of % trichloroacetic acid equal to that of the resulting solution was added to precipitate proteins. after being vortexed for min, the samples were cooled for min and centrifuged at , × g for min. the naflu concentration in the supernatant was measured with a spectrophotofluorometer (excitation wavelength, nm; emission wavelength, nm). a calibration curve was created by assaying solutions with controlled naflu concentrations under identical assay conditions. all experimental measurements were within the detection range established with the calibration curve, which had an r -value of . - . . naflu levels were calculated as micrograms per gram of spinal cord tissue. to assess t cell entry into the spinal cord after cci, flow cytometry was used to measure cd -positivity levels in mononuclear cells extracted from the dorsal horn, as cd is a well-known t cell marker ( ) . on day of cci, after an overdose intraperitoneal injection of urethane ( g/kg), the rats' lumbar spinal cord segments were harvested. the dorsal horn tissues ipsilateral to the site of cci or sham surgery were isolated, placed in . -m pbs, and homogenized into single-cell suspensions with a cell strainer. homogenates were washed with . -m pbs, suspended in a %/ % discontinuous-gradient percoll solution (sigma-aldrich), and centrifuged at × g for min. mononuclear cells were collected, washed with . -m pbs, and resuspended in a fluorescence-activated cell sorting buffer solution for min at • c. the cells were labeled with fluorescein isothiocyanate-conjugated mouse anti-cd antibodies ( : dilution; ebioscience, san diego, ca, usa) for min at room temperature, and ≥ , -cell samples were analyzed with the facscalibur platform running with cellquest software (becton dickinson; franklin lakes, nj, usa) to determine the percentage of mononuclear cells that were cd -positive. statistical analyses were performed with prism . software (graphpad software; la jolla, ca, usa). data were expressed as means ± standard errors. behavioral data were analyzed with two-way repeated-measures analysis of variance (anova) followed by bonferroni post-hoc testing. bscb permeability evaluation and flow cytometry data were analyzed with the independent t-test. mann-whitney u-test was used if equal-variance assumptions were not made. statistical significance was defined as p < . . we initially performed immunohistochemistry for specific cell markers to determine the profile of cxcr expression in the spinal cord. the results revealed that cxcr is expressed abundantly in the spinal cord, where it colocalized with neun (neuron marker), but not with gfap (astrocyte marker) or iba (microglia marker; figure ). relative to the sham surgery group rats, the cci group rats had decreased ipsilateral hindpaw pwts on experimental days , , , , and (p < . ; n = , anova, figure a ), indicating cci-induced mechanical hyperalgesia. no significant between-group differences were observed for contralateral hindpaw pwts (figure a) . the cci group rats also had dramatically elevated lumbar spinal cord naflu concentrations on experimental day (p = . ; n = , mann-whitney u-test, figure b ), which suggests that cci increased bscb permeability. compared to the sham surgery group, we observed increased cxcl expression in the ipsilateral spinal cord after cci injury (p = . ; n = , independent t-test, figure a ). the immunohistochemical results revealed that cci induced cxcr activation, as shown in figure b . this was further confirmed by western blotting, which demonstrated that the cxcr protein level was increased from to days after cci (p < . ; n = , anova, figure c ). relative to the rats treated with cci + saline group, rats in the cci + anti-cxcl antibody group exhibited a marked increase in pwts from experimental day to day or from day to (p < . , n = , anova, figures a,b) . this indicates that the anti-cxcl antibody attenuated cciinduced mechanical hyperalgesia in both the developmental and established stages. on the other hand, anti-cxcl antibody dramatically reduced naflu concentrations in the lumbar spinal cord on experimental day (p = . ; n = , independent t-test, figure c) . these results suggest that the cxcl /cxcr signaling pathway is involved in the pathophysiology of cci-induced hypernociception and increased bscb permeability. the percentage of ipsilateral dorsal horn mononuclear cells that were cd -positive was more than two times higher in the cci group rats than in the sham surgery group rats (p < . ; n = - , independent t-test, figures a,b) and was lower in the cci + anti-cxcl antibodies group rats than in the cci + ns group rats (p < . ; n = , independent t-test, figures b,c) . these results suggest that cci promotes t cell entry into the spinal cord, and that blocking the cxcl /cxcr signaling pathway counteracts this effect, which provides further evidence for the cxcl /cxcr signaling pathway being involved in the pathophysiology of cci-induced bscb disruption ( figure d ). in this study, we investigated the putative link between cxcl /cxcr signaling-mediated bscb disruption and neuropathic pain. as in our previous studies, cci group rats exhibited robust post-operative behavioral hypersensitivity to mechanical stimuli, and this hypersensitivity persisted throughout the experimental period ( , ) . we also determined that cci induced cxcl /cxcr signaling, increased bscb permeability, and promoted t cell migration into the spinal dorsal horn. moreover, intrathecal administration of anti-cxcl antibodies attenuated the rats' behavioral hyperalgesia and reduced the cci-induced increases in bscb permeability and t cell infiltration into the dorsal horn. to the best of our knowledge, it is the first study to report that blocking cxcl /cxcr signaling attenuates the increases in bscb permeability and t cell infiltration of the spinal cord induced by peripheral nerve injury. researchers have attempted to unravel the mechanisms underlying cci-induced inflammatory reactions in the spinal cord, and have made considerable progress ( , ) . myriad inflammatory mediators in the spinal cord may contribute to the development of neuropathic pain, with interleukin- , tumor necrosis factor alpha, and c-x-c motif chemokines being possible examples ( , , ) . among the chemokines, cxcl has been identified as a potentially important trigger ( , ) , and our results provide further evidence for its importance. previous studies have indicated that increased bscb permeability is a prerequisite for immune cell infiltration of the spinal cord during the development of neuropathic pain ( ) , and we found that blocking cxcl /cxcr signaling with anti-cxcl antibodies reduced the bscb's permeability to naflu, which suggests that cxcl /cxcr signaling plays a critical role in cci-induced bscb dysfunction. the chemokines cxcl , cxcl , and cxcl compose a subfamily of chemokines that bind to cxcr and have various roles in nociceptive signaling. past investigations have suggested that cxcl /cxcr signaling contributes to the pathophysiology of neuropathic pain, although spinal cxcl and cxcl levels do not seem to have important roles in the development of chronic pain ( , ) . other reports have shown that t cell infiltration of the dorsal horn may contribute to the onset of neuropathic and inflammatory hyperalgesia ( , , ) . our present findings further this line of research by elucidating the spinal cxcr protein level increased during days - after cci as shown by western blotting (**p < . ; ***p < . ; anova, n = ). the data are presented as means ± standard errors. gapdh was used as a loading control. anova, analysis of variance; cci, chronic constriction injury; cxcl , c-x-c motif chemokine ; contra, contralateral to cci. figure | neutralizing cxcl alleviated hyperalgesia and reduced cci-induced bscb disruption. (a,b) hindpaw pwts in cci + anti-cxcl antibodies group and cci + saline group rats on various experimental days (*p < . , **p < . , ***p < . ; anova, n = - ). (c) lumbar spinal cord naflu concentrations in the cci + anti-cxcl antibodies group, cci + saline group, and sham surgery group rats on experimental day (*p < . , cci + ns group vs. sham surgery group; mann-whitney u-test, n = - ; ## p < . , cci + anti-cxcl antibodies group vs. cci + ns group; independent t-test, n = ). the data are shown as means ± standard errors. anova, analysis of variance; bscb, blood-spinal cord barrier; cci, chronic constriction injury; cxcl , c-x-c motif chemokine ; naflu, sodium fluorescein; pwt, paw withdrawal threshold; ns, normal saline. the potential mechanistic role of cxcl /cxcr signaling in the development of neuropathic pain following peripheral nerve injury. our observation that blocking cxcl /cxcr signaling reduced cci-induced t cell migration into the spinal cord is consistent with past reports suggesting that cxcl /cxcr signaling plays a role in the migration of t cells from the periphery into the cns ( ) . within the spinal cord, neurons, and glia can secrete cxcl , which in turn promotes the entry of circulating immune cells into the spinal cord ( ) ( ) ( ) ( ) . studies have shown that type t helper cells secrete interferon gamma, and elevated spinal cord interferon gamma levels can induce cxcl secretion. cxcl in turn increases bscb permeability and promotes the migration of t cells into the spinal cord ( , ) . this creates a positive feedback system that favors ever-increasing migration of activated t cells into the spinal cord. this implies that blocking the contribution of cxcl /cxcr signaling to increased bscb permeability, as we did by administering anti-cxcl antibodies to the cci group rats, may disrupt this positive feedback loop. in conclusion, our study suggests that cxcl /cxcr signaling triggers a positive feedback loop involving bscb permeabilization and t lymphocyte infiltration of the spinal cord. intrathecal administration of anti-cxcl antibodies prevents the development of cci-induced neuropathic pain. our 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expression of cd and cd : skin involvement revealing systemic disease neuroinflammation and the generation of neuropathic pain an inflammation-centric view of neurological disease: beyond the neuron cytokine mechanisms of central sensitization: distinct and overlapping role of interleukin- beta, interleukin- , and tumor necrosis factor-alpha in regulating synaptic and neuronal activity in the superficial spinal cord chemokines, neuronal-glial interactions, and central processing of neuropathic pain chemokine cxcl /cxcr signaling contributes to neuropathic pain in spinal cord and dorsal root ganglia after chronic constriction injury in rats delayed functional expression of neuronal chemokine receptors following focal nerve demyelination in the rat: a mechanism for the development of chronic sensitization of peripheral nociceptors quantifying blood-spinal cord barrier permeability after peripheral nerve injury in the living mouse promoted interaction of c/ebpalpha with demethylated cxcr gene promoter contributes to neuropathic pain in mice spinal cxcl and cxcl are not involved in neuropathic pain despite an upregulation in the spinal cord following spinal nerve injury expression of neuronal cxcl induced by rabies virus infection initiates infiltration of inflammatory cells, production of chemokines/cytokines and enhancement of blood-brain barrier permeability cytokine regulation of cc and cxc chemokine expression by human astrocytes innate stat -dependent genomic response of neurons to the antiviral cytokine alpha interferon induction of the genes for cxcl and cxcl is dependent on ifn-gamma but shows differential cellular expression in experimental autoimmune encephalomyelitis and by astrocytes and microglia in vitro spinal ifn-gamma-induced protein- (cxcl ) mediates metastatic breast cancer-induced bone pain by activation of microglia in rat models c motif) ligand (cxcl) in autoimmune diseases the authors would like to thank editage (www.editage.com) for english language editing. key: cord- -bjs oliw authors: jin, yilin; jia, kuntong; zhang, wanwan; xiang, yangxi; jia, peng; liu, wei; yi, meisheng title: zebrafish trim promotes innate immune response to rgnnv infection by targeting card and rd regions of rig-i for k -linked ubiquitination date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: bjs oliw rig-i-like receptors (rlrs) play important roles in response to virus infection by regulating host innate immune signaling pathways. meanwhile, the rlr signaling pathway is also tightly regulated by host and virus to achieve the immune homeostasis between antiviral responses and virus survival. here, we found that zebrafish trim (zbtrim ) functioned as a positive regulator of rlr signaling pathway during red spotted grouper nervous necrosis virus (rgnnv) infection. post-rgnnv infection, zbtrim expression was obviously inhibited and ectopic expression of zbtrim led to enhanced expression of rlr signaling pathway-related genes. overexpression and knockdown analysis revealed that zbtrim promoted zebrafish rig-i (zbrig-i)-mediated ifn signaling and inhibited rgnnv replication. mechanistically, zbtrim bound to zbrig-i; in particular, the spry domain of zbtrim interacted with the tandem caspase activation and recruitment domains ( card) and repressor domain (rd) regions of zbrig-i. zbtrim promoted the k polyubiquitination of card and rd regions of zbrig-i. furthermore, zbtrim -mediated zbrig-i activation of ifn production was enhanced by k -linked ubiquitin, indicating that zbtrim -mediated zbrig-i polyubiquitination was essential for rig-i-triggered ifn induction. in conclusion, these findings reveal a novel mechanism that zbtrim positively regulates the innate immune response by targeting and promoting the k -linked polyubiquitination of zbrig-i. the innate immune system recognizes pathogen-associated molecular patterns (pamps) by pattern recognition receptors (prrs) as against microbial pathogen invasion ( ). retinoic acid inducible gene-i (rig-i)-like receptors (rlrs), as intracellular prrs, composed of rig-i, mda , and lgp , recognize non-self signatures of viral rnas in the cytosol of cells. after activated by viral rna, rig-i and mda recruited the downstream adaptor molecule, mavs, to their n-terminal caspase-recruitment domains (cards). then, tumor necrosis factor receptor-associated factors (traf) and tank-binding kinase /iκ-b kinase ε interacted with mavs, which in turn leads to the phosphorylation and cytoplasm-to-nucleus translocation of interferon (ifn) regulatory factor (irf ), and the activation of type i ifn. subsequent ifns activated a variety of ifn-stimulated genes (isgs) to limit the virus replication ( ) . nervous necrosis virus (nnv) is a non-enveloped, singlestranded rna virus belonging to the family nodaviridae. increasing evidence has shown that nnv can infect more than fish species and causes mass mortalities of infected fish worldwide ( ) . it has been revealed that rlrs respond in vivo or in vitro to the stimulation of nnv and possess capacities in the induction of ifns and isgs in a variety of fish species. for example, in zf cells, expression of rlrs was significantly enhanced post-nnv infection and rig-i knockdown significantly restrained group ii type i ifn activation ( ) . our previous studies also suggested that rlr signaling pathway was activated during red spotted grouper nervous necrosis virus (rgnnv) infection in sea perch and its key components possessed anti-rgnnv activities ( , ) . however, regulation mechanisms of rlr signaling pathway during rgnnv infection is still unclear. rlr-mediated antiviral signaling pathway is tightly regulated at multiple steps in the signaling cascade. several studies demonstrated that posttranslational modifications, including ubiquitination, isgylation, and phosphorylation, were important mechanisms that regulated the rlr signaling pathway, of which ubiquitination was a key regulatory mechanism for rlr pathway ( ) . for instance, rnf negatively regulated rlr signaling pathway by targeting rig-i ( ) . mda and mavs were targeted for k -linked ubiquitination by trim and rnf , respectively, which induced mda and mavs degradation and rlrs signal termination ( , ) . trim e ubiquitin ligase induced the k linked ubiquitination of rig-i, which activated rlr signaling pathway to elicit host antiviral innate immunity ( ) . trim , an ifn-inducible e ligase, is associated with all kinds of cellular processes, such as the immune response, cancer, and so on ( ) . it is becoming evident that trim has a dual role in rig-i regulation, since trim not only induces k -linked ubiquitination of rig-i to positive regulate rlr signaling activation but also negatively regulates rig-i activation through inhibiting hla-f adjacent transcription degradation, a negative regulator of rig-i-mediated inflammatory response ( ) . multiple fish trim homologs have been reported, including rhodeus uyekii ( ), epinephelus coioides ( ) , and larimichthys crocea ( ) . increasing evidence showed that fish trim was involved in antiviral immunity and played a pivotal role in rlr antiviral signaling pathway ( ) . however, the mechanism by which fish trim regulates rlr signaling pathway has not been explored. in the present study, zebrafish trim (zbtrim ) was involved in rgnnv infection and was identified as a positive mediator of rlr signaling pathway by binding to and ubiquitinating the caspase activation and recruitment domain ( card) and repressor domain (rd) regions of rig-i, which is different with the findings in mammals. our findings reveal a novel mechanism of trim to activate rlr signaling pathway and will help to develop new treatments for viral nervous necrosis disease. all procedures with zebrafish were approved by the ethics committee of sun yat-sen university and the methods were carried out following the approved guidelines. zebrafish wild-type ab line was purchased from china zebrafish resource center. fish were raised with h darkness and h light at • c and were fed with commercial pellets twice a day. all embryos were obtained by natural spawning and staged as previously reported ( ) . zbe cells derived from zebrafish embryos were cultured at • c as previously described ( ) . hek t cells were cultured in dmem (invitrogen) enriched with % fbs (invitrogen) at • c under a humidified atmosphere of air containing % co . rgnnv was propagated in zbe cells and stored at − • c until use. anti-flag (m ), anti-myc (m ), anti-his (m l), and anti-ha antibodies (m ) were purchased from abmart. anti-α-tubulin (ab ) and anti-gfp antibodies (g ) were purchased from abcam and sigma, respectively. goat anti-rabbit igg-hrp, goat anti-mouse igg-hrp, alexa fluor -labeled goat anti-mouse igg, and alexa fluor -labeled goat anti-rabbit igg secondary antibodies were purchased from invitrogen. for in vitro infection, zbe cells were challenged with rgnnv [multiplicity of infection (moi) = ] for , , and h, respectively. subsequently, rna from cells was extracted to detect the expression of zbtrim mrna by quantitative realtime pcr (qrt-pcr). for in vivo infection, rgnnvs ( tcid /ml) were injected into the egg yolk of embryos at the single-cell stage in the experimental group. in the mock group, embryos were injected with dmem. a total of nl of solution was microinjected into each embryo using a microinjector. rna from zebrafish embryo was extracted to detect the expression of zbtrim mrna by qrt-pcr at h post-injection. knockdown of zbtrim by sirna zbtrim sirna ( ′ -gaatccagttgaagagaaa- ′ ) and control sirna were synthesized by ribobio company (guangzhou, china). zbe cells were transfected with zbtrim sirna or control sirna according to the manufacturer's protocol using lipofectamine as previously described ( ) . twenty-four hours after transfection, zbe cells were infected with rgnnv (moi = ) for h and total rnas were extracted for qrt-pcr analysis. the orf of zbtrim (genbank accession no. nm . ) was sub-cloned into pcmv-flag or pcmv-myc vectors (invitrogen) to generate recombinant plasmid pcmv-flag-zbtrim or pcmv-myc-zbtrim , respectively. full-length zbrig-i and zbrig-i deletion mutant cdnas encoding amino acids - (zbrig-i- card), - (zbrig-i- card), - (zbrig-i-rd), and - [zbrig-i-( card+rd)] were inserted into the pegfp-n vectors. full-length zbrig-i was inserted into the pet- a(+) (clontech) vector to generate recombinant plasmid pet- a(+)-zbrig-i. zbtrim deletion mutant zbtrim -spry and zbtrim -spry were generated using the pcmv-flag-zbtrim plasmid as a template. primers used for amplifying these genes are listed in table s . ha-k ub plasmid was purchased from rebio (shanghai, china). rna extraction and cdna synthesis were performed using trizol (invitrogen) and primescript tm st strand cdna synthesis kit (takara) according to the manufacturer's instructions. qrt-pcr analyses of zbtrim , zbrig-i, rna dependent rna polymerase (rdrp), rlr signaling pathway related genes (mavs, traf , irf , and ifn ), and isg were performed as previously described ( ) . relative expression levels of target genes were normalized to s rrna using − ct methods. data represent the mean ± sd from three independent experiments, each performed in triplicate. primers sequences used for qrt-pcr are listed in table s . hek t cells, pre-seeded in -well plates, were transfected with ng of pgl -drifn -pro-luc plasmid or pgl -basic empty vector with ng of prl-tk vector (promega) together with pcmv-myc-zbrig-i or pcmv-myc and pcmv-flag or pcmv-flag-zbtrim ( ng per well) for h. then, cells were incubated with poly i:c for h and lysed. luciferase activities were measured using the dual-luciferase reporter assay system (promega). relative luciferase activities were expressed as the ratio of firefly to renilla luciferase activity. the results were the representative of three independent experiments in triplicate. hek t cells, pre-seeded in -well plates, were transfected with ng of pgl -drifn -pro-luc plasmid or pgl -basic empty vector with ng of prl-tk vector (promega). meanwhile, pcmv-myc-zbtrim , mutant zbrig-i or empty control plasmids were co-transfected. after being incubated with poly i:c for h, cells were lysed for luciferase assay as described above. at least three independent experiments were performed. hek t cells, seeded on glass cover slips, were transfected with pcmv-myc-zbtrim and pcmv-flag-zbrig-i plasmids. twenty-four hours post-transfection, cells were washed with pbs three times and fixed with prechilled methanol and then permeabilized using % triton x- in pbs for min and blocked with % normal goat serum for min at room temperature (rt). cells were incubated with anti-myc and anti-flag antibodies for min at rt. finally, cells were washed with pbs and incubated with the appropriate alexa fluor or conjugated secondary antibodies for h. after cell nucleus was stained with hoechst , cells were observed by a confocal microscope (zeiss, germany). co-ip and western blotting experiments were performed as described previously ( ) . hek t cells in -cm flasks were co-transfected with µg of different plasmid combinations for h. then, the cells were lysed on ice with lysis buffer for min and were immunoprecipitated with the indicated antibodies. the precipitated samples and whole-cell lysates (input) were analyzed by immunoblotting with the indicated antibodies. escherichia coli bl (de ) cells were transformed with pet- a(+)-zbrig-i or pet- a(+) plasmids, respectively. then, cells were grown in ml of lb medium (beyotime) containing . mm isopropyl- -thio-β-d-galactopyranoside (iptg) (sigma) at • c overnight with shaking at rpm. cells were pelleted by centrifugation at , rpm for min and lysed in ml of lysis buffer ( mm sodium phosphate, ph . , mm nacl, and . % tween- ) (beyotime) via sonication on ice. the sonicated mixture was centrifuged at , rpm at • c for min, and then the supernatant was affinity-purified with dynabead his-tag magnetic beads (invitrogen) according to the manufacturer's instruction. his pull-down assays were performed as described previously with some modifications ( ) . his-zbrig-i-magnetic beads were incubated with the lysates of hek t cells transfected with pcmv-flag-zbtrim or pcmv-flag empty vectors on a roller, respectively. after incubation at • c overnight, the magnetic beads were washed three times with lysis buffer to remove unbound his-zbrig-i and then analyzed via western blotting using anti-flag or anti-his antibodies. his tag protein alone was served as a negative control. ubiquitination assays were performed as described previously with some modifications ( ) . hek t cells, pre-seeded in -cm flasks overnight, were co-transfected with µg of different plasmid combinations. cells were lysed at h after transfection, and then gfp-zbrig-i mutants were immunoprecipitated with anti-gfp antibodies as described above. immunoprecipitates or input were analyzed by immunoblotting with the indicated antibodies. all statistics were calculated using spss version . differences between control and treatment groups were assessed by one-way anova. p < . is considered statistically significantly different. p < . was considered highly significant. as shown in figure a , mrna level of zbtrim was downregulated within h after rgnnv infection. meanwhile, we also investigated the expression of zbtrim in rgnnvinfected zebrafish embryos at h, and the results were concordant with zbe cells (figure b) . these data indicated a potential role of zbtrim in innate immune response to rgnnv infection. in mammals, trim has been suggested to promote ifnβ production by functioning as a key upstream activator of rig-i to activate the rlr signaling pathway ( ) . to investigate whether zbtrim regulated rlr signaling pathway in zebrafish, the expression of several rlr signaling pathwayrelated genes was measured in zbtrim overexpressing zbe cells. as shown in figure a , overexpression of zbtrim markedly enhanced the expression of rig-i, mavs, traf , irf , ifn , and isg during rgnnv infection. similar results were detected in zbtrim overexpressing zbe cells treated with poly i:c ( figure b) . furthermore, coexpression of zbtrim with zbrig-i induced a dosedependent increase in ifn activation compared with the zbrig-i overexpression alone (figure c) . overexpression of zbtrim dose-dependently inhibited rgnnv replication ( figure d) . on the contrary, knockdown of zbtrim using sirna increased the level of rdrp in rgnnv-infected zbe cells (figures e,f) . all these results demonstrate that zbtrim is a positive regulator of rlr signaling pathway and functions as an antiviral factor during rgnnv infection in zebrafish. to elucidate the mechanism by which zbtrim participates in rlr signaling pathway in zebrafish, the interaction of zbtrim and zbrig-i was investigated. we co-expressed myc-zbtrim and flag-zbrig-i plasmids in hek t cells, and immunofluorescence imaging showed zbtrim and zbrig-i colocalized in the cytoplasm of hek t cells (figure a) . co-ip against the myc tag revealed that zbtrim could interact with full-length zbrig-i but not with flag-vector ( figure b) . his pull-down analysis showed that zbtrim was directly bound to zbrig-i ( figure c ). all these data suggest that zbtrim interacts with zbrig-i. to identify the region involved in the zbrig-i/zbtrim interaction, firstly, zbrig-i deletion mutants [pegfp-zbrig-i- card, pegfp-zbrig-i- card, pegfp-rig-i-rd, and pegfp-rig-i-( card+rd)] were constructed and cotransfected with flag-zbtrim in hek t cells ( figure a) . zbrig-i- card, zbrig-i- card, and zbrig-i-rd could bind to zbtrim individually (figures b-d) ; however, zbrig-i-( card+rd) failed to co-precipitate with zbtrim ( figure e) . these results indicate that zbrig-i binds to zbtrim through its n-terminal card region and the c-terminal rd region. furthermore, we constructed two truncations of zbtrim (zbtrim -spry and zbtrim -spry) co-transfected with zbrig-i- card or zbrig-i-rd in hek t cells, respectively ( figure f) . we found that the spry domain of zbtrim interacted with card and rd regions of zbrig-i (figures g-j) . collectively, these results indicate that the spry domain of zbtrim is responsible for its interaction with card and rd regions of zbrig-i. to investigate whether the e ligase activity of zbtrim is involved in the regulation of zbrig-i, the ubiquitination of zbrig-i was tested in zbtrim overexpressing cells. we found that zbtrim markedly promoted the k polyubiquitination of zbrig-i ( figure a) . furthermore, hek t cells were transfected with flag-tagged zbtrim , zbrig-i deletion mutants [gfp-zbrig-i- card, gfp-zbrig-i-( card+rd), and gfp-zbrig-i-rd], and ha-tagged k ubiquitin, and our results showed that zbtrim obviously enhanced the ubiquitination of zbrig-i- card and zbrig-i-rd (figures b,c) , but not zbrig-i-( card+rd) (figure d) . these data suggest that zbtrim ubiquitinates both n-terminal card and c-terminal rd regions of zbrig-i. it has been reported that ubiquitination of rig-i by trim is vital for ifn signaling. thus, the effect of zbtrim -mediated zbrig-i ubiquitination on zbrig-i's ifn-inducing activities was assessed. our results showed that ectopic expression of zbrig-i- card and zbrig-i-rd could enhance ifn promoter activity (figure a) , and this activation was markedly enhanced by zbtrim overexpression (figures b,c) . furthermore, overexpression of k -linked ubiquitin dose-dependently increased the promotion effect of zbtrim on zbrig-i- card and rd mediated ifn promoter activation (figures b,c) . these data confirm the importance of zbtrim -mediated k ubiquitination in the n-terminal card region and c-terminal rd region of zbrig-i for zbrig-i-mediated ifn induction. rlr signaling pathway plays crucial roles in recognizing viral infections and initiating the antiviral immune response. rig-i, as an important component of rlr signaling pathway, can detect viral dsrnas in the cytoplasm and induce type i ifn production and the secretion of pro-inflammatory cytokines to suppress virus spread during virus infection ( ) . multiple studies have demonstrated that the ubiquitination of rig-i plays an important role in the rig-i-mediated antiviral signaling pathway. for instance, trim , trim , and mex c positively regulate rig-i-mediated signaling by targeting rig-i for k linked polyubiquitination ( , ) . trim , well-known as an ubiquitin e ligase and an isg e ligase, is widely involved in the regulation of innate immunity ( , ) . in mammals, previous reports showed that trim enhanced rlrs antiviral pathway by binding viral rna-activated rig-i to induce its k -linked polyubiquitination and subsequent ifns and isgs production ( ) . in teleost fish, several trim homologs were reported to play a pivotal role in innate immunity ( , ) ; however, the mechanisms by which fish trim modulates the innate immune response against viruses remain elusive. here, we found that zbtrim positively regulated rlr signaling pathway and facilitated zbrig-i-mediated ifn promoter activation, and overexpression of zbtrim inhibited rgnnv infection, indicating the conservative antiviral properties of trim in fish and mammals. several reports showed that trim was involved in the regulation of antiviral innate immunity by targeting rig-i ( , ) . the mammal rig-i protein contains two n-terminal cardlike domains, a c-terminal rd region and an rna helicase region ( ) . in zebrafish, rig-ia (an insertion variant of rig-i) and rig-ib (the typical rig-i) were identified as two transcripts of rig-i, and overexpression of rig-ib in cultured fish cells, but not rig-ia, activated zebrafish type i ifn and induced antiviral response ( ) . thus, in this report, we investigated the interaction of zbtrim and zbrig-i (rig-ib), and our results showed that zbtrim was directly associated with zbrig-i and especially the card or rd region of zbrig-i was sufficient for its interaction with zbtrim . trim is characterized by an n-terminal region containing a catalytic ring domain, one or two b-box domains, a coiled-coil dimerization domain, and a c-terminal spry domain ( ) . among these domains, spry was associated with protein-protein interactions and/or rna binding ( ) . gack et al. reported that the c-terminal spry domain of trim interacted with the first card of rig-i, but not the helicase region and rd of rig-i, and this interaction delivered the k -linked ubiquitin moieties to the second card of rig-i, which facilitated the dimerization of rig-i and subsequent interaction with mavs to induce antiviral signal transduction ( ) . we further investigate whether the spry domain of zbtrim was responsible for its interaction with zbrig-i. unlike previous studies, we found that the spry domain of zbtrim interacted not only with card but also with rd regions of zbrig-i. in non-infected cells, rd covered the rna-binding and helicase domains and cards folded over one another, which made rig-i to exist in an auto-repressed conformation. upon virus infection, viral rnas interacted with the rd and the helicase domain of rig-i, which in turn exposed the cards for mavs interaction, thereby triggering antiviral responses ( , ) . considering the interaction between rd of rig-i and viral rnas, we speculated that the interaction of zbtrim and zbrig-i rd might inhibit zbrig-i sensing of viral rnas. meanwhile, it has been known that card domains of rig-i are widely involved in its interaction with other proteins, such as mavs, trim , and virus proteins ( , , ) . thus, the interaction of zbtrim and zbrig-i rd might also make room for other proteins to bind to card of zbrig-i, zbtrim , and other proteins and will work cooperatively in regulation of rlr signaling pathway. the differences between the findings for zbtrim and trim in mammals indicate that zbtrim may regulate rlr signaling pathway in various ways. ubiquitination is a vital post-translational modification for the modulation of rig-i activity. several e ubiquitin ligases that mediate k -linked ubiquitination of rig-i for its activation have been identified. for instance, mex c overexpression caused the k -linked ubiquitination of rig-i- card but not rig-i- card, and lysines , , and of rig-i were required for rig-i ubiquitination by mex c ( ) . rnf mediated the k -linked polyubiquitination of rig-i-rd, and lysines and residues of rig-i were crucial for rnf -mediated ubiquitination ( ) . in contrast to rnf , trim mediated the k -linked polyubiquitination of rig-i- card, but not rig-i- card, and the lysine residue of rig-i was critical for efficient trim -mediated ubiquitination and the ability of rig-i to activate antiviral signal transduction ( ) . our results indicated that zbtrim mediated k -linked polyubiquitination of both card and rd regions of zbrig-i, which is distinct from the findings in mammals that trim only targeted and promoted the k -linked polyubiquitination of rig-i card. in addition, our reporter analysis showed that overexpression of zbrig-i- card led to the activation of ifn promoter, which is similar with other reports ( ) . overexpression of zbrig-i-rd also resulted in the activation of ifn promoter. furthermore, k -linked ubiquitin is essential for the zbtrim -mediated enhancement of zbrig-i card and rd-dependent ifn promoter activation. zbrig-i possessed capacities in the induction of ifns and isgs to enhance the antiviral response ( ) . taken together, these findings suggest that zbtrim mediated ubiquitination of card and rd regions of zbrig-i is crucial for its antiviral innate immune response. however, due to the lack of trim or rig-i-knockout zebrafish, we cannot assess the impact of the zebrafish trim /rig-i pathway at the in vivo level. a recent study demonstrated that zebrafish rnf also interacted with and ubiquitinated zbrig-i ( ) . further studies will be performed to determine the precise architecture of the zebrafish trim /rnf /rig-i protein complex and the mechanism by which zbtrim and zbrnf worked together to regulate ubiquitination of zbrig-i. it was known that several virus proteins could positively or negatively regulate rlr signaling pathway by targeting its key components or regulatory proteins ( ) . for instance, paramyxovirus v proteins interacted with the rig-i/trim regulatory complex and inhibited rig-i signaling ( ) . influenza a virus ns protein bound to trim to block ubiquitination of the rig-i ( ) . severe acute respiratory syndrome nucleocapsid inhibited trim -mediated rig-i ubiquitination, causing the inhibition of ifn production ( ) . the rgnnv genome encodes a structural (capsid protein, cp) and a nonstructural (rna-dependent rna polymerase, rdrp) protein ( ). huang et al. reported that rdrp from ognnv induced ifn by activating irf , the key regulatory component of rlrs-ifn signaling ( ) , indicating that rdrp might be a positive rlr signaling pathway. whether rdrp targets the key components of rlr signaling pathway to exert its positive regulation role is a question that deserves further research. in addition, some mirnas could target critical regulatory proteins of rlr pathway for immune evasion ( , ) ; whether rgnnv infection-related mirna was also involved in the regulation of rlr signaling pathway needs to be further investigated. in summary, zbtrim is identified as a positive regulator of rlr signaling pathway by targeting zbrig-i. the spry domain of zbtrim is required for its interaction with card and rd regions of zbrig-i. zbtrim promotes k polyubiquitination of both zbrig-i card and rd regions, which subsequently induces the activation of downstream signaling event via mavs and thereby inhibits viral infection (figure ) . these findings represent a new mechanism underlying the regulation of rlr signaling pathway. all datasets generated for this study are included in the article/supplementary material. the animal study was reviewed and approved by the ethics committee of sun yat-sen university. shaping the landscape of host immunity regulation of rlr-mediated innate immune signaling -it is all about keeping the balance understanding the interaction between betanodavirus and its host for the development of prophylactic measures for viral encephalopathy and retinopathy rig-i specifically mediates group ii type i ifn activation in nervous necrosis virus infected zebrafish cells identification and characterization of the melanoma differentiation -associated gene in sea perch, lateolabrax japonicus characterization and expression analysis of laboratory of genetics and physiology gene in sea perch rnf suppresses antiviral type i interferon production by targeting rig-i cards to mediate rig-i degradation trim is a negative regulator of mda -mediated type i interferon production the e ubiquitin ligase rnf targets virus-induced signaling adaptor for ubiquitination and degradation trim ringfinger e ubiquitin ligase is essential for rig-i-mediated antiviral activity trim in the regulation of the antiviral innate immunity. front immunol ubiquitin-like modifier fat attenuates rig-i mediated antiviral signaling by segregating activated rig-i from its signaling platform molecular characterization of tripartite motif protein (trim ) involved in erα-mediated transcription in the korean rose bitterling rhodeus uyekii ring domain is essential for the antiviral activity of trim from orange spotted grouper the two trim isoforms were differentially induced in larimichthys crocea post poly (i:c) stimulation. fish shellfish immun stages of embryonic development of the zebrafish establishment of a cell line with high transfection efficiency from zebrafish danio rerio embryos and its susceptibility to fish viruses mandarin fish caveolin interaction with major capsid protein of infectious spleen and kidney necrosis virus and its role in early stages of infection interferon regulatory factor from sea perch (lateolabrax japonicus) exerts antiviral function against nervous necrosis virus infection the eukaryotic translation initiation factor subunit e binds to classical swine fever virus ns a and facilitates viral replication ubiquitination is essential for avibirnavirus replication by supporting vp polymerase activity rig-i-like receptor regulation in virus infection and immunity pivotal role of rna-binding e ubiquitin ligase mex c in rig-i-mediated antiviral innate immunity trim modulates type i interferon induction and cellular antiviral response by targeting rig-i for k -linked ubiquitination the ubiquitin-specific protease usp promotes rig-i-mediated antiviral signaling by deubiquitylating trim mechanism of trim catalytic activation in the antiviral rig-i pathway paramyxovirus v proteins interact with the rig-i/trim regulatory complex and inhibit rig-i signaling structural features of influenza a virus panhandle rna enabling the activation of rig-i independently of '-triphosphate higher antiviral response of rig-i through enhancing rig-i/mavs-mediated signaling by its long insertion variant in zebrafish trim/rbcc, a novel class of 'single protein ring finger' e ubiquitin ligases structure and function of the spry/b . domain proteins involved in innate immunity structural insights into rna recognition and activation of rig-i-like receptors the structural basis of ' triphosphate double-stranded rna recognition by rig-i c-terminal domain the e ubiquitin ligase trim attenuates antiviral immune responses by targeting mda and rig-i. cell rep identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf riplet/rnf , a ring finger protein, ubiquitinates rig-i to promote interferon-beta induction during the early phase of viral infection involvement of zebrafish rig-i in nf-kappab and ifn signaling pathways: insights into functional conservation of rig-i in antiviral innate immunity retinoic acid-inducible gene i (rig-i)-like receptors (rlrs) in fish: current knowledge and future perspectives rnf is a positive regulator of ifn expression and involved in rig-i signaling pathway by targeting rig-i. fish shellfish immun influenza a virus ns targets the ubiquitin ligase trim to evade recognition by the host viral rna sensor rig-i the severe acute respiratory syndrome coronavirus nucleocapsid inhibits type i interferon production by interfering with trim -mediated rig-i ubiquitination protein a from orange-spotted grouper nervous necrosis virus triggers type i interferon production in fish cell downregulation of microrna mir- a by enterovirus inhibits rig-i-dependent innate immune response microrna- a feedback inhibits rig-i-dependent type i ifn production in macrophages by targeting traf , irak , and irak yj and kj performed all experiments with assistance from yx, wz, pj, and wl analyzed data. kj and my conceived the study and designed experiments. kj and my wrote the manuscript. all authors read and approved the final manuscript. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © jin, jia, zhang, xiang, jia, liu and yi. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -ss vdpox authors: wang, shenghua; geng, na; zhou, dong; qu, yi; shi, mengke; xu, yuliang; liu, kangping; liu, yongxia; liu, jianzhu title: oral immunization of chickens with recombinant lactobacillus plantarum vaccine against early alv-j infection date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: ss vdpox in this study, a novel oral vaccine of recombinant lactobacillus plantarum (l. plantarum) containing the gp protein was explored, and the effects of this vaccine on the prevention of subgroup j avian leukosis virus (alv-j) infection were assessed. in the current study, the gp protein of alv-j was expressed on the surface of l. plantarum with the surface-display motif, pgsa, by constructing a shuttle vector pmg e:pgsa:gp . surface localization of the fusion protein was verified by western blotting and flow cytometry. subsequently, specific pathogen free hy-line brown layer chickens were orally vaccinated with the recombinant l. plantarum and presented with high levels of serum immunoglobulin g (igg) and secretory immunoglobulin a (siga) titers in bile and duodenal-mucosal fluid. after challenged with alv-j of a × ( ) % tissue culture infective dose (tcid ), serum samples of the chickens were collected and viremia was analyzed. results showed that, compared to the l. plantarum and pbs control group, the recombinant l. plantarum group showed a significant rise in antibody levels after inoculation, and provide improved protection against alv-j according to viremia detection. these results indicate that oral immunization with the recombinant l. plantarum provided an effective means for eliciting protective immune response against early alv-j infection. the j subgroup avian leukosis virus (alv-j) is a subgroup of retrovirus that was first reported and isolated from broiler chickens in the united kingdom in the early s ( ) . however, numerous cases of alv-j infection and tumors in commercial layer chickens and broiler breeders have emerged in recent years ( ) ( ) ( ) ( ) . this resulted in serious economic losses to breeding farms, including growth retardation, high mortality, tumor production and cost for eradication. therefore, here is an urgent need to take measures to protect chickens against alv-j infection. as we know, alv-j spreads via vertical and horizontal infections, with horizontal transmission accounting for most alv-j infections during the brood period ( ) . vertical transmission from broiler breeders to progeny is frequent with alv-j ( ); this is due to the current lack of an effective vaccine against alv. consequently, researchers have been trying to explore a vaccine that will increase the proportion of positive antibodies against alv through incubating breeder flocks, thereby reducing the chance of infection from infected chickens and improving the resistance of chickens by increasing alv maternal antibodies to later infection. however, the inactivated vaccine has no significant effect, because the antigenicity of the inactivated virus is commonly destroyed ( ) . furthermore, considering the complexity of alv, the existing vaccine did not work well ( , ) . the glycoprotein (gp) with mr of , (gp ) is encoded by the envelope (env) gene of alv, and that the gp protein forms globular structures on the surface of the virus, and is closely associated with viral neutralization which determines the antigenicity of alv-j ( ) . previous researches have shown that the protein could be used as a vaccine to help alv-j infect susceptible cells and induce the production of specific antibodies among infected chickens ( , ) . therefore, this study set out to search for a live vehicle that could carry the surface protein gene gp to prevent the alv-j infection. the mucosal immune barrier is the first line of defense of the immune system, and mucosal inoculation is a practical, noninvasive and efficacious method for the simultaneous induction of systemic and mucosal immune responses ( , ) . oral vaccination is more favored and convenient than other routes of mucosal vaccination ( ) ( ) ( ) . the edible lactic acid bacteria, lactobacillus plantarum, is well-known as a safe candidate live vector vaccine to deliver antigen ( ). many recent investigations have analyzed and validated the potentiality of recombinant lactic acid bacteria for the delivery of heterologous antigens to the mucosal immune system ( , , ( ) ( ) ( ) . poly-γ-glutamate synthase a (pgsa) is a constituent protein of bacillus subtilis polyglutamate synthetase (pga), which can be used as a bacterial surface display element to immobilize the enzyme system on the surface of the cell membrane. in view of the characteristics of pgsa protein, it has been applied to various prokaryotic proteins on the surface display, in particular the lactic acid bacteria and other gram-positive receptor strains ( ) . these findings provided a theoretical basis for studying the process of immobilizing exogenous proteins on the cell wall of l. plantarum. in the current study, the recombinant l. plantarum harboring pmg e-pgsa-gp was constructed using genetic engineering technology, and then expressed on the surface of l. plantarum. the live recombinant l. plantarum was then used to orally vaccinate chickens. igg and iga antibodies against alv-j, as well as viremia were detected to assess the effect of the recombinant l. plantarum. the alv-j-nx strain, pmd t-env recombinant vector (containing the gp gene), gp -specific mouse monoclonal anti-body (mab je ), alv-j antibody test kit, and alv p antigen enzyme-linked immunosorbent assay (elisa) test kits (idexx usa inc., beijing, china) were donated by prof. zhizhong cui. the alv iga antibody test kit was purchased from lanpai biotechnology company (shanghai, china). the pgsa gene and purified gp protein were stored in our laboratory. the pmg e expression vector was obtained commercially (invitrogen, shanghai, china). l. plantarum hq was purchased from china center of industrial culture collection (cicc) and grown anaerobically at • c, without agitation in mrs broth medium ( g, hopebiol, qingdao, china). the e. coli strains were cultured at • c with continuous shaking in luria-bertani (lb) medium ( g, hopebiol). hy-line brown layer chickens ( -day-old) were purchased from the hylan breeder company (shandong province, china) and housed in a specific-pathogen-free environment at the laboratory animal and resources facility, shandong agricultural university. the animals had free access to water and commercial standard pellet diet from liuhe jingwei farming and animal husbandry co., ltd. (taian, china). each chicken was confirmed negative for the alv-j antibody and virus by alv-j-antibody elisa initiation of the experiment. the experimental procedure was approved by the animal care and use committee of the shandong agricultural university and performed in accordance with animal welfare and ethics guidelines (sdaua- - ). the primers were designed according to the bacillus subtilis pgsa genes, complete cds sequence published in genebank (number ab . ) by primer . and synthesized in sangon biotech (shanghai, co. ltd). the forward and the reverse primer of the pgsa gene was ′ -cgagctcgcgaactgagctttcatgaaaag- ′ and ′ -ct agtctagactatgatcaatatcaaacgtca- ′ , containing the saci and xbai sites (underlined) respectively, with the plasmid t -pgsbca as the template. pcr amplification was performed as follows: • c for min, cycles of • c for s, • c for s and • c for s, and • c for min of final extension. the pcr product was confirmed by dna sequencing. the gp gene was amplified using the forward primer ( ′ -tcatctagagggagttcatctgttg- ′ ) and the reverse primer ( ′ -tccaagcttattagcgcctgctac- ′ ) containing the xbai and hind iii sites (underlined), respectively, with the pmd t-env as the template. pcr amplification was performed as follows: • c for min, cycles of • c for s, • c for s and • c for min, and • c for min of final extension. the pcr product was confirmed by dna sequencing. electroporation was performed according to the method of josson et al. ( ) . briefly, electrotransformation was performed in a . cm cuvette which was subjected to one single electric pulse ( . kv/cm, , µf) using a gene pulser (bio-rad, richmond, ca, usa). recombinant strains were selected on mrs medium plates with µg/ml of erythromycin (ery; sigma, st. louis, mo, usa), which was incubated anaerobically at • c for h. the recombinant l. plantarum containing the plasmid of the pmg e-pgsa-gp was identified via antibiotic selection and pcr experiments using the primers pgsa and gp . sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) and western blotting were used to analyze the expression of the target protein, and western blotting was developed using the gp -specific mouse monoclonal antibody je as the primary antibody. horseradish peroxidase (hrp)-conjugated goat antimouse igg (sigma, : , ) was used as the secondary antibody. for flow cytometry, l. plantarum cells were cultured in mrs broth overnight at • c. the cell pellets were sequentially incubated with gp -specific mouse monoclonal antibody ( : ) and fluorescein isothiocyanate (fitc)-conjugated antimouse igg secondary antibodies ( : , ; sigma). finally, × cells were analyzed with a facs calibur platform (becton dickinson, oxnard, ca, usa) equipped with cellquest software. the layer chickens ( in total) were randomly divided into three groups: control group, recombinant l. plantarum group and natural l. plantarum group. each group contained chickens that were kept in separated isolators receiving filtered positive-pressure air. the recombinant l. plantarum group was immunized intragastrically with recombinant strains of l. plantarum (harboring plasmid pmg e-pgsa-gp ), while the chickens in the control and negative control group were inoculated with pbs and natural l. plantarum, respectively. the suspensions of strains for administration were prepared as follows: recombinant strains cultured overnight were collected by centrifugation at , × g for min, washed three times with sterile pbs, and then resuspended in sterile pbs to a concentration of × cfu/ml ( µl/chicken). the control group and negative control group received equal doses of sterile pbs and natural l. plantarum. the immune protocol was administered on five consecutive days (days - ). a booster immunization was given between days to , the second booster was given between days to and the third booster was given between days - . blood samples were collected from the wing vein on days (before immunization), , , , , , and . blood samples without additives were centrifuged and the serum was stored at − • c for subsequent analysis. to obtain bile and intestinal lavages samples, chickens were randomly selected from each group, and were killed on day (pre-immune), , , , , , and . moreover, using a previously described method based on that of wu and russell ( ) , intestinal lavage fluids were obtained by flushing the excised small intestine with ml of pbs containing mm ethylenediaminetetraacetic acid (edta) and . mg /ml of soybean trypsin-chymotrypsin inhibitor (sigma). the contents were collected and retained on ice for processing, whereupon the fluids were vortexed and centrifuged at × g for min at • c. a µl volume of mm phenylmethylsulfonyl fluoride (pmsf, sigma) was added to the supernatants before they were vortexed and spun at , × g for min at • c. a further µl of pmsf, µl of fetal bovine serum (fbs), and µl of % sodium azide (sigma) were added to the supernatants before they were dispensed into aliquots and then stored at − • c. the anti-alv-j antibody in serum, bile and duodenal lavages were analyzed by elisa. briefly, the titers of igg were tested using an alv-igg elisa kit (idexx usa inc., beijing, china) according to the requirements of the specification. the optical density (od) of the igg were measured at nm and determined by the sample-to-positive (s/p) method following the formula below: s/p = [(mean of od nm of sample-mean of od nm of negative control)]/[(mean of od nm of positive control-mean of od nm of negative control)]. the samples from each group were tested in three parallel trials, and the s/p values of any sample higher than . were considered as alv-j antibody positive. furthermore, the commercial alv-iga antibody test kit (lanpai bio., shanghai, china) was used to detect the positive ratio of the anti-alv-j iga in serum, bile and duodenal lavages according to the manufacture's protocol. the od of the iga was measured at nm and determined by the critical value (cut off) method following the formula below: cut off = (mean of od nm of sample) + . . the samples from each group were tested in three parallel trials, and the mean of od nm of each sample higher than cut off were considered to be alv-j antibody positive. in our study, the test was considered effective if the mean of the negative control wells was < . and the mean of the positive control was > . . indirect elisa assay was used to assess the titers of anti-gp protein antibody. a -well elisa plate was coated overnight with gp protein of appropriate concentration ( µg/ml) and kept at • c. after blocking of the plates, the samples of serum, bile and duodenal lavages were used as the primary antibody and incubated at • c for h. then hrp-conjugated goat antichicken siga at a dilution of : , (sigma) at • c for h was used to detect bound antibodies. following washing the plate thice with pbs- . % tween , tetramethylbenzidine (qiagen, germany) was added as the colorimetric substrate. finally, the optical density (od) was measured at nm. alv-j nx strain was selected as the challenge virus and transfected into chicken embryo fibroblasts (cef) for amplification and detection of virulence. the tcid of alv-j was judged following the method described by fadly ( ) . all of the chickens were challenged intraperitoneally with × tcid of the alv-j nx strain at day . the plasma samples from the chickens were aseptically collected and then inoculated into the cef cells in -wells plates. the cell supernatant of viremia was collected weekly to check for the presence of the virus using alv p antigen elisa test kits (idexx usa inc.). the relative antigen titer level was determined by calculating the s/p ratio using the formula mentioned above. the samples from each group were tested in triplicate, and plasma samples with s/p ratios higher than . were considered virus-positive. statistical analysis was performed using the spss software (version . , spss inc., usa). meanwhile, one-way anova was used to identify the significant values. all values were expressed as mean ± standard error of the mean (sem). all measurements were replicated three times. the differences were considered significant when the p-value was < . . the amplification of the gp gene from the pmd t-env recombinant vector (expected size bp) is shown in figure a . the amplification of the pgsa gene from the t -pgsbca recombinant vector (expected size , bp) is shown in figure b . the recombinant gene pgsa-gp was expressed in l. plantarum, and confirmed by sds-page (figure a) and western blotting using the gp -specific mab (figure b) . flow cytometry was used to analyze the cell surface display of l. plantarum (figure a) . the cell surface-displayed pgsa-gp was performed using mouse je antibody as the primary antibody and fitc-conjugated goat anti-mouse igg as the secondary antibody. l. plantarum and l. plantarum cells harboring the plasmid pmg e-gp were used as control for flow cytometry. the cells displaying pgsa-gp showed a significantly greater intensity of fluorescence signals than the control cells. this result is consistent with the data shown in figure b . one week after vaccination, the bw of chickens in recombinant l. plantarum group were significantly higher than control group at , , , , and d (p < . ). meanwhile, bw of chickens in the natural l. plantarum group were also higher than control, but without significant differences to the recombinant group. results showed that the recombinant pmg e-pgsa-gp l. plantarum significantly triggered specific igg and iga antibodies against alv-j, and enhanced the levels of igg and siga compared to the control group (figures , ) . the level of systemic igg in serum increased significantly after oral inoculation as shown in figure . the sera of chickens in the orally immunized recombinant l. plantarum group were taken at random intervals each weekly after day (pre-immune) and then analyzed by elisa. a low level of igg titers was detected after the primary immunization. the igg level increased slowly after the first booster immunization, and increased statistically after the second booster immunization on day ( weeks, p < . ). peak igg titers were reached after the third booster immunization on day ( weeks). however, no significant specific antibodies were observed in the negative l. plantarum group until day the virus was challenged. samples were taken from three chickens in each group at random, at weekly intervals, from day (pre-immune) until day ( weeks) and then analyzed by elisa. the specific alv-j iga antibodies in bile, duodenal lavages and sera were detected during each test period ( table ) . the siga level increased consistently after oral inoculation in all samples of the recombinant l. plantarum group as shown in figure . results of the siga levels in bile ( figure a) showed that, compared to the control groups (groups and ), the immunized group (group ) started to increase rapidly after days of the first immunization (p < . ). the profile showed a steady increase before day ( weeks) and achieved the highest level on the th day after the rd booster immunization ( weeks, p < . ). there were no significant differences between the l. plantarum group and pbs control group (p > . ). the results of the siga level in duodenal lavages and sera (figures b,c) showed that, compared to the control groups (group and ), there was a significant increase in the immunized group (group ) from the st day after nd booster immunization. it also reached the highest level on the th day ( weeks, p < . ). in addition, there was no significant difference between the l. plantarum group and pbs control group (p > . ). therefore, these findings indicated that the recombinant pmg e-pgsa-gp l. plantarum significantly enhanced the siga antibody response and produced local mucosal immune responses. viremia were determined weekly after challenge until the th day, the positive viremia ratios were calculated, and the results were shown in table . the positive ratio of viremia in the natural l. plantarum+alv-j and pbs+alv-j groups were obviously higher than those in the recombinant l. plantarum+alv-j group. before , alv-j was widely recognized and reported in many regions of the world; it was predominantly prevalent in white feather broiler chickens ( , , ) . however, in recent years, alv-j has also occurred in commercial layer flocks ( ) , and it has become a serious threat to local breeder flocks. for many pathogens, initial infection occurs in the mucosa of animals. chickens with alv-j can infect other healthy individuals via their excrements or cloacal secretions. therefore, our study was devoted to seeking an effective vaccine that would protect chickens from alv-j infection. compared to routine styles of antigen delivery, mucosal immunization still offers some advantages including those of being more convenient, lower cost, and less stress ( , ) . in addition, lactic acid bacteria have been proposed as a live vehicle for the delivery of exogenous antigen proteins for mucosal immunization or for other therapeutic molecules. the biological functions of lactic acid bacteria are effectively combined with the immunogenicity of exogenous antigen genes ( , , ) . l. plantarum, a lactic acid bacteria, that can be used as a probiotic for animal gastrointestinal tracts, has many functions, such as maintenance of intestinal flora, enhancement of immunity, and promotion of nutrient absorption. l. plantarum can also act as a potential delivery vehicle for mucosal vaccines because it is generally regarded as safe and is able to protect antigens from premature disintegration and degradation in animal gastrointestinal tract ( , ) . however, the inherent immunogenicity of vaccine antigens in many cases is insufficient to elicit an effective immune response, which led to the suggestion that displaying antigens on the bacterial surface maybe a practical method for enhancing antigen immunogenicity ( ) . to this end, alv-j gp protein was expressed on the surface of l. plantarum using the surface-display motif, pgsa, which was adopted from the poly-γ-glutamic synthetase complex. by using this approach, we hoped to address the aforementioned challenges. in the present study, the results indicated that pgsa effectively displayed the gp protein on the surface of l. plantarum (figures , ) . l. plantarum as a carrier can successfully express exogenous antigens, resist damage from the extreme gastrointestinal environment, and significantly enhance the immunogenicity of pgsa-gp antigens. thus, it plays a double role of adjuvant and vector of the recombinant vaccine. because alv-j is able to cause growth retardation in infected chickens, body weight is considered as a valuable indicator to evaluate the protective effect of our established recombinant vaccine ( , ) . as expected, in our study, body weight in the recombinant l. plantarum group was significantly higher than that in the control group (p < . ) at , , , , d, indicating that vaccination with recombinant l. plantarum can prevent body weight loss caused by alv-j infection in chickens. this study described the mucosal and systemic immune responses specific to alv-j induced by an orally administered recombinant l. plantarum vaccine. efficient mucosal immunity is mediated predominately by secretory iga ( , ) . some studies also show that recombinant l. plantarum can induce an intestinal mucosal immune response, promote the secretion of intestinal mucosal siga, and strengthen intestinal mucosal immunobarrier function ( ) . iga is the predominant antibody on the mucosal surface, as it is produced locally at a level that exceeds those of all other immunoglobulins ( , ) . therefore, an efficient gp oral vaccine can induce a specific mucosal immune response of iga (table ) . in our study, we found that the siga levels in bile, duodenal lavages, and sera of the vaccinated chickens were dramatically increased after the first immunization compared to the natural l. plantarum and pbs groups, it reached the highest level on the th day after the third booster immunization (p < . ). these results indicated that recombinant pmg e-pgsa-gp l. plantarum effectively enhanced the siga antibody response and produced strong local mucosal immune responses ( figure ) . thus, l. plantarum could act as an excellent mucosal adjuvant and vector of recombinant vaccines ( ) . moreover, mucosal delivery of vaccines should also elicit specific immunity in systemic lymphoid tissues because most infections that gain entrance through mucosal surfaces will become systemic ( ) ( ) ( ) , and in this study recombinant l. plantarum can induce high igg in sera. igg is the main antibody produced by the humoral immune response, and it plays an important role against infection, including neutralizing toxins and internal conditioning in body defense mechanisms ( ) . some researchers have succeeded in detecting the specificity of igg by using recombinant lactic acid bacteria to express exogenous genes ( , ) . igg is an important indicator for assessing the immunity efficiency of exogenous vaccines. in our study, results showed that igg titers of the recombinant l. plantarum group were significantly higher than those in the natural l. plantarum and control group from day to (figure ) . in addition, further study of alv viremia was performed to verify the protection created by this vaccine. compared to the natural l. plantarum and control groups, after challenge, better protection was observed in the recombinant l. plantarum group against alv-j. in conclusion, our study demonstrated that the alv-j gp protein was present on the surface of the non-pathogenic l. plantarum, this can be administered orally to animals, tolerate gastric acidity, elicit both mucosal and systemic immune responses, and effectively alleviate viremia. a novel subgroup of exogenous avian leukosis virus in chickens host range of rous sarcoma virus pseudotype rsv(hprs- ) in avian species: support for a new avian retrovirus envelope subgroup, designated detection and localization of naturally transmitted avian leukosis subgroup j virus in eggtype chickens by in situ pcr hybridization molecular epidemiology of avian leukosis virus subgroup j in layer flocks in china isolation and characterization of emerging subgroup j avian leukosis virus associated with hemangioma in egg-type 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specific antibody production induction of immune responses in mice after intragastric administration of lactobacillus casei producing porcine parvovirus vp protein jl and yl designed the study. sw, yx, and ng contributed analytic tools. sw, yq, and ng analyzed the data. jl, kl, and ms wrote the paper. dz, yl, and ms revised the manuscript. all authors reviewed the results and approved the final version of the manuscript. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © wang, geng, zhou, qu, shi, xu, liu, liu and liu. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - i nv t authors: caracciolo, massimo; macheda, sebastiano; labate, demetrio; tescione, marco; la scala, stefano; vadalà, eugenio; squillaci, rosalba; d’aleo, francesco; morabito, antonella; garreffa, cristina; marciano, maria concetta; oliva, esther n. title: case report: canakinumab for the treatment of a patient with covid- acute respiratory distress syndrome date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: i nv t severe cases of covid- present with serious lung inflammation, acute respiratory distress syndrome and multiorgan damage. sars-cov- infection is associated with high cytokine levels, including interleukin- and certain subsets of immune cells, in particular, nk, distinguished according to the cell surface density of cd . cytokine levels are inversely correlated with lymphocyte count, therefore cytokine release syndrome may be an impediment to the adaptive immune response against sars-cov- infection. canakinumab, a monoclonal antibody targeting il- β is under investigation for the treatment of severe sar-cov- infection. an year old male presenting in our hospital with covid- , whose condition was complicated by acute respiratory distress syndrome and cardiac and renal failure (with oliguria) after days of hospitalization, was intubated and received canakinumab for compassionate use. on the next day, diuresis recovered and conditions improved: high il- levels and nk cells expressing cd (bright) (associated with cytokine relase) were significantly reduced giving rise to nk cd (dim). patient died on day with pulmonary bacterial superinfection and persistent sars-cov- positivity. in conclusion, canakinumab rescued a high risk, very elderly patient, from multiorgan damage complicating covid- . it may represent an useful treatment in severe cases. introduction sars-cov- is responsible for the current pandemic of coronavirus disease . patients present with fever, dry cough, dyspnea, and pneumonia ( ) . some patients (approximately %), prevalently elderly and with comorbidities, develop serious multiple organ inflammation and acute respiratory distress syndrome (ards) ( ) ( ) ( ) and require intensive care unit (icu) admission ( , ) . the immune response, including the release of pro-inflammatory cytokines and activation of t cells, are essential for controlling the viral spread, inflammation, and tissue renewal ( , ) . the damged host cell releases proteins induce the production of pro-inflammatory cytokines by nearby cells. monocytes, macrophages and t cells are attracted to the site of infection, establishing a pro-inflammatory feedback circuit. when the immune response is hampered, the excessive proinflammatory cytokines in the lungs are responsible for lung tissue damage and the cytokine storm (cs), or macrophage activation syndrome (mas), leading to multi-organ damage. severe cases with cs progress to ards ( , ) . the mechanisms leading to such complications are complex and still under investigation. many features of covid- resemble mas triggered by viral infection ( , ) . in fact, sars-cov- infection is associated with high levels of cytokines and, interestingly, levels of il- have been reported to be correlated with mortality ( ) . furthermore, in severe cases, a reduction of natural killer cells and other t lymphocytes, has been observed. cytokine levels are inversely correlated with lymphocyte count, therefore cs may be an impediment to the adaptive immune response against sars-cov- infection ( , ) . moreover, the severity of covid- is correlated with nk subsets distinguished according to the cell surface density of cd . in fact, cd bright subset increases with severity. cd dim nk cells physiologically comprise around % of nk cells in peripheral blood and are frequently described as the most cytotoxic, whereas cd bright nk cells are abundant cytokine producers ( , ) . interleukin- (il- ) activates the expression of several pro-inflammatory genes. il- β induces inflammation during infection and autoimmunity ( ) . il- β is released by various cell types, including macrophages ( , ) . canakinumab, a monoclonal antibody targeting il- β, is approved for use in rheumatologic disorders . based on the mechanism of action of canakinumab, the drug is under investigation for the treatment of severe sar-cov- infection. we present a case of an year old male presenting with covid- , complicated by ards and cardiac and renal failure, rescued by canakinumab. an indication for compassionate use for covid- during the current pandemic and approval from the local ethics committee was obtained in our center. patient was admitted to hospital on march , , presenting with fever ( . • c), hypoxemia (p = mmhg), cough, and dyspnea. medical history revealed only mild arterial hypertension treated with amlodipine and prostatic hypertrophy not requiring treatment. sars-cov- swab was positive. chest x-ray showed an interstitial lung pattern and small left pleural effusion. renal and liver biochemistry were normal. noteworthy, the patient presented lymphopenia. reactive c protein (rcp) was mg/l. coagulation tests were normal except for fibrinogen mg/dl and d-dimers ng/ml. he was at first treated in the covid ward and received broad spectrum antibiotics, hydroxychloroquine, and oxygen therapy with venturi mask with https://www.drugs.com/pro/ilaris.html#s- - % fio setting. on day , a chest computerized tomography (ct) without contrast showed severe lung injury (figure ) . on day , though the fever had subsided, his respiratory condition deteriorated and continuous positive airway pressure (cpap) non-invasive ventilation with % fio setting and positive end-expiratory pressure (peep) cmh was initiated, together with azitromicin, enoxaparin sodium and lopinavir/ritonavir. on day , tocilizumab mg/kg was administered intravenously (within a clinical trial) repeated after h, while continuing hydroxychloroquine, azitromicin and enoxaparin. on day , his conditions precipitated with presentation of ards, a pao /fio ratio (pf) of (fi setting %, p mmhg) and severe arterial hypertension. he was transferred to the intensive care unit (icu) in an obnubilated and non-collaborative condition, so that he was sedated with dexmedetomidine while continuing cpap ventilation. on day , patient presented oliguria with acute renal and cardiac failure and progressive respiratory failure. he was intubated and received assisted mechanical ventilation together with furosemide continuous intravenous infusion and vasopressor amines. on day , the patient's son was informed of the severity of the patient's clinical conditions and of the risks and benefits of canakinumab treatment. he signed informed consent to administer treatment, to process and publish all relevant clinical research data and potentially identifying information. canakinumab was administered at a single mg s.c. dose on days and . to evaluate the biochemical effects of canakinumab, general laboratory chemistry, il- , and immunophenotype were collected before and after first and second administration. the drug was well tolerated in the short term, and on the day following the first administration, the patient's diuresis normalized and renal function improved gradually without full recovery (on day , creatinine level reached µmol/l). the findings are summarized in table . during hospitalization, the patient underwent periodical microbiological surveillance tests. sars-cov- genome was evaluated by the microbiology and virology laboratory of our hospital. samples from upper (nasopharyngeal) and lower (bronchoalveolar, bronchoaspirate, and tracheal aspirate) airways were collected and processed within h. rna-covid was evaluated using an allplex -ncov assay that identifies three different target genes: e (envelope), rdrp (rna-dependent rna polymerase), and n (nucleoprotein gene). based on the interpretation criteria, detection of one or more genes was interpreted as positive covid- . there was a high viral replication persisting on day . on day , as the respiratory conditions did not improve significantly, the film array pneumonia detected the presence of bacterial infection caused by acinetobacter c.b. complex - copies/ml -and pseudomonas aeruginosa - copies/ml, treated initially with the association of piperacillin + tazobactam together with cotrimoxazole provided intravenously qid, followed by colistin aerosol bid, then with ceftazidime/avibactam intravenous tid and finally with doxycycline mg intravenous bid (the latter ongoing). the initial chest ct scan on day and x-rays performed before first administration (day ) and after second administration (day ) are shown in figure . canakinumab is an il- antagonist indicated to treat autoinflammatory disorders. severe covid- cases show symptoms associated with an excessive release of cytokines ( , ) . the il- cytokine family plays an important role in regulating inflammation and is produced in response to inflammatory stimuli and infections. il- production requires inflammasome/caspase- -dependent processing. it mediates its effects by binding to its receptor to activate downstream signaling which activates mapks and nf-kappa b, leading to the expression of pro-inflammatory mediators that drives the il- signaling pathway. il- significantly contributes to mas. its levels increase with the severity of covid- ( , , ) and the area of pulmonary infiltration (> %) in patients with ards, together with specific lymphocyte subsets ( ) . though the present case had received tocilizumab prior to canakinumab, il- level remained high, postulating that tocilizumab be insufficient to rescue the patient from the subsequent cytokine storm. in our patient, il- and nk cd bright both decreased after treatment with canakinumab, suggesting that canakinumab, by interfering with il- , reduces il- . following the second administration, il- and crp levels increased which can be explained with the development of superimposed pulmonary bacterial infection. several immunotherapeutic drugs are promising for the treatment of the cytokine storm associated with covid- ( ) . amongst these, anakinra, an il- receptor antagonist, saltuximab, an il- antagonist, and sarilumab, an il- receptor antagonist, are undergoing phase stage development ( ) . however, canakinumab treatment is associated with adverse events, mainly an increased incidence of serious infections. in fact, il- β physiologically contributes to host defense against infection by enhancing the antimicrobial action of phagocytes and inducing th and th adaptive immune responses ( ) . in our case, the patient survived the mas, but developed bacterial pulmonary superinfection, which was the final cause of death on day . this case represents the first published report of canakinumab for the treatment of multiorgan damage associated with covid- . excessive cytokine release induces severe complications and worsens the prognosis in covid- . there are numerous ongoing trials to find treatments that target virus and/or inflammation. until an effective treatment is found, in this scenario, canakinumab due to its blocking action of proinflammatory activity by il- β can constitute a potential useful treatment in the modulation of hyperinflammatory symptoms, for a subgroup of patients with covid- , that resemble the cytokine storm in patients with mas. the active involvement of immunologists in the clinic and in clinical trials may improve patient outcomes. the studies involving human participants were reviewed and approved by comitato etico sezione sud -regione calabria. the patients/participants provided their written informed consent to participate in this study. mc, eo, and dl drafted the initial manuscript. cg performed flow cytometry. fd'a performed microbiological testing. mm performed cytokine tests. all authors reviewed and revised the manuscript and have read and agreed to the published version of the manuscript to the work reported. a pneumonia outbreak associated with a new coronavirus of probable bat origin pathological findings of covid- associated with acute respiratory distress syndrome clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan clinical characteristics of coronavirus disease in china regulation of type i interferon responses coronavirus infections and immune responses hemophagocytic lymphohistiocytosis: review of etiologies and management dysregulation of immune response in patients with covid- in wuhan, china characteristics of lymphocyte subsets and cytokines in peripheral blood of hospitalized patients with novel coronavirus pneumonia (ncp) the anti-viral facet of anti-rheumatic drugs: lessons from covid- sars-cov- : a storm is raging coronavirus infections and immune responses the biology of human natural killer-cell subsets functions of natural killer cells therapeutic strategies to reduce il- activity in treating local and systemic inflammation glial proinflammatory cytokines mediate exaggerated pain states: implications for clinical pain role of interleukin- beta during pain and inflammation clinical course and risk factors for mortality of adult in patients with covid- in wuhan, china: a retrospective cohort study hlh across speciality collaboration, uk. covid- : consider cytokine storm syndromes and immunosuppression secretion of il- β from imatinib-resistant chronic myeloid leukemia cells contributes to bcr-abl mutation-independent imatinib resistance the immunology of macrophage activation syndrome the definition and risks of cytokine release syndrome-like in covid- -infected pneumonia critically ill patients: disease characteristics and retrospective analysis adjunct immunotherapies for the management of severely ill inflammasome activation and il- β and il- processing during infection novartis provided compassionate use of canakinumab for the case described. conflict of interest: eo reports personal fees from novartis, during the conduct of the study.the remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © caracciolo, macheda, labate, tescione, la scala, vadalà, squillaci, d'aleo, morabito, garreffa, marciano and oliva. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -j ot lt authors: ahmed-hassan, hanaa; sisson, brianna; shukla, rajni kant; wijewantha, yasasvi; funderburg, nicholas t.; li, zihai; hayes, don; demberg, thorsten; liyanage, namal p. m. title: innate immune responses to highly pathogenic coronaviruses and other significant respiratory viral infections date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: j ot lt the new pandemic virus sars-cov- emerged in china and spread around the world in < months, infecting millions of people, and causing countries to shut down public life and businesses. nearly all nations were unprepared for this pandemic with healthcare systems stretched to their limits due to the lack of an effective vaccine and treatment. infection with sars-cov- can lead to coronavirus disease (covid- ). covid- is respiratory disease that can result in a cytokine storm with stark differences in morbidity and mortality between younger and older patient populations. details regarding mechanisms of viral entry via the respiratory system and immune system correlates of protection or pathogenesis have not been fully elucidated. here, we provide an overview of the innate immune responses in the lung to the coronaviruses mers-cov, sars-cov, and sars-cov- . this review provides insight into key innate immune mechanisms that will aid in the development of therapeutics and preventive vaccines for sars-cov- infection. severe acute respiratory syndrome coronavirus (sars-cov- ) reportedly emerged at a live animal market in the chinese city of wuhan is currently causing a pandemic and negatively affecting global health ( ) ( ) ( ) . there are more than million confirmed sars-cov- infections with an mortality rate that widely varies by country and region ( ) . even in industrialized countries, sars-cov- led healthcare systems approach the brink of collapse by overwhelming their capacity and straining resources. governments and local leaders ordered the shutdown of cities, regions, countries leading to massive disruptions in the local and global economy. unlike previous coronavirus (cov) infections, the rapid global spread, high transmission rate, longer incubation time, and disease severity of sars-cov- requires a better understanding of the epidemiology and immunopathogenesis of this viral outbreak in order to learn from this experience and to manage future pandemics. sars-cov- is a highly pathogenic cov ( ) (case-fatality rate of . - . %) ( , ) that is related to severe acute respiratory syndrome cov (sars-cov) (case-fatality rate of - %) and the middle east respiratory syndrome cov (mers-cov) (case-fatality rate of . %), see also table ( , ) . sars-cov, sars-cov- and mers-cov target the lower respiratory system, causing respiratory illnesses, including severe pneumonia, acute lung injury (ali) and acute respiratory distress syndrome (ards) ( , ) . sars-cov- infection results in higher hospitalization rates in the elderly (> ) and persons with pre-existing conditions including hypertension, diabetes and obesity compared to rates among younger populations without pre-existing conditions ( table ) ( , ) . in addition to an age disparity, males with covid- appear to have higher risk for worse outcomes and death ( , ) . epidemiological research of the sars and mers infections also showed that males had a higher mortality rate than females ( ) ( ) ( ) . while sars-cov- is a novel coronavirus, several important insights have already been made about its basic mode of transmission. virus particles are inhaled in respiratory droplets expelled from the airways of infected individuals. angiotensinconverting enzyme (ace ), expressed on the ciliated bronchial cells, endothelial cells, and type i and ii alveolar cells, is the host receptor for cell entry into the respiratory tract by both sars-cov- and sars-cov ( table ) ( ) ( ) ( ) ( ) . the spike protein (s) of cov is responsible for the entry of the virus into the target cell (figure ) ( , ) . ace is a type i transmembrane metallocarboxypeptidase that plays a vital role in the renin-angiotensin system (ras) ( , ) , which in turn is critical in maintaining blood pressure homeostasis as well as fluid and salt balance in mammals. ace is found in vascular endothelial cells, in the renal tubular epithelium, and in leydig cells of the testes ( ) . studies have shown that ace is expressed in gastrointestinal (gi) tissues, making it a potential site for harboring sars-cov ( ) . this may be one of the reasons for gi pathology reported in some patients with covid- and viral shedding in stool. in contrast, mers-cov uses dipeptidyl-peptidase (dpp ) as an entry receptor, which is expressed on endothelial cells and some epithelial tissues ( table ) ( , ) . accumulating data suggest that the innate anti-viral response and adaptive immunity may contribute to a cytokine storm leading to systemic hyper inflammation and exacerbation of the disease in patients with (a) comorbidities (b) older than years of age (c) of the male sex. the exact role of the innate immune system in disease pathogenesis and prevention between the sexes and the impact of age is not fully elucidated. in addition, the potential dysregulation of the innate immune response by sars-cov and sars-cov- is not completely understood which warrants further research. the cells of the airway epithelium are the first line of defense, providing a mechanical barrier (mucociliary escalator) that expels particles and pathogens via cilia, mucus, and induced coughing ( , ) . this barrier includes cells of the pulmonary epithelium and tissue-resident macrophages and dendritic cells (dcs). the macrophages and dcs express pattern recognition receptors (prrs) that can detect molecules from pathogens (pathogen-associated molecular patterns-pamps) or molecules released from damaged cells (damage or danger-associated molecular patterns-damps) ( ) ( ) ( ) . in the lung, there are two populations of macrophages, alveolar and interstitial macrophages ( ) . in addition to these macrophages, dcs play a vital role in facilitating the host defenses against respiratory diseases ( ) ( ) ( ) . dcs can be divided into plasmacytoid (pdc) and myeloid types (mdc) ( ) ( ) ( ) . macrophages and the two dc subtypes trigger antiviral responses by generating a substantial amount of type i interferon and these cells play important roles in immune surveillance in the airways and the distal lung ( , ( ) ( ) ( ) ( ) ( ) . during steady state, the dc density in lung associated tissue declines from the trachea to the alveoli ( ) while the representation of cells in macrophage compartment seems more complex ( ) . if a virus infects airway epithelial cells, the viral rna would be sensed via intrinsic innate receptors, including rig- , mda , nlrp inflammasome, and the rna sensing tlrs and . in the case of influenza a infection, triggering the prrs causes a strong induction of type interferon (ifn) in epithelial cells ( , ) . in other viral infections, such as respiratory syncytial virus (rsv), alveolar macrophages are the predominant source of type ifns ( ). furthermore, respiratory epithelial cells and lung macrophages are capable of secreting a broad range of chemokines like il- , macrophage inflammatory protein- (mip- ), rantes and cytokines including tnf-α, il- , il- β that influence the types of immune cells being recruited to the area in response to acute viral infections ( , ) . macrophages, depending on their polarization status of either m or m , and in a similar way as airway epithelial cells, can further elicit a th or th response ( , , ) . in the case of influenza virus infection, the magnitude of epithelial cell response can be proportional to the amount of virus which result in paracrine induction of ifn λ ( ) . not only can airway epithelial cells produce a large array of cytokines/chemokines in response to an acute viral infection, but, depending on the magnitude of prr engagement and the combination of pamps and damps triggered, these epithelial cells can modulate the type of chemokines/cytokines produced and influence the influx of innate and adaptive immune cells ( , ) . the response to different viral infections is generally similar, however, the response can be tailored in timing, magnitude and the induced gene signatures in response to each virus ( ) . unlike rsv and mers-cov, which both productively infect alveolar multiple cell types in the lower respiratory tract were found to be infected, including type i alveolar epithelium, macrophages, and putative cd + oct- + stem/progenitor cells in human lungs ( ) ( ) ( ) ciliated bronchial epithelial cells and type ii pneumocytes ( , ) un-ciliated bronchial epithelial cells and type ii pneumocytes ( ) ( ) ( ) infect mostly human type i and type ii pneumocytes and alveolar macrophages ( ) respiratory, nasal, corneal and intestinal epithelial cells ( ) club cells, ciliated cells, type i and type ii alveolar cells ( ) ciliated epithelial cells of the upper and lower respiratory tract ( ) the ciliated cells of the human airway epithelium are the main target, it also infects basal cells ( ) and immune cells, such as macrophages, b cells cd + and cd + t cells ( ) upper and lower airways epithelial cell ( no specific antiviral drugs ( ) antiviral drugs may be a treatment option ( ) no antiviral agents symptomatic treatment supportive care ( ) there are no approved antiviral medications ( ) ace macrophages ( , ) , seasonal influenza and sars-cov usually lead to non-productive infections in these cells ( ) . in addition, sars-cov infection of primary monocytes yielded little virus, likely due to the suppressive effects of ifn-α ( ) . thus, the initial cell type(s) involved in propagating a viral infection intensifies the complexity of the immune response. another key factor that determines the magnitude of the immune response is the induction and rate of cell death. although related, mers-cov induces widespread cell death when compared to sars-cov in human bronchial epithelial cell cultures ( ) . however, the sars-cov open reading frame a (orf a) protein can induced necrotic cell death in a variety of cell lines ( ) . the same orf a protein can activate the nlrp inflammasome, leading to activation of nf-κb and elevated secretion of active il- β in cell culture ( ) . cytokine ( ), with slightly different cytokine/chemokine profiles. this delay in cytokine induction was confirmed in another study using the same epithelial cell lines ( ) as well as in human alveolar type ii cells ( ) . in both cell lines and primary alveolar type ii cells, sars-cov induced ifn-β, ifn-λ, cxcl , cxcl , il- , ip- , and tnf-α ( , ) . mers-cov did not induce ifn-β but induced higher level of il- transcript in cell culture. however, no difference in il- production was observed between sars-cov and mers-cov at h post-infection ( ) . this was confirmed in-vivo using a non-human primate model comparing the immune responses to sars-cov infection between young adult cynomolgus macaques ( - yrs) and older macaques ( - yrs) ( ) . interestingly, the high induction of il- was observed on a transcript level in the older animals, while the younger once showed higher levels of ifn-β transcript ( ) . in all animals, the expression of ifn-β was inversely correlated with the pathology score, supporting the role of ifn-β in controlling disease severity ( ) and introducing a potential area of research to define age disparity observed in patients infected with sars-cov- . both older age and male sex are important factors associated with high mortality of sars-cov and sars-cov- infection ( , ) . many viruses have evolved to disrupt and subvert the immune responses. a common virus that is well-known to affect the lower airway and counteract the immune response is rsv ( , ) . the rsv genome encodes non-structural proteins ns and ns that can block type ifn production and signaling in cell cultures ( ) . similar to rsv, the measle virus v protein blocks ifn-α and β signaling by inhibiting stat and stat signaling in cell lines ( ) , whereas mers-cov m protein also suppresses type ifn by inactivating irf ( ) , leading to the low expression of ifn-β. in contrast to reports in epithelial cell lines or primary alveolar type ii cell culture and observations in non-human primates, sars-cov nucleocapsid (n) protein and membrane (m) protein, as well as nsp , can suppress ifn response via various mechanisms in cell lines ( ) ( ) ( ) . to bridge the dichotomy of inhibition of ifn signaling in cell lines, and the ifn expression in vivo, cells recruited by the infection need to be considered as a potential source. as previously discussed, infected epithelial cells via paracrine signaling to neighboring cells and resident macrophages, secrete chemokines and cytokines to attract other immune cells. in general, monocytes/macrophages are recruited by ccl (mip- a), ccl (mcp- ), and neutrophils are recruited by il- (cxcl ), cxcl , and cxcl chemokines, all of which can be secreted by airway epithelial cells ( , , ) . both monocytes and neutrophils are also recruited by complement fragment (anaphylatoxin) c a (figure ) . both influenza and sars virus can induce acute lung injury (ali) which is accompanied by high levels of c a, leading to the influx and activation of innate immune cells ( ) (figure ) . serum samples and lung tissue of sars patients showed high-level expression of cxcl (ip- ), which is also found to be induced by sars-cov in the epithelial cell line calu- ( ) . significant neutrophil, macrophage and cd t-cell infiltration can be found in the lung of sars patients by immunohistochemistry ( , , ) . in addition to post-mortem lung histology analysis in patients with sars-cov, experiments using rhesus macaques infected with sars-cov found monocyte and macrophage recruitment. the accumulation of pathogenic inflammatory monocytemacrophages (imms) was also observed in a sars-cov mouse model. the accumulation of imms resulted in heightened lung inflammatory cytokine/chemokine levels, extensive vascular leakage, and impaired virus-specific t cell responses ( ) . a strong infiltration of cd and mac positive monocytes/macrophages were found in the human and animals lung samples ( , ) . macrophages further stimulate fibroblasts to deposit extracellular matrix leading to pulmonary fibrosis ( ) , which was also observed in patients who recovered from sars ( , ) . autopsy samples acquired from patients with sars-cov- patients contained viral nucleocapsid protein (np) positive cd + macrophages in the capillaries of the spleen and in lymph nodes, indicating that sars-cov- might migrate into the spleen and lymph nodes through macrophages. this study also found that cd + macrophages appear to mediate sars-cov- into these tissue sites, contributing to virus dissemination ( ) . similar to sars-cov- , sars viral particles and genomic sequences were detected in monocytes, macrophages as well as within different organs of sars patients ( ) . sars-cov was shown to infect both immature and mature human monocyte-derived dcs by electron microscopy and immunofluorescence. the detection of negative strands of sars-cov rna in dcs suggests viral replication, but no increase in viral rna was observed ( ) . as mentioned above, there was no perceivable increase to sars-cov replication in primary monocytes ( ) . another study looked at sars-cov and mers-cov replication in human macrophages, human monocyte-derived macrophages, and dendritic cells (mddcs) and found that both viruses replicated poorly. mers-cov induced ifn-λ , cxcl , and mxa mrnas in both macrophages and mddcs, however, sars-cov was unable to induce such responses ( ) . interestingly, depletion of inflammatory monocyte-macrophages in the mouse model partially protected from lethal sars infection ( ) . these data suggest that monocytes, macrophages and dendritic cells have essential roles in cov infection. the severity of disease and the response to these viruses seems to be dependent on the induced cytokine/chemokine profiles and the amplification of the immune response by the recruited cells. growing evidence of dysregulation of an innate anti-viral response originates from studies using clinical samples ( ) and murine models ( , , ) . in addition to dysregulated cellular responses, the complement system may play an important role in sars-cov- infection (figure ) . evidence comes from sars infected patients who had lower levels of mannan binding lectin (mbl) in serum compared to healthy controls ( ) . the sars patients with a higher frequency of mbl gene polymorphisms were associated with lower serum levels or deficiency of mbl ( ) . it is still unknown if this is also true for covid- patients, which requires further investigation. in cell culture experiments sars-cov was able to bind and activate the complement cascade and block viral infection ( ) . preliminary findings in a limited number covid- patients found significant deposits of the membrane attack complex (mac), c d and mbl-associated serine protease (masp) in the microvasculature indicating sustained, systemic activation ( ) . the sars-cov- spike protein was co-localized with c d and mac ( ) . in a non-peer reviewed publication by gao et al., mers-cov, sars-cov and sars-cov- n protein are able to bind to masp- leading to enhanced complement activation ( ) (figure ) . in a later phase of the infection, the complement system might be also triggered via antibodies bound to the virus (classic activation pathway, figure ). this excessive complement activation might play a role in multi organ damage in severe covid- cases ( ) . in a mers-cov mouse model the blockade of the c a-c ar axis alleviated not only lung damage but also spleen damage ( ) . mice treated with a monoclonal antibody to c a showed reduced lung infiltration of cd + cells and significantly lower cytokine levels of il- β, tnf-α, inf-γ and il- ( ) . complement blockade might be an important way to curb part of the immune dysregulation associated with covid- . overall, we need to look closer at the role of the complement system, the recruited innate immune cells and their combined role in pathogenesis, viral clearance and the eventual resolution of the infection. the most abundant leukocytes, neutrophils, play a critical role in clearing viral infections. neutrophils, attracted by chemokines/cytokines released by tissue-resident macrophages and dcs, swarm to the site of infection. they recognize and release bioactive compounds, including cytokines, chemokines and ros, as well as nos in the very early phase of the infection ( , ) . neutrophils modulate other innate and adaptive immune responses via cytokine/chemokine release and cell death and, therefore, can ameliorate or exacerbate disease progression. neutrophils infiltrate tissues infected by cov, including sars-cov, rat coronavirus (rcov), and mouse hepatitis virus (mhv). a high neutrophil count in the blood of sars patients at the time of hospital admission is associated with a poor prognosis ( , ) . gao et al. suggested that patients with sars-cov- pneumonia can be stratified by neutrophil to lymphocyte ratio (nlr) and age ( ) . patients older than years of age and having an nlr ≥ . had more severe illness, so rapid access to the intensive care unit is required ( , ) . experiments in mice showed that sars-cov disease severity in older mice correlated with increased pulmonary inflammation and influx of neutrophils ( , ) . infection of rats with rcov could lead to neutrophil infiltrating into the respiratory tract early after inoculation, followed by the recruitment of macrophages and lymphocytes ( ) . infection of mice with a neurotropic murine cov (mhv-jhm) showed infiltration of neutrophils into the brain as early as the first day after inoculation, which then promoted the recruitment of other types of inflammatory cells into the brain, likely through the loss of the blood-brain barrier integrity ( ) . gene expression analysis in experimentally infected rhesus macaques with mers-cov revealed the recruitment of neutrophils into infected lung tissue ( , ) . angiotensin-converting enzyme inhibitors (ace-is) could serve as a potential risk for fatal covid- through the upregulation of ace ( ) and may provide a direct linkage to neutrophils and disease progression. investigators found that ace modulates il- -mediated neutrophil influx by impacting stat activity ( ) . animal models used to study the pathogenesis of sars-cov- have revealed important roles of neutrophils in infection and confirmed findings observed in patients. a new aspect in sars-cov- infection is the potential role of neutrophil extracellular traps (nets). the process of net formation is a specific type of cell death that can be triggered under inflammatory conditions ( , ) , such as gm-csf+c a, il- , ifn-α+c a or other tlr response mediators; all conditions present in severe sars-cov- infection ( , ) . the net formation has been observed in covid- patients and may contribute to thrombotic complications in covid- patients ( , ) . microvascular injury and thrombosis have been reported in covid- patients, increasing the likelihood that neutrophil net formation might play a role ( , , ) . net formation was reported to be involved in clot formation and thrombosis and can further increase inflammation ( , ) . therefore, neutrophils can attract a second wave of immune cells to the site of infection by cytokine/chemokine secretion as well as via netosis ( , ) , which included monocytes and natural killer cells. on the other hand aggregated nets were reported to limit inflammation by degrading cytokines and chemokines and disrupting neutrophil recruitment and activation ( ) . despite the presence of neutrophils in sars-cov- -infected tissues, their role in the clearance and/or immunopathology of the viral infection remains unclear. future studies on the responses of neutrophils to sars-cov- -infection or infected cells in vitro may elucidate the role of neutrophils in the pathogenesis of respiratory cov infections. natural killer (nk) cells are a heterogenic immune cell subset that acts promptly to combat viral infections. they produce significant amounts of ifn-γ, kill virus-infected cells, provide direct support to other innate immune cells, and aid in the adaptive immune response to counter viruses. nk cells express activating receptors that detect viral antigens, enabling the destruction of infected cells ( ) ( ) ( ) ( ) . lectin-like receptor cd and killer immunoglobulin-like receptors, such as cd b, regulate the function of nk cells. a study of patients with sars explored the relationship of the number of nk cells and the expression level of their immunoglobulin-like receptor cd b in the peripheral blood to the severity of sars ( ) . the overall count of nk cells and cd + nk cells and the percentage of cd + nk cells in patients with sars were significantly lower than counts in healthy subjects ( ) . a separate study that analyzed lymphocytes and lymphocyte subsets in a cohort of patients with sars observed reduced nk cell counts in patients ( %) ( ) . clinical reports reveal that children appear to have a milder form of sars-cov- , with peripheral blood lymphocyte levels remaining in the normal range, suggesting less immune dysfunction following the disease ( ) . this could be attributed to healthy children expressing lymphocytes, especially nk cells, in a greater quantity compared to healthy adults ( ) . interestingly, previous studies found rapid and significant restoration of lymphocyte subsets including, nk cells, in peripheral blood in patients recovering from the initial stages of sars infection ( ) . although the primary mechanism for the decrease in nk cells and other subsets during disease onset remains unknown, their contribution to sars-cov- needs further study especially from a treatment perspective. innate lymphoid cells (ilcs) are a family of innate immune cells that include ilc , ilc and ilc . although ilc facilitates lung repair after injury, the role of ilcs during respiratory viral infection is not clearly defined ( ) . evidence for the potential involvement of ilc cells in the lung during viral infection was reported in a murine model ( ) . this study found a rapid accumulation of ilc cells in the lung after an influenza virus infection, however their initial contribution to exacerbation of the disease was limited ( ) . a recent study identified an interaction between ace -expressing sars-cov- target cells and ilcs in the colon ( ) . thus, elucidating the role of ilc subsets will be important in understanding the pathogenesis of sars, sars-cov- and mers infections. there is distinct evidence indicating an important role of ifns in sars and other cov infections ( , ) . the sera of patients with sars revealed the presence of high levels of il- , il- , infγ, ccl , cxcl , and il- and products of interferon stimulated genes ( , ) . high expression levels of isgs such as cd , ifnar , and ifngr and ifn-stimulated chemokines cxcl and ccl were observed in another cohort of sars patients and were correlated with the severity of pathogenesis ( ) . significant upregulation of cxcl gene expression was observed in the severe phase of patients who died from sars. this data is corroborated by studies in patients with mers that found upregulation of cxcl in the serum of patients who developed pneumonia ( ) . cxcl and infα were also correlated with severity of disease ( ) . the importance of ifn signaling in response to cov infection has been well-demonstrated in several knockout mouse models. type i, ii, and iii ifn signaling deficient mice have increased susceptibility to mouse-adapted sars-cov strains ( , ) . studies using mice lacking the ifnar and ifnlr or stat identified higher sars-cov replication in the lungs and delayed virus clearance ( , ) . another study with mers-cov in mice expressing the human dpp receptor showed a role for the ifnar in viral clearance and lung inflammation ( ) . these mouse models suggest an important role of ifn response for cov clearance. this quick expanding medical literature is very suggestive of an important role of ifn responses for cov control and clearance. many viruses have evolved to disrupt and subvert the immune response. rsv counteracts the immune response ( , ) ; as discussed earlier, the rsv genome encodes non-structural proteins (ns and ns ) that are able to block type ifn production and signaling in cell cultures ( ) . similar to rsv, the measle virus v protein blocks ifn-α and β signaling by inhibiting stat and stat signaling in cell culture lines ( ) . covs have developed several ways to escape from innate immune pressure. mers-cov m protein suppresses type ifn by blocking the irf activation ( ) , explaining the low expression of ifn-β. in various cell lines, sars-cov nucleocapsid (n) protein, membrane (m) protein, as well as nsp , were reported to suppress ifn response ( , , ) . the nucleocapsid protein (n) of sars-cov interferes with the function of irf . although it does not form a complex with rig-i or mda , rna binding activity at the initial recognition stage of viral rna potentially contributes to immune evasion ( , ) . aside from the hcov, structural proteins, accessory, and non-structural proteins (nsp) are involved in innate immunity modulation. in both sars-cov and mers-cov, host mrna endonucleolytic cleavage is promoted by nsp protein, which modifies capped non-viral rnas ( , ) . nsp in sars-cov prevents host mrna translation by interacting with the s subunit of the ribosome; in turn, transcription and translation of viral rna is given preference over the host mrna ( ) . another study found that additional sars-cov nsp residues interfered with ifn-dependent signaling ( ) . ifn production is affected by nsp proteins in sars-cov and mers-cov. these proteins have both papain-like protease (plpro) and a plp domain, and the plpro domains in both sars-cov and mers-cov downregulate mrna levels of ccl , infβ, cxcl , and other pro-inflammatory cytokines ( ) . the suppression of ifn responses by sars-cov plpro is due to the inhibition of phosphorylation of ifn-regulatory factor (irf ) and its subsequent translocation to the nucleus where it enhances ifn gene transcription ( ) . mers-cov plpro also suppresses rig-i and mda and antagonizes ifn induction ( , ) . in hcov- e and sars-cov suppression of ifn responses, the key molecule is a adp-ribose- -monophosphatase macrodomain encoded within nsp ( ) . accessory proteins are not key in viral replication; however, in human cov, this group of proteins are involved in diverse cellular signaling, including cell proliferation, apoptosis, and ifn signaling ( ) . by downregulating phosphorylation and nuclear translocation of irf , open reading frame orf b and - interfere with ifnβ synthesis and prevent ifnβ-induced activation of ifnstimulated response element (isre) in the promoter of isg in sars-cov ( ) . in cells transfected with orf a, - b, and - of mers-cov, ifnβ promoter-driven luciferase activity is significantly reduced, and it may follow a similar pattern of suppression of irf nuclear translocation ( ) . therefore, the suppression of signaling events in infected immune and airway epithelial cells, as well as the magnitude of suppression due to elevated expression levels of these accessory proteins, needs to be further elucidated to understand delayed or hyperimmune responses and cytokine storm that occurs in cov infection. in addition to revealing our unpreparedness of handling a worldwide pandemic by a viral infection, covid- exposed our lack of understanding of the pathogenesis of diseases as well as the host immunity. the interaction of the host innate immune system and other factors including age, sex, and pre-existing conditions need further investigation regarding disease severity and morbidity of sars/mers and covid- . disease severity and 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antiviral signaling mers-cov papain-like protease has deisgylating and deubiquitinating activities regulation of irf- -dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease the adp-ribose- ''-monophosphatase domains of severe acute respiratory syndrome coronavirus and human coronavirus e mediate resistance to antiviral interferon responses accessory proteins of sars-cov and other coronaviruses the remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © ahmed-hassan, sisson, shukla, wijewantha, funderburg, li, hayes, demberg and liyanage. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - udb uc authors: hunegnaw, ruth; mushtaq, zuena; enyindah-asonye, gospel; hoang, tanya; robert-guroff, marjorie title: alveolar macrophage dysfunction and increased pd- expression during chronic siv infection of rhesus macaques date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: udb uc hiv infected individuals have been shown to be pre-disposed to pulmonary infections even while receiving anti-retroviral therapy. alveolar macrophages (ams) play a critical role in lung innate immunity, but contradictory results have been reported regarding their functionality following hiv infection. here, using the siv rhesus macaque model, we document the effect of siv infection on the phenotypic and functional properties of ams. following infection with siv(mac ), ams in bronchoalveolar lavage (bal) sampled over - to -weeks post-infection (wpi) were compared to those in bal samples from naïve macaques. am expression of proinflammatory cytokines tnf-α, il- , il- β, and chemokine rantes drastically increased -wpi compared to ams of naïve macaques (p < . for all), but dropped significantly with progression to chronic infection. phagocytic activity of ams -and -wpi was elevated compared to ams of naive animals (p = . , p = . , respectively) but significantly decreased by -wpi (p = . , p = . , respectively). by -wpi the ability of ams from chronically infected animals to perform siv-specific antibody-dependent phagocytosis (adp) was also diminished (p = . ). acute siv infection was associated with increased fcγriii expression which subsequently declined with disease progression. frequency of fcγriii(+) ams showed a strong trend toward correlation with siv-specific adp, and at -wpi fcγriii expression negatively correlated with viral load (r = − . ; p = . ), suggesting a contribution to viremia control. importantly, pd- was found to be expressed on ams and showed a strong trend toward correlation with plasma viral load (r = . ; p = . ), indicating that similar to over-expression on t-cells, pd- expression on ams may also be associated with disease progression. further, ams predominantly expressed pd-l , which remained consistent over the course of infection. pd- blockade enhanced siv-specific adp by ams from chronic infection indicating that the pd- /pd-l pathway may modulate functional activity of ams at that stage. these findings provide new insight into the dynamics of siv infection leading to am dysfunction and alteration of pulmonary innate immunity. our results suggest new pathways to exploit in developing therapies targeting pulmonary disease susceptibility in hiv-infected individuals. hiv infected individuals have been shown to be pre-disposed to pulmonary infections even while receiving anti-retroviral therapy. alveolar macrophages (ams) play a critical role in lung innate immunity, but contradictory results have been reported regarding their functionality following hiv infection. here, using the siv rhesus macaque model, we document the effect of siv infection on the phenotypic and functional properties of ams. following infection with siv mac , ams in bronchoalveolar lavage (bal) sampled over -to -weeks post-infection (wpi) were compared to those in bal samples from naïve macaques. am expression of proinflammatory cytokines tnf-α, il- , il- β, and chemokine rantes drastically increased -wpi compared to ams of naïve macaques (p < . for all), but dropped significantly with progression to chronic infection. phagocytic activity of ams -and -wpi was elevated compared to ams of naive animals (p = . , p = . , respectively) but significantly decreased by -wpi (p = . , p = . , respectively). by -wpi the ability of ams from chronically infected animals to perform siv-specific antibody-dependent phagocytosis (adp) was also diminished (p = . ). acute siv infection was associated with increased fcγriii expression which subsequently declined with disease progression. frequency of fcγriii + ams showed a strong trend toward correlation with siv-specific adp, and at -wpi fcγriii expression negatively correlated with viral load (r = − . ; p = . ), suggesting a contribution to viremia control. importantly, pd- was found to be expressed on ams and showed a strong trend toward correlation with plasma viral load (r = . ; p = . ), indicating that similar to over-expression on t-cells, pd- expression on ams may also be associated with disease progression. further, ams predominantly expressed pd-l , which remained consistent over the course of infection. pd- blockade enhanced siv-specific adp by ams from chronic infection indicating that the pd- /pd-l pathway may modulate functional activity of ams at that stage. these findings provide new insight into the dynamics of siv infection leading to am dysfunction and alteration of pulmonary innate immunity. our results suggest new pathways to exploit in developing therapies targeting pulmonary disease susceptibility in hiv-infected individuals. keywords: alveolar macrophage, siv, rhesus macaque, antibody-dependent phagocytosis, pd- , fcγriii, bronchoalveolar lavage introduction macrophages are found in tissues of infected individuals and play a continuous role in pathogenesis. although earlier studies reported that macrophages were the major cell type initially infected by hiv ( ), more recent studies have shown that cd + t cells are preferentially targeted by hiv/siv at the site of transmission ( , ). however, macrophages still serve as targets for the virus in vivo ( ) . they can sustain viral replication, disseminate virus, and serve as a viral reservoir post-infection ( ) ( ) ( ) . cells of the macrophage/monocyte lineage vary greatly in phenotype, longevity, and in phagocytic, immunoregulatory, and secretory properties ( ) ( ) ( ) ( ) . macrophages are categorized as classically (m ) or alternatively (m ) activated based on surface markers and functional role ( , ) . m macrophages mediate inflammatory responses against pathogens while m macrophages have anti-inflammatory properties, promoting tissue repair and remodeling ( ) . alveolar macrophages (am) in the lung uniquely express both m and m phenotypic markers, indicating ability to quickly respond to pathogens but also prevent immune activation in response to harmless antigens that enter the alveolar lumen ( ) . am express cd and chemokine receptors making them vulnerable to hiv infection ( ) . however, macrophages may be poorly susceptible to hiv induced cytopathic effects. minimal consequences of hiv infection on the macrophage transcriptome were observed ( ) ; in contrast expression of hiv nef and gp envelope induced macrophage activation ( , ) . indirect activation can also occur by exposure of uninfected macrophages to viral gene products or cytokines from other infected cells ( ) . am are important lung phagocytes ( ) , yet am obtained from hiv-infected individuals have shown contradictory results regarding the impact of viral infection on phagocytosis. some studies have shown impaired phagocytosis of opportunistic pathogens by am ( ) ( ) ( ) ( ) ( ) ; others have reported no change in am phagocytic activity during infection ( , ) . such variations in outcome may result from differences in length of infection or reliance on the use of monocyte derived macrophages (mdm) and infected cell lines which may not ideally represent clinical situations. macrophages can utilize fcγ receptors to internalize antibody-opsonized virions or infected cells, potentially leading to antibody-mediated clearance of infectious material ( ) . antibody-dependent phagocytosis (adp) by am contributes to protection against viral infections such as influenza ( ) , west nile ( ), adenovirus ( ) , and sars coronavirus ( ) . however, adp-mediated protection by macrophages against hiv infection has not been observed. here we investigated the dynamics of siv-related changes in am activity and function by sampling bronchoalveolar lavage (bal) from siv infected rhesus macaques during acute and chronic infection. the am response to siv infection consisted of phenotypic changes and alterations in proinflammatory responses, ability to respond to gp antigen, and phagocytic activity. fcγriiib expression on am was linked to siv-specific adp and viral control during acute infection. novel results showed increased expression of programmed cell-death- (pd- ) on am from chronically infected macaques and positive correlations between pd- expressing ams and siv viremia. we believe this is the first report of pd- expression on ams of siv infected macaques. our results suggest associations between pd- expression, macrophage dysfunction, and lack of viremia control. forty-nine female indian rhesus macaques used in this study were housed in the nci animal facility, bethesda, md, under protocol vb- . the nci facility is accredited by the association for assessment and accreditation of laboratory animal care international, and its animal care and use committee approved all animal experiments prior to study initiation. standard practices followed recommendations made in the guide for the care and use of laboratory animals of the united states, national institutes of health, and the weatherall report. thirty macaques were used as naïve controls and macaques were challenged weekly intravaginally using a repeated low-dose of siv mac ( tcid ). infection was confirmed for of the macaques with plasma viral loads of ≥ siv rna copies/ml as assessed by droplet digital pcr (chung et al., manuscript in preparation). the remaining five macaques were infected intrarectally with a single high dose of siv mac ( tcid ). out of the infected macaques, were euthanized after wpi and the remaining infected macaques were monitored longitudinally for up to wpi. bal samples were obtained using standard techniques ( ) . briefly, rhesus macaques were anesthetized, and an endotracheal tube was inserted through which sterile saline solution ( ml/kg) was instilled. suction was applied to recover the instilled fluid and the lung lavage was collected in sterile conical tubes. cells were pelleted by centrifuging at rpm for min at • c and washed in ml cold pbs. centrifugation was repeated, and cells were resuspended for counting. cells showed ∼ % viability as determined by trypan blue staining. bal cells were enriched for ams by depleting epcam + , cd + , and cd + cells. the following reagents were used: rabbit polyclonal anti-epcam (n c ) (genetex, irvine, ca); cd and cd microbeads for non-human primate (miltenyi biotec, auburn, ca). depletion was performed using macs cell separation technology according to the manufacturer's instructions (miltenyi biotec, auburn, ca). enriched ams were stimulated for h in µl r- media (rpmi + % fbs + % pen-strep + % anti-anti) containing purified native hiv- envelope gp (advanced bioscience laboratories, inc., rockville, md) at a concentration of nm or lps (thermofisher scientific, waltham, ma) at ng/ml concentration. for experiments assessing cytokine production by pd- + ams, cells were stimulated with µg/ml lps in the presence of golgistop ( µl, bd biosciences) for h prior to intracellular staining. adp activity was measured as previously described ( ) . siv mac gp was biotinylated with a biotin-xx microscale protein labeling kit (life technologies, grand island, ny) and incubated with a -fold dilution of µg avidin coated sky blue fluorescent beads ( . µm diameter; spherotech, lake forest, il) overnight at • c. enriched ams were plated in a u-bottom well plate at , cells/well and undiluted bal-f was added. to determine phagocytic activity of am independent of siv specific antibody, bal-f from naïve macaques was used. for siv-specific adp, autologous bal-f from infected macaques was used. the bead-gp mixture was brought to a final -fold dilution in r- media and µl was added to the cells and incubated for h at • c. for pd- blocking experiments, bal samples collected from macaques at wpi were enriched for ams as described above and incubated with either µg/ml ultraleaf purified anti-human pd- antibody (eh . h , biolegend) or µg/ml migg isotype control (sigma-aldrich). cells incubated with naïve bal-f were used for normalization. in all cases, after h of incubation, µl of % paraformaldehyde was added for fixation. fluorescent bead uptake was assessed using a bd biosciences lsrii flow cytometer. bead uptake specifically by am was made possible by focusing on cells that were autofluorescent in the fitc channel. the phagocytic score of each sample was calculated by multiplying the frequency of bead-positive cells by the degree of phagocytosis measured as mean fluorescence intensity (mfi) and dividing by . values were normalized to background values (cells and beads with either pbs or naïve bal-f) by dividing the phagocytic score of the test sample by the phagocytic score of the background samples. mouse anti-non-human primate or anti-human fluorochromeconjugated monoclonal abs (except where specified otherwise) known to cross-react with rhesus macaque antigens were used in this study. the following antibodies were used for surface staining: bv anti-cd (l ), bv anti-cd (d - ), buv anti-cd (sp - ), buv anti-cd ( g ), cells were then washed with bd permwash, and incubated with intracellular staining antibodies for min at rt. finally, cells were washed with pbs and staining data were acquired using the laser bd facsymphony (bd biosciences, san jose, ca). fluorescence minus one (fmo) and isotype controls were used to confirm the phenotype and cytokine expressing population of ams ( figure s ). data analyses were performed with flowjo (version . , treestar, inc ashland, or) software. antibodies in bal-f were measured as follows: wells of greiner high-binding ½ area -well plates were coated overnight at • c with ng/well of siv mac gp (for determining gp specific igg) or ng/well of anti-rhesus igg (alpha diagnostics) (for determining total igg) in sodium bicarbonate buffer (ph . ) (sigma-aldrich, st. louis, mo). wells were blocked with µl of % bsa diluent/blocking solution (kpl) in distilled water for h at rt. bal-f ( µl) was added and incubated for h at • c. env-specific igg derived from purified serum igg obtained from siv mac -infected macaques and quantified as previously described ( ) was used to generate a standard curve for env-specific igg. purified rhesus igg (non-human primate reagent resource) was used to generate a standard curve for total igg. plates were washed times with x wash solution (kpl). horseradish peroxidase-labeled goat anti-monkey igg ( µl at a : , dilution; alpha diagnostics) was added, and plates were incubated for h at • c. after washing as described above, µl of , ′ , , ′ -tetramethylbenzidine (tmb) peroxidase substrate (kpl) was added for - min at rt. color development was stopped with µl m phosphoric acid (sigma), and plates were read at nm by using a biotek plate reader. siv gp -specific antibody levels were expressed as ng gp -specific igg/µg total igg. statistical analysis was performed using one-way anova or way anova with the tukey multiple comparisons test as indicated in the figure legends. the wilcoxon matched-pairs signed rank test was also used where indicated. correlation analyses were performed by non-parametric spearman correlations. statistics were generated using graphpad prism. rhesus macaques were infected with siv mac and bal was collected at , , , , and -wpi. nineteen macaques were sampled at -wpi and for the subsequent time points. bal samples were also obtained from naïve macaques. ams from bal were characterized by flow cytometry as hla-dr hi , cd b int , and cd + cd + ( ) (figure a) . the mean frequency of ams was > % of leukocytes found in the lavage samples, consistent with other reports on bal specimens from naive rhesus macaques ( ) . no significant change in the frequency of ams was observed over the weeks of infection ( figure b) . expression of cd c and cd has been shown to identify am phenotypes with changes in expression occurring during the fibrotic stage of lung disease ( ) here, no significant changes in am expression of cd c or cd over the time period studied were observed (figures c,d) . we also did not observe changes in the percentage of ams expressing the costimulatory molecules, cd and cd , indicating that any inflammatory responses recorded may not be associated with the expansion of these cell subsets (figures e,f) . macrophages secrete proinflammatory cytokines and chemokines in response to hiv infection ( , ) . however, a clear dynamic of the cytokine and chemokine response by macrophages over the course of hiv/siv infection has not been shown. here we characterized cytokine levels in am collected from siv infected rhesus macaques between and -wpi and compared them to levels in ams from naïve macaques. a strong proinflammatory response was seen during the acute phase of infection. expression of tnf-α, il- , and il- β in ams was significantly increased by -wpi (p < . for all; figures a-c) . by -wpi expression levels were significantly lower. cytokine expression remained low until the -wpi time point except for il- expression, which increased significantly by -wpi compared to -wpi ( figure b) . the il- expression level at -wpi was not significantly higher than levels seen in naïve populations. similarly, a higher level of the chemokine rantes was found in ams from macaques at -wpi ( figure e) . expression decreased to levels comparable to naïve samples by -wpi and remained low until the -wpi time point (figure e ). mip- α expression did not change over the course of infection ( figure d ). overall, we observed a transient increase in the proinflammatory cytokine responses, which decreased to levels comparable to those of naïve macaques during the chronic phase of infection. to assess whether ams were leaning toward an anti-inflammatory role during chronic infection, we evaluated the intracellular expression of il- by ams ( figure f ). ams play an important role in maintaining an anti-inflammatory environment in the lung ( , ) . indeed, il- expressing ams were detected in naïve animals and the frequency of il- + cells was significantly higher compared to that of acutely infected animals ( figure f) . interestingly, the percentage of il- expressing ams further decreased significantly as infection progressed into the chronic stage ( figure f) . thus, siv infection was associated with a significant spike in the am proinflammatory response by -wpi, which waned in chronic infection. in addition, the low proinflammatory cytokine response in chronic infection was not associated with an increase in il- -expressing ams. to investigate am activation, bal cells obtained from naïve and acute and chronically infected macaques at weeks , , , , and wpi, were incubated with native gp from r tropic siv or lps for h, and intracellular expression of mip- β and il- was assessed (figures a,b) . both gp and lps induced comparable levels of mip- β and il- in ams from naive and acutely-infected animals (figures a,b) . however, in chronically infected macaques gp did not induce intracellular il- or mip- β expression in ams (figures a,b) , and lps did not stimulate expression of either factor to the level induced in naïve animals suggesting a dysfunctional state of the ams (figures c,d) . to assess whether ams contribute to clearance of siv, we evaluated their phagocytic activity and ability to perform adp over the course of siv infection using bal samples from naïve animals and siv-infected macaques sampled between and -wpi. while the bal samples contained a high percentage of ams (figure b) , the cells were further enriched for ams using macs by depleting cd , cd , and epcam positive cells ( figure a) . to assess phagocytic capacity independent of siv antibody, enriched cells were incubated with siv mac gp -coated fluorescent beads and bal-f from naïve macaques prior to quantification of bead uptake. a significant increase in phagocytic activity was detected during the acute phase, and -wpi (figure b) . at -wpi, phagocytosis decreased and was comparable to naïve cells. activity further decreased significantly at the and -wpi time points compared to the acute and naïve time points (figure b ). prior to evaluating adp by ams, we assessed siv gp specific igg in bal-f ( figure c) . specific igg was detected at -wpi with levels maintained or continuing to rise until -wpi. to determine siv-specific adp, ams from naïve macaques or sampled over the course of siv infection were incubated with antigen-coated beads along with autologous bal-f. phagocytosis scores were normalized against the score obtained using naïve bal-f to highlight phagocytic activity associated with the presence of siv-specific antibody ( figure d) . significantly higher phagocytic scores were detected at -wpi compared to those at naïve and -wpi time points, indicating that ams could perform siv-specific adp ( figure d) . to further assess adp by ams in chronic infection, phagocytic scores at chronic time points (figure d ) were normalized against siv-specific antibody levels ( figure c) . results showed that in the presence of comparable antibody levels, ams mediated adp activity at -wpi. however, this activity was significantly compromised by week pi ( figure e) . thus, am are capable of gp specific adp for a limited time period during chronic infection. this diminished functional capacity of ams in the chronic stage further highlights the dysfunction observed upon lps and gp stimulation (figure ) . three classes of fcγr are constitutively expressed on ams with an active role in protecting against pathogens in the airway: high-affinity fcγri (cd ) and low-affinity fcγrii (cd ) and fcγriii (cd ) ( ) . fcγriii occurs in two isoforms: fcγriiia (expressed mainly on nk cells and macrophages) and fcγriiib (expressed on neutrophils) ( ) ( ) ( ) . infected mdms have shown compromised expression of the γ signaling chain of fcγriiia, possibly leading to altered phagocytic activity ( ) . monocytes from chronically infected individuals have also shown reduced expression of fcγriiia and diminished phagocytic activity ( ) . however, thus far no correlations have been found between adp and hiv/siv viral load, cd count or fcγr expression. given the unique features of ams compared to other tissue macrophages and monocytes, and the importance of phagocytic function in the lung, we investigated whether there was altered fcγr expression on ams over the course of siv infection and any association with adp. bal from naive macaques contained over % of fcγri + and fcγrii + ams (figures b,c) . both the frequencies and mfi of fcγrii and fcγri remained high and unchanged over the course of infection (figures b,c,e,f) . in contrast, the mean frequency of fcγriii + cells was % in naïve macaques. at -wpi there was a significant increase in fcγriii + am frequency but levels significantly decreased by -wpi (figure a) . the low frequency of fcγriii + ams was maintained until -wpi. a similar increase in am fcγriii mfi was not observed in the acute phase of infection compared to naïve animals ( figure d ). however, expression levels were significantly reduced at the and -wpi time points compared to levels at -wpi ( figure d ) in concert with the decreased frequencies observed. the siv-specific adp observed at -wpi ( figure e ) exhibited a strong trend toward positive correlation with the percentage of fcγriii + ams at the same time point ( figure g) . further, the fcγriii mfi at -wpi ( figure d ) negatively correlated significantly with acute plasma viral load ( figure h) . these data suggest that fcγriii plays an important role in siv-specific adp and that its expression is associated with early viral load control. to characterize the dysfunction observed in ams from chronically infected macaques, we looked for parallels with t cell exhaustion. since the initial description of t cell exhaustion in chronic viral infections ( , ) , common phenotypic and functional properties have been attributed to a dysfunctional state regardless of the type of pathogen. high and prolonged expression of inhibitory receptors are a key feature of t cell exhaustion in chronic viral infections ( ) . in particular, overexpression of the pd- receptor on cd + t-cells plays a major role in t cell dysfunction associated with hiv infection ( ) ( ) ( ) . here, we identified pd- + ams in rhesus macaque bal and assessed changes in its expression on ams from macaques over the course of siv infection (figure a) . when the frequency of pd- + ams was assessed in naive macaques and compared to frequencies in siv infected animals, five out of six siv + macaques showed an increase in frequency (figure b) . the pd- + am frequency at -wpi was significantly different from both naïve and -wpi time points: p = . and p = . , respectively. ams at -wpi also displayed a significant increase in pd- mfi signifying increased expression of the receptor ( figure c) . furthermore, the percentage of pd- + ams correlated positively with viral load at -wpi ( figure d) . we also observed a strong trend toward correlation between pd- mfi and viral load at -wpi (figure e) , indicating an association between pd- expression in ams and siv disease progression. pd- has two well-known ligands via which inhibitory effects have been reported to occur: programmed death -ligand (pd-l ) and programmed death ligand (pd-l ). while pd-l has been shown to be expressed broadly in both hematopoeitic and non-hematopoeitic cells, a more restricted expression pattern has been documented for pd-l , namely antigen presenting cells (apc) ( ) . in order to identify a potential role for the pd- pathway on am function, we looked for expression of these ligands on ams after siv infection. ams from naïve macaques predominantly expressed high levels of pd-l (figure b) . this expression decreased modestly, but not significantly, after siv infection and remained consistent thereafter. on the other hand, we observed minimal expression (∼ fold lower than that of pd-l ) of pd-l on ams from naïve macaques ( figure a) . further, pd-l expression significantly decreased over the course of siv infection. these results indicate that am dysfunction is more likely associated with increased pd- expression rather than changes in the expression of its ligands and may occur through the pd- /pd-l pathway. to identify whether pd- blockade can rescue am phagocytosis, we examined the phagocytic activity of ams from chronically infected macaques in the presence of pd- blocking antibody or mouse igg as control. interestingly, we found higher phagocytic scores in the pd- blockade group in out of the animals (p = . ), indicating that pd- expression is likely mechanistically linked to impairment of phagocytic activity (figure c) . we further assessed cytokine expression of pd- expressing ams. we stimulated ams from chronically infected macaques with µg/ml lps and compared cytokine expression between pd- + and pd- − populations. although not significant, we found lower expression of tnf-α on pd- expressing cells compared to the pd- − population ( figure d) . comparisons with additional cytokines and chemokines (il- , il- β and rantes) are not reported here as expression of these cytokines could not be detected in the ams from the infected macaques that were tested. taken together, these data strongly suggest that pd- expression on ams during siv infection may be a marker of dysfunction. our results show that in the siv macaque model, the pro-inflammatory response of ams to siv infection peaks in the acute phase of infection and is diminished in the chronic phase as observed following stimulation with lps or gp . we report that ams from siv infected macaques can perform adp early in infection, but this activity diminishes in the chronic phase. in novel findings, we report an elevated percentage of pd- + ams in chronically infected macaques, which positively correlated with viral load. during the same period diminished inflammatory responses and adp were observed. the percentage of ams stayed consistent over the course of siv infection (figure b) . viral infections can lead to activation of macrophages and subsequent release of factors that recruit additional cells of the innate and adaptive immune system ( , ) . the lack of detection of macrophage recruitment to the lung may have resulted from a concurrent lymphocyte recruitment, making the relative number of macrophages appear consistent. indeed, a higher percentage of lymphocytes in bal of hivinfected patients has been reported ( ) . here, estimating actual am numbers by multiplying cell count with percentage of ams did not result in significant differences ( figure s ) . including absolute counting beads as part of the initial analysis might have provided more definitive answers. variations in surface marker expression of ams over weeks of siv infection were also not seen, except for changes in fcγriii expression (figures , ) . phenotypic comparisons of ams from the bal of hiv-infected and uninfected individuals also have not shown differences ( ) . proinflammatory cytokines il- and il- β, tnf-α, and chemokine rantes was induced in ams during acute infection (figures a-e) . the rapid secretion of cytokines and chemokines was expected as the innate immune system initiates lymphocyte recruitment to establish adaptive immunity prior to viral spread. although the hiv/siv infection rate of ams may be low, macrophages respond to exposure to viral particles or virus-derived gene products such as nef, tat, and the gp envelope ( , ) . following the acute phase, the elevated cytokine and chemokine responses by ams were not sustained despite exposure to viral products and likely lps from the gut ( ) . further, stimulation with lps or native gp ex vivo showed an impaired il- and mip- β response of ams from chronically infected macaques (figure ) , indicating a potentially compromised inflammatory response against lung pathogens. this novel observation is consistent with data showing inhibition of tnf-α release by macrophages in response to toll-like receptor (tlr)- stimulation during hiv infection, even though the expression level of tlr- remained unchanged ( ) . desensitization of tlr ligands on ams as a result of viral respiratory infections has also been reported ( ) . the β-chemokines mip- α, mip- β, and rantes can bind to the hiv- coreceptor ccr indicating a potential protective effect against entry of r -tropic viruses ( ) ( ) ( ) ( ) . the pattern of chemokine expression here shows that any protective effect of β-chemokines expressed by ams is likely limited to the acute phase of infection (figures , ) . the lowering of cytokine and chemokine responses after the acute phase of infection was not associated with increased il- expression (figure f ). in fact, ams of naïve macaques expressed the highest level of intracellular il- ( figure f) . some studies have shown no induction of il- secretion or mrna in macrophage-tropic hiv- infected mdm compared to uninfected cells ( ) . others have shown increased il- secretion and mrna levels for in vitro hiv-infected pbmc, monocytes and mdms [reviewed in ( ) ]. differences in virus tropism or target cells could account for the variations observed. increased levels of il- have been detected in the bal-f of hiv infected patients ( ) , however the il- in these bal samples could have originated from other lymphocytes that upregulate il- during hiv infection ( ). in sum, our data indicate that siv disease progression is not associated with increased am il- expression, suggesting that ams are not switching to a more regulatory phenotype. ams are essential for lung microbial clearance. despite reports that am function is not affected by hiv- infection ( , ) , recent studies have shown that phagocytic activity can be impaired ( , ) . as viral load and activation status of macrophages can vary over the course of infection, here we tracked the phagocytic activity of ams following siv infection, providing new insights into the dynamics of am function. ams readily internalized antigen coated beads during acute infection, but this ability markedly decreased in chronically infected animals ( figure b ). the initial phagocytic activity was non-specific, as no gp -specific antibody was present, and was likely due to the rise in proinflammatory responses during acute infection (figures a-e) . adp was observed in the chronic stage of infection when gp specific antibody levels increased in bal-f (figures c,d) . however, a continued rise in gp antibody levels was not associated with increased adp. in fact, decreased adp activity was seen later in chronic infection. therefore, even though the pattern of chemokine expressing ams over the course of siv infection (figures a-e) suggested that frequencies returned to naive levels during the chronic phase of infection, the diminished functional activity observed suggested am dysfunction. fcγriii expression was elevated during acute siv infection but decreased as infection progressed (figures a,d) . these data are partly in contrast to data from hiv-infected mdms that showed either elevated or no change in fcγr expression [reviewed in ( ) ]. the fcγriii isoform expressed in macrophages (fcγriiia) consists of a ligand bindingα-chain associated with disulfide-linked γ-chains ( , ) . the γ signaling subunit of fcγrs has been shown to be downregulated as a consequence of hiv infection, resulting in aberrant downstream signaling required for phagocytosis ( , ) . the fcγriii antibody used here recognizes the ligand binding fcγriii α-chain ( ); thus the loss of fcγriii expression cannot be explained by downregulation of the γ-chain alone. our data suggest that in spite of an initial increase in the frequency of fcγriii + ams during acute infection (figure a) , prolonged hiv infection may lead to diminished expression of fcγriii. this outcome may not have been observed previously as in vitro mdm infection experiments mainly mimic acute stages of infection. nevertheless, although the frequency of fcγriii + ams at -wpi had dwindled to < % of the level observed in naïve animals, a correlative trend between the frequency of fcγriii + ams and adp was observed ( figure g) indicating the importance of this receptor in adp activity mediated by ams. furthermore, fcγriii mfi negatively correlated with acute viremia suggesting an important role for fcγriii-mediated phagocytosis in initial viral load control ( figure h) . the pd- /pd-l pathway plays a key role in negative regulation of adaptive immunity in hiv and other viral infections [reviewed in ( ) ], but few studies have explored the role of pd- in innate immunity, particularly by macrophages. pd- expression on t cells following immune activation and its role in t-cell exhaustion when highly expressed during hiv- and other chronic viral infections have been described ( , ). regarding macrophages, pd- expression has been linked to diminished ability to clear microbial invasion in septic mice ( ) , inhibition of phagocytic activity and tumor immunity ( ) , and inability to perform phagocytosis and intracellular killing in patients with tuberculosis ( ) . unlike on t cells, pd- expression on macrophages in these studies described a single positive population. here, we also identified a single pd- + population of ams derived from chronically infected macaques (figures c,d) . the macaques were clear of lung infections at the time of bal collection, suggesting pd- expression was only associated with siv infection. until now, no direct evidence has implicated pd- in am dysfunction. our data show a direct correlation between pd- and siv viremia and suggest that in keeping with correlations described with t cells ( ) , disease progression can also be associated with pd- expression in ams ( figure e) . while pd- ligands were found to be expressed on ams from naïve macaques, pd-l expression levels were low and subsequently declined after siv infection ( figure a ). this result was unexpected and in contrast to a study on mdms that showed up-regulation of pd-l and pd-l after exposure to inactivated hiv virions ( ) . however, the alveolar environment is indeed unique and it is unsurprising that am responses to siv infection differ from in vitro exposure of mdms to virions. rodriguez-garcia et al. further indicated differential regulation of pd-l and pd-l by il- , whereby presence of il- increased pd-l expression and its blockade increased pd-l expression ( ) . analysis of il- expression by ams in our study showed a gradual decrease after siv infection and may be one explanation as to why we observed decreased pd-l expression ( figure c) . pd-l expression, however, remained high and did not increase with decreased il- expression. future analysis of the factors found in bal-f could provide further insight into the dynamics of pd-l expression on ams. pd- blockade experiments have shown enhancement of siv/hiv-specific responses, proliferative ability and cytokine production by exhausted pd- high t cells ( ) ( ) ( ) . in keeping with this, blockade of the pd- /pd-l pathway has been reported to ameliorate phagocytic function in macrophages found in the tumor microenvironment and in active tuberculosis ( , ) . here, we also found that blockade of pd- could significantly improve phagocytic activity further highlighting pd- as a factor playing a role in dysfunction of ams ( figure c ). in addition, although not significant potentially due to small sample size, we found lower abundance of tnf-α expressing pd- + ams compared to the pd- − population during chronic infection ( figure d) . these data suggested that the presence of pd- on ams is likely a factor in any inhibitory role exerted on ams through the pd- /pd-l pathway. in summary, our longitudinal investigation has provided important new information about the consequences of siv infection on ams, and in novel findings, propose a role for pd- , a well-recognized inhibitor of adaptive immune responses, on innate immunity against siv infection. all datasets generated for this study are included in the manuscript and/or the supplementary files. forty-nine female indian rhesus macaques used in this study were housed in the nci animal facility, bethesda, md, under protocol vb- . the nci facility is accredited by the association for assessment and accreditation of laboratory animal care international, and its animal care and use 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the binding epitopes of human cd (fc gamma riii) monoclonal antibodies. implications for ligand binding t cell exhaustion pd- expression in acute hepatitis c virus (hcv) infection is associated with hcv-specific cd exhaustion pd- expression by macrophages plays a pathologic role in altering microbial clearance and the innate inflammatory response to sepsis pd- /pd-l pathway inhibits m.tb-specific cd + t-cell functions and phagocytosis of macrophages in active tuberculosis expression of pd-l and pd-l on human macrophages is upregulated by hiv- and differentially modulated by il- enhancing siv-specific immunity in vivo by pd- blockade pd- blockade in rhesus macaques: impact on chronic infection and prophylactic vaccination responsiveness of hiv-specific cd t cells to pd- blockade pd- expression by tumour-associated macrophages inhibits phagocytosis and tumour immunity the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material key: cord- -zvc s authors: choudhary, ishita; vo, thao; bathula, chandra s.; lamichhane, richa; lewis, brandon w.; looper, jayme; jeyaseelan, samithamby; blackshear, perry j.; saini, yogesh; patial, sonika title: tristetraprolin overexpression in non-hematopoietic cells protects against acute lung injury in mice date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: zvc s tristetraprolin (ttp) is a mrna binding protein that binds to adenylate-uridylate-rich elements within the ′ untranslated regions of certain transcripts, such as tumor necrosis factor (tnf) mrna, and increases their rate of decay. modulation of ttp expression is implicated in inflammation; however, its role in acute lung inflammation remains unknown. accordingly, we tested the role of ttp in lipopolysaccharide (lps)-induced acute lung injury (ali) in mice. lps-challenged ttp-knockout (ttp(ko)) mice, as well as myeloid cell-specific ttp-deficient (ttp(myeko)) mice, exhibited significant increases in lung injury, although these responses were more robust in the ttp(ko). mice with systemic overexpression of ttp (ttp(Δare)) were protected from ali, as indicated by significantly reduced neutrophilic infiltration, reduced levels of neutrophil chemoattractants, and histological parameters of ali. interestingly, while irradiated wild-type (wt) mice reconstituted with ttp(ko) hematopoietic progenitor cells (hpcs) showed exaggerated ali, their reconstitution with the ttp(Δare) hpcs mitigated ali. the reconstitution of irradiated ttp(Δare) mice with hpcs from either wt or ttp(Δare) donors conferred significant protection against ali. in contrast, irradiated ttp(Δare) mice reconstituted with ttp(ko) hpcs had exaggerated ali, but the response was milder as compared to wt recipients that received ttp(ko) hpcs. finally, the reconstitution of irradiated ttp(ko) recipient mice with ttp(Δare) hpcs did not confer any protection to the ttp(ko) mice. these data together suggest that non-hpcs-specific overexpression of ttp within the lungs protects against ali via downregulation of neutrophil chemoattractants and reduction in neutrophilic infiltration. acute lung injury (ali) and its severe form, acute respiratory distress syndrome (ards), are serious health concerns due to a high rate of mortality ( ) . ali is characterized by elevated levels of proinflammatory mediators, exaggerated neutrophil recruitment, and compromised pulmonary epithelial-endothelial barrier, resulting in increased vascular permeability ( ) . non-cardiogenic pulmonary edema, characterized by excessive accumulation of protein-rich edematous fluid and inflammatory cells in the alveolar spaces, results in hypoxemia in ards that requires aggressive clinical management including mechanical ventilation ( ) . despite significant health burdens posed by these diseases, the identity of key cellular and molecular players of host defense against ali remains unclear. zinc finger protein (zfp ), commonly known as tristetraprolin (ttp), is an mrna binding protein that binds to adenylate-uridylate-rich elements (ares) within the untranslated regions ( utrs) of target mrnas and increases their rate of decay ( ) . germline ttp-knockout (ttp ko ) mice exhibit the spontaneous development of a systemic inflammatory syndrome characterized by cachexia, erosive arthritis, myeloid hyperplasia, dermatitis, conjunctivitis, and autoimmunity ( ) . these phenotypes were shown to be essentially completely prevented in ttp ko mice with either tnf receptor deficiency, or when ttp ko mice were treated with anti-tnf antibodies ( ) . biochemical studies demonstrated that ttp binds to ares within the tumor necrosis factor (tnf ) mrna utr and results in tnf mrna degradation under normal conditions ( , ) . subsequent reports have shown that a number of other proinflammatory mediators including cxcl ( , ) , cxcl ( ), il- ( ), il- ( ) , ccl ( ) , and il- ( ) are also regulated by ttp ( ) . recently, using a systemic ttp overexpression (ttp are ) mouse model, we demonstrated protective effects of enhanced ttp levels in chronic immune-mediated inflammatory diseases including mouse models of arthritis, psoriasis, and autoimmune encephalomyelitis ( ) . ttp are mice lack ares in the utr of the endogenous ttp gene (zfp ) that results in increased stability of ttp mrna and, in turn, moderately increased expression of ttp protein in essentially all the tissues ( ) . together, these studies have indicated that ttp may be an endogenous anti-inflammatory protein and that enhancing its levels may be beneficial against various chronic inflammatory diseases. in the present study, we investigated the role of ttp in regulating lung inflammation in a mouse model of ali. using an oropharyngeal aspiration approach, lipopolysaccharide (lps)induced ali was modeled in adult mice, and the animals were monitored for signs of ali. to identify the protective role of ttp in a cell-specific manner, we performed bone marrow irradiation and reconstitution experiments in wild-type (wt), ttp ko ( ) , and systemic ttp overexpression (ttp are ) mice ( ) . our findings elucidate cell-specific roles of ttp in protection against ali, and indicate that ttp is an important modulator of endotoxin-induced ali. zfp floxed mice (zfp flox/flox ) ( ) were crossed with lysmcre recombinase expressing mice ( ) to generate mice for experimental (cre +/+ /zfp flox/flox ; ttp myeko ) and control (cre −/− /zfp flox/flox ; cre +/+ /zfp wt/wt ) groups. genotype status of progeny was determined by polymerase chain reaction (pcr) as described previously ( ) . ttp knockout mice (ttp ko ) and ttp overexpression mice (ttp are ) have been described before ( , ) . all the animal experiments were performed in accordance with principles and procedures outlined in the national institute of health guide for the care and use of laboratory animals and were approved by the louisiana state university animal care and use committee. both male and female adult ( - -week-old) mice were used for experiments. mice were anesthetized with isoflurane/oxygen followed by administration of µg lipopolysaccharide (lps) from escherichia coli o :b (l , sigma-aldrich) per mouse dissolved in sterile endotoxin-free saline ( µl total volume), or an equivalent volume of sterile endotoxin-free saline as a vehicle control, via oropharyngeal aspiration ( ) . mice were observed for signs of distress including anorexia, weight loss, hunched posture, ruffled haircoat, labored breathing, and dehydration every - h post lps challenge. mice exhibiting at least four of these clinical signs were humanely euthanized before the end of the study. following saline or lps treatment, mice were anesthetized with , , -tribromoethanol (sigma-aldrich, st. louis, mo, united states) at the indicated time points, and mid-line laparotomy was performed. briefly, bronchoalveolar lavage fluid (balf) was harvested from the right lung. recovered balf was processed and analyzed for total and differential cell counts by routine methods ( ) . unlavaged left lung lobes were fixed in % neutral buffered formalin (nbf) and used for preparation of slides for histopathological evaluation. right lung lobes were snap-frozen and stored at − • c. bronchoalveolar lavage fluid was harvested and centrifuged at × g for min, and the supernatant was stored at − • c for further analyses. the cell pellet was resuspended in µl of pbs and total cell counts were determined using a hemocytometer (brightline, horsham, pa, united states). cytospins were prepared using µl of cell suspension (statspin cytofuge ; hemocue, brea, ca, united states) followed by differential staining (modified giemsa kit; newcomer supply, middleton, wi, united states). mouse cytokine and chemokine levels were assayed in cell-free balf supernatant using luminex-xmap-based assay (mcytomag- k), according to the manufacturer's instructions (emd millipore, billerica, ma, united states). five micrometer sections of lung were stained with hematoxylin and eosin (h&e) for routine histology. histology: a semiquantitative histopathological scoring system was used to analyze the sections as follows: ( ) consolidation (percent of total surface area of lung section affected); ( ) bronchiolitis ( , no bronchioles affected; , one bronchiole affected; , between - bronchioles affected; , more than bronchioles affected); ( ) perivascular edema ( , minimal; , mild; , moderate; , severe); ( ) perivascular inflammation/inflammatory cells ( , absent; , minimal; , mild; , moderate; , severe); ( ) airspace edema ( , minimal; , mild; , moderate; , severe); ( ) airspace hemorrhages ( , absent, , patchy, mild; , extensive, moderate; , extensive, severe). slides were graded in a blinded manner without knowledge of sex and treatment groups. bone marrow transplantation experiments were performed as described previously ( ) . briefly, - -week old recipient mice were irradiated with megavolt x-rays from a linear accelerator (varian clinac ex) with two (dorsal and ventral) -rad ( cgy) doses. to prepare bone marrow cells for transplantation, femur bones of donor mice were flushed to collect bone marrows, and single cell suspensions were prepared. a total of × cells were injected into the tail vein of lethally irradiated recipient mice. reconstituted recipient mice were given . % neomycin sulfate dissolved in acidified water for the first weeks post-transplantation. lps-challenge experiments were performed weeks post bone marrow reconstitution, which has been previously shown to be an optimal period for repopulation of resident alveolar macrophages with donor cells following total body irradiation ( ) . lung tissue was lysed using pierce tm ripa buffer (thermo fisher scientific, waltham, ma, united states) supplemented with pierce tm protease inhibitor mixture (thermo fisher scientific, waltham, ma, united states) and phosphatase inhibitors ( mm sodium fluoride and mm sodium orthovanadate). tissues were mechanically homogenized using a bead beater (thermo fisher scientific, waltham, ma, united states). tissue lysates were centrifuged ( , × g, min, • c) to remove insoluble material and protein concentration of the supernatants was measured through bradford assay (bio-rad laboratories, hercules, ca, united states). equivalent amounts of denatured protein was separated on a - % bis-tris plus precast gels (invitrogen, carlsbad, ca, united states), transferred on to pvdf membrane (invitrogen, carlsbad, ca, united states) and probed with a : dilution of rabbit antiserum raised against a recombinant mouse ttp-maltose binding protein fusion ( ) followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit igg (bio-rad). signal was determined using supersignal west pico chemiluminescent substrate (pierce) on x-ray film. significant differences among groups were determined by oneway analysis of variance (anova) followed by tukey's post hoc test for multiple comparisons except for cytokine assays where two-way anova was used. measurements from two groups were compared using student's t-test assuming unequal variance. all data were expressed as mean ± sem. a p-value < . was considered statistically significant. statistical analyses were performed using graphpad prism . (graphpad software, la jolla, ca, united states). in order to explore the role of ttp in ali, ttp ko and littermate control wt mice were subjected to ali through oropharyngeal aspiration of endotoxin (lps) (single dose; µg lps/mouse) for a period of h. while saline-treated ttp ko and wt groups had comparable numbers of total immune cells in the balf (saline-treated wt; × ± × , saline-treated ttp ko , × ± × ), lps administration resulted in increased infiltration of immune cells in both the ttp ko and the wt groups. the total number of recovered immune cells in lps-challenged ttp ko mice ( × ± × ) were ∼ fourfold higher as compared to lps-challenged wt ( × ± × ) mice ( figure a) . increases in total cell counts in lps-challenged ttp ko mice were attributed to a significant increase in neutrophil ( figure b) , macrophage ( figure c) , and lymphocyte counts ( figure d ). these increases were associated with an increased injury to the pulmonary vascular barrier, as depicted by the presence of red blood cells in the cytospins prepared from the balf fluid of ttp ko mice ( figure e ; right panel, black arrow) versus control lps-challenged wt mice ( figure e ; left panel). histologically, the lungs of lps-challenged wt mice were characterized by mild to moderate consolidation (∼ % of total area of lung section), two-to fourfold increase in alveolar septal thickening (broken green arrow), moderate perivascular and airspace edema, and perivascular inflammation (figures f-h) . in contrast, the lung injury in lps-challenged ttp ko mice was characterized by severe consolidation (> % of total area of lung section) (figures f,g) that included infiltration of neutrophils, edema, fibrin, and airspace hemorrhage within the airway and alveolar lumen, multifocal loss of bronchiolar epithelium with infiltration of neutrophils and red blood cells within the bronchiolar lumen, and moderate to severe perivascular edema and inflammation (figures f-h) . of note, ∼ % lps-challenged ttp ko mice succumbed to lps challenge before -h and these had to be excluded from the analysis. these data suggest that systemic loss of ttp results in extreme susceptibility of mice to lpsinduced ali. in order to explore the role of myeloid cell-specific ttp on inflammatory response in ali, myeloid cell-specific ttp deficient mice (ttp myeko ; cre +/+ /zfp flox/flox ) and control (cre control; cre +/+ /zfp wt/wt and flox control; cre −/− /zfp flox/flox ) mice were challenged with lps. similar to saline-treated wt and ttp ko groups, saline-treated control and ttp myeko groups had comparable numbers of total . data are represented as mean ± sem. statistical analysis was performed by one-way anova followed by tukey's correction for multiple comparison test. *p < . ; **p < . ; ***p < . . n = each for wt and ttp ko saline controls; n = , wt group; n = , ttp ko group for lps-challenge group. three lps-challenged ttp ko mice succumbed to lps-challenge before h and were not lavaged for further analyses. representative photomicrographs of wright-giemsa stained balf cytospins of lps-challenged wt (e; left panel) and lps-challenged ttp ko (e; right panel) mice. neutrophil (red arrow), macrophage (green arrow), lymphocyte (blue arrow), red blood cell (black arrow) (original magnification × ). representative photomicrographs (f) from h&e-stained left lung lobe sections from adult lps-challenged wt (f; left panel) and lps-challenged ttp ko (f; right panel) mice. septal thickening (green broken arrow), intra-alveolar neutrophilic infiltrates (green arrow), intra-alveolar red blood cells (red arrow), bronchiolar lumen neutrophilic accumulation (blue arrow), and perivascular cellular infiltration (black arrow). asterisk represents alveolar space that is minimally affected (f; left) or severely consolidated with blood and neutrophils (f; right) (original magnification × ). semiquantitative histopathological scoring for consolidation (g) is shown as a percent of total surface area of the lung section affected in lps-challenged wt and lps-challenged ttp ko mice. semiquantitative histopathological scoring of lung sections for bronchiolitis, perivascular edema, perivascular inflammation, airspace hemorrhage, and airspace edema in lps-challenged wt and lps-challenged ttp ko mice (h). statistical analysis in g and h was performed using student's t-test. *p < . ; **p < . ; ***p < . ; ****p < . . immune cells (flox control; × ± × , ttp myeko ; × ± × ). lps administration resulted in increased numbers of immune cells in ttp myeko as well as both the control groups. this increase in total cell counts was, however, significantly greater in ttp myeko ( × ± × ) compared to cre control ( × ± × ) mice. the increase in cellular recovery did not reach statistical significance in lps-challenged ttp myeko when compared to the lps-challenged flox control group (p = . ) (figure a ). this effect was comparable in both sexes (data not shown). of note, the increase in cellular infiltration was ∼ threefold less in lps-challenged ttp myeko ( × ± × ) (figure a ) when compared to lps-challenged ttp ko mice ( × ± × ) ( figure a) . while neutrophil counts were comparable between lps-challenged ttp myeko and lpschallenged control mice (figures b,e) , macrophage counts were significantly elevated in the balf obtained from lps-challenged ttp myeko mice compared to the two groups of control mice (figures c,e) . lymphocyte counts did not differ between the lps-challenged ttp myeko and the two groups of control mice (figures d,e) . histopathological analysis revealed comparable levels of lung consolidation with widespread inflammatory cellular infiltrates within the airspaces of both lps-challenged ttp myeko and flox control mice; however, perivascular edema, perivascular inflammation, and airspace edema were somewhat exaggerated in lps-challenged ttp myeko mice compared to the flox control group (figures f-h) . unlike lps-challenged ttp ko mice, airspace hemorrhage was not observed in any of the groups. all the lps-challenged ttp myeko mice survived lps challenge, as compared to the lps-challenged ttp ko mice, in which ∼ % mortality was observed. these data indicate that myeloid cell-specific ttp is essential for protection against ali. mitigates lps-induced ali in mice during acute and sub-acute course of lung injury next, we examined whether the systemically ttp overexpressing (ttp are ) mice exhibit protection against ali. in experimental ali, while time points earlier than h of lps-challenge represent acute phases of ali, later time points represent somewhat sub-acute to chronic or resolution phases of endotoxin-induced ali in mice ( ) . therefore, we examined both lps-challenged wt and lps-challenged ttp are mice over time points representing acute to subacute phases, i.e., ± . × , . × ± . × , and . × ± . × cells at h, h, h, days, and days, respectively) ( figure a) . lipopolysaccharide administration resulted in increased numbers of total cells in balf from both wt and ttp are mice when compared to saline administration ( figure a) . total cell counts in balf from lps-challenged wt mice were . × ± . × , . × ± . × , × ± × , × ± . × , and . × ± . × at h, h, h, days, and days time points, respectively ( figure a) . interestingly, significantly reduced numbers of immune cells were recovered from the lungs of lps-challenged ttp are mice compared to lungs of lps-challenged wt mice at all time points examined post lpschallenge ( . × ± . × , . × ± . × , . × ± . × , . × ± . × , and . × ± . × at h, h, h, days, and days, respectively) ( figure a) . the decrease in total cell counts in lps-challenged ttp are mice was contributed by significantly reduced numbers of neutrophils at , , and h (figures b,e) . interestingly, however, the total numbers of macrophages were only significantly different between lps-challenged wt and lps-challenged ttp are mice at h and days time points (figures c,e) . lymphocyte counts were not significantly different in lps-challenged ttp are mice compared to lps-challenged wt mice (figures d,e) . cellular counts followed the same trend in both the sexes (data not shown). reduced cellular infiltration was also evident in cytological slides prepared from lps-challenged wt and lps-challenged figure | myeloid-ttp deficiency exacerbates lps-induced ali in mice. total cell counts (a) in the harvested balf of adult saline-(white bars; n = each for flox control and ttp myeko ) or lps-challenged cre control (cre +/+ /zfp wt/wt , gray bar; n = ), flox control (cre -/-/zfp flox/flox , purple bar; n = ), and ttp myeko (cre +/+ /zfp flox/flox , green bar; n = ) mice. differential cell counts are shown for neutrophils (b), macrophages (c), and lymphocytes (d). data are represented as mean ± sem. statistical analysis was performed by one-way anova followed by tukey's correction for multiple comparisons. ns, non-significant; *p < . ; ***p < . ; ****p < . . representative photomicrographs of wright-giemsa stained balf cytospins from lps-challenged flox control (e; left) and ttp myeko (e; right) mice. neutrophil (red arrow), macrophage (green arrow), lymphocyte (blue arrow) (original magnification × ). representative photomicrographs from h&e-stained left lung lobe sections from post- h lps-challenged flox control (f; left) and ttp myeko (f; right) mice (original magnification × ). asterisk represents alveolar spaces minimally obliterated in flox control (f; left) and severely obliterated in lps-challenged ttp myeko mice (f; right). bronchiolar lumen neutrophilic infiltrates (blue arrow), absent in lps-challenged flox control (f; left) but present in lps-challenged ttp myeko (f; right). semiquantitative histopathological scoring for consolidation (g) is shown as a percent of total surface area of the lung section affected in lps-challenged flox control and lps-challenged ttp myeko mice. semiquantitative histopathological scoring of lung sections for bronchiolitis, perivascular edema, perivascular inflammation, and airspace edema in lps-challenged flox control and lps-challenged ttp myeko mice (h). n = (flox control); n = (ttp myeko ). statistical analysis in g and h was performed using student's t-test. *p < . . ttp are bal fluid, which showed the sparsely present neutrophils, macrophages, and lymphocytes in lps-challenged ttp are mice compared to dense presence of these cells in lps-challenged wt mice ( figure e) . histologically, lpschallenged wt lungs exhibited ∼ % lung consolidation with significantly increased infiltration of immune cells within the airspaces, bronchiolitis, perivascular edema, and inflammation (figures f-h) . in contrast, bronchiolitis and perivascular edema were significantly attenuated in lps-challenged ttp are mice. lung consolidation, perivascular inflammation, and airspace edema were not significantly different between the two groups, and airspace hemorrhage was not observed in any group (figures f-h) . we next analyzed the changes in the protein expression levels of ttp over the course of ali in wt and ttp are mice whole lung tissue. as shown in figure i , basal expression levels of ttp were higher in ttp are versus wt lungs. upon lps administration, the expression levels of ttp increased in both wt and ttp are mice lungs by h post lps administration. this increase was significant in ttp are mice lungs as compared to unchallenged wt mice lungs. the levels of ttp started reducing at subsequent time points in both wt and ttp are lungs. while the ttp expression decreased to basal levels in wt mice, the ttp expression remained at relatively higher levels in the ttp are mice lungs at all subsequent time points. these data show that the presence of higher levels of ttp under basal conditions, combined with a significant increase at h post-lps challenge and maintenance of higher expression levels at later time points post lps challenge, protects ttp are mice from ali. cellular recruitment within the lung in response to lps challenge is mediated by the production of chemoattractants. therefore, next we sought to examine the levels of inflammatory cytokines and chemokines within the balf of lps-challenged wt and lps-challenged ttp are mice. of the cytokines/chemokines analyzed, four cytokines/chemokines, including g-csf, kc, il- , and il- p , were found to be significantly different between the lps-challenged wt and the lpschallenged ttp are mice. g-csf was significantly reduced in lps-challenged ttp are compared to lps-challenged wt mice at and h; kc and il- were significantly reduced at h; and il- (p ) was significantly reduced at h post-lps challenge (figures a-d) . interestingly, the levels of tnfα ( figure e ) and mip (figure f) , two known ttp targets, were not significantly different between the two groups. to determine the cell-specific role of ttp levels in ali, we modulated ttp levels in hematopoietic progenitor cells (hpcs) and non-hpcs. in order to test whether donor hpcs repopulate the recipient mouse lungs, we first made bone marrow chimeras in which total body irradiated wt mice were transplanted with hpcs from a mouse expressing green fluorescent protein (gfp) in their somatic cells. we found that greater than % of the cells recovered from the balf of these mice were gfp positive (data not shown). next, we generated three bone marrow chimeras in which irradiated wt recipient mice received hpcs from either wt (wt→wt), ttp are (ttp are →wt), or ttp ko (ttp ko →wt) mice ( figure a) . while saline-treated ttp ko →wt chimeras had no signs of cellular infiltration that included total cells (figure b) , neutrophils (figure c) , macrophages (figure d) , and lymphocytes (figure e) , lps-challenged wt→wt, ttp are →wt, and ttp ko →wt chimera mice had significant cellular recruitment, as indicated by balf total cellular counts (figure b) , neutrophils (figure c) , and macrophages ( figure d) . while lpschallenged ttp are →wt mice had somewhat reduced cellular infiltration as compared to lps-challenged wt→wt chimeras (figures b-d) , the lps-challenged ttp ko →wt chimera mice had remarkably higher number of bal cellular counts (figures b-d) . lymphocyte counts did not differ significantly between the three groups ( figure e) . histologically, ∼ , , and % of the lung parenchyma was consolidated in lps-challenged wt→wt, lps-challenged ttp are →wt, and lps-challenged ttp ko →wt group, respectively (figures a top panel, c) . consolidated parenchyma was characterized by the presence of large infiltrates of neutrophils and macrophages within the airspaces (alveolar and airway). other histological findings included the presence of edema and immune cells within the perivascular spaces, bronchial lumen cellular infiltrates, airspace edema, and occasional bronchoalveolar lymphoid aggregates (figures d-g) . these data suggest that while baseline expression of ttp in hpcs is essential for protection against exaggerated ali, its overexpression in these cells does not confer significant additional advantage. next, we generated bone marrow chimeras in which irradiated ttp are recipient mice received hpcs from either wt (wt→ttp are ), ttp are (ttp are →ttp are ), and ttp ko (ttp ko →ttp are ) mice ( figure a) . as compared to wt recipient chimera counterparts, lps-challenged wt→ttp are , lps-challenged ttp are →ttp are , and lps-challenged ttp ko →ttp are chimeras had significantly lower degrees of cellular recruitment, that included total cells ( figure b , blue solid bar, orange solid bar, green solid bar), neutrophils ( figure c , blue solid bar, orange solid bar, green solid bar), and macrophages ( figure d , blue solid bar, orange solid bar, green solid bar). lymphocyte counts were significantly reduced in lps-challenged ttp are →ttp are compared to the lps-challenged wt→wt chimeras ( figure e ). although the lps-challenged ttp ko →ttp are chimeras had significantly higher cellular recruitment as compared to lps-challenged wt→ttp are and lps-challenged ttp are →ttp are chimeras, the extent of recruitment was much diminished in lps-challenged ttp ko →ttp are chimera as compared to lps-challenged ttp ko →wt chimera ( figure b) . histologically, ∼ , %, % of the lung parenchyma was consolidated in lps-challenged wt→ttp are , lpschallenged ttp are →ttp are , and lps-challenged ttp ko →ttp are , respectively (figures a bottom panel, figure c ). histologically, mild increases in septal thickening with cellular infiltration, mild bronchiolitis, perivascular edema, inflammation, and airspace edema, and occasional balts were evident in all the three groups (figures d-g, solid blue, orange, and green bars). these data suggest that enhanced expression of ttp in lung non-hpc populations conferred significant protection against ali. further, this protection is somewhat compromised in the absence of baseline levels of ttp in hpcs. a tabular summary of differential cellular and ali responses in various chimeric mice is included in supplementary table . finally, we generated bone marrow chimeras in which irradiated ttp ko recipient mice received hpcs from ttp are (ttp are →ttp ko ). as expected, the cellular counts were significantly higher than any of the other lps-challenged chimeras (figures b-e, red solid bar) . however, total cellular counts in lps-challenged ttp are →ttp ko chimera were ∼ twofold lower than the lps-challenged ttp ko mice (figure ) . additionally, none of the lps-challenged ttp are →ttp ko chimeras succumbed to ali, as compared to % mortality in lps-challenged ttp ko mice (figure ) . these data suggest that complete loss of ttp in lung non-hpc populations significantly exaggerates ali, and that overexpression of ttp in hpcs may provide partial protection in severe ali. histologically, this group exhibited the most severe lung injury, characterized by ∼ % lung consolidation with neutrophils, macrophages, fibrin, and edema, severe bronchiolitis, and moderate to severe bronchiolar and alveolar hemorrhages (figures b-g) . tristetraprolin knockout (ttp ko ) mice exhibit a systemic inflammatory syndrome that is characterized by cachexia, polyarticular synovial arthritis, dermatitis, and myeloid hyperplasia ( ). however, the lungs of ttp ko mice exhibit very little spontaneous inflammation, characterized by the presence of rare foci of leucocytic infiltrates within the pulmonary parenchyma ( ) . these rare leucocytic infiltrates are abrogated upon combined deletion of ttp and tnf receptors, indicating the role for tnf activity in leucocytic infiltration ( ) . myeloid cell-specific loss of ttp (ttp myeko ) does not recapitulate the ttp ko phenotype, indicating that non-myeloid cells are required for the overall manifestation of the ttp deficiency syndrome ( ) . however, ttp myeko mice were found to be hypersensitive to endotoxin-induced systemic inflammation, particularly through exaggerated tnf production, delineating the critical role of myeloid cell-specific ttp in protection against systemic injury and inflammation ( ) . the role of ttp in localized lung inflammation, however, has remained unexplored. therefore, in this study we sought to explore the role of ttp in ali. we hypothesized that ttp modulates acute lung inflammation and that cell-specific modulation of ttp levels will differentially affect the outcome of acute lung inflammation. the unchallenged ttp ko mice display minor degrees of leukocytic infiltration in lung parenchyma that were thought to be contributed by tnf activity ( ) . here, in lps-challenged ttp ko mice, immune cell infiltration was ∼ fourfold higher as compared to lps-challenged wt mice. in fact, the susceptibility of ttp ko mice to lps-induced ali was so severe that ∼ % ttp ko mice succumbed to lps-challenge within - h post-lps administration, while no mortality was observed in lpschallenged wt mice. the surviving ttp ko mice displayed severe pulmonary pathology with exaggerated airspace and interstitial neutrophilic infiltration, exaggerated edema, vascular congestion, and lung injury that included epithelial and endothelial damage. it is likely that the increased production of inflammatory mediators, that are otherwise regulated by ttp, in lpschallenged ttp ko mice, contribute to their worsened pulmonary pathology. one such mediator, tnf, is already established to be highly secreted in ttp ko mice ( , ) . macrophages are the primary source of tnf in ali ( , ) although alveolar epithelial cells have also been suggested to release tnf in lps-induced lung inflammation ( ) . accordingly, we reasoned that, if macrophages are the primary source of tnf, deletion of ttp in myeloid cells would enhance its activity, leading to worsening of pulmonary pathology. although the lps-challenged ttp myeko mice had exaggerated pulmonary pathology as compared to lps-challenged wt mice, the extent of tissue damage and neutrophilic infiltration was not as severe as in lps-challenged ttp ko mice. these differences in the sensitivity of systemic versus myeloid cell-specific ttpdeficient mice indicate that ttp in cells other than myeloid-cells may play critical roles in modulating endotoxin-induced ali. these data suggest that while loss of myeloid cell-specific ttp worsens the ali, ttp expression in non-myeloid cells confers significant protection. clinically, the numbers of neutrophils within the balf of patients with ards have been shown to correlate with the severity of disease and poor outcome ( , ) . despite being the first line of defense against pathogenic insults, excessive recruitment and activation of neutrophils leads to lung tissue damage and loss of lung function ( , ) . therefore, targeting neutrophilic recruitment through suppressed release of key neutrophil chemoattractants may be an attractive therapeutic strategy against ali. we observed correlations between ttp deficiency or ttp overexpression and neutrophilic inflammation. for example, balf counts and tissue infiltration of neutrophils were overwhelming in the lps-challenged ttp ko mice. these outcomes were also exaggerated, but to a lesser degree, in lps-challenged ttp myeko mice. on the other hand, these outcomes were significantly attenuated in the lps-challenged ttp are mice. our findings are in line . data are represented as mean ± sem. statistical analysis in c-g was performed by one-way anova followed by tukey's correction for multiple comparisons within the three recipient groups and student's t-test between the three recipient groups. *p < . ; **p < . ; ***p < . ; ****p < . . with previous reports in other tissues. for instance, massive infiltration of neutrophils has been shown to occur in the skin of ttp ko mice subjected to psoriasis-like inflammation ( ) , whereas reduced airway neutrophilic infiltration was shown in mice genetically modified to express constitutively active endogenous unphosphorylated ttp following challenge with cigarette smoke ( ) . a number of studies have shown that ttp is phosphorylated at multiple sites by p -regulated kinase mapk-activated protein kinase (mapkapk ) and that ttp activity is significantly affected by its phosphorylation status ( ) ( ) ( ) . ttp phosphorylation has been shown to result in reduced ttp activity/reduced binding of ttp to ares, thus resulting in stabilization of its target mrnas ( ) . regulation of ttp activity by p mapk was in fact shown to result in a biphasic response of tnfα-induced il- expression in human bronchial smooth muscle cells ( ) . conversely, dephosphorylation of ttp resulted in reduced expression of il- and il- in a lung epithelial cells ( ) . since lps is an inducer of both ttp and p mapk, ttp would be expected to undergo phosphorylation and inactivation shortly after lps challenge. however, the effect of ttp phosphorylation is transient and would be expected to be reversed upon dephosphorylation when p is turned off. although we did not track the phosphorylation status of ttp in various lung cells during ali, we speculate that the p -mediated phosphorylation of ttp occurs well before h post lps challenge and is reversed by h time point. consistent with our speculation, we found differential effect of ttp overexpression on late phase cytokines (kc) versus early phase cytokines (tnf) (figure ) . tristetraprolin is a known regulator of key neutrophil chemoattractants including cxcl /kc ( , ) and cxcl /mip ( , ) . cxcl /kc is a central chemokine in neutrophil recruitment into the airspace in ards ( ) ( ) ( ) . clinically, increased concentrations of il- (cxcl /kc homolog in human), disease severity, and neutrophil migration into airspaces have been shown to be correlated ( ) ( ) ( ) . cxcl mrna utr contains ttp-binding sites and has been previously demonstrated to be a direct target of ttp ( , ) . consistent with this, the lps-challenged ttp are mice had significantly lower cxcl /kc concentrations. in contrast, the two well characterized ttp targets, tnf and mip /cxcl , were not significantly different between lps-challenged wt and lpschallenged ttp are mice. these observations are in line with our previous report, in which no differences were observed in the levels of tnf and mip /cxcl in the serum of wt and ttp are mice systemically challenged with lps ( ). these two inflammatory mediators peak early (before h in the serum) ( ) , and we speculate that overexpressed ttp may be less effective at mrna decay due to its phosphorylation status at these early time points. g-csf, another neutrophil chemoattractant, is also consistently detected within the balf of ali and ards patients and has been suggested to be associated with the accumulation and activation of neutrophils in ards ( ) . plasma g-csf levels have also been shown to correlate with clinical outcome in patients with ali ( , ) . g-csf has also been shown to exacerbate bleomycin induced lung injury in rats through marked infiltration of activating neutrophils ( ) . g-csf levels have been found to be increased in ttp ko mouse plasma ( ) . although g-csf has not been shown to be a direct ttp target, g-csf mrna utr in mouse possesses two ttp binding sites, uauuuau. the balf g-csf levels were significantly reduced in lps-challenged ttp are mice. g-csf levels were also found to be significantly reduced in ttp are mice in a previous study where we showed ttp are mice to be significantly protected from exhibiting collagen-antibody induced arthritis ( ) . lipopolysaccharide-challenged ttp myeko mice exhibited milder ali as compared to lps-challenged ttp ko mice, indicating that total loss of ttp expression in non-myeloid as well as myeloid cells contributes to severe ali. on the other hand, systemic ttp overexpression conferred significant protection against lps-induced ali. here, we addressed two logical questions: ( ) whether enhanced levels of ttp in non-hpcs would ameliorate endotoxin-induced ali, ( ) whether enhanced levels of ttp in hpcs would confer protection against endotoxin-induced ali. towards this, employing various bone marrow chimeras, we investigated cell-specific roles of ttp in protection against ali. in these experiments, we modulated ttp levels in hpcs by using bone marrow donors that were either wt, ttp are , or ttp ko . to modulate ttp levels in non-hpcs, wt, ttp are , or ttp ko recipients were used. one limitation of this study was that since hpcs also include various non-myeloid populations, we were not able to specifically investigate the effect of ttp modulation in myeloid cells. the bone marrow irradiation and reconstitution experiments revealed somewhat unexpected but interesting findings. as compared to wt→wt chimera, the ali in ttp are →wt chimeras was somewhat attenuated, whereas the ali in ttp ko →wt chimeras had worsened significantly. these data suggest that, when the ttp expression in non-hpcs is genetically unaltered (wt ttp in the recipient's non-hpcs), the ttp overexpression in hpcs is partially protective against lps-induced ali. however, when the ttp expression was ablated in non-hpcs [ttp depleted in the recipient's non-hpcs (ttp are →ttp ko chimera)], the overexpression of ttp in the hpcs was not sufficient to confer protection against ali. as compared to ttp are →ttp ko chimeras, in ttp ko →ttp are chimeras, the mere overexpression of ttp in non-hpcs (ttp overexpression in the recipient's non-hpcs) provided significant protection, even though the hpcs were ttp-deficient. however, this protection was further enhanced when the normal levels of ttp were restored in the hpcs (wt→ttp are chimera). based on these data, we conclude here that the ttp levels in non-hpcs are critical in protection against ali. further, while the wt levels of ttp in hpcs are essential for additional protection, the overexpression of ttp in hpcs is not significantly advantageous. since the non-hpcs population in lungs consists of a multitude of cell types including epithelial cells, endothelial cells, and fibroblasts, the cell-specific role of ttp still remains elusive. contribution of non-hpc-specific ttp toward modulation of inflammatory responses has also been suggested in previous reports. for instance, ttp regulation of tnf in keratinocytes, but not in myeloid or dendritic cells, was shown to protect mice from exacerbation of psoriasis-like pathology, development of spontaneous systemic inflammation, and dactylitis ( ) . another study suggested a role of intestinal epithelial cell-specific ttp in exacerbation of acute colitis ( ) . along similar lines, ttp depletion in myeloid cells did not replicate the ttp-deficiency phenotype ( ) , and the spontaneous reactive granulopoiesis seen in ttp ko mice was suggested to be caused by a non-cell autonomous mechanism likely contributed by liver cells ( ) . all these studies indicated an essential role of ttp in non-hpcs in regulating inflammation. among various non-hpc cell types in the lung, alveolar epithelial cells produce pro-inflammatory cytokines and chemokines ( ) . in in vitro conditions, lpschallenged pulmonary type ii epithelial cells have been shown to produce greater levels of neutrophil chemoattractants [il- , a human homolog for cxcl (kc), cxcl (mip ), and cxcl (lix)] indicating that ttp in lung alveolar epithelial cells may play an important role in regulating ali ( ) . consistent with these reports, while our data indicate critical roles of non-hpcs in lungs in mediating neutrophil recruitment to the airspaces, the exact identity of these non-hpcs remain unknown. in conclusion, our results show that (a) enhancing the levels of ttp is protective against endotoxin-induced ali; (b) the protective effect seen in ttp are mice is attributable to reduced neutrophilic recruitment and, in turn, reduced lung damage; (c) reduced neutrophilic recruitment is attributed to reduced secretion of chemoattractants, particularly kc and g-csf; and that, (d) ttp in non-hpcs plays an essential role in protection against endotoxin-induced ali. based on these results, we propose a model in which endotoxin damages epithelial cells within the lung, which then initiates a cascade of events leading to exaggerated release of proinflammatory mediators and neutrophilic chemoattractants, resulting in further lung damage and neutrophilic infiltration (figure ) . in this process, ttp acts as an intracellular regulator for the expression of proinflammatory mediators and neutrophilic chemoattractants. ttp expression in the non-hpcs, i.e., most likely epithelial and endothelial cells, confers protection against endotoxin-induced ali via suppression of mrnas encoding proinflammatory mediators and neutrophilic chemoattractants. hence, strategies to increase ttp expression or activity in non-hpcs together with hpcs population may prove beneficial for patients with ali/ards. in future studies, ttp expression within the lung could also be investigated as a prognostic indicator for the severity of ali/ards. our studies could also have implications for the lung hyper-inflammation and potentially life-threatening cytokine storms in the severe coronavirus disease (covid- ). the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. the animal study was reviewed and approved by lsu iacuc. sp conceived and designed the study, maintained the animal colony, and performed histopathological analyses. ic, tv, and bl conducted animal necropsies. ys, cb, and rl performed balf cellularity assays. rl performed the intravenous injections. ys, tv, ic, and cb performed cytokine assays. jl performed the irradiations. sj provided technical expertise on bone marrow transplantations and reviewed the manuscript. pb provided various transgenic and knockout mice and edited the manuscript. sp and ys wrote and reviewed the manuscript for intellectual contents. all authors contributed to the article and approved the submitted version. the acute respiratory distress syndrome: pathogenesis and treatment acute lung injury: a clinical and molecular review tristetraprolin as a therapeutic target in inflammatory disease a pathogenetic role for tnf alpha in the syndrome of cachexia, arthritis, and autoimmunity resulting from tristetraprolin (ttp) deficiency roles of tumor necrosis factor-alpha receptor subtypes in the pathogenesis of the tristetraprolin-deficiency syndrome feedback inhibition of macrophage tumor necrosis factor-alpha production by tristetraprolin evidence that tristetraprolin binds to au-rich elements and promotes the deadenylation and destabilization of tumor necrosis factor alpha mrna tristetraprolin (ttp) coordinately regulates primary and secondary cellular responses to proinflammatory stimuli tristetraprolin regulates cxcl (kc) mrna stability genome-wide analysis identifies interleukin- mrna as target of tristetraprolin tristetraprolin down-regulates il- through mrna destabilization zinc finger protein tristetraprolin interacts with ccl mrna 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obstructive pulmonary disease mitogenactivated protein kinase p controls the expression and posttranslational modification of tristetraprolin, a regulator of tumor necrosis factor alpha mrna stability mapkap kinase phosphorylates tristetraprolin on in vivo sites including ser , a site required for - - binding the role of ttp phosphorylation in the regulation of inflammatory cytokine production by mk / mitogen-activated protein kinase-activated protein kinase regulates tumor necrosis factor mrna stability and translation mainly by altering tristetraprolin expression, stability, and binding to adenine/uridine-rich element temporal regulation of cytokine mrna expression by tristetraprolin: dynamic control by p mapk and mkp- activating protein phosphatase a (pp a) enhances tristetraprolin (ttp) anti-inflammatory function in a lung epithelial cells bi-phased regulation of the post-transcriptional inflammatory response by tristetraprolin levels expression and biologic characterization of the murine chemokine kc il- ) in the bronchoalveolar lavage fluid from patients with the adult respiratory distress syndrome (ards) and patients at risk for ards interleukin -related neutrophil elastase and the severity of the adult respiratory distress syndrome increased interleukin- concentrations in the pulmonary edema fluid of patients with acute respiratory distress syndrome from sepsis elevated levels of nap- /interleukin- are present in the airspaces of patients with the adult respiratory distress syndrome and are associated with increased mortality alveolar granulocyte colony-stimulating factor and alpha-chemokines in relation to serum levels, pulmonary neutrophilia, and severity of lung injury in ards plasma granulocyte colony-stimulating factor levels correlate with clinical outcomes in patients with acute lung injury effects of granulocyte colony-stimulating factor (g-csf) on bleomycin-induced lung injury of varying severity deletion of tristetraprolin caused spontaneous reactive granulopoiesis by a non-cellautonomous mechanism without disturbing long-term hematopoietic stem cell quiescence tristetraprolin targets nos expression in the colonic epithelium differential regulation of cytokine release and leukocyte migration by lipopolysaccharide-stimulated primary human lung alveolar type ii epithelial cells and macrophages we thank thaya stoufflet for assistance with multiplex cytokine assays, sherry ring for histological tissue processing, and deborah stumpo for helping with the ttp mutant mice and their genotyping. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © choudhary, vo, bathula, lamichhane, lewis, looper, jeyaseelan, blackshear, saini and patial. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - ltb dpa authors: li, xiangru; hao, zhenhua; liu, xiaorong; li, wei title: deficiency of mouse fhr- homolog, fhr-e, accelerates sepsis, and acute kidney injury through enhancing the lps-induced alternative complement pathway date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: ltb dpa alternative complement pathway (ap) plays an important role in the development of sepsis, which is life threatening. deficiency of factor h-related protein (fhr- ), which is a regulator of ap, has been considered as a susceptible factor for atypical hemolytic uremic syndrome (ahus) and other types of nephropathy when an inducer such as infection exists. however, the underlying mechanism of the disease development is largely unknown. there is no report on cfhr gene knockout in any animal infection model and its function in vivo is still unclear. here, a cfhr knockout mouse was generated for investigating ap in sepsis and sepsis-induced acute kidney injury (aki). we found that murine fhr- homolog (fhr-e) deficiency enhanced lipopolysaccharide (lps)-induced ap activation both in vitro and in vivo and that cfhr knockout mice exhibited more severe sepsis and aki in response to lps challenge. these results indicated that fhr-e deficiency promoted lps-induced sepsis and aki through ap over-activation, providing a mouse model for studying ap regulation and sepsis. this study revealed the function of fhr-e in vivo, which may further provide hints to the pathogenesis of fhr- deficiency-related diseases by enhancing lps-induced ap activation. sepsis is a critical health condition with high mortality rate ( , ) and acute kidney injury (aki) is a common severe complication of sepsis ( ) . gram-negative bacteria infection is a predominant cause of severe infection-triggered sepsis ( ) . sepsis is an intricate process during which complement system is activated and proved to be double-edged with benefits and harms ( ) . the complement system is crucial in immune surveillance ( ) and has extensive cross-talk with coagulation system and inflammation for homeostasis ( ) . it is triggered through three pathways among which the alternative complement pathway (ap) activation is responsible for more than % of terminal complement activation ( , ) . ap plays an important role in endotoxin clearance during the process of sepsis ( ) . lack of ap activation in individuals predisposes to infection ( ) ( ) ( ) . however, it is also life-threatening when complement is excessively activated, and inhibition of its over-activation prevents organ injuries ( ) . therefore, uncovering the precise regulatory mechanism of ap during sepsis may shed light on sepsis intervention. however, the mechanism of ap regulation during sepsis remains elusive. factor h (fh) and its related proteins (fhrs) are regulators of ap ( ) and responsible for determining complement activating surfaces ( ) . fh, the major regulatory factor of ap ( ) , has been widely investigated, while functions of fhrs are unclear or controversial. fh and fhrs consist of different number of complement control protein modules (ccps). all fhrs contain homologous ccps to ccp , of fh which are responsible for ligand recognition ( ) . in general, fh functions as a cofactor of factor i (fi) to cleave c b and accelerate the decay of c convertase ( , ) , while fhrs act as the competitor of fh ( ) . however, different fhrs may have different functions. fhr- inhibits c convertase ( ) . fhr- displays the cofactor activity for fi ( ) . fhr- inhibits c convertase in fluid phase and displays cofactor activity for fi ( ) . nevertheless, it remains elusive for the physiological functions of fhrs. mutations in fh and fhrs are associated with various diseases ( ) . cfhr -cfhr deletion increased the risk of atypical hemolytic uremic syndrome (ahus) and systemic lupus erythematosus (sle) ( ) ( ) ( ) . cfhr -cfhr deletion was proved to protect against iga nephropathy (igan) ( ) and age-related macular degeneration (amd) ( ) . the precise mechanism of this contradictory effect is unclear. in vitro studies of factor h-related protein (fhr- ) have shown that fhr- interferes with the regulation of fh by competing with fh and inhibits the activity of c convertase and the formation of terminal complement complex (tcc) ( ) . no obvious c and c regulatory activity at physiological concentration has been found in fhr- ( ) , albeit it is the most abundant fhr protein ( , ) . healthy individuals with cfhr -cfhr deletion showed higher frequency of patrolling monocytes, plasmacytoid dendritic cells (dcs), and lower frequency of classical monocytes, myeloid dcs. monocytes with cfhr -cfhr deletion secreted higher level of il- β in response to lps challenge ( ) . necrotic cells bound to fhr- promotes the secretion of tnf-α, il- β, il- , and il- by monocytes ( ) . these reports suggest multiple effects of cfhr deletion and highlight its complexity. the existence of relatively high frequency of healthy individuals with cfhr -cfhr deletion ( ) indicates that other triggers are essential to amplify the effect of fhr- deficiency, such as infections ( , ) . however, there is no report on fhr- deficiency in any animal infection model. three genes, fhrb, fhrc, and fhre (alias cfhr ), and two pseudogenes, fhra and fhrd, were identified in mouse ( , ) . recombinant fhr-a, fhr-b, and fhr-c were reported to interact with human c d, suggesting that murine fhrs function as homologs of human fhrs ( ) . none of fhr-a, fhr-b, and fhr-c contain the critical dimerization domain that exists in human fhr- , fhr- , and fhr- ( ), while fhr-e is predicted to contain it ( ) . thus, fhr-e is more likely the murine homolog of human fhr- or fhr- ( ) . nomenclature of human and mouse fhrs differs and there exist discrepancies in literature and existing database ( ) . inferred from the homology analysis (figure ) , we use mouse fhr- homolog fhr-e (np_ ) for the protein encoded by cfhr (refseq# at ncbi: nm_ , and a locus at mgi database: ) . homology analysis has shown the difference between human and murine fhrs ( , ) , suggesting that murine fhrs may function differently from their human counterparts. fhr-b, fhr-c, and fhr-e are functional fhrs in mouse. fhr-b and fhr-c were detected in mouse plasma with anti-fh antibody ( ) . mouse fhr-b interacts with c b and pentraxin to enhance complement activation and necrotic cells promoted this activation ( ) . fhrc mrna was found completely absent in the liver of three lupus-prone mouse strains and one diabetic-prone mouse strain ( ) . fhr-c may function on specific surfaces ( ) . it is unknown whether fhr-e exists in murine plasma and how it functions in ap. its human homolog, fhr- , was reported to function as a competitor of fh ( , , ) and its deletion was associated with various autoimmune diseases. however, its function in vivo and whether it has a regulatory role alone in the activation of ap remain unclear. in this study, cfhr was deleted on c bl/ mouse to study the function of fhr-e on ap and the effect of fhr-e deficiency on lps-induced sepsis. we found that fhr-e deficiency increased the mortality rate of lps-induced sepsis and potentiated kidney injury through enhancing ap activation. our data demonstrated a protective role of fhr-e during lps-induced sepsis in vivo and highlighted its importance in ap regulation. cfhr was deleted on c bl/ mouse by using the crispr/cas system developed by biocytogen co. (beijing, china). the heterozygous mouse was backcrossed with wild-type c bl/ for more than six generations. all the mice were bred in the animal facility of the institute of genetics and developmental biology (igdb), chinese academy of science. all procedures were approved by the institutional animal care and use committee of igdb. the age-matched wild-type (wt) or heterozygous littermates were used as controls. all experiments were conducted on the offspring of mice backcrossed more than six generations. the polyclonal antibody anti-fhr-e was generated in new zealand white rabbit. the dna fragment of the last three ccp domains (amino acids to ) of fhr-e was inserted into the expression vector pgex- t- and expressed in e. coli bl host cells. the recombinant protein was expressed as insoluble protein and separated by sds-page. the separated recombinant protein was used as an antigen to immunize the new zealand white rabbit every other week. the rabbit was sacrificed week after the fourth immunization and the sera was used as the anti-fhr-e polyclonal antibody. the rat anti-c (ab ), rat anti-c (ab ), sheep anti-fh (ab ), and rabbit anti-fibrinogen (ab ) were obtained from abcam (cambridge, uk). purified the upper bands were amplified with primers f and r . these bands can only be amplified in wild-type and heterozygous mice and cannot be amplified in homozygous mice because of the absence of the sequence of primer r . the lower bands were amplified with primers f and r . these bands can only be amplified in heterozygous and homozygous mice. the sequence between primers f and r is too long to be amplified in wild-type mice. (c) verification of cfhr deletion from rna level. hepatic rna of mice of different genotypes was extracted and reversely transcribed. primers spanning the open reading frame (orf, , bp, nm_ ) of cfhr were used for rt-pcr. actb was used as a semi-quantitative control. (d) verification of fhr-e deficiency at the protein level. plasma proteins of mice with different genotypes were separated by % sds-page gel. proteins were transferred to the membrane and analyzed by western blotting using anti-serum to mouse fhr-e, which was generated in rabbit immunized with a recombinant peptide of ccp - of fhr-e. an about kda specific fhr-e band was detected in wt and heterozygous mice, but not in homozygous mice. "unknown" indicates an unknown protein recognized by anti-fhr-e antibody. western blotting of fh was regarded as an internal control. rat anti-mouse c a ( ) and biotin rat anti-mouse c a ( ) were obtained from bd pharmingen (california, usa). mice were marked by cutting different toes and the toes were lysed by µl of lysis buffer (tris-hcl ph . mm, edta ph . mm, nacl mm, . % sds, and . mg/ml proteinase k) at • c for h. after transient centrifugation, µl of m nacl was added and mixed thoroughly. the mixture was centrifuged at , rpm for min and the supernatant was collected by adding double volume of ethanol and mixed thoroughly. the mixture was centrifuged at , rpm for min and then the supernatant was discarded. the pellet was washed with % ethanol. after the liquid was evaporated, µl of ddh o was added to dissolve the precipitated dna. one forward primer located on the upstream of the deleted sequence, one reverse primer located on the deleted region, and the other reverse primer located on the downstream of the deleted sequence were used for genotyping pcr assays. the primer's sequences are ′ -cagtaagactgcaagagacatatg- ′ , ′ -ctaagagcaacaggcgacag- ′ , and ′ -cattttaa aagaaaaataagccagcca- ′ , respectively. the amplified products are depicted in figure a . mouse hepatic rna was extracted using rneasy mini kit (qiagen, germantown, germany) and reversely transcribed with bio-rad transcript cdna synthesis kit (bio-rad, california, usa). the cdna was used as templates to amplify cfhr and actb (gene for β-actin). primers of cfhr were ′ -atggggtt ctgtcgcctgttgc- ′ and ′ -tcaatgaataaacgtattg tga- ′ . primers of actb were ′ -tgatggtgggaatggg tcaga- ′ and ′ -ccgctcgttgccaatagtgat- ′ . immunoblotting equal volume of serum or plasma of mice was diluted : with pbs (hyclone, utah, usa). equal volumes of diluted samples were separated with % sds-page gel and transferred onto polyvinylidene difluoride (pvdf) membrane (millipore, massachusetts, usa). the membrane was blocked with % defatted milk (bd, california, usa) for h at room temperature and immunoblotted with specific antibody overnight at • c followed by incubation with hrp-conjugated secondary antibody (zsgb-bio, beijing, china) for h at room temperature. the membrane was visualized by ecl (thermo fisher scientific, massachusetts, usa or ge healthcare, usa) and exposed with chemiluminescence apparatus (beijing sage creation, beijing, china). protein bands' gray values were measured by nih image j. serum was diluted by ap buffer ( . mm barbital, . mm sodium barbital, mm nacl, mm mgcl , and mm egta, ph . - . ). ten-fold diluted serum was incubated with . mg/ml lps o :b (sigma-aldrich, missouri, usa) or equal volume of pbs as control at • c for min. the reactions were stopped by mm edta. the samples were analyzed with % sds-page under reducing condition and protein bands' gray values were measured by nih image j. lps-stimulated ap activity was performed as previously published by elisa ( ) . briefly, lps ( µg/well) was coated on plates. ten-fold diluted serum with ap buffer was incubated on plates at • c for h. serum diluted with edta buffer ( . mm barbital, . mm sodium barbital, mm nacl, and mm edta, ph . - . ) was used as a negative control. after washing, the plates were incubated with rat anti-mouse c antibody and followed by incubation with hrp-conjugated antirat igg. tetramethylbenzidine (tmb, solarbio, beijing, china) was used as substrate and m h so was used as a stop solution. the absorbance was measured with multiskan go microplate spectrophotometer (thermo scientific, massachusetts, usa). for the detection of lps-induced c a production, -fold diluted serum was incubated with . mg/ml lps or equal volume of pbs as control at • c for h and the reactions were stopped by mm edta. the reaction mixtures were added to the plate, which was coated with purified rat anti-mouse c a antibody ( µg/ml) and incubated at • c for h. the mixtures were discarded and biotin rat anti-mouse c a ( . µg/ml) was added into the according plates as detection antibody. after washing, streptavidin hrp ( : , ) (bd pharmingen, california, usa) was added and incubated at • c for h. tmb (solarbio, beijing, china) was used as substrate and m h so was used as a stop solution. the absorbance was measured with multiskan go microplate spectrophotometer (thermo scientific, massachusetts, usa). six-to eight-week-old male mice were injected intraperitoneally (i.p.) with mg/kg lps dissolved in pbs and the control group mice were injected i.p. with equal volume of pbs. the edta anticoagulant blood was extracted from the tail tip at different time points and the plasma was collected to analyze with western blotting. mice were sacrificed at different time points and the edta anticoagulant blood was harvested from heart puncture. partial whole blood was used for complete blood counting using tek-ii mini full-automatic animal blood analyzer (tecom science, jiangxi, china). the plasma was collected for c a, c a, il- β and tnf-α concentration detection using elisa kit as described below. plasma c a, c a, il- β, and tnf-α measurements lps challenged mice were anesthetized and edta anticoagulant blood was collected by heart puncture. the whole blood was centrifuged at , rpm • c for min and the supernatant was collected. plasma levels of c a and c a were determined using elisa kits (mybiosource, california, usa) according to the manufacturer's instructions. the dilution factor of c a and c a was : . plasma il- β and tnf-α were measured with elisa kits (r&d systems, minnesota, usa) according to the manufacturer's instructions. about -week-old mice were injected i.p. with mg/kg lps. sixteen hours later, these mice were observed every half hour and the death of mice was recorded. the data were analyzed by graphpad prism version . using the log-rank (mantel-cox) test. mice were sacrificed at h after lps challenge and the left kidneys were harvested. half of the kidney was fixed in % paraformaldehyde for h at • c, dehydrated using graded ethanol, cleared with xylene, and embedded in paraffin. paraffin blocks were sliced into -µm sections. hematoxylin and eosin (he) staining was performed on the sections using hematoxylin and eosin staining kit (solarbio, beijing, china). pictures were captured with nicon ci-l microscope (nicon, tokyo, japan) at × magnification. tubular damage was assessed by tubular dilation, necrosis, and apoptosis. tubular dilations were counted in every non-overlap field and averaged. cell necrosis and apoptosis were determined using in situ cell death detection kit (roche, basel, switzerland). the fluorescent pictures were captured with an lsm confocal fluorescence microscope (zeiss, oberkochen, germany) at × magnification. only dots overlapped with nuclei were counted and the whole cell number of each field was counted by image j. proportion of apoptotic and necrotic cells was computed. paraffin slides that were processed by xylene de-waxing and gradient ethanol hydrating were blocked by ready-to-use goat serum (boster, wuhan, china) at • c for h and incubated with rabbit anti-fibrinogen ( : ) overnight at • c. after washing three times with pbs, the slides were incubated with alexa fluor goat anti-rabbit igg ( : , ) (invitrogen, california, usa) at • c for h. the slides were washed three times and mounted with mounting medium with dapi (zsgb-bio, beijing, china). images were captured using leica tcs sp confocal laser scanning microscope (leica microsystems, wetzlar, germany) at × magnification. the fluorescence intensity and fluorescence positive area were analyzed by image j. the protein sequences were downloaded from ncbi-protein (https://www.ncbi.nlm.nih.gov/protein/). amino acid sequences were aligned by clustalw with mega . ( ). the alignment diagram was drawn with genedoc. the evolutionary tree was constructed using the neighbor-joining method with mega . ( , ) . the domain structures of fhrs were analyzed by smart (http://smart.embl-heidelberg.de/). domain similarities among different fhrs were computed with pblast. data were presented as mean ± sem. student's t-test was used to compare the two groups. time course of factors of different time points after lps challenge was analyzed with one-way anova by spss software. comparison of survival curves was analyzed by mantel-cox test. significant differences were considered when the p-value was less than . and extremely significant differences were considered when the p-value was less than . . to ascertain the mouse fhr-e homolog of human fhr proteins, homology analysis between mouse and human fhrs was conducted. the evolutionary tree of fhrs showed that fhr-e converged with human fhr- , fhr- , and fhr- ( figure a) . the overall homology between mouse fhr-e and human fhr- / / is about %, while fhrb and fhrc have about % homology to fhr- / / ( ) . structure analyses of fhr-e and human fhr- , fhr- , and fhr- by smart showed that they all consist of ccp modules. amino acid sequence of each ccp in fhr-e was compared with human fhr- , fhr- , and fhr- by pblast and amino acid sequence alignment was analyzed by mega . . results showed that fhr-e shared more similarity with human fhr- (figures b-e) . with this regard, we selected the mouse cfhr gene that encodes the fhr-e protein for the generation of a gene knockout colony. the exons from to of cfhr were deleted by using the crispr/cas system on c bl/ mouse (figure a) . verification of cfhr deletion was conducted at dna, mrna, and protein levels (figures b-d) . the results demonstrated that fhr-e was detectable in murine plasma and was depleted in the knockout mice. our homemade anti-fhr-e antibody recognized murine fh as well due to the high epitope identities to scrs and of fh (data not shown). this is the first report of fhr-e in murine plasma. fhr-b and fhr-c has been detected in mouse plasma by using fh-specific antiserum ( ) . the unrecognized fhr-e in their experiment may be because of the antibody specificity. both heterozygotes (cfhr −/+ ) and homozygotes (cfhr −/− ) were viable and healthy bred in spf (specific pathogen free) mouse facility. under physiological condition, ap keeps at a low level of activation by spontaneous hydrolysis of c ( ) . to study whether cfhr deletion can trigger complement dysregulation under physiological condition, the plasma levels of c , c , and fh, central molecules of complement and main soluble regulator of ap, were detected on -to -week-old male sibling mice. western blotting results showed no obvious changes among mice of different genotypes (figure a) . the relative gray values of mice were analyzed and results showed that there were no significant differences (figures b-d) . furthermore, the concentrations of c a and c a, which are small cleaved fragments generated by complement activation, and the key mediators of inflammation were measured. neither the level of c a nor c a had significant differences among different genotypes (figures e,f) . these results suggested that under physiological condition, the deletion of cfhr was not sufficient to impact the ap regulation. ap activation is enhanced in cfhr −/− mice in response to lps treatment in vitro lps, the principal component of gram-negative bacteria cell wall, is an activator of ap ( ) and responsible for gramnegative bacteria induced sepsis ( ) . to test the effect of fhr-e deficiency on lps-induced ap, in vitro lps-induced ap activation experiments were performed. lps was incubated with serum from mice of different genotypes and the mixtures were analyzed by western blotting. a higher amount of c activated fragment in cfhr −/− mice was observed compared to the wild-type group (figures a,b) . ap activity measured by elisa assay ( , ) was also conducted to verify this effect. as expected, cfhr −/− mice showed more deposited c b on lps ( figure c) . furthermore, lps-induced c a production, an ensuing event of ap activation, was determined and significantly higher concentration of c a was detected in cfhr −/− mice compared to wild-type mice ( figure d ). an increasing tendency was observed in the heterozygotes, but there was no significant difference between wt and heterozygotes, suggesting that dose effects of fhr-e in heterozygotes may exist. thus, fhr-e deficiency promoted lps-induced ap activation in vitro. to further study the regulatory function of fhr-e, in vivo challenge of lps was applied. edta anticoagulant blood of different time points was collected from the tail tip to monitor the content changes of main complement or coagulation factors using western blotting. our results showed that the level of c decreased over time and that the level of c increased at first and decreased from h after lps challenge. no significant difference was observed among different genotypes at different points. however, more significant decrease of c content over time was observed in cfhr −/− mice compared with wild-type mice (figures a-c) . the levels of fh and von willebrand factor (vwf), which is involved in hemostasis, were also determined. the content of vwf increased at first and decreased from h post lps challenge (figures a,e) while the content of fh did not have any observable change over time (figures a,d) . this demonstrated that the hemostasis was promptly triggered and gradually vanished as the exhaustion of clotting factor, and that fhr-e deficiency and lps stimulation did not affect the level of fh. the contents of fhr-e in wild-type and heterozygous mice were also measured. interestingly, we found that fhr-e increased significantly at h post-lps challenge (figures a,f) . the data in figure were obtained from the same batches of mice. furthermore, another group of mice were challenged with lps and edta anticoagulant blood of different time points was harvested from heart puncture. the contents of plasma c a and c a of cfhr −/− mice were significantly higher than wild-type controls at h post-challenge (figures a,b) . these results demonstrated that fhr-e deficiency accelerated lps-induced ap. the plasma il- β and tnf-α contents at h post-challenge were detected and (c) detection of ap activation by plate bounded lps in diluted serum with elisa method. equivalent lps was coated on the plate. ap buffer and edta buffer diluted serum was added into the plate and incubated for h at • c. edta buffer diluted serum was used as a negative control for background subtraction. after washing, the lps-bound c b was detected with c antibody. twelve mice in each group were used. (d) measurement of the concentration of c a produced at h after lps in vitro stimulation. ap buffer diluted serum was incubated with lps or pbs for h at • c. equal volume of pbs was used as a negative control for background subtraction. the quantity of c a was detected with sandwich enzyme-linked immunosorbent assay. ten pairs of mice were used. n.s., not significant. *p < . . elevated concentrations of il- β and tnf-α were found in cfhr −/− mice (figures c,d) . subsequently, we observed that the proportion of granulocytes significantly increased and lymphocytes mildly decreased at h after challenge, which suggests that cfhr −/− mice had more severe inflammation (figures e,f) . the quantity of red blood cells in cfhr −/− mice at h after lps challenge was dramatically lower than the wild-type mice, which indicates that much more congestion could happen in cfhr deletion mice (figure g) . in summary, the cfhr −/− mice had features of enhanced complement activation and inflammation, which may lead to organ injury. to explore the endpoint effect of fhr-e deficiency on lpsinduced sepsis, the mortality assay was performed. cfhr −/− and wild-type mice were administrated i.p. injection of mg/kg lps. the survival data were recorded and analyzed. compared to the wild-type mice, the average survival time of cfhr −/− mice was significantly shorter and the mortality rate of cfhr −/− mice was significantly higher (figure h human cfhr deletion was associated with nephropathy ( , ) and kidney injury can be induced by complement overactivation ( ) . urea and creatinine, which are indicators of renal function, were tested at h post-challenge and significant increases were observed in both wt and ko groups (figures a,b) . however, we did not observe an apparent increase in the ko group compared with the wt group after lps treatment. this suggests that renal function was not worsened dramatically in fhr-e deficiency at the time of measurement. we then performed histological examination of kidney. renal tissue sections of h after lps challenge were prepared and he staining was conducted. kidney injury degree was assessed by the tubular dilations, apoptosis, and necrosis. tubular dilations were quantified, and cell apoptosis and necrosis were determined through tunel assay. more tubular dilations (figures c,d) and more cell apoptosis and necrosis (figures e,f) were exhibited in cfhr −/− mice. fibrin, which is the marker of vascular injury, was also stained to testify tissue injury. we found that cfhr −/− mice had significantly more fibrin deposition than wild-type mice (figures a-c) . plasma fibrin was also analyzed by western blotting and higher concentration of plasma fibrin was found in cfhr −/− mice ( figure d ). twenty non-overlapped fields were counted and averaged. (c) comparison of fluorescence intensity between wild-type and cfhr −/− mice. fluorescence intensity of each field was calculated by image j. twenty non-overlapped fields were counted and averaged. (d) western blotting analysis of plasma fibrin of h after lps challenge. western blotting of fhr-e was performed to determine the genotypes. equal volumes of plasma were loaded. the western blotting of fh on the same blot was regarded as an internal control. *p < . . in this study, cfhr knockout mice were generated to further investigate its role in ap regulation. no obvious changes of c a and c a concentration in plasma were found in cfhr knockout mice. these results suggest that the basal level of fhr-e plays negligible roles in spontaneously activated ap pathway. this may explain why many individuals with cfhr homozygous deletion are healthy ( ) . surprisingly, with fhr-e deficiency, increased c and c cleavage were found after lps stimulation in vitro, suggesting that fhr-e itself inhibits lps-induced ap activation. this may be explained by the conformation switch model in which soluble fh exists in a low-affinity latent conformation and transits to high-affinity activated conformation by interacting with self-surface targets ( ) . based on this model, fh may play negligible inhibition on lps-induced ap and fhr-e may compete c b with positive regulators of ap, like properdin, which plays an essential role in lps induced ap activation ( ) . when fhr-e is absent, ap activity is enhanced by the positive regulators. the inhibitory effect of fhr- on c convertase has been reported ( ) and was regarded as a therapeutic target ( ) . the result that more c a production after lps challenge in cfhr −/− mice was observed supports the idea that fhr-e has functional homology to human fhr- . cfhr deletion has been reported to be associated with renal disease in an incomplete penetrance and a second hit such as infection is considered to be essential for disease development ( ) . it is unknown whether cfhr homozygous deletion alone would trigger the onset of these conditions upon infection. one reported case showed that shiga toxin was a potential trigger of cfhr deletion-related thrombotic microangiopathy ( ) . here, we chose lps, which is the main constituent of the wall of gram-negative bacteria as a stimulator to study the effect of cfhr deletion in mice for possible pathological changes. both in vitro and in vivo assays showed that fhr-e deficiency significantly promoted lps-induced ap activation. these results suggested that fhr-e may play a coordinated role with fh in determining complement activation. however, the mechanism of selective activation of ap remains enigmatic. in many ahus patients with cfhr deletion, fh autoantibody was detectable ( ) . it was believed that ahus may result from the blocking of fh function by fh auto-antibody. however, this cannot explain those ahus patients with cfhr deletion without fh auto-antibody ( ) . our results suggest that fhr-e deficiency promotes infection-induced damage through enhancing ap activation. this may provide a hint for fhr- deficiency-related nephropathy. nevertheless, how fhr-e functions in this complex process warrants further investigation. in addition, we did not see much difference of fh level in fhr-e deficiency mice. to the best of our knowledge, there has been no report of fhrs on any inflammatory disease mouse models. our results revealed that cfhr knockout mice challenged with lps served as a good model to study the intricate network of complement, inflammation, and coagulation in sepsis and aki. we found that cfhr −/− mice showed higher content of c a and c a, more severe inflammation, more blood coagulation, and more severe renal injury after lps challenge compared with the wild-type group. however, we did not see significantly higher levels of urea and creatinine in cfhr −/− mice compared to the wildtype mice upon lps challenge. c a and c a, which are two main products of complement activation, have been shown to have anti-inflammatory ( , ) and pro-inflammatory function ( ) , respectively. administration of c a receptor antagonist peptide in thrombotic glomerulonephritis model significantly reduced leucocyte accumulation and thrombus formation in glomeruli ( ) and improved survival of mice with sepsis ( ), while administration of c a receptor antagonist exacerbated mortality of mice with sepsis ( ) . the regulation mechanism of c a and c a on sepsis development, which seemed to have converse effect, is still unclear. in our study, the deficiency of fhr-e promoted complement activation with more c a and c a production, which in turn induced more inflammation and eventually accelerated coagulation and tissue injury. these results suggested that c a may play a more potent role on sepsis development. our cfhr −/− mouse model provides a unique tool to study the underlying mechanism in which ap is activated upon lps challenge to induce tissue injury. beyond the scope of this study, it is speculative that patients with fhr deficiency may be susceptible to severe conditions under sars-cov- infection when sepsis is induced in covid- patients. the datasets generated for this study are available on request to the corresponding author. the animal study was reviewed and approved by institutional animal care and use committee of igdb (institute of genetics and developmental biology, chinese academy of science). xli, zh, and wl designed the experiments, analyzed the data, and supervised the project. xliu contributed to the initiation and design of the project. xli and wl wrote the manuscript. xli and zh performed the assays. this study was partially supported by grants from the ministry of science and technology of china ( yfc ) and from national natural science foundation of china ( ; ). severe sepsis in pre-hospital emergency care: analysis of incidence, care, and outcome incidence and trends of sepsis in us hospitals using clinical vs claims data acute renal failure and sepsis epidemiology of sepsis and infection in icu patients from an international multicentre cohort study complexity of complement activation 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molecular evolutionary genetics analysis version . for bigger datasets the neighbor-joining method: a new method for reconstructing phylogenetic trees acquisition of c b-like activities by spontaneous hydrolysis of the putative thioester in native c acylation of the lipid a region of a klebsiella pneumoniae lps controls the alternative pathway activation of human complement endotoxins and other sepsis triggers murine systemic thrombophilia and hemolytic uremic syndrome from a factor h point mutation mini review: a unique case of crescentic c glomerulonephritis role of c a in multiorgan failure during sepsis functional anatomy of complement factor h the mfhr fusion protein is a novel synthetic multitarget complement inhibitor with therapeutic potential rare functional variants in complement genes and anti-fh autoantibodies-associated ahus shiga toxin as a potential trigger of cfhr deletion-associated thrombotic microangiopathy characterization of genetic predisposition and autoantibody profile in atypical haemolytic-uraemic syndrome cutting edge: targeted disruption of the c a receptor gene demonstrates a novel protective anti-inflammatory role for c a in endotoxin-shock is the complement activation product c a a proinflammatory molecule? re-evaluating the evidence and the myth in vitro and in vivo dependency of chemokine generation on c a and tnf-alpha the role of c a in the development of thrombotic glomerulonephritis in rats carboxypeptidase b deficiency reveals opposite effects of complement c a and c a in a murine polymicrobial sepsis model the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © li, hao, liu and li. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - ex ow authors: qun, sen; wang, yulan; chen, jun; huang, xiang; guo, hui; lu, zhaohui; wang, jinquan; zheng, changcheng; ma, yan; zhu, yuyou; xia, daqing; wang, yinzhong; he, hongliang; wang, yong; fei, mingming; yin, yihong; zheng, mao; xu, yehong; ge, wei; hu, fuyong; zhou, jian title: neutrophil-to-lymphocyte ratios are closely associated with the severity and course of non-mild covid- date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: ex ow background: severe acute respiratory syndrome coronavirus (sars-cov- ) infection is spreading worldwide. measuring the prevention and control of the disease has become a matter requiring urgent focus. objective: based on coronavirus disease (covid- ) clinical data from wuhan, we conducted an in-depth analysis to clarify some of the pathological mechanisms of the disease and identify simple measures to predict its severity early on. methods: a total of patients with non-mild covid- were recruited, and information on their clinical characteristics, inflammatory cytokines, and t lymphocyte subsets was collected. risk factors for severity were analyzed by binary logistic regression, and the associations of neutrophil-to-lymphocyte ratios (n/lrs) with illness severity, disease course, ct grading, inflammatory cytokines, and t lymphocyte subsets were evaluated. results: our results showed that the n/lrs were closely related to interleukin (il)- and il- (p < . , p = . ) and to cd (+) and cd (+) t lymphocytes (p < . , p = . ). in particular, the n/lrs were positively correlated with the severity and course of the disease (p = . , p < . ). compared to the values at the first test after admission, il- and il- were significantly decreased and increased, respectively, as of the last test before discharge (p = . , p < . ). more importantly, through binary logistic regression, we found that male sex, underlying diseases (such as cardiovascular disease), pulse, and n/lrs were all closely related to the severity of the disease (p = . , p = . , p = . , p = . ). conclusions: as a quick and convenient marker of inflammation, n/lrs may predict the disease course and severity level of non-mild covid- ; male sex, cardiovascular disease, and pulse are also risk factors for the severity of non-mild covid- . at present, severe acute respiratory syndrome coronavirus (sars-cov- ) infection ( ) is largely under control in china, but it is still spreading rapidly worldwide, seriously threatening and damaging global health ( ) . the prevention and control of coronavirus disease (covid- ) is currently the most urgent issue facing the globe. many studies have confirmed that inflammatory storms are the key pathological basis determining the clinical symptoms, course, and prognosis of covid- ( ) . according to the diagnosis and treatment protocol for covid- (seventh edition) ( ), the pathologic anatomy of patients with covid- revealed that the lungs, spleen, cardiovascular system, liver, kidney, and other organs were markedly infiltrated by a large number of inflammatory cells, which resulted in functional damage and functional failure. many studies have confirmed that the impairment of immune function directly affects the course and prognosis of covid- ( ) . by autopsy, a decrease was observed in the numbers of cd + and cd + t cells in the spleen and lymph nodes, and denaturation and necrosis of the spleen and lymph nodes were also found, in addition to the proliferation of spleen macrophages. it has been speculated that t lymphocytes and macrophages may also be important targets for covid- ( ) . severe pneumonia and acute respiratory distress syndrome are the worst outcomes in patients with covid- , resulting in cytokine release syndrome and multiorgan failure; the role of il- in the pathological development of patients with covid- has been a focus of research ( , ) , but the function of il- in the course of the disease is still controversial ( ) ( ) ( ) ( ) . lymphocytopenia is a common feature of covid- and may be a key factor associated with disease severity and mortality ( ) . it is critically important to identify at the early stage of the infection those patients who would be prone to developing the most adverse effects. based on clinical data from wuhan (which had a severe outbreak of covid- early in the pandemic), we conducted an in-depth analysis to clarify some of the pathological mechanisms of the disease and identify simple measures to predict its severity early on. this study was approved by the research ethics committee of the first affiliated hospital of the university of science and technology of china (ustc) and was therefore performed in accordance with the ethical standards laid down in the declaration of helsinki and its later amendments. all participants gave their informed consent prior to their inclusion in this study. consent was obtained directly from each patient or from a family member or other legal guardian. if a patient was considered incapable of giving informed consent, a family member or other legal guardian was contacted to give informed consent on behalf of the patient. all patients or their close relations were informed of the purpose of the study and signed informed consent. the consent procedures were approved by the ethics committee. we collected the clinical data of all inpatients with covid- in the cancer center of wuhan union hospital from february , to march , . the diagnostic criteria for suspected cases of covid- pneumonia included the following ( ): ( ) epidemiological history included: (i) pre-onset history of travel or residence in wuhan or other areas where local cases continue to spread, (ii) fever or respiratory symptoms in patients who were from wuhan city or other areas with continued local spread and were exposed days before onset, and (iii) a cluster or epidemiological association with covid- infection; ( ) the following clinical features were present: (i) fever, (ii) imaging features indicative of pneumonia, and (iii) a normal or decreased total number of white blood cells in the early stage of the disease or a reduced lymphocyte count; or ( ) if there was any epidemiological history, a suspected case could be diagnosed in the presence of any two of the listed clinical manifestations. the inclusion criteria were as follows: ( ) the case met two criteria: (i) suspected covid- and (ii) detection of covid- nucleic acids by real-time fluorescent rt-pcr using sputum, pharyngeal swabs, or lower respiratory tract exudate; ( ) nonmild severity. the exclusion criteria were as follows: mild cases were defined according to the national protocol: "the clinical symptoms were mild, and there was no sign of pneumonia on imaging" ( ) . given the state of emergency at the time, only patients with moderate or severe cases were admitted to the cancer center of wuhan union hospital; patients with mild symptoms were admitted to the mobile cabin hospital ( ) . clinical data on patients with covid- pneumonia in the isolation ward of the cancer center of wuhan union hospital who met the above criteria were collected from february to march , . all confirmed patients were clinically classified according to the "novel coronavirus pneumonia treatment scheme of the national health commission of the people's republic of china (version )" at the time of admission ( ), as follows: ( ) general type: symptoms such as fever and respiratory tract complaints were present, and manifestations of pneumonia could be seen on imaging; ( ) serious type: any of the following criteria were met: ( ) respiratory distress, respiratory rate (rr) ≥ times/min; ( ) resting oxygen saturation ≤ %; or ( ) arterial partial oxygen pressure (pao )/oxygen absorption concentration (fio ) ≤ mmhg ( mmhg = . kpa); ( ) critical type: any of the following criteria were met: ( ) respiratory failure and a need for mechanical ventilation, ( ) shock, or ( ) a combination of factors with other organ failure requiring icu care. the course of disease was defined as the time (days) between the onset of clinical symptoms of covid- and the time of discharge from the hospital (symptoms recovery). all images were independently read by three senior radiological specialists. the location, shape, number, and size of the abnormalities on chest cts were carefully observed and recorded. in case of discordant reading, consensus was reached during another reading session. ct grading was defined according to the "novel coronavirus pneumonia treatment scheme of the national health commission of the people's republic of china (version )"and chinese expert consensus ( , ) . the confirmation of covid- was achieved by real-time reverse transcription polymerase chain reaction (rt-pcr) testing of throat sputum, pharyngeal swabs, and lower respiratory tract exudates of suspected patients. following the recommendation of the china national centre for disease control, two target genes were targeted as described previously ( ) , namely, open reading frame ab (orf ab) and nucleocapsid protein (n), and these genes were simultaneously amplified and tested during the real-time rt-pcr assay. the primers for target (orf ab) were forward primer ccctgtgggttttacacttaa; reverse primer acgattgtgcatcagctga; and probe -fam-ccgtctgcggtatgtggaaaggttatgg-bhq - . the primers for target (n) were forward primer ggggaact tctcctgctagaat; reverse primer cagacattttgct ctcaagctg; and probe -famttgctgctgcttgacag att-tamra- . a cycle threshold value (ct value) less than was defined as a positive result, and a ct value exceeding was defined as a negative test. venous blood was taken in the emergency department at admission and sent to the laboratory for examination of routine blood parameters and inflammatory cytokines. however, t lymphocyte subsets and some biochemical indicators were obtained by testing fasting blood the morning after admission. serum inflammatory cytokines were detected by enzyme-linked immunosorbent assays for il- , il- , il- , il- , il- , and tumor necrosis factor-α (tnf-α). the tests were carried out strictly according to the instructions, and the test kits were purchased from ebioscience company (epx - - ). in order to quantify t lymphocyte subsets, µl of whole blood was incubated in µl of tris-nh cl buffer (thermo fisher scientific) at room temperature for min to lyse erythrocytes. after two washes with phosphate-buffered saline (pbs), cells were incubated with cd -apc, cd -percp, and cd -fitc antibodies ( µg/ml each, all from bd biosciences) for min on ice. after another two washes with pbs, cells were resuspended in µl of pbs. samples were analyzed on a bd facs canto plus flow cytometer. among all collected events, single events were gated between forward scatter (fsc)-a and fsc-h. cell debris was excluded, and intact cells were then gated from single events based on fsc-a and side scatter (ssc). each cell population was then detected based on antibody staining. all statistical analyses were conducted with statistical package for the social sciences version . (spss, company, chicago, il, united states). all continuous data were tested for normal distributions using the kolmogorov-smirnov test. normally distributed variables were described as the mean ± standard deviation (m ± sd), and non-normally distributed variables were presented as the median and the interquartile range [median (iqr)]. categorical variables were expressed as constituent ratios and percentages in each category. one-way anova or kruskal-wallis analysis was applied to continuous variables, and the chi-square test or fisher's exact test was applied to categorical variables. differences in cytokine and t lymphocyte subsets tested twice for each patient were assessed by paired t-test or wilcoxon signed rank test. correlation analysis was used to determine the relationship between n/lrs and inflammatory cytokines, t lymphocyte subsets, severity of illness, course of disease, or ct grading. n/lrs in different patient condition groups were tested by kruskal-wallis analysis and the jonckheere-terpstra trend test. binary logistic regression analysis was used to test the risk factors for the severity level of patients with covid- . a p value < . was considered statistically significant. ( ) clinical characteristics of patients with covid- . we collected patients with covid- ; ( %) patients were male, the average age was ( ) years, and the total course of disease was ( ) days. the distribution of severity categories was as follows: ( . %) patients had the general type, ( . %) patients had the serious type, and five ( . %) patients had the critical type. according to ct grading, ( . %) patients were in the early stage, nearly half ( patients, . %) were in the progressive stage, and ( . %) were in the critical stage. the underlying (comorbid) diseases of the patients were as follows: ( . %) patients had respiratory system diseases, ( . %) patients had immune-related diseases, ( . %) patients had cardiovascular system diseases, ( . %) patients had liver or kidney diseases, and ( . %) patients had other comorbidities. the patients' initial symptoms were as follows: ( . %) patients had fever, ( . %) patients had respiratory system symptoms, ( . %) patients had general symptoms, nine ( . %) patients had cardiovascular system symptoms, ( . %) patients had digestive system symptoms, ( . %) patients had nervous system symptoms, and five ( . %) patients were asymptomatic. the damage to physiological systems was as follows (multiple system damage were directly caused by covid- ): ( . %) patients had respiratory system damage, ( . %) patients had cardiovascular system damage, ( . %) patients had liver function damage, ( . %) patients had renal function damage, ( . %) patients had digestive system damage, ( . %) patients had nervous system damage, and ( . %) patients had muscle damage. a total of ( . %) patients had general symptoms. of the patients, three ( . %) were asymptomatic. the variables with statistical significance were identified by univariate analysis. these variables were sex (male) (p = . ), underlying disease (cardiovascular system diseases) (p = . ), multisystem damage (muscle) (p = . ), course of disease (p = . ), pulse (p = . ), systolic pressure (p = . ), l (p = . ), and n/lrs (p = . ) (see table ). ( ) there were significant differences between the first test and last test regarding the cytokines and classification of leukocytes. we collected the data regarding the cytokines and t lymphocyte subsets and classification of leukocytes at the time of admission (first test) and the time of discharge (last test), and there were statistically significant differences in cytokine levels between admission and discharge. for example, il- and il- were significantly decreased and increased, respectively (p = . , p < . ). the il- /il- ratio was also significantly different (p < . ). additionally, there were statistically significant differences in lymphocytes and n/lrs between admission and discharge (p = . , p < . ). however, there were no statistically significant differences in t lymphocyte subsets between admission and discharge ( table ) . ( ) risk factors for the severity of covid- . the risk factors for the severity of covid- were analyzed by binary logistic regression. due to the small number of critical-type samples, the critical type and the serious type were grouped together. variables with statistically significant differences among different groups of diseases by univariate analysis were substituted into the regression equation, and finally, four variables with statistical significance were selected: male sex, underlying disease (cardiovascular disease), pulse, and n/lrs were all closely related to the severity of the disease (p = . , p = . , p = . , p = . ) ( table ) . ( ) n/lrs are closely related to severity level and disease course. we found that n/lrs were closely related to the severity level and disease course of covid- according to the kruskal-wallis test, p = . and p < . , respectively ( table ) . the difference between each pair of groups was statistically significant. patients with higher n/lrs had more severe disease. a jonckheere-terpstra trend test was performed and showed a statistically significant difference between each group; the more severe the disease, the higher the n/lr (figure ) . patients with higher n/lrs had longer disease courses (figure ) (p < . ). ( ) n/lrs were closely related to il- and il- . we found that n/lrs were positively related to il- and il- , p < . and p = . , respectively. however, there was no significant correlation with other cytokines (figure ) . the trend of the relationship between n/lrs and different inflammatory cytokines is shown in figure . ( ) n/lrs are closely related to cd + and cd + cells (t lymphocyte subsets). we found that n/lrs were negatively related to cd + and cd + t lymphocytes (p < . and p = . , respectively; figure ). the trend of the relationship between n/lrs and different t lymphocyte subsets is shown in figure . in this study, we analyzed the clinical data of patients diagnosed with non-mild covid- in the cancer center of wuhan union hospital and summarized the clinical characteristics of this disease. we found the following: ( ) n/lrs, male sex, underlying disease (cardiovascular disease), and pulse were all closely related to the severity of the disease; ( ) n/lrs were positively correlated with the levels of cytokines (il- , il- ); ( ) n/lrs were negatively correlated with the proportion of cd + and cd + t lymphocyte subsets; ( ) n/lrs were positively correlated with the severity of the disease, as the higher n/lrs were, the more serious the disease; ( ) n/lrs were related to the course of disease, as the higher n/lrs were, the longer the course of disease; and ( ) most patients had multisystem symptoms and injury and had underlying multisystem comorbidities. as the covid- epidemic continues to spread across the globe, effective treatment is urgent, and it is particularly crucial to explore the pathogenesis of the disease. covid- infection activates innate and adaptive immune responses. however, uncontrolled damage from the natural inflammatory response and adaptive immune response may lead to local and systemic tissue damage. lymphocytopenia is a common feature in patients with severe covid- , with a dramatic decrease in the number of cd + and cd + t cells, b cells, and natural killer (nk) cells ( , ( ) ( ) ( ) and lower proportions of monocytes, eosinophils, and basophils among leukocytes ( , ) ; in particular, patients with higher n/lrs have poorer outcomes ( ) . yang et al. found that nlrs can be considered independent biomarkers for indicating poor clinical outcomes ( ) . consistent with the above data, our research found that the nlrs were closely related to the severity and course of patients with non-mild covid- and that higher n/lrs were related to longer courses and more severe non-mild covid- . currently, n/lrs are a well-known marker of systemic inflammation and infection, and they have been studied as predictors of bacterial infection, showing superior predictive value over conventional inflammatory markers ( ) ( ) ( ) . in addition, the n/lrs have displayed good predictive power for pneumonia, as well as dose-response information relating to the burden of community-acquired pneumonia, such as pneumonia severity or mortality ( , ) . neutrophilia and lymphocytopenia are physiological responses of the innate immune system to systemic inflammation. lymphocytopenia consists of accelerated apoptosis and margination of lymphocytes within the reticuloendothelial system, liver, and splanchnic lymphatic system and of the redistribution of lymphocytes within the lymphatic system ( ) ( ) ( ) . as immune changes and inflammation are the core pathological basis of covid- , compared with imaging and biochemical examination, n/lrs may be a simpler and more specific way to determine the prognosis of covid- . most patients with severe covid- show significantly elevated serum levels of inflammatory cytokines, such as il- and il- , as well as il- , il- , g-gsf, gm-gsf, ip- , mcp , mip- a (also known as ccl ), and tnf, known as a cytokine storm ( , ( ) ( ) ( ) . in our research, we found that cytokines, such as serum il- , il- , il- , il- , tnf-α, and infγ, were significantly elevated at admission, and in some patients, these inflammatory cytokines were reviewed before discharge. there were statistically significant differences in cytokine levels at admission and discharge. interestingly, we found that in the process of treatment, the levels of some inflammatory cytokines were decreased, while others were increased. this phenomenon may be because some inflammatory cytokines are pro-inflammatory and others are anti-inflammatory; these changes may reflect the dynamic transformation process of inflammation from damage to repair during homeostasis and inflammation ( ) . more importantly, correlation analysis indicated that n/lrs were closely related to il- and il- . a recent study showed that m macrophages produce proinflammatory cytokines, such as il- , and that m macrophages produce anti-inflammatory cytokines, such as il- ( ). our research found that, compared to those at the first test, il- significantly increased and il- significantly decreased at the last test (p < . ), which described the patient's transition from the acute phase to the convalescent phase, and these phenomena may indicate that n/lrs were closely related to injury in the acute stage and repair in the recovery stage in patients with covid- . the source, function, and interaction of various inflammatory cytokines are complex. severe acute respiratory syndrome coronavirus -infected macrophages demonstrate upregulation of il- production and low expression of interferons ( , ) . th cell-derived ifn-γ is essential for an effective antiviral immune response. however, il- may inhibit th polarization by stimulating cd + cells to differentiate into th cells or by suppressing ifn-γ expression ( , ) . however, il- can cooperate with transforming growth factor (tgf)-β to induce il- production in th cells ( ) ; il- is also produced by regulatory t cells (tregs), and tgf-β is critical to enable human tregs to express il- ( ). il- can stimulate lymphocytes, leading to a decrease in the secretion of il- and ifn-γ by t cells ( ) . studying the complex inflammatory cytokine network after covid- infection is of great significance for the treatment and prediction of the disease. most patients with covid- exhibit mild to moderate symptoms, but approximately % progress to severe pneumonia, and approximately % eventually develop acute respiratory distress syndrome (ards), septic shock, and/or multiple organ failure ( , ) . wei's group study showed that after covid- infection, cd + t lymphocytes are rapidly activated to become pathogenic t helper (th) cells and generate gm-csf. the cytokine environment induces inflammatory cd + cd + monocytes with high expression of il- and accelerates inflammation ( ). these aberrant pathogenic th cells and inflammatory monocytes may enter the pulmonary circulation in large numbers and play an immune damaging role to cause lung functional disability and quick mortality ( , ) . their team launched a clinical trial using tocilizumab to block inflammatory storms (il- receptor inhibitor) in severely ill patients and achieved some results ( ) . partly corresponding to wei's team ( ), our clinical study found that the serum level of il- decreased significantly before discharge, which may reflect the pro-inflammatory role of il- in the course of covid- from another perspective. however, our team did not find that inflammatory cytokines (such as il- ) were significantly different among different severity level groups. part of the explanation is that there are no mild cases in this study (see section "materials and methods"). more importantly, although the role of il- in the pathological development of covid- has attracted much attention ( ), its specific role remains controversial ( ) ( ) ( ) ( ) , and several experimental models of viral lung infections suggest that il- demonstrates either pathogenic ( )or protective ( ) effects in vivo; the consequences of il- induction in covid- may vary depending on the stage of infection and the immune status of the host. the role of these cytokines in sars-cov- infection should be carefully evaluated. severe acute respiratory syndrome coronavirus might act mainly on lymphocytes, especially t lymphocytes ( ) , and decreases in the levels of cd + and cd + t lymphocytes are associated with immunosuppression ( ). our research found that n/lrs were negatively related to cd + and cd + t lymphocyte levels, suggesting that the elevated n/lrs reflect the degree of lymphatic impairment, which may support the hypothesis that n/lrs are a sensitive and simple biomarker of immune function in patients with covid- . additionally, our results suggest that male sex, underlying disease (cardiovascular disease), and pulse are risk factors for covid- . the risk associated with male sex raises a topic of great interest: the effects of estrogens on il- and on covid- progression. estrogens can suppress lipopolysaccharide (lps)-mediated il- expression in mouse macrophages by both blocking nuclear factor kappa b (nf-kb) activation ( , ) and inhibiting p mitogen-activated protein kinase (mapk) phosphorylation ( ) by acting on estrogen receptors to decrease the production of pro-inflammatory cytokines ( ) . however, the immunomodulatory effects of estrogens in covid- require further study. cardiovascular disease and pulse are risk factors for covid- , which may suggest that cardiovascular disease has a critical impact on covid- patient mortality ( ) and requires more attention as a comorbidity. we found that there were statistically significant differences in cytokines and n/lrs at admission and discharge, while there were no differences in t lymphocyte subsets (cd + /cd + /cd + ) in the research. we wanted to know whether the indicators of lymphocyte subset recovery lagged relatively over time and what their significance was. further work is needed to understand the specific characteristics of the various inflammatory cytokines and t cell subsets in covid- , the specific relationship between n/lrs and different inflammatory cytokines and t cell subsets in covid- , and the prognostic value for the disease, especially during immunotherapy. post-covid- inflammation is a very complex network system that has a decisive influence on the prognosis of patients. some inflammatory cytokines (such as il- ), whose mechanisms and effects are still controversial, need further study. as a quick and convenient marker of inflammation, n/lrs may predict the disease course and severity level of non-mild covid- ; male sex, cardiovascular disease, and pulse are also risk factors for the severity of non-mild covid- . first, the sample size of critical type patients was relatively small, which may have influenced the results. second, due to the emergency situation of the epidemic outbreak, there is a certain lack of clinical data, including inflammatory cytokines. we are also collating clinical data from other centers and investigating the mechanisms of inflammatory factors in animal models. the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. a novel coronavirus outbreak of global health concern the outbreak of coronavirus disease (covid- )-an emerging global health threat pathogenic t cells and inflammatory monocytes incite inflammatory storm in 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assessment, diagnosis, and treatment options sq was involved in the study design, data interpretation, and manuscript writing and was a recipient of the obtained funding. jz participated in the analysis, interpretation, and collection of the data. fh participated in the study design and the statistical analysis. zl, jw, jc, hg, cz, ym, yz, dx, yiw, hh, yow, mf, yy, mz, and yx were involved in the data collection. yuw and jc participated in the data analysis and data collection. xh participated in the data analysis. wg participated in the data analysis and manuscript writing. all authors contributed to the article and approved the submitted version. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © qun, wang, chen, huang, guo, lu, wang, zheng, ma, zhu, xia, wang, he, wang, fei, yin, zheng, xu, ge, hu and zhou. this is an openaccess article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - td efjw authors: zhou, yanrong; wu, wei; xie, lilan; wang, dang; ke, qiyun; hou, zhenzhen; wu, xiaoli; fang, ying; chen, huanchun; xiao, shaobo; fang, liurong title: cellular rna helicase ddx is involved in transmissible gastroenteritis virus nsp -induced interferon-beta production date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: td efjw transmissible gastroenteritis virus (tgev), an enteropathogenic coronavirus (cov) of porcine, causes lethal watery diarrhea and severe dehydration in piglets and leads to severe economic losses in the swine industry. unlike most covs that antagonize type i interferon (ifn) production, previous studies showed that tgev infection induces ifn-i production both in vivo and in vitro. however, the underlying mechanism(s) remain largely unknown. in this study, we found that tgev infection significantly facilitated ifn-β production as well as activation of the transcription factors ifn regulatory factor (irf ) and nuclear factor-kappab (nf-κb) in porcine kidney (pk- ) cells. screening of tgev-encoded proteins demonstrated that non-structural protein (nsp ) was the most potent ifn-β inducer and induced ifn-β production mainly by activating nf-κb but not irf . further analysis showed that nsp interacted with ddx , a member of the dexd/h helicase family. knockdown of ddx by specific small interfering rna (sirna) significantly decreased nsp -induced ifn-β production and nf-κb activation. furthermore, tgev-induced ifn-β production and ifn-stimulated gene (isg) expression were decreased in cells transfected with ddx -specific sirna, indicating the vital role of ddx to tgev-induced ifn-β responses. in summary, our data revealed a potential coactivator role of host rna helicase ddx to the induction of ifn-β response initiated by tgev and demonstrated that nsp is an important ifn inducer among the tgev-encoded proteins. transmissible gastroenteritis virus (tgev), an enteropathogenic coronavirus (cov) of porcine, causes lethal watery diarrhea and severe dehydration in piglets and leads to severe economic losses in the swine industry. unlike most covs that antagonize type i interferon (ifn) production, previous studies showed that tgev infection induces ifn-i production both in vivo and in vitro. however, the underlying mechanism(s) remain largely unknown. in this study, we found that tgev infection significantly facilitated ifn-β production as well as activation of the transcription factors ifn regulatory factor (irf ) and nuclear factor-kappab (nf-κb) in porcine kidney (pk- ) cells. screening of tgev-encoded proteins demonstrated that non-structural protein (nsp ) was the most potent ifn-β inducer and induced ifn-β production mainly by activating nf-κb but not irf . further analysis showed that nsp interacted with ddx , a member of the dexd/h helicase family. knockdown of ddx by specific small interfering rna (sirna) significantly decreased nsp -induced ifn-β production and nf-κb activation. furthermore, tgev-induced ifn-β production and ifn-stimulated gene (isg) expression were decreased in cells transfected with ddx -specific sirna, indicating the vital role of ddx to tgev-induced ifn-β responses. in summary, our data revealed a potential coactivator role of host rna helicase ddx to the induction of ifn-β response initiated by tgev and demonstrated that nsp is an important ifn inducer among the tgevencoded proteins. keywords: transmissible gastroenteritis virus, non-structural protein , ddx , interferon-beta, innate immune response, pattern-recognition receptors introduction transmissible gastroenteritis virus (tgev) is a member of the alphacoronavirus genus within the family coronaviridae in the order nidovirales. tgev infection mainly causes acute enteric disease characterized by lethal watery diarrhea, severe dehydration, and high mortality in suckling piglets less than weeks old, which has led to severe economic losses in the global swine industry since gene name sirna sequence hddx (sense) ggagcuucugauaauuggagguguu hddx (anti-sense) aacaccuccaauuaucagaagcucc pddx (sense) gaaagaccuuggucuggcauuugaa pddx (anti-sense) uucaaaugccagaccaaggucuuuc the first outbreak in in ilinois, usa ( , ) . tgev contains a single-stranded, positive-sense rna genome of about . kb ( ) , including at least nine open reading frames (orfs). two slightly overlapping orfs, orf a and orf b, located at the ' two-thirds of the viral genome, encode a replicase complex that is proteolytically processed into non-structural proteins (nsp to ) ( ). structural proteins, nucleocapsid (n) protein, membrane (m) glycoprotein, spike (s) glycoprotein, a small envelope (e) glycoprotein, and accessory proteins a, b, and are encoded by genes located at the ′ end ( ) . as early as , the presence of high levels of type i interferon (ifn-i) activity in the digestive tract of tgev-infected newborn piglets was first observed ( ) . thereafter, charley et al. reported that tgev infection in human or bovine peripheral blood mononuclear cells also induced high ifn-α production ( ) . subsequently, bosworth et al. demonstrated that ′, ′-oligoadenylate synthetase (oas), a well-known ifn-stimulated gene (isg), was increased in tgev-infected pigs ( ) . our previous quantitative proteomics analysis revealed that tgev infection induced canonical ifn-i signaling through janus kinase signal transducer and activator of the transcription (jak-stat ) pathway, and eight tested isgs, including ifninduced protein with tetratricopeptide repeats (ifit ), ifit , ifit , oas , oas , mx , mx , and isg were upregulated after tgev infection ( ) . these early results indicated that tgev infection activated the ifn-i pathway in vitro and in vivo. however, the underlying mechanism(s) utilized by tgev to induce ifn-i, and especially which viral protein(s) contribute to it, remain largely unclear. the ifn-i response is a well-known innate immune reaction that occurs in response to virus infection and considered as an important bridge between innate and adaptive immunity. nuclear factor-kappab (nf-κb) and ifn regulatory factor (irf ) are two critical transcription factors for the regulation of ifn-i production. secreted ifn-i then stimulates the jak-stat signaling pathway to induce the expression of numerous isgs, which collaborate to regulate the replication of virus ( ) . many viruses antagonize ifn responses to benefit their propagation, and some viruses such as human immunodeficiency virus-type ( ), vesicular stomatitis virus (vsv) ( ), influenza a virus (iav) ( ) , encephalomyocarditis virus ( ), reovirus ( ), herpes simplex virus (hsv ) ( ), respiratory syncytial virus ( ), newcastle disease virus ( ) , and sendai virus (sev) ( ) initiate innate immune responses. different viruses employ different mechanisms to regulate innate immune responses. for example, hsv induces ifn-α/β production through toll-like receptor (tlr ), dna-dependent activator of ifn regulatory factors (dai), and ifn-inducible ( ) . for iav infection, at least tlr , tlr , retinoic acid-inducible gene i (rig-i), and pyrin domain-containing (nlrp ) are responsible for the detection of iav and subsequent innate immune responses ( ) . elucidating the mechanisms through which viruses regulate innate immune responses will help us understand the interactions between virus and host. this study sought to identify tgev-encoded protein(s) involved in the induction of ifn-β production. our results revealed that tgev nsp was the best inducer of the ifn-β pathway among the tgev-encoded proteins. mechanistically, nsp activates nf-κb but not irf , and it interacts with rna helicase ddx , which in turn activates ifn-β production. porcine kidney (pk- ) cells and hek- t cells were cultured in dulbecco's modified eagle's medium (invitrogen, carlsbad, ca, usa) supplemented with % fetal bovine serum in a humidified incubator with °c/ % co . tgev strain wh- (genbank accession no. hq ) was propagated and titered in pk- cells. recombinant vsv-expressing green fluorescent protein (vsv-gfp) was generously provided by prof. zhigao bu from the harbin veterinary research institute, china. rabbit polyclonal antibodies against p , irf , phosphorylated irf (p-irf ), and ddx were purchased from abclone (wuhan, china). rabbit polyclonal antibody against phosphorylated p (p-p ) was purchased from cell signaling technology (beverly, ma, usa). anti-β-actin antibody was purchased from beyotime (nantong, china). mouse monoclonal antibodies (mabs) against hemagglutinin (ha) and flag were purchased from medical and biological laboratories (mbl, nagoya, japan). mab against tgev n protein was prepared by our laboratory. horseradish peroxidase (hrp)-conjugated goat anti-rabbit antibody and hrp-conjugated goat anti-mouse antibody were purchased from mbl. alexa fluor -conjugated donkey anti-rabbit igg, alexa fluor -conjugated donkey anti-mouse igg, and alexa fluor -conjugated donkey antimouse igg were obtained from santa cruz biotechnology inc. (santa cruz, ca, usa). expression plasmids of tgev-encoded proteins used in this study were constructed by rt-pcr amplification from the genomic rna of tgev strain wh- and cloned into expression vector pcaggs-ha. the details of primers used for pcr clone are available on request. the p gene was derived from human rela cdna and cloned into pegfp-c vector. the ddx expression plasmid was constructed by rt-pcr amplification from the cdna of pk- cells and cloned into pcmv-tag b vector. luciferase reporter plasmids p -luc (ifn-β-luc), × prdiii/i-luc (referred to as irf -luc), × prdii-luc (referred to as nf-κb-luc), and the internal control plasmid prl-tk have been described previously ( ) . small interfering rna (sirna) targeting ddx or negative control sirna (sinc, invitrogen) were each transfected at a final concentration of nm. the sirna sequences used here are listed in table . np- , nm phenylmethylsulfonyl fluoride], and protein concentration was measured and adjusted. for each immunoprecipitation, µg of cell lysate protein was incubated with µg of indicated antibody and µl of protein a/g-agarose (beyotime) overnight at °c. after three washes with ml lysis buffer, precipitates were subjected to % sds-page and subsequently analyzed with immunoblot analysis using the indicated antibodies. porcine kidney (pk)- cells were fixed with % paraformaldehyde for min followed by permeabilization with pre-cooled methanol for min, blocking with % bovine serum albumin for min, incubated with the indicated primary antibodies for h, followed by staining with specific alexa fluor-conjugated secondary antibodies for h. the cells were subsequently stained with ', -diamidino- -phenylindole (beyotime) for min. after washing with pbs, fluorescent images were obtained using an olympus fv laser scanning confocal microscope (olympus, japan). total rnas were extracted using trizol reagent (invitrogen). real-time rt-qpcr was performed using sybr green real-time pcr master mix (toyobo biologics, osaka, japan) in the abi prism sequence detection system (applied biosystems). individual transcripts in each sample were assayed three times. the fold change in gene expression relative to normal was calculated using the delta-delta cycles to threshold (ΔΔct) method. primers ( table ) were designed using primer express software (version . ; applied biosystems, carlsbad, ca, usa). all experiments were performed at least three times with reproducible results. data are presented as the mean ± sd. statistical analysis was performed using one-way anova without interaction terms followed by dunnett's for multiple comparisons. all animal experiments were approved by the hubei administrative committee for laboratory animals (permission number ) and complied with the guidelines of hubei laboratory animal welfare and ethics of hubei administrative committee of laboratory animals. previous studies demonstrated that tgev infection potently induced ifn-α ( , , ) , as well as ifn-β in ipec-j and swine testicular (st) cells ( ) ( ) ( ) . however, whether tgev infection induces ifn-β in pk- cells remains unknown. to explore the effect of tgev on ifn-β, dual luciferase assays were performed. pk- cells were transfected with ifn-β-luc and prl-tk. after the induction of ifn-i is reliant on the co-regulation of transcription factors irf and nf-κb. to investigate the potential mechanism(s) involved in the ifn-β production by tgev infection, the effect of tgev on irf and nf-κb promoters were also tested. as displayed in figures c,d , tgev infection also upregulated irf and nf-κb promoter activity dosedependently, indicating that irf and nf-κb are involved in the ifn-β production by tgev infection. because of the high sensitivity of vsv-gfp to ifn, vsv-gfp expression is commonly monitored for ifn detection. to further evaluate the ifn-β response in tgev-infected cells, a vsv-gfp-based ifn detection assay was performed. pk- cells were infected or mock infected with sev or tgev (moi = . , . , . ). then, cell supernatants were collected, uv-irradiated, and then transferred onto fresh pk- cells. after h, cells were infected with vsv-gfp and observed under a fluorescence microscope at hpi. as a positive control, supernatants collected from sev-treated cells suppressed vsv-gfp replication prominently compared with the negative control group (mock). in accordance with the results of dual luciferase assays described above, tgev infection limited the replication of vsv-gfp in a dose-dependent manner (figure e) . these results suggested that tgev infection increased ifn-β production. transmissible gastroenteritis virus encodes non-structural proteins (nsp - ), four structural proteins (n, m, s, e), and three accessory proteins (orf , orf a, orf b) . to identify the key viral protein(s) involved in ifn-β induction, an ifn promoterreporter system was employed to screen tgev-encoded proteins for their relative capacities to activate the ifn-β promoter. hek- t cells were cotransfected with ifn-β-luc, prl-tk, and different tgev protein expression vectors. as shown in figure a , nsp was the most significant inducer of ifn-β production. furthermore, similar to sars-(coronavirus) cov ( ), tgev m glycoprotein also potently mediated ifn-β induction. because nf-κb and irf are necessary transcription factors for ifn-β production, we examined the effects of all tgev-encoded proteins on irf and nf-κb promoter activity. interestingly, nsp upregulated an approximately . -fold change in nf-κb promoter activity, but only an approximately . -fold change in irf promoter activity (figures c,e) . m glycoprotein induced a higher fold change in irf , but a lower fold change in nf-κb compared with nsp . because m glycoprotein has been investigated previously ( ), we focused on nsp . to confirm the ability of nsp to induce ifn-β production, large-scale screen experiment was also conducted in pk- cells, a permissive cell line of tgev infection. as shown in figures b,d,f, nsp was also a potent ifn inducer that mainly induced activation of nf-κb but not irf promoter in pk- cells. to confirm the above large-scale screen results, increasing doses ( . , . , . µg) of pcaggs-ha-nsp and ifn-β-luc, irf -luc or nf-κb-luc together with prl-tk were cotransfected into hek- t cells. in line with the results in figure , nsp enhanced the activation of ifn-β and nf-κb in a dose-dependent manner (figures a-c) . interestingly, nsp induced the activation of nf-κb to a greater extent than that of irf , indicating nf-κb has a fundamental role in nsp induced ifn-β activation. similar results were also obtained in pk- cells (figures d-f) . nf-κb and irf activation are characterized by the phosphorylation and subsequent translocation of p (nf-κb subunit) or irf to the nucleus, respectively ( ) ( ) ( ) . next, we investigated the role of nsp in the phosphorylation of p and irf . hek- t cells were transfected with increasing amounts of pcaggs-ha-nsp , and cell lysates were examined for the expression levels of p-p or p-irf and total p or irf at h post-transfection. as shown in figure g , nsp overexpression had no significant effect on the amount of p , irf , and p-irf ; however, markedly increased p phosphorylation levels, indicating the activation of nf-κb, rather than irf is associated with nsp -induced ifn-β production. therefore, the subcellular location of p was further investigated after nsp overexpression. as shown in figures h,i , ectopic expression of nsp resulted in the nuclear translocation of overexpressed p in pk- cells ( figure h ) and endogenous p in hek- t cells (figure i ). early studies suggested that nsp of cov ibv and sars-cov interacts with host protein ddx ( ) . therefore, we investigated whether tgev nsp also interacts with ddx by co-ip. hek- t cells were cotransfected with pcaggs-ha-nsp and pcmv-tag b-ddx . because ddx is a dexd/h-box helicase and is associated with rna metabolism, the lysates were treated with rnase to avoid the effect of rna on the co-ip experiment. as shown in figure a , flag-tagged ddx was coprecipitated by ha-tagged nsp , indicating the interaction between nsp and ddx . reversed ip with flag antibody further confirmed this interaction ( figure b) . to test whether the colocalization of nsp and ddx occurs, a prerequisite for the interaction, pk- cells were transfected with pcaggs-ha-nsp or empty vector and fixed at h posttransfection. ha-tagged nsp protein was detected with mouse anti-ha antibody, and ddx was detected with rabbit anti-ddx antibody. the results revealed that nsp was colocalized with ddx and distributed both in the cytoplasm and nucleus (figure c) , which further confirmed the interaction between tgev nsp and ddx . because nsp activates ifn-β and interacts with ddx , we investigated whether ddx is involved in nsp -induced ifn-β production. synthesized sirna targeting human ddx (hsiddx ), which efficiently decreases the expression of endogenous ddx mrna ( figure a ) and protein (figure b) , was selected. next, hek- t cells were transfected with hsiddx or sinc, followed by co-transfection of ifn-β-luc or nf-κb-luc, prl-tk, along with pcaggs-ha-nsp or empty vector. the results revealed that knockdown of ddx significantly decreased nsp -induced promoter activity of ifn-β ( figure c ) and nf-κb ( figure d) . moreover, mrna expression levels of nsp -induced ifn-β were downregulated values are the mean ± sd of three independent tests. **p < . or ***p < . compared with empty vector group. (g) hek- t cells were transfected with increasing quantities ( , , µg) of pcaggs-ha-nsp or empty vector for h, and then subjected to immunoblotting with antibodies specific for endogenous irf , phosphorylated irf (p-irf ), p , or p-p . anti-ha mouse antibody was used to confirm the expression of nsp . β-actin expression was used as a loading control. the ratio of phosphorylated/total p and phosphorylated/total irf was analyzed using imagej software. (h) pk- cells were cotransfected with plasmids encoding ha-tagged nsp protein or empty vector together with plasmids encoding egfp-tagged p protein. then, the cells were fixed and immunostained with anti-ha monoclonal antibodies (mabs) to observe the nuclear translocation of overexpressed p using confocal microscopy. (i) hek- t cells were transfected with plasmids encoding ha-tagged nsp protein or empty vector. then, the cells were fixed and immunostained with anti-p antibody to observe the nuclear translocation of endogenous p using confocal microscopy. by ddx deficiency (figure e) , suggesting the involvement of ddx in ifn-β induction by nsp . to determine whether ddx is involved in tgev-induced ifnβ activation, we designed three pairs of sirnas targeting porcine ddx (psiddx ) and selected one with the best knockdown efficiency as demonstrated by rt-qpcr ( figure a ) and western blot assay (figure b) , for subsequent experiments. pk- cells were cotransfected with ifn-β-luc or nf-κb-luc, prl-tk, together with psiddx or sinc. at h post-transfection, cells were infected with tgev for h, followed by dual luciferase assay. ddx depletion had no effect on the basal activity of ifn-β and nf-κb promoter, but significantly decreased tgev-induced activation of ifn-β and nf-κb (figures c,d) . we also detected the expression level of p-p in tgev-infected pk- cells when ddx was silenced. as shown in figure e , knockdown of ddx reduced tgev-induced p phosphorylation. these results suggested that ddx is associated with tgev-induced ifn-β and nf-κb activation. interferon-i initiates a series of signaling cascades through the jak/stat pathway, resulting in the expression of numerous isgs ( ) . furthermore, nf-κb activation plays a pivotal role in regulating the transcription and expression of many proinflammatory cytokines. because ddx is involved in tgev-induced ifn-β and nf-κb activation, theoretically, it should have an impact on tgev-induced isg and pro-inflammatory cytokine expression. the expression levels of some isgs (ifit , ifit , ifit ) and proinflammatory cytokines (il- , il- ) in ddx -knockdown cells were analyzed after tgev infection. as expected, ddx depletion inhibited the expression of ifit , ifit , ifit , as well as il- and il- to some degree, compared with that in cells transfected with sinc (figures f-j) . the innate immune response characterized by the synthesis of ifn and proinflammatory cytokines is the first line of antiviral defense. multiple studies have reported the involvement of covs in the regulation of innate immune responses. the majority of covs decreased dsrna-mediated ifn-β production, including porcine epidemic diarrhea virus (pedv) ( ), severe acute respiratory syndrome coronavirus (sars-cov) ( ) , and infectious bronchitis virus (ibv) ( ) . interestingly, mouse hepatitis virus (mhv)-induced ifn-α/β and established an antiviral state in plasmacytoid dendritic cells and macrophages, but failed to produce ifn in neurons, astrocytes, and hepatocytes ( , ) , indicating that mhv induction of ifn-α/β is cell type dependent. a previous study showed that tgev-induced ifn-α secretion in vitro and in vivo ( ) . here, we found that tgev infection increased the production of ifn-β in a dose-dependent manner in pk- cells, in line with previously reported data ( ) . the significant roles of many cov proteins in the regulation of innate immune response have been identified. for example, nsp , nsp , nsp , papain-like protease (plpro), orf b, orf , ( ) . this indicated the potential effect of tgev nsp on ifn-β induction, which is in line with our data and further confirms our conclusion using a reverse genetic system. although nsp was identified as a key ifn-β activator among tgev-encoded proteins, it remains unclear which prr(s) is involved in its detection and induction of ifn-β production. indeed, viral proteins, similar to viral nucleic acids or replication intermediates, can in some cases also function as pamps, specifically recognized by certain host prrs, such as tlr and tlr , to modulate the ifn responses during viral infection ( , ) . for example, the m glycoprotein of sars-cov has been reported to function as a novel cytosolic pamp to promote ifn-β production by activating a non-canonical tlr signaling cascade ( ) . in addition to the four major prr groups reported previously, including tlrs, rig-i-like receptors, nod-like receptors and cytoplasmic dna receptors ( ) , multiple dexd/h-box helicases, such as ddx , ddx , ddx , and ddx , were reported recently to act as prrs and sense viral pamps to activate the nf-κb signaling pathway and induce ifn-β production ( ) ( ) ( ) ( ) ( ) . previous study revealed that ddx , ddx , and dhx form a complex with the adaptor molecule trif to sense dsrna in dendritic cells ( ) . in this paper, ddx interacted with tgev nsp in a rna-independent manner and enhanced both tgevand nsp -induced activation of ifn-β responses. however, the direct interactions between nsp and ddx or dhx were not observed. we speculated that nsp may be sensed by ddx / ddx /dhx complex by interacting with ddx . these results suggest that nsp may be recognized as pamp by ddx , (e) pk- cells were transfected with psiddx or sinc, and h later, cells were mock infected or infected with tgev (moi = . ) for h. then, the cells were collected for western blot assay with specific antibodies against p , p-p , ddx , or tgev n protein, using β-actin expression as a loading control. the ratio of phosphorylated/total p was analyzed using imagej software. (f-j) pk- cells were treated as described for (e) and collected at hpi. cell rnas were extracted for rt-qpcr to examine the mrna expression levels of ifit (f), ifit (g), ifit (h), il- (i), and il- (j). the mrna expression levels were normalized to porcine gapdh transcripts. values are the mean ± sd of three independent tests. *p < . or **p < . compared with the sinc group. which triggers an antiviral response. further studies are required to investigate this in more detail. ddx is a dexd/h helicase family member composed of the dead-box and related deah, dexh, and dexd protein family and is involved in multiple cellular processes of rna metabolism ( , ) . besides these traditional roles, it appears that multiple proteins of the dexd/h-box helicase family are associated with viral components and/or have alternative effects on viral propagation ( ) ( ) ( ) . for instance, ddx shows antiviral functions against vaccinia virus, denv, and hbv ( - ), but is of benefit for hcv and hiv infection ( , ) . in addition, ddx interacts with the nsp of venezuelan equine encephalitis virus and enhances viral multiplication ( ) . the interaction of ddx with human immunodeficiency virus type rev protein is involved in the regulation of virus replication ( ) . in the present study, the knockdown and ectopic expression of ddx demonstrated that ddx had antiviral activity against tgev replication (data not shown). interestingly, earlier studies also showed an interaction between ddx with coronavirus (ibv and sars-cov) nsp , and in contrast to tgev, this interaction might enhance the replication of ibv ( ) . this difference suggests that ddx is not likely to be a general target against cov infection. furthermore, it should be noted that the effect of ddx on progeny tgev production was moderate (data not shown). difference from other covs, such as sars-cov and mers-cov which antagonize ifn-i, tgev infection induces ifn-i production, and a most recent paper showed that poly(i:c)induced ifn-i responses could only inhibit tgev replication in the early infection stage, but failed in the late infection stage ( ) . they also demonstrated that the activation of ifn-i responses by tgev infection cannot inhibit viral replication. our results are consistent with the conclusion proposed by zhu and colleagues. in addition, it is surprising that the expression levels of ifn-β are paralleled with the increase of viral rna during tgev infection. these may explain why the effect of ddx on tgev replication was moderate accompany with its significant role in ifn-β induction by tgev. however, more studies are required to investigate the complex interaction between tgev and ifns. in conclusion, our data demonstrate that tgev infection induces ifn-β production and nsp is the most significant ifn-β inducer among the tgev-encoded proteins. nsp interacts with cellular dexd/h helicase ddx to activate ifn-β in a nf-κb dependent manner, and ddx is associated with tgev-induced ifn-β production, revealing a potential coactivator role of host rna helicase ddx on virus and viral protein induced innate immune responses. all animal experiments were approved by the hubei administrative committee for laboratory animals (permission number ) and complied with the guidelines of hubei laboratory animal welfare and ethics of hubei administrative committee of laboratory animals. author contributions yz, ww, and lf designed research; yz, ww, and lx performed research; yz, yf, dw, and sx analyzed data; yz and lf wrote the first draft of the manuscript. hc, qk, zh, and xw contributed to modify the manuscript. all the authors read and approved the manuscript. acknowledgments simulation of the economic impact of transmissible gastroenteritis on commercial pig production in australia effects of virulent and attenuated transmissible gastroenteritis virus on the ability of porcine dendritic cells to sample and present antigen complete sequence ( kilobases) of the 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genome-wide, proteomic, and molecular studies ddx dead-box rna helicase inhibits hepatitis b virus reverse transcription by incorporation into nucleocapsids viral targeting of dead box protein reveals its role in tbk /ikkepsilon-mediated irf activation dead-box rna helicase ddx x inhibits denv replication via regulating type one interferon pathway understanding the interaction of hepatitis c virus with host dead-box rna helicases requirement of ddx dead box rna helicase for hiv- rev-rre export function venezuelan equine encephalitis virus non-structural protein (nsp ) interacts with rna helicases ddx and ddx in infected cells ddx is an rna-dependent atpase involved in hiv- rev function and virus replication key: cord- -p rvupjo authors: schmidt, megan e.; varga, steven m. title: the cd t cell response to respiratory virus infections date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: p rvupjo humans are highly susceptible to infection with respiratory viruses including respiratory syncytial virus (rsv), influenza virus, human metapneumovirus, rhinovirus, coronavirus, and parainfluenza virus. while some viruses simply cause symptoms of the common cold, many respiratory viruses induce severe bronchiolitis, pneumonia, and even death following infection. despite the immense clinical burden, the majority of the most common pulmonary viruses lack long-lasting efficacious vaccines. nearly all current vaccination strategies are designed to elicit broadly neutralizing antibodies, which prevent severe disease following a subsequent infection. however, the mucosal antibody response to many respiratory viruses is not long-lasting and declines with age. cd t cells are critical for mediating clearance following many acute viral infections in the lung. in addition, memory cd t cells are capable of providing protection against secondary infections. therefore, the combined induction of virus-specific cd t cells and antibodies may provide optimal protective immunity. herein, we review the current literature on cd t cell responses induced by respiratory virus infections. additionally, we explore how this knowledge could be utilized in the development of future vaccines against respiratory viruses, with a special emphasis on rsv vaccination. introduction given its continuous exposure to the outside environment, the respiratory mucosa is highly susceptible to viral infection. the human respiratory tract can be infected with a variety of pulmonary viruses, including respiratory syncytial virus (rsv), influenza virus, human metapneumovirus (hmpv), rhinovirus (rv), coronavirus (cov), and parainfluenza virus (piv) ( ) . the severity of disease associated with respiratory viral infection varies widely depending on the virus strain as well as the age and immune status of the infected individual. symptoms can range from mild sinusitis or cold-like symptoms to more severe symptoms including bronchitis, pneumonia, and even death. rsv is the leading cause of severe lower respiratory tract infection in children under years of age ( ) . rsv is commonly associated with severe lower respiratory tract symptoms including bronchiolitis, pneumonia, and bronchitis and is a significant cause of hospitalization and mortality in children, the elderly, and immunocompromised individuals ( ) ( ) ( ) ( ) ( ) . similarly, piv commonly infects children and is a major cause of croup, pneumonia, and bronchiolitis ( , ) . seasonal influenza infections, most often of the influenza a virus (iav) subtype, are responsible for - million cases of severe infection annually ( ) . seasonal iav infections also result in approximately , - , deaths per year, most commonly in either young children or elderly populations ( ) ( ) ( ) . however, infection with emerging pandemic iav strains, such as the h n pandemic strain, primarily induces severe disease and mortality in otherwise healthy adults younger than years of age ( ) . in contrast, respiratory infection with hmpv, rv, and cov are most commonly associated with symptoms of the common cold ( ) ( ) ( ) . two notable exceptions are severe acute respiratory syndrome (sars) cov and middle east respiratory syndrome (mers) cov, which cause acute respi ratory distress and mortality in infected individuals ( ) ( ) ( ) . despite their profound impact on human health, most common respiratory viruses lack an approved vaccine. the strategy employed most often in vaccine development is the induction of robust neutralizing antibody responses. however, the hallmark of many respiratory viral infections, including rsv, hmpv, and rv, is the ability for reinfections to occur frequently throughout life ( ) ( ) ( ) . this suggests that the antibody response to these respiratory viruses may wane over time. indeed, despite a correlation between pre-existing nasal iga and protection from reinfection, the development of long-lasting rsv-and rv-specific mucosal iga responses was poor in infected adults ( , ) . although iav-specific neutralizing antibodies are elicited efficiently through either infection or vaccination, iav vaccine formulations must be redeveloped annually to account for the rapid mutations of ha and na genes in seasonal strains ( ) . therefore, vaccinations that solely promote the induction of neutralizing antibodies may not be optimal in providing protection against many respiratory virus infections. the induction of cellular immune responses has thus far received little attention in respiratory virus vaccine development. cd t cells play a critical role in mediating viral clearance following many respiratory virus infections including rsv, iav, and hmpv ( ) ( ) ( ) . in addition, recent murine studies utilizing cd t cell epitope-specific immunization strategies observed significantly reduced lung viral titers following iav, rsv, or sars challenges ( ) ( ) ( ) . therefore, the induction of virus-specific cd t cell responses has the potential to improve upon the efficacy of current vaccination strategies. here, we review the current literature on cd t cell responses following respiratory virus infections and discuss how this knowledge may best be utilized in the development of future vaccines. following an acute respiratory infection, dendritic cells (dcs) that have taken up viral antigen stimulate the activation of naive cd t cells in the lung draining lymph node to induce robust virus-specific cd t cell responses [reviewed in ref. ( ) ( ) ( ) ]. respiratory virus infection in mouse models results in an increase in the frequency and number of total and antigenspecific cd t cells in the lungs and airways. rsv-specific cd t cell responses typically reach peak numbers in the lung at approximately day following an acute infection ( ) ( ) ( ) ( ) . the kinetics of virus-specific cd t cells are slightly more delayed following other respiratory virus infections with peaks occurring at approximately day for iav, days - for hmpv, and days - for pneumonia virus of mice (pvm), a model respiratory virus ( ) ( ) ( ) ( ) ( ) . the peak of the antigen-specific cd t cell response generally corresponds to lung viral clearance following rsv, iav, hmpv, and pvm infections ( , ( ) ( ) ( ) ( ) . human cd t cell kinetics following respiratory virus infections are less well known given the lack of identified cd t cell epitopes and the difficulty in obtaining respiratory tract samples from children following initial virus exposure. the frequency of total activated cd t cells in tracheal aspirates peaked at approximately days after the onset of symptoms in children with rsv, iav, rv, or cov infections ( ). rsv-specific cd t cells were detected in the tracheal aspirates of children; however, the evaluated epitopes were present at very low frequencies, comprising up to only % of the total cd t cell response ( ). following peak expansion, cd t cell contraction occurs and a memory population of virus-specific cd t cells remains within the lung. the majority of virus-specific cd t cells are located within the lung parenchyma, rather than the pulmonary vasculature, following localized respiratory infections in mice ( ) . similarly, human rsv-and iav-specific cd t cells were enriched within the lung compared to the blood ( ). rsv-specific cd t cells in tracheal aspirates of children remain elevated during convalescence following a severe rsv infection, in contrast to murine studies ( ). these studies suggest that cd t cell responses in the airways may be more prolonged following viral clearance in humans compared to mice. following respiratory virus infection, cd t cells become activated and develop the ability to produce inflammatory cytokines. virus-specific cd t cells in the lung and airways of mice upregulate expression of markers associated with activation including cd a, cd , nkg a, and cd as well as downregulate expression of the lymphoid homing receptor cd l ( , , , ) . activated cd t cells also acquire effector functions following viral infection. virus-specific cd t cells rapidly produce cytokines including ifn-γ and tnf as well as degranulate, as measured by cd a expression, following ex vivo peptide stimulation ( , , , ) . human virusspecific cd t cells also acquire an activated phenotype and effector functions following a respiratory virus infection. cd t cells from the tracheal aspirates of children following rsv, rv, or cov infections expressed elevated levels of the activation markers cd and hla-dr and the proliferation marker ki- ( ). expression of effector molecules such as granzyme b and perforin were also increased. similarly, cd t cells from bronchiolar lavage (bal) fluid samples exhibited increased expression of ki- , granzyme b, cd , and hla-dr following either experimental rsv infection of adults or severe, natural rsv infection of infants ( , ). additionally, human virusspecific cd t cells produce cytokines following respiratory virus infection, as peripheral blood cd t cells secreted ifn-γ, tnf, and il- following stimulation with peptides derived from rsv, iav, hmpv, or rv ( - ). following contraction, a subset of virus-specific cd t cells remain in the host to form a long-lasting memory population that provides protection against subsequent infection. cd t cell contraction to form long-term memory populations in the lung is regulated in part by inflammatory chemokine signaling ( ). mice deficient in either cxcr or cxcr and ccr exhibit a significant increase in the number of memory cd t cells following iav infection, suggesting that chemokine signaling through cxcr and ccr plays a critical role in t cell memory generation ( ). following respiratory viral infections in mice and humans, virus-specific cd t cells can be detected up to several months post-infection ( , , , ). however, respiratory virus-specific memory cd t cell populations decline in magnitude with age in the peripheral blood ( ) . interestingly, adult rsv-specific cd t cell responses are significantly reduced compared to iav-specific cd t cell responses in the peripheral blood, suggesting that memory cd t cell responses to iav in humans may be more stable than rsv ( ) . memory cd t cells rapidly expand in the lung following a secondary respiratory virus infection in both mice and humans ( , , , , ) . the observed expansion is primarily due to the migration of circulating cd t cells into the lung and airways, rather than proliferation of resident cells ( ) . the expansion of virus-specific cd t cells in the lung and airways following infection corresponds with an increase in cxcr -and ccr -binding chemokines, supporting a role for chemokine-mediated migration of cd t cells following secondary infection ( ) . indeed, ccr expression on memory cd t cells is required for their early recruitment into the airways after secondary infection, but not to the lung parenchyma ( ) . following secondary expansion, memory cd t cells rapidly produce effector cytokines such as ifn-γ and tnf ( , , ) . additionally, virus-specific memory cd t cells express high levels of cd a and produce cytolytic molecules, such as granzyme b, after infection ( , ) . these effector functions of respiratory virus-specific memory cd t cells are critical for mediating viral clearance and protecting against infection, as discussed below. based on the expression of activation marker cd ra and lymphoid homing receptor ccr , human memory cd t cells have been broadly separated into four major subsets: ( ) naive (cd ra + ccr + ), ( ) central memory (tcm; cd ra -ccr + ), ( ) effector memory (tem; cd ra − ccr − ), and ( ) late effector memory (temra; cd ra + ccr − ) ( ) . due to their expression of ccr , tcm home primarily to secondary lymphoid organs, while tem migrate to peripheral tissues and rapidly exert effector functions. temra are a subset of tem cells that have re-expressed cd ra. they exhibit reduced proliferative and functional capacity, and thus are considered to be terminally differentiated cells. human virus-specific memory cd t cell populations are typically composed of a combination of tem and temra within the peripheral blood ( , , , , ). alternatively, rsv-specific memory cd t cells located in the airways in both adults and infants are primarily of tem phenotype and also express high levels of cd , cd , and ccr and low levels of cd l ( , ). together, these studies indicate that tem cd t cells are dominant following respiratory virus infection in humans. given the frequent exposure to viruses in the respiratory tract, tem cells may be critical for the rapid employment of cd t cell effector mechanisms following reinfection. recently, an additional population of memory cd t cells that persist within peripheral tissues has been identified, termed tissue-resident memory cd t cells (trm) ( ) . trm have been observed within several peripheral organs including the intestine, skin, female reproductive tract, and lung. trm generated following a respiratory virus infection represent a non-circulating population of memory cd t cells that are maintained within the lung parenchyma ( ) . virus-specific trm are located along the wall of large airways and within pulmonary tissue surrounding bronchioles and alveoli ( , ) . respiratory virus infection also induces trm within the airway lumen ( , ) . airway trm downregulate cd a expression and can be distinguished from recently trafficked cd t cells that express high levels of cd a ( , ) . the localization of lung and airway trm following respiratory virus infection is distinctly different from that of tcm, which traffic through the pulmonary vasculature and accumulate in the lung-draining lymph node ( , ) . virus-specific tem are also differentially located from trm residing primarily in the pulmonary vasculature or within the lung tissue near blood vessels, spacially distinct from regions that contain trm ( , ). following either rsv or iav infection in mice, lung and airway trm are induced and can be identified by their expression of the canonical resident memory markers cd and cd , which promote their migration to and retention within the lung tissue ( , , ( ) ( ) ( ) . iav-and rsv-specific trm are also generated in the lungs of mice that have been locally vaccinated via an intranasal route, but not mice that have been immunized systemically ( , ( ) ( ) ( ) . importantly, iav-specific trm expressing cd were also detected in human lung tissue sections but were absent from the spleen ( ) . similarly, rsv-specific trm expressing both cd and cd were identified in the human bal but were not present in the peripheral blood ( ). following secondary viral infection, trm expand prior to the recruitment of circulating memory cd t cell populations from the peripheral blood and rapidly produce ifn-γ ( , ). thus, trm provide a crucial first-line of defense for protecting the host from re-infection with a respiratory virus. however, in contrast to other memory cd t cell subsets that remain stable for long periods of time, iavspecific trm exhibit limited longevity and enhanced apoptosis with time following infection ( , ) . the loss of iav-specific trm corresponds to an increase in viral titers and weight loss following a heterosubtypic iav infection ( , ) . interestingly, infant mice generate fewer lung trm following iav infection or vaccination and exhibit reduced heterosubtypic protection compared to mice initially infected as adults ( ) . given the role of lung trm in providing protection against respiratory virus infections, identifying strategies to promote the generation of long-lived trm will be critical for future vaccines, particularly for infant populations. it has been well established that cd t cells are critical for viral clearance following an acute respiratory virus infection in mice. adoptive transfer of cd t cell clones resulted in significantly reduced viral titers in the lung following rsv, iav, or hmpv infections ( , , ( ) ( ) ( ) ( ) . similarly, the transfer of either rsv-or iav-immune splenic cd t cells accelerated viral clearance in the lung following infection ( ) ( ) ( ) ( ) ( ) . accordingly, rsv infection of mice depleted of cd t cells resulted in significantly increased lung viral titers at day post-infection, although the virus was ultimately cleared by day ( ) . in contrast, depletion of cd t cells alone did not alter clearance of hmpv ( ). instead, depletion of both cd and cd t cells together elevated lung virus titers at day following infection with hmpv. importantly, cd t cells have been shown to be sufficient to mediate viral clearance in the lung following acute respiratory infections ( , ) . athymic nude mice, which lack t cells, fail to clear either rsv or iav resulting in persistent infections ( , ) . however, the transfer of either rsv-or iav-immune splenic cd t cells into athymic mice resulted in significantly reduced lung viral titers by day and , respectively ( , ) . together, these studies indicate that cd t cells play a critical role in mediating viral clearance following acute respiratory infections in mice. although studies are limited, a role for cd t cells in the elimination of respiratory viruses has also been established in humans. early studies demonstrated that immunocompromised children with t cell defects experienced prolonged viral shedding following rsv, iav, or piv infections compared to immunologically normal children ( , , ) . following bone marrow transplantation of an rsv-infected child with severe combined immunodeficiency, a marked reduction in nasal viral load was observed that correlated with an elevation of cd t cell counts ( ) . recently, it has been demonstrated that the number of pre-existing virus-specific cd t cells in the airway of adults experimentally infected with rsv correlated with reduced overall viral load in the nasal cavity and bronchial brushings ( ). in addition to pre-existing cd t cell numbers, cd t cell effector functions also correlate with reduced viral load. cd t cell target cell lysis activity measured by chromiumrelease assay correlated with a lack of viral shedding in the nasal washes of adults experimentally infected with h n iav ( ) . additionally, individuals with the lowest frequencies of ifn-γ + cd t cells exhibited the highest viral titers following natural h n iav infection ( ) . these studies support the role of cd t cells in respiratory virus clearance in humans, consistent with the numerous murine studies. cd t cells mediate viral clearance by utilizing a variety of effector mechanisms to induce the apoptosis of virus-infected cells ( ) . cd t cells can use direct cell-cell contact to eliminate target cells through the interactions of surface molecules such as fas (cd ) and fasl (cd l). additionally, trail expressed on cd t cells can interact with its receptors dr and/or dr to induce the destruction of infected cells. cd t cells can also secrete perforin and granzymes to cause membrane pore formation and induce apoptosis. lastly, cd t cells produce inflammatory cytokines, such as ifn-γ and tnf, which may either directly or indirectly promote the cell death of virus-infected cells. while the exact mechanism utilized is unclear, many of these effector functions have been associated with cd t cell-mediated clearance of respiratory viruses. fas/fasl interactions and the perforin pathway have been established as the primary mechanisms by which cd t cells eliminate infected cells following an iav infection ( , ) . studies utilizing trail-deficient mice and antibody-mediated trail blockade have also demonstrated a role for trail in cd t cell-mediated clearance of iav ( , ) . similarly, fas/ fasl and perforin pathways have also been associated with virus elimination following rsv infection. perforin-deficient and fasl-deficient gld mice exhibit significantly delayed viral clearance ( , ) . however, both perforin-deficient and gld mice achieve complete viral clearance by day post-infection, suggesting that cd t cells compensate for those deficiencies through alternative mechanisms. one such mechanism is likely tnf production, as neutralization of tnf in perforin-deficient and gld mice significantly increased viral titers compared to igg-treated controls ( ) . this is in contrast to studies following pvm and iav infections, where viral clearance occurs independently of tnf ( , ) . ifn-γ does not appear to play a prominent role in cd t cell-mediated viral clearance, as both ifn-γ-deficient mice and mice that received ifn-γdeficient cd t cells exhibit equivalent viral titers to wild-type mice following rsv, pvm, or iav infections ( , ( ) ( ) ( ) . together, these studies demonstrate that cd t cells use multiple complementary mechanisms to eliminate virally infected cells following a respiratory virus infection. given the ability of cd t cells to mediate viral clearance following an acute viral infection, it is no surprise that memory cd t cells also play a critical role in protecting against secondary respiratory virus infections. the adoptive transfer of airway iav-specific memory cd t cells resulted in significantly reduced lung titers following iav challenge compared to pbs transfer controls ( ) . similarly, transfer of airway rsv-specific memory cd t cells reduced lung viral load and weight loss following subsequent rsv infection ( ) . these studies indicate that transferred memory cd t cells are capable of providing protection against secondary respiratory virus challenge. memory cd t cell-mediated protection against secondary infection has been shown more convincingly in mouse models through the use of vaccination strategies to generate virus-specific memory cd t cells. recombinant baculovirus or murine cytomegalovirus (mcmv) vectors expressing the rsv m protein induced m -specific cd t cells that mediated the reduction of lung viral titers following rsv challenge ( , ) . whole protein vaccination with hmpv virus-like particles containing f and m proteins elicited hmpv-specific cd t cells that reduced viral titers in μmt mice, which lack antibodies ( ) . cd t cell epitope vaccines against either rsv or hmpv have also demonstrated cd t cell-mediated protection following challenge by reducing lung viral load and histopathology compared to unimmunized controls ( , ) . a similar strategy utilizing dc-peptide vaccination to generate pre-existing pvm-specific memory cd t cells also resulted in enhanced viral control following pvm infection ( ). recently, several studies have utilized dc-prime, recombinant listeria monocytogenes-(dc-lm) or vaccinia virus-boost (dc-vv) vaccination protocols to generate a high frequency of pre-existing antigen-specific memory cd t cells in the absence of virus-specific cd t cell memory and antibodies ( ) ( ) ( ) . prime-boosted mice exhibited significantly reduced lung viral titers following rsv, iav, or sars infections compared to controls lacking virus-specific memory cd t cells. additionally, memory cd t cells were able to reduce weight loss and mortality following lethal challenges with either iav or sars. overall, these studies provide clear evidence that memory cd t cells provide protection against secondary respiratory virus infection by reducing viral titers. the most well studied example of memory cd t cellmediated protection from a secondary respiratory virus infection is heterosubtypic immunity to iav subtypes. iav-specific neutralizing antibody responses recognize the surface glycoproteins hemagglutinin (ha) and neuraminidase (na), which vary between subtypes as a result of genetic reassortment, known as antigenic drift. however, the internal proteins of the virus are often conserved between iav subtypes. therefore, memory cd t cells that recognize epitopes within conserved viral proteins may be capable of providing cross-protection between iav viruses of differing subtypes. evidence of heterosubtypic immunity was first demonstrated by the protection of h n iav-immune mice from a lethal h n iav challenge without the induction of a neutralizing antibody response ( ) . since then, a memory cd t cell-mediated role in accelerating clearance of a heterosubtypic iav strain has been well-established in mouse, chicken and non-human primate models ( , ( ) ( ) ( ) ( ) . recently, it has been demonstrated that trm are essential in providing cross-protection against secondary iav infection with a heterosubtypic strain ( , , ) . mice with cd + trm in the lung exhibit more efficient viral clearance and reduced weight loss following heterosubtypic challenge than mice lacking a trm response ( ) . importantly, the protection was provided solely by lung-resident memory cd t cells, as blocking the ability of recently proliferated tcm cells from trafficking to the lung did not impact protection ( ) . consistent with the limited lifespan of iav-specific trm, heterosubtypic protection by memory cd t cells wanes over time, with a decline observed as early as days following the initial infection ( , ) . interestingly, systemic immunization with cognate antigen is capable of boosting the trm pool by expanding the circulating tem population that seeds the lungs ( ) . therefore, it is possible that trm-mediated heterosubtypic protection could be re-established by vaccination after a waning of the protective trm population in the lung. while protection in mouse models is well established, whether memory cd t cells play a critical role in protection following secondary respiratory infection in humans is currently unclear. similar to studies in murine models, evidence for heterosubtypic immunity mediated by memory cd t cells has also been demonstrated in humans. individuals lacking h n -specific neutralizing antibody titers exhibited an inverse correlation between memory cd t cell activity and viral shedding following their first exposure with h n iav ( ) . more recently, it was demonstrated that the frequencies of pre-existing crossreactive memory cd t cells correlated with reduced symptoms, including fewer patients with fever, sore throat, and cough, following infection with the pandemic h n iav strain ( ). similarly, a correlation between pre-existing h n -specific memory cd t cells and reduced risk of viral shedding following pandemic h n iav infection was observed ( ) . thus, memory cd t cell-mediated heterosubtypic protection is also likely to be critical in humans. following experimental rsv infection in humans, the frequency of pre-existing rsv-specific memory cd t cells in the airways correlates with a reduction in both cumulative and lower respiratory tract symptom scores, suggesting a possible role for memory cd t cells in protection against rsv in humans ( ). however, evidence has also been provided suggesting that memory cd t cells may not contribute to protection following respiratory virus infections in humans. natural reinfection of infants with rsv did not result in a boosting of the cd t cell response ( ) . similarly, the frequency of rsv-specific memory cd t cells in the peripheral blood of healthy adults is significantly reduced compared to iavspecific memory cd t cells ( ) . therefore, the extent to which memory cd t cells play a role in providing protection against rsv infection in humans remains unclear. despite their beneficial role in mediating viral clearance and protecting against secondary infection, cd t cells have also been associated with the induction of immunopathology following respiratory virus infection. although mice depleted of cd t cells exhibited elevated lung viral titers, weight loss and symptom illness scores were significantly reduced in cd t cell depleted mice following acute rsv infection ( ) . similarly, the adoptive transfer of cd t cell lines exacerbated weight loss following an acute rsv infection, despite accelerating viral clearance ( ) ( ) ( ) . similar reduction in disease severity was also demonstrated following either hmpv or pvm infection of cd t cell depleted mice or mice genetically deficient in cd t cells, respectively ( , ). in addition to the induction of immunopathology following acute respiratory virus infections, we recently demonstrated that memory cd t cells also mediate severe immunopathology following secondary rsv infection ( ) . large frequencies of systemic, pre-existing rsv-specific memory cd t cells generated through dc-lm immunization induced significant weight loss, pulmonary dysfunction, and mortality following rsv challenge, despite a significant reduction in lung viral titers. this result was in contrast to studies using similar immunization strategies to either iav or sars, in which memory cd t cells mediated protection against lethal viral challenge in the absence of immunopathology ( , ) . interestingly, the immunopathology induced by rsv-specific memory cd t cells occurred only in the context of an rsv infection, as mice challenged with a recombinant iav strain expressing an rsv-derived cd t cell epitope exhibited significantly reduced morbidity and were protected from mortality ( ) . this result is consistent with several studies that demonstrate cd t cells enhance viral clearance while preventing mortality following iav infection ( , , , , ) . together, these studies demonstrate a clear role for cd t cells in the development of immunopathology following primary and secondary infections with some respiratory virus infections, particularly rsv. antiviral mechanisms utilized by cd t cells to mediate viral clearance following respiratory virus infection also contribute to the development of immunopathology. removal of the fas/ fasl pathway in gld mice resulted in significant amelioration of weight loss and symptom illness scores following rsv infection ( ) . similarly, rsv-infected perforin-deficient mice exhibited prolonged weight loss and symptom illness scores compared to wild-type mice ( ) . tnf contributes substantially to immunopathology, as antibody-mediated depletion of tnf prior to either rsv or iav infection significantly reduced weight loss ( , , ) . additionally, mice that are ifn-γ-deficient, depleted of ifn-γ, or received an adoptive transfer of ifn-γ-deficient cd t cells prior to rsv infection lost significantly less weight than controls ( ) . cd t cell production of tnf and ifn-γ following pvm infection also induced pulmonary immunopathology by initiating a cytokine storm ( ) . in addition to causing disease in acute respiratory infections, ifn-γ produced by memory cd t cells mediated the severe and fatal immunopathology following rsv infection of dc-lm prime-boosted mice ( ) . the role of cd t cells in the development of pathology following respiratory infections in humans remains unclear. the best evidence supporting a pathogenic role for cd t cells in humans infected with respiratory viruses comes from a study evaluating an rsv-infected severe-combined immunodeficiency patient after bone marrow transplantation ( ) . the patient exhibited increased cd t cell counts following bone marrow transplant, which corresponded to a sharp reduction in rsv nasal titers. however, the appearance of cd t cells also correlated with a marked increase in respiratory rate indicative of reduced pulmonary function. also supporting a pathogenic role of cd t cells is the finding that children requiring mechanical ventilation due to severe rsv infection expressed significantly increased levels of activated granzymes and more cd t cells producing granzyme b compared to healthy controls ( ) . in contrast, a study of infants following either fatal iav or rsv infections revealed a near absence of cd t cells from affected lung regions by immunohistochemical staining ( , ) . similarly, infants with severe rsv infection exhibited an underexpression of genes related to cd t cells in the peripheral blood ( ) . in support of a protective, rather than pathogenic, role of cd t cells, correlations have been identified between increased cd t cell cytolytic activity and cytokine production with reduced symptom score, faster recovery, and fewer fatalities following h n or h n iav infections ( , ) . therefore, whether cd t cells play a primary role in mediating pathology versus protection following human respiratory virus infection remains controversial and is an important topic of future investigation. given the potential of cd t cell effector functions to cause immunopathology following respiratory virus infection, the immune system has evolved critical regulatory mechanisms to prevent prolonged cd t cell effector activity following viral clearance. cd t cell effector functions, including production of ifn-γ and tnf, are suppressed in the lung following the resolution of iav and rsv infections ( ) ( ) ( ) ( ) . one of the primary mechanisms utilized to limit the cd t cell response is through suppression by regulatory cd t cells (tregs). tregs accumulate in the lungs following either rsv or iav infection peaking at approximately day post-infection, prior to the peak of the cd t cell response ( , ( ) ( ) ( ) . antibody-mediated depletion of cd + tregs prior to rsv infection resulted in exacerbated weight loss, pulmonary dysfunction, and lung inflammation ( ) . this enhanced illness corresponded to an increased frequency of rsv-specific cd t cells and elevated levels of ifn-γ and tnf protein in the lung ( , ) . consistent with the treg depletion studies, increasing rsv-specific tregs prior to rsv infection using rsv peptide-immunization resulted in an amelioration of weight loss and a reduction in cd t cell numbers in the blood and spleen, but not the lung ( ) . tregs also can suppress cd t cell effector functions following a secondary infection with iav ( ) . antibody-mediated cd + treg depletion prior to heterosubtypic iav challenge resulted in enhanced inflammation and pulmonary dysfunction corresponding to an increase in cd t cell numbers and ifn-γ production. one mechanism through which tregs may suppress cd t cell responses is through the production of the anti-inflammatory cytokine il- . foxp + tregs secrete il- following primary infection with rsv or iav ( , ( ) ( ) ( ) . infection of either il- -deficient mice or mice treated with il- receptor blocking antibody resulted in increased numbers of either ifn-γ + or ifn-γ + tnf + cd t cells, suggesting that il- suppresses cd t cell effector functions following respiratory virus infection ( ) ( ) ( ) . interestingly, il- production by foxp − cd t cells and cd t cells following either rsv or iav infection has also been reported, indicating that effector t cell responses may self-regulate their effector functions ( ) ( ) ( ) ( ) . together, these studies demonstrate that tregs and il- production play a critical role in regulating cd t cells following primary and secondary respiratory virus infections to prevent immunopathology. interactions between inhibitory receptors on cd t cells with their ligands represents another important mechanism mediating the inhibition of cd t cell effector functions following infection. regulation of cd t cells through the pd- :pd-l pathway is a common inhibitory pathway utilized following respiratory virus infection. expression of pd- on pulmonary cd t cells is upregulated following rsv, iav, or hmpv infection in mice ( , , ) . blockade of pd-l in primary rsv, iav, or hmpv and secondary hmpv infections results in enhanced cd t cell effector functions, including ifn-γ, tnf, and granzyme b production ( , [ ] [ ] [ ] . cd t cell effector functions are also enhanced following either hmpv or iav infections in pd- -deficient mice ( ). importantly, the pd- :pd-l pathway has also been associated with human cd t cell responses. human cd t cells in the nasal cavity significantly upregulated pd- following rsv infection compared to cd t cells from the blood of either healthy or rsv-infected individuals ( ) . pd- and pd-l are also both upregulated in the lung figure | critical factors for an optimal cd t cell-mediated respiratory syncytial virus (rsv) vaccine. a future rsv vaccine designed to elicit a cd t cell response will require a balance between cd t cell-mediated protection and immunopathology, which may be achieved through the consideration of three important aspects: ( ) magnitude, ( ) localization, and ( ) regulation. an optimal magnitude of the cd t cell response will be one that achieves efficient viral clearance in the absence of immunopathology. the vaccination route will be critical in determining the localization of the cd t cell response. a pulmonary route of vaccination will induce trm in the lung that provides superior protection compared to a systemic immunization that would likely not generate protective trm. lastly, regulation of the cd t cell response generated through vaccination will be crucial, as uncontrolled effector functions, particularly ifn-γ production, can result in immunopathology. frontiers in immunology | www.frontiersin.org april | volume | article tissue following severe infections with either rsv or the h n iav pandemic strain ( ). in vitro human studies have demonstrated that pd-l is constitutively expressed on human airway and bronchial epithelial cells, but expression is significantly upregulated following either iav or rsv infection ( , ) . similar to in vivo mouse studies, in vitro pd-l blockade resulted in significantly increased cd t cell production of ifn-γ, il- , and granzyme b following rsv infection ( ) . together, these studies demonstrate a critical role for pd- in the suppression of cd t cell-mediated immunopathology and cytokine production in both mice and humans. in the absence of pd- signaling following hmpv infection, cd t cell ifnγ production remains impaired, suggesting the involvement of compensatory inhibitory pathways ( ) . antigen-specific lung cd t cells express inhibitory receptors tim- , lag- , and b following hmpv infection and exhibit enhanced cytokine production following in vitro blockade of each receptor individually ( ) . in vivo blockade of lag- partially restored cd t cell ifn-γ production in pd- -deficient mice following hmpv infection ( ) . tim- has also been demonstrated to be critical in suppressing cd t cell responses in vivo, as tim- receptor (galectin- )-deficient mice exhibited significantly enhanced cd t cell responses following both primary and secondary iav infections ( ) . together, these studies indicate that multiple inhibitory receptor pathways are utilized following pulmonary virus infection to dampen the pathogenic cd t cell response and prevent immunopathology. successful vaccinations against the majority of respiratory viruses remain elusive. the goal of most vaccination strategies is to induce robust virus-specific neutralizing antibody responses. however, the antibody response generated by infection with many respiratory infections, including rsv and rv, wanes over time. therefore, neutralizing antibody responses as the sole mediator of a vaccine against most respiratory viruses may not provide long-term protection without yearly vaccination. vaccination strategies that include the induction of virus-specific cd t cell responses, either alone or in combination with humoral immunity, may be advantageous by providing many benefits associated with cellular immune responses. cd t cells are critical for the elimination of virusinfected cells, and viral clearance was prolonged in the absence of cd t cells following acute respiratory virus infections. additionally, robust memory cd t cell responses efficiently reduced lung viral titers in the absence of neutralizing antibodies following rsv, iav, or sars secondary infections. an important property of cd t cells is that they often recognize conserved viral proteins, allowing for cross-protection between different virus strains. this is particularly important for heterosubtypic protection of iav strains, as neutralizing antibodies are not capable of recognizing iav strains of differing subtypes. despite their benefits in mediating viral clearance and providing protection against secondary infections, memory cd t cell responses have been associated with the induction of immunopathology following respiratory virus infections. the same antiviral mechanisms employed by memory cd t cells to accelerate viral clearance also contribute to immunopathology, including the fas/fasl pathway-and perforin-mediated cytolysis and ifn-γ and tnf cytokine secretion. thus, the efficient elimination of respiratory viruses by memory cd t cells comes at a cost of disease for the host. cd t cell-mediated immunopathology appears to be virus-specific. although high frequency, systemic, antigen-specific memory cd t cells induced severe disease and mortality following rsv infection, no pathology was observed using similar systems for iav and sars infections. therefore, induction of memory cd t cells as the sole immune mediator may be particularly dangerous for an rsv vaccine, but significantly less so in either an iav or a sars vaccine. to be able to include cd t cell responses within a future respiratory virus vaccine, it will be extremely important to determine how to balance cd t cell-mediated protection versus immunopathology following respiratory infection. for rsv in particular, three critical aspects to consider in this balance include magnitude, localization, and regulation of the rsv-specific cd t cell response (figure ) . dc-lm immunization generated m - -specific cd t cells at a frequency of approximately % in the peripheral blood, but induced fatal immunopathology following rsv challenge ( ) . however, dc-prime only and trivax immunizations generated a much lower frequency of total m -specific cd t cells, and rsv induced significantly reduced disease in these mice ( , ) . thus, identifying the optimal magnitude of rsv-specific cd t cells for protection in the absence of immunopathology is crucial. it is also clear from recent studies in mouse models that localization of rsv-specific cd t cells is a significant factor for both their efficacy of mediating viral clearance and their ability to induce immunopathology following infection. intranasal immunization with mcmv-m generated trm within the lung tissue that accelerated viral clearance. in contrast, mice administered mcmv-m systemically did not generate trm and exhibited delayed viral clearance ( ) . similar results were observed with local immunization with dc-iav-m ( ) . m -specific lung trm generated by pulmonary immunization did not induce immunopathology following rsv infection, in contrast to systemic dc-lm immunization, which resulted in severe pathology in the absence of trm cells. thus, vaccination strategies against rsv will likely be the most effective when administered through a pulmonary route to generate trm that will provide protection within the lung following reinfection without inducing immunopathology. lastly, identifying ways to regulate vaccine-generated cd t cell responses will likely reduce immunopathology following subsequent infection. ifn-γ produced by cd t cells was the primary mediator of immunopathology following rsv infection of dc-lm vaccinated mice ( ) . however, neutralization of ifn-γ had no effect on lung viral titers, suggesting that cd t cells utilize other antiviral mechanisms to mediate viral clearance in this system. since cd t cells are able to reduce viral titers in the absence of ifn-γ, reducing the amount of ifn-γ produced by cd t cells would likely result in ameliorated disease following rsv infection. if vaccination strategies can identify mechanisms by which cd t cell cytokine production, particularly ifn-γ, can be attenuated without altering their ability to eliminate virusinfected cells, the pathology induced by cd t cells would likely also be decreased. the development of a cd t cell-mediated vaccine should be pursued given the limitations of antibody responses to respiratory viruses. it is possible that the ideal vaccine for respiratory virus infections will include the induction of both virus-specific cd t cells and neutralizing antibodies. a vaccination approach combining both arms of the adaptive immune response may allow for optimal viral control in the absence of disease symptoms. however, before cd t cells can be developed further as a mediator of protective immunity, the balance between protection and pathology must be achieved. future studies evaluating aspects of memory cd 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infection regulation of cytokine production by virus-specific cd t cells in the lungs foxp + cd regulatory t cells limit pulmonary immunopathology by modulating the cd t cell response during respiratory syncytial virus infection influenza a virus infection results in a robust, antigen-responsive, and widely disseminated foxp + regulatory t cell response antigen-specific memory regulatory cd +foxp + t cells control memory responses to influenza virus infection epitope-specific regulatory cd t cells reduce virus-induced illness while preserving cd t-cell effector function at the site of infection effector t cells control lung inflammation during acute influenza virus infection by producing il- multiple cd + t cell subsets produce immunomodulatory il- during respiratory syncytial virus infection il- regulates viral lung immunopathology during acute respiratory syncytial virus infection in mice cd + treg cells suppress cd + t cell-responses by il- -dependent mechanism during h n influenza virus infection local blockade of epithelial pdl- in the airways enhances t cell function and viral clearance during influenza virus infection control of pathogenic effector t-cell activities in situ by pd-l expression on respiratory inflammatory dendritic cells during respiratory syncytial virus infection programmed death- impairs secondary effector lung cd + t cells during respiratory virus reinfection rsv-induced bronchial epithelial cell pd-l expression inhibits cd + t cell nonspecific antiviral activity multiple inhibitory pathways contribute to lung cd + t cell impairment and protect against immunopathology during acute viral respiratory infection t cell immunoglobulin and mucin protein- (tim- )/ galectin- interaction regulates influenza a virus-specific humoral and cd t-cell responses key: cord- -btb oodz authors: liu, yiliu; olagnier, david; lin, rongtuan title: host and viral modulation of rig-i-mediated antiviral immunity date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: btb oodz innate immunity is the first line of defense against invading pathogens. rapid and efficient detection of pathogen-associated molecular patterns via pattern-recognition receptors is essential for the host to mount defensive and protective responses. retinoic acid-inducible gene-i (rig-i) is critical in triggering antiviral and inflammatory responses for the control of viral replication in response to cytoplasmic virus-specific rna structures. upon viral rna recognition, rig-i recruits the mitochondrial adaptor protein mitochondrial antiviral signaling protein, which leads to a signaling cascade that coordinates the induction of type i interferons (ifns), as well as a large variety of antiviral interferon-stimulated genes. the rig-i activation is tightly regulated via various posttranslational modifications for the prevention of aberrant innate immune signaling. by contrast, viruses have evolved mechanisms of evasion, such as sequestrating viral structures from rig-i detections and targeting receptor or signaling molecules for degradation. these virus–host interactions have broadened our understanding of viral pathogenesis and provided insights into the function of the rig-i pathway. in this review, we summarize the recent advances regarding rig-i pathogen recognition and signaling transduction, cell-intrinsic control of rig-i activation, and the viral antagonism of rig-i signaling. to ensure successful antiviral defenses and to avoid aberrant or dysregulation of host immune signaling, antiviral pathways need to be tightly regulated at each level. in this review, we will summarize the cell-intrinsic regulation of rig-i receptor activity, as well as the viral strategies to subvert the rig-i signaling machinery. the three members of the rlr family: rig-i, mda (melanoma differentiation factor ), and lgp (laboratory of genetics and physiology ) are expressed in most cell and tissue types. they function as cytoplasmic sensors for the recognition of a variety of rna viruses and subsequent activation of downstream signaling to drive type i ifn production and antiviral gene expressions. these three rlr proteins are rna-dependent atpases belonging to the dexd/h-box family of helicases ( ) . structurally, rlrs have a similar central helicase core that is comprised of two helicase domains, hel and hel with an insertion termed hel i. in addition, they all have a c-terminal domain (ctd). however, only rig-i and mda contain two n-terminal caspase activation and recruitment domains (cards) ( ) (figure a ). among these three, rig-i is the founding member and hence the most intensively studied member of this family. each domain of rig-i plays unique roles during rig-i autorepression and activation. in brief, the ctd and helicase domain are involved in rna ligand binding and atp hydrolysis-involved conformational changes ( ) ( ) ( ) , whereas the rig-i cards facilitate interaction with other downstream card containing molecules ( ) . retinoic acid-inducible gene-i has been shown to be involved in the recognition of a variety of rna viruses in the cytoplasm, such as the sendai virus, influenza a and b viruses (iav, ibv), vesicular stomatitis virus, measles virus (mv), newcastle disease virus, ebola virus (ebov), dengue virus (denv), and hepatitis c virus (hcv) ( ) ( ) ( ) ( ) . the short double-stranded (ds) rna with a triphosphate (ppp) motif at the ′-end, as found in these viral genomes, were shown to be a key signature recognized by rig-i ( , ) . the ′ppp dsrna of viral nucleocapsids has also been characterized as stimulating rig-i ( ) . ′-diphosphate-bearing rna ( ′pprna), either naturally contained in viruses, produced by in vitro transcription, or via chemical synthesis, were all shown to bind to rig-i and were sufficient to activate rig-i ( , ) . physiologically, the control of in vitro and in vivo infections of reoviruses, which bear the ′pprna genome, relies on rig-i functionality ( ) . it is worth noting that the in vitro-synthesized ′ppprna sequences also trigger rig-i activation ( ) . these agonists have demonstrated their therapeutic potential as broadspectrum antiviral agents and could be optimized as vaccine adjuvant candidates ( ) ( ) ( ) ( ) ( ) . furthermore, the recognition of several dna viruses, including herpes simplex virus type (hsv- ), epstein-barr virus (ebv), vaccinia virus (vacv), and adenovirus, via the rna polymerase iii were found to be rig-idependent ( , ) . interestingly, the rig-i-mediated upregulation of sting is required for protection against the hsv- by the rig-i agonist, offering new evidence of the overlapping between rig-i signaling and the host response to dna viral infection ( ) . notably, viral rna triggered rig-i signaling also mediates the inflammatory response via distinct pathways. the first involves the formation of the rig-i inflammasome through interactions between rig-i, asc, and caspase- and the stimulation of il- β release. the second involves the adaptor proteins card , bcl- , mitochondrial antiviral signaling protein (mavs), and the activation of nuclear factor-κb (nf-κb) ( , ) . upon rna ligand binding, rig-i undergoes a series of conformational changes and posttranslational modifications (ptms) to achieve full activation (further detail below). activated rig-i recruits its downstream adaptor molecule mavs (also known as ips- , cardif, and visa) through card-card-mediated interactions ( , ) . the oligomeric rig-i card assembly and the polymeric formation of mavs, together serve as a signaling platform for protein complexes that mediate the bifurcation of signaling into two branches. one branch recruits tumor necrosis factor receptor-associated factors (traf)- / and the receptor-interacting protein to subsequently activate the ikk complex, resulting in nf-κb activation ( ) . the other branch signals through traf and activates the tank/ikkγ/ikkϵ/tbk complex, leading to the phosphorylation and dimerization of interferon regulator factors (irf)- and - ( , ) . activated irf / and nf-κb then translocate to the nucleus, together with atf , c-jun, and the transcription coactivator creb-binding protein/p , to coordinate the ifn and pro-inflammatory gene expressions ( ) . once secreted, ifns bind to specific cell surface receptors and activate the jak-stat pathway. the activated transcription factors stat , stat , and irf form the interferon-stimulated gene factors (isgf ) complex. isgf then translocates to the nucleus and coordinates the transcription of hundreds of isgs including rig-i, thus generating an amplifying loop leading to the accumulation of rig-i during several types of infections ( ) (figure b ). structural and biochemical studies have demonstrated that the activation of rig-i is a multi-step process and is primarily regulated by conformational changes and ptms. when initially identified as a dsrna sensor, it was hypothesized that rig-i was under negative regulation in physiological conditions. the over expression of the card domain of rig-i alone demonstrated superior signaling activity than full length rig-i in absence of viral pamps ( ) . studies by saito et al. showed that the deletion of card was dominant-negative for rig-i signaling. by contrast, the deletion of repressor domain (rd) resulted in constitutive signaling, whereas rd expression alone ablated rig-i signaling actions. together, these findings provided the model of rig-i autoregulation in which the rd is predicted to mask cards for signaling transduction in uninfected cells ( ) . the crystal structural analysis further delineated the models of autorepressed and ligand activated states of rig-i, respectively. in a ligand-free state, cards and hel i interactions hinder dsrna binding and inactivate rig-i ( ) . the binding of ′ppp dsrna to rd leads to a conformational switch of rig-i, which releases the autorepressed cards and exposes the helicase domain for atp binding ( , ) . atp hydrolysis is essential for rig-i signaling. it enables rig-i to translocate along the dsrna, and further promotes the oligomerization of rig-i cards. these processes assemble rig-i into a filamentous architecture which facilitates the card-card interactions with the mitochondrial mavs, leading to the subsequent signaling transduction for ifn production ( , ) . importantly, rig-i atpase activity also plays a role in distinguishing self-rna from non-self-rna ( ) . it was reported that rig-i atp hydrolysis increases the binding affinity of rig-i and dsrna ligands; whereas the rig-i mutants deficient in atp hydrolysis promotes the interaction of rig-i and self-dsrna and results in unintentional immune signaling ( ) . one of the first ptms of rig-i following the initial ligand recognition is performed by the robust ubiquitination machinery (figure ) . mass spectrometry analysis revealed that trim , a member of the tripartite motif (trim) protein family possessing e ligase activity, induces the covalent lys -linked ubiquitination of rig-i. mechanistically, the c-terminal spry domain of trim interacts with card and facilitates the ubiquitination of card at k ( ) . the rig-i-trim ubiquitination complex, associates with the adaptor protein - - ϵ and translocates to mitochondria for mavs binding ( ) . mutation figure | regulation of retinoic acid-inducible gene-i (rig-i) activation. (a) in resting cells, rig-i is kept inactivated through the phosphorylation of caspase activation and recruitment domains (cards) and c-terminal domain (ctd) mediated by casein kinase ii and protein kinase c-α/β, respectively. (b) following the binding of ′ triphosphate ( ′ppp) rna and atp hydrolysis, rig-i is dephosphorylated by phosphoprotein phosphatase -α/γ and results in a conformational change that opens cards. hdac -mediated deacetylation of rig-i ctd is critical for rig-i and ′ppprna binding. the lys -linked ubiquitination of rig-i mediated by trim , riplet, oligoadenylate synthetases-like protein, and mex c at both cards and ctd further activate rig-i and facilitate its tetramerization. (c) interactions between rig-i-trim complex and - - ϵ promote rig-i translocation to mitochondrial mitochondrial antiviral signaling protein (mavs) for downstream signaling, leading to interferon production. interactions between trim , rig-i, and mavs are further negatively regulated by the lys -linked ubiquitination, which is meditated by lubac, rnf , and rnf . sec l and atg -atg both inhibit the signaling by interrupting rig-i-mavs interactions, whereas sumoylation promotes rig-i-mavs binding. of k disrupts the interaction between rig-i and mavs thus abrogating downstream signaling and ifns production ( ) . furthermore, a rig-i splice variant which lacks the trim interaction domain acts as a feedback inhibitor of rig-i signaling transduction upon viral infections ( ) . in addition, riplet (ring-finger protein leading to rig-i activation, also named rnf or reul), another e ubiquitin ligase, also promotes rig-i ubiquitination. multiple sites within the cards, as well as within the ctd of rig-i, were identified as the crucial ubiquitin anchoring residues ( - ). among which, k -linked polyubiquitination (pub) at lys , is demonstrated as being critical for rig-i activation. however, unlike trim -induced ubiquitination, riplet induced rig-i pub is dispensable for rig-i-rna binding but is essential for releasing card from its autorepressed state. this enhances trim functionality as well as promoting the oligomerization of rig-i and the activation of mavs ( ) . mex c (mex- rna binding family member c), another recently identified e ligase, also mediates lys -ub at k and k of card, playing a critical role in rig-i activation ( ) . in addition, the oligoadenylate synthetases-like (oasl) protein, although not an e ubiquitin ligase itself, contains a dsrna-binding groove and enhances rig-i activation by mimicking the k -linked pub through its ubiquitin-like (ubl) domain ( , ) . non-covalent binding of k -ubiquitin chains to cards also potently activates rig-i ( ) . recent structural analysis suggests that covalent and non-covalent binding of ubiquitin synergistically stabilize rig-i tetramerization and enhance polymerization of mavs cards ( ) . on the other hand, several deubiquitinating enzymes (dubs) were identified to remove k -linked pub chains from rig-i, thus dampening rig-i signaling. the tumor suppressor protein cylindromatosis (cyld) removes k -linked pub chains from rig-i as well as tbk and ikkϵ to inhibit the irf response, serving as a pathway negative regulator ( ) . syndecan- , a newly identified negative regulator of rig-i, functions through attracting cyld to rig-i complex, thus potentiating the k -mediated deubiquitination of rig-i ( ) . in addition, the ubiquitin-specific protease (usp) family members, such as usp and usp , were also identified as inhibitors of rig-i activation by deubiqutinating rig-i ( , ) . in contrast to k -linked ubiquitination, which promotes protein activation, k -linked ubiquitination triggers proteasomal degradation of its target. for instance, the ring-finger protein (rnf ), together with the ubiquitin e ligase ubch , conjugate k -linked ubiquitin to rig-i and mavs, targeting them for proteasomal degradation and thereby inhibiting downstream signaling ( ) . similarly, rnf was recently demonstrated to mediate the proteasomal degradation of rig-i by delivering the k -linked ubiquitin to rig-i cards ( ) . the linear ubiquitin assembly complex (lubac) has been shown to promote k pub of trim , leading to its degradation ( ). conversely, the deubiquitinase usp antagonizes lubac by removing k linked ubiquitin from trim , leading to its stabilization and thereby promoting rig-i-mediated antiviral signaling ( ) . in parallel with ubiquitination, phosphorylation has emerged in the past several years as a critical regulator of the rig-i signaling transduction (figure ) . protein purification and mass spectrometry analysis identified that phosphorylation of thr in the cards antagonizes rig-i signaling by inhibiting trim -mediated lys ubiquitination and mavs binding ( ) . ser phosphorylation of cards also serves as a negative regulator of rig-i ( ). in addition, the ctd of rig-i is constitutively phosphorylated at thr and ser / by casein kinase ii to promote intermolecular interactions between ctd and cards, thereby maintaining rig-i at an autorepressive state to prevent premature downstream signaling ( ) . a recent mass spectrometry analysis revealed that ikk phosphorylates rig-i at ser , thereby providing a negative feedback regulation of rig-i ( ). furthermore, conventional protein kinase c-α (pkc-α) and pkc-β have also been shown to phosphorylate cards, thus suppressing rig-i-trim interaction and subsequent antiviral responses ( ) . in fact, rig-i signaling activity is controlled by a dynamic balance between phosphorylation and dephosphorylation. dephosphorylation of rig-i occurs rapidly with the presence of viral rna. a functional sirna screen identified phosphoprotein phosphatase -α (pp α) and pp γ as essential phosphatases responsible for cards dephosphorylation at ser and thr , leading to rig-i signal activation and viral inhibition ( ) . in addition to the ubiquitination and phosphorylation described above, acetylation modulation has recently started to gain more acknowledgment for controlling rig-i activity (figure ) . mass spectrometry has identified the acetylation of two lysine residues (k and k ) in the ctd of rig-i at its inactivate state and are deacetylated during viral infection ( ) . the mutation of these two sites restricts rig-i from undergoing the virusinduced interaction with mavs. k and k acetylation of rig-i has also been shown to control the pamp rna-induced rig-i oligomerization ( ) . the cytoplasmic deacetylase hdac -mediated removal of k acetylation has been shown as critical for rig-i binding to dsrna during viral infections ( ) . furthermore, hdac -dependent rig-i deacetylation also regulates rig-i oligomerization upon ligand binding, thus facilitating rig-i activation ( ) . rig-i signal transduction is further regulated by additional ptms, regulatory proteins, and other cellular processes (figure ) . it is worth noting that a number of ubl proteins including sumo, isg , fat , and atg -atg are involved in these positive or negative regulatory mechanisms ( ) . sumoylation serves as a positive regulator of rig-i by enhancing the rig-i and mavs binding ( ) . on the contrary, the hla-f adjacent transcription (fat ), an ubl modifier protein, was shown to negatively regulate rig-i by modulating rig-i solubility through a non-covalent association with cards ( ) . in addition, ifn-induced isg negatively regulates the rig-i mediated signaling in a feedback-loop control manner ( ) . sec l has been observed competing with mavs for rig-i card binding ( ) . furthermore, autophagy has been reported to be involved in rig-i modulation through its key regulator, the atg -atg conjugate. atg -atg has been found to suppress rig-i-mavs interaction, thereby inhibiting downstream signaling ( ) . recently, deamidation of ctd has been described as a distinct means to induce rig-i activation. for examples, vgat (glutamine amidotransferase), from kshv (kaposi's sarcomaassociated herpesvirus) and γhv (murine gamma herpesvirus ), recruits cellular phosphoribosylformyglycinamide synthase to deamindate and activate rig-i ( , ). in order to establish infections, viruses have developed sophisticated mechanisms to counteract host immune responses. with regard to rig-i signaling, these include mechanisms such as altering viral genomes and their intermediate transcripts to avoid detection, manipulating the activation and degradation of rig-i and mavs, as well as modulating downstream signaling cascades. studying these antagonistic viral strategies has greatly broadened our understanding of rig-i activation and regulation. since ′ triphosphate ( ′ppp) is an important feature recognized by rig-i, modification of this motif has long been described as one of the major mechanisms for viruses to antagonize rig-i signaling. crimean-congo hemorrhagic fever virus, borna disease virus (bdv), and hantavirus (htnv) remove the ′ppp group on their genome posttranscriptionally, make rig-i unable to bind to viral rna, and therefore incapable of triggering rig-i activation ( ) . mechanistically, htnv uses the "prime and realign" strategy to generate a ′-terminal monophosphorylate ( , ) . bdv on the other hand, employs genome trimming to form a ′-terminal overhang as well as convert ′ppp to ′p to avoid detection by rig-i ( ) . the arenavirus presents an unpaired ′ppp-nucleotide overhang to evade recognition by rig-i ( ) . the ′-end of viral rna can also be modified through rna-capping pathways. for example, the genomic rna of polioviruses linked to vpg (viral protein genome-linked) to cap the ′-end from exposure to rig-i ( ). the ′-end capping with -methyl guanosine and methylation of ′ppp dsrna at the ′-o position makes viral rna non-distinguishable from the host mrnas, and therefore does not stimulate rig-i ( , ) . by contrast, some viruses encode viral proteins to prevent rna recognition. the ebov utilizes its vp protein to sequester viral rna ( ) . the crystal structural analysis indicates that the vp interferon inhibitory domain competes with rig-i for dsrna binding by forming an "end-cap" complex with dsrna, resulting in substantially diminished activation of rig-i ( ) . similarly, the marburg virus vp spirals around the dsrna backbone and end-caps the dsrna to escape from rig-i detection ( , ) . the iav non-structural protein (ns ) possesses dsrna-binding properties to shield viral rna from rig-i ( ). iav has also been shown to antagonize rig-i activation via its viral polymerase subunit pb . pb position k in the mammalian strain increases pb -nucleocapids binding affinity, thus inhibiting rig-i interaction with the nucleoprotein-encapsidated ′ppp rna ( , ) . in addition to altering and concealing their genome to prevent rna binding, viruses also re-localize viral rna to specific cellular compartments, such as mitochondria, endoplasmic reticulum (er), and golgi, to avoid cytosolic surveillance by rig-i. for instance, the denv conceals dsrna in the intracellular membrane as an escape strategy ( ) . er is an important organelle for viral entry, replication, and assembly. the severe acute respiratory syndrome (sars) coronavirus (sars-cov) has been shown to induce a modified er to hide its replicating rna from detection ( ) . these viral antagonism strategies highlight the importance of cellular organelle localization in viral-host interactions during innate antiviral responses. as reviewed above, ubiquitination represents one critical ptm mechanism of rig-i activation and, not surprisingly, is an attractive target for viral manipulation (figure a) . viruses have evolved ways to inhibit k -linked ubiquitination of rig-i by interacting with the e ligases trim and riplet. for instance, iav ns from various strains has been shown to suppress trim -mediated rig-i cards ubiquitination. among all the trim binding amino acids identified in ns , r /k and e /e were described as critical in interfering with the coil-coiled domain of trim . these interactions resulted in an inhibition of trim multimerization and therefore blocked the rig-i cards ubiquitination ( ) . intriguingly, ns -trim binding is found to be preserved in human and avian, but lost in mouse, indicating a species-specific manner of inhibition. this study further demonstrates that the ns suppression of rig-i ubiquitination in mouse is riplet-dependent ( ) . conversely, phosphorylation of ns at thr was recently identified as impairing the ns -trim interaction, thereby suppressing its antagonistic activity of rig-i signaling ( ) . phosphorylation of another site on ns , thr , has also been reported to disrupt ns binding affinity with rig-i ( ) . similar to iav, the ibv non-structural ns protein (ns -b) has recently been described as inhibiting rig-i ubiquitination, which involves trim -ns c-terminal effector domain interaction and the rig-i/trim / ns -b complex formation ( ) . by contrast, the protease ns - a of hcv functions differently, rather than inhibiting trim , it is thought to target the e ligase riplet. ns - a directly disrupts riplet, abolishes riplet-mediated rig-i ubiquitination, and further reduces the interaction between trim and rig-i ( ). on the other hand, some viruses encode enzymes that directly deubiquitinate rig-i. for instance, kshv encoded deubiquitinase orf cleaves lys -ubiquination chains on cards, blocks cards interaction between rig-i and mavs, thereby downregulating rig-i signaling ( ) . other viruses including arterivirus, nairovirus, sars-cov, and foot-and-mouth disease virus (fmdv) have also been reported to downregulate rig-i ubiquitination through their viral encoded dubs ( , ) . few viruses have been shown to manipulate rig-i regulation with regards to targeting the phosphorylation or dephosphorylation process of rig-i. nevertheless, it was reported that mv efficiently escapes antiviral response via suppressing rig-i dephosphorylation in dendritic cells (dcs). in this study, the growth arrest and dna damage protein (gadd ) was shown to form complexes with pp to facilitate rig-i activation. the mv infection induced dc-sign signaling results in an inhibition of gadd -pp phosphatases activity and thereby impairs rig-i activation ( ). another distinct strategy used by viruses to antagonize rig-i signaling is the direct cleavage or degradation of the receptor and multiple members of the signaling cascade ( figure a) . rig-i has been reported in some studies to be cleaved by the proteinase c pro during infections with picornavirus, coxsackievirus b (cvb ), and enterovirus (ev ) ( , ) . the encephalomyocarditis virus directs both caspase-and proteasome-dependent degradation of rig-i ( ) . intriguingly, the ns -ns degradasome of the respiratory syncytial virus (rsv) has been shown to mediate the proteasomal degradation of rig-i ( ) . mitochondrial antiviral signaling protein is also a well-studied molecule which is often targeted by many types of viral-induced cleavage. for example, the hepatitis a virus (hav) cleaves mavs for proteolysis by its protease c pro ( ) . both cvb proteinase a pro and c pro trigger mavs cleavage at different sites during infection, and the cleavage of mavs by ev is accomplished via its a pro activity ( , ) . in addition, serine protease ns - a of hcv cleaves mavs, removing it from the mitochondria, thereby inhibiting downstream signaling ( , ) . in a parallel fashion, many viruses mediate cellular proteolytic degradation of mavs to attenuate rig-i antiviral responses. hepatitis b virus viral protein hbx triggers the proteasome-mediated degradation of mavs through lys ubiquitination ( ) . another study reported that the hav cysteine protease abc targets mavs for proteolysis at mitochondrial membrane ( ) . additionally, viral modulation of cellular organelles such as mitochondria also affects rig-i-mavs signaling. the pb -f of iav, for instance, has been described as decreasing the mitochondrial membrane potential, resulting in the acceleration of mitochondrial fragmentation, thereby inhibiting rig-i-mavs signaling ( ) ( ) ( ) . it is important to note that the proper localization of rig-i and mavs is a prerequisite for effective signaling transduction. mavs resides on the mitochondrial membrane, peroxisomes, and mitochondria-associated membranes for antiviral signaling. in fact, a rig-i translocon has been identified to direct rig-i redistribution from cytosol to membranes during viral infection ( ) . studies have shown that several viruses encode proteins to disrupt the proper localization of rig-i or mavs as a novel mechanism of regulation, such as ns of denv ( ) , nucleoprotein of rsv ( ), and non-structural proteins of thrombocytopenia syndrome virus (sftsv) ( ) . to ensure successful rig-i signaling transduction, the kinase activities of tbk and ikkϵ are tightly controlled via various regulatory mechanisms and are common targets of viruses ( figure b) . for example, both the leader proteinase (l pro ) of fmdv ( ) and the non-structural protein (ns ) of the mouse hepatitis virus a ( ) inhibit ubiquitination of tbk . dengue virus serotype non-structural proteins, ns a and ns b, as well as the flips proteins encoded by the molluscum contagiosum virus (mcv), all reduce tbk phosphorylation, thereby preventing its activation ( , ) . several viruses have been shown to prevent the formation of functional tbk -containing complexes. the k protein of the vacv prevents tbk /ikkϵ complex-induced irf activation by targeting host dead box protein (ddx ) ( ) . two other viruses, the ny- htnv and sars-cov, disrupt the tbk -traf and tank-tbk /ikkϵ complex, respectively ( , ) . moreover, sftsv has been shown to irreversibly re-localize tbk and ikk from mitochondria and sequester the tbk /ikkϵ/irf complex via the formation of inclusion bodies, causing signaling cascade termination ( ) . viral regulation of the transcription factors, irfs and nf-κb, further serve as points of control in rig-i signaling ( figure b) . one of the best studied examples is the inhibition of irf activity by the iav ns protein ( ) . besides this, the hsv- , rabies virus, sars-cov, as well as several paramyxoviruses have been demonstrated to interfere with the phosphorylation state of irf , thereby blocking ifn induction ( ) ( ) ( ) ( ) . the ebv conjugates sumo to irf at lysine to decrease irf transcriptional activity ( ) . the rotavirus ns , targets both irf and irf for degradation to prevent irfs from undergoing dimerization ( ) . viruses have also developed various means to suppress the irf dna binding ability. herpes simplex virus, thogoto virus, and kshv, all developed strategies to downregulate irf transcriptional activity by either disrupting irf binding complex formations or competing binding regions on the ifnb promoter ( ) ( ) ( ) . viral strategies in inhibiting cytoplasmic or transcriptional activities of nf-κb have been extensively studied during the vacv infection. studies reported that multiple proteins encoded by vacv and hsv- suppress nf-κb activation ( ) ( ) ( ) ( ) . viruses have also developed multiple inhibitory mechanisms to counteract the ifn stimulation of isgs by targeting stat and/or stat ( figure b ). for example, the langat virus was shown to inhibit the phosphorylation of both stat and stat ( ) . varicella viruses and the japanese encephalitis virus, both block the jak/stat pathway through multiple mechanisms including inhibiting stat proteins phosphorylation and nucleotranslocation ( , ) . the non-structural protein ns of several flaviviruses, have been shown to target stat proteins via distinct mechanisms. for example, mnv ns inhibits stat phosphorylation, whereas denv ns interacts with ubr to promote stat degradation ( , ) . by contrast, the zika virus ns induced proteasomal degradation of stat was recently identified as ubr independent ( ) . furthermore, other viruses, such as hcv ( ) , rsv ( ) , and paramyxovirus ( ) , also demonstrate negative regulation of the jak-stat pathway. studies from the past decade have well established rig-i as one of the principal prrs for the recognition of cytoplasmic viral rna, as well as defining its critical role in the induction of ifns during viral infections. our understanding of the rig-i-mediated antiviral response has been greatly expanded with the key discoveries made regarding the molecular mechanism of rig-i regulation, such as ubiquitination, phosphorylation, and acetylation. meanwhile, investigating viral strategies to manipulate rig-i responses not only allow us to understand the viral pathogenesis, but also significantly contributed to our knowledge of how rig-i is activated and regulated. these new insights into the viral-mediated rig-i regulations are important for vaccine and drug development aiming to suppress infectious diseases and enhance immune responses. yl wrote the manuscript. rl and do revised and approved the manuscript. the authors would like to thank alexandre sze for his critical reading and editing of the manuscript. this research was supported by grant from canadian institutes of health research (mop ) to rl; do was supported by a peter quinlan mcgill postdoctoral fellowship. the authors would like to acknowledge all the colleagues in the field and apologies to those whose important contributions could not be included in the review due to space constraints. the figures of the review were illustrated using the servier medical art library, http://www. servier.com/powerpoint-image-bank. the role of pattern-recognition receptors in innate immunity: update on toll-like receptors the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses immune signaling by rig-i-like receptors tlr signaling pathways regulation of the antimicrobial response by nlr proteins cyclic gmp-amp synthase is a cytosolic dna sensor that activates the type i interferon pathway ifi is an innate immune sensor for intracellular dna mechanisms of type-i-and type-ii-interferon-mediated signalling viral evasion and subversion of patternrecognition receptor signalling viral evasion of intracellular dna and rna sensing pattern recognition receptors and inflammation structural insights into rna recognition by rig-i structural basis of rna recognition and activation by innate immune receptor rig-i structural basis for the activation of innate immune pattern-recognition receptor rig-i by viral rna orchestrating the interferon antiviral response through the mitochondrial antiviral signaling (mavs) adapter recognition of viral nucleic acids in innate immunity rna recognition and signal transduction by rig-i-like receptors ebola virus vp protein binds double-stranded rna and inhibits alpha/beta interferon production induced by rig-i signaling rig-i, mda and tlr synergistically play an important role in restriction of dengue virus infection ′-triphosphate rna is the ligand for rig-i recognition of ′ triphosphate by rig-i helicase requires short blunt double-stranded rna as contained in panhandle of negative-strand virus incoming rna virus nucleocapsids containing a ′-triphosphorylated genome activate rig-i and antiviral signaling rig-imediated antiviral responses to single-stranded rna bearing ′-phosphates antiviral immunity via rig-i-mediated recognition of rna bearing ′-diphosphates systems analysis of a rig-i agonist inducing broad spectrum inhibition of virus infectivity inhibition of dengue and chikungunya virus infections by rig-i-mediated type i interferon-independent stimulation of the innate antiviral response enhanced influenza virus-like particle vaccination with a structurally optimized rig-i agonist as adjuvant defining new therapeutics using a more immunocompetent mouse model of antibody-enhanced dengue virus infection sequence-specific modifications enhance the broad-spectrum antiviral response activated by rig-i agonists cutting edge: the rig-i ligand prna potently improves ctl cross-priming and facilitates antiviral vaccination early innate recognition of herpes simplex virus in human primary macrophages is mediated via the mda /mavs-dependent and mda /mavs/ rna polymerase iii-independent pathways rna polymerase iii detects cytosolic dna and induces type i interferons through the rig-i pathway rig-i mediated sting up-regulation restricts hsv- infection recognition of rna virus by rig-i results in activation of card and inflammasome signaling for interleukin beta production type i ifn triggers rig-i/tlr /nlrp -dependent inflammasome activation in influenza a virus infected cells cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf regulation and function of nf-kappab transcription factors in the immune system a functional c-terminal traf -binding site in mavs participates in positive and negative regulation of the ifn antiviral response the nemo adaptor bridges the nuclear factor-kappab and interferon regulatory factor signaling pathways direct triggering of the type i interferon system by virus infection: activation of a transcription factor complex containing irf- and cbp/p regulation of innate antiviral defenses through a shared repressor domain in rig-i and lgp the c-terminal regulatory domain is the rna ′-triphosphate sensor of rig-i atpase-driven oligomerization of rig-i on rna allows optimal activation of type-i interferon rig-i forms signaling-competent filaments in an atp-dependent, ubiquitin-independent manner rig-i atpase activity and discrimination of self-rna versus non-self-rna correction: atp hydrolysis by the viral rna sensor rig-i prevents unintentional recognition of self-rna roles of rig-i n-terminal tandem card and splice variant in trim -mediated antiviral signal transduction the mitochondrial targeting chaperone - - epsilon regulates a rig-i translocon that mediates membrane association and innate antiviral immunity trim ringfinger e ubiquitin ligase is essential for rig-i-mediated antiviral activity riplet/rnf , a ring finger protein, ubiquitinates rig-i to promote interferon-beta induction during the early phase of viral infection the ubiquitin ligase riplet is essential for rig-i-dependent innate immune responses to rna virus infection reul is a novel e ubiquitin ligase and stimulator of retinoic-acid-inducible gene-i a distinct role of ripletmediated k -linked polyubiquitination of the rig-i repressor domain in human antiviral innate immune responses pivotal role of rna-binding e ubiquitin ligase mex c in rig-i-mediated antiviral innate immunity antiviral activity of human oasl protein is mediated by enhancing signaling of the rig-i rna sensor structural and functional analysis reveals that human oasl binds dsrna to enhance rig-i signaling reconstitution of the rig-i pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity structural basis for ubiquitin-mediated antiviral signal activation by rig-i the tumour suppressor cyld is a negative regulator of rig-i-mediated antiviral response syndecan- negatively regulates antiviral signalling by mediating rig-i deubiquitination via cyld usp inhibits type i interferon signaling by deubiquitinating rig-i-like receptors usp negatively regulates antiviral response by acting as a rig-i deubiquitinase negative regulation of the rig-i signaling by the ubiquitin ligase rnf rnf suppresses antiviral type i interferon production by targeting rig-i cards to mediate rig-i degradation linear ubiquitin assembly complex negatively regulates rig-i-and trim -mediated type i interferon induction the ubiquitin-specific protease usp promotes rig-i-mediated antiviral signaling by deubiquitylating trim phosphorylationmediated negative regulation of rig-i antiviral activity negative role of rig-i serine phosphorylation in the regulation of interferon-beta production phosphorylation of rig-i by casein kinase ii inhibits its antiviral response ikk negatively regulates rig-i via direct phosphorylation conventional protein kinase c-alpha (pkc-alpha) and pkc-beta negatively regulate rig-i antiviral signal transduction dephosphorylation of the rna sensors rig-i and mda by the phosphatase pp is essential for innate immune signaling lysine acetylation targets protein complexes and co-regulates major cellular functions regulation of retinoic acid inducible gene-i (rig-i) activation by the histone deacetylase hdac regulates cellular viral rna sensing by deacetylation of rig-i ubiquitin-like proteins sumoylation of rig-i positively regulates the type i interferon signaling ubiquitin-like modifier fat attenuates rig-i mediated antiviral signaling by segregating activated rig-i from its signaling platform negative feedback regulation of rig-i-mediated antiviral signaling by interferon-induced isg conjugation negative regulation of rig-i-mediated innate antiviral signaling by sec l the atg atg conjugate associates with innate antiviral immune responses viral pseudo-enzymes activate rig-i via deamidation to evade cytokine production emerging roles of protein deamidation in innate immune signaling processing of genome ′ termini as a strategy of negative-strand rna viruses to avoid rig-i-dependent interferon induction the ′ ends of hantaan virus (bunyaviridae) rnas suggest a prime-and-realign mechanism for the initiation of rna synthesis old world hantaviruses do not produce detectable amounts of dsrna in infected cells and the ′ termini of their genomic rnas are monophosphorylated genome trimming: a unique strategy for replication control employed by borna disease virus unpaired ′ ppp-nucleotides, as found in arenavirus double-stranded rna panhandles, are not recognized by rig-i a protein covalently linked to poliovirus genome rna conventional and unconventional mechanisms for capping viral mrna structural basis for m g recognition and ′-o-methyl discrimination in capped rnas by the innate immune receptor rig-i structural basis for dsrna recognition and interferon antagonism by ebola vp marburg virus vp can both fully coat the backbone and cap the ends of dsrna for interferon antagonism structural basis for marburg virus vp -mediated immune evasion mechanisms a recombinant influenza a virus expressing an rna-binding-defective ns protein induces high levels of beta interferon and is attenuated in mice influenza virus adaptation pb - k modulates nucleocapsid inhibition by the pathogen sensor rig-i the dengue virus conceals double-stranded rna in the intracellular membrane to escape from an interferon response sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum influenza a virus ns targets the ubiquitin ligase trim to evade recognition by the host viral rna sensor rig-i species-specific inhibition of rig-i ubiquitination and ifn induction by the influenza a virus ns protein phosphorylation of influenza a virus ns protein at threonine suppresses its interferon antagonistic activity threonine phosphorylation of non-structural protein regulates the replication of influenza a virus by reducing the binding affinity with rig-i robust lys -linked ubiquitination of rig-i promotes cytokine eruption in early influenza b virus infection inhibition of rig-i-mediated signaling by kaposi's sarcoma-associated herpesvirusencoded deubiquitinase orf regulation of rig-i-like receptor signaling by host and viral proteins arterivirus and nairovirus ovarian tumor domain-containing deubiquitinases target activated rig-i to control innate immune signaling measles virus suppresses rig-i-like receptor activation in dendritic cells via dc-sign-mediated inhibition of pp phosphatases rig-i is cleaved during picornavirus infection enterovirus apro targets mda and mavs in infected cells the viral rna recognition sensor rig-i is degraded during encephalomyocarditis virus (emcv) infection viral degradasome hijacks mitochondria to suppress innate immunity disruption of innate immunity due to mitochondrial targeting of a picornaviral protease precursor the coxsackievirus b c protease cleaves mavs and trif to attenuate host type i interferon and apoptotic signaling hepatitis c virus protease ns / a cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity the hepatitis b virus x protein disrupts innate immunity by downregulating mitochondrial antiviral signaling protein influenza virus protein pb -f inhibits the induction of type i interferon by binding to mavs and decreasing mitochondrial membrane potential the influenza virus protein pb -f inhibits the induction of type i interferon at the level of the mavs adaptor protein influenza a virus protein pb -f translocates into mitochondria via tom channels and impairs innate immunity human respiratory syncytial virus nucleoprotein and inclusion bodies antagonize the innate immune response mediated by mda and mavs hijacking of rig-i signaling proteins into virus-induced cytoplasmic structures correlates with the inhibition of type i interferon responses the leader proteinase of foot-and-mouth disease virus negatively regulates the type i interferon pathway by acting as a viral deubiquitinase plp , a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production dengue virus ns proteins inhibit rig-i/mavs signaling by blocking tbk /irf phosphorylation: dengue virus serotype ns a is a unique interferon-regulating virulence determinant inhibition of interferon gene activation by death-effector domain-containing proteins from the molluscum contagiosum virus viral targeting of dead box protein reveals its role in tbk /ikkepsilon-mediated irf activation the ny- hantavirus gn cytoplasmic tail coprecipitates traf and inhibits cellular interferon responses by disrupting tbk -traf complex formation sars coronavirus papain-like protease inhibits the type i interferon signaling pathway through interaction with the sting-traf -tbk complex evasion of antiviral immunity through sequestering of tbk /ikkepsilon/irf into viral inclusion bodies activation of interferon regulatory factor is inhibited by the influenza a virus ns protein inhibition of interferon regulatory factor activation by paramyxovirus v protein the sars coronavirus papain like protease can inhibit irf at a post activation step that requires deubiquitination activity genetic dissection of interferon-antagonistic functions of rabies virus phosphoprotein: inhibition of interferon regulatory factor activation is important for pathogenicity herpes simplex virus serine/threonine kinase us hyperphosphorylates irf and inhibits beta interferon production epstein-barr virus latent membrane protein regulates the function of interferon regulatory factor by inducing its sumoylation rotavirus nsp mediates degradation of interferon regulatory factors through targeting of the dimerization domain thogoto virus ml protein suppresses irf function binding of kaposi's sarcoma-associated herpesvirus k-bzip to interferon-responsive factor elements modulates antiviral gene expression recruitment of activated irf- and cbp/p to herpes simplex virus icp nuclear foci: potential role in blocking ifn-beta induction the vaccinia virus k ankyrin repeat protein inhibits nf-kb activation by preventing rela acetylation vaccinia virus protein c inhibits nf-kappab activation and promotes virus virulence herpes simplex virus -encoded tegument protein vp abrogates the production of beta interferon (ifn) by inhibiting nf-kappab activation and blocking ifn regulatory factor to recruit its coactivator cbp herpes simplex virus protein kinase us hyperphosphorylates p /rela and dampens nf-kappab activation inhibition of interferon-stimulated jak-stat signaling by a tick-borne flavivirus and identification of ns as an interferon antagonist blocking of interferon-induced jak-stat signaling by japanese encephalitis virus ns through a protein tyrosine phosphatase-mediated mechanism varicella viruses inhibit interferon-stimulated jak-stat signaling through multiple mechanisms the ns protein of the virulent west nile virus ny strain is a potent antagonist of type i interferon-mediated jak-stat signaling dengue virus co-opts ubr to degrade stat and antagonize type i interferon signaling zika virus targets human stat to inhibit type i interferon signaling hepatitis c virus targets the interferon-alpha jak/stat pathway by promoting proteasomal degradation in immune cells and hepatocytes respiratory syncytial virus ns protein degrades stat by using the elongin-cullin e ligase paramyxovirus disruption of interferon signal transduction: status report key: cord- -btf ju authors: sun, zhiheng; pan, yuchen; qu, junxing; xu, yujun; dou, huan; hou, yayi title: β-estradiol promotes trained immunity in females against sepsis via regulating nucleus translocation of relb date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: btf ju sepsis is more common among males than females, and the unequal estrogen levels have been suspected to play a vital role in gender differences. recently, trained immunity is reported to be a novel strategy for the innate immune system to fight infection. however, it has not been clarified whether β-glucan-induced trained immunity causes different responses to early sepsis between male and female mice. in this study, sepsis was induced in mice by intraperitoneal injection of escherichia coli (e. coli). the changes of inflammatory cytokines expression, and macrophage polarization in male, female, and ovariectomized c bl/ mice in sepsis model were investigated. for in vitro studies, different macrophages were treated with lps. the function of estradiol (e ) on macrophage cell lines was verified and the mechanism of e affecting trained immunity was explored. we demonstrated that β-glucan-induced trained immunity was more resistant to sepsis in female than male mice. macrophage polarization toward the m phenotype, which exhibited enhanced trained immunity, was related to the difference in sepsis resistance between female and male mice. moreover, ovariectomized (ovx) mice manifested serious sepsis consequences with a weaker trained immunity effect than female mice. female bone marrow-derived macrophages (bmdms) were also apt to be polarized to the m phenotype in response to trained immunity in vitro. furthermore, e promoted trained immunity in macrophage cell lines j and raw . . e was also verified to facilitate trained immunity in primary bmdms from female and male mice. mechanistically, we found that e inhibited the nuclear translocation of relb, which is a member of non-canonical pathway of nfκb and contributes to macrophage polarization to change the intensity of trained immunity. this study is the first to indicate the role of e in the trained immunity induced by β-glucan to protect against e. coli-induced sepsis via the non-canonical nfκb pathway. these results improve our understanding of the molecular mechanisms governing trained immunity in gender differences. sepsis is a systemic reaction, which can even be caused by an ordinary infection ( ) . the infection is most commonly bacterial but also can be fungal, viral, or parasitic. since many organs are affected, the mortality rate of sepsis is - % ( ) . with the development of modern medicine, the mortality rates have declined to about % ( ), but this is still far from acceptable. the biomarker of sepsis is widely discussed by a number of reviews ( ) ( ) ( ) ( ) . several important signs of sepsis are damage to the kidney, liver, and lung, as well as an increased bacterial load in the kidney and elevated levels of transaminase and lactate in serum ( ) . moreover, the common model of sepsis is an immune response to escherichia coli or endotoxin, lipopolysaccharide (lps), found in the cell wall of gram-negative bacteria. endotoxin is an excellent example of a pathogen-associated molecular pattern (pamp). of note, a number of studies indicate gender dimorphism in terms of response to sepsis ( ) . the intuitive result is that the incidence of female sepsis is much lower than that of males ( , ) . furthermore, some studies attributed these differences to the prevailing hormonal milieu of the victim ( ) ( ) ( ) . thus, it is necessary to explore the mechanism of estrogen in modulating immunity, which allows females to resist sepsis. the nfκb (nuclear factor kappa-b) family of transcription factors constitutes five members (rela or p , relb, crel, nfκb or p , and nfκb or p ), all of which play important roles in cell homeostasis, especially in the immune process ( ) . apart from the well-known canonical signaling pathway, the noncanonical pathway is also involved in vital immune processes, which could be triggered by signaling from a subset of tnfr members. this pathway mediates the persistent activation of relb/p complex with the ability to modulate a series of gene expression, including macrophage polarization-associated cytokines. to date, a growing number of studies indicated that estrogen is involved in some immune processes. estrogen can bind to and activate estrogen receptors (ers), which regulate the expression of downstream genes ( ) . the interaction between ers and nfκb is mainly discussed in breast cancer articles ( ) . a highly significant negative correlation between the expression of nfκb target genes and er activation was found. some studies abbreviations: e , -β-estradiol; ti, trained immunity; lps, lipopolysaccharide; pbs, phosphate buffer saline; ast, aspartate aminotransferase; alt, alanine aminotransferase; il- , interleukin- ; tnfα, tumor necrosis factor α; il- , interleukin- ; il- , interleukin- ; m-csf, macrophage colony-stimulating factor. demonstrated the interaction of er with nfκb; ers can compete with nfκb for binding to transcriptional coactivators (ie, creb) or ers to recruit co-suppressors into nfκb complexes ( ) . ers can inhibit de novo relb synthesis in breast cancer tissues and cell lines. in addition, e inhibits the nucleus translocation of p , c-rel, and relb without affecting p in mouse splenocytes ( ) . these data suggest that estrogen-er signaling regulates the nfκb pathway at the transcriptional level of its constituents. the host's immune defense mechanisms can be divided into innate immunity and adaptive immunity. adaptive immunity, although slower than the innate immune response, has good specificity and produces immunological memory ( ) . recently, researchers discovered an ability in innate immunity, similar to immune memory in adaptive immunity, called trained immunity ( ) . trained immunity is found in mammals and its basic features have been identified by several researchers. trained immunity mainly involves a set of cells (myeloid cells, natural killer cells, and innate lymphoid cells) ( ) . antituberculosis vaccine bacillus calmette-guérin (bcg) is a wellknown immune modulator that induces trained immunity. it was reported that estrogen did not influence the induction of trained immunity by bcg, and did not induce training or tolerance in monocytes themselves, indicating that the estrogen is unlikely to explain the sex-differential effects after bcg vaccination ( ) . in fact, β-glucan is a major cell wall component of c. albicans and can induce trained immunity in monocytes. the initiation of trained immunity is associated with enhanced signaling of the akt (protein kinase b)-mtor (mammalian target of rapamycin)-hif- α (hypoxia-inducible factor- α) pathway ( ) , modifications in metabolic pathways (conversion to glycolysis), and epigenetic rewriting ( , ) . however, the effect of estrogen on β-glucan-induced trained immunity to resist sepsis has not been clarified. β-glucan (g- ) and β-estradiol ( - - ) were purchased from xiensi biotechnology company (tianjin, china). m-csf (#cb ), ifn-γ (#c ), and tnfα (#cf ) were purchased from novoprotein (shanghai, china). e and lps was purchased from sigma (st. louis, mo, usa). antibody against p-akt (ser , # ), akt (# ), p- ebp (# ), ebp (thr / , # t), p-s (ser / , # ), s (# ), pcna (# ), p (# ), estrogen receptor α ( s), gapdh (# ), and β-actin (# ) were purchased from cell signaling (boston, mo, usa). relb (sc- ) was purchased from santa cruz biotechnology (dallas, tx, usa). f / -apc, f / -fitc, cd -pe, cd -apc, cd -fitc, inos-pe were all from biolegend (san diego, ca, usa). raw . , j , and hek t cells were obtained from the type culture collection of the chinese academy of sciences, shanghai, china. cells were cultured in phenol red-free dmem with % fbs, % ( u/ml penicillin and ug/ml streptomycin) at • c in an atmosphere of % air and % co . cells were seeded onto different types of plates for further experiments when the cell density reached ∼ %. the cells were used within a maximum of five passages. the in vitro trained immunity model was established with raw . and j . cells were challenged with µg/ml βglucan for h. the cells were then washed and rested in culture medium for days. next, cells were treated with ng/ml lps for h, and then the cytokines were measured in mrna level. × bmdms were seeded in -well plates ( µl final volume; corning) and stimulated with µg/ml β-glucan for h. then cells were washed and rested for days in culture medium. on day , bmdms were washed again and treated with ng/ml ifn-γ for h. on day , a final wash was performed, and cells were primed with µg/ml lps. the supernatants were harvested for elisa assay after h of lps stimulation. other experiments were all based on the same model but with different numbers of bmdms. to assess mrna expression and cell viability, × bmdms were plated in non-treated well plates ( , ml final volume; corning) and followed the training scheme described above. for western blotting (wb) assays × bmdms were plated in six-well plates ( ml final volume; corning). male and female c bl/b mice were purchased from the model animal research center of nanjing university. mice used in the model of trained immunity and sepsis were weeks old. all animal procedures were performed in accordance with guidelines of the us nih with specific pathogen free conditions. soyfree standard rodent chow and water were provided ad libitum. female mice were anesthetized with % chloral hydrate and then underwent ovariectomy (ovx) at weeks of age. when they were weeks old, the same model of trained immunity and sepsis was established. peripheral blood mononuclear cells (pbmcs) were separated from mouse plasma by ficoll centrifugation using lymphocyte separation medium from mp biomedicals (solon, usa) according to the standard procedures. mice were sacrificed via cervical dislocation and sterilized by soaking in % ethanol. dissected the legs and bone marrow was extracted from tibia and femur bones by using a -gauge needle and a ml syringe filled with pbs following removal of surrounding muscle. blow the bone marrow gently and spread it through a µm cell strainer. the cell suspension was centrifuged at g for min at room temperature. cells were cultured in phenol red-free dmem with % ultra-low endotoxin fbs and m-csf ( ng/ml). after changing the fresh medium on the third and fifth day, bmdm was induced. the e. coli strains were purchased from atcc, collected, and identified by the medical laboratory center of zhongda hospital in nanjing jiangsu, china, and stored at − • c. bacterial strains were prepared in lb medium. a nuclear protein extraction kit was purchased from biyuntian (wuhan, china) and used according to the manufacturer's instructions. sirna transfection sirna was transfected according to the product instructions (ruibo company, china). the concentration of sirna used in the study was nm. the erα sirna target sequence is tgcacattgaagatgctga. the target sequence of non-coding (nc) sirna was a random sequence with no biological effects. to assess the effect of e on cell viability, a cck- assay was used according to the manufacturer's instructions (bioss company, china). raw . /j were seeded onto -well plates at a concentration of ∼ × cells/well. different concentrations of e were used to treat cells for different length of time as described in the article. total rna was extracted from cells using trizol reagent, and reverse transcriptions were performed in a µl mixture with µg of total rna according to the manufacturer's instructions (vazyme company, china). the oligonucleotide primers used for pcr amplification are listed in table . pcr amplification consisted of cycles of denaturation at • c for min, annealing at • c for s, and extension at • c for min. all reactions were run in triplicate. the gene expression levels were normalized to ß-actin. the protein samples were obtained from lysis buffer treated cells. cell lysates were put on ice for min and then centrifuged at , × g for min. subsequently, µg of protein per lane was separated on % polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (millipore, billerica, ma, usa). membranes were blocked with % bovine serum albumin (bsa) in tris-buffered saline containing . % tween , and then the membranes were incubated with specific antibodies. the values were normalized to the β-actin/gapdh intensity levels. mice were trained with two intraperitoneal (i.p.) injections of mg β-glucan particles on days − and − . sterile pbs was used as a control. on day , mice were challenged with . × e. coli. the lung, kidney, and serum were harvested h after e. coli treatment. the fresh lung tissues were fixed in % paraformaldehyde (pfa). then, the samples were gradually dehydrated and embedded in paraffin. after that, the samples were cut into µm sections and stained with hematoxylin and eosin for further light microscopy observation. scores were evaluated by a pathologist based on the lung tissue integrity, alveolar integrity, and mononuclear infiltration ( = none; = mild; = moderate; = severe). cultured cells were seeded on glass coverslips in six-well plates. after three pbs washes, the samples were fixed for min at room temperature with % paraformaldehyde. fixed cells were rinsed with pbs and then incubated for min at • c with . % triton x- and . % bsa in pbs. following permeabilization, non-specific binding in the cells was blocked by % bsa in pbs for h at room temperature. cell samples were incubated with anti-erα, anti-relb, and anti-p primary antibodies at a : dilution for h at room temperature. samples were further incubated with alexa fluor -conjugated and alexa conjugated secondary antibody at a : dilution for . h in the dark. after washed with pbs, the nuclei were stained by dapi. slides were visualized using a nikon eclipse ti-u fluorescence microscope equipped with a digital camera (ds-ri , nikon). the protein concentration of il- , lactate, tnfα, and estrogen in cell supernatant or mouse serum were detected using the corresponding mouse enzyme-linked immunosorbent assay (elisa) kit according to the manufacturer's instructions (biolegend, china). the kidney e. coli burden at indicated time points was measured by plating organ homogenates obtained mechanically over µm cell strainers (bd biosciences) following slicing the tissue, in serial dilutions on lb agar plates; colony-forming units (cfus) were counted after growth at • c for h, and data are shown as cfus in total kidney. pbmcs or bmdms were filtered through a µm cell strainer and then washed with complete rpmi medium to generate single-cell suspensions. an fc-receptor blocker (cd / , ebioscience) was used to reduce non-specific antibody binding. antibodies used in these experiments included f / -apc, f / -fitc, inos-pe, cd -apc, and cd b-pe. stained samples were detected by a facs calibur flow cytometer (bd bioscience) and data were analyzed using flowjo software (treestar, ashland, or). the statistical analysis was performed using prism (prism for windows, graphpad software inc., usa). unless specified, statistical significance for comparison between two sample groups with a normal distribution (shapiro-wilk test for normality) was determined using two-tailed paired or unpaired student's t-test. differences were considered significant at p < . as indicated. except when specified, only significant differences are shown. as indicated in figure legends, either a representative experiment or a pool is shown, and the number of repetitions of each experiment and number of experimental units (either cultures or mice) is indicated. the results are presented as the means ± standard error (sem). several studies have shown that women have better survival and tolerance to sepsis than men ( , ) . thus, we further verified this phenomenon by establishing a sepsis model in mice ( figure a) . the difference in lung injury between male and female mice in sepsis was determined by h&e analysis of lung sections (figure ba) . the histochemical scores (figure bb ) indicated that male mice had more severe lung damage than female mice; trained immunity prevented lung injury in mice and the protective effect of trained immunity was better for females than males. notably, we observed a decreased renal e. coli burden in females than males. in addition, in trained immunity mice, females had less renal e. coli burden than males (figures c,d) . in addition, liver damage markers, aspartate aminotransferase (ast), and alanine aminotransferase (alt) were increased in males than females; and trained immunity markedly decreased ast and alt concentration, with a greater decrease in females than in males (figures e,f) . we found that in the sepsis model, the serum lactate concentration of the male mice was higher than that of the female mice. in the trained immunity group, serum lactate was decreased, with the level in the females lower than males ( figure g ). as expected, male serum e content is much lower than female mice ( figure h) . our results showed that females expressed higher il- and tnfα than males in sepsis, and trained immunity exacerbated this trend (figures i,j) . macrophages are the most significant cells in the body in regard to trained immunity ( ) . the promoted trained immunity is related to the increased m phenotype macrophage. in order to understand the different response to sepsis between the different genders, we further investigated the polarization of macrophages during the process. thus, we focused on macrophage content and polarization. by detecting the percentage of macrophages in the spleen, we found that macrophages in female mice are more abundant than male mice, and trained immunity significantly increased the content of female macrophages (figure a) . sepsis promoted the polarization of macrophages to m and trained immunity aggravated this trend. notably, females showed more changes than males (figure b) . on the other hand, females inhibited the polarization of m macrophages ( figure c) . trained immunity simulated m type polarization of macrophages, and this effect was more pronounced for females. by analyzing the experiments above, we found that the difference between male and female responses to sepsis was due to the difference in macrophage polarization in vivo, and the difference in estrogen content in the different genders was very significant. to verify that differences in the estrogen level play a vital role, ovx mice were made, since the ovary is the main organ to produce e . the same sepsis model was established with ovx mice (figure a) . firstly, the serum e concentration was significantly decreased (figure a) . the degree of lung injury was much higher than that of female mice as well as ti + e. coli group (figure b) . the kidney e. coli burden was also evaluated. the results showed that ovx mice also had more severe kidney injury ( figure c ) as well as lung injury, as measured by serum alt (figure d ) and ast ( figure e ) concentrations, in comparison to female mice. with a lower serum il- and tnfα concentration in ovx mice in the ti + e. coli group, the data suggested that ovx mice have a lower trained immunity response than female mice (figures f,g) . finally, similar to male mice, ovx mouse pbmcs began polarizing to m -type at h after i.p. e. coli ( figure h ). taken together, these results demonstrated that e does facilitate trained immunity in the body to resist sepsis, and simultaneously inhibits macrophage polarization to m . since the macrophages are mostly derived from bone marrow cells, and also the cytokines up-regulated in trained immunity are pro-inflammatory cytokines. although the upregulation of inflammatory cytokines after trained immunity of macrophages is determined, no research has studied the polarization of macrophages during this process. therefore, exploring the polarization of bmdm as an important primary macrophage in trained immunity is very significant and necessary to understand the nature of trained immunity. to determine whether bmdms are similar to macrophage polarization in pbmcs, we tested the polarization of microphages from male or female mice when challenged with lps or trained immunity plus lps. we considered f / + inos + bmdms as m -type bmdms and f / + cd + bmdms as m -type bmdms. the results demonstrated that female bmdms are more polarized to m in both the lps group ( . - . %) and ti + lps group ( . - . %) ( figure a) . also, male bmdms began to transform to m in h, which did not occur in female bmdms. trained immunity also prevented both male and female bmdms from m polarization as well as pbmcs (figure b ). macrophage cell lines j and raw . in order to examine the effect of estradiol on trained immunity, we decided to pre-treat the macrophage cell lines with estradiol before trained immunity model. since the concentration of estradiol that can stimulate the macrophage cell lines raw . and j is unknown, we made a concentration gradient of estradiol on the activity of the macrophage cell lines. the results showed that the response of the macrophage cell lines to the stimulation of estradiol is not particularly sensitive, and the viability of j begins to increase significantly under the stimulation of nm estradiol. thus, the concentration of e used in cell experiments was verified as nm (figures a,b) . the in vitro trained immunity model was established with raw . and j ( figure c ) cell lines derived from male and female mice, respectively. using tnfα and il- β as the marker of inflammation, our data suggested that e can induce stronger trained immunity both in raw . and j (figures d,e) . we also made a trained immunity model of bmdms (figure a ). m-csf was used to induce bmdm with a purity of over % from both male and female mice ( figure b) . then, the mrna levels ( figure c ) and protein levels ( figure d ) of tnfα and il- were measured. the results suggested that female bmdms can have a more intensive response to lps as well as a trained immunity response than male bmdms. phosphorylation of akt and mtor targets (s and ebp ) are considered to be key hallmarks of trained immunity ( ) . to clarify the effect of e on trained immunity, these hallmarks are checked by western blot, which indicated that e as well as β-glucan induced trained immunity in bmdms ( figure e ). in addition, the data showed that e further boosted the polarization of m -type macrophage in trained immunity. consistently, this effect of e is more apparent in female bmdms, and e inhibited m polarization in trained immunity (figure f ). nfκb signaling pathway is involved in the regulation of many cellular physiological processes. previous articles have reported that relb −/− mice, or inhibiting the nucleus translocation of relb, led to a higher basal inflammatory level ( , ) . other reviews declared that e could regulate nuclear translocation of nfκb through ers. since it has been widely studied that tnfα can combine with tnfr on the cell membrane surface to promote nfκb translocate into the nucleus. also, because tnfα as a representative inflammatory cytokine is secreted by macrophages and will stimulate back to macrophage. we use tnfα as a positive control to study nfκb entry into the nucleus. we use immunofluorescence to explore the intracellular localization of nfκb protein. in immunofluorescence, the results indicated that when tnfα is used to stimulate cells, it does not affect the colocalization of erα and the nucleus but increases the colocalization area of p and relb with the nucleus. on the contrary, e treatment appeared to induce nuclear positioning of erα and kept relb distribution in the cytoplasm in both hek t and bmdms (figures a,b) . however, stimulation of e did not affect the distribution of p in cells. next, we used the rna interference system to further verify the impact of e on the nuclear translocation of relb. the silencing performance of interfering small rna sequence was evaluated both at the mrna level ( figure c ) and protein level ( figure d) . by extracting nuclear protein, we found that estrogen could reduce the content of relb protein in the nucleus; by silencing the expression of erα, the content of relb protein in the nucleus was also restored ( figure e ). as markers of m macrophage polarization, il- and il- are also the downstream genes regulated by relb ( , ) . we found that e inhibited the mrna expression level of il- and il- by regulating nuclear translocation of relb ( figure f ). in conclusion, these results indicated that e could block relb translocation to the nucleus; to further inhibit m -associated gene expression, e suppressed bmdms m polarization. during the development of sepsis, many systemic organ abnormalities occur including damage to the lung, liver and kidney; the normal operation of the body's neurosensory system may even be affected ( ) . since lps is a component in the cell wall of gram-negative bacteria, it is wildly used as an ideal stimulus to establish sepsis models. when tlr on figure | female bmdms are apt to be polarized to m phenotype in responses to trained immunity in vitro. (a) female bmdms m polarization was stronger than male bmdms treated with lps or ti + lps groups. (b) very few female bmdms to polarized to m in lps group, while male bmdms showed the polarization of m phenotype. ti suppressed bmdms to m polarization from both male and female mice (n ≥ /group). ***p < . , paired student's t-test comparing lps group and ti + lps group in the same gender. # p < . , ### p < . , paired student's t-test comparing same group between different genders. the macrophage cell membrane recognizes lps, a series of signaling pathways will be initiated to stimulate macrophages to produce pro-inflammatory cytokines, which can induce potent systemic inflammatory responses in vivo. therefore, tnfα, il- β, and il- are currently considered to be markers of the early phase of sepsis ( ) . the development of sepsis begins with systemic inflammatory response syndrome (sirs) ( ) , which is considered as the early phase of sepsis. elevated expression of pro-inflammatory cytokines could help the body return to a normal state in early sepsis ( ) . after sirs, the body enters the cars (compensatory anti-inflammatory response syndrome) stage. in the cars stage, the immune system will undergo the decline-regulation ( ) . the hyper-inflammatory response of sirs will normally be restored by the subsequent cars stage. the patients can return to normal state after the cars stage ( ) . however, if the cars phase fails, it can cause death from sepsis. as one of the important ways to protect the body from foreign microbes and antigens, the role of trained immunity is very significant. this study focused on the effects of e on early sepsis by promoting trained immunity. in the development of early sepsis, estrogen induced a stronger trained immunity response of macrophages to remove antigens and microorganisms from the body. simultaneously, estrogen promoted the polarization of macrophages to m in vivo and inhibited the polarization of m , which is considered anti-inflammatory in the early phase of sepsis. in summary, estrogen can maintain the higher proinflammatory state of macrophages. this also illustrates the reason why females are more tolerant to sepsis than males in one aspect. however, further research is needed to understand the role of estrogen in the later stages of sepsis. the mrna levels of tnfα and il- β in raw . were detected by qpcr to determine the trained immunity effect with or without e (n ≥ /group). # p < . , ### p < . , paired student's t-test comparing β-glucan + lps group and lps group. *p < . , ***p < . , paired student's t-test comparing with control group. ∧p < . , ∧∧p < . , and ∧ ∧ ∧p < . , paired student's t-test comparing between β-glucan + lps groups with or without e . here, we demonstrated that β-glucan-induced trained immunity was more resistant to sepsis in female than male mice. ovx mice manifested serious sepsis consequences with a weaker trained immunity effect than female mice. macrophage polarization toward m phenotype is thought to exhibit the enhanced trained immunity. e promoted trained immunity in vitro and in vivo. inhibition of e on the nucleus translocation of relb contributed to macrophage polarization to change the intensity of trained immunity. our results indicated that e is involved in the trained immunity in gender differences. since trained immunity was discovered in , although there are many articles about trained immunity, there is no article to find the fundamental way of trained immunity activation but only about the changes of immune cell characterization after trained immunity activation. such as epigenetic changes, upregulation of glycolysis, autophagy level, akt phosphorylation, and so on. in this study, we found that e promotes trained immunity by suppressing relb entry into nucleus, while inhibiting macrophage polarization to m in inflammation (a way reverse the inflammatory response). it is possible that e can change the epigenetics of macrophages through estrogen receptors to affect the trained immunity. this hypothesis needs further researches. trained immunity is a way of immunization that occurs in the innate immune cells through the first stimulation to fight the later secondary infections. by obtaining trained immunity, the body can fight various infections from bacteria, fungi and even viruses timely. it is worth mentioning that in countries and regions with underdeveloped scientific research capabilities, it may be an economical and quick means of protection against covid- to enhance people's trained immunity ability. sepsis is a clinically common infectious disease with obvious gender differences. in this article, sepsis model is used as an infection model to evaluate the role of estradiol in enhancing trained immunity. the mechanism and effects of estradiol in promoting trained immunity also need subsequent study in other types of infectious diseases. the purpose of this paper was to explore the reasons why different genders have different tolerance effects on sepsis. an article reported that estradiol and dht (dihydrotestosterone) did not influence bcg-induced trained immunity, and the article only focused on the inflammatory cytokines of monocytes induced by bcg in vitro ( ) . apart from this, almost no articles study the effects of e on trained immunity and its biological significance. our research attempted to explain its effects on trained immunity and understand how it causes women to be more tolerant of sepsis than men. however, in terms of gender differences, many factors, such as androgen concentration, genetic differences, metabolic differences, and physiological differences may all be the result of different tolerances for sepsis between genders ( , , ) . gender differences are a very complex and exquisite scientific issue ( ) , and all of the factors that contribute to gender differences are worth exploring for subsequent experiments. in summary, we demonstrated that as one of the key factors for gender differences, e plays a vital role in the stronger tolerance of females to sepsis than males. one the one hand, e can better facilitate trained immunity by increasing expression of inflammatory cytokines and inducing macrophage m -type polarization; on the other hand, e can inhibit or delay the macrophage m -type polarization by regulating nucleus translocation of relb and its down-stream m -associated genes. thus, this may provide a new idea for the clinical treatment of sepsis. drugs with a structure similar to estrogen may be developed to treat sepsis. more importantly, the treatment of some diseases may able to find better solutions by addressing the different characteristics of different genders. the datasets generated for this study are available on request to the corresponding author. the animal study was reviewed and approved by model animal research center of nanjing university. all authors reviewed the manuscript, contributed to data analysis, drafting and revising the article, gave final approval of the version to be published. zs conceived and designed the project. zs, yp, jq, and yx participated in the animal experiments. zs and jq wrote the manuscript. yh and yx revised it. biomarkers of sepsis two decades of mortality trends among patients with severe sepsis: a comparative meta-analysis incidence and trends of sepsis in us hospitals using clinical vs claims data biomarker cruises in sepsis: who is the captain? discussion on "circulating biomarkers may be unable to detect infection at the early phase of sepsis in icu patients: the captain prospective multicenter cohort study sepsis pathophysiology, chronic critical illness, and persistent inflammationimmunosuppression and catabolism syndrome penkid: a novel biomarker of reduced gfr in sepsis adaptation of a biomarker-based sepsis mortality risk stratification tool for pediatric acute respiratory distress syndrome reconsidering lactate as a sepsis risk biomarker gender differences in sepsis: genetically determined? shock effects of gender on the severity of sepsis sexual dimorphism in bacterial 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injury and neurology, renal failure and endocrinology, metabolism and nutrition, sepsis, infections and pneumonia sepsis and septic shock: a review sepsis: a review of advances in management review: immunology of sinusitis, trauma, asthma, and sepsis review on sepsis in children did not mention important trial clinical outcome and risk factors of neonatal sepsis among neonates in felege hiwot referral hospital systematic review of gender differences in sepsis management and outcomes the influence of gender on the epidemiology of and outcome from severe sepsis age and sex influence the hippocampal response and recovery following sepsis the authors appreciate the warm-hearted help from all the co-authors and the professor. key: cord- -qy b l f authors: picard-sánchez, amparo; estensoro, itziar; perdiguero, pedro; del pozo, raquel; tafalla, carolina; piazzon, m. carla; sitjà-bobadilla, ariadna title: passive immunization delays disease outcome in gilthead sea bream infected with enteromyxum leei (myxozoa), despite the moderate changes in igm and igt repertoire date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: qy b l f passive immunization constitutes an emerging field of interest in aquaculture, particularly with the restrictions for antibiotic use. enteromyxum leei is a myxozoan intestinal parasite that invades the paracellular space of the intestinal epithelium, producing a slow-progressing disease, leading to anorexia, cachexia and mortalities. we have previously demonstrated that gilthead sea bream (gsb, sparus aurata) that survive e. leei infection become resistant upon re-exposure, and this resistance is directly related to the presence of high levels of specific igm in serum. thus, the current work was aimed to determine if passive immunization could help to prevent enteromyxosis in gsb and to study in detail the nature of these protective antibodies. serum from a pool of resistant (sur) or naïve (nai) animals was intracoelomically injected h prior to the e. leei-effluent challenge and at days post-challenge (dpc). effluent challenge lasted for days, and then the injected groups were allocated in separate tanks with clean water. a non-lethal parasite diagnosis was performed at dpc. at the final sampling ( dpc), blood, serum and tissues were collected for histology, molecular diagnosis and the detection of circulating antibodies. in parallel, we performed an immunoglobulin repertoire analysis of the fish generating sur and nai sera. the results showed that, fish injected with parasite-specific antibodies (spabs) became infected with the parasite, but showed lower disease signs and intensity of infection than the other groups, indicating a later establishment of the parasite. repertoire analysis revealed that e. leei induced a polyclonal expansion of diverse igm and igt subsets that could be in part an evasion strategy of the parasite. nonetheless, gsb was able to produce sufficient levels of parasite-spabs to avoid re-infection of surviving animals and confer certain degree of protection upon passive transfer of antibodies. these results highlight the crucial role of spab responses against e. leei and set the basis for the development of effective treatment or prophylactic methods for aquaculture. pathogens are an important cause of economic losses in aquaculture, and the partial effectiveness of the available treatments increases the chances of drug-resistant pathogens in fish and in the aquatic environment ( ) . the therapeutic use of specific antibodies (spabs) is an attractive alternative to provide immunity against pathogens. opposite to chemicals and antibiotics, which have a broad-spectrum of action, antibodies constitute a very specific defense mechanism. antibody mediated immunity can be active or passive. active immunity depends on the production of spabs after direct contact with the pathogen or antigen (vaccination or random encounter), it takes days/weeks to develop and results in the formation of immunological memory that can last for months or years. in passive immunity, spabs obtained from a previously infected or immunized donor, are introduced in a naïve individual to confer resistance or to combat a specific pathogen. passive immunity is faster, shortlived and does not involve memory ( ) . passive immunization can be allogeneic or xenogeneic, if the origin of the transferred abs is the same or different than the recipient species, respectively. this technique has been applied successfully in human medicine ( ) . it has also been tested in fish-parasite models showing promising results. allogeneic therapies have partially protected carp (cyprinus carpio) against trypanoplasma borreli ( ), or tomato clownfish (amphiprion frenatus) against amyloodinium ocellatum ( ) . however, passive immunization failed to protect in other fish-parasites models like rainbow trout (oncorhynchus mykiss) against gyrodactylus derjavini infection ( ) . of note, the first two experiments required repeated doses of serum to induce protection, whereas in the latter, only one dose was administered. xenogeneic therapies have also been applied successfully. mouse monoclonal antibodies against surface proteins of ichthyophthirius multifiliis protected channel catfish (ictalurus punctatus) against infection ( ) . clearly, a key aspect for the success of the procedure is a deep knowledge about the host-parasite model, especially the time for parasite spreading in the host and the time that transferred antibodies remain in the circulation of the recipient animal ( ) . teleost fish have three different immunoglobulin (ig) isotypes: igm, igt/z, and igd ( ) . the basic structure of fish igs is the same as that of mammals. the heavy chain is constituted of an isotype-specific constant domain (ch) and a variable domain (vh) which, together with the variable domain of the light chain (vl), are responsible for the great diversity of antigen binding sites. each v domain has three hyper variable regions termed complementarity-determining regions (cdr), which constitute the majority of the antigen binding sites of the ig molecule. different combinations of cdr in vl and vh are responsible for the amazing diversity in binding sites among the millions of antibodies in an individual ( ) . in fish, igm is the highest expressed ig in all organs and it is essential for immune protection against different pathogens upon different routes of infection ( ) ( ) ( ) ( ) ( ) . igm is highly abundant in fish serum with concentrations between and , µg/ml ( ) . the teleost specific isotype igt is considered the most important ig in mucosal surfaces, but is also found in serum at much lower concentrations than igm, so its role in systemic responses should not be discarded ( ) ( ) ( ) ( ) . the role of igd is still not well defined in mammals or fish, however, recent studies performed in fish have established that it is also secreted ( ) and might have a relevant role in some mucosal surfaces such as gills ( ) or intestine ( ) . like mammals, fish can develop immunological memory. a secondary exposure to an antigen will induce faster and greater responses than primary response, with larger numbers of antigen-specific b cells originated from the expansion of the pool of memory b cells. detailed repertoire analyses have helped to define fish ig responses upon infections of different etiology ( , ) . enteromyxum leei is a myxozoan intestinal endoparasite, that can be transmitted from fish-to-fish and causes a slow progressing catarrhal enteritis in gilthead sea bream (gsb, sparus aurata) ( , ) . the parasite lives and divides in the paracellular space between enterocytes causing chronic infections with intestinal inflammation leading to dysfunctional absorption and anorexia, reflected in weight loss and decreased specific growth rate (sgr), condition factor (cf), and fat deposits in liver ( ) . currently, this parasite cannot be cultured in vitro and there are no preventive or curative measures against this disease. however, there is plenty of background knowledge on the host response against the parasite ( ) ( ) ( ) ( ) ( ) ( ) ( ) . we have recently demonstrated that gsb is able to develop acquired immunity against e. leei. fish that survived an infection did not get re-infected upon re-exposure, even months after the initial exposure, and this resistance was correlated with high levels of spabs (igm) in serum ( ) . therefore, the aim of the present study was to demonstrate the protective role of spabs against enteromyxosis in gsb by passive immunization of naïve animals. we also aimed to characterize the type of ig response produced by this parasite, analyzing and comparing the ig repertoire in naïve and resistant animals reexposed to the parasite. the results obtained in this study provide novel information to understand the role of antibodies during the response of gsb to enteromyxosis, and will lead us a step further toward developing successful vaccines and/or treatments against this economically important disease in aquaculture. specific-pathogen-free (spf) and clinically healthy gsb juveniles from a commercial fish farm, were kept in µm-filtered and uv irradiated sea water (salinity . g/l) between and . • c, with the natural photoperiod at latitude • n; • e. the spf status was confirmed by qpcr according to the protocol previously described ( ) . fish were fed ad libitum once a day a commercial diet (biomar, palencia, spain). all experimental protocols involving fish were approved by the ethics and animal welfare committee of iats, csic, and generalitat valenciana. they were carried out in a registered installation facility in accordance with the principles published in the european animal directive ( / /eu) and spanish laws (royal decree rd / ) for the protection of animals used in scientific experiments. all efforts were made to minimize the suffering of animals. fish sera used for the passive immunization trial were obtained from a previous experimental infection ( ) . briefly, gsb that had survived and cleared an infection with e. leei were reexposed to the parasite months after the beginning of the first infection by exposure to parasite-infected water effluent. these fish showed to be resistant to re-infection (sur) and tested negative for the presence of the parasite and days after the second exposure. sera from the four fish that showed the highest levels of parasite-specific igm (immunoreactivity = , see below) were selected and pooled. sur serum and tissue samples were obtained days after the second exposure to the parasite. in parallel, sera from four naïve (nai) fish from the same batch were tested to be negative for parasite-specific igm and pooled to be used as a control. before injection, the complement was heat-inactivated ( • c min). from the same eight animals, rna from the anterior intestine was extracted as previously described ( ) . the anterior intestine was chosen because it is the tissue where immunoglobulin gene expression was significantly increased upon re-exposure to the parasite in sur animals ( ) . fifty naïve gsb ( . g) were individually tagged with passive integrated transponders (pit-tags) and kept together in the same tank during the first part of the trial involving serum injections and effluent challenge. one day before and nine days after the challenge, fish per group were injected intracoelomically (i.c.) with µl/g of nai or sur serum. the remaining fish were injected with µl/g of sterile pbs. in addition, naïve fish from the same stock were kept separately, constituting the nonchallenged control group. a preliminary trial was conducted to determine the duration of injected antibodies in the recipient fish blood circulation. for that purpose: µl/g of rabbit serum (dako) were i.c. injected and rabbit igs were detected by dot blot in serum as soon as h post-injection and remained in circulation at least days (supplementary figure s ). one day ( h) after the first injection, fish were challenged with e. leei by exposure to infected water effluent, mimicking one of the natural routes of infection in farmed fish ( ) . briefly, the tank holding fish, already injected with pbs, nai or sur serum, received water effluent from a donor tank holding heavily e. leei-infected gsb, as previously described ( ) . at days post-challenge (dpc), fish were weighed and measured, and the different injected groups were moved in groups of to individual tanks receiving clean water, ending the effluent challenge. serum-injected fish (n = for each type of serum) were moved to two replicate tanks (n = ) for each group. the -day exposure was selected as the minimum time needed for all fish to be challenged with the parasite without exerting a too high infection pressure that will mask the results at the end of the trial ( ) . at dpc, fish were non-lethally sampled to evaluate the progression of the infection and the final lethal sampling was performed at dpc. a schematic representation of the trial and samplings can be found in figure . before all samplings ( days before challenge, , , and dpc), fish were starved for two days and biometric parameters measured. additionally, at the second sampling ( dpc), fish were also sampled for parasite diagnosis by non-lethal rectal probing and qpcr with specific primers for the parasite s rrna gene, as previously described ( ) . finally, in the last sampling ( dpc), fish were killed by overexposure to anesthetic (ms- , . g/l; sigma), and blood and tissue samples for molecular and histological procedures were taken. blood samples ( ml) were drawn from the caudal vessels by puncture with heparinized sterile needles and serum was obtained after overnight clotting at • c and centrifugation at , × g for min, and maintained at - • c until further use. intestine (anterior-immediately after the pyloric caeca-, middle and posterior-immediately before the anal ampoule), head kidney, spleen, and liver samples from all fish were taken in % buffered formalin for standard histological procedures. condition factor (cf = [ × body weight]/fork length ) and specific growth rate (sgr = [ln (final weight) -ln (initial weight) × ]/days) were calculated for each animal individually. infection intensity was semiquantitatively evaluated on giemsa stained histological sections of anterior (ai), middle (mi), and posterior intestine (pi) using a scale from (lowest) to (highest) ( ) . the remaining intestinal tissue was processed for molecular diagnostic (qpcr), as previously described ( ) . in addition, pas-staining was carried out on liver sections. one fish of the nai group that died between the scheduled sampling points was checked postmortem for the presence of the parasite and was no longer included in the results. total igm and igt were measured by elisa as previously described ( ) . specific igm against e. leei in serum samples was immunohistochemically detected using paraffin embedded sections of e. leei-infected gsb intestines obtained from previous and independent infection trials, following an immunohistochemical sandwich elisa protocol, as described previously ( ) . intensity of immunoreactivity of each fish serum against the parasite was evaluated by microscopic examination of the immunolabelled tissue sections according to a semiquantitative scale ranging from to (scaling: = no immunoreactivity against the parasite; = very slight reactivity; = slight reactivity; = medium reactivity; = medium-intense reactivity; = intense reactivity; = very intense reactivity). the detection of t cells (zap + ) in the intestine of experimental individuals was performed by immunohistochemistry (ihc), as previously described ( ) . in the former study, we have reported that intestinal t cells increase in numbers upon e. leei infection and these cells are one of the key mechanisms to clear the parasite. briefly, paraffin sections ( µm thick) were deparaffinized, hydrated and the endogenous peroxidase activity was blocked by incubation in hydrogen peroxide ( . % (v/v) for min). an antigen retrieval step was performed by boiling the samples in citrate buffer ph for min. incubations were performed in a humid chamber at room temperature and all washing figure | schematic representation of the passive immunization trial. seven days before the challenge by effluent exposure, fish were tagged, weighted, measured and allocated together in the same tank. one day before the challenge and nine days post-challenge (dpc) fish were injected with the different sera or pbs. at dpc, the effluent challenge was terminated and fish were separated in three different tanks ( fish/tank, sur, nai, and pbs injected groups). at dpc, a non-lethal diagnosis of the parasite by pcr was performed, and at dpc all fish were sacrificed and samples for histology and diagnosis were collected. i.c., intracoelomic. procedures consisted of successive min immersions in ttbs ( mm tris-hcl, . m nacl, . % tween , ph . ) and tbs ( mm tris-hcl, . m nacl, ph . ). slides were washed, blocked for min with . % normal goat serum (vector laboratories). after washing, samples were then incubated with a monoclonal rabbit anti-zap antibody ( f cell signaling technologies) diluted : in tbs % bsa for h and washed again. slides were incubated with a biotinylated goat anti-rabbit antibody (vector labs.) diluted : in tbs . % normal goat serum for h, washed and the avidin-biotin-peroxidase complex (abc) (vector labs.) was applied for h before washing slides again. bound peroxidase was developed by adding , 'diaminobenzidine tetrahydrochloride chromogen (sigma) for min and the reaction was stopped with deionized water. tissue sections were counterstained with gill's hematoxylin, dehydrated and mounted with di-n-butyl phthalate in xylene. incubation of tissue sections with abc alone served as control to discard the presence of endogenous biotin-binding proteins. negative controls omitting the primary antibodies, the secondary antibody and the abc were carried out and were negative. to perform repertoire analysis of gsb immunoglobulins, genomic regions potentially encoding ighv genes were identified along gsb draft genome ( ) , searching for conserved domains using the online tool https://www.ncbi.nlm.nih.gov/ structure/cdd/wrpsb.cgi from ncbi. transcriptional activity of identified regions were further confirmed by blasting against a gsb transcriptome from a previous study ( ) . nucleotide sequences were aligned using clustalw in order to identify conserved regions among genes to design primers which may amplify all potential genes. a total of seven forward primers, recognizing the fr region, were designed and used in pcr amplifications in combination with reverse primers designed in igm or igt constant region (cµ and cτ , accession numbers kx and kx , respectively) (supplementary table s ). https://www.genome.jp/tools-bin/clustalw to study the igm and igt repertoire in the fish used as serum donors for passive immunization, cdna from anterior intestine of sur and nai donor fish were used as template in pcr amplifications for each primer combination (f + r). mix of reagents for pcr reaction was: µl of cdna as a template, . µm of each forward and reverse primer combination, and . u/µl dna polymerase (accuprime tm taq polymerase high fidelity, invitrogen) in × accuprime pcr buffer i, containing mm mgcl and a mix of . mm of each dntp (invitrogen). the thermal cycling regime was as follows: • c for min, followed by cycles ( • c for s, • c for min, and • c for min) and the final extension at • c for min. negative controls without cdna were also included. for the visualization of pcr products, a µl aliquot from each pcr product was mixed with µl of loading buffer and loaded into a % agarose with ethidium bromide staining. at the same time, samples for sequencing were composed by µl from each reaction belonging to the same individual pooled together. samples were quality checked using an agilent bioanalyzer (agilent technologies) and sequenced by life sequencing (valencia, spain). one library per individual was constructed with the truseq dna pcr-free library prep kit (illumina, san diego, ca, united states) according to the manufacturer's protocol. libraries were pooled together, and paired-end sequencing was performed on an illumina miseq with a miseq reagent kit v ( × cycles) cartridge (illumina, san diego, ca, united states). raw sequencing data were demultiplexed and sequencing adapters and barcodes were removed from the sequences using the miseq analysis pipeline. the first nucleotides from the reverse primers were used as a barcode for the identification of ' ends corresponding to each constant ig gene. reads that matched a primer sequence with a maximum of three mismatches allowing an overlap of two nucleotides (-mismatches -partial ) were classified into the corresponding igm or igt isotype using the fastq/a barcode splitter tool . the opposite paired end reads, corresponding to the end of pcr products, were extracted with the fastq interlacer tool implemented in galaxy ( ) . the paired forward and reverse reads were merged using pear software ( ) with a minimum overlap size of nucleotides. finally, a quality filter was applied keeping sequences with a phred base quality ≥ in at least the % of sequence. in the current study, in absence of a v-d-j germline for our species, sequences for each isotype from the eight individuals were annotated by comparative genomics according the germline of zebrafish (danio rerio), used as model species, which is available at international immunogenetics information system databases ( ) . thus, gsb repertoire was annotated according the best match identified for each gene (v,d, or j) from zebrafish using the imgt/highv-quest tool ( ) . imgt/highv-quest results were parsed using convert tool from vdjtools software ( ) . non-functional clonotypes were filtered out using filternonfunctional tool from vdjtools software. retained functional clonotypes were further analyzed using the repexplore, repclonality, repdiversity, geneusage, and getkmers tools from the r package immunarch (v . . ) ( ) . prior to analysis, data were normalized by subsampling. this analysis allowed a first preliminary glimpse on the diversity of the clonotypes and the type of response that is being elicited by the parasite by comparison of two sampling groups (nai vs. sur). differences among groups were evaluated with one-way anova followed by tukey test. data which failed the normality or equal variance test were analyzed with kruskal-wallis anova-i on ranks followed by dunn's method. differences between two groups were determined by student's t-test or mann-whitney test when normality conditions were not met. a chi-square test was run to determine differences in the presence/absence of liver fat and glycogen deposits. in all cases differences were considered significant at p < . . statistics and visualizations were performed with the r package immunarch (v . . ) ( ) and graphpad prism (v . ). the typical clinical signs of enteromyxosis include weight loss and decreased cf and sgr ( ) . the current results show that while cf of some challenged fish was not affected until the final sampling at dpc, sgr was already affected at the intermediate sampling ( dpc) in the pbs-injected group (figures a-d) . fish injected with pbs or nai serum always showed lower cf and sgr than non-challenged fish. interestingly, fish injected with sur serum did not show significant differences with the nonchallenged controls and always displayed intermediate values. no significant differences were found in the prevalence of infection among the different challenged groups in any of the sampling points. however, the prevalence of infection in fish injected with parasite-spabs (sur serum) was always the lowest ( figure e) . histological diagnosis allowed assessing the percentage of fish with a progressed infection, that is, fish that have more than one intestinal segment parasitized. progression of infection also showed lower values in fish injected with sur serum (figure e) . interestingly, all infected passively immunized fish (sur serum) showed low intensity of infection (ct values > ), whereas, roughly half of the individuals from the other two groups had high intensity of infection (ct values < ) . the intensity of infection of sur-injected fish was significantly lower than that of the nai serum group (figure f , higher ct values correspond to lower intensity of infection). all parasite-challenged fish showed the typical intestinal reaction against e. leei, although some differences could be observed among groups. overall, , . , and % of the pbs, nai, or sur fish, respectively, had abundant lymphocyte-like cells at the base of the intestinal epithelium, particularly at the anterior segment ( figure a) . ihc revealed that most of these cells were zap + , identifying, at least part of this increased cell population, as t cells (figures c,d) . all challenged groups showed a noteworthy infiltration of eosinophilic granular cells in the epithelial layer ( figure b) . interestingly, the prototypical hyperplasia of the submucosa upon e. leei infection was observed in fewer animals injected with parasite-spabs ( % in sur vs. % in nai) (figures e,f) . the most striking difference was found in the liver. the liver of heavily infected fish, due to the anorexia and the nutrient absorption impairment induced by the parasite, had clearly less accumulation of fat and glycogen deposits. this was the case for most pbs ( %) and nai ( %) challenged fish, whereas all sur challenged fish, except one ( %), had normal liver histological features with abundant fat and glycogen deposits (figures g,h,i,j) . this difference was statistically significant (chi-squared test, p = . ). total serum igm, igt and parasite specific igm of the different groups were measured at dpc. total circulating igm was significantly higher in the group injected with serum with parasite-spabs than that of all the other groups, which did not show differences among them (figure a) . circulating igt was higher in the pbs injected group than in the control non-challenged group. the serum injected groups showed intermediate values (figure b) . no parasite-specific igm was found in the non-challenged group, while all the other groups showed no differences among each other ( figure c ). the next step we took was an attempt to characterize the type of ig response elicited by the parasite. thus, we studied the igm and igt repertoire in the gsb that acted as sur and nai serum donors for the passive immunization trial. by in silico analysis using immunoglobulin conserved domains, up to regions showing homology with v genes were identified along the gsb genome (supplementary data s ). following this identification, intensity of infection is shown as ct values (mean ± sd) obtained from a parasite specific qpcr of the intestine at dpc (f). low ct values indicate high infection intensity. pbs is the group injected with pbs, whereas nai represent the fish injected with serum without specific antibodies and sur the fish injected with parasite-specific antibodies. the negative control (ctrl-) was the group not challenged with the parasite. different letters indicate significant differences among groups (p < . ). seven forward primers were designed in v regions which in combination with reverse primers located in igm or igt constant regions allowed for the first time the amplification of a global gsb repertoire (supplementary figure s ) . using illumina miseq ( × ), an average of . million paired end reads per sample were obtained and assembled. using the isotype specific primers as a barcode, . and . % of the sequences were cataloged as igm and igt, respectively. due to the absence of gsb reference sequences for vdj genes, imgt/highv-quest analysis was performed using zebrafish as a reference. this analysis resulted in the identification of productive sequences as well as their cdr , and served to assign vdj genes and unique clonotypes according to homology to zebrafish, which allowed the comparison between experimental groups. after analysis, . % of the igm sequences and . % of the igt sequences were classified as productive (supplementary table s ). analysis with immunarch revealed igt (e) was also evaluated using the chao diversity index. the differential usage of vh-gene families for igm (c) and igt (f) was calculated with the assignment against zebrafish sequences. bars represent the average number (mean ± sd) on unique sequences for n = fish. asterisks represent statistical differences between nai and sur groups at p < . . that the number of unique clonotypes was significantly higher for both isotypes in immunized (sur) fish than in naïve (nai) individuals (figures a,d) . the clonotype diversity (estimated with the chao index) was also always significantly higher in sur animals than in nai fish (figures b,e) . sequences classified as similar to ighv and ighv zebrafish subgroups were the most expressed in both isotypes. ighv like sequences were significantly overexpressed in igt, whereas ighv like sequences were overrepresented in both isotypes in sur fish (figures c,f) . around % (igm) and % (igt) of the different clonotypes were found one to five times, indicating a broad diversity of ig repertoire in gsb intestines. interestingly, the percentage of igt clonotypes found one to five times decreased in sur animals, whereas igt clonotypes, which were represented more than times, increased in sur fish. these results demonstrate a higher frequency of expanded igt clonotypes in sur fish, indicating a clear response. no significant changes were found for igm (figures a,b) . when analyzing the cdr amino acids length of the different clonotypes, a shift to longer cdr lengths was observed for igm, but no clear differences appeared in igt distribution (figures c,d) . none of the cdr length histograms fitted the expected gaussian bell distribution bars show the average percentage (mean + sd) of clonotypes observed n times in the datasets. asterisks represent statistical differences between nai and sur groups at p < . . cdr length distributions for all detected igm (c) and igt (d) clonotypes. the graphs represent the average number of clonotypes of a given amino acid (aa) length (+sd) for sur and nai fish. kolmogorov-smirnoff tests reported that none of the curves followed a bell-shaped gaussian distribution (p > . ). normally expected for naïve animals (kolmogorov-smirnov normality test p > . ). analysis of the five most frequent k-mers (k = ) from the cdr sequences revealed that the k-mer yfdyw is very frequent in both, igm and igt sequences, with a frequency increase in sur individuals. the other detected k-mers were not common for both isotypes (figure ) . in mammals, passive immunization represents a prophylactic strategy when vaccination is not possible, such as in immunosuppressed or very young individuals. also, in the case of certain cancers or emerging human diseases, when vaccines or treatments have not yet been developed ( , ) . in fish, passive transfer of spabs by injection has been successful against some infectious diseases, including parasites ( , , ) . opposite to other animal production sectors, aquaculture animals live in the aquatic environment, which hinders individual treatment approaches. thus, passive immunization through injection is not the most feasible strategy for fish. however, research is nowadays being conducted on encapsulated antibodies to be delivered in-feed ( ) , as it was previously described for pigs ( ) . in any case, experimental injection trials, such as the one presented in this manuscript, allow to test whether spabs could be effective for treating a particular infection and can serve as a base to develop future control strategies such as vaccination or in-feed approaches ( ) . in passive immunization trials two main considerations are important: the time of the injected antibodies to reach the bloodstream, and the half-life of these antibodies in circulation ( ) , which will indicate the time-frame of protection. in salmonids, i.c. injected allogeneic igm against vibriosis were detected in serum min post-injection and the uptake was complete after h. these antibodies remained protective for up to days ( , ) . in carp, the half-life of allogeneic igm was much shorter, . days ( ) . it is clear that the half-life of serum antibodies varies depending on the species, but teleost igs are assumed to remain in circulation from to days still retaining the antigen binding efficiency ( ) . in the current study, we performed a preliminary test that revealed that i.c. injected rabbit igg could be detected in gsb serum from h to days post-injection in high concentrations. however, igg besides being xenogeneic, which could elicit a host response against the injected molecules, is also a monomer, so faster absorption times could be expected. in any case, these results, together with literature data, allowed us to plan the injection timings. fish were challenged with the parasite h after serum injection, which is a safe window to ensure complete uptake of the antibodies. a second injection was performed dpc to ensure that injected antibodies remained in circulation during the whole challenge period ( days). the current passive immunization trial did not show significant protection of gsb against e. leei. however, the results indicate that injection with parasite-spabs delayed the initial propagation of the parasite in naïve gsb. the group that received serum with parasite-spabs (sur) showed minor signs of infection, particularly in terms of sgr and intensity of infection than fish injected with serum without spabs or pbs. e. leei invades and proliferates in the intestinal epithelium, causing anorexia, impaired nutrient absorption and weight loss, leading to emaciation ( ) . decreased cf and sgr have been linked to e. leei infection in several studies ( , , ) . this effect was evident in the pbs and nai-injected groups, showing a higher prevalence of infection. sur-injected fish also were infected, reaching % of prevalence of infection dpc. however, these animals showed less disease signs than the other two challenged groups, in coincidence with lower prevalence and progression of the infection. e. leei starts invading the posterior part of the intestine, slowly progressing to the anterior and finally to the middle segment of the intestine ( , ) . hence, the number of intestinal segments parasitized serves as a measure of the progression of the infection and provides a hint of the time a fish has been parasitized. in fact, the number of intestinal segments parasitized was significantly correlated (negatively) with disease signs such as cf ( ) . in the same study, intensity of infection, measured as ct values of the parasite s rrna, was significantly correlated with cf, sgr and extension of the infection. lower ct values indicate more copies of parasite s rrna and coincided with stronger disease signs and more intestinal segments parasitized ( ) . the current results are in agreement with this previous observation. enteromyxum leei induces a massive hyperplasia of the intestinal lamina propria-submucosa due to recruitment and proliferation of heterogeneous leukocytes ( ) . igm + cells massively proliferate in the submucosa and within the epithelium ( ) , whereas zap + t cells aggregate mainly at the base of the epithelium ( ) . this inflammatory response, together with enterocyte apoptosis and necrosis, hamper intestinal barrier integrity producing nutrient malabsorption and osmotic intestinal failure ( ) . as expected, these signs were also observed in the current experiment, where challenged fish showed an inflammatory response in the submucosa with abundant zap + t cells at the base of the epithelium and infiltrated in it. nutrient absorption impairment was also reflected in the reduction of lipid and glycogen deposits in the liver, which were probably used as an energy source to compensate the low energy intake. again, fish injected with parasite-spabs, even though being infected, showed lower signs than the other groups, probably denoting a later onset of the disease. antibodies have already been described as main actors in gsb immune response against e. leei ( ) , and in other fish-myxozoan models ( ) ( ) ( ) ( ) ( ) . the presence of high levels of circulating parasite-specific igm in gsb that survived an infection with e. leei has been linked to their resistance to re-infection ( ) . e. leei induces igm and igt gene expression both at local (intestine) and systemic levels (head kidney), with a potent local effect. this increased gene expression has been correlated with increased serum levels of parasite-specific igm ( , ) . regretfully, no information is available on levels of parasite-specific igt or whether these spabs can be found in circulation, as igt has been described mainly as a mucosal ig ( ) . b cell responses against e. leei have been detected from dpc onward ( , , , ) , and this response is temperature dependent ( , ) . delayed b cell responses against myxozoan parasites have already been described, with spabs being detectable only after - weeks ( ) . in the current challenge, we did not observe major differences on circulating igm and igt among injected groups. the higher presence of total circulating igt in the pbs injected group could be related to a higher reaction to the antigen, as this group was the one showing the higher prevalence and progression of the infection. however, the mechanism behind this and the specificity of the antibodies should be checked in future studies. fish injected with specific-antibodies showed an increase in total igm, but this increase was not reflected as a parasite-specific response, therefore, this difference remains to be explained. in any case, as sur injected fish seem to have been protected in the first weeks of infection, a lower amount of parasite-spabs would support the latter onset of the disease. however, all groups challenged with the parasite showed an increase in parasite-specific igm in serum after days, with no difference among groups. the overall low antibody response detected in the current experiment could be attributed to the low temperature as previously described ( ) . the current results suggest that the injected antibodies may be acting at the initial steps of parasite invasion probably by blocking the parasite host-specific receptors (opsonization) and/or promoting phagocyte activity ( , ) . however, probably due to the long-term nature of the infection, the injected antibodies were not enough against the parasite pressure. similar results have been found in other fish slow-progressing parasites models. passively immunized rainbow trout showed a lower, although not significant, abundance of the monogenean gyrodactylus derjavini one month after infection ( ) . also in rainbow trout, passive immunization against the microsporidian parasite loma salmonae was not enough to protect the fish against infection, but delayed its arrival at the heart by one week ( ) . in our experiment, fish were challenged with parasite containing effluent water for days and then each group was separated to tanks with clean water to avoid excessive parasite pressure. nonetheless, cohabitation is also an effective infection route for e. leei ( ) . thus, even with one animal getting infected during the challenge phase, the disease can still be transmitted to the cohabitants at later time points, when passively transferred antibodies are no longer viable. future experiments using repeated injections of serum throughout the whole period, injecting higher amounts of immune serum, and/or separating the fish individually after the challenge phase, will help to determine the exact degree of protection that can be acquired by passive immunization against e. leei. the current results support the hypothesis from previous studies that a spab response is key against enteromyxosis in gsb ( ) . thus, we further analyzed the antibody response being elicited in the animals that acted as serum donors for the passive immunization trial. these animals had high levels of circulating parasite-specific igm and were resistant to reinfection. the repertoire analysis was performed on the anterior intestine, as previous studies revealed that in this particular organ igm and igt transcript expression increased upon re-exposure to the parasite in resistant fish ( ) . the cdr of the vh domain was targeted because this region is the key determinant of specificity in antigen recognition in antibodies and the t cell receptor, whereas cdr and cdr sequences are much more cross-reactive ( ) . a strong and specific response against a pathogen would induce a decrease in the clonotype diversity in favor of the expansion of a specific pathogen-specific subset. this phenomenon has been described in fish in some viral infections ( ) . parasites, however, are much more complex organisms, in particular metazoans with different life stages like myxozoans. parasites can harbor vast amounts of antigenic peptides and some even demonstrated specific strategies to avoid the host's antibody responses. for instance, some parasites are known to induce hypergammaglobulinemia, resulting in a polyclonal expansion with significantly increased antibody titers but a limited proportion of parasite-spabs. this has been reported for human and fish protozoan parasites, such as trypanosoma cruzi ( ) or trypanoplasma borreli ( ), but also in some myxozoan infections caused by sphaerospora molnari ( ) or tetracapsuloides bryosalmonae ( ) . the current results, showing a significant increase in clonotype diversity for both igm and igt, together with previous findings on this host-parasite model ( , ) , seem to indicate a similar strategy for e. leei. close to % of igt and igm clonotypes were found - times, indicating a high degree of repertoire diversity in gsb anterior intestine. this high clonotype diversity, with very few clonotypes expressed more than times, has been described in naïve rainbow trout spleen and, upon viral ( ) or parasitic ( ) infection, having certain clonotypes a high frequency, and indicating a clear response against the pathogens. the results of the current study deal with the ig repertoire of intestinal b cells, thus the pattern is expected to be different from that found in spleen. intestine is a major mucosal tissue that is in close contact with the environment and harbors a very diverse community of symbiont microorganisms, the microbiota. hence, intestinal cells are constantly exposed to a myriad of antigens. this probably explains the different patterns found in naïve animals in our study with more abundant high frequency clonotypes and non-gaussian cdr length distributions. the cdr length distribution analysis is an estimate of the overall diversity and deviations from a bell-shaped gaussian distribution are indicative of clonal expansions ( ) . the different distribution of igm cdr lengths, and the significantly more abundant igt clonotypes with frequencies of - in sur animals indicate that e. leei is inducing a clear response that involves both isotypes but probably through different mechanisms. a complete characterization of a gsb specific reference germline throughout the definition of the gsb igh locus following the identification of all segments encoding v, d and j genes will allow further deep analyses of this response at gene level. thus, the definition of a gsb specific germline will allow a better dissection of clonotypes specifically selected during the response to myxozoans as well as will allow to further explore somatic hypermutation patterns in selected clonotypes. to conclude, e. leei induces a differential igm and igt response in gsb characterized by a polyclonal expansion of diverse ig subsets. this could be part of an immuneevasion strategy elicited by the parasite aiming to dilute parasite-spabs by increasing the proportion of irrelevant antibodies, as has been described in similar host-parasite models ( , ) . however, increased levels of parasite-spabs (igm) have been detected in the serum of gsb when challenged with the parasite, and the presence of high levels of these antibodies has been related with acquired resistance. in addition, the current results show that passive transfer of parasite-spabs can delay the establishment of the infection and the appearance of disease signs. taken together, these findings indicate that, regardless of the probable immuneevasion strategy of the parasite, antibodies are key molecules against enteromyxosis in gsb. future studies should be conducted to further evaluate the role of specific igt, a specialized mucosal immunoglobulin, in this model. also, more in depth studies on the protective antibody response will be key to develop effective treatments and prophylactic methods against e. leei. the datasets presented in this study can be found in online repositories. the names of the repository/repositories and accession number(s) can be found here: https://www.ncbi.nlm. nih.gov/, prjna . the animal study was reviewed and approved by ethics and animal welfare committee of iats, csic and generalitat valenciana. the authors thank p. boudinot (inrae) for his help in designing and interpreting the immunoglobulin repertoire study and results, j. pérez-sánchez (iats-csic) for providing access to the gilthead sea bream genome sequences to perform the repertoire analysis, and j. monfort and l. rodríguez (iats-csic) for histological processing. the supplementary material for this article can be found 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follicular splenic b-cell response which is a high source of non-parasite-specific antibodies the immune response of carp to trypanoplasma borreli: kinetics of immune gene expression and polyclonal lymphocyte activation the kinetics of cellular and humoral immune responses of common carp to presporogonic development of the myxozoan sphaerospora molnari statistical analysis of cdr length distributions for the assessment of t and b cell repertoire biases key: cord- - k f tw authors: parker, elaine l.; silverstein, rachel b.; verma, sonam; mysorekar, indira u. title: viral-immune cell interactions at the maternal-fetal interface in human pregnancy date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: k f tw the human decidua and placenta form a distinct environment distinguished for its promotion of immunotolerance to infiltrating semiallogeneic trophoblast cells to enable successful pregnancy. the maternal-fetal interface also successfully precludes transmission of most pathogens. this barrier function occurs in conjunction with a diverse influx of decidual immune cells including natural killer cells, macrophages and t cells. however, several viruses, among other microorganisms, manage to escape destruction by the host adaptive and innate immune system, leading to congenital infection and adverse pregnancy outcomes. in this review, we describe mechanisms of pathogenicity of two such viral pathogens, human cytomegalovirus (hcmv) and zika virus (zikv) at the maternal-fetal interface. host decidual immune cell responses to these specific pathogens will be considered, along with their interactions with other cell types and the ways in which these immune cells may both facilitate and limit infection at different stages of pregnancy. neither hcmv nor zikv naturally infect commonly used animal models [e.g., mice] which makes it challenging to understand disease pathogenesis. here, we will highlight new approaches using placenta-on-a-chip and organoids models that are providing functional and physiologically relevant ways to study viral-host interaction at the maternal-fetal interface. pregnancy is a unique immunological phenomenon in which the semiallogenic fetus is able to grow in the maternal uterine environment. in order for a successful pregnancy to occur, healthy placentation is necessary to create an environment that is protective for the developing fetus and promotes growth. how immune balance is maintained by maternal and fetal cells to promote the survival of the genetically distinct fetus, while preventing infection by a large number of pathogens, is yet to be fully elucidated ( ) . this little understood enigma has been the subject of interest and research for decades ( ) . fertilization leads to the creation of single celled embryo which undergoes several successive divisions to form a blastocyst. the blastocyst is made up of two types of cells: the outer trophoblast or trophoectoderm (te) layer forming the placenta and chorion, and the inner layer or inner cell mass (icm) forming the embryo proper and amnion ( ) . the decidua underlying the embryo is called the decidua basalis, which composes the maternal side of the placenta. the maternal-fetal interface is made up of the maternal decidua and fetally-derived placenta. during implantation, the blastocyst attaches to the decidualized endometrium and the outer layer of the blastocyst differentiates into different lineages. the te gives rise to cytotrophoblast cells (ctbs) which follow villous and extravillous pathways to form the placenta. in the villous pathway, the mononuclear ctbs fuse, creating multinucleated syncytiotrophoblasts (stbs) that establish floating villi (fv). the fv are surrounded by maternal blood, with stbs aiding the provision of nutrients by enabling gas exchange and exchange of secreted pregnancy-related hormones (human chorionic gonadotropin, hcg, human placental lactogen, hpl) at the maternal-fetal interface. furthermore, ctbs act as anchoring villi for the attachment of the embryo to the uterus. the ctbs present in the cell column of the anchoring villi follow the extravillous pathway and differentiate into interstitial (ictbs) and endovascular extravillous trophoblast cells (ectbs). the ictbs further invade up to the inner third of the myometrium and ectbs remodel the spiral arteries in low resistance high blood flow to provide nutrients to the developing embryo ( ) ( ) ( ) ( ) . the invasion of trophoblast cells at the maternal-fetal interface occurs in the presence of a large population of maternal immune cells ( ) . this includes % decidual natural killer (dnk) cells, %- % macrophages, %- % t cells and . % dendritic cells ( ) ( ) ( ) . the abundance of decidual cytotoxic t cells and macrophages can vary through the course of pregnancy ( ) . the abundance of nk cells in the decidua during the first trimester, and through the pregnancy (albeit at lower abundance), implicates them as an essential element in both the promotion of an immunotolerant environment and the control of pathogenic infection during pregnancy ( figure ) . thus, the paradoxical maternal-fetal interface is admired for both its immunotolerance to semiallogeneic trophoblastic invasion (leading to a successful pregnancy) while remaining remarkably resilient to pathogenic infections. nevertheless, several pathogen, termed torch pathogens (described below), successfully cross the placental barrier and cause devastating infection in the developing fetus ( ) . in this review, we will look at the interactions between decidual immune cells and specific viral torch pathogens and review known mechanisms which may enable viral pathogenesis within the placental environment. torch is an acronym defining some of the most common infections associated with vertical transmission. initially described in , this group contained just pathogens; toxoplasmosis, rubella, cytomegalovirus (cmv) and herpes simplex type and ( ) . since then this group has been broadened to comprise a host of other infections including listeria monocytogenes, syphilis, varicella zoster virus, human immunodeficiency virus (hiv), enteroviruses and parvovirus b ( ) . most recently, following the zika virus (zikv) epidemic in south america resulting in observed congenital anomalies, this group has been further expanded to include zikv, with some suggesting renaming this group "torchz" ( ) . the mechanism by which these "torchz" pathogens are able to circumvent typical clearance by groups of immune cells (e.g. nk cells, macrophages and others) has been studied by many groups over the last few decades in order to elucidate not only routes of pathogenicity but also roles of immune cells within this immune-privileged environment ( ) . it remains to be proven whether the new emerging viral threat by sars-cov which causes covid- including in pregnant women, will be included in this group of vertically transmitted pathogens ( ) . in this review, we will focus on maternal and fetal macrophages, t cells, and nk cells and their relationship with each virus. we will focus on the viruses human cytomegalovirus (hcmv) and zikv, which are known causes of adverse pregnancy outcomes and delve into how they interact with various decidual immune cells to promote their survival and replication. we will examine the timings of pregnancy that appear to be most permissive to pathogenic infection by these viruses and we will look at the role of various immune cells in this context ( figure ). in the early first term decidua, %- % of resident leukocytes are t cells with approximately %- % of these t cells being cd + (t helper cells) and %- % being cd + (cytotoxic) t cells ( ) ( ) ( ) . further studies have estimated the decidual cd + population to be comprised of about % activated memory cd dim t cells and % cd + cd bright foxp + treg cells. unlike the peripheral circulation, the decidua has a higher ratio of cd + t cells to cd + t cells and an overall higher number of cd + t cells ( ) . in addition, approximately % of the decidual cd + population are effector-memory t cells with reduced perforin and granzyme b in comparison to their peripheral counterparts ( ) . one study published in described a small percentage of cd + t cells found in uncomplicated term decidua to be viral specific. though these populations of viral specific cd + t cells were . % and . % in the decidual basalis and decidual parietalis respectively, they demonstrated that this was higher than that seen in peripheral blood and postulated a role for their presence in the decidua as one of immunoprotection for the fetus. this study could not conclude upon the origin of these t cells, and whether they were recruited from the periphery or activated in the decidua. in addition, more work remains to be done to establish whether these virus specific cd + t cells exist early in pregnancy ( ) . another study has described the presence of a small population of cd + hla-g+ t cells which are thought to acquire hla-g through trogocytosis from decidual dendritic cells. it is thought that these t cells promote immunotolerance at the maternal-fetal interface, and they have been shown to be downregulated in pathologies such as preeclampsia (pe) ( ) . therefore, it appears t cells play specific roles in immunity and tolerance. to this end we will look at the role that various populations of t cells may play in either enabling or preventing infection by torch pathogens at the maternal-fetal interface. macrophages constitute - % of all leukocytes in the first trimester decidua and play an important role in tissue remodeling, angiogenesis, host defense and immunotolerance ( ) . macrophages are considered a key link between adaptive and innate immunity, communicating to other immune cells and modulating their activity ( , ) . these cells are therefore vital throughout pregnancy, adapting their phenotype to address the changing requirements of the evolving decidua ( ) . tissue resident decidual macrophages are thought to be recruited from monocytes in the peripheral circulation ( ) . distinct subtypes of macrophages have been shown to be present in first-trimester decidual tissue exhibiting immunomodulatory, proinflammatory, and tissue remodeling phenotypes and play key roles in protective immunity as well as fetal tolerance ( ) . decidual macrophages are known for their highly immunosuppressive phenotype at the maternal-fetal interface, expressing cd , dc-sign and tim- among other receptor markers ( , ) . in addition to these maternally derived macrophages exist fetal-derived macrophages called hofbauer cells (hcs), which sit in the stroma of the chorionic villi ( ) . these hcs are resident in close proximity to fetal vessels and trophoblast cells from the first trimester until birth. hcs could serve as a portal of entry for pathogens from the infected mother ( ) . initially during implantation, they appear to have an inflammatory m phenotype which has both microbicidal activity and promotes a cell-mediated th cytokine response. later, they shift to a mixture of both m and m phenotypes following trophoblastic invasion and remodeling ( , ) . several studies have implicated hcs in host viral interactions. here, we look at the reciprocal interactions between hcs, maternal macrophages, and hcmv and zikv. the nk cell population in the peripheral circulation is predominately made up of cd dim cd + cells, which are believed to have a more cytotoxic phenotype ( ) . approximately % of the peripheral circulation is constituted by cd bright nk cells, which have a more immunotolerant phenotype ( ) . in the decidua, these nk cell proportions are reversed; - % of the total lymphocytes are cd bright cd - ( ) . research has demonstrated a number of dnk subsets within the cd +cd -population. it is believed that this distinct immunotolerant population is fundamental to the maintenance of a successful pregnancy, with research postulating both an ability to enable the semiallogenic fetus to thrive while at the same time responding to pathogenic infections. these nk cells reside in the decidua basalis close to invading evts and express specific receptors (e.g. kir receptors, cd /nkg a, ilt ) to activate or inhibit evt function ( ) . this large population of dnk cells are known to be sustained during the first and second trimester, with their numbers declining toward term ( , ) . despite the unique immunotolerant phenotype demonstrated by dnk cells, it is evident that this cell population displays a high level of plasticity, gaining cytotoxic function in the presence of specific pathogens ( ) . one way by which this happens is through activation of dnk cell cytotoxcity via killer cell ig-like receptor ds (kir ds ). reduced expression of this receptor has been associated with adverse pregnancy outcomes such as miscarriages and fetal growth restriction and individuals with increased kir ds expression have shown better outcomes post-viral infections ( ) . we will explore further the role that nk cells play in specific viral infections in pregnancy torch pathogens hcmv human cytomegalovirus (hcmv) was first described in by margaret smith, who replicated a virus from two newborn babies who had died from cytomegalic inclusion disease (cid) ( ) . what we now know as hcmv first came to the attention of ribbert et al. in , where intranuclear inclusions within large cells were noted in renal and parotid gland cells of stillborn fetuses. these inclusions, often described as 'owl's eye inclusions', were noted to be surrounded by a clear halo ( ) . hcmv was identified in the s when smith, weller and rowe isolated and cultured hcmv from salivary glands, adenoid tissue and liver biopsies respectively ( , ) . mechanisms of vertical transmission of hcmv can either be transplacental during gestation or transvaginal during parturition; additionally, there is some evidence for breastmilk transmission ( ) . hcmv infection is most likely to occur in the third trimester, demonstrating a % risk of mother to child transmission in the first trimester compared to a % risk in the third trimester ( ) ( ) ( ) . congenital hcmv has been estimated to affect - in every , live births, with % of hcmv positive infants suffering neurological consequences from birth ( ) . hcmv infection during pregnancy therefore poses a substantial risk to the developing fetus, leading to congenital disease including cerebral abnormalities such as periventricular calcifications, microcephaly, visual impairment, sensorineural hearing loss, neurodevelopmental delay and hepatomegaly ( ) . congenital hcmv affects , - , pregnancies annually in the united states and accounts for % of all incidents of pediatric sensorineural hearing loss ( ) ( ) ( ) . it is estimated that the burden of morbidity associated with congenital hcmv infection is greater than that of other common congenital pediatric conditions such as down's syndrome or fetal alcohol syndrome ( ) ( ) ( ) . hcmv is also associated with intrauterine growth restriction and miscarriage. there is a great need to understand maternal immunity pathways involved in hcmv infection to develop effective vaccines ( ) . hcmv is associated with asymptomatic infection of most of the world's population and subclinical illness in pregnant mothers. in the us, an estimated % of unexposed pregnant women experience primary infection during pregnancy, resulting in congenital infection in % of cases from this population ( , ( ) ( ) ( ) ( ) ( ) . however, vertical transmission of hcmv is not only seen in mothers with primary infection but also igg seropositive mothers, who exhibit a % rate of congenital hcmv infection. mechanisms of infection have been studied through analysis of placental tissue from all three trimesters of human gestation. in placental tissues from those suffering from hcmv, necrosis and oedema has been noted associated with severity of congenital disease symptoms. it has also been noted that hcmv infection is often associated with bacterial coinfection with a potentially pathogenic synergism ( ) . hcmv resides in the chorionic villi, specifically infecting ctbs, stbs and hcs. it is believed that the ability to travel between stbs in the decidua is key to hcmv pathogenesis ( ) . many studies have explored the role of the adaptive and innate immune system in hcmv infection. below we review established interactions between hcmv and immune cells (figure ). hcmv's ability to infect different populations of macrophages has been demonstrated by several studies. hcmv has been shown to be sequestered by hcs, with placentas from confirmed cases of hcmv infection demonstrating significant hyperplasia of this cell population ( ) ( ) ( ) . a study investigating vaccine development showed that when neutralizing antibodies are produced against hcmv, rates of hcs infection are decreased ( ) . a different study utilizing placental explants showed hcmv-igg immune complexes to undergo fc receptor mediated transcytosis as a mechanism to traverse the syncytium to ctbs. hcmv is then taken up by hcs in the placental villi ( ) . furthermore, another study by loenen et al., supports the idea that hcmv genes are able to increase fcr expression on infected cells ( ) . another study suggested that hcmv replication in stbs is upregulated in the presence of macrophages ( ) by analyzing hcmv replication in stbs alone or when infected stbs were cultured with uninfected placental macrophages. this study also demonstrated elevated levels of hcmv viral titres in co-cultured supernatants when compared to those from stbs cultured alone. this demonstrates that not only do macrophages have the capacity to be infected by hcmv, but also that they may amplify hcmv infection of surrounding cells in the decidua. some studies have depicted a role for latently infected maternal decidual macrophages in congenital hcmv infection, describing how microbial infections or insults in the placenta may reactivate these macrophages and in turn reactivate hcmv infection ( ) ( ) ( ) . the maternal-fetal interface is unique in respect to allogenic interactions with cd + t cells. evts are known to invade the decidua, evading destruction despite the intrinsic ability of cd + t cells to recognize foreign antigen via mhc class i molecules. as discussed previously, one mechanism by which evts are believed to evade cd + t cell recognition is through a lack of expression of hla-a and hla-b, which are key to cd + cytotoxic activity. during pregnancy, many viruses have been shown to upregulate maternal cd + t cell activity, leading to migration of highly differentiated effector memory t cells to the decidua. despite many descriptions regarding the role of t cells in hcmv infection in the fetus and the mother, there are few studies identifying their tissue specific role at the maternal-fetal interface ( ) . hcmv is thought to limit cd + t cell activity through restriction of mhc class ii expression on apcs, which in turn may prevent activation of cd + and cd + t cells ( ) . this is thought to be mediated through the hcmv protein gpus , which may degrade mhc class ii glycoproteins or disrupt downstream ciita/jak/stat signaling pathways ( ) . crespo et al., in demonstrated that hcmv did not induce a significant difference in hla-g expression on either jeg- cells or primary evts. hla-g expression has been associated with immunotolerance, and therefore its persistence despite infection may act to protect infected trophoblast cells from cytotoxic destruction ( ) . studies looking at the role of t cells in viral infection at the maternal-fetal interface demonstrated lower t-cell numbers and response in mothers who vertically transmitted hcmv to their offspring when compared to infected mothers who did not transmit hcmv, potentially suggesting an active role for t cells in vertical hcmv transmission ( ) . more specifically, a reduction in the number of cd +cd ra +ifn-g+ treg cells and cd +cd ra+ifn-g+ t cells in mothers who transmitted hcmv to their fetus was noted when contrasted with mothers who were hcmv positive but did not transmit the infection. there was also a measurable blunted t cell response in hcmv infected mothers who vertically transmitted infection, compared to infected mothers who did not transmit the virus ( , ) . in infected mothers, hcmv virus specific t cells have been shown to be elevated in the final trimester when compared to uninfected mothers ( ) . congenital hcmv infection risk is highest for the fetus in the third trimester, with a % transmission risk compared to a % risk in first trimester ( ) . this is despite the abundance of immune cells, specifically nk cells, in early pregnancy. dnk cells exposed to hcmv infected decidual fibroblasts are known to alter their phenotype to express higher levels of activating receptors (such as nkg d and cd /nkg c or nkg e). uniquely, utilizing in vitro studies, it was noted that decidual nk cells had targeted cytotoxic activity against hcmv infected autologous decidual fibroblasts and heterologous uninfected fibroblast cells, but appeared to spare trophoblast cells ( , ) . this demonstrates a clear cytotoxic effector response by decidual nk cells to hcmv, switching from their typically immunotolerant phenotype with high levels of inhibitory receptor expression (cd /nkg a, lir- , kirs), to a cytotoxic phenotype ( , ) . this group also studied the interaction between dnk and hcmv-infected cells using hcmv positive and hcmv negative decidual villous explants. this investigation revealed through fluorescent staining of dnk cells that colocalisation of dnk cells to cells throughout the hcmv positive explant occurred, including synaptic connections which was not seen in hcmv negative explants. this was thought to suggest that the dnk cells were unable to connect with uninfected trophoblasts. this also demonstrates that dnk cells are able to localize and target hcmv infected cells while sparing fetal derived semiallogenic trophoblast cells ( ) . dnk cells are unique in their function, both contributing to immunotolerance at the maternal-fetal interface, thereby enabling invasive trophoblastic activity, as well as controlling pathogenic infection ( ) . this is thought to be mediated by secretion of specific cytokines ( , ( ) ( ) ( ) ( ) . the relatively limited vertical transmission of hcmv during the first trimester of pregnancy, when the population of nk cells is abundant, has led many to speculate about the role nk cells may play in hcmv control ( ) . tilburgs and colleagues have recently demonstrated distinct cytotoxic responses in dnk cells to hcmv in first trimester versus at term wherein term pregnancy dnks harbor reduced efficacy in responding to hcmv-infected cells ( ) . siewera et al., suggested that dnk cells undergo a phenotypic transformation to acquire cytotoxic function in the presence of hcmv-infected cells ( ) . this study proved, through antibody mediated abrogation of the fas ligand (fasl) and tumor necrosis factor-related apoptosis-induced ligand (trail) on dnk cells, that death of hcmv infected cells is not initiated by dnk cells through these death receptor-ligand pathways. however, this study demonstrated that dnk cells form immunological synapses with hcmv infected fibroblasts, enabling the delivery of perforin/granzyme for cellular destruction. furthermore, the ability of dnk cells to degranulate in the presence of hcmv infected fibroblasts was demonstrated to be through high levels of cd a expression, a key cell surface molecule in the mechanism of lytic granule release. dnk cells have also been found to secrete higher quantities of granulysin when compared to peripheral blood nk cells. upon incubation with infected fibroblast cells, it was noted that cd bright nk cells decreased from . % to %, while there was an elevation in cd expression by nk cells, denoting a transformation to a cytotoxic phenotype. hcmv infected cells have been noted to upregulate expression of natural cytotoxicity receptor (ncrs) nkp by almost -fold on dnk cells as well as increasing expression of nkg c. ncrs are associated with activation of the cytotoxic profile of nk cells. accompanying this was a reduction in nkg a, kir dl , kir dl , and ilt receptor expression, receptors aligned with nk effector inhibitory function ( ) . activating dnk cell receptors such as kir ds , kir ds , kir ds and kir ds have been correlated with antiviral activity ( ) . a study by crespo et al., demonstrated an increased population of kir ds + nk cells in the decidua, suggesting an increased activating dnk cell capability in response to hla-c , and thereby increased cytotoxic potential. these cells also displayed higher levels of cytolytic molecules when compared to peripheral nk cells. this study demonstrated that kir ds + dnk cells showed increased cytotoxicity to hcmv infected decidual stromal cells (dscs) positive for hla-c when compared to kir ds -dnk cells. this was not the case for infected jeg- and primary evt cells, which did not appear to initiate degranulation or cytokine secretion from dnk cells. despite this, a reduction in the number of infected evts in the presence of co-cultured dnk cells was noted, suggesting that dnk may be clearing virus infected evts by other means ( ) . hcmv has been seen to reduce expression of mhc class i, thereby potentially evading cd + t cell destruction ( ) ( ) ( ) . one study reports an initial reduction in hla-c expression on evts in hcmv infection. the possible reason for this is not clear, however this study suggests it could prevent inhibition of nk cells through the hla-c/kir dl route, with an additional suggestion of potentially other unknown ligands being upregulated for activation of kir ds , leading to cytotoxic action against infected cells ( ) . another study showed that the potential effect of dnk cell activation on t-cell activation could be mediated via an upregulation in hla-dr expression upon exposure to hcmv infected fibroblasts ( ) . therefore, dnk cells may play a role in congenital hcmv infection by potentially protecting the first trimester fetus from infection via activation of t cell function. collectively, these studies indicate varied interactions between dnk cells and hcmv, with many routes by which hcmv may evade clearance as well as a number of ways through which dnk cells may be activated in the presence of hcmv infected cells. additionally, dnk cells are seen specifically to modulate activity in the context of t cell activation. zika virus (zikv) was first isolated in in zika forest, uganda, from infected rhesus monkey serum during epidemiological yellow fever research ( , ) . however, the first case of human infection was not reported until , when three patients presented with jaundice and were later confirmed to have rising levels of zika antibodies ( ) . initially, zikv was associated with innocuous prodromal illness on the african and indian subcontinents transmitted by the aedes aegypti mosquito, leading to an asymptomatic or self-limiting course of infection ( ) . in , a mild disseminated infection was identified to be zikv in over % of the population of the island of yap ( ) . concerns regarding human zikv infections were not aroused until when incidences of neurological deficits associated with zikv infection were first described, with almost , recorded infections noted in french polynesia ( , ) . shortly following this in , a zikv epidemic began in south america where not only were neurological deficits such as guillain-barreś yndrome seen, but also spontaneous abortion and congenital malformations such as microcephaly in infants from infected mothers ( ) . by the end of the epidemic in brazil, there were more than , notifications of zikv cases ( ) . estimates for infants born with congenital zika syndrome (czs) after the - epidemic ranged from to in every , births ( ) . the threat of a zikv epidemic lingers, with who reporting countries affected by aedes aegypti mosquitoes, therefore carrying the potential for zikv infection and transmission ( ) . zikv demonstrated continuing global epidemic capacity in india in ( ) . zikv belongs to the flavivirus family alongside west nile virus, dengue virus, and yellow fever virus. zikv is an enveloped and icosahedral virus with a nonsegmented, . kb single stranded positive sense rna genomes ( ) . this virus is composed of several proteins, categorized as three structural (capsid, pre-membrane and envelope) and seven nonstructural proteins. the seven nonstructural proteins (ns , ns a, ns b, ns , ns a, ns b, and ns ) are essential for viral replication and assembly, as well as being responsible for the pathogenicity of the virus by binding to transcription and restriction factors ( ) . the biggest risk of congenital zikv infection is for mothers infected during their first trimester ( ) . zikv infection demonstrates wide tissue tropism, with zikv successfully infecting the central nervous system, blood, retinal, genital and reproductive tissues including placenta ( ) ( ) ( ) ( ) ( ) . zikv was thought to be exclusively arthropod transmitted until cases of human-human transmission emerged in nonendemic regions, illustrating a role for sexual transmission ( ) ( ) ( ) . the presence of zikv rna has also been found in breast milk of zikv infected mothers ( ) ( ) ( ) . however, there are reports which suggest that vertical transmission of zikv by breastmilk does not occur in most cases, which suggests the possibility that breastmilk does not have a high enough viral load to infect the newborn ( , ) . despite some knowledge regarding zikv pathogenesis, its mechanism of infection in placental immune cell types remains limited ( ) ( ) ( ) ( ) . histopathology of zikv infected placentae has shown zikv infection in first trimester villous stromal tissue cells, which includes immune cells in the chorionic villi ( , , ) . uniquely, zikv was also found to infect ctbs, endothelial cells, fibroblasts and hc in chorionic villi, as well as amniotic epithelial cells and trophoblast progenitor cells ( , ( ) ( ) ( ) ( ) ( ) ( ) (figure ). similar to hcmv, zikv has also been shown to infect hcs and ctbs ( , , ) . during the first trimester of pregnancy, zikv infects hcs, entering the fetal blood stream in order to reside in the placenta. zikv uses hcs as a "trojan horse". this strategy is utilized by several viruses in order to cross the blood brain barrier, where the virus infects leukocytes, leading to them being carried across barriers and thereby enabling the propagation and spread of infection ( ) ( ) ( ) . hcs have been associated with zikv spread to the fetus through the "trojan horse" route ( ) . the presence of zikv-specific antigen was demonstrated in hcs in confirmed maternal infection. multiple studies suggest hcs are a crucial step in vertical transmission of zikv to fetal cells, demonstrating that hcs are preferentially infected when compared to ctbs ( , , ) . infection of hcs with zikv is thought to propagate infection through hyperplasia and proliferation of these cells, leading to persistence of this hc population into later trimesters ( , , ) . a study performed on first trimester fetal and maternal tissue showed that zikv can replicate in different cell types, such as decidual fibroblasts and macrophages. it can also infect trophoblasts and hcs as well as umbilical cord mesenchymal stem cells, suggesting that the route of zikv infection may move from the decidua basalis to the anchoring villi ( ) . a study performed using blood from + asian zikv infected pregnant women shows that cd + monocytes are the primary target of zikv infection. these monocytes are resistant to change in m phenotype and downregulate type ifn signaling, which induces the expression of different host genes involved in pregnancy complications ( ) . in the decidua basalis, zikv infects evts, macrophages and stromal cells. zikv also targets proliferative ctbs in the anchoring villi, however is unlikely to infect stbs due to ifnl-mediated antiviral defense mechanisms ( ) . zikv achieves replication within macrophages through fcr, tlr and dc-sign receptors ( , ) . in vitro studies have demonstrated zikv infection to be augmented in hcs by igg from prior flavivirus exposure through antibody dependent enhancement (ade) ( ) . there remain many gaps in knowledge regarding the role for macrophages targeted and infected by zikv. a study performed using decidual and chorionic villous tissue from early and mid-gestation human pregnancy shows that zikv appears to elevate type i and iii ifn expression, which does not occur in hcmv infection ( ) . studies looking at the interaction between zikv and t cells in humans are scarce although zikv infection has been demonstrated to activate both cd and cd t cells ( ) with specific increases in vd tcr+ cells which have been implicated in recurrent miscarriages but not associated with zikv-induced fetal complications. there have not been notable studies looking specifically at t cell zikv communication at the human maternal-fetal interface ( ) . a recent study examined peripheral t cell responses of confirmed cases of zikv infection that had been stimulated with pooled zikv peptides from all viral components ( ) . this study demonstrated responses from both cd + and cd + t cells to both structural and nonstructural zikv components. however, this study particularly showed that cd + t cells exhibited a strong response to nonstructural proteins ns , ns and ns , and cd + t cells a strong response to cap and env proteins. this response was demonstrated by marked ifn-g production from both cell subtypes indicating cell activation ( ) . another case looking at a zikv infected individual from the united states demonstrated interactions between the zikv ns and env proteins with cd + and cd + t cells, respectively. ( ) . furthermore, in a different study, cd + t cells of two a b d c fibroblasts occurs through fcgr and results in increased expression of some interferons, such as ifna, and decreased expression of others, such as ifn-g and ifnb. infection is associated with increased secretion of proinflammatory cytokines such as ip- , il- , and mcp- . ns viral proteins are thought to downregulate interferon-stimulated genes (isgs) and reduce interferon signaling via stat degradation, while the viral proteins ns and ns b inhibit ifn signaling by downregulating tbk . (c) cd + t cells exhibit a strong response to nonstructural ns , ns , and ns zikv proteins, while cd + t cells respond to cap and env zikv proteins. in both cases, response to zikv proteins was characterized by increased ifn-g production. (d) systemic dnk cells exhibit increased activation, including increased ifng production and cd a expression when incubated with zikv infected monocytes. frontiers in immunology | www.frontiersin.org october | volume | article zikv infected individuals showed activity in response to nonstructural proteins (ns , ns and ns ). consistently, cd + t cells were seen to raise activity against the structural protein env ( , ) . these studies demonstrate consistency in the response of cd + t cells and cd + t cells to zikv proteins, revealing cd + t cells to specifically respond to particular nonstructural proteins and cd + t cells to react to structural proteins, particularly cap and env ( , , ) . several studies have looked at the ability of denv-specific cd + and cd + t cells to be stimulated by the presence of zikv peptides in humans ( , ) . these studies showed viral epitopes for specific peptides located in similar regions and structurally conserved across flaviviruses; however, they displayed differences in their sequences ( ) . nonetheless, these studies indicated cross-reactivity between the viruses regarding their cd + and cd + t cell activity. one study demonstrated that cd + and cd + t cells from denv positive donors reacted to zikv viral peptides, resulting in an upregulation of ifng secreting cells. this group also showed that stimulation with zikv peptides for those in acute phase of zikv infection resulted in recruitment of elevated levels of cd + ifn-g+ t cells ( ) . a recent transcriptomics study investigated transcriptional signatures in cd /cd t cells, b, and nk cells and plasmacytoid dendritic cells in patients (nonpregnant) infected with zikv ( ) . interestingly, they did not note significance transcriptional changes in nk or cd t cells in a zikv infected background but noted significance alterations in pdcs. whether pregnancy plus zikv infection would affect the immune cell transcriptome in humans remains to be determined. studies specifically analyzing interactions between dnk cells and zikv in humans once again are lacking. however, studies have looked at zikv and its communication with peripheral nk cells. one such study postulated crosstalk between monocytes and nk cells in zikv infected patients. the activation of nk cells was associated with the presence of monocytes, which induced expression of ifn-g and cd a, key markers of nk cell function. depletion of monocytes in the peripheral blood reduced the levels of these markers and thus the activation of nk cells ( ) . there are few studies showing the interaction between zikv with nk cells. glasner et al., showed that zikv infection led to activation of mhc class i, which was somehow not sensed by dnk cells and their activating receptors, allowing the virus to escape nk cell-mediated killing. mhc class i expression is triggered through the ifn-b pathway via activation of rigi-irf ( ) . however, the mechanism by which nk cells may promote an immunosuppressive environment in the face of zikv infection is not clear. some studies have indicated that interactions between other aspects of the innate immune system and nk cells may be at play in zikv pathogenesis. there are several studies suggesting that pathogenesis of zikv is not mediated through decidual immune cells alone but rather conducted, at least in part, through the activation of interferonstimulated genes (isgs), which in turn leads to activation of innate host cell immunity ( ) . isgs act to specifically target viral replication. multiple studies have indicated zikv stimulation of interferons (ifn) to vary depending on the type of ifn. while type i and iii ifns have been shown to be inhibited by zikv, specifically the ns component of the pathogen, type ii ifns have been shown to be upregulated by the virus ( , ) . one study demonstrated that when type iii ifns were upregulated, specifically ifn-l , trophoblast cells were infected with zikv at a lower rate. further, ns , ns a and ns b have been demonstrated to inhibit ifn type i response. this leads to suppression of the tank binding kinase (tbk )/irf and jak-stat pathway, which in turn results in reduced activation of innate immune responses ( ) . interferon induced transmembrane protein (ifitm ) and ifitm specifically are isgs which act as restriction factors to inhibit zikv replication. the mechanism by which the inhibition and activation of innate immunity impacts the recruitment of innate immune cells to the site of infection is not clear. little is known about the role of nk cells in human zikv infection. one study has noted interactions between tlr , cd and ifitm , postulating that the restriction of zikv is associated with inhibitory activity of ifitm , potentially through activation of nk cells ( , ) . another group looking at isgs showed that viperin played a role in zikv pathogenesis, with data revealing that when viperin levels were high, zikv mrna levels were low and vice versa ( ) . ns is seen to target directly the akt-mtor pathway, leading to reduced signaling from this pathway and subsequent activation of autophagy in host cells ( ) . zikv has been shown to co-opt the autophagy pathway for post-rna replication capacity and survival ( , ) . importantly, the ns b-ns protease activity of zikv can be blocked by an inhibitor of autophagy, hydroxychloroquine (hcq) ( ) . hcq is an fda approved drug considered safe to use during pregnancy and could serve as an effective treatment for preventing zikv congenital syndrome ( ) . the relationship between zikv infected cells and attenuated ifn production has been extensively reported, leading to questions regarding the mechanism underlying this association. it has been proposed that zikv may infect cells through ade of infection. many cells express the fcg receptor, and it is thought that viral particles may complex with antibodies and thereby enter into cells via fcg receptors ( ) . host cells (such as trophoblasts and fibroblasts) infected with zikv demonstrate innate immune system activation with a rise in specific ifns (e.g. ifn-a), but falling levels of others such as ifn-l and ifn-b ( ) . the elevated levels of proinflammatory cytokines and chemokines, namely il- , mcp- and ip- which are linked to recruitment of immune cells such as monocytes and t cells ( ) . zikv has been shown in multiple studies to downregulate type i ifn signaling and to be active in suppression of antiviral signaling. zikv nonstructural proteins ns and ns b inhibit ifn signaling by downregulating levels of tbk . however, ns b downregulates the jak-stat pathway and inhibits apoptosis of zikv, and hence inhibits innate antiviral responses ( ) . one study specifically has implicated the role of the nonstructural zikv protein ns in promoting zikv propagation by targeting stat for degradation, thereby reducing isg levels ( ) . this is thought to promote viral replication through a dampened host innate immune cell response. there remains much to be elucidated in terms of zikv infection in human pregnancy. new studies are identifying metabolic reprogramming pathways underpinning innate immune responses to zikv which opens additional avenues of investigation ( ) . we refer readers to recent reviews highlighting zikv-immune interactions in adverse pregnancy outcomes ( ) and ade ( ) . the limited availability of placental tissues during early pregnancy has always been a challenge for the reproductive biologists, hampering the study of placental physiology and cell to cell interactions. in vitro cell line models can often be biologically distinct and therefore unable to demonstrate enough similarity to replicate the conditions of human pregnancy. in addition, the use of cell line models can fail to reproduce the complexity of the number of cell types and cell interactions present within the decidua. therefore, functional in vitro d models being are developed, for example placenta-on-a chip and organoid cultures, which can mimic in vivo conditions and would be useful to understand the mechanisms of viral host interactions. the 'placenta-on-a-chip' is a microfluidics model utilizing human trophoblast cells (bewo) and fetal derived cells (huvecs and hpvecs) ( , ) . these cell lines are cultured and separated by a semipermeable membrane within flow conditions with the purpose of understanding placental mechanisms and barrier function ( ) . recent reports have described the faithfulness of placenta-on-a-chip model to in vivo placental conditions ( ) . for example, glucose transport using a placenta-on-a-chip model was demonstrated by lee et al., and blundell et al., highlighting significant similarity to in vivo glucose transport in the human placenta ( , ) . placenta-on-a-chip models have also been used to investigate the transport of heparin and anti-hyperglycemic agents such as glyburide using bewo and human placental villous endothelial cells ( ) . recently, the transport of the xenobiotic compound caffeine across the placenta has been studied using this model system, providing new insights into the extent of caffeine transfer from mother to fetus ( ) . bacterial infections have also been studied using this model. zhu et al., showed that in the presence of escherichia coli (e. coli), trophoblast cells (bewo) activated the circulating macrophages on the "maternal" side of the chip to secrete several inflammatory cytokines that mimicked in vivo conditions during pregnancy ( ) . the impact of common environmental exposures such as titanium dioxide nanoparticles (tio -nps) has also been studied using this d placental model showing a series of different placental responses (barrier permeability, oxidative stress, cell apoptosis, and maternal immune cells behavior ( ) . they showed placental barrier permeability and maternal immune cells to be influenced by even low concentration of nps ( ) . therefore, this simple in vitro model can prove useful in understanding the environmental exposure of nps during pregnancy and can help in a range of biological studies ( ) . recent studies report generation of an organ-on-a-chip model, wherein decidualized human endometrial stromal cells and macrophage cell lines are co-cultured in a microfluidic device and shown to inhibit secretion of tnf-a in response to lps stimulation ( ) . these devices have also been used to determine the impact of cytokine secretion by dnk cells on the migration of primary trophoblast cells. these studies illustrate the functionality of microfluidic organ on chip devices to elucidate importance of maternal immune cells in the placenta ( ) . thus, the use of fetal membrane on organ-ona-chip provides a suitable model to explore the impact of pathogenic infections during pregnancy ( , ) . the use of in vitro trophoblast organoids as a d culture model also provides a new tool to understand the mechanism of implantation at the maternal-fetal interface. recent studies have shown the characterization of these organoids derived from sttrimester ctbs ( to weeks) and suggest their resemblance to primary trophoblast cells ( ) ( ) ( ) . due to similarity with the placental architecture, these organoids could be used to study physiological, metabolic and hormonal changes that occur during pregnancy. the viruses we highlighted in this review, hcmv and zikv, do not naturally infect commonly used animal models [e.g., mice] which makes it challenging to understand disease pathogenesis. in particular, there remains a paucity of understanding zikv-immune cell interactions during pregnancy. thus, the employment of placenta-on-a-chip or organ-on-a-chip, and organoid models will be pivotal in providing functional and physiologically relevant ways to study the interaction of immune cells at the maternal-fetal interface with viral pathogens that affect pregnancy. both hcmv and zikv can be sequestered into fetal macrophages. hcmv implicates hcs in the potential infection of other decidual cells, leading to the promotion of hcmv transcytosis in trophoblasts. zikv preferentially infects hcs, persisting in this cell population and potentially mediating infection of other fetal-derived cells. more poignant is the suggestion that decidual macrophages may mediate reactivation of hcmv by acting as a latent reservoir for infection. these studies collectively indicate a central role for macrophages in the pathogenesis of torch viruses. dnk cells have been seen to alter their phenotype to express higher levels of various activating receptors when in the presence of infected decidual fibroblast cells. they also are known for their plasticity in the face of specific pathogens, acquiring more cytotoxic function. kir dl /hla-c has been identified as a mechanism by which dnk cells are activated and display cytoxicity toward hcmv infected cells. it has also been suggested that dnk cell activation may trigger activation of t-cells through upregulating hla-dr expression on infected fibroblast cells. zikv viral components demonstrate capacity to elicit strong responses from peripheral cd + and cd + t cells, with ns , ns , and ns being associated with cd + stimulation whereas cap and env proteins being associated with cd +. we also see in this review the importance of interferon stimulating genes in the restriction of zikv replication. thus, the implications and outcomes of viral interactions with immune cells at the maternal-fetal interface are varied. we see the importance of the host immune response and recognize the importance of studying mechanisms of pathogenesis in detail to enable targeted therapeutic interventions including vaccines to mitigate the adverse outcomes of viral infections during pregnancy ( figure ) . finally, we posit that better understanding of the immunological underpinnings of infections at the maternal fetal interface can support the inclusion of pregnant women in trials testing vaccines and therapeutics to compact existing and emerging viral infections. ep, sv, and im wrote 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interactions self-renewing trophoblast organoids recapitulate the developmental program of the early human placenta trophoblast organoids as a model for maternal-fetal interactions during human placentation derivation of trophoblast stem cells from naive human pluripotent stem cells conflict of interest: im serves on the scientific advisory board of luca biologics.the remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © parker, silverstein, verma and mysorekar. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -tn hc jv authors: khawaja, akif a.; chong, deborah l. w.; sahota, jagdeep; mikolasch, theresia a.; pericleous, charis; ripoll, vera m.; booth, helen l.; khan, saif; rodriguez-justo, manuel; giles, ian p.; porter, joanna c. title: identification of a novel hif- α-α(m)β( ) integrin-net axis in fibrotic interstitial lung disease date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: tn hc jv neutrophilic inflammation correlates with mortality in fibrotic interstitial lung disease (ild) particularly in the most severe form, idiopathic pulmonary fibrosis (ipf), although the underlying mechanisms remain unclear. neutrophil function is modulated by numerous factors, including integrin activation, inflammatory cytokines and hypoxia. hypoxia has an important role in inflammation and may also contribute to pulmonary disease. we aimed to determine how neutrophil activation occurs in ild and the relative importance of hypoxia. using lung biopsies and bronchoalveolar lavage (bal) fluid from ild patients we investigated the extent of hypoxia and neutrophil activation in ild lungs. then we used ex vivo neutrophils isolated from healthy volunteers and bal from patients with ild and non-ild controls to further investigate aberrant neutrophil activation in hypoxia and ild. we demonstrate for the first time using intracellular staining, hif- α stabilization in neutrophils and endothelial cells in ild lung biopsies. hypoxia enhanced both spontaneous (+ . -fold, p < . ) and phorbol -myristate -acetate (pma)-induced (+ . -fold, p < . ) neutrophil extracellular trap (net) release, neutrophil adhesion (+ . -fold, < . ), and trans-endothelial migration (+ . -fold, p < . ). hypoxia also increased neutrophil expression of the α(m) (+ . -fold, p < . ) and α(x) (+ . -fold, p < . ) integrin subunits. interestingly, net formation was induced by α(m)β( ) integrin activation and prevented by cation chelation. finally, we observed net-like structures in ipf lung sections and in the bal from ild patients, and quantification showed increased cell-free dna content (+ . -fold, p < . ) and mpo-citrullinated histone h complexes (+ . -fold, p < . ) in bal from ild patients compared to non-ild controls. in conclusion, hif- α upregulation may augment neutrophil recruitment and activation within the lung interstitium through activation of β( ) integrins. our results identify a novel hif- α- α(m)β( ) integrin axis in net formation for future exploration in therapeutic approaches to fibrotic ild. neutrophilic inflammation correlates with mortality in fibrotic interstitial lung disease (ild) particularly in the most severe form, idiopathic pulmonary fibrosis (ipf), although the underlying mechanisms remain unclear. neutrophil function is modulated by numerous factors, including integrin activation, inflammatory cytokines and hypoxia. hypoxia has an important role in inflammation and may also contribute to pulmonary disease. we aimed to determine how neutrophil activation occurs in ild and the relative importance of hypoxia. using lung biopsies and bronchoalveolar lavage (bal) fluid from ild patients we investigated the extent of hypoxia and neutrophil activation in ild lungs. then we used ex vivo neutrophils isolated from healthy volunteers and bal from patients with ild and non-ild controls to further investigate aberrant neutrophil activation in hypoxia and ild. we demonstrate for the first time using intracellular staining, hif- α stabilization in neutrophils and endothelial cells in ild lung biopsies. hypoxia enhanced both spontaneous (+ . -fold, p < . ) and phorbol -myristate -acetate (pma)-induced (+ . -fold, p < . ) neutrophil extracellular trap (net) release, neutrophil adhesion (+ . -fold, < . ), and trans-endothelial migration (+ . -fold, p < . ). hypoxia also increased neutrophil expression of the α m (+ . -fold, p < . ) and α x (+ . -fold, p < . ) integrin subunits. interestingly, net formation was induced by α m β integrin activation and prevented by cation chelation. finally, we observed net-like structures in ipf lung sections and in the bal from ild patients, and quantification showed increased cell-free dna content (+ . -fold, p < . ) and mpo-citrullinated histone h complexes (+ . -fold, p < . ) in bal from ild patients compared to non-ild controls. in conclusion, hif- α upregulation may augment neutrophil recruitment and activation within the lung interstitium through activation of β integrins. our results identify a novel hif- αα m β integrin axis in net formation for future exploration in therapeutic approaches to fibrotic ild. the interstitial lung diseases (ild) are a group of diffuse parenchymal lung disorders that can result in pulmonary fibrosis (pf) ( ) . despite recent advances in diagnostics and therapeutics, ild is still associated with substantial morbidity and mortality ( ) . neutrophil activation may be important in ild, particularly the most severe fibrotic form, idiopathic pf (ipf). the pathogenesis of ipf is unknown but is thought to involve a "frustrated repair" response to repetitive epithelial injury, with associations to genes and proteins linked to epithelial function, integrity and repair. progressive epithelial damage, and abnormal wound repair leads to extensive scar formation and correlates, clinically, with worsening hypoxia. increasing desaturation during exercise ( ) or sleep ( ) is a significant predictor of mortality. further evidence from animal models suggests that hypoxia may actually contribute to a vicious cycle of disease progression ( ) . this evidence has led to the view that hypoxia itself may contribute to worsening of pf but the mechanistic pathway is unknown. hypoxia, a state in which oxygen supply is inadequate for tissue demands, modulates gene expression via transcriptions factors called hypoxia inducible factors (hif). there are members of the hif family, hif- α, hif- α, and hif- α, which bind conserved dna sequences or hypoxia response elements (hre). although it seems plausible that the ipf lung is hypoxic much of the evidence is indirect. levels of lactic acid, a metabolite generated in response to hypoxia, are high in ipf lung tissue supporting the concept of a hypoxic microenvironment ( ) and hif- α and - α have been shown, ex vivo, to be expressed in lung biopsies from patients with ipf, in some but not all reports ( , ) . additional genomic studies in ipf patients show upregulation of hypoxia-related gene signatures, including tgfβ ( ), the key fibrotic cytokine in pf, and of the hif- α pathways ( , ) . the contribution of neutrophils to ild has also been relatively less studied compared to other inflammatory and fibrotic diseases. early studies began to explore the potential role of neutrophils in ipf ( ) ( ) ( ) ( ) , however research focus has since shifted to other cell types. the number of neutrophils in the bronchoalveolar lavage (bal) fluid has been shown to predict both disease severity in ipf ( ) and the development of pf in patients with hypersensitivity pneumonitis ( ) . in addition, neutrophil extracellular traps (nets) have been shown to indirectly drive pf by stimulation of collagen production from fibroblasts in vitro ( ) , and net release has been associated with pf in older mice in vivo ( ) with loss of peptidyl arginine deiminase (pad)- , a key neutrophil enzyme for net formation, being protective ( ) . neutrophils are also associated with disease severity in acute lung injury and acute respiratory distress syndrome (ards) ( , ) however, their precise contribution remains uncertain ( ) . neutrophil depletion can ameliorate disease features in mouse models of ards ( ) and a reduction in neutrophil infiltration ( ) , or knock-down of neutrophil elastase (ne) attenuates fibrosis in bleomycin-induced mouse models of pf ( ) . taken together, these studies implicate a contributory role of neutrophils to fibrotic ild. neutrophil survival is a tightly regulated process. prolonged survival can delay resolution of inflammation and can cause damage to surrounding cells and tissues; however, if apoptosis is premature, antimicrobial function can be compromised ( ) . hypoxia drives neutrophil survival via hif- α-dependent nf-κb activation ( ) . in addition, hif- α has also been shown to be important in regulating neutrophil function ( ) . few reports address the effects of hypoxia upon net formation. inhibition of hif- α can reduce net release ( ) , whilst pharmacological stabilization of hif- α increases phagocyte bactericidal activity ( ) and net release ( ) , implicating a role for down-stream targets of hif- α in leukocyte function. given the importance of hypoxia and hif signaling in neutrophil function and the emerging role of neutrophils as key drivers of ild, we sought evidence for hypoxia and nets in the lungs of patients with ild and the functional effects of low oxygen levels upon ex vivo neutrophil function and activation. fiber-optic bronchoscopy with bal was performed in line with the american thoracic society guidelines ( ) . bal was frozen for later analysis. none of the patients undergoing bronchoscopy had any infections at the time of procedure. bal was obtained from patients with fibrotic ild and seven non-ild controls undergoing diagnostic bronchoscopies. demographics, clinical history and treatments at the time of sample collection are listed in table . within the ild cohort: ( %) had ipf, ( %) had nonspecific interstitial pneumonia, ( %) had chronic hypersensitivity pneumonitis (hp) and ( %) had unclassifiable ild. our non-ild control group underwent diagnostic bronchoscopy due to: ( %) investigation of haemoptysis, ( . %) right middle lobe collapse and ( . %) previous tracheal schwannoma patients undergoing yearly bronchial surveillance. only the ild group had lung function tests, as part of standard patient care. none of the patients recruited were taking anti-fibrotic drugs at the time of bronchoscopy. differential cell counts obtained from bal from patients are listed in supplementary table . lung biopsies were collected as part of routine clinical care. ethical approval was given by the uk national research ethics committee ( /lo/ ). ihc was performed using the automated bond-max system (leica biosystems ltd., newcastle) with µm ffpe sections. hif- α (clone ep y, abcam, : dilution), myeloperoxidase (mpo) (polyclonal, dako, : dilution) or ne (clone np , dako, : dilution) was incubated in epitope retrieval solution for min and stained using the , , protocol. test antibodies were controlled clinical details were recorded for all subjects at the time of bronchoalveolar lavage. our ild cohort had a mean age of ± . years and consisted of four patients with ipf, three patients with fibrotic nsip, three patients with chronic hp and one patient with unclassifiable ild. our non-ild control group had a mean age of ± . years and consisted of five patients with haemoptysis, one patient with rml collapse and one patient with tracheal schwannoma. lung function was only obtained for the ild cohort as part of standard care. fvc, forced vital capacity; hp, hypersensitivity pneumonitis; ild, interstitial lung disease; ipf, idiopathic pulmonary fibrosis; nsip, nonspecific interstitial pneumonia; rml, right middle lobe; tlco, transfer factor for carbon monoxide. for using species-and isotype-matched control antibodies. slides were scanned on a nanozoomer digital slide scanner and images analyzed using ndp viewer software (hamamatsu corportation). a "blinded" reviewer analyzed five randomly selected areas from each subject. representative images were chosen from those selected. neutrophils were isolated as previously described ( ) . in brief, neutrophils were isolated by percoll plus density centrifugation from sodium citrate anticoagulated blood obtained by informed consent from healthy volunteers. neutrophils were diluted to × neutrophils/ml in phenol-free rpmi (thermo scientific, uk) supplemented with % heat-inactivated fbs (thermo scientific, uk) and mm l-gluatamine (lonza, uk). to induce hypoxia, neutrophils were cultured under % oxygen in a coy oxygen control glove box (coy laboratory products inc., usa) in a temperature controlled and humidified incubator. human umbilical cord vein endothelial cells (huvec) (lonza, switzerland) were cultured in endothelial growth media (lonza, switzerland) supplemented with % fbs (thermo scientific, uk) and mm l-glutamine (lonza, switzerland) and used at passage . for endothelial activation, huvec were treated with ng/ml tnf-α (r&d systems, uk) for h prior to experimentation. to induce hypoxia, huvec were cultured under % oxygen in a coy oxygen control glove box (coy laboratory products inc., usa) in a temperature controlled and humidified incubator. nets were quantified using the quanti-it tm picogreen r dsdna kit (invitrogen, uk) and using a capture elisa. streptavidin-coated plates (fisher scientific, uk) were coated with an anti-mpo capture antibody (abcam, uk) overnight at • c and blocked with . % bovine serum albumin for h at • c. neutrophil supernatants were incubated for h at • c. further h incubations were performed with an anticitrullinated histone h detection antibody (abcam, uk) and hrp-conjugated secondary antibody (dako, uk). sureblue tmb microwell peroxidase substrate (kpl, uk) was then added and incubated in the dark at • c for min and then stopped by the addition of tmb stop solution (kpl, uk). absorbance was read at nm using a tecan genios spectra fluor plate reader (tecan uk ltd., uk). nets were stained for immunofluorescence microscopy as described ( ) using methodology modified from ( ) . in brief, × neutrophils were added to fibrinogen-coated coverslips, stimulated for h with nm pma, . mm mncl or varying concentrations of leukadherin- (la- ; sigma, uk), and fixed with % pfa. coverslips were blocked and sequentially incubated with an anti-histone h antibody (abcam, uk) and alexa fluor r -conjugated goat anti-rabbit igg secondary antibody (life technologies, uk). coverslips were washed, mounted, and sealed using with prolong tm gold antifade mountant with dapi (invitrogen, uk). slides were visualized using a zeiss axio imager.a inverted fluorescence microscope (zeiss, germany) and images analyzed using image j. lung sections were stained using a modified protocol based on published reports ( , ) . five micrometer sections from paraffin-embedded lung biopsies from control or ipf patients were dewaxed prior to heat-induced epitope retrieval with tris-edta buffer, ph . . sections were blocked with fc block (bd biosciences, uk) before incubation with a blocking buffer ( % goat serum/ . % bsa/pbs/ . % tween- ) for h. slides were then washed and incubated with anti-citrullinated histone h (abcam, uk) and anti-mpo (r&d systems, uk) antibodies diluted in . x blocking buffer overnight at • c. anti-rabbit alexa fluor r -conjugated and anti-mouse alexa fluor r conjugated secondary antibodies (invitrogen, uk) and dapi (sigma, uk) diluted in . x blocking buffer were then added for min. stained sections were washed, mounted, sealed and visualized using an olympus inverted fluorescence confocal microscope and analyzed using fluoviewer software (olympus). bal fluid was filtered using a µm cell sieve. bal cells were pelleted, counted and × viable cells were used to produce cytospin slides (thermo shandon cytospin , thermo scientific). cytospin slides were fixed in % pfa, washed, and blocked overnight in blocking solution ( % goat serum/ % bsa/ mm edta/hbss/ . % tween- ). slides were then washed and incubated with anti-histone h a.x antibody (abcam, uk) for h before washing. anti-rabbit alexa fluor r -conjugated secondary antibody (invitrogen, uk) and dapi (sigma, uk) were then diluted in blocking buffer for h. stained slides were then washed, mounted, sealed and visualized using an olympus inverted fluorescence confocal microscope and analyzed using fluoviewer software (olympus). cell surface expression of neutrophil integrins was evaluated by flow cytometry. following isolation and culture under either normoxia or hypoxia, neutrophils were washed and resuspended in a sodium hepes buffer ( mm hepes, mm nacl, mg/ml glucose, . % bsa). cells were then stained using integrin subunit specific antibodies or appropriate isotype control for min at room temperature. stained cells were then washed twice, fixed in % pfa and assessed using a facs verse (bd biosciences, uk). data was analyzed using flowjo (treestar inc., uk). huvec were cultured in -well black tissue culture plates (thermo scientific, uk). twenty-four hours prior to experimentation, huvec were subjected to normoxia or hypoxia in the absence or presence of ng/ml tnf-α. neutrophil adhesion in response to nm pma or ng/ml lipopolysaccharide (lps) were measured as previously described ( ) . briefly, neutrophils were cultured under normoxia or hypoxia for h, labeled with ′ , ′ -bis-( -carboxyethyl)- -(and- )-carboxyfluoresceinacetoxymethyl ester (life technologies, uk) and then added to wells under normoxia or hypoxia. fluorescence was measured using a tecan genios spectra fluor plate reader (tecan uk ltd., uk). adhesion was calculated by comparing the fluorescence of washed wells to initial fluorescence. trans-endothelial migration assays were performed as previously described ( ) . in brief, huvec were grown on transwell inserts (millipore, uk). twenty-four hours prior to experimentation, huvec were cultured under normoxia or hypoxia in the absence or presence of ng/ml tnf-α. neutrophils were cultured under normoxia or hypoxia for h and then labeled with celltracker (invitrogen, uk). × neutrophils were added to the upper chamber of transwells and allowed to migrate in the absence or presence of ng/ml il- in the lower chamber for min. percent transmigration was calculated by comparing the number of cells in the lower chamber and the number of neutrophils added to the upper chamber. cell lysates ( µg protein) were resolved by electrophoresis and transferred to a polyvinylidene fluoride membrane (ge healthcare, uk). membranes were blocked for h in % skimmed milk/tbs/ . % tween- and incubated with primary antibodies ( : , dilution) overnight at • c. membranes were then washed, incubated with hrp-conjugated secondary antibodies, and visualized using the luminata western hrp substrate system (millipore, ireland). data were evaluated using graphpad prism. data were tested for normality using a kolmogorov-smirnov test. in experimental data sets only comparing two groups, a mann-whitney test was performed or a wilcoxon matched pairs test. in data sets with two variables, data were assessed by two-way anova with a dunnet's or sidak's multiple comparison test. correlations were determined by two-tailed pearson correlation coefficients. a p value below . was considered significant. given reports of localized hypoxia in pulmonary disease ( ) , biopsies from four patients with fibrotic ild, performed to determine a clinical diagnosis of etiology, were examined for evidence of hypoxia. in this representative patient, diagnosed with ipf, hif- α staining demonstrated positive staining in the endothelium and polymorphonuclear cells, with very little staining in the fibrotic interstitium and overlying epithelium and no staining in control sections (figures a,b) . as aberrant net formation has been implicated in several immunopathologies, we also stained lung sections for mpo and ne (figures c,d) , highlighting the presence of neutrophils within the pulmonary vasculature. taken together, this staining pattern suggests that tissue-specific hypoxia and neutrophil recruitment may be a feature of the ild lung. these findings led us to examine the effects of hypoxia upon neutrophil function. pharmacological hif- α stabilization has been reported to enhance bacterial killing and net release ( ) ( ) ( ) , however, these studies were performed using atmospheric oxygen levels. we therefore assessed for any alteration in function, described below, of healthy neutrophils under normoxia ( % oxygen) and hypoxia ( % oxygen). first, we verified hypoxia by examining neutrophil cell lysates for the presence of hif- α and hif- α. we observed rapid stabilization of hif- α under hypoxia, with delayed hif- α stabilization (figure a) . having demonstrated induction of hypoxia, we assessed neutrophil supernatants for mpo-citrullinated histone h complexes, which are specific for nets. hypoxic neutrophils displayed greater levels of both spontaneous (+ . -fold, p < . ) and pma-induced (+ . -fold, p < . ) net release ( figure b) . as reactive oxygen species generation is thought to drive net formation ( , ) , we also examined hydrogen peroxidase (h o ) production. rates of h o generation however, were comparable between oxygen states for both unstimulated ( . having found an effect on net release, we next examined integrin activation and neutrophil adhesion, which are also implicated in net induction ( ) ( ) ( ) . we measured neutrophil adhesion to primary human endothelial cells in the absence or presence of pma (a general integrin activator) or lps (to mimic infectious stimuli), stimuli that induce nets via distinct pathways ( ) . hypoxia increased both unstimulated ( . ± . % vs. . ± . %, p < . ) and lps-stimulated ( . ± . % vs. . ± . %, p < . ) adhesion to resting endothelium, whilst pma-stimulated adhesion, which was already high, was unaffected (figures a-c) . we then looked at adhesion to endothelium pretreated with tnf-α, to mimic an inflammatory event. whilst unstimulated neutrophil adhesion to tnf-α activated endothelial cells was not altered by hypoxia, there was a . -and . -fold increase in pma-( . ± . % vs. . ± . %. p < . ) and lps-stimulated ( . ± . % vs. . ± . %, p < . ) adhesion, respectively (figures d-f) . next, we evaluated neutrophil trans-endothelial migration in the absence and presence of il- , which has been shown to induce neutrophil migration. basal transmigration (in the absence of il- ) across both resting and tnf-α activated endothelium was unaffected by hypoxia. in contrast, in the presence of il- , hypoxia enhanced neutrophil trans-endothelial migration across both resting and tnf-α activated endothelium (p < . ) (figures g,h) . hypoxia increases expression of neutrophil β integrins, but not β integrins given the role of integrins in leukocyte extravasation and reports documenting reduced nets following integrin blockade ( ), we assessed surface integrin expression. whilst α l expression was unaffected (figure a) , significantly higher levels of α m (+ . -fold, p < . ) and α x (+ . -fold, p < . ) were observed under hypoxia (figures b,c) . there were no significant differences in β expression (figure d) . hypoxia did not have an effect upon α , α , α , or β integrin subunit expression (figures e-h) . given reports of reduced net formation following integrin inhibition and our data showing increased α m β and, to a lesser extent, α x β integrin expression, we tested whether integrin engagement induced the release of nets, using an established model in which neutrophils adhere to fibrinogen. although there is some base-line adhesion ( . %), stimulation with pma increases binding to . % that can be blocked with specific α m β inhibition (supplementary figure ) . as expected, no nets were observed in unstimulated neutrophils, whilst pma stimulation induced the externalization of histone h to form net-like structures (figures a,b) . cation chelation through the use of edta, which abolishes integrinmediated adhesion, suppressed pma-induced net formation ( figure c) . finally, global integrin activation by means of manganese chloride treatment, induced some histone h externalization that could also be suppressed with edta treatment (figures d,e) . to confirm the role of α m β , neutrophils were stimulated with la- , a compound that specifically activates the α m β integrin ( ). la- stimulation showed a dose dependent effect upon neutrophil adhesion figure | hypoxia enhances neutrophil adhesion and trans-endothelial migration. bcecf-am labeled neutrophil adhesion to endothelial monolayers over min was assessed under normoxia ( % oxygen) and hypoxia ( % oxygen). we first examined neutrophil adhesion to resting huvec using (a) unstimulated neutrophils, (b) neutrophils stimulated with nm pma and (c) cells stimulated with ng/ml lps. next, we examined the effects of hypoxia upon neutrophil adhesion to activated huvec, which had been stimulated with ng/ml tnf-α for h prior to experimentation. we evaluated (d) unstimulated neutrophil adhesion, (e) neutrophil adhesion in response to nm pma and (f) neutrophil adhesion in response to ng/ml lps. finally, the effects of hypoxia upon trans-endothelial migration of celltracker tm green labeled neutrophils over min was evaluated. data are presented as the mean ± sem from four independent experiments and analyzed by a wilcoxon matched pairs test. (g) neutrophil transmigration across resting endothelial monolayers was measured under both normoxia and hypoxia in the absence or presence of ng/ml il- . (h) huvec were stimulated with ng/ml tnf-α for h. neutrophil trans-endothelial migration was subsequently measured in the absence or presence of ng/ml il- under normoxic or hypoxic conditions. data are presented as the mean ± sem from three independent experiments and analyzed by two-way anova with a dunnett's multiple comparison test. * = p < . . hif, hypoxia-inducible factor; pma, phorbol -myristate -acetate. ( figure a) . whilst low concentrations of la- failed to induce the formation of net-like structures (figures b,c) , high levels of la- produced dna-histone structures similar to pma-stimulated cells (figures d-f) . these results confirmed that α m β integrin activation can induce nets in human neutrophils. having found evidence of hypoxia within the ild lung and shown that hypoxia augments neutrophil activation, we evaluated lung tissue sections for evidence of nets. in non-ild control lung sections, we observed normal lung architecture with the absence of cellular infiltrates and lung tissue remodeling at both low and high magnification (figures a,b) . in contrast, we noted cellular infiltration in ipf lungs accompanied with the presence of mpo and histone citrullination (figures c,d) . co-localization of extracellular dna, mpo and citrullinated histones is suggestive of net formation within the ild lung. finally, we examined bal for evidence of neutrophil activation. we generated slides with bal and stained with dapi and citrullinated histone h a to identify the presence of neutrophils initiating the production of nets. control bal neutrophils displayed punctate dapi staining with the absence of citrullinated histones (figures a,b) . in contrast, we observed the presence of citrullinated histones and more diffuse dna staining in bal polymorphonuclear cells obtained from patients with ild, indicative of neutrophils forming net-like structures (figures c,d) . we then obtained bal from ild patients (ild-bal) and seven non-ild controls (control bal) and quantified levels of cell-free dna. we found ild-bal had . -fold greater cell-free dna content compared to control bal (p < . ) (figure e) . cell-free dna content positively correlated with neutrophil counts (% of total cells) isolated from ild-bal (p = . ) (figure f) , but not in control bal (figure g) . to verify that these were nets, we also examined bal for the presence of mpocitrullinated histone h complexes. similar to total cell-free dna, we observed significantly greater values in ild-bal (+ . -fold, p < . ), indicating greater levels of nets ( figure h ). neutrophil dysfunction and aberrant activation have been implicated in the pathology of numerous diseases including autoimmune rheumatic diseases ( ) ( ) ( ) ( ) ( ) and cancer ( ) ( ) ( ) ( ) . more recently the release of nets, essential for robust immune defense against pathogens, has also been linked to increased immunopathology in patients with covid- , a disease characterized by neutrophilic inflammation and endothelial activation ( , ) . the precise mechanism in which neutrophils contribute to ild pathogenesis is unknown. early work from the s began to explore whether neutrophils might contribute to ipf pathology ( ) ( ) ( ) ( ) , however this avenue of research lost momentum. since then, reports have associated increased neutrophil migration and activation with severe pulmonary disease both in animal models ( , ) and man ( , , ) . we report, for the first time, that neutrophils and endothelial cells in ild lung biopsies display hif- α expression and provide evidence of the extracellular release of nuclear dna, citrullinated histones and mpo, indicative of net formation in the ild lung. given the profound effects hypoxia exerts upon neutrophil survival and function ( , ) , these findings led us to investigate whether hypoxia affects neutrophil extravasation and activation, thus contributing to ild pathology. integrins are adhesive molecules that enable leukocytes to interact with their external environment. similar to a previous report ( ), we found increased β integrin expression in neutrophils under hypoxia, but specifically found increased α m and, to a lesser extent, α x integrin subunit expression. interestingly, the α m β and α x β integrins also function as complement receptors, which may be relevant to ild pathology given that increased levels of complement c a and c a and roles for their receptors have been reported in ipf ( , ) . moreover, studies using the bleomycin-induced mouse model of ipf highlight roles for both c and c in pulmonary fibrosis ( , ) . upregulation of β integrins has been described under hypoxia ( ) , however, there are no reports assessing expression in neutrophils. a lack of effect may be explained by the relatively low β integrin expression in human neutrophils. taken together, the evidence indicates that neutrophils predominately engage via β integrins, a mechanism which is enhanced under hypoxia. early studies demonstrated that hypoxia enhances neutrophil adhesion to endothelial cells ( ) , epithelial cells ( ) , and trans-epithelial migration ( ) . in support of these findings, our results show altered function of healthy neutrophils with increased neutrophil adhesion and trans-endothelial migration under hypoxia. in addition, we report that hypoxia enhances net formation. given that the gold standard markers or methods for the induction and detection of nets have not been established ( ), we used several different techniques to confirm the release of nets by cultured neutrophils: the co-localization of nuclear dna and histone h complexes by immunostaining; figure | bal isolated from ild patients contain more nets and net-releasing neutrophils. bal from ild patients and non-ild controls was examined for evidence of neutrophil activation. we first performed confocal microscopy to identify nets as defined by nuclear dna (dapi, blue staining) and citrullinated histone expression (citrullinated histone h a, green staining). (a,b) non-ild control bal cells displayed punctate nuclear staining and lack of citrullinated histones, whilst (c,d) bal cells obtained from patients with ild showed degrees of dna externalization from polymorphonuclear cells, along with the presence of citrullinated histones indicative of neutrophils undergoing net release (white arrowheads). representative images from two non-ild or ild donors are shown and the size is denoted by the scale bar. (e) bal fluid was then assessed for the presence of nets by quant-it tm picogreen ® dsdna assay (thermo fisher, uk), which found that bal fluid from ild patients (n = ) has significantly more cell free dna compared to non-ild controls (n = ). from differential cell counts, we found that (f) cell free dna positively correlated with the proportion of bal neutrophils in patients with ild, (g) but not in non-ild controls. (h) finally, we also tested bal fluid using an optimized capture elisa detecting mpo-citrullianted histone h complexes, demonstrating that bal fluid from ild patients (n = ) has significantly more nets compared to non-ild controls (n = ). data were analyzed by either a mann-whitney test or two-tailed pearson correlation coefficients. ** = p < . . bal, bronchoalveolar lavage; ild, interstitial lung disease. confocal imaging of dna, citrullinated histones and mpo; quantification of cell-free dna; and the detection of neutrophilderived proteins (mpo) and citrullinated histone h complexes. our observations complement studies in the literature showing that pharmacological hif- α stabilization enhances net release and inhibition of hif- α reduces nets and bactericidal activity ( , ) . whilst hif- α stabilization has been shown to promote net production ( ) , the opposing effect of hypoxia upon net formation has also been described. in contrast to our findings, branitzki-heinemann et al. found hypoxia reduced levels of both spontaneous and pma-induced net release ( ) . whilst the definitions of hypoxia and normoxia were identical ( % oxygen vs. % oxygen), key methodological differences may explain the contrasting conclusions. branitzki-heinemann et al. isolated neutrophils using gradient centrifugation with polymorphprep, whilst this study used percoll plus. whilst a minor difference, comparative analysis of isolation procedures found reduced cd and cd b expression in neutrophils isolated with polymorphprep ( ), which may have further implications on ex vivo function. both reports used pma to initiate net formation, however at different concentrations. in this study, neutrophils were stimulated with nm pma for h, whilst the earlier branitzki-heinemann study treated cells with nm pma for h. it is possible that stimulating neutrophils with a higher concentration of a potent pkc activator may account for the differing response to hypoxia. finally, when quantifying nets, branitzki-heinemann et al. seeded neutrophils on coverslips coated with poly-l-lysine whilst neutrophils in this study were exposed to either nunclontreated or fibrinogen-coated surfaces. this difference may result in varying degrees of α m β engagement and alter neutrophil responses. our data suggest that α m β engagement may induce net formation, which is supported by recent work demonstrating α m β triggering net release ( ) . moreover, several studies have shown that α m β blockade reduced net release ( , , , ) , which indirectly supports our work. our findings that α m β interaction with ligand may regulate net formation builds on earlier findings, which showed that pma stimulation led to high levels (> %) of chromatin decondensation (a prelude to net formation) and was not affected by substrate ( ) . in contrast, lps stimulation led to lower basal levels of decondensation (∼ %) and levels increased with matrix stiffening and increased cell spreading on β and β integrin ligands. this effect was inhibited by pi k inhibition suggesting a dependence on integrin outside-in signaling. the impact of matrix stiffness is highly relevant in fibrotic ild as lung fibrosis changes tissue elasticity. there are some methodological differences between this work and that of erpenbeck and colleagues. in particular, our neutrophils were rested in hypoxia or normoxia overnight before pma stimulation. our background net levels were lower in response to pma rather than the dramatic increase from < to > % previously reported. it is possible that when the level of nets is lower, integrin activation related to matrix stiffness plays more of a role regardless of the stimulus. this finding further emphasizes the importance of a complete description of the experimental system ( ) . this finding may have particular relevance not only to ild but also to other fibrotic lung diseases. the finding that hypoxia drive net formation may also be of relevance to non-fibrotic pathologies including covid- , a disease characterized by severe hypoxia, net release and hyperinflammation ( , ) . further work could build on the level of hypoxia required to produce these effects. in our study we used % oxygen, however, normal oxygen levels can range from . to . % in the circulation and . to . % in tissues ( , ) . further experiments could determine whether lesser degrees of hypoxia, seen in clinical practice, also enhance neutrophil adhesion, trans-endothelial migration and net formation. these studies could determine the relative importance of hif transcription factors to net formation and release, through the use of previously identified small molecule inhibitors ( ) ( ) ( ) , and better understand the relationship between neutrophil activation and hypoxia. we observed tissue-specific hif- α expression in ild lung tissue, mainly restricted to pulmonary endothelial cells and neutrophils, with only minimal upregulation in areas of epithelium and fibrosis, which may hold pathological relevance. previously, markers of hypoxia have been variably reported in the epithelium of patients with ipf. several authors have found hif- α and ca-ix within the ipf fibrotic reticulum and hif- α in the overlying epithelium with ihc ( ), [albeit sometimes in a single patient ( ) ]. hif- α is more readily found in the mouse bleomycin model of pf raising the question of differences between the two species and insults ( ) . whilst epithelium-specific hif- α deletion has no effect upon radiation-induced enteritis, mice with endothelium-specific hif- α deletions present with reduced intestinal damage ( ) . hif- α is known to contribute to the pathology of pulmonary hypertension ( , ) , with some work specifically interrogating endothelial hif signaling ( ) . neutrophilic inflammation has also been associated with pulmonary hypertension ( ) , and believed to drive angiogenesis via net release ( ) . therefore, the pulmonary pathology in ild patients may in part be attributed to endothelial and neutrophil hif- α expression enhancing neutrophil recruitment and net formation within the lung. in this paradigm, enhanced nets would initiate angiogenic signals and drive lung pathology. interestingly, the model of neo-angiogenesis underlying ild pathology has attracted interest, and the powerful angiogenic inhibitor, nintedanib, shown to have therapeutic benefits in a range of fibrotic ilds ( ) and endothelial reactivity with angiogenesis is also noted in covid- . our findings of hypoxia driving net formation complements the increasing evidence that nets may play a role in many acute and chronic lung diseases ( ) , including ild by stimulation of fibroblasts ( ) . in pf, we propose that elevated net release may cause epithelial cell damage, dysfunction and death, drive innate and adaptive immune cells activation, and promote a pro-fibrotic environment that ultimately facilitates the progression of pulmonary fibrosis. for our experiments, we used neutrophils isolated from peripheral blood donated from healthy volunteers. further work could examine cells isolated from patients with ild, isolated from either peripheral blood or bal, to determine whether hypoxia has similar or an enhanced effect within this patient population. in addition, it would also be interesting to examine neutrophil function using autologous human serum from patients with ild to provide further insight under more physiological conditions in vitro. hypoxia is also thought to have differential integrinindependent effects upon net formation ( ) . further experiments exploring the effects of hypoxia upon neutrophil activation by examining neutrophil responses to a wider range of stimuli, such as bacterial/fungal antigens, ionomycin or monosodium urate crystals, which also activate β integrins ( ), may therefore provide further insight into the relationship between neutrophil function, integrin activation and hypoxia. interestingly, the recent consensus article written by opinion leaders to present prevailing concepts and state of the science in net-related research and elaborate on open questions and areas of dispute does not specify which stimulus should be used to induce net formation ( ) . instead, this consensus article suggests that researchers should specify in detail culture conditions, including base medium, use of serum, absence of platelets and surface constitution of the cell culture plate, as well as stimulus and source/preparation of inducer used. our analysis of net formation ultimately focuses on the end stages culminating in net release into cell supernatants. further work could explore the effects of hypoxia upon the preceding stages such as cell spreading and chromatin decondensation/nuclear swelling ( , ) , to better understand how hypoxia affects the entire net formation process. in conclusion, we report that the ild lung contains molecular features of hypoxia, mainly localized to neutrophil and endothelial cells, which may contribute to disease pathology. hypoxia enhanced neutrophil β integrin expression, which translated to augmented adhesion and migration across endothelial cells, and net release. our findings are further supported by demonstration of nets within the human fibrotic lung seen through imaging of ipf lung sections and bal cells, as well as detecting cell-free dna and mpo/citrullinated histone complexes in bal obtained from patients with ild. taken together, our work begins to elucidate a potential role of hypoxia in driving neutrophil recruitment and activation within the airspace to promote a pro-fibrotic environment. these findings offer a rationale for future translational medicine exploration of a novel neutrophil hif- α-integrin axis as a potential therapeutic target in fibrotic ild. all datasets generated for this study are included in the article/supplementary material. the studies involving human participants were reviewed and approved by uk & national research & ethics committee. the patients/participants provided their written informed consent to participate in this study. ak designed and performed experiments, analyzed the data, and contributed to writing the manuscript. dc, js, and hb obtained and analyzed bal samples and performed microscopy. tm performed lung cyrobiopsies that were used for lung staining. sk and mr-j performed and analyzed ihc images. cp and vr contributed to experimental design and analysis. ig and jp conceived the study, designed the experiments, were involved in data analysis, and contributed to the writing of the manuscript. all authors contributed to the article and approved the submitted version. this work was supported by a ucl impact studentship, the rosetrees trust (m ) and breathing matters, and undertaken at uclh/ucl who received a proportion of funding from the department of health's nihr biomedical research centres funding scheme. jp was funded by an mrc new investigator research grant (mr/k / ). open access publication fees were 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potential of nintedanib in treatment of progressive fibrosing interstitial lung diseases the emerging role of neutrophil extracellular traps in respiratory disease crystal-induced neutrophil activation vi. involvment of fcgammariiib (cd ) and cd b in response to inflammatory microcrystals chromatin swelling drives neutrophil extracellular trap release identification of a novel hif- α-αmβ integrin-netosis axis in fibrotic interstitial lung disease. biorxiv conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material key: cord- -qu o iu authors: vlasova, anastasia n.; butler, john e. title: editorial: porcine anti-viral immunity date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: qu o iu nan the swine industry is an important part of agriculture in many countries, generating over $ billion annually in the us alone. viral diseases in pigs pose a serious risk for swine health and significantly impact the economy of the global swine industry. pigs also serve as zoonotic reservoirs for viruses transmittable to humans and other species, including influenza a virus, nipah virus, and hepatitis e virus ( ) . these problems are made worse by globalization and industrial livestock rearing ( , ) . unlike bacterial pathogens, antibiotics do not protect against viral infections and cannot be used to control viral outbreaks or epidemics. since viruses can alter their genome > times faster than their mammalian hosts ( ), the latter would have succumbed to microbial infection were it not for their adaptive immune system that uses somatic gene rearrangement and mutation to counter the rapid diversification of microbes. immediately following viral infection, neonatal survival depends on innate immunity and passive protection by lactogenic immune factors such as pathogen-specific antibodies, until an adaptive immune response can develop. thus, intervention against these moving targets, depends on availability of effective maternal and neonatal vaccines and strict biosecurity measures. wide-spread porcine reproductive and respiratory syndrome virus (prrsv) and swine influenza virus (siv) represent major health challenges in the large us swine production systems and possibly worldwide. in the us alone, economic annual losses due to prrsv are estimated to be million u.s. dollar (usd) ( ) . while the exact costs associated with siv and other endemic viruses, including rotavirus, are hard to evaluate, they are economically important due to their ubiquitous nature, which can reduce growth and increased morbidity ( ) . emerging and re-emerging coronaviruses in pigs are quite often associated with diarrheal disease and high morbidity and mortality ( ) . the emergence of seneca valley virus (svv) and increased incidence of svv-associated vesicular disease alarmed the swine industry in several countries, including the us, brazil, china, thailand, and colombia ( ) . finally, a deadly swine disease, caused by african swine fever virus (asfv), that has been plaguing africa for decades, has now spread to south eastern europe and asia, and has severely impacted the world's largest swine industries in china ( ) . in this special volume of frontiers in immunology, comparative immunology section, lager and buckley provide an overview of the importance of swine in the world food supply and a review of the major viral infection that threaten this species (lager and buckley). in introducing the topic of anti-viral immunity, we emphasize the genetic diversity of viruses, the virus life cycle and the pathology that viral infection can cause. a total of articles are contained in this volume to which researches have contributed. despite the advantage of internal offspring development and the provision for passive immune protection, fetuses and neonates of higher vertebrates still remain vulnerable to environmental pathogens. piglets in utero are protected from eukaryotic parasites and bacteria, but are vulnerable to viruses (including parvovirus, prrsv, and porcine circovirus ) since they are able to cross the placental barrier ( ) ( ) ( ) . these cause pathological injury and often fetal abortion. in a number of mammals, viruses infect the thymus and can also cause hypergammaglobulinemia. butler et al. discuss the implication of thymic atrophy and apoptosis of thymocytes and hypergammaglobulinemia with regard to the persistence of prrsv. however, apoptosis is also a host mechanisms used to eliminating virus-infected cells ( ) , but can also promote spread of the virus ( ) . fernandes et al. demonstrated that svv developed a c protease-dependent mechanism for late apoptosis that facilitates virus release from infected cells. thus, the role and importance of virus-induced apoptosis awaits further research. since the adaptive immune system of fetal piglets is underdeveloped, the innate immune system is very important. schäfer et al., emphasize its importance with the specific focus on porcine invariant natural killer t cells (inkt) and their role in the pathogenesis of asf and si. they discuss inkt agedependence, levels and distribution in relationship to various porcine viral infections. one of the first host responses to viral infection is the production of interferons, which are needed to drive other elements of innate immunity and adaptive immunity. likai et al. show how a porcine deltacoronavirus escapes from the immune system by suppressing ifn-α production, while shi et al. describe a novel immune evasive mechanism that depends on prrsv non-structural protein a which antagonizes tbk -irf -ifn signaling. while the initial antibody response depends on broadly specific natural igm antibodies, effective anti-viral immunity depends on an adaptive immune response that delivers igg and iga antibodies. since adaptive immune responses depend on stimulation through the innate immune system, viruses that impair or interfere with the innate immune response, also impair adaptive immunity. nedumpun et al. discuss how prrsv-induced il- ra downregulates innate immune responses, t lymphocyte differentiation and proliferation. piglets, unlike humans, but like the offspring of other artiodactyls and perissodactyls (horses), are especially dependent on lactogenic immunity since there is no transplacental transfer of passive antibodies in this group of mammals. thus, providing ways to deliver anti-viral antibodies via colostrum/ milk is important. this topic is the focus of studies by langel et al.. antibodies can intercept and neutralize a virus before it infects additional cells and can prevent further infection by blocking the viral receptor; these are called virus neutralizing (vn) antibodies. naturally, a critical step in viral vaccine design is the identification of viral epitopes targeted by effective vn antibodies. this become increasingly important in the case of prrsv in which effective vn antibodies are slow to appear and current vaccines are of questionable efficacy. this is also an ongoing challenge in the case of asfv which is rapidly spreading and for which no vaccine exists. goldeck et al. describe a novel b cell cloning procedure to identify these epitopes on several strains of prrsv. while vn antibodies can block or delay infection, the ultimate elimination of a virus is to kill the cells in which the virus replicates which is the job of cytotoxic t cells. portions of the virus are displayed on infected cells. these are called a t cell epitope, i.e., a molecular structure that binds to the t cell receptor (tcr). in this volume, pan et al. describe their efforts to identify such epitopes for their potential use in stimulating expansion of the cytotoxic t cells that recognize them. viral vaccines take various forms, the simplest being the use of killed virus. a more tedious procedure is to use only parts of the virus as the vaccine (subunit vaccines) that target the immune response to those viral epitopes that elicit vn antibodies. killed viruses and subunit vaccines typically generate immediate responses that wane faster, making it imperative for long-lived mammals like humans, to receive booster vaccinations. a second approach to vaccine development is use of live attenuated virus that has been genetically modified or cell culture adapted and cannot produce a disease in the host but can still replicate. these are often more likely to stimulate virus-specific cytotoxic t cells and to induce longer term immunity. sharma et al. developed a recombinant svv strain using reverse genetics and tested its immunogenicity and protective efficacy in pigs. toman et al. provide comparative data on four vaccines for prrsv, two killed and two modified live. we believe that veterinary immunovirology should place more emphasis on how each viral pathogen effects and/or avoids the host immune system and on the identification of viral epitopes that are effective targets of vn antibodies and t cell epitopes that can promote the action of cytotoxic t cells. jb wrote the first draft and then both co-authors contributed equally to the final draft. reservoir host immune responses to emerging zoonotic viruses current drivers and future directions of global livestock disease dynamics the future of pork production in the world: towards sustainable, welfare-positive systems what contemporary viruses tell us about evolution: a personal view assessment of the economic impact of porcine reproductive and respiratory syndrome virus on united states pork producers impact of health challenges on pig growth performance, carcass characteristics, and net returns under commercial conditions emerging and reemerging coronaviruses in pigs emergence of a novel recombinant seneca valley virus in central china african swine fever virus in asia: its rapid spread and potential threat to unaffected countries apoptosis and necroptosis as host defense strategies to prevent viral infection viruses and apoptosis fetal infections and antibody profiles in pigs naturally infected with porcine circovirus type (pcv ) effect of wean-tofinish management on pig performance transplacental infection and embryonic death following maternal exposure to porcine parvovirus near the time of conception the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © vlasova and butler. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -wbwlfx q authors: gómez-rial, jose; currás-tuala, maria josé; rivero-calle, irene; gómez-carballa, alberto; cebey-lópez, miriam; rodríguez-tenreiro, carmen; dacosta-urbieta, ana; rivero-velasco, carmen; rodríguez-núñez, nuria; trastoy-pena, rocio; rodríguez-garcía, javier; salas, antonio; martinón-torres, federico title: increased serum levels of scd and scd indicate a preponderant role for monocytes in covid- immunopathology date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: wbwlfx q background: emerging evidence indicates a potential role for monocytes in covid- immunopathology. we investigated two soluble markers of monocyte activation, scd and scd , in covid- patients, with the aim of characterizing their potential role in monocyte-macrophage disease immunopathology. to the best of our knowledge, this is the first study of its kind. methods: fifty-nine sars-cov- positive hospitalized patients, classified according to icu or non-icu admission requirement, were prospectively recruited and analyzed by elisa for levels of scd and scd , along with other laboratory parameters, and compared to a healthy control group. results: scd and scd levels were significantly higher among covid- patients, independently of icu admission requirement, compared to the control group. we found a significant correlation between scd levels and other inflammatory markers, particularly interleukin- , in the non-icu patients group. scd showed a moderate positive correlation with the time lapsed from admission to sampling, independently of severity group. treatment with corticoids showed an interference with scd levels, whereas hydroxychloroquine and tocilizumab did not. conclusions: monocyte-macrophage activation markers are increased and correlate with other inflammatory markers in sars-cov- infection, in association to hospital admission. these data suggest a preponderant role for monocyte-macrophage activation in the development of immunopathology of covid- patients. emerging evidence from sars-cov- infected patients suggests a key role for monocyte-macrophage in the immunopathology of covid- infection, with a predominant monocytederived macrophage infiltration observed in severely damaged lungs ( ) , and morphological and inflammation-related changes in peripheral blood monocytes that correlate with the patients' outcome ( ) . an overexuberant inflammatory immune response with production of a cytokine storm and t-cell immunosuppression are the main hallmarks of severity in these patients ( ) . this clinical course resembles viral-associated hemophagocytic syndrome (vahs), a rare severe complication of various viral infections mediated by proinflammatory cytokines, resulting in multiorgan failure and death ( ) . a chronic expansion of inflammatory monocytes and over-activation of macrophages have been extensively described in this syndrome ( ) ( ) ( ) . viral-associated hemophagocytic syndrome has been identified as a major contributor to death of patients in past pandemics caused by coronaviruses ( ) , including previous sars and mers outbreaks ( ) , and currently suggested for sars-cov- outbreak ( ) . cd and cd are both myeloid differentiation markers found primarily on monocytes and macrophages, and detection of soluble release of both in plasma is considered a good biomarker of monocyte-macrophage activation ( , ) . elevated plasma levels of soluble cd (scd ) are associated to poor prognosis in vih-infected patients, are a strong predictor of morbidity and mortality ( , ) , and associated with diminished cd +-t cell restoration ( ) . in addition, soluble cd (scd ) plasma levels are a good proxy for monocyte expansion and disease progression during hiv infection ( ) . in measles infection, a leading cause of death associated with increased susceptibility to secondary infections and immunosuppression, scd and scd levels have been found to be significantly higher, indicating an important and persistent monocyte-macrophage activation ( ) . we hypothesized that monocytes/macrophages may be an important component of immunopathology associated to sars-cov- infection. in this paper, we analyze serum levels of soluble monocyte activation markers in covid- patients and their correlation with severity and other inflammatory markers. we recruited patients with confirmed pcr-positive diagnosis of sars-cov- infection, classified according to icu admission requirement (n = patients), or non-icu requirement (n = ), and age-matched healthy individuals (n = ) as a control group. demographic data, main medication treatment and routine lab clinical parameters including inflammatory biomarkers were collected for all infected patients. leftover sera samples from routine analytical controls were employed for the analysis, after obtaining the corresponding informed consent. time elapsed from hospital admission to sample extraction was also recorded. to determine levels of soluble monocyte activation markers in serum specimens, appropriate sandwich elisa (quantikine, r&d systems, united kingdom) were used following manufacturer indications. briefly, diluted sera samples were incubated for h at room temperature in the corresponding microplate strips coated with capture antibody. after incubation, strips were washed and incubated with the corresponding human antibody conjugate for h. after washing, reactions were revealed and optical density at nm was determined in a microplate reader. concentration levels were interpolated from the standard curve using a four-parameter logistic ( -pl) curvefit in prism graphpad software. final values were corrected applying the corresponding dilution factor employed. data are expressed as median and interquartile range. all statistical analyses were performed using the statistical package r. mann-whitney tests were used for comparison between icu and non-icu groups versus healthy controls. pearson's correlation coefficients were used to quantify the association between scd and scd concentration and other lab parameters in non-icu patients. data outliers, falling outside the . interquartile range, were excluded from the statistical analysis. the nominal significance level considered was . . bonferroni adjustment was used to account for multiple testing. patients in the icu group showed significant differences when compared to non-icu group in several clinical laboratory parameters: lymphocytes, ferritin, d-dimer, lactate dehydrogenase (ldh), procalcitonin (pct), and interleukin- (il- ). the absolute value for circulating monocytes did not show significant differences between groups. however, these values may have been distorted by the use of tocilizumab, an il- blocking drug extensively employed in the icu group which interferes with monocyte function. age and time elapsed from admission to sample extraction did not show differences between groups. values are summarized in table . median levels for scd in sera from icu patients were . ( %ci: . - . ) ng/ml, compared to . ( %ci: . - . ) ng/ml in non-icu patients. the healthy control group median value was . ( %ci: . - . ) ng/ml. we observed significant statistical differences when comparing infected patients against controls (p-value < . ), however no significant differences were observed between icu and non-icu groups. median levels for scd in sera from icu patients were . ( %ci: . - . ) ng/ml, and . ( %ci: . - . ) ng/ml in non-icu patients. the healthy control group value was . ( %ci: . - . ) ng/ml. as with scd , we observed significant differences for values from infected patients compared to control group (p-value < ), but no differences between icu and non-icu infected patients. values are summarized in table and figure . we assessed the correlation between scd and scd levels and time elapsed from hospital admission to sample extraction (figure ) . we found a significant positive correlation between scd levels and time elapsed (r = . , p-value = . ) we did not observe a significant correlation between scd levels and time elapsed from hospital admission to sample extraction. we found significant correlations between scd and scd levels and several clinical laboratory parameters in infected patients (in these analysis, adjusted significance under bonferrori correction is . ), but only in the non-icu group, possibly reflecting an interference of the use of tocilizumab or corticoids in the icu group. levels of scd showed a negative correlation with the absolute value of lymphocytes (r = − . , p-value = . ) and a positive correlation with levels of ldh (r = . , p-value = . ), crp (r = . , p-value < . ); pct (r = . , p-value = . ), and ferritin (r = . , p-value = . ) (figure ) . no other significative associations were found with other lab parameters. levels of scd did not show significant correlation with clinical laboratory parameters (figure ) . particularly, il- also showed significant positive correlation with scd (r = . , p-value = . ) (figure ) . we analyzed possible interference of different treatments on scd and scd serum levels for all patients. we found an interference of corticoid treatment on scd , levels with median values of ( %ci: - ) ng/ml for treated group, and values of ( %ci: - ) ng/ml for nontreated group. values were significantly lower in corticoid-treated group (p-value = . ) (figure ) . no impact was found for corticoids on scd levels. likewise, hydroxychloroquine and/or tocilizumab were not found to have an impact on scd and scd serum levels. levels of scd and scd did not show association with length of hospital stay in both groups. also, these biomarkers did not show association with the number of days of onset of symptoms. we analyzed for possible age-dependence of scd and scd levels. values did not show association between these biomarker levels and the age of patients. our results show, for the first time, increased levels of scd and scd in sera from sars-cov- infected patients admitted to hospital. we did not observe statistical differences when comparing icu versus non-icu patients. this is probably due to the interference on monocyte function and scd levels produced by the use of corticoid treatment in icu patients, as shown here and previously by others ( , ) . however, levels of scd showed a strong correlation with clinical laboratory parameters, including acute phase reactants (ferritin, ldh, c-reactive protein, procalcitonin) and a strong correlation with il- levels in the non-icu patient group, where no corticoids treatments were used. hydroxychloroquine and tocilizumab treatment did not show interferences on scd and scd levels. furthermore, scd levels showed a correlation with the time elapsed from hospital admission to sample extraction, suggesting a potential indicator of disease progression. monocytes and macrophages constitute a key component of immune responses against viruses, acting as bridge between innate and adaptive immunity ( ) . activation of macrophages has been demonstrated to be pivotal in the pathogenesis of the immunosuppression associated to several viral infections (such as vih, measles), where expansion of specific subsets of monocytes and macrophages in peripheral blood are observed, and considered to be drivers of immunopathogenesis ( ) . our results support the hypothesis of a preponderant role for monocytes in sars-cov- immunopathology, associated to an overexuberant immune response. increased levels of monocytemacrophage activation markers, and their correlation with other inflammatory biomarkers (particularly il- ), indicate a close relationship between monocyte activation and immunopathology in these patients. inflammatory markers are closely related to severity in covid- pathology ( ) and selective blockade of il- has been demonstrated to be a good therapeutic strategy in covid- pathology ( ). our results thus suggest that monocyte-macrophage activation can act as driver cells of the cytokine storm and immunopathology associated to severe clinical course of covid- patients. further, monitorization of monocyte activity trough these soluble activation markers and/or follow-up of circulating inflammatory monocytes in peripheral blood, could be useful to assess disease progression in the same way as in other viral infections ( ) . in addition, our results identify monocyte-macrophage as a good target for the design of therapeutic intervention using drugs that inhibit monocyte-macrophage activation and differentiation. in this sense, anti-gm csf inhibitor drugs, currently under clinical trials for rheumatic and other auto-inflammatory diseases, might provide satisfactory results in covid- patients. other drugs targeting monocyte and/or macrophage could also be useful in covid- , as in other inflammatory diseases ( ) . the strategy of inhibiting monocyte differentiation has proved useful in avoiding cytokine storm syndrome after car-t cell immunotherapy ( ), suggesting a possible therapeutic application to covid- immunopathology ( , ) . the present study has several limitations, including a relatively low sample size and the interference of corticoids in icu patients' results. however, these preliminary results are strongly suggestive of an important implication of monocytemacrophage in covid- immunopathology, as highlighted by the correlations found between these biomarker levels and inflammatory parameters. further studies using broader series are needed to confirm our findings. in summary, our data underscore the preponderant role of monocyte and macrophage immune response in covid- immunopathology and provide pointers for future interventions in drug strategies and monitoring plans for these patients. the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. the studies involving human participants were reviewed and approved by comité de Ética de la investigación con medicamentos de galicia (fast-track approval -march- ). written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. the landscape of lung bronchoalveolar immune cells in covid- revealed by single-cell rna sequencing. medrxiv covid- infection induces readily detectable morphological and inflammation-related phenotypic changes in peripheral blood monocytes, the severity of wich correlate with patient outcome. medrxiv speciality collaboration, covid- : consider cytokine storm 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events immunologic failure despite suppressive antiretroviral therapy is related to activation and turnover of memory cd cells increased monocyte turnover from bone marrow correlates with severity of siv encephalitis and cd levels in plasma persistent high plasma levels of scd and scd in adult patients with measles virus infection modulation of human monocyte/macrophage activity by tocilizumab, abatacept and etanercept: an in vitro study effects of corticosteroids on human monocyte function co-ordinating innate and adaptive immunity to viral infection: mobility is the key soluble cd , a novel marker of activated macrophages, is elevated and associated with noncalcified coronary plaque in hiv-infected patients correlation analysis between disease severity and inflammation-related parameters in patients with covid- pneumonia. medrxiv the cytokine release syndrome (crs) of severe covid- and interleukin- receptor (il- r) antagonist tocilizumab may be the key to reduce the mortality gm-csf inhibition reduces cytokine release syndrome and neuroinflammation but enhances car-t cell function in xenografts a strategy targeting monocyte-macrophage differentiation to avoid pulmonary complications in sars-cov infection role of monocytes/ macrophages in covid- pathogenesis: implications for therapy the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © gómez-rial, currás-tuala, rivero-calle, gómez-carballa, cebey-lópez, rodríguez-tenreiro, dacosta-urbieta, rivero-velasco, rodríguez-núñez, trastoy-pena, rodríguez-garcía, salas and martinón-torres. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -e zjxjox authors: lee, cheryl yi-pin; lin, raymond t. p.; renia, laurent; ng, lisa f. p. title: serological approaches for covid- : epidemiologic perspective on surveillance and control date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: e zjxjox since december , the novel coronavirus, sars-cov- , has garnered global attention due to its rapid transmission, which has infected more than two million people worldwide. early detection of sars-cov- is one of the crucial interventions to control virus spread and dissemination. molecular assays have been the gold standard to directly detect for the presence of viral genetic material in infected individuals. however, insufficient viral rna at the point of detection may lead to false negative results. as such, it is important to also employ immune-based assays to determine one's exposure to sars-cov- , as well as to assist in the surveillance of individuals with prior exposure to sars-cov- . within a span of months, extensive studies have been done to develop serological systems to characterize the antibody profiles, as well as to identify and generate potentially neutralizing antibodies during sars-cov- infection. the vast diversity of novel findings has added value to coronavirus research, and a strategic consolidation is crucial to encompass the latest advances and developments. this review aims to provide a concise yet extensive collation of current immunoassays for sars-cov- , while discussing the strengths, limitations and applications of antibody detection in sars-cov- research and control. the ongoing pandemic, which originates from a newly emerged coronavirus, sars-cov- , was discovered in the city of wuhan in china's hubei province in december ( ) . to date, due to rapid transmission globally, there are more than two million laboratory-confirmed human infection cases, with a few hundred thousand deaths across countries and territories (https://www.who.int/emergencies/diseases/novel-coronavirus- / situation-reports/). this unprecedented crisis led to a worldwide effort to rapidly characterize the immunobiology of sars-cov- , while mitigating further spread of this deadly pathogen. sars-cov- is a single stranded, positive sense rna virus that belongs to the coronaviridae family of the betacoronavirus genus ( ) . it has a genome size of ∼ kilobases that encodes for multiple structural proteins comprising the spike (s), the envelope (e), the membrane (m), and the nucleocapsid (n), as well as non-structural proteins ( ) (figure ) . infection by sars-cov- causes an acute respiratory disease termed the coronavirus disease . the clinical manifestations of covid- form a spectrum, from being asymptomatic to fever with mild respiratory illness, to acute respiratory distress syndrome, and death from respiratory failure or associated complications ( ) ( ) ( ) . as the reported incubation period varies among different patient cohorts, it is often difficult to ascertain the actual day of onset, and infected subjects who are asymptomatic or pre-symptomatic may go undetected ( ) ( ) ( ) . early detection of sars-cov- infection is one of the crucial interventions to control virus transmission. with the discovery of the genome organization of sars-cov- viral rna, which is adapted from genbank accession number: mn , is characterized by sequence alignment against two representative members of the betacoronavirus genus. the entire genome sequence is ∼ kilobases (kb) long. the virus, numerous diagnostic assays using quantitative reverse transcriptase pcr (qrt-pcr) were developed ( ) . qrt-pcr is the reference standard for diagnosing infections with high sensitivity and accuracy in the acute phase of illness. sars-cov- viral rna has been detected in both throat and nasal swabs of infected individuals by qrt-pcr, which becomes almost undetectable by days post-illness onset (pio) (or symptom onset) ( , ) (figure ) . apart from being costly and time consuming to perform, false negative results may arise due to improper handling of nucleic acid samples, inadequate and variable sampling resulting in insufficient viral genetic material at the point of detection (after days pio), or biological variation on when viral rna is detectable by qrt-pcr ( , ) . with the limitations of qrt-pcr, immunoassays may offer another figure | schematic illustration on the window period of detection for either viral rna or antibodies in sars-cov- -infected individuals. presence of sars-cov- viral rna (boxed in pink) in throat or nasal swab of patients are typically undetectable by day post illness onset (pio) ( , ) . sars-cov- -specific antibodies (boxed in blue): igm is detectable as early as days pio, and peaks between and weeks pio ( , ) . igm response was still detectable after more than month pio ( ) . both iga and igg are present as early as days pio, and peaks after weeks pio in serum samples ( , , , ) . there are currently no reports on the presence of these sars-cov- -specific antibodies in the later phase pio, as indicated by dotted lines. this depicts the importance of serological studies to identify individuals with current or prior exposure to sars-cov- that went undetected, by testing for either igm, igg, or iga antibodies against sars-cov- . illustration was created using biorender. avenue to reduce undiagnosed cases, with the advantage that rapid test formats may deliver results in a relatively shorter time and lower cost ( ). immunoassays are another diagnostic approach that can provide information on both active viral infections and past exposures (figure ). to date, many commercial companies and research institutes have developed serological assays to detect sars-cov- antibodies from patient serum or plasma samples ( , ) . closely related to another pathogen, severe acute respiratory syndrome coronavirus (sars-cov), these assays mainly target immunogenic coronavirus proteins: s protein, which is the most exposed viral protein, and n protein, which is abundantly expressed during infection ( , , ) . in addition, the receptor-binding domain (rbd), which is located along the s protein, is also a target of interest to detect the presence of sars-cov- -specific antibodies ( , ) . in recent pre-prints deposited in medxriv and bioxriv, it was shown that both anti-sars-cov- -igm and igg levels increase gradually along with infection phases, with igm being detected as early as days pio, which peaks between two to three weeks pio ( , ) . one study has reported that sars-cov- -specific igm is still present in the serum after month pio ( ) . sars-cov- -specific igg antibodies, on the other hand, can be present as early as days pio, and peak after days pio ( , ) (figure ) . these observations are similar to what was previously reported during a sars-cov infection ( ) . however, interestingly, one study demonstrated that longitudinal profiling of both antibodies in a population of covid- patients showed no specific chronological order in terms of igm and igg seroconversion ( ) , which was also observed in patients infected with sars-cov and another human coronavirus, middle east respiratory syndrome coronavirus (mers-cov) ( , ) . in addition, there seems to be no correlation between seroconversion rates with age, gender or time of hospitalization ( ) . these findings on sars-cov- -specific antibodies seroconversion against the s viral protein suggest the importance to test for both igm and igg antibodies to confirm a positive infection. expectedly, similar to what was reported for sars-cov and mers-cov, both igm and igg levels seems to be correlated with disease severity, with a higher level of both antibodies present in patients with more severe sars-cov- infection ( , , , ( ) ( ) ( ) . in contrast to other flu-like infections such as influenza, instead of igg , igg appears to be the dominant igg subtype during sars-cov- infection ( , , ) . as a majority of the human population has prior exposure to endemic human coronavirus infections including alphacoronaviruses ( e and nl ), and other betacoronaviruses (oc and hku ) ( ) , it is crucial to validate the specificity and sensitivity of current immunoassays against sars-cov- to avoid false positive outcomes. within the s protein antigen, cross-reactivity was observed when samples were tested against sars-cov s and s subunit proteins, and to a smaller extent, with mers-cov s protein ( table ) . interestingly, there was no cross-reactivity with the s subunit of mers-cov ( ) . the high level of crossreactivity between sars-cov and sars-cov- can be attributed to the high degree of genetic homology ( , , ) . furthermore, detailed analysis revealed a highly conserved s subunit domain across coronaviruses, which may explain for the cross-reactivity observed with only the s protein of mers-cov, and not with the s subunit ( , ) . these data suggest that using an s subunit-based immunoassay may be more specific than the entire s antigen for diagnosing sars-cov- infections. another immunogenic target, the rbd, which lies along the s protein is usually the target of many neutralizing antibodies against sars-cov ( ) . a substantial level of cross-reactivity by sars-cov rbd-induced antibodies to sars-cov- rbd was described ( table ) ( ) . of clinical relevance, these antibodies were also able to cross-neutralize sars-cov- pseudovirus infection, signifying the potential of an immunotherapy-based treatment ( ) . while one non-peer reviewed study has shown that rbd-based serological assays are more sensitive than s subunit-based assays in identifying antibodies in mild covid- patients ( ), other non-peer-reviewed studies have described a lower degree of antibody response to the rbd as compared to full-length s protein, plausibly reflecting the larger number of epitopes present on the larger s antigen ( , ). due to a high level of similarity of % between sars-cov and sars-cov- n proteins, the n antigen of sars-cov was also used for serological detection of sars-cov- -specific antibodies ( table ) ( ) . these n-based assays were reported to be more sensitive than s subunit-based tests ( ) . the use of sars-cov antigens to diagnose sars-cov- infections may be reliable, given that sars-cov has not circulated in the human population since ( ). in addition, an earlier report has demonstrated waning of sars-cov-specific antibodies, therefore being undetectable in % of patient serum samples after years ( ) . since respiratory diseases are the hallmark of coronavirus infections, which activate mucosal immunity, several studies have exploited the detection of iga to diagnose sars-cov- infection in patients (table ) ( , ) . although a strong iga response was also detected in covid- patients where peak seroconversion was achieved by two weeks pio (figure ) , igabased immunoassay has been hypothesized to be less specific than igg-based elisa due to cross-reactivity with serum samples from patients infected by other coronaviruses ( ) . with the availability of immunoassays utilizing various coronavirus structural proteins, the use of more than one different antigen-based serological approach may be essential to establish a true positive sars-cov- infection. in addition, frontiers in immunology | www.frontiersin.org the use of saliva samples and other bodily fluid swabs as a less invasive alternative, which have been done for other viral infections including hiv and measles, should also be explored for serological testing of sars-cov- infections ( , ). apart from using immunoassays for the early detection of sars-cov- infected individuals, it is also critical to determine the regions where sars-cov- -specific antibodies bind to help guide vaccine designs. using sars-cov-derived b-cell epitopes that have been experimentally identified from positive b-cell assays ( ), out of linear b-cell epitopes have an identical match with sars-cov- protein sequences without any mutations ( ) . notably, majority of these matches were located at both the s and n viral antigens, with only from the m protein, and none in the e protein ( ). on the other hand, conformational b-cell epitopes identified from the same database were located on the s antigen. however, unlike the linear epitopes, none of these mapped identically to the sars-cov- protein ( ). further mapping the residues of linear b-cell epitopes onto available sars-cov s protein structure revealed several regions on the s subunit that may allow cross-neutralization of both sars-cov and sars-cov- ( , ) . in contrast, conformational b-cell epitopes mapped onto the s subunit, resulting in very few identical residues within sars-cov and sars-cov- ( ). these findings indicate that sars-cov-specific antibodies targeting these discontinuous regions may not be able to cross-react with sars-cov- ( , ) . as these regions are computationally predicted, serological studies using patient samples are necessary to validate the importance of these regions for serology and in controlling sars-cov- infection. it also remains imperative to identify other sars-cov antibodies that may recognize the conformational epitopes of sars-cov- s protein, which can greatly reduce the amount of time needed to develop novel neutralizing antibodies. the findings derived from serological assays can provide valuable information that would help to support the diagnosis, treatment, and prevention of sars-cov- infections. characterization of antibody profiles suggested that any suspected individuals with undetectable antibody levels against sars-cov- after days pio may be a true negative case, since both anti-sars-cov- igm or igg seroconversion should have already occurred ( , ) . however, these findings may be limited to the relatively small sample size (< patients) and may require further validation with a larger cohort. in order to reinforce diagnosis, it would be advisable to perform multiple assays against different viral antigen. in addition, the information of antibody seroconversion is crucial in determining the optimal timepoints to collect serum or plasma samples for immunoassay screening, as well as obtaining peripheral blood b cells for the generation of therapeutic monoclonal antibodies ( ) . currently, in order to rapidly generate neutralizing monoclonal antibodies against sars-cov- , repurposing of existing sars-cov-specific antibodies was demonstrated. to date, two human sars-cov-specific antibodies, cr and d , have been shown to recognize sars-cov- ( , ) . cr recognizes an epitope along the rbd of sars-cov- , which differs largely at the c-terminus residues to the rbd of sars-cov ( ) . unfortunately, this variation in sequence impacted the ability of cr to crossneutralize sars-cov- . monoclonal antibody d , on the other hand, targets the rbd along the s subunit of both sars-cov and sars-cov- with similar affinities, thereby enabling cross-neutralization against sars-cov- infection ( ) . while combinatory therapy has exhibited a stronger neutralization capability against sars-cov infection ( ), a cocktail antibody approach for sars-cov- could be explored. surprisingly, reports on antibodies against the coronavirus e protein are scarce, possibly due to it being the smallest protein. however, the e antigen is involved in viral assembly, release of virions, as well as virus pathogenesis ( ) . it was demonstrated that recombinant coronaviruses lacking the e protein displayed significantly reduced viral titers, impaired viral maturation and produced avirulent virus progenies, suggesting a similar importance of e protein during sars-cov- infection ( , ) . thus, it would be worthwhile to identify or generate neutralizing antibodies that are specific against the viral e protein. during the course of an epidemic, one of the main challenges is the identification of asymptomatic infection. since these individuals do not present any distinguishable symptoms, they could be the major source of transmission ( ) . immunoassays may be able to detect mildly infected cases ( ) , which is important to ascertain the extent of community spread. while it is fast, robust and easy to perform, there are several limitations to serological assays. one of the major setbacks of immunoassays is the inability to detect the presence of infection during the early stage of disease, as antibodies take several days to be generated after exposure to foreign material ( ) . as such, a recent infection may provide false negative results during serological testing. thus, the use of rt-pcr may be more suitable to diagnose an early acute sars-cov- infection. furthermore, due to the unique genetic makeup of each individual, there would be an inherent variability of the antibody response ( ) . this could possibly explain the difference in antibody profiles elicited among individuals infected with sars-cov- ( ). cross-reactivity could potentially be a limitation of immunoassays as it severely impacts the specificity and sensitivity of the test. although the phylogenetically closest coronavirus, sars-cov, has not been reported to be circulating in the human population since ( ), other endemic human coronaviruses may still pose a problem to accurately diagnose patients with true sars-cov- infection. while a recent study has demonstrated negligible cross-reactivity from human coronavirus, nl , to sars-cov- ( ), validation with other human coronaviruses remains to be investigated. in addition, prior findings on the s protein sequence and neutralization antigenicity of other coronaviruses suggest that antibodies neutralizing clinical human coronavirus isolates may not have the same degree of cross-reactivity with laboratory strains of human coronaviruses, thereby affecting the sensitivity of immunoassays ( ) ( ) ( ) . given the rapid increase in the number of confirmed covid- cases coupled with the shortage in test kits to meet rising demands, decentralized point-of-care tests (poct) may be another alternative to facilitate sars-cov- diagnosis. such tests include lateral flow assay (lfa), which is a paper-based platform for the detection and quantification of analytes in complex mixtures ( ) . to design lfa for sars-cov- detection, an antibody specific to the viral antigen, or a viral antigen that is detectable by patient serum or plasma samples can be immobilized on a nitrocellulose membrane. detection of binding between the analyte and capture antibody by a detector antibody will give rise to a colored line, closely resembling home pregnancy kits ( ) . poct is advantageous as it is usually designed to be rapid, sensitive, highly accessible, and easily performed, requiring only a small amount of sample ( ) . meanwhile, several hundreds of candidate pocts are being evaluated for their applicability toward identifying sars-cov- -infected individuals ( ) . however, pocts can't replace rt-pcr and it is crucial that these developing tests are rigorously assessed prior to use. it is important to note that wrong use and interpretation could lead to disastrous public health consequences ( ) . rapid development of diagnostic tools and immune-based assays are important early interventions against the ongoing sars-cov- pandemic. the availability of serological assays that target a diverse range of viral antigen has no doubt assisted in the accurate diagnosis of covid- patients. essentially, data generated through serological studies can greatly aid in supplementing the results from qrt-pcr, as well as contribute to seroepidemiology, which has been shown to help in the design of virus elimination programs ( ) . moving forward, this extensive collation of the current immunoassays against sars-cov- will provide insights toward monoclonal antibodies discovery and characterization for the development 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( ) seroepidemiology: an underused tool for designing and monitoring vaccination programmes in low-and middle-income countries diagrams are created with biorender. the authors wish to thank drs. siew-wai fong and yi-hao chan for critical comments of this manuscript. key: cord- - rrh gg authors: stryhn, anette; kongsgaard, michael; rasmussen, michael; harndahl, mikkel nors; Østerbye, thomas; bassi, maria rosaria; thybo, søren; gabriel, mette; hansen, morten bagge; nielsen, morten; christensen, jan pravsgaard; randrup thomsen, allan; buus, soren title: a systematic, unbiased mapping of cd (+) and cd (+) t cell epitopes in yellow fever vaccinees date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: rrh gg examining cd (+) and cd (+) t cell responses after primary yellow fever vaccination in a cohort of volunteers, we have identified and tetramer-validated cd (+) and cd (+) t cell epitopes, many inducing strong and prevalent (i.e., immunodominant) t cell responses. restricted by and hla-class i and ii allotypes, respectively, these responses have wide population coverage and might be of considerable academic, diagnostic and therapeutic interest. the broad coverage of epitopes and hla overcame the otherwise confounding effects of hla diversity and non-hla background providing the first evidence of t cell immunodomination in humans. also, double-staining of cd (+) t cells with tetramers representing the same hla-binding core, albeit with different flanking regions, demonstrated an extensive diversification of the specificities of many cd (+) t cell responses. we suggest that this could reduce the risk of pathogen escape, and that multi-tetramer staining is required to reveal the true magnitude and diversity of cd (+) t cell responses. our t cell epitope discovery approach uses a combination of ( ) overlapping peptides representing the entire yellow fever virus proteome to search for peptides containing cd (+) and/or cd (+) t cell epitopes, ( ) predictors of peptide-hla binding to suggest epitopes and their restricting hla allotypes, ( ) generation of peptide-hla tetramers to identify t cell epitopes, and ( ) analysis of ex vivo t cell responses to validate the same. this approach is systematic, exhaustive, and can be done in any individual of any hla haplotype. it is all-inclusive in the sense that it includes all protein antigens and peptide epitopes, and encompasses both cd (+) and cd (+) t cell epitopes. it is efficient and, importantly, reduces the false discovery rate. the unbiased nature of the t cell epitope discovery approach presented here should support the refinement of future peptide-hla class i and ii predictors and tetramer technologies, which eventually should cover all hla class i and ii isotypes. we believe that future investigations of emerging pathogens (e.g., sars-cov- ) should include population-wide t cell epitope discovery using blood samples from patients, convalescents and/or long-term survivors, who might all hold important information on t cell epitopes and responses. examining cd + and cd + t cell responses after primary yellow fever vaccination in a cohort of volunteers, we have identified and tetramer-validated cd + and cd + t cell epitopes, many inducing strong and prevalent (i.e., immunodominant) t cell responses. restricted by and hla-class i and ii allotypes, respectively, these responses have wide population coverage and might be of considerable academic, diagnostic and therapeutic interest. the broad coverage of epitopes and hla overcame the otherwise confounding effects of hla diversity and non-hla background providing the first evidence of t cell immunodomination in humans. also, double-staining of cd + t cells with tetramers representing the same hla-binding core, albeit with different flanking regions, demonstrated an extensive diversification of the specificities of many cd + t cell responses. we suggest that this could reduce the risk of pathogen escape, and that multi-tetramer staining is required to reveal the true magnitude and diversity of cd + t cell responses. our t cell epitope discovery approach uses a combination of ( ) overlapping peptides representing the entire yellow fever virus proteome to search for peptides containing cd + and/or cd + t cell epitopes, ( ) predictors of peptide-hla binding to suggest epitopes and their restricting hla allotypes, ( ) generation of peptide-hla tetramers to identify t cell epitopes, and ( ) analysis of ex vivo t cell responses to validate the same. this approach is systematic, exhaustive, and can be done in any individual of any hla haplotype. it is all-inclusive in the sense that it includes all protein antigens and peptide epitopes, and encompasses both cd + and cd + t cell epitopes. it is efficient and, importantly, reduces the false discovery rate. the unbiased nature of the t cell epitope discovery approach presented here should support the refinement of future peptide-hla class i and ii predictors and tetramer technologies, which eventually should cover all hla class i and ii isotypes. we believe that future investigations of emerging the immune system attempts to protect its host against invading pathogens; yet, it can also cause serious pathology. the ability to discriminate between foreign and self is key to exerting immune protection without inflicting immune pathology. immune recognition is therefore of immense interest and efficient methods to identify and validate immune epitopes are a high priority. in this context, t cells, which effectively orchestrate the overall immune response, are of particular interest. t cells are specific for compound ligands consisting of peptides, generated intracellularly by proteolytic degradation of protein antigens, which are presented in the context of major histocompatibility complex (mhc) [or human leucocyte antigens (hla)] molecules on the surface of antigen presenting cells (apc) ( ) . the interaction between peptide and hla is specific; the resulting hla-mediated t cell epitope selection process being greatly diversified by the polygenic and polymorphic nature of the hla. this significantly affects the peptide-binding specificity of the set of hla molecules that are available to any given host; something that effectively individualizes our immune responses. although other events are also involved in antigen processing and presentation, the single most selective event is that of peptide-hla binding. it is estimated that ca. . % of all possible peptide-hla combinations are of a sufficiently high affinity that they potentially, but not necessarily, could be immunogenic ( ) . major efforts have been devoted to understand, quantitate and preferably predict peptide-hla binding as a means to identify t cell epitopes. proposed in , the "human mhc project" aims at mapping all human mhc (or hla) specificities ( , ) . established in , the "immune epitope database" (iedb) has become an authoritative repository of hla binding peptides and t cell epitopes, and of methods to predict these ( ) . the recent breakthrough in cancer immunotherapy has reinforced the interest in fast and efficient methods to identify t cell epitopes with special emphasis on identifying immunogenic neoepitopes for personalized cancer immunotherapy. thus, several recent international research efforts, such as the "human immunopeptidome project and consortium, " "tumor neoantigen selection alliance" and others, have focused on t cell epitope discovery. employing recent advances in mass spectrometry to perform large-scale identification of peptides eluted of hla molecules, these efforts promise to identify natural ligands thereby capturing information on both antigen processing and hla binding ( ) . over the past decades, substantial progress has been made on predicting peptide-hla interactions, particularly for hla class i (hla-i), which restricts cd + cytotoxic t cells (ctl's), and to a lesser degree on predictions for hla class ii (hla-ii), which restricts cd + helper t cells (th) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . state-of-theart predictors such as netmhcpan, an artificial neural network method based on a large collection of experimental peptide-hla-i binding data, can successfully identify . % of cd + t cell epitopes, while rejecting . % of non-epitopes ( ) . however, considering that only of , ( ) to , ( ) random peptides is a t cell immunogen in the context of a given hla molecule, even a rejection rate as high as . % translates into a high false discovery rate (fdr) ( , , , ) . this is a general problem of current peptide-hla binding predictors ( , ) , and it is particularly problematic when trying to develop a neoepitope-specific, personalized cancer immunotherapy where timely delivery of a few unique cancer neoepitopes is of paramount importance; something that potentially could be achieved with even better predictors ( , ( ) ( ) ( ) . yellow fever virus (yfv) is a mosquito-borne flavivirus (i.e., a ssrna virus) ( , ) . it remains an important human pathogen despite the existence of an effective live attenuated vaccine ( ) . particularly relevant to this study, previous analyses of the cd + t cell response against a limited number of epitopes have revealed that vaccination with this live vaccine represents an excellent model for studying the host response to a viral infection ( , ) . the main advantages are that the precise time and the exact identity of the immune challenge are both known [note that the vaccine strain used here is known to be stable ( ) ]; issues that otherwise might complicate the interpretation of immune responses observed in patients that are naturally infected with a variable pathogen. here, we have generated a comprehensive, population-wide t cell epitope discovery approach with a much-reduced fdr, and used it to identify and validate immunodominant cd + and cd + t cell epitopes in a cohort of hla-typed, primary yfv vaccinees. this involves using a "forward (or direct) immunology" approach, where you start with a specific t cell response of interest and then search for the epitope(s) being recognized ( , ) , to perform an initial identification of t cell stimulatory peptides. subsequently, a "reverse immunology" approach, where you start by predicting possible t cell epitopes and then search for a t cell response of the corresponding specificity ( , ) was used, to perform a final identification and validation of the underlying specific t cell epitopes and their hla restriction elements. from here on, this approach is denoted as a "hybrid forward-reverse immunology" (hfri) approach. briefly, in the "forward immunology" step, pbmc's obtained - weeks after primary yfv vaccination were ex vivo stimulated with an overlapping peptide library representing the entire , amino acid yfv proteome and tested by an ifnγ-specific intracellular cytokine secretion (ics) assay thereby identifying cd + and cd + t cell stimulatory yfv-derived peptides. in the subsequent "reverse immunology" step, predictors were used to select appropriate peptide-hla combinations for the generation of peptide-hla tetramers, which then were used to identify and validate the underlying t cell epitopes and their hla restriction elements. applying this hfri approach to t cell epitope discovery in yfv vaccinees, we identified and tetramervalidated cd + and cd + t cell epitopes covering hla-i and hla-ii allotypes, respectively (note that he tetramer-validation step could not be performed exhaustively for the cd + t cell epitope discovery process and that the true number of cd + t cell epitopes probably was many times larger than the validated cd + t cell epitopes reported here). with a cohort of yfv vaccinees, the prevalence of responses against the cd + t cell epitopes could be examined. about a third ( %) of these epitopes were recognized in > % of the individuals expressing the hla-i in question. by this token, they could be considered strongly immunodominant. we conclude that t cell epitope discovery using this hfri approach is highly efficient, in particular when examining larger populations responding to the same pathogen (e.g., an infectious pathogen e.g., sars, ebola, zika, sars-cov- ). furthermore, we suggest that the hfri approach is unbiased and that the resulting t cell epitopes should serve as a valuable benchmark for future improvements of predictive algorithms of immunogenicity. primary vaccination with the attenuated yfv vaccine, d- , is known to trigger a prompt and vigorous cellular immune reaction ( , ) . here, vaccinees were recruited, and peripheral blood mononuclear cells (pbmc) were prepared from -to -ml blood samples obtained before and ca. weeks after primary vaccination, respectively ( ) . the typical yield from the latter was ca. million pbmc. all vaccinees were hla typed at high-resolution (i.e., digit) including all nine classical, polymorphic hla loci (i.e., hla-a, b, c, drb , drb / / , dqa , dqb , dpa , and dpb ) ( ) . the d- vaccine encodes a single polyprotein precursor of , amino acids (aa), which is processed into proteins. the full genome (genbank accession# x ) and proteome (swiss-prot accession# p ) sequences of the d- have been determined ( ) . a library of overlapping mer peptides overlapping by aa, spanning the entire yfv precursor protein (essentially the yfv proteome), was generated. additionally, peptides representing potentially aberrant yfv translation products were selected. of the resulting peptides, synthesis failed for peptides ( %) leaving peptides for analysis. since testing each of these peptides individually would exhaust the available pbmc's, the peptides were tested in pools. initially, the peptides were organized into a single × matrix from which "column pools" and "row pools" were generated leading to a total of pools each containing ca. different peptides. each peptide would be present in two pools: one column and one row pool (supplementary figure s a) . the intersections of stimulatory column and row pools should ideally identify which peptide might be immunogenic and therefore should be further investigated on an individual basis. this × matrix strategy was initially tested using an exvivo ifnγ elispot assay as readout. after the first primary vaccinated donors had been recruited, the average number of positive column/row intersections was found to be (range - ) ( figure a) suggesting that the hit rate from pools containing peptides was too high, at least in the setting of this acute viral response, to be effective in eliminating nonstimulatory peptides from further consideration. to reduce the hit rate per peptide pool, the peptides were re-organized into four smaller matrices, three × matrices and one × . for each matrix, to column pools and row pools were generated leading to a total of pools, which each contained to different peptides (supplementary figure s b) . to further reduce the number of relevant intersections, the ifnγ elispot assay was replaced by an ifnγ intracellular cytokine staining (ics) assay, which can discriminate between cd + and cd + t cells and therefore eliminate intersections with mismatched cd + and cd + t cell responses. furthermore, to increase the number of t cells available for the ics assay, pbmc were expanded in four separate in vitro cultures containing a pool of ca. peptides corresponding to each of the four matrices, respectively (the potential bias introduced by this in vitro culture is discussed in supplementary results and discussion). after days, each matrix-expanded pbmc culture was tested against the appropriate row and column pools using ifnγ ics as readout. for comparison, donors, which had already been analyzed using the × -matrix, elispot-based screening strategy, were re-screened using the ×( × )-matrix, icsbased screening strategy (figures b-d) . the aggregated cd + and cd + t cell responses were calculated for ics responses (denoted "all t cells" in figure ) and compared to those from elispot responses. the total number of intersections needing deconvolution was significantly lower for the ×( × ics strategy [average intersections (range - )] than for the × elispot strategy [average (range - ), (p < . , n = , mann-whitney u-test), figures a,b] . the intersections, which on average were detected by the ics-based screening strategy, could further be broken down into an average of (range - ) ( figure c ) and (range - ) ( figure d ) intersections representing cd + and cd + t cell responses, respectively ( of these intersections were shared). the peptides corresponding to these intersections were subsequently tested individually to identify which of the intersections truly represented cd + and/or cd + t responses. figure | comparing the number of peptide-containing peptides identified by the two different approaches. t cells obtained ex vivo from primary yfv vaccinees were stimulated with matrix-derived pools of yfv peptides and responses were read by ifnγ-specific elispot or ics. the peptides were distributed into matrixes, column and row pools of peptides were generated, and used to test t cell stimulation. intersections of stimulatory column and row pools putatively identified single stimulatory peptides for further analysis (supplementary figure s ) . (a) peptides were distributed into one × matrix generating + = pools, which were used to stimulate t cell responses in donors using an ifnγ-specific elispot assay as readout of all (i.e., cd + and cd + ) t cell responses [average positive intersections (range ]. (b,c) peptides were distributed into four ca. × matrices generating x(ca. + ) ca. pools, which were used to stimulate t cell responses in donors using an ifnγ-specific ics assay as readout of (b) all t cell responses [average intersections (range - )], (c) cd + t cell responses [average intersections (range - ), and (d] cd + t cell responses average intersections (range - ). the symbols representing the donors that were examined by both elispot and ics have been shaded. mann whitney u test was used to determine the significance of the difference between the indicated groups (***p < . ). the complete screening and validation procedure is illustrated using donor yf . the blood sample for donor yf was collected at day post vaccination, which is within the time span of optimal post vaccination yfv responses ( , ) . it gave a relatively high yield of × pbmc's for the subsequent epitope discovery effort. donor yf was initially analyzed by the × -matrix, ifnγ elispot-based screening strategy where positive intersections were identified. a total (i.e., cumulative) yfv-specific response of sfu were obtained suggesting a t cell response of considerable breath and magnitude. re-analyzing this donor using the four-matrix, ics-based screening strategy, the number of intersections for figure | t cell epitope screening strategy. (a) identification of stimulatory mer peptides: pbmc's from yfv vaccinated donors were divided into four cultures and in vitro stimulated with peptide sublibraries corresponding to each of the four × peptide matrices. after days, each sublibrary expanded pbmc culture was tested by ics against the matrix-specific row and column peptide pools. subsequently, individual peptides representing stimulatory matrix intersections were analyzed to identify single t cell stimulatory -mer peptides. (b) identification of t cell epitopes and their hla restriction elements: cd + t cell epitope deconvolution: single cd + t cell stimulatory mer peptides were tested for binding to the donor's hla-dr molecules using a biochemical hla class ii binding assay, positive interactions were used to generate peptide-hla class ii tetramers, and these tetramers were used to stain expanded t cells, and the resulting epitopes were eventually validated by ex vivo elispot analysis. cd + t cell epitope deconvolution: single cd + t cell stimulatory mer peptides were submitted to the netmhcpan . predictor together with the donor's hla class i haplotype to identify optimal epitopes and their hla-restriction elements. these optimal epitopes were subsequently synthesized and validated by ex vivo peptide-hla class i tetramer staining. follow-up analysis could be reduced to ; representing cd + t cell responses and representing cd + t cell responses ( of the intersections contained both cd + and cd + t cell responses). peptides corresponding to the intersections were tested individually by ics; this identified and cd + and cd + t cell stimulatory mer peptides, respectively (for a detailed listing of these peptides, see tables , below). the next steps aimed at identifying the underlying cd + and cd + t cell epitope(s) and their hla restriction element(s), preferably by generating the corresponding tetramer(s), and validate the epitope(s). for a general outlined of this epitope discovery scheme, see figure . identification and validation of cd + t cell epitopes exemplified by donor yf the sequences of the mer peptides, which stimulated cd + t cell responses in donor yf , were submitted, along with the donor's hla-i allotypes (in casu hla-a * : , -a * : , -b * : , -b * : , -c * : and -c * : ), to our webserver netmhcpan (version . at http://www.cbs.dtu.dk/ services/netmhcpan- . was available at the time of this analysis). in silico, this predictor considered all submer peptides of - mer length, which could possibly be generated from a mer peptide, and predicted their binding to all six hla-i allotypes of donor yf , a total of × = submer-hla-i combinations per mer peptide, and returned a ranked list across all six hla-i allotypes of the most likely epitope(s) and their hla-i restriction element(s). for all cd + t cell stimulatory peptides, this amounted to predicting the binding affinities of × = , submer-hla-i combinations. for each mer peptide, submers representing the top one to three predicted affinities were synthesized and the stabilities of the corresponding peptide-hla-i interactions were measured experimentally ( table ) . fluorochrome-labeled tetramers corresponding to the most stable peptide-hla-i interactions were generated and used to label relevant cd + t cells. when available, surplus t cells from the initial expansion cultures were used as a first line of identification of cd + t cell epitopes and their restriction elements, however, ex vivo tests were always used for the final cd + t cell epitope validation, and for enumerating and characterizing epitope-specific cd + t cells. the matrix-identified cd + t cell stimulatory mer peptides and the corresponding tetramer-validated optimal the cd + t cell stimulatory mer peptides are given including their sequences and ics stimulation values. the epitopes eventually identified are given in red. as shown, many epitopes were found in consecutive mer peptides. these mer peptides were submitted along with the donors hla-i haplotype to netmhcpan . , which returned suggested epitopes and restriction elements. in most cases, the epitope was found as a top ranking prediction. the stabilities of the suggested peptide-hla-i complexes were measured and the corresponding tetramers generated. finally, these tetramers were used to validate the cd + t cell epitopes and to enumerate the responding cd + t cells ex vivo. cd + t cell epitopes and their restriction elements are listed ( table ) . for each of the cd + t cell stimulatory mer peptides identified in donor yf , one or more cd + t cell epitopes and their hla-i restriction elements were identified. some of the epitopes were present in two consecutive overlapping mer peptides and should therefore only be counted as epitopes once. with this in mind, unique cd + t cell epitopes were recognized by donor yf ( epitopes restricted by hla-a * : , by hla-a * : , by hla-b * : , by hla-b * : , and none by hla-c * : or -c * : ) ( table ) . cd + t cell specific for the unique yfv-derived epitopes were readily detectable and enumerable ex vivo during the acute primary response of donor yf . the frequencies of total, as well as activated, tetramer-positive cd + t cells were determined ( table ) . the most frequently and immunodominant epitope of all, the hla-a * : -restricted ns b − epitope, was recognized by . % of cd + t cells in donor yf . the frequencies of cd t cells recognizing each of the other epitopes ranged from . to . %; the total frequency of cd + t cells recognizing the yfv epitopes was ca. %. the yfv vaccine induced a measurable increase in the overall frequency of activated cd + t cells (i.e., cd + hla-dr + cd + t cells) ( ) . in donor yf , the yfv vaccine induced an increase in activated cd + t cell from . % preto % post-vaccination. notably, the hla-a * : -restricted ns b − -epitope was recognized by % of the activated cd + t cells in donor yf . the frequencies of activated cd + t cells recognizing each of the other epitopes ranged from . to . %. in total, the identified yfv-specific cd + t cell epitopes accounted for the majority (ca. %) of activated cd + t cells observed during the acute response following primary yfv vaccination. the cd + t cell epitope discovery strategy described above for donor yf was extended to randomly selected donors, who were sampled at day - after vaccination i.e., at the peak of a primary anti-yf vaccine response ( , ) . cd + t cell responses specific for different peptide hla-i combinations were identified and validated by ex vivo tetramer staining (for an overview, see figure , and for details, see supplementary table si) . this represented different cd + t cell epitopes restricted by different hla-i molecules; , and epitopes were restricted by , , and different hla-i molecules, respectively. the hla-a, -b and -c allotypes covered by the donors were, respectively , , and of which the majority, , , and , were available to us for tetramer validation. thirteen of the different hla-a allotypes tested served as restriction elements of different cd + t cell peptide epitopes leading to the presentation of immunogenic peptide-hla-a combinations; of the different hla-b allotypes tested served as restriction elements of different epitopes leading to the presentation of immunogenic peptide-hla-b combinations; whereas only one of the different hla-c allotypes tested served as restriction elements of different epitopes leading to the presentation of immunogenic peptide-hla-c combinations. the average number of cd + t cell epitopes identified per hla-a and -b allotype, . and . , respectively, were not significantly different [p > %, fishers exact test, two-tailed (graphpad)]. to the best of our knowledge, of the yf-specific cd + t cell epitopes, and of the epitope-hla-i combinations reported here and in previous publications ( , ) , were first identified as a result of this hfri project. for the previously reported epitopes or epitope-hla-i combinations, minor adjustments of the already available information could be made: some had not been tetramer validated before, and others were also found to be restricted by other, albeit closely related, hla-i allotypes than those previously reported. in a few cases, tetramers representing the exact epitope-hla-i combinations previously reported failed to label cd + t cells in our donors despite expressing the appropriate hla-i allotype (for details see supplementary table si) . our in-house peptide repository included yfv-derived peptides from previous hla mapping efforts ( ) . using the contemporary netmhcpan . at %rank cut-off of . % to select putative binders from this repository, we generated additional peptide-hla-i tetramers (i.e., tetramers that had not already been prepared in the course of the present hfri approach). we included these tetramers in the immunodominance analysis described below. nine additional peptide-hla-i combinations, which had not been observed previously, were identified; four representing previously identified epitope presented by an alternative hla-i restriction element, and five representing new yfv-specific cd + t cell epitopes (supplementary table sii) . thus, the total number of cd + t cell epitopes discovered and tetramer validated here was of which (or %) were identified by the hfri approach. we systematically extended the analysis of ex vivo responses to additional donors expressing relevant hla-i restriction elements and evaluated them in terms of prevalence (the frequency of responders in donors with the hla-i restriction element in question) and response magnitude (the average ex vivo frequency of tetramer positive, activated cd + t cells of the responding donors) supplementary tables si, sii. to allow for a reasonable assessment of prevalence, the final analysis included epitopes restricted by hla-i molecules represented by at least donors, who had donated blood samples - days after vaccination. this involved a total of peptide-hla combination representing epitopes presented by hla-i allotypes (figure ) . immunodominance was frequently observed. from an epitope point of view, (or %) of the epitopes had a prevalence of ≥ % and a median magnitude > . %, and (or %) had a prevalence of ≥ % and a median magnitude > . %. from an hla point of view, (or %) of the hla-i molecules presented at least one epitope with ≥ % prevalence, and all hla-i molecules presented at least one epitope with at least % prevalence. in terms of hla-i coverage and immunodominance, the vast majority of our cohort, , , and %, carried at least one, two or three hla-i allotypes, respectively, which presented at least one epitope with ≥ % prevalence. a selection of immunodominant epitopes representing the most frequent hla-a and -b allotypes would cover % of the caucasian population. the "all cd " bar indicates the positions of each cd + t cell stimulatory mer peptide relative to the yfv polyprotein. each mer is shown as a frame that horizontally indicates the starting and ending positions of the mer peptide, vertically indicates the number of donors, of the donors tested, who responded to the peptide, and the color-coded is according to the polyprotein coloring scheme (to enhance the visualization of overlapping peptide sequences, this coloring is translucent). the lower hla-i allotype-designated bars indicate the tetramer-validated epitopes and their hla-i restriction elements (e.g., a* : is shorthand for hla-a* : ). again, the frame horizontally indicates the starting and ending positions of the epitopes; however, for visual clarity, all frames have the same vertical dimension. the details of each epitop (epitope sequence, cd + t cell stimulation, ex vivo tetramer staining frequency, and response prevalence is given in supplementary table si) . figure | prevalence and magnitude of cd + t cell responses. to determine the prevalence and the median magnitude of the cd + t cell responses toward the epitopes discovered by the hfri approach in donors were extended to additional donors expressing the relevant hla-i restriction elements. only donors sampled - days after vaccination were included. the prevalence (gray columns) and the median magnitude of the responses (black diamond) were determined for each epitope-hla combination. only epitope-hla combinations analyzed in or more donors were included. the epitopes are organized according to restriction elements. the top figure shows the hla-a restriction elements; the bottom figure shows the hla-b and -c restriction elements. it has been suggested that immunodominant epitopes can curtail responses to other epitopes (reviewed in ). the hla-a * : restricted, yfv ns b − -epitope may represent a unique opportunity to address this in an outbred human population: it represents an exquisitely dominant cd + t cell response as all hla-a * : -positive donors examined here responded to this epitope and an average of % of all activated cd + t cells from ex vivo blood samples obtained - weeks after yfv vaccination were specific for this epitope. it has recently been suggested that this massive response can be explained by the invariant cdr α loop of trav - taking part in the recognition of this epitope ( ) . in donors, who had donated blood samples at the peak of the response ( - days after vaccination), we examined whether the presence of hla-a * : , -a * : , or -a * : , could be correlated to the strength of cd + t cell responses restricted by other restriction elements, in casu all available hla-b allotypes. we included donors, which, respectively, could be split into and hla-a * : positives and negatives, and hla-a * : positives and negatives, or and hla-a * : positives and negatives; and used tetramers to examine the ex vivo frequencies of up to different hla-b-restricted responses. in the presence or absence of each of the three hla-a restriction elements, the average frequencies of each of the hla-b-restricted responses were determined leading to the generation of up to matched-pairs per hla-a. the frequencies, or magnitude, of the hla-b-restricted responses were significantly reduced in the presence vs. absence of hla-a * : (median reduction of . %, p < . ). in contrast, in the presence vs. absence of hla-a * : , which have lesser immunodominant cd + t cell responses, there was a smaller and not significant reduction (median reduction of . %, p = . ); in the presence vs. absence of hla-a * : , which have even fewer immunodominant cd + t cell responses, there was a very small and non-significant increase (median increase of . %, p = . ) (wilcoxon signed-rank test, figure ). we suggest that this may be the first demonstration of immunodomination in an outbred human population. the length of the discovered cd + t cell epitopes ranged from to mers with a predominance of mers ( ( %) mers, ( %) mers, ( %) mers, and ( %) mers) (supplementary figure s ) . this matches well with available data for peptides eluted of hla-a and -b molecules ( ) . some of the epitopes were size variants of the same peptide sequence. in six cases, such size variants were presented by the same hla-i restriction element (four cases involving two size variants each and two cases involving three size variants each, supplementary table si). we reasoned that cd + t cell recognition of these identically restricted size variants could either involve cross-recognition of shared epitope structure(s) by the same tcr(s), or involve recognition of genuinely different epitope structures by different tcr(s). to evaluate this, tetramers of the different epitope size variants and the relevant hla restriction elements were produced with unique fluorochrome labels and used to determine whether the epitope size variants were recognized by the same or different t cell populations. in some cases, distinctly defined and shared subpopulations were observed (figures a,d,i,j) indicating that one or more unique shared epitope structures were presented and recognized; in other cases, we observed shared subpopulations merging with populations that were single-stained with one of the lengthvariant tetramers (figures c,g,h,i) ; and finally, in some cases, no shared subpopulations were observed suggesting that the corresponding length-variants were presented and recognized as being distinctly different (figures b,e,f) . accommodating length-variants by extending the peptide-binding groove or by one or more aa's bulging out of the groove ( ) could affect the presented epitopes dramatically, whereas accommodating length-variants by protruding out of the n-or c-terminal ends of the groove could leave the non-protruding end of the epitope unaltered. elucidating the structural basis of these various recognition modes is beyond the scope of this paper. one of the more frequent hla allotypes, hla-b * : , offered an opportunity to compare the hfri approach with a strictly reverse immunology approach. theoretically, a total of , peptides of - mer size could be generated from the yfv proteome. netmhcpan . predicted of these as being strong hla-b * : binders at a %rank of < . %. we selected of those for further examination (supplementary table siii) . with one exception, all of these predicted binders supported hla-b * : tetramer generation, which subsequently were used to examine ex vivo obtained pbmc's from at least hla-b * : + donors. apart from the epitopes that had already been described (supplementary tables si, sii) , no additional hla-b * : -restricted cd + t cell epitopes were identified. thus, a final count can be made: combining the hfri and a strictly reverse immunology approach, a total of unique hla-b * : -restricted cd + t cell epitopes were found; the hfri strategy identified nine of these, whereas the reverse immunology strategy identified eight; seven ( %) of these epitopes were shared. assuming that the number of true positive hla-b * : epitopes is ten, then both strategies were sensitive (correctly identifying - % of the epitopes) and at the same time very specific (correctly rejecting . % of the , non-epitopes); the hfri approach being slightly more sensitive and specific than the reverse immunology approach. the major performance difference between the two strategies arose from the lower false discovery rate (fdr) where the hfri screening strategy required peptides to identify nine of the ten epitopes found (a fdr of %; albeit some of these apparently false positive peptides were eventually identified as epitopes restricted by other hla-i restricting elements expressed by the donors suggesting that the true false discovery rate of the hfri approach was even smaller), whereas the reverse immunology approach required peptides to identify eight of the ten epitopes suggesting a false discovery rate of %. in conclusion, the present hfri approach ranks epitope at the very top of the list of candidates while decimating the false discovery rate (further comparisons of hfri vs. reverse immunology is described in the discussion and detailed in supplementary results and discussion). the unbiased nature of our cohort of different hfriidentified peptide hla-i combinations covering hla-i restriction elements provided an opportunity to evaluate the performance and discriminatory power of various prediction methods such as the authoritative netmhcpan [both the contemporary version . ( ) and the most recent version . ( ) trained on both eluted ligands (el) and peptide binding affinity (ba)], and the recent mhcflurry ( ) (trained either only on ba data or on both el and ba data) ( ) and mixmhcpred (trained only on el data) ( ) . in addition to these peptide-hla-i affinity predictors, we also included a stability predictor, netmhcstabpan . ( ) . for each of these methods, predictions scores of all , peptides of length - aa that could be generated from the , aa yfv proteome were predicted for the relevant hlas (using %rank scores allowing comparisons across hla allotypes and predictors as read-outs). subsequently, a receiver operating characteristics (roc) analysis was performed and the area under the curve (auc) was determined. a non-discriminatory predictor has an auc of . , whereas a perfectly discriminating predictor has an auc of . . applied to this unbiased and validated set of epitopes, all of these predictors gave highly discriminatory auc's of . to . (supplementary figure s ) . these impressive auc's are heavily influenced by the many nonimmunogenic peptides being correctly rejected; however, this may still leave considerable room for false positive discovery rates (fdr). in this case, a more fdr-averse way to visualize the performance is to use the frank score, which is the number of false positive predictions (fp) relative to the total number of peptides (n) that can be generated from the source protein (i.e., frank = fp/n). a frank score of indicates a "perfect prediction" where a true epitope receives the highest prediction value of all peptides within the source protein and avoids any false positive predictions, whereas a frank score of . indicates a random prediction where half of the predictions are false positives. frank values were calculated for each epitope-hla pair and predictor (supplementary figure s ) . the best predictors were netmhcpan . el and mixmhcpred, which, respectively, scored and "perfect" predictions, obtained an average frank score of . and . , and a median frank score of . and . , respectively. the median, being a more "outlier-resistant" measure, would, respectively, indicate that the netmhcpan . el and mixmhcpred methods would place and false-positive non-epitopes ahead of each epitope, corresponding to a false discovery rate of and %. these numbers should be appreciated in the context of a random predictor, which would yield a fdr of %, and a perfect predictor which would yield an fdr of ca. % [assuming that only % of hla-presented peptides are immunogenic ( )]. in line with earlier work ( ) , comparing the predictive power of the various predictors in terms of the frank values, netmhcpan . el was found to significantly outperform all other predictors (p < . in all cases, wilcoxon matched-pairs signed rank test) (supplementary figure s ) . identification and validation of cd + t cell epitopes (exemplified by donor yf ) in donor yf , the ics-based screening analysis identified mer peptides as stimulating cd + t cell responses. at face value, these mer peptide sequences qualified as cd + t cell epitopes (the iedb epitope curation manual . defines a cd + t cell epitope of residues or less in length as an "exact epitope"). to identify the underlying hla class ii restriction elements, the binding of each of the mer peptides to each of the hla-dr molecules of donor yf (in casu hla-drb * : , -drb * : , -drb * : and -drb * : ) was tested in a biochemical binding affinity assay ( ) . nine ( %), eleven ( %), four ( %) and four ( %) of the epitopes bound with an affinity better than nm to one, two, three and four of the donor's hla-dr molecules, respectively ( table ) , whereas three ( %) bound to none of them. secondly, we generated tetramers for of the ( × + × + × + × ) = strongly interacting peptide-hla combinations and used these to label in vitro expanded cd + t cells from donor yf . twenty-two of the tetramers successfully identified cd + t cell epitopes and their hla-dr-restriction elements (figure ) . the final validation and enumeration of specific cd + t cell was performed by an ex vivo ifnγ elispot analysis ( table ) . in one case, the same epitope was presented by two different hla-drb allotypes and should therefore only be counted as epitope once. thus, of the different hla-dr-restricted cd + t cell epitopes observed in donor yf were identified at the tetramer level; the remaining eleven epitopes were not resolved. the latter could potentially be explained as being restricted by hla-dq or dp molecules; something that could not be readily addressed by our tetramer capabilities at the time; albeit, in one case, we successfully generated a ns b − -dpa * : -dpb * : tetramer and identified an hla-dprestricted epitope. although of the cd + t cell stimulatory peptides bound to more than one of the four hla-dr allotypes of donor yf , there was only one epitope that exploited more than one of the available hla-dr allotypes as restriction element: the ns − epitope, which was recognized by cd + t cells in the context of both hla-drb * : and hla-drb * : . that this was not a case of tcr cross-recognition was shown by double staining with the two tetramers showing two distinctly different cd + t cell populations recognizing the ns − -epitope presented by either hla-drb * : or hla-drb * : (see section recognizing the same cd + t cell epitope presented by two to three different hla-dr allotypes below). thus, in donor yf , a total of cd + t cell epitopes were identified; of these could be hla-dr or -dp tetramer validated. the cd + t cell epitope discovery strategy was extended to the same donors used for cd + t cell epitope discovery. a total of cd + t cell stimulatory mer epitopes were identified (for an overview, see figure "all cd ", and for details, see supplementary table siv) . some of these epitopes were frequently recognized. thus, the single most recognized cd + t cell epitope, enve − , was recognized in ( %) of tetramer-tested donors tested, and another epitopes were recognized in - ( - %) of donors. however, most of the epitopes were much less frequently recognized; in fact, of the peptides were recognized in only one ( %) of the donors. we suggest that the strongest and most immunodominant cd + t cell epitopes have been found. an important objective was to identify and validate the hla-dr restriction element(s) used to present these epitopes (for an overview, see figure , and for details, see supplementary table sv) . we have evaluated the restriction elements for of the epitopes. for each epitope, the most likely hla-dr restricting element was selected based on its affinity to one or more of the hla-dr allotypes available to the donor. guidance was also obtained from which hla-dr allotypes were shared amongst the epitope-responding donors. in some cases, more than one strong binding hla-dr allotype and/or more than one shared hla-dr allotype were found highlighting that multiple hla-dr allotypes would have to be considered as potential restriction elements. in total, peptide-hla-dr tetramers were generated and used to validate the cd + t cell epitopes. of these, the cd + t cell stimulatory mer peptides are given including their sequences and ics stimulation values. overlaps between two consecutive peptides are given in red. the binding affinity of the mer peptides to the four hla-drb allotypes of donor yf were measured. tetramers corresponding to the strongest binders were generated. the resulting tetramers were used to stain and analyze expanded cd + t cells by flow cytometry gating on cd + cd + t cells. in cases, staining with a hla-dr tetramer was demonstrated ( figure ) . note, that no hla-dr-restricted cd + t cell responses were found for the ns b ( - ) epitope, yafvgvmynlwkmkt. eventually, it was found to be a dpa * : -dpb * : binder, the corresponding tetramer was generated, and cd + t cell staining could be demonstrated (figure ) . finally, an ex vivo elispot assay was performed to validate the cd + t cell epitopes. tetramers were tested positive for cd + t cell staining in one or more donors. this covered cd + t cell peptide epitopes restricted by different hla-dr molecules (some epitopes were presented by more than one hla-dr allotype) and one hla-dp molecule. for of the epitopes, the hla-dr molecules available to us for tetramer generation did only partially cover the hla-dr molecules observed in one or more of the responding donors. as an example, the most frequently recognized epitope, enve − , was found in donors (supplementary tables siv, sv) . using appropriate tetramers, two restriction elements, hla-drb * : and -drb * : , were identified, however, four of the enve − responding donors expressed neither the drb * : nor the drb * : . this suggested that one or more additional, not yet identified, restriction element(s) existed for this epitope; something that could apply to more of the epitopes. for each peptide, a biochemical hla class ii binding assay was used to identify which of donor yf 's hla-ii molecules could bind the peptide and therefore could serve as restriction elements. productively interacting peptide-hla-ii combinations were used to design and generate peptide-hla class ii tetramers. the resulting tetramers were used to stain and analyze expanded cd + t cells by flow cytometry gating on cd + t cells. note, that the selective hla class ii tetramer staining of cd + , not cd + , t cells is a demonstration of the specificity of the tetramer staining. the identities of the epitopes and their restricting hla-ii elements are indicated. some of the mer peptides could stimulate cd + t cell responses restricted by two or three different hla-dr restriction elements (supplementary table sv) . no donor happened to possess three appropriate hla-dr molecules, but some did possess two and could generate appropriate cd + t cell response restricted by both of these restriction elements. in these cases, staining cd + t cells with two uniquely labeled tetramers, representing either of the two restriction elements, allowed us to address whether the same epitope presented by two different restriction elements were recognized by the same, or by distinctly different, cd + t cells. (figures a-e) . the remaining three epitopes were presented by the closely related hla-dr allotypes (hla-drb * : and -drb * : (one amino acid difference, a v g, a part of the peptide binding site interacting with p of the core sequence), ns − , ns − , and ns − , showed various degrees of cross-recognition. the ns − peptide presented by hla-drb * : and -drb * : is mostly recognized by separate t cell populations; only a small population recognized the peptide presented by both molecules (figure f ). for peptides, ns − and ns − about half of the t cells recognizing the peptides presented by hla-drb * : cross-recognized the peptides presented by hla-drb * : , with none or a very small t cell population recognizing the peptides presented only by hla-drb * : (figures g,h) . we speculate that a peptide presented by two restricting hla-dr molecules with only a few polymorphic amino acid differences may be cross-recognized by some, but not necessarily all, cd + t cells of appropriate specificity, whereas, presentation by two restricting hla-dr molecules with many polymorphic amino acid differences are more likely to be recognized as being distinctly different. in cases, two consecutive overlapping mer peptides stimulated cd + t cell responses restricted by the same hla-dr restriction element. if the two peptides of such an overlapping mer peptide pair were presented through two different core regions, one for each, then the two neighboring epitopes should be perceived as being distinctly different and should be recognized by two disparate cd + t cell populations. alternatively, if the two peptides were presented through the exact same core region, then the two neighboring epitopes could potentially be perceived as being identical and be recognized by the same cd + t cell populations. to examine this, cd + t cells were double-stained with hla-dr tetramers, which had been prepared with each of the overlapping peptides of a mer pair and labeled with a unique fluorochrome. we analyzed such pairs and found a wide variety of staining patterns. in no case did two peptides of an overlapping pair engage two distinctly different cd + t cell populations; rather, in all cases observed, the two peptides engaged at least some shared cd + t cell populations suggesting usage of shared core regions. in most cases, a plethora of shared, yet subtly different, cd + t cell populations were observed (figure ) . by way of examples, tetramers representing the overlapping hla-drb * : -restricted mer peptides, capc − and capc − , revealed multiple distinct cd + t cell subpopulations, which recognized one, the other, or both tetramers at various efficiencies ( figure a) ; whereas tetramers representing the overlapping hla-drb * : -restricted mer peptides, ns − and ns − , revealed almost exclusively cd + t cell subpopulations recognizing both tetramers, albeit clearly comprising multiple distinct subpopulations ( figure b) . we argue that this phenomenon increases and diversifies cd + t cell responses. apart from two small proteins, the aa er anchor and the aa prm proteins, all yfv proteins contained both cd + and cd + t cell epitopes. on average, the frequencies of cd + and cd + t cell epitopes were ca. and per aa, respectively (table ) . notably, the cd t cell epitopes, which have been tetramer mapped exhaustively, exhibited stretches of overlapping epitopes restricted by several different hla class i molecules: twelve stretches encompassing two epitopes, five stretches encompassing three epitopes, and three larger hot-spots areas encompassing four to six epitopes, many of which were presented by several different hla molecules. thus, the frequently recognized enve − sequence comprised six peptide epitopes and ten hla-restriction elements giving a total of twelve epitope-hla combinations (figure ) . these three hot-spot regions accounted for about of the ( %) cd + t cell epitopes identified, and encompassed hla-i restriction elements covering ca. % of the caucasian race. although the yf protein was generated as one long precursor polyprotein, no epitopes were found in any of the overlaps between the different processed proteins. no epitopes were found in any of the peptides representing products of alternative translation initiation codons. in order to understand the complexity of the human t-cell response to a circulating pathogen, and its potential impact on population dynamics of both pathogen and host, knowing a wide range of epitopes relevant for t-cell/pathogen interplay is essential. however, identifying the exact epitope sequence and the exact hla allotype involved in t cell recognition of a specific pathogen is a demanding challenge. over the years, a plethora of methods have been used to identify t cell epitopes. there are two major and principally different approaches of t cell epitope discovery. the "forward immunology" approach ( , ) uses specific t cell responses as a starting point to search for the underlying t cell epitope and its mhc restriction, whereas the "reverse immunology" approach ( , ) uses predictions (e.g., of peptide-mhc interactions) to suggest possible t cell epitopes and then screen them for their ability to stimulate specific t cell responses [reviewed in ( , , the frequencies of epitopes per aa are also given. ]. the experimental procedures involved in both of these epitope discovery modes tend to involve slow, low throughput, cumbersome and expensive processes [e.g., expression cloning of antigen libraries and/or hla genes ( , [ ] [ ] [ ] [ ] , synthesis of peptide libraries etc.]. in contrast, the bioinformatics component of a reverse immunology approach offers a process that is fast, of high capacity and throughput, yet very easy and inexpensive; a process, which is well-suited to support systematic analyses of genomic and proteomic information ( , , , ) . it is not surprising that reverse immunology has become the preferred approach to t cell discovery. the need for high speed and capacity is of obvious importance in emerging infectious diseases (including bioterrorism), and even more so in personalized cancer immunotherapy where fast and high-throughput methods are essential for the selection of relevant and safe cancer neoepitopes in real time. current peptide-mhc predictors are highly sensitive and specific [ . and . %, respectively ( ) ]. however, despite continued improvements of these predictors, the false discovery rate (fdr) is very high ( , , ) ; something that compromises the successful inclusion of one, or preferably more, t cell epitopes in cancer immunotherapy even if these encompass up to - predicted epitopes ( , ) . reducing the fdr while maintaining the sensitivity will be needed if reverse immunology in the future should fully support neoantigen discovery and secure timely, personalized immunotherapy of cancer ( ) . indeed, most of the larger cd + and cd + t cell epitope submissions to the iedb have been identified by "reverse immunology." thus, sette and coworkers used "reverse immunology" to identify dengue virus-specific t cell epitopes and have, as of july , contributed with the single largest submissions of cd + and cd + t cell epitopes (iedb reference id , , and ). in contrast, the "forward immunology" approach has fallen relatively into disuse. an innovative approach pioneered by koelle and co-workers, which has resulted in larger iedb submissions of cd + and cd + t cell epitopes (e.g., iedb reference id ), have used a "forward" component where co-transfecting panels of apc with cdna encoding antigen and hla class i or ii, each apc representing a single antigen and a single hla restriction element, were used to interrogate cd + and cd + t cell responses of virus infected donors ( ) . the "forward" component of this approach identified intact immunogenic protein antigens and their restriction element(s); however, for the epitope discovery part of this work, the entire antigen was subjected to a "reverse" component predicting the epitope(s) and its hla restriction element(s). another innovative approach, tetramer guided epitope mapping (tgem), pioneered by kwok and james, which has resulted in large cd + t cell epitope submissions [iedb references id ( ) , , , and ], have also used a "forward" component. longer overlapping peptides representing entire antigens were offered to single hla class ii molecules and the resulting peptide-hla class ii complexes were multimerized and the ensuing tetramers used to interrogate cd + t cell responses of appropriate donors. using shorter overlapping peptides suitable as cd + t cell epitopes, maeurer and coworkers established a tetramer-based approach for cd + t cell epitope discovery, which also resulted in larger iedb submissions [iedb id ( ) ]. this latter approach would obviously be very peptide intensive if every relevant peptide was to be tested in that way (e.g., the yellow fever proteome would require peptides to represent all possible - mer peptides). here, we have generated a "hybrid forward-reverse immunology" (hfri) approach capable of doing concurrent cd + and cd + t cell epitope discovery and demonstrated that it can perform large-scale epitope discovery and at the same time decimate the false positive discovery rate. for the initial "forward immunology" screen, we used an overlapping peptide library of mer peptides overlapping by aa, which represented the entire , aa yellow fever virus proteome, to stimulate pbmc's obtained ex vivo from primary yellow fever virus vaccinees at the peak of the resulting t cell response. since mer peptides are further processed during in vitro ics and/or elispot assays, this peptide library represented all possible yfv-specific cd + and cd + t cell epitopes of up to aa in length; some, but not all, epitopes of - aa in length; and no epitopes of a length longer than aa. distributing this peptide library into matrices, the initial screening effort could be reduced to testing ca. peptide pools for their ability to stimulate t cell responses preferably by ics analysis. the matrix design subsequently allowed us to home in on the individual t cell stimulatory peptides. following the "forward immunology" screening, a "reverse immunology" approach was applied to all the mer peptides containing cd + t cell epitopes. in silico, the affinities of all possible - mer peptides that could be generated from the mer were predicted in the context of up to different hla-a, -b and -c allotypes per individual. this reduced the number of potential peptide-hla-a, -b or -c combinations from per stimulatory mer peptide to typically one to three combinations. the most likely peptide binders were synthesized and used to generate appropriate peptide-hla-i tetramer(s), which subsequently were used to validate cd + t cell epitope(s). for the vast majority of t cell stimulatory mer peptides, at least one epitope was identified per mer peptide. once the stimulatory mer peptides had been identified, predicting the exact epitope and its restriction element was a highly efficient process; typically, the epitopes ranked first, second or third amongst the many potential epitope-hla combinations. as a cost-saving measure, if the predictions clearly discriminated between the candidates, a consecutive process was applied whereby the top peptide(s) were synthesized and tested before any next tier peptides were synthesized and tested. this hfri approach was extended to primary yfv vaccinees, where it identified and tetramer-validated cd + t cell epitopes (predominantly of size - mer, range - mer) covering hla-i allotypes (representing a total of peptide-hla-i combinations). before this work, the iedb had registered ten yfv-specific cd + t cell epitopes as being "exact epitopes" (i.e., length from to aa) and restricted by an hla allotype defined at high ( -digit) resolution; however, none of them were tetramer validated. four of the ten already registered yfv-specific, cd + t cell epitopes were included in the epitopes identified here. thus, the present approach identified and validated - = new, or ca. % of all currently known, yfv-specific, cd + t cell epitopes. the total number of exact cd + t cell epitopes with high resolution hla-i restriction, which are currently registered in the iedb is , of which , have been tetramer validated (extracted from the iedb, july ). thus, this study accounts for > % of these tetramer-validated human cd + t cell epitopes. to evaluate the prevalence of the different yfv-specific cd + t cell immune responses, the tetramer analysis was extended to additional vaccinees with the appropriate hla-i allotypes. many epitopes were frequently observed (i.e., were highly prevalent) in vaccinees with the appropriate hla allotype. thus; (ca. %) and (ca. %) of cd + t cell epitopes were observed in ≥ and ≥ %, respectively, of vaccinees with the appropriate hla-i allele. conversely, (ca. %) of hla-i allotypes presented at least one cd + t cell epitope with a prevalence of ≥ %. thus, the hfri approach identified a cohort of immunodominant yellow fever-derived peptides, which could be of broad diagnostic and therapeutic interest. large-scale t cell epitope discovery could also address more fundamental issues in immunobiology. pertinent examples of phenomena that are poorly understood include the closely related immunodominance (that the immune response is focused on just a few of the many available determinants expressed by a pathogen) and immunodomination (that the immune response of one specificity can suppress the response of another specificity). not surprising, these phenomena are closely related to antigen processing and presentation including mhc and t cell repertoire ( ) . the vast majority of experimental data on immunodominance and immunodomination emanates from studies involving inbreed mice. few studies in humans address immunodominance [e.g., ( ) ]; to the best of our knowledge none involve immunodomination. the latter is particularly difficult to address in an outbreed system like the human where the extremely diverse hla creates context dependent effects that confounds attempts to address immunodomination. assuming that the context-dependent effects hla could even out in larger donor cohorts, we exploited the size of our study to ask whether the presence of hla-a * : , which restricts a strongly immunodominant, ns b − -specific t cell response, would correlate with a reduction of responses restricted by other hla allotypes. indeed, under these conditions, we could demonstrate such a correlation in the presence of hla-a * : , but not in the presence of hla-a * : or -a * : . note that the hla-a * : or -a * : allotypes themselves featured a hierarchy of immunodominant t cell responses i.e., they are valid hla restricting elements. this may be the first demonstration of primary anti-virus responses being subjected to immunodomination in humans. a further analysis of the mechanism of behind these phenomena is beyond the scope of this paper. hla-c restricted, cd + t cell epitopes were scarcely represented (< %) in the iedb; something that potentially could be explained by hla-c being insufficiently investigated. a priori, we expected that the unbiased nature of our approach would reveal several hla-c restricted cd + t cell epitopes, however, we only found one case of a strong and highly prevalent cd + t cell response, which could not be explained by any of the hla-a or -b allotypes available to the responding donors. instead, a strongly predicted binding to a shared hla-c allotype amongst the responding donors suggested an hla-c * : restricted response. eventually, two hla-c * : restricted epitope length variants; ns − (trrflpqil) and ns − (rrflpqil), were tetramer validated. these were the only hla-c restricted cd + t cell epitope identified; all other identified cd + t cell epitopes were validated as being either hla-a or -b restricted. hla-c is less polymorphic and is known to be expressed at a lower level than hla-a and -b ( ) ( ) ( ) ( ) ; something that has been correlated with reduced cytotoxic t lymphocyte responses ( , ) . in the case of the hla-c * : -restricted ns − (trrflpqil) epitope identified here, any reduced expression level of hla-c * : might have been compensated by the very strong predicted binding affinity for ns − . although weaker hla-c-restricted cd + t cell responses may have been missed, we would argue that it is unlikely that we have missed strong and prevalent hla-c restricted cd + t cell epitopes. thus, we suggest that the paucity of strong hla-c restricted cd + t cell responses, at least in an acute viral infection like yellow fever virus, is not due to hla-c having been neglected in the scientific literature, but rather reflects a true biological phenomenon. notwithstanding, future cd + t cell discovery efforts should include hla-c, in particular if one or more hla-c restricted epitopes can be suggested in a situation where there are no obvious hla-a or -b restricted candidates. concurrent with cd + t cell discovery, the "forward-reverse immunology" approach also allowed hla-ii-restricted cd + t cell epitope discovery. the initial matrix-driven "forward" analysis of donors identified cd + t cell stimulatory yfv-derived mer peptides. this suggests that cd + t cell epitopes are more numerous than cd + t cell epitopes, perhaps as much as - times greater. if generalizable, this would have important implications for cd + t cell immunity since, everything else being equal, it would be more difficult for a microorganism to escape many cd + t cell epitopes than fewer cd + t cell epitopes. addressing the number of immunogenic open reading frames, other have also hinted at a greater preponderance of cd + than cd + t cell epitopes ( , ) ; to the best of our knowledge, ours is the first proteome-wide study that have made this observation at the epitope level. the identification of the restricting hla class ii element(s) is a serious challenge in part due to different hla-ii allotypes having overlapping peptide binding repertoires ( ) . in fact, this problem is so manifest that sette et al. have developed a panel of different single hla-ii transfected cell lines to identify hla-ii restriction elements ( ) . it would be ideal if hla-ii restrictions could be identified by predictors and then validated by tetramer analysis. unfortunately, the contemporary cd + t cell epitope discovery tools were immature (e.g., the early netmhciipan predictors were relatively inefficient and focused solely on the hla-dr isotypes), and access to peptide-mhc class ii tetramers was very limited. moreover, ex vivo frequencies of tetramer-positive cd + t cells tend to be < . %, which make them difficult to detect. thus, our cd + t cell epitope discovery process was not exhaustive; however, as cd + t cell discovery tools mature, we believe that the efficiency of cd + t cell epitope discovery eventually should approach that of cd + t cell epitope discovery. here, using a panel of recombinant hla-dr molecules, we measured the binding affinity of the overlapping mer peptides to the most common hla-dr allotypes. for each stimulatory mer peptide, this suggested which of the donor's hla-dr molecules should be used to generate peptide-hla-dr tetramers for validation of cd + t cell epitopes. this "brute force" approach was extended to donors, where we tetramervalidated cd + t cell epitopes covering different hla-dr allotypes (and one hla-dp allotype). as of july , the iedb has registered a total of , yfv-specific cd + t cell epitopes as being "exact cd + t cell epitopes" (i.e., length aa, or less) and restricted by an hla-ii defined at high (i.e., -digit) resolution; of which have been tetramer-validated. thus, the tetramer-validated yfv-specific cd + t cell epitopes reported here represents a significant increase in the number of tetramer-validated cd + t cell epitopes. it should be noted that james and coworkers have identified and tetramer-validated different yfv-specific cd + t cell epitopes [iedb reference id ( ) ] that are aa long and therefore fall just outside the definition of an exact cd + t cell epitope. a detailed examination of cd + t cell responses revealed a phenomenon that could have profound biological and practical implications for cd + t cell recognition. in many cases, two consecutive overlapping mer peptides stimulated cd + t cell responses, which were restricted by the same hla-dr restriction element. when the responding cd + t cells were double-stained with hla-dr tetramers, which had been prepared with each of the overlapping peptides of a mer pair and labeled with a unique fluorochrome, we observed a plethora of different, yet partially shared, cd + t cell specificities. situations where overlapping peptides are presented must occur regularly in vivo since experiments sequencing natural peptides eluted of hla-ii molecules frequently find large series of staggered peptides surrounding each core region ( ) . exploiting this wealth of closely related peptides to engage a large number of different cd + t cell specificities recognizing the same core region in slightly different ways [something that actually was noted years ago ( ) ], may represent a biologically significant diversification mechanism of cd + t cell responses reducing the risk of pathogen escape and increasing the chances of recognizing a given target. this phenomenon is also important for how cd + t cell responses should be analyzed. a single peptide-hla-ii tetramer is likely to engage a range of t cells of various avidities for the tetramer; something that might explain why single hla-ii tetramers often appear to label a poorly defined, non-base line separated, mono-specific cd + t cell population of low frequency. if examined with two "overlapping" tetramer, this could in reality turn out to be a heterogeneous collection of better defined and separated cd + t cell populations of a higher accumulated frequency. from a practical perspective, this implies that double staining involving two overlapping peptide-hla-ii tetramers will be needed to faithfully enumerate and monitor any cd + t cell response. from a technical perspective, this will increase the observed frequencies of specific cd + t cells for a given specificity (i.e., increase the sensitivity of the analysis), and it will increase the resolution of the flow cytometric analysis since it may separate various positively staining subpopulations and provide a better discrimination against negatively staining cd + t cells. the t cell epitope discovery approach described here has several advantages. in the forward immunology component, an overlapping peptide library is used to search for peptides containing cd + or cd + t cell epitopes. in the subsequent reverse immunology component, pan-specific predictors are used to identify the underlying epitope and its hla restriction element. these steps can be done in any (obviously outbred) individual of any hla haplotype using simple and standardized conditions. this reduces the number and combinations of peptides and hla allotypes that should be considered for peptide-hla tetramer generation and used in the final validation of the discovered t cell epitopes. as shown here, this approach is efficient and, not surprisingly, it reduces the false discovery rate. as peptide-hla class i and ii predictors and tetramer technologies mature, this approach will eventually be able to cover all frequently found hla class i and ii iso-and allotypes. this approach is systematic, all-inclusive, complete, and global in the sense that it includes all protein antigens and peptide epitopes, encompasses both cd + and cd + t cell epitopes, and can be extended to all individuals of all populations. this approach could be extended to other attenuated live virus vaccines (e.g., those targeting measles, mumps, rubella, polio, and hpv). compared to a strictly reverse approach, significant disadvantages of the hfri approach include the time and cost associated with establishing a complete overlapping peptide library as well as using a cellular readout as an initial selection step. therefore, this will probably not be justified if the aim is to identify epitopes in an urgent effort involving one donor (e.g., for cancer immunotherapy purposes); rather, it would be appropriate if the aim is to examine a large panel of donors in order to get population-wide data including immunodominance, candidates for diagnostics and vaccine development for infectious disease purposes (examples include a range of emerging and re-emerging infectious diseases like hiv, sars, mers, chikungunya, dengue, west nile, zika, ebola and sars-cov- ). for all of the above examples, the proteomes are small enough that their entire proteomes could be addressed by an overlapping peptide strategy using the number of pbmc's that could be obtained from a donor. addressing larger pathogen proteomes (e.g., herpes virus, bacteria or parasites; or smaller highly variable virus like hiv and dengue) in their entirety would require either a selection process downsizing the source target protein antigens, or the development of novel miniaturized, yet high-capacity, technologies. one could envision that future investigations of emerging diseases would include population-wide t cell epitope discovery efforts using blood samples from patients, convalescents and/or longterm survivors, which all possess important information on t cell epitopes and responses. similarly, one could envision that approval and registration of new vaccines could include population-wide analysis of t cell epitopes and responses. one could also envision that population-wide information on t cell epitopes and immunodominance could be used to design subunit vaccines. another important advantage of the forward-reverse approach presented here is the unbiased nature of the t cell epitope discovery process. whereas, current data-driven bioinformatics peptide-mhc predictors are quite accurate, the need for even better predictors stresses not only the need for high-quality training data, but also the need for high-quality validation data. in this context, there is an inherent problem in most epitope discovery efforts being dependent on peptide-mhc predictors since this effectively means that current t cell epitope discovery submissions tend to be biased by current predictors; something that might compromise the validation and benchmarking of predictors. having reasoned that our forward-reverse approach captures about % of the true t cell epitopes, we would like to propose that the resulting data is largely unbiased and should serve as an appropriate benchmark [others have reached similar conclusions ( )]. as an example, we used the cd + t cell epitopes identified here to benchmark current predictors. all current predictors were quite efficient and accurate. the newer predictors, some of which included immunopeptidomics and therefore may also reflect antigen processing, were better than the older predictors [as also noted by others ( ) ]. however, these improvements are incremental and even the newest predictors were afflicted by high fdr's. taken together, this could be interpreted as a need for a change in how we predict t cell epitopes that is more fundamental than merely acquiring more peptide-mhc affinity and/or stability data e.g., by including t cell receptor specificities and repertoire propensities. a source of unbiased t cell epitope data would be instrumental in improving predicting tools. in conclusion, for smaller proteomes, it is possible to design a limited set of overlapping peptides spanning the entire proteome and use these to reveal the vast majority, if not all, specific cd + and cd + t cell responses concurrently; use predictors to identify the underlying combination of peptide epitopes and mhc restriction elements; and finally use this information to construct suitable peptide-mhc multimers and validate the t cell epitopes discovered. performing this in cohorts of patients or vaccinees allows for a systematic, global and cost-efficient analysis of cd + and cd + t cell epitopes, and evaluation of their immunodominance. as previously described ( ) , this study was approved by the danish national committee on health research ethics (protocol # h- - - ), and the collection of data and cells was approved by the danish data protection agency (permission - - ). all volunteers gave written informed consent prior to participation. based on previous yfv vaccination history and their international card of vaccination, healthy volunteers, who for traveling purposes were about to receive a primary yfv vaccination, were recruited. the attenuated yfv vaccine, d- (sanofi pasteur; marketed as stamaril in more than countries globally and as yf-vax in the usa) was administered intramuscularly. about % of the volunteers received an yfv vaccination only, whereas the remaining % received additional vaccines, typically killed, inactivated or subunit vaccines; in no case was the yfv vaccine co-administered with another live attenuated vaccine. as previously described ( ) , blood samples were obtained just prior to and after the yfv vaccination (typically day - post vaccination, range - days). peripheral blood mononuclear cells (pbmc) were isolated by density gradient centrifugation using ficoll-paque tm plus (ge healthcare europe, brøndby, denmark). they were either examined directly ex vivo or cryopreserved in % dmso and % fcs at − • c for later in vitro analysis. chromosomal dna was prepared from the pbmc's and sequence-based typing (sbt) was used to perform highresolution (i.e., digit) hla-typing (genome diagnostics, utrecht, the netherlands). all loci encoding classical hla molecules were typed i.e., the three class i ioci, hla-a, -b, -c and the six class ii loci, hla-drb , -drb / / , -dqa , -dqb , -dpa , and -dpb . the pbmcs were analyzed ex vivo for the t cell markers, cd , cd , and cd , and the extracellular t cell activation markers, cd and hla-dr as previously described ( ) . briefly, pbmcs were incubated with fluorochrome-conjugated anti-cd , -cd , -cd , -cd , and -hla-dr antibodies for min at room temperature, washed, fixed with % formaldehyde, and analyzed by flow cytometry (lsr-ii, bd biosciences) using diva software. all antibodies were obtained from biolegend (san diego, ca, usa). all peptides were synthesized by standard fluorenylmethyloxycarbonyl (fmoc) chemistry and purified by reversed-phase high-performance liquid chromatography (purity at least %, usually > %), mass spectrometry validated and lyophilized (schafer-n, copenhagen, denmark). an overlapping peptide library systematically covering the entire proteome of the vaccine strain, yf- d- (uniprot# p ), was synthesized. this encompassed the entire yf precursor protein of aa, which could be represented by peptides, each aa long and overlapping its neighboring peptides by aa. in addition, peptides, which were predicted to be binders to hla-a or -b supertype representatives by our netmhcpan predictor, were selected from putative products of alternative translation initiation codons ( ) . one hundred and seven ( , or . %) of the total of selected peptides were difficult to synthesize and/or purify; many of which had long stretches of hydrophobic aa. adding one or two lysine to their n-or c-termini allowed the successful synthesis and purification of of these "difficult to synthesize" peptides leaving peptides that could not be synthesized and/or purified. thus, ( %) of the selected peptides could be included in this epitope screening effort. these peptides were initially organized in a × matrix, and eventually in four × matrices (supplementary figure s ). fresh or thawed pbmcs were tested using an interferon-γ (ifnγ) specific eli spot assay as previously described ( ) . briefly, - × cells/well were plated in an eli spot plate (mahas , merck millipore, usa) and in vitro cultured for - h in media supplemented with or without peptide at . µm [or, as positive control, with µg/ml staphylococcal enterotoxin b (seb, sigma aldrich, st. louis, usa)]. an ap conjugate substrate kit (bio-rad) was used for visualization of spots. eli spots were counted using a ctl immunospot series uv analyzer. immunospot . . software (c.t.l., shaker heights, usa) was used for analysis. wells with spotforming units spu > times the background wells were considered positive. pbmcs were incubated overnight ( • c, % co ) in x-vivo media (fisher scientific) supplemented with % ab serum (invitrogen) and a mixture of relevant peptides at a concentration of . µm of each peptide. the cells were harvested and washed, and subsequently plated in -well plates at a concentration of × /ml supplemented with u/ml il- for expansion. fresh media and il- were supplemented every second day until the cells were harvested at day , and il- ( ng/ml) was added the last days. in vitro cultured pbmc's were harvested, washed, and aliquoted at - × cells/well in a round bottom well-plate. the in vitro cultured pbmc's were stimulated for h at • c, % co with, for the matrix analysis, each of the column and row mixes ( µm/peptide), and for the epitope identification with a single peptide ( . µm). after h of stimulation µg/ml brefeldin a (bd biosciences) was added. the cells were subsequently permeabilized (becton dickinson permeabilizing solution ) and stained with anti-cd -pacific blue, anti-cd -percp, anti-cd -apc, anti-cd -pe, and anti-ifnγ-fitc, according to the "fastimmune" protocol (becton dickinson). the cells were subsequently analyzed by flow cytometry using a lsrii (bd biosciences). the gating strategy is illustrated in supplementary figure s . staining of more than . % of cd + or cd + t cells was considered positive. hla class i tetramers were produced as previously described ( ) . briefly, recombinant, biotinylated hla class i heavy chain, human β -microglobulin and peptide were incubated in mm tris-maleate ph . hla-dra and hla-drb chains were produced as previously described ( ) . for tetramer production, hla-dra and hla-drb chains were mixed in a : . ratio and incubated in µm peptides in pbs (ph . ) with % glycerol and . % pluronic f for h at • c. the resulting monomers were buffer changed into pbs with % glycerol and concentrated on kd vivaspin (satorius) and quantitated by luminescent oxygen channeling immunoassay (loci)-driven assay ( ) . the resulting monomers were tetramerized by addition of fluorochrome labeled streptavidin (streptavidin-phycoerythrin (sa-pe) or streptavidin-allophycocyanin (sa-apc); both from biolegend) sequentially over min at a : molar ratio of streptavidin to monomer. for t cell analysis, pellets of × cells obtained from in vitro peptide stimulated cell cultures, were re-suspended in a µl tetramer diluted in media to a final concentration of ca. nm, and incubated for h at • c, followed by min incubation with fluorochrome conjugated anti-cd , -cd , -cd , and -hla-dr antibodies. the cells were analyzed by flowcytometry (fortessa or lsr-ii, bd biosciences) using diva software. for each donor, all mer peptides eliciting a cd + t cell response were submitted to our bioinformatics predictor, netmhcpan ( ) , which returned a prioritized list of predicted optimal epitopes, which could bind to any of the up to six hla-a, -b, or-c molecules of the donor in question. for each donor, all mer peptides eliciting a cd + t cell response were submitted to our bioinformatics predictor, netmhciipan ( ) , which returned a prioritized list of predicted epitopes including a predicted core-region, which could bind to any of the up to four hla-drb , or-drb / / molecules of the donor in question. the stability of peptide-hla class i complexes was measured using dissociation of i radiolabelled β m in a scintillation proximity assay (spa) as previously described ( ) . briefly, recombinant, biotinylated hla class i heavy chains were diluted into a refolding buffer containing the test peptide and trace amounts of i radiolabeled β m, and allowed to refold at • c for h in a streptavidin-coated scintillation microplate (flashplate plus, perkin elmer, boston, ma). dissociation was initiated by adding excess unlabeled β m and placing the microplate in a scintillation counter (topcount nxt, packard) adjusted to • c. reading the microplate continuously for h allowed determination of the dissociation of radiolabeled β m. peptide-hla-ii binding affinities were determined using a previously described luminescent oxygen channeling immunoassay (loci)-driven assay ( ) . briefly, denatured and purified recombinant hla-ii alpha and beta chains were diluted into a refolding buffer (tris-maleate buffer, ph . ) with graded concentrations of the test peptide, and incubated for h at • c to allow for equilibrium to be reached. the peptide concentration leading to half-saturation (ed ) was determined as previously described ( ) . under the limited receptor concentrations used here, the ed reflects the affinity of the interaction. graphpad prism was used for statistical analyses (unpaired and paired mann-whitney-wilcoxon tests, unpaired and paired t-tests, fishers exact test, and roc analysis). all datasets presented in this study are included in the article/supplementary material. towards a systems understanding of mhc class i and mhc class ii antigen presentation immunodominance in major histocompatibility complex class i-restricted t lymphocyte responses description and prediction of peptide-mhc binding: the human mhc project identifying cytotoxic t cell epitopes from genomic and proteomic information: the human mhc project the immune epitope database and analysis resource program - : reflections and outlook a case for a human immuno-peptidome project consortium computational tools for the identification and 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peptide binding to any hla-dr molecule of known sequence: netmhciipan real-time, high-throughput measurements of peptide-mhc-i dissociation using a scintillation proximity assay the authors thank doctors and nurses at the two recruiting centers and the blood bank of the university hospital, and members of the buus laboratory for expert technical assistance. the studies involving human participants were reviewed and approved by danish national committee on health research ethics (protocol # h- - - ). the patients/participants provided their written informed consent to participate in this study. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material key: cord- -wpub krf authors: benmamar-badel, anouk; owens, trevor; wlodarczyk, agnieszka title: protective microglial subset in development, aging, and disease: lessons from transcriptomic studies date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: wpub krf microglial heterogeneity has been the topic of much discussion in the scientific community. elucidation of their plasticity and adaptability to disease states triggered early efforts to characterize microglial subsets. over time, their phenotypes, and later on their homeostatic signature, were revealed, through the use of increasingly advanced transcriptomic techniques. recently, an increasing number of these “microglial signatures” have been reported in various homeostatic and disease contexts. remarkably, many of these states show similar overlapping microglial gene expression patterns, both in homeostasis and in disease or injury. in this review, we integrate information from these studies, and we propose a unique subset, for which we introduce a core signature, based on our own research and reports from the literature. we describe that this subset is found in development and in normal aging as well as in diverse diseases. we discuss the functions of this subset as well as how it is induced. the term "microglia" was brought to the scientific community's attention a century ago with its first use by pio del rio-hortega ( ), who strived to distinguish them from oligodendrocytes. his early work also highlighted their phagocytic ability, as well as their potential to undergo morphological changes. this early description led the community to consider microglial cells as a homogeneous population, even though the first description of a microglial subset ("satellite microglia") appeared as early as ( ) . microglia originate from yolk-sac progenitors that start migrating toward the fetus around midpregnancy. these progenitors reach the embryonic brain around embryonic day (e) . -e . ( , ) until the formation of the blood-brain barrier around e . -e . in the mouse, and between the th gestational week to the th gestational week in the human ( , ) . as such, they are among the first cells to colonize the developing brain, and they participate in central nervous system (cns) development. for instance, they contribute to refine brain wiring through enhancing both synapse formation ( , ) and elimination ( , ) , they modulate axonal growth ( , ) , they secrete factors promoting neuronal progenitors survival ( ) helping with neuronal positioning ( , ) , and they participate in the clearance of live and apoptotic cells during development ( ) . microglia also take on physiological functions in the adult cns, as they constantly sense their immediate environment, in a so-called "never-resting state" ( , ) . our knowledge of microglial physiology and process motility relies heavily on studies in anesthetized animals. understanding of microglial functions in the steady state is challenged by a recent study showing that microglial process motility and morphology are affected by the wakefulness state of mice ( ) . aside from this surveillance immune function, they are also fundamental for regulation of social behavior, learning, and memory, as these functions are impaired upon their depletion and restored after repopulation ( ) . microglial roles in injury and disease contexts have been investigated extensively, with new advances contributing to deepen our understanding of microglia and their effect on other glial cells [reviewed in greenhalgh et al. ( ) ]. these physiological functions advanced our view of microglia, from being initially thought of as exclusively sentinel cells reacting in the context of injury. this dated view on microglia led to the superposition of macrophage m /m phenotypes onto them ( ), which was an early attempt to grasp the extent of microglial diversity. this classification is however mostly obsolete nowadays, as it was proved to be simplistic and disconnected from in vivo reality ( ) . indeed, the variety of functions microglia take on in space, time, and health states along with reports of sex differences in microglial function have led the community to infer a greater microglial heterogeneity than initially thought. with the progress of technology, investigating such diversity has become possible, notably through the development of high-throughput techniques such as mass cytometry and with the recent advances in transcriptomic studies with single-cell rna-sequencing (rnaseq). these technologies allowed the identification of microglial signatures linked to their "activation state." in , butovsky et al. described a "homeostatic" microglial signature, comparing microglia with monocytic populations and other cns cells ( ) . this signature includes genes such as p ry , fclrs, tmem , hexb, mertk, cx cr , csf r, etc. that have been used in numerous studies thereafter to identify microglial cells. this was a fundamental step in distinguishing resident microglia from other tissue-resident macrophages and infiltrates in disease context. this "homeostatic" signature was more recently revised and extended to developmental stages in addition to adulthood by matcovitch-natan et al. ( ) . in this study, single-cell rna-seq helped associate the microglial signature identified at each different age to the potential functions these cells take on during life. they pinpointed three different temporal stages of development, each linked to a particular signature: early microglia associated with proliferation and differentiation, pre-microglia related to neuronal development, and adult microglia. it has recently been suggested that microglial heterogeneity peaks early during development and then reaches a minimum in the homeostatic adult brain, only to regain diversity in old age ( ) . in addition, some microglial subtypes have been based on surface markers and sometimes function [discussed in stratoulias et al. ( ) ]. this has been mostly achieved through systematic transcriptional investigation of microglia in different contexts. however, because every study is done with different techniques (microarrays, bulk rna-seq, single-cell rna-seq, etc.), on different kinds of samples (whole brain, sorted microglia based on different gating strategies, microdissected microglia, sorted nuclei, etc.), and in different animal models, there is a risk for confusion of data. we believe that there is a need for an overview-by looking at the big picture, common patterns can be identified between studies that might otherwise have been overlooked. in this review, we summarize and interpret transcriptomic studies on microglia from development, homeostasis, and disease states to bring to light a subpopulation common to all these different states. we discuss the factors inducing this subpopulation and its functional importance in all of the studied conditions. finally, we provide a core signature for this subset and propose to systematize and unify the naming of this microglial subpopulation to clarify the literature and avoid redundancy in future studies. we propose to use a name already used in numerous studies and that accounts for these cells' expression signature: cd c+ microglia. for long, microglia have been considered simply as macrophages, due to the belief that all macrophages emerged from the bone marrow. consensus that a subset of microglia expressed cd c was therefore at first difficult to achieve. cd c was widely accepted as a marker for dendritic cells (dcs), to the extent that some studies have used it as the sole identifier for dcs. added to this was the constant difficulty of discriminating cnsresident parenchymal microglia from blood-derived myeloid cells, with which they share many markers [reviewed in amici et al. ( ) ]. until recently, it was indeed not possible to reliably discriminate microglia, especially activated microglia, from blood-derived monocytic myeloid cells, using morphology or routine myeloid markers. panels of differentially expressed genes that can be used to distinguish microglia including tmem ( ) and the homeostatic marker p ry ( ) were however recently identified and validated in both homeostatic and disease conditions ( ) . to our knowledge, the first observation of microglia expressing cd c was made in human multiple sclerosis (ms) tissue by immunohistochemical analysis ( ) . one, however, cannot be completely certain of the exclusive microglial nature of the cells identified in this study based on the markers used and our current knowledge of myeloid cell marker expression patterns. the first report to explicitly identify cd c+ cells in the cns as microglia came from butovsky et al. in ( ) . they identified populations of cd c+ cells in a mouse model for alzheimer's disease (ad) as microglia, based on their location and co-expression of isolectin b and cd b, although these cells showed a dendritic morphology. the major point of interest in that study was the observation that all mhc-ii+ microglia that engulfed amyloid β in the brain of glatiramer acetate (ga)vaccinated transgenic (tg)-ad mice co-expressed cd c. also, relevant to our subsequent studies, these cells could be stained with an antibody specific for insulin-like growth factor (igf ). a "gold standard" for microglial identification remains their relatively low level of expression of cd in flow cytometry analyses ( ) . in the course of study of glial responses in the dentate gyrus to axonal transection in the entorhinal cortex (the perforant path lesion model), we noted a subpopulation of cd low cd b+ cd c+ cells in flow-cytometry profiles of cells isolated from lesion-reactive hippocampus. their functional significance and whether they derived intraparenchymally or by immigration from bone marrow were not determined (babcock and owens, unpublished). exactly similar cells were then observed in cuprizone-demyelinated corpus callosum ( , ) . these were described to express slightly higher levels of cd than their cd c− counterparts, while remaining within the cd low gate ( , ) . in addition, they did not express ccr characteristic for infiltrating leukocytes and expressed high levels of cx cr supporting their microglial status ( ) . further analysis showed that cd c+ microglia were also induced in experimental autoimmune encephalomyelitis (eae) ( ) ( ) ( ) ) and a mouse model for neuromyelitis optica (nmo) ( ) , as well as during postnatal development ( , ( ) ( ) ( ) . in older studies, ambiguity in assigning cd levels resulted in cd b+ cd c+ populations in cns of mice with eae or infected with toxoplasma gondii being identified as dcs ( ) , although, with hindsight, consideration of bimodal cd profiles allows that at least some of them may have been microglia. the fact that cd c+ microglia express slightly higher cd levels than resting microglia may have contributed to uncertainty, and claims that dcs derived from microglia ( , ) may need re-evaluation. relative cd levels as detected by flow cytometry are not as useful for histological discrimination. depending on the antibodies and staining protocols used, microglia may even not be detected as cd + cells, or else cannot be distinguished from other cd hi cells. similarly, cd c promoter-driven fluorescent reporter transgenic mice cannot discriminate between the many cell types that can express or upregulate cd c without co-staining for lineage-specific markers. identification of cd c+ microglia in such mice relies on interpretation of sometimes fortuitous observations that include consideration of a cell's morphology and location. using an eyfp-cd c transgenic strain, bulloch et al. identified a small fraction of cd c+ microglia that were immunoreactive for mac- , iba , cd , and f / ( ) . the parenchymal juxtavascular iba + cd b+ gfp-cd c+ cells described by prodinger et al. in a cd c-gfp reporter mouse likely included microglia, although in a non-diseased mouse, they would only account for around % of them ( ) . flow-cytometric analysis confirmed cd low gfp-cd c+ cells in the cns of these mice ( ) . the fact that they were mhc ii-negative likely reflects that they derived from non-diseased tissue, unlike the eae-derived cells that we described ( ) . typical microglia markers and their functions are listed in table . even before microglia were formally identified, the presence of fat-laden cells had been reported and suggested to be a part of ( ) the normal developing cns ( ) ( ) ( ) , and to participate in either cell death processes ( ) or myelin formation ( ) ( ) ( ) . early after the initial description of microglial cells, neuroanatomists began to track and map microglia in the cns. del rio-hortega was the first to describe "fountains of microglia" in the developing brain, having amoeboid morphology and being preferentially located in the white matter ( ) . already in , penfield reported that what he describes as "neuroglia of mesodermal origin" "were variously considered to be normal and having to do with myelination or to indicate an abnormal inflammatory process" ( ) . in the mid-to late s, with del rio-hortega's "fountains of microglia" in mind, these cells were investigated again using light and electron microscopy. most studies describe round, amoeboid, highly vacuolated cells with fat-containing granules, which are found in developing white matter, particularly along unmyelinated axonal tracts in the corpus callosum of rabbits ( ) , rats ( ), mice ( ) , birds ( ) , fish ( ) , and humans ( ) , as opposed to more highly ramified cells present in the gray matter. in all these studies, amoeboid or ovoid-shaped microglia invade the white matter before disappearing when increasing numbers of ramified microglia colonize the gray matter (peaking around postanatal day (p) and disappearing around p to p in rodents). multiple studies support this finding and extrapolate their potential function, stating either that they have enhanced phagocytic abilities for the elimination of apoptotic material coming from normal developmental cell death or that they participate in myelination ( , , ( ) ( ) ( ) ( ) . this involvement in myelination was reinforced by a study by pont-lezica et al. showing that microglial alteration early in development leads to impaired corpus callosum fasciculation ( ) . their phagocytic abilities along with their morphology provoked debates regarding their origin ( ) , their fate ( ), and even their microglia status with some studies modifying the nomenclature by referring to them as "brain macrophages" rather than "amoeboid microglia" ( , ) . with the new notion of microglial phenotypes emerging, these early amoeboid microglia were hypothesized to have higher "activation" levels before becoming "deactivated" in a controlled manner, as this was believed to be temporarily helpful to scavenge debris coming from developmental cellular death. to corroborate this hypothesis, hristova et al. attempted the first phenotypic analysis of these cells, and reported expression of high levels of integrins alpha x (itgax, cd c), alpha (itga ), alpha (itga ), and beta (itgb ) in microglia from periventricular white matter in comparison to cortical microglia at p by staining quantification in iba + cells ( ) . in addition, in situ hybridization clearly showed transient igf and colonystimulating factor (csf ) mrna expression within microglial cells in the corpus callosum and periventricular white matter until approximately two postnatal weeks ( ) . in this study, expression of igf and csf by microglia were hypothesized to play a protective role, preventing axonal damage for instance, which has since then been confirmed in a study by ueno et al. ( ) . this finding was reinforced by our own study showing that microglial cells expressing high levels of itgax and igf are present in the white matter (cerebellum and corpus callosum) of developing mouse brains particularly between p and p where they make up almost % of all microglia and decrease in numbers already at p before being almost completely undetectable by p ( ) . presence of igf -expressing microglia in these locations in p brains was further confirmed by in situ hybridization ( ) . we performed rna-seq on these cells between p and p after facs-sorting based on cd dim cd b+ cd c+ gating comparing them to their cd c− counterparts. we identified a robust neurodevelopmental gene signature for developmental cd c+ microglia, including factors involved in astrocyte and neuronal differentiation, tissue remodeling, and myelinogenesis accompanied by downregulation of immune function-related genes. of note, itgax, itga , csf , and igf , which were highlighted in the hristova study, were also part of this signature. importantly, we demonstrated that igf expression by cd c+ microglia during development is crucial for primary myelination. indeed, selective deletion of igf specifically from cd c+ cells led to myelination defects in p brains ( ) . interestingly, all neonatal microglia expressed neuroectodermal genes including nestin. a concomitant study by hagemeyer et al. similarly identified amoeboid microglia in the developing white matter of the corpus callosum and cerebellum particularly between p and p before being almost undetectable by p ( ) . interestingly, they used a mac- staining to identify these cells, reminiscent of a study by valentino and jones who reported mac- expression in "fountain microglia" in a footnote ( ) . they identified a signature akin to the one we found ( genes in common out of upregulated genes including itgax, csf , and igf ) by comparing "fountain microglia" from corpus callosum at p with cortical microglia at the same age by whole-genome microarray ( ) . of note, the study underscores that many of the most upregulated genes were related to a primed or activated microglial phenotype and they confirmed cd c expression in the "fountain of microglia" cells with a reporter mouse. in addition, by depleting all microglia during the critical period of the first postnatal week, they showed that the number of oligodendrocyte progenitor cells was reduced and a long-lasting effect on myelination was induced into adulthood ( ) , in line with our own results. two recent studies used single-cell rna-seq to elucidate microglial heterogeneity during development ( , ) . the barres lab study used deep single-cell rna-seq on microglial cells sorted based on cd b+ gating and cd levels from six different brain regions at e . , p , and p ( ). they found a cluster of cells they named "proliferative region-associated microglia" (pam), mainly found at p in the white matter, that have an amoeboid morphology and phagocytose newly formed oligodendrocytes ( ) . in addition, they reported enhanced expression of igf and itgax in this cluster compared to any other at p or other time points. these cells were observed as early as e . in the embryonic brain, their numbers peaking around p and were almost absent from p brains ( ) . all these features fit with cd c+ microglia from our study and the historical "fountain of microglia" cells. the stevens lab used high-throughput rna-seq on microglial cells from the whole brain sorted based on a cd dim cd b hi cx cr hi gating at e . , p - , p , p , and p and in injury contexts, prioritizing high numbers of cells over depth of sequencing ( ) . they identified a cluster of cells exclusive for the p - time point, which have an amoeboid morphology, express phagocytosis-related genes, and are restricted to the corpus callosum and cerebellum, associating closely with axonal tracts, which they named "axon tractassociated microglia" (atm) ( ) . again, the features of this subset resembled closely the features of cd c+ microglia and "fountain of microglia" cells described above. interestingly, their study showed no evidence for a sex bias, the number of cells associated to this cluster being similar for neonatal female and male pups ( ) . in addition, anderson et al. ( ) described gene signatures of retinal microglia in p mice, % of which were found to express cd c. the microglial signature in the p retina fit the signature associated to developmental cd c+ microglia as itgax, lpl, clec a, and igf were enriched in sorted cd c hi vs. cd c low cells at p , whereas p ry and tmem were downregulated ( ) . we therefore hypothesize that cd c+ microglia, fountains of microglia, pams, and atms, although described in different studies by different methods under different names, actually represent the same population of cells. comparison of the transcriptomic signature found in each of these studies leads to a core signature of genes found in all four studies (gpnmb, itgax, spp , fam c, fabp , hpse, igf , folr , csf , and anxa ) and additional genes found in at least three of these studies (atp v d , slpi, cd , crip , lgals , anxa , vat , ifitm , gm , plaur, s a , colec , clec a, atf , atp a , ephx , nceh , lpl, pld , plin , aplp , ccl , bnip , ccl , gpx , slc a , lag , and lilrb ) (figure ) . interestingly, csf , one of the genes of the core signature, has been identified as one of the prominent genes characteristic of the pre-microglia homeostatic signature ( ) . these genes constitute the "developmental signature" of the microglial population described in this section. of note, homeostatic microglia markers, such as tmem , p ry , sall , tgfbr , fcrls, and cx cr , have been shown to be expressed by this subset, although in most reports at slightly lower levels than in adult microglia or other neonatal microglia ( , , , ) . later in this review, we will refer to this population as "developmental cd c+ microglia". features of this population include peak numbers between p and p , amoeboid morphology, phagocytic abilities, and location in white matter (figure ). in addition, studies mentioned in this section clearly reveal a critical functional role of developmental cd c+ microglia in the myelination process. their presence in high numbers in the white matter makes them strategically placed in both space and time to take on that role. the aforementioned data support their involvement in phagocytosis of newly formed oligodendrocytes, probably linked to the proper establishment of primary myelination ( , , , ) . two of the studies show the long-term importance of these cells on oligodendrocytes and myelination later in life ( , ) . although the number of common genes in the developmental signature might appear low, we would argue that this is probably due to discrepancies in the transcriptomic techniques used (microarray, bulk rna-seq, high-throughput single-cell rna-seq, deep single-cell rna-seq), as well as the isolation techniques used (facs-sorting based on various gatings, presence or absence of perfusion, whole brain dissection, or region microdissection) (see table ) [discussed in ( ) ]. however, similarities in the localization, colonization kinetics, morphology, and functional role leave little room for doubt regarding the uniqueness of the population described. recent studies have described the homeostatic adult brain as the state with lowest microglial heterogeneity ( ) . in addition, most high-throughput studies investigating adult microglia in steady state generally report very homogeneous populations in the homeostatic clusters, whether by mass cytometry ( ) or single-cell rna-seq ( ) , characterized by robust expression of classical microglial homeostatic markers. however, in a cd c-eyfp reporter mouse, yfp-expressing cells have been found throughout the brain and retina in adulthood. although initially thought to be dcs ( ), they have since then been shown to exhibit a phenotype resembling microglia ( , ) . interestingly, a particular abundance of these cells is found in ventral areas of the brain, white matter tracts, and areas of adult neurogenesis ( ) . this is in line with a report that clec a+ microglia are found in neurogenic niches in the adult mouse ( ) , showing that in the homeostatic adult brain, microglia with a phenotype similar to developmental cd c+ microglia could remain in low number in selected areas. consistent with this, a subset of microglia (also positive for tmem and p ry ) expressing higher levels of cd c was found in the human subventricular zone and thalamus ( ) . in reporter mice, expression of cd c has been shown to not always follow the expression of the yfp reporter and should therefore be taken cautiously ( ) . the existence of cd c-expressing microglia has however been confirmed in the adult homeostatic brain (around % of total microglia) ( - , , , ) . similarly, a small population of cells from the choroid plexus of adult mice was shown to be transcriptionally distinct from other choroid plexus cells and border-associated brain macrophages. this population named "kolmer's epiplexus cells" closely resembles microglial cells and was associated with enriched expression of spp , apoe, and igf ( ). although itgax was not among the significantly upregulated genes in this study, cd c+ cells expressing low levels of cd have previously been described in the choroid plexus of adult mice ( ) . change in microglial gene expression and phenotype in steadystate aging has been studied extensively. although reports agree on the changes in morphology and general phenotype of microglia toward dystrophic microglia (deramification, cytorrhexis, and fragmentation) in aging [reviewed in ( ) ], genomic studies have given discrepant results, with some arguing for shift toward neuroprotection ( ) and others highlighting a "primed phenotype" with higher immune activation ( ) . that said, having a second look at datasets from various studies brings to light common highly expressed genes in aged microglia compared to young microglia: spp , clec a, igf , lpl, axl, apoe, lgals , itgax, cst , etc. are indeed found across several studies ( ) ( ) ( ) , although not all and not always in the same range of upregulation ( ) . in a later study, holtman et al. related the "primed" microglial signature they found from two aging models (one physiological aging model and one accelerated aging model) to the study by hickman et al. and found a high correlation between the datasets ( ) . high-throughput single-cell methods are a good way to decipher complex populations with mixed subsets. a masscytometry study revealed that a specific subset of microglia emerges during aging that overexpresses surface cd c and cd , clec a, and cd as compared to other microglia figure | cd c+ microglia signature in developmental stages. during development, cd c+ microglia have an amoeboid morphology and localize close to white matter tracts, essentially in the corpus callous and cerebellum. they are present early during embryonic development and their numbers peak between p and p . comparison of genes upregulated in four studies ( , , , ) reveals a common signature for developmental cd c+ microglia of genes upregulated in at least three of the studies (bold dark outline). genes shared with the disease signature in figure are in bold. the venn diagram was generated using the online tool venny ( ). at the same age, although they downregulate cx cr and mertk ( , ) . cd c expression of microglia in the white matter and caudal areas of the cns of aged mice was also shown using immunohistochemistry ( ) . this study also reports expression of clec a in white matter tracts of aged animals and reports numerous changes in white matter microglia associated with aging. similarly, single-cell rna-seq revealed that several populations of microglia that were present in younger age at very low numbers become increasingly prevalent with aging. one of these populations (referred to as oa ) is characterized by genes from the developmental signature and genes classically associated with neurodegeneration (spp , lpl, lgals , lilrb , ( ) , possibly indicating the presence of cd c+-microglia-like cells in the repopulating clusters they described. this is reinforced by our study in which cd c+ microglia could be found in repopulating microglia clusters after genetic microglial depletion ( ) . however, in contrast to zhan et al. our analysis did not show neonatal-like, neurodevelopmental gene signature in repopulated microglia ( ) . the low extent of overlap between these studies and our newly defined neonatal cd c+ microglia could be explained by heterogeneity of repopulating microglia, diluting the signal from cd c+ microglia in bulk rna-seq studies. microglia activation is a common feature in many neurological disorders including inflammatory, demyelinating, and degenerative diseases, as well as glioma and injury. although microglia activation may have deleterious consequences, it has also been shown in many instances to exert protective and regenerative effects. it is now becoming clear that there is an emergence of cd c+ microglia population in pathological conditions. in this section, we will discuss the importance and the role of this cell subset in several neurological diseases. for decades, it has been known that microglia localize around aβ plaques, and engulf aβ in ad, showing their importance in the disease. in recent years, interest in these cells has increased, largely due to a wave of transcriptomic and genome-wide association (gwas) studies. in addition, a majority of ad risk genes are related to microglia, including triggering receptor induced on myeloid cells (trem ) [reviewed in mcquade and blurton-jones ( )]. despite the enormous amount of data generated, no consensus has yet been reached on whether microglia are protective or detrimental in neurodegeneration. some of the attempts to resolve this issue involved comparing transcriptomes of microglia sorted from healthy, aged, and diseased brains. the study by holtman et al. cited above identified a microglial signature found not only in aging models but also in disease models including the app/ps ad model and the sod model for amyotrophic lateral sclerosis (als) ( ) . the common genes included itgax, clec a, axl, lgals , and apoe, indicating the presence of a cd c-expressing microglial population in these models. the gene module described in this study mostly contained genes related to phagocytosis and cell proliferation, with tissue protective elements ( ) . with a similar strategy, other studies demonstrated that microglia from aging brains and from amyloidosis (app/ps ) and tauopathy (aav-tau p l) shared a common gene signature including cst , itgax, gpnmb, clec a, lpl, lgals , apoe, and spp ( ). similar results were also obtained by krasemann et al. in the app/ps model. such shared microglial characteristics led to the term "microglial neurodegenerative phenotype (mgnd) signature" ( ) . this is also in line with the presence of cd c-expressing microglia in these models, with a phenotype similar to the one found in physiological aging. the presence of cd c+ microglia around aβ plaques has been shown in several studies ( , , , , ) . a recent study by kamphuis et al. extensively investigated the localization, proliferation status, and transcriptome of cd c+ vs. cd c− microglia in app/ps mice ( ) . importantly, this study also highlighted a steady increase in cd c transcripts in brains of app/ps and xtg-ad mice with aging as plaques appear, as well as in hippocampal samples from ad patients, although it declines in the later stages of the disease ( ) . the transcriptomic signature of cd c+ microglia, when compared to their cd c− counterparts, showed increased expression of gpnmb, fabp , spp , igf , itgax, gm , cst , cox a , apoe, ch h, clec a, lilrb , csf , axl, lpl, sulf , egr , anxa , cd , timp , and ctsb among others. many of these genes are common with the developmental signature of cd c+ microglia described above or with the signatures found in whole brain "primed" microglial signatures ( ) . these findings further support that the "primed" microglia phenotype described in many studies recapitulates the cd c+ microglia signature diluted among cd c− counterparts. the robustness of the signature is hardly surprising, considering that cd c+ microglia make up for % of all iba + cells in the aged app/ps brain ( ) . of note, strong upregulation of some cd c+ microglia signature genes, including itgax, clec a, and cst , was even detectable in whole tissue samples from cortex and hippocampus in ad models ( , ) . high-throughput single-cell studies also contributed to our understanding of microglial populations in ad rodent models. the same study that identified cd c and cd surface expression by mass cytometry on a microglia population emerging in aging also identified a similar population in app/ps brains ( ) . single-cell rna-seq studies identified three microglial signatures in neurodegeneration models: the disease-associated microglia (dam) signature ( ), the late response microglia signature ( ) , and the activated response microglia (arm) signature ( ) that emerge in the xfad, ck-p , and app nl−g−f models for ad, respectively. all three studies described cell clusters showing nearly identical microglia populations, similar to the cd c+ microglia signature observed in the kamphuis study. importantly, all of the dam cells were cd c+ ( ) with highly overlapping gene signatures uncovered by bulk sequencing of sorted cd c+ microglia ( ) . microglia with characteristics from the arm cluster are present in low numbers (ca. %) even in wild-type mice at young age, increasing as part of normal aging to reach up to about % of all microglia ( ), consistent with observations discussed above of cd c+ microglia in the steady state in adult and aging mice. arm microglia are however most evident in app nl−g−f mice where they outnumber all other microglial clusters reaching % of all microglia at months of age ( ) . this is in line with increases in cd c+ microglia reported in other studies. importantly, the signature observed in cd c+/dam/mgnd/arm microglia is enriched for known ad risk genes ( ) . of note, this transcriptomic signature is similar to that induced by retinal degeneration ( ) . cd c+ microglia have been demonstrated to be beneficial for and to correlate with increased aβ uptake and induction of igf -mediated neurogenesis in an animal model of ad ( ) . in addition, abundance of igf -expressing microglia around aβ plaques was recently confirmed by in situ hybridization in an ad model ( ) . functional analyses led to discrepant results suggesting either protective, immunosuppressive function as well as enhanced capacity for uptake and lysosomal degradation of aβ ( ) , or pathogenicity via possible contribution to local arginine deprivation and subsequent neurodegeneration ( ) . butovsky's group also proposed a detrimental role for these cells due to ameliorated aβ deposition in -month-old trem -deficient mice that lack cd c+ microglia ( ) . however, the role of trem is not clear, since other data show either protective or detrimental roles for this protein depending on the age of the animals ( , ( ) ( ) ( ) . nonetheless, all these studies demonstrate lack of microglial proliferation and clustering around plaques in trem -deficient animals, thus allowing for more dispersed aβ localization in ad models ( , ( ) ( ) ( ) ( ) ( ) . this can be detrimental due to aβ spreading that is not limited by microglia clusters, ultimately leading to severe axonal dystrophy ( ) . moreover, it has been demonstrated that in trem -deficient animals older than months, the aβ burden is enhanced as compared to -month-old animals, suggesting that trem signaling is necessary for limiting advanced stage pathology ( ) . thus, cd c+ microglia may actually be beneficial and protective in later stages of the disease as proposed by keren-shaul et al. ( ) . human data further support this hypothesis since loss-of-function mutations in trem have been identified as a strong risk factor for the development of ad and other neurodegenerative diseases [reviewed in mcquade and blurton-jones and ulland and colonna ( , ) ]. collectively, cd c+ microglia (also referred to as primed microglia, late response microglia, dam, arm, or mgnd) are a well-defined population of cells that show adaptation predominantly for phagocytic clearance of apoptotic/necrotic neurons and limiting aβ spreading. given that ad risk genes are enriched in this population ( ) , mutations in such genes may have an impact on the ability of cd c+ microglia to cope with aβ plaque burden, either promoting or limiting ad pathology. als is a disease affecting motor neurons leading to their degeneration. microglial contribution to the disease has been established since a robust microglial activation has been found in both patient and transgenic mouse tissue ( , ) . in addition, many risk factors for the disease have been shown to be expressed by microglia in the cns, reinforcing the idea of an involvement of these cells in the disease ( ) . microglial activation in the disease arises from accumulation of misfolded protein, and, similarly to observations made in other disease contexts, microglia have been reported to play a beneficial role in the pre-symptomatic phase of the disease before shifting to detrimental roles in the advanced disease state ( ) . however, microglial depletion in the context of als has not been found to increase survival ( ) , leading to the idea that both functions might be concomitant, constantly counteracting each other. interestingly, a study from analyzed the transcriptome of microglia sorted from mice carrying an als-associated mutation and found a particular signature for these cells at the end stage of the disease compared to microglia from healthy brains ( ) . once again, among the top regulated genes were genes related to huntington's disease, ad, and parkinson's disease (mapt, psen , apoe, etc.). the signature found in this study includes both factors reported to be beneficial in the context of als (igf , grn, trem , tyrobp, etc.), and factors known to be detrimental (mmp , optn, cybb, etc.), as well as some like spp , gpnmb, and itgax recurrently found in neurodegenerative diseases. microglia were also found to upregulate surface cd c. microglia from sod mice were also found to fit the abovementioned mgnd signature, in addition to expressing clec a levels increasingly during disease progression ( ) . neuron degeneration and nerve injury have been linked to microglia in various models for traumatic brain injury (tbi) ( ) , spinal cord injury (sci) ( ) , nerve injury ( ) , and ischemic stroke ( ) . much like in inflammation models, microglial contribution in all of these models is still rather unclear and they may play a double role considering their association with both beneficial and detrimental effects. studying microglia in context of inflammation can get quite complicated due to massive infiltration of peripheral immune cells, notably monocytes and macrophages, occurring subsequently to tbi ( ) , sci ( ) , and stroke ( , , ) . in a study comparing the transcriptomics of microglia and macrophages after ischemia in rats, it was reported that microglia played a detrimental role and macrophages played a beneficial role with regard to recovery, based on their expression of classical inflammation markers ( ) . investigation of the genes enriched in microglia three days after middle cerebral artery occlusion compared to sham controls, however, revealed spp , gpnmb, lgals , fabp , and axl among others, fitting with the potential presence of cd c+ microglia-like cells in this context, diluted among other microglia. consistent with this, ccl mrna was found to be increased in microglia and macrophages at this time point ( ) , an aspect that has been associated with the emergence of cd c+ microglia ( ) . another study, conducted in a model of phototrombic stroke on whole tissue, actually showed upregulation of gpnmb, itgax, and clec a in a cluster associated with early response ( ) , which the authors related to the dam phenotype ( ) . in a study of facial nucleus axotomy, the authors also related the observed microglial phenotype ( ) to the dam phenotype, as well as to a phenotype found in the ck-p model ( ): genes were regulated in common between all three studies representing almost % of all genes upregulated in the facial nucleus axotomy model. interestingly, in an sci transcriptomic study, a profile of microglia reminiscent of the cd c+ phenotype was identified (with upregulation of gpnmb, spp , lpl, apoe, igf , lgals , and itgax among others) and persisted in a full transection model, whereas it contracted concomitantly to recovery in a hemisection model ( ) , indicative of the transitory nature of this subset. conversely, in tbi, the microglial signature was further from the cd c+ microglia signature, although itgax was among the upregulated genes and days post-injury, possibly indicating once again a dilution of the signature in all microglia ( ) . in addition, considering the difficulty associated with gating out macrophages from microglia in a context of extensive infiltration, macrophage contamination of the sorted samples cannot be excluded in these studies, potentially complicating interpretation of the observed transcriptomes. ms is an inflammatory, demyelinating disease of the cns that can be modeled by eae or toxin-induced demyelinating models. recent advancement in our understanding of the disease points toward important roles for microglia in the pathomechanism. although the evidence supporting their implication in initiation and facilitation of the disease is strong ( ) , there is a growing body of evidence for their protective functions including involvement in remyelination ( ) . we have identified cd c+ microglia during eae accounting for around % of total microglia in whole cns ( , ) . of note, this subset is even more abundant in the spinal cord at the peak of the diseases reaching up to % of total microglia (wlodarczyk, unpublished) . the emergence of the cd c+ microglia is a dynamic process starting at the onset, reaching a maximum at the peak and contracting in the chronic phase of eae ( , ) . these cells are localized in the demyelinated spinal cord lesions ( ) . cd c+ microglia from eae again showed upregulation of similar genes as in neurodegenerative models including itgax, gpnmb, spp , etc. ( ) . a similar signature was confirmed by krasemann et al. ( ) . in addition, deep analysis of genes that were upregulated in cd c+ microglia population pointed to their involvement in immune responses ( ) . a key aspect of neuroinflammation in eae is the recruitment and reactivation of encephalitogenic t cells to express their effector functions. many cell types are implicated in this process, including blood-derived dcs and monocytes/macrophages but also parenchymal microglia ( ) . in eae, cd c+ microglia express mhci, mhcii, and costimulatory molecules cd /cd ( , ) , which is in line with recent highthroughput mass-cytometry reports ( , ) . we have provided evidence that cd c+ microglia are able to induce similar proliferative response of encephalitogenic cd + t cells as blood-derived professional antigen-presenting cells ( , ) . interestingly, in contrast to cd c+ blood-derived cells and cd c− microglia, cd c+ microglia completely lacked mrna expression for il- ( ) that is known to induce gm-csf-producing cd + t cells, critical for eae pathology ( ) . this indicates that although cd c+ microglia alone might contribute to t cell expansion, they are unlikely to induce pathogenic t cell responses. importantly, a subsequent study showed that they were a major source of message for myelinogenic igf , suggesting that they might exert protective roles in eae ( ) . this is supported by our recent study showing that stimulation of csf r with its ligands during symptomatic eae significantly reduced demyelination and ameliorated disease progression most likely through induction of cd c+ microglia ( ) . moreover, decreasing cd c+ microglia by blocking of trem signaling (as discussed below) led to increased severity of eae and exacerbated demyelinating lesions in the spinal cord ( ) , further supporting protective roles of cd c+ microglia. microglia are known to contribute to remyelination by creating an environment supporting opc recruitment and differentiation by phagocytosing myelin debris, secreting growth factors and modulating extracellular matrix [reviewed in lloyd and miron ( ) ]. circumstantial evidence for remyelinating properties of cd c+ microglia includes our first demonstration of the expansion of these cells in cuprizone-demyelinated corpus callosum ( ) . a microarray study by olah et al. identified a pro-remyelinating microglial signature that includes several genes reminiscent of the cd c+ microglia characteristics described above (itgax igf , clec a, apoe, spp ) ( ) . moreover, cd c immunoreactive microglia were present in remyelinating corpus callosum ( ) . a similar microglial signature was later confirmed in both demyelination and remyelination phases ( ) . conversely, microglia expressing the cd c+ microglia signature including apoe, axl, igf , lyz , itgax, and gpnmb were identified by single-cell transcriptomics in both deand remyelinated lesions ( ) . recently, cuprizone-mediated demyelination was shown to be alleviated in mice lacking microglial sirpα that have increased numbers of cd c+ microglia, pointing to their protective role ( ) . in line with the induction of cd c+ microglia ( ) , stimulation of csf r ameliorated cuprizone-induced demyelination ( ) . another line of evidence comes from the influence of trem deficiency, which leads to absence of cd c+ microglia in adult mice ( , ) , on remyelination after cuprizone demyelination. the data indicate that trem deficiency had no impact on the initial demyelination, but affected subsequent remyelination when the cuprizone treatment was prolonged, most likely by impairing myelin removal as well as myelin regeneration, which further supports a protective role for cd c+ microglia in this paradigm ( , ) . additionally, it was reported that microglial necroptosis in circumstances of lysophosphatidylcholine demyelination leads to repopulation by pro-regenerative cd c+ microglia, as blocking of this mechanism prevented remyelination ( ) . of note, demyelination induced by mouse hepatitis virus also led to enrichment of cd c+ microglial gene signature in the spinal cord ( ) . taken together, association of cd c+ microglia to white matter ( ) as well as their role in primary myelination strongly support their importance in induction and facilitation of remyelination. this opens the possibility for induction of innate repair programs in diseased cns via promotion of the emergence of cd c+ microglia. very early studies identified microglial cells close to gliomas to resemble the amoeboid form described during development and to take on phagocytic functions ( ) . more recent studies have shown that parenchymal microglia are attracted to the tumor in glioma-affected brains, representing up to % of the tumor mass ( ) . microglia associated to the tumor have been termed glioma-associated microglia/macrophages (gam). these cells initially exhibit beneficial anti-tumor abilities but have been found to be hijacked by the tumor to exert tumor-promoting functions [reviewed in li and graeber ( ) ]. a study from identified a signature for gams, and emphasized their high expression of spp and gpnmb ( ) . they compared this signature to classical macrophage activation markers (m /m ) and concluded a lack of overlap between the gam signature and these classical phenotypes. of note, the signature also includes genes such as itgax, fabp , and clec a among others recurrently found in disease signatures ( ) . considering the similarities observed in gene expression from the different studies aforementioned, we compared the transcriptomic signatures obtained in studies comparing specifically microglia sorted based on a typical marker for this specific subset of microglia or from single-cell rna-seq (three of the ad studies and one eae study, figure ). we found a core disease signature for microglia consisting of genes shared between all four studies (figure ) . itgax being once again a part of this signature and with clarity in mind, we will refer to this signature as the "cd c+ microglia disease signature" henceforth. once again, the microglial nature of this subset is supported by expression, although slightly lower than in homeostatic microglia, of tmem , cx cr , p ry , sall , and tgfbr among other homeostatic genes ( , ( ) ( ) ( ) . over the years, advancements in technology have allowed the scientific community to investigate cells and cell populations in increasingly detailed ways, particularly at the molecular level. this investigation has been done using a multiplicity of different conditions and models, leading to increasing amounts of data generated. although invaluable, this work has also led to redundancy in the microglial profiles that were identified ( ) . our investigation led us to define two particularly strong signatures for cd c+ microglia in development (figure ) and in disease (figure ) . interestingly, li et al. ( ) as well as anderson et al. ( ) related the developmental microglia signature observed in their studies to the dam microglial signature. these similarities prompted us to compare the signatures we identified from the literature. comparison of the developmental signature and the disease signature resulted in defining of a "core" signature common to cd c+ microglia across all contexts, which consists of genes: ank, anxa , aplp , atp a , clec a, colec , csf , ephx , fabp , fam c, gm , gpnmb, hpse, igf , itgax, lilrb , lpl, nceh , plaur, pld , plin , and spp (figure and supplementary table ) . interestingly, the protein network linked to these genes had significantly more links than what can be expected, indicating at least a partial biological connection between these genes (figure ) . further investigation of the physiological function of the proteins related to the genes present in the core signature revealed their involvement in lipid metabolism, cell migration and proliferation, and, to a lesser extent, immune function (supplementary table ) . as expected, all of these proteins had been associated with various brain diseases (supplementary table ). of note, many of these proteins assume similar function or have been found to interact directly or indirectly with each other (supplementary table ). further investigation of these genes and proteins in link with one another would most likely unveil interesting mechanisms underlying cd c+ microglia function. although described previously as different microglial subsets, we argue that the robust core signature we have identified can be found for this subset across all these different stages. we suggest that the differences in this subset observed between conditions reflect methodological discrepancies ( table ) or microenvironment-linked context-specific changes and the subset's own phenotypic plasticity in coping with these variations, rather than fundamental differences in cell lineage. the dynamics of cd c+ microglia seem tightly spatiotemporally regulated. they first emerge during the first postnatal week, peaking at p and gradually decreasing as animals age, being barely detectable in the healthy adult cns ( - , , , ) to increase again in aging or disease ( , , , , ) . importantly, none of the studies that have investigated induction of inflammation by means of lipopolysaccharide, poly(i:c), or other immune challenges could recapitulate the robust cd c+ signature found in steady state and disease and injury contexts ( , , , , , ) . below, we present factors that participate in controlling the induction of this population (figure ) . one candidate that has been extensively studied with regard to cd c+ microglia is the trem pathway. trem -deficient animals were shown to downregulate the cd c+ microglia signature in cuprizone-induced demyelination ( ) and in an ad model ( ) . in addition, in the study from the amit lab, trem deficiency in an ad mouse model led to an arrest of microglia in an intermediate state between the homeostatic state and the cd c+ microglia stage. barely any microglia in these mice exhibited the cd c+ microglial signature ( ) . this suggests that cd c+ microglia induction is a two-step process, where the first step, to leave the homeostatic state, is trem -independent and the second step, to reach the complete cd c+ microglia phenotype, is trem -dependent. these observations were confirmed by krasemann et al. in another trem -deficient ad model ( ) . similarly, apoedeficient mice exhibit lower numbers of cd c+ microglia in ad, als, and ms mouse models ( , ) . this is suggestive of a positive feedback loop, as this population itself strongly upregulates apoe ( ) . surprisingly, the barres lab showed that induction of cd c+ microglia during postnatal development in contrast to adulthood is trem -apoe-independent ( ) . a similar trem independence of cd c+ microglia induction was shown in the developing retina ( ). figure | cd c+ microglia signature in disease states. in diseased cns, cd c+ microglia adopt an amoeboid, reactive morphology. in ad, they are found surrounding aβ plaques. similarly, in ms and als models and in injury, they are found around and in the lesions. in glioma, they are found mixed with tumor cells. cd c+ microglia numbers in diseased cns vary considerably, ranging from to % of all microglia. comparison of genes upregulated in four studies ( , ( ) ( ) ( ) reveals a common signature for cd c+ disease microglia of genes upregulated in at least three of the studies (bold dark outline). genes shared with the developmental signature in figure are in bold. raw data for the krasemann study were obtained using the gene expression omnibus database and the differential gene expression analysis was performed using the debrowser package in r ( ) . the venn diagram was generated using the online tool venny ( ). krasemann et al. highlighted phagocytosis of apoptotic neurons and monocytes as a trigger for the induction of the cd c+ microglia phenotype ( ) . of note, induction of this phenotype was not observed upon microglia exposure to escherichia coli, zymosan particles ( ), or microparticles (marczynska et al., unpublished), suggesting that induction of cd c+ microglia is a tightly controlled reaction to local cell damage or apoptosis, rather than to phagocytosis itself. interestingly, microglial necroptosis in demyelination models leads to brain repopulation with cd c+ microglia from nestin+ resident microglia ( ) . similarly, nestin+ microglia colonizing the brain after microglia ablation expressed surface cd c ( ) . the gene expression in repopulating microglia highly overlapped with the cd c+ microglia signature. we showed that genetic or toxin-induced ablation of neonatal cd c+ cells led to their instant repopulation ( ) . whether the observed concomitant decrease of cd c− microglia ( ) reflects induction of cd c+ phenotype in cd c− cells by phagocytosis of dying microglia has not been determined. interestingly, a dramatic decrease in cd c+ microglia was observed in the postnatal retina of mice deficient in bax, a pro-apoptotic gene that is essential for developmental death of neurons ( ) . this emphasizes that apoptotic cells are a strong and common inducer of cd c+ microglia regardless of age and condition. this is also in line with several studies where developmental cell death has been linked figure | core cd c+ microglia signature. considering similarities between the transcriptomic signatures and functions in the cd c+ microglia subset in development and in disease, we compared both signatures to obtain a core of genes upregulated in this subset across all conditions. we observe overlap of % of the genes between both signatures, corresponding to shared genes. upon interrogating the string database (szklarczyk d, gable al, lyon d, junge a, wyder s, huerta-cepas j, simonovic m, doncheva nt, morris jh, bork p, jensen lj, von mering c. string v : protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. nucleic acids res. nov; :d - .), we observed that the network formed by the proteins corresponding to the genes in the core signature had significantly more interactions than expected from a similar set of random proteins, indicating that these proteins related to the genes in the core signature are at least partially biologically connected. the thickness of the edges linking the different genes is proportional to the strength of the evidence linking the two proteins. the venn diagram was generated using the online tool venny ( ) . to microglial entry in the developing cns ( ) . in addition, retinal cd c+ microglia were resistant to depletion induced by either csf r deficiency or blocking, contrary to their cd c− counterparts. in line with this, our own data showed that despite using several depletion regimens, cd c+ microglia could not be depleted from postnatal brain as they were immediately repopulated ( ) . we have shown that both populations of adult microglia (cd c+ and cd c−) express equal levels of csf r ( ) . importantly, stimulation of this receptor by its ligands, interleukin (il)- and csf , induced a significant increase in cd c+ microglia numbers, with faster kinetics for il- ( ) . moreover, such stimulation induced ccl in the brain, and we showed that overexpression of ccl leads to a dramatic expansion of cd c+ microglia in a ccr independent manner ( ). butovsky et al., on the other hand, showed that another cytokine, il- , can induce cd c+ expression on aβ pretreated microglia ( , ) . moreover, they demonstrated that ga vaccination leads to an increase of cd c+ microglia surrounding aβ plaques and suggested that this was induced by t-cell-derived il- ( ). recently, the emergence of cd c+ microglia in the adult brain has been shown to be homeostatically controlled by sirpα/cd interaction. genetic ablation of sirpα in microglia or global lack of cd equally resulted in increased numbers of cd c+ microglia, suggesting that microglial sirpα suppresses cd c expression in the same cells ( ) . here, we have demonstrated that the subpopulation of microglia described in many recent studies (and named pam, atm, fountain of microglia, dam, arm, mgnd, and late response microglia) indeed reflects the characteristics of cd c+ microglia, originally identified over a decade ago. thus, we believe that a unification of the nomenclature by referring to the microglial subset expressing the described signature, from development to old age, as cd c+ microglia is a necessary step to progress our understanding of microglia biology. this subset emerges in development before contracting during adulthood but is triggered to re-emerge in aging as well as in the context of disease or tissue injury (figure ) . the summary of the data that mentioned microglia showing the aforementioned signature strongly points to the importance figure | cd c+ microglia as a subset of microglia present through life and across conditions. our investigation leads us to believe that cd c+ microglia represent a subset of microglia characterized by a robust signature of genes expressed by this subset at any age and in various disease states. emergence of this subset is induced by various factors including signaling through the trem -apoe pathway, cell death, il- signaling, and cytokine signaling through csf r inducing ccl , and is inhibited by cd /sirpα signaling. in physiological conditions, cd c+ microglia account for around % of all microglia, before contracting to % in adulthood and being re-induced by aging at levels similar to development. in disease states, their numbers oscillate between and %. we argue that despite the numerous names given to this subset across conditions, it is unique and should be referred to as "cd c+ microglia". of cd c+ microglia in primary myelination during cns development as well as their protective, remyelinative, and regenerative 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attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -b sbvy g authors: marques neto, lázaro moreira; kipnis, andré; junqueira-kipnis, ana paula title: role of metallic nanoparticles in vaccinology: implications for infectious disease vaccine development date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: b sbvy g subunit vaccines are safer but less immunogenic than live-attenuated vaccines or whole cell inactivated vaccines. adjuvants are used to enhance and modulate antigen (ag) immunogenicity, aiming to induce a protective and long-lasting immune response. several molecules and formulations have been studied for their adjuvanticity, but only seven have been approved to formulate human vaccines. metallic nanoparticles (menps), particularly those containing gold and iron oxides, are widely used in medicine for diagnosis and therapy and have been used as carriers for drugs and vaccines. however, little is known about the immune response elicited by menps or about their importance in the development of new vaccines. there is evidence that these particles display adjuvant characteristics, promoting cell recruitment, antigen-presenting cell activation, cytokine production, and inducing a humoral immune response. this review focuses on the characteristics of menps that could facilitate the induction of a cellular immune response, particularly t-helper and t-helper , and their potential functions as adjuvants for subunit vaccines. (tlr agonists) are immunomodulatory molecules, capable of generating a th response ( ) . there is a demand for safe adjuvants capable of inducing efficient cellular immunity, especially th and th , to be used against tuberculosis, leishmaniasis, malaria, and other diseases caused by intracellular microorganisms ( , ) . the majority of molecules with this type of adjuvanticity (th driven) are related toward the response of danger receptors to trigger inflammation, thus safety and tolerance could be major barriers that prevent their use in human vaccines ( ) . however, comparing alum and cpg/dna adjuvants in human trials, only common adverse effects, including local site reaction, flu-like symptoms and headache were observed when cpg/dna was used ( ) . also, verstraeten et al. ( ) , analyzing more than , individuals, who received vaccine-containing as , observed that only common side effects occurred. nanoparticles (nps) are classically described as structures smaller than nm and can be classified, based on their composition, as polymeric, inorganic, liposomes, immunostimulating complexes, virus-like particles, emulsions, or self-assembled proteins ( ) . they are made of different materials and differ in size, shape, and surface properties; interactions with biological systems, therefore, are varied, with several applications in modern medicine. in vaccinology, they are classically thought to have delivery and deposit properties. however, many nps have been shown to stimulate immune responses, including cell recruitment, activation of antigen (ag)-presenting cells (apcs), and induction of cytokine and chemokine release. the development of nanostructures and nanoadjuvants may therefore offer alternatives to currently used adjuvants once studies establish ways for them to elicit innate immune response and support the development of adaptive immune response in the context of vaccine formulations ( ) . metallic nanoparticles (menps) are relatively non-biodegradable, have rigid structures, and possess simple synthesis methodology. many have been studied for their immunological properties ( ) . however, there are still gaps in understanding the immune response generated by nps, especially menps. few studies have compared nps of different types and there is no standardization among published methodologies, which hampers comparisons of immunostimulatory characteristics. several important characteristics, therefore, have not been well studied. for example, how chemical and physical properties (including material composition, size, shape, surface charge, and hydrophobicity) impact vaccine immune response ( ) . this review focuses on the use of menps in formulations against infectious diseases, aiming to assess progress of their use in vaccinology and their possible applications as adjuvant. the immune response generated by menp-formulated vaccines table summarizes the articles that report the use of menps as part of vaccine formulations against infectious diseases and the immune responses they elicited. a range of immune responses is required to fight a diverse group of microorganisms. the type of protective immune response can be simplistically divided based on the type of microorganism: extracellular bacteria and toxin, intracellular bacteria, viruses, fungi, and protozoa. among the vaccines targeting extracellular bacteria and toxin, two were formulated with lipopolysaccharide (lps) in glycopeptide ag. the use of glycoantigen and lps can trigger an intense response through tlr and b cell receptor activation; the presence of gold nps (aunps) may have minimal influence on this response. however, in the work of gregory et al. ( ) and torres et al. ( ) , the use of aunps in the formulation generated a different response, improving anti-lps immunoglobulin g (igg) response, decreasing bacterial burden, generating a more efficient humoral response, and improving animal survival, showing that aunps may influence immune response and protection. using protein ag, barhate et al. ( ) formulated a vaccine using aunps and toxoid ag and demonstrated that their formulation could induce a mucosal and systemic igg and iga response. when co-administered with asparagus racemosus extract, a botanically derived adjuvant, the response was further enhanced ( ) . dakterzada et al. ( ) developed a vaccine against pseudomonas aeruginosa using the flagellin subunit and aunps that elicited an igg response comparable to that induced by freund adjuvant. flagellin is a tlr agonist but the recognition and signaling is structure dependent. this study, however, used only the - aa from flagellin and its ability to activated tlr could not be maintained ( ) . gregory et al. ( ) used an f yersinia pestis ag conjugated to aunps that induced an ab response with higher igg a associated with higher levels of interferon gamma (ifnγ), suggesting activation of th cells. among the studies that used menps in vaccine formulation, only one targeted intracellular bacteria (listeria monocytogenes). the protective immune response against intracellular bacterial infections requires th activation and, therefore, apcs activation and ag presentation through major histocompatibility complex ii (mhc ii). to generate a th response, an aunp and listeria ag formulation were used in different strategies. although the authors tested direct vaccination, when dendritic cells (dc), in vitro loaded with aunp plus listeria ag, were adoptively transferred to a naïve animal, they induced th , cd +, and natural killer (nk) cells that provided better protection against l. monocytogenes than the traditional vaccine approach ( ) . in evaluating vaccines developed with menps against viral infections, niikura et al. ( ) ( ) also evaluated the addition of cytosine and guanine linked by phosphodiester unmethylated (cpg/ dna) and found that it improved ab levels and animals' survival rates. another important feature of studies by niikura et al. ( ) and chen et al. ( ) was the use of various np sizes and the demonstration that all different np shapes were capable of inducing a humoral response. the levels of ab were size dependent, but the results were inconsistent: the first study found that a nm sphere was the most efficient ab inducer and the second study found that the nm and nm spheres performed best. a special case of the use of menps was the use of nickelfunctionalized nanolipoprotein particles (ninlps) by yan et al. ( ) and wadhwa et al. ( ) in combination with hiv ag. ninlps are nanometer-sized nanolipoprotein particles with nickel incorporation into their surface in order to induce polyhistidine tagged proteins adsorption ( ) . they demonstrated that specific igg (igg and igg a) levels were greater than those obtained when alum was used in the formulation. fischer et al. ( ) used truncated wnv envelope protein ag and found that a single dose vaccination induced a superior anti-wnv igg response and improved protection against a wnv challenge ( ) . these responses were associated with nickel functionalization, described as a hapten, and triggered responses through activation of human tlr and intracellular transduction signals through myeloid differentiation primary response (myd- ), nuclear factor-κb (nf-κb), and mitogenactivated protein kinase (mapk), inducing pro-inflammatory responses [tumor necrosis factor (tnf)-α and interleukin (il)- ] ( , ) . for protozoan infections, parween et al. ( ) , using plasmodium falciparum merozoite surface protein subunit and aunps, evaluated the humoral immune response (igg , igg a, igg b, and igg ) and found an intense igg response compared with the alum formulation ( ). kaba et al. ( ) , using p. berghei circumsporozoite protein and aunps, generated long-lasting protective immunity with th that produced il- and mixed high avidity igg /igg a (th /th ) ( ) . in other studies, these authors replaced ag with p. falciparum circumsporozoite protein; vaccination was shown to induce protective cytotoxic (cd +) cells, high avidity ab titers, and specific effector memory, central memory, and long-term central memory cd + t cells in draining lymph nodes, spleen, and liver ( ) . this response was shown to be generated by dc cross-presentation, which had delayed fusion and interaction of endosomes with lysosomes caused by the aunp formulation ( ) . finally, pfmsp was used with dextrancoated iron oxide nps (ionps) and was capable of inducing a humoral response in two animal models (mouse and monkey). this response was also shown to inhibit parasite growth by - % ( ). most studies evaluated immunogenicity through measurement of the humoral immune response. according to their findings, the use of nps was efficient in inducing an ab-based response. based on heavy chain structure, there are five types of ab, each with a different role: igg, igm, iga, igd, and ige. igg and iga can be subdivided as igg , igg , igg , igg , iga , and iga based on additional small differences in their heavy chain. with regard to vaccination, humoral immunity is especially important in responding to infection by extracellular pathogens, toxins, protozoa, and viruses. its importance is associated with the biological activities of immunoglobulins, including microorganism opsonization and phagocytosis; complement activation ( ); toxins and microorganism neutralization ( ) ; and mast cells and basophil activation ( , ) . in addition, immunoglobulins can help target cytotoxicity against infected cells (ab-dependent cell cytotoxicity of cd t cells and nk). in some cases, however, the pathogens have the ability to evade the humoral system or can even use immunoglobulins as a way to facilitate cell invasion, as in the cases of mycobacterium tuberculosis and leishmania spp. ( , ) . the studies described above clearly show that menps (gold, iron, and nickel) can be used for vaccine development. different menps were used in conjunction with several ag for distinct microorganisms and showed the ability to generate humoral and cytotoxic responses. although the generation of igg a and ifn-γ shown in some studies are indicators of th responses using menps as adjuvant, further research is needed to specifically assess the role of different menp vaccines in th induction. to understand the possible uses of menps as platforms for vaccines against infectious diseases, analysis is needed of the impact of different physicochemical characteristics of nps on the innate immune response (figure ) . several strategies have included menps as vaccine platforms, involving menps of different materials (including gold, iron oxide, and nickel); shapes (including spheres, cubes, rods, and disks); sizes (from nm to over nm); and types of coating [e.g., citrate, chitosan, dextran, or cetyltrimethylammonium bromide/ -styrenesulfonic acid-comaleic acid (ctab/pss-ma)]. the material from which an np is made has a direct influence on the functions of apcs; gold nps (aunps) have been most commonly used in vaccinology ( table ) . the most recent studies involving aunps demonstrate the effects of gold sodium thiomalate on macrophage function, showing lysosomal enzyme inhibition and reducing phagocytosis ( ) . similar effects were seen in macrophages of several origins, which, when stimulated with aunps, showed diminished bactericidal activity against staphylococcus aureus ( ) and low or absent cytokine production il- , il- , and tnf-α ( , ) . moreover, when splenocytes were stimulated with lps, the addition of aunp reduced il- and tnf-α release ( ) . some of these results raise the concern on the use of aunps as adjuvants, since these immunomodulatory properties can act inhibiting the generation of th . however, the response to aunps is also correlated with other physicochemical characteristics that will be discussed below, which may be tailored to improve immunostimulatory or immunomodulatory capacity. iron oxide nanoparticles have also been used as adjuvants. iron is an important ion in the homeostasis of all cells and in generating immune responses to several microorganisms. the effect of ionps phagocytosis have been explored in several studies, for example, m macrophages after exposure to ionps induced reactive oxygen species (ros), but after h induced il- production ( ) . the use of ionps in balb/c mice demonstrated the immunomodulatory capacity of this np by diminishing splenocyte cytokine production (il- and ifn-γ) ( ) as well as suppressing the response to pancreatic ag in diabetic mice ( ) . sindrilaru et al. ( ) , however, showed that macrophages, under iron overloaded conditions, became unrestrained m (with an incomplete switch to m macrophages) and produced more tnf-α, which impaired wound healing and had an important role in the immunopathology of chronic venous leg ulcers. consequently, ionp response seems to have direct correlation with time and dose, once iron overload seems to be a requisite to developed pro-inflammatory response and this aspect must be evaluated to avoid the inhibition of the desired immune response. other critical characteristics are the shape and size of nps, which have a direct impact on vaccine efficiency, ag load capacity, and interaction with cells (phagocytes and apcs). these characteristics have been studied in different nps; shah et al. ( ) published a review focusing on the impact of size for alum, oil-in-water, emulsion, polymeric particles, and liposome adjuvanticity, but did not evaluated menps. in the studies reviewed here, np sizes range from nm nanospheres to nm nanocapsules. two authors have evaluated the impact of size and shape for menps ( table ) : chen et al. ( ) evaluated differences in immune response based on aunp sizes (ranging from to nm nanospheres) and found that and nm were the most drained np ( ); niikura et al. ( ) went further and, using four different shapes of np ( nm sphere, nm sphere, cube, and rod), showed that ab responses and tnf-α were directly correlated with the specific np surface area (the ratio of the total surface area per single np volume). furthermore, nm spheres appear to be the most efficient in generating immune responses (il- and il- ) and granulocyte macrophage colony-stimulating factor production. surface charge and hydrophobicity are additional important np characteristics for immune response induction and are directly influenced by np functionalization (chemical modification of nps surface by adding or replacing functional groups) and coating (ag) ( ) . most studies used citrate-coated nps, but dextran and ctab/pss-ma have also been used; all three result in negatively charged (anionic) particles. only one np, revised here, used positive charged (cationic) functionalization [( ); table ]. the higher hydrophobicity of aunp was shown to activate the innate immune system (tnf-α secretion) ( ) . although the surface charge of other non-metallic nps has been studied ( ) , to our knowledge the studies using menps did not address the other characteristics associated with immune response induction. for non-metallic nps, it appears that a positive charge signified a greater ability to induce immune responses than a negative charge. interestingly, negatively charged non-metallic nps were associated with ag-specific tolerance ( ) . further studies are needed to investigate whether or not the charge imputed by np coating influences the immune response. though the size and shape of menps had little to no impact on the innate response elicited, coating modifications may improve the capacity of these molecules to influence immune responses. finally, it is important to note that the majority of adjuvant characteristics were evaluated using non-metallic nps. t-helper cells are associated with immunity against intracellular pathogens and the secretion of ifn-γ, which, in turn, is essential for the activation of mononuclear phagocytes, including macrophages, resulting in enhanced phagocytic activity ( ). th cells (il- a and il- f producer cells) are associated mainly with stimulation and chemotaxis of neutrophils to the site of inflammation. however, their function goes beyond this and includes the targeting of various cells types, including nonlymphoid cells and the stimulation of cytokine, chemokine, and prostaglandin production. another characteristic of these cells is their memory effector subset, which is maintained in mucosal tissues for extended periods. this subset has high plasticity and is able to transform into th or th phenotypes depending on the cytokine milieu at mucosal sites. this diversity of function and actuation make th cells very important in defense against several microorganisms, mainly those acquired through mucosal routes ( , ) . t-helper and th cells have their own distinct sets of functions and differentiation factors. both cell types require t cell receptor downstream activation by ag presentation cells through mhc ii and co-stimulatory molecules ( ) . consequently, cytokine release during ag presentation is correlated with the type of adaptive immune response generated. while th differentiation requires stimulation by il- , th generation requires transforming growth factor-β and il- . however, this generation is influenced by other factors and how menp are involved in the possible induction of th or th will be discussed below. in this review, only one study investigated the development of the direct th (type t helper cell) and th response. using a listeria ag, rodriguez-del rio et al. ( ) showed that in contrast to advax™ adjuvant alone, a combination of nm aunps and advax™ was capable of inducing the highest th response. pusic et al. ( ) immunized mice with ionps covered with rmsp , a p. falciparum merozoite ag, and showed that after immunization (intramuscular, subcutaneous, or intraperitoneal), production of il- was greater than that of ifn-γ, suggesting a predominant th response (although the cellular immune response was not directly evaluated). the first major determinant in generating th and th populations is the route of vaccine administration, which dictates the cell dynamic and initial response to the vaccine. for example, mohanan et al. ( ) , in a cross-sectional study using a liposome plus ag (ova) vaccine formulation, compared intradermal (high igg ; intermediate igg ; and ifn-γ), intralymphatic (high igg , igg , and ifn-γ), intramuscular (high igg ; intermediate igg and ifn-γ), and subcutaneous (high igg ; low igg and ifn-γ) routes of administration ( ) . the predominant th response to administration through the intradermal route was most likely due to the cooperation between langerhans cells, the primary innate immune response cells and keratinocytes that may also be stimulated by the formulation. these elicited the production of cytokines and chemokines that helped in the activation of other apcs ( ) . the early phase of vaccination is characterized by recruitment of neutrophils and monocytes to the site of inoculation. both cell types can also act as apcs, delivering ag-specific and co-stimulatory signals to t cells. their collaborative endeavors have been found to modulate (positively or negatively) the activity of different effector t cell subsets ( , ) . neutrophils are the first cell lineage to migrate to inflammation sites and, when stimulated, they produce cytokines and chemokines that will attract and activate other cell types. for example, neutrophils were shown to be an important inducer of th and th cells ( ) , but their role in cytokine secretion is much broader ( ) . moreover, signals may elicit different function in neutrophils and therefore, influence the quality of t cell responses. for example, aunps have been described as capable of inducing neutrophil extracellular traps, which act as damage-associated molecular patterns and stimulate immune system through dna receptors such as tlr ( ) . upon stimulation by nps (tio -titanium dioxide-and alum), duffin et al. ( ) demonstrated neutrophil influx to the lungs and also induced production of il- . silver nps were also shown to be capable of interacting with neutrophils, inducing apoptosis of these cells, and inducing caspase- \ caspase- partially dependent il- β secretion ( ) . in another study, cobalt and nickel nps were shown to induce higher nitric oxide, tnf-α, and cxcl chemokine production, by human peripheral blood neutrophils, than titanium nps (tio np) ( ) . nonetheless, tio nps also induced polymorphonuclear cell activation through phosphorylation of several proteins, including p mapk and extracellular signal-regulated kinases- / (erk- / ), which were associated with increased neutrophil life-span and production of several cytokines and chemokines ( ) . classically, apcs, macrophages, and dcs act at the site of vaccine inoculation by sensing foreign agents, through tlrs and other receptors, and triggering inflammation. apcs play a key role in the initiation, maintenance, and selectivity of inflammation, through their three major functions: endocytosis, ag presentation, and production of various cytokines and growth factors ( ) . the main family of pattern recognition receptors in microbial recognition is the tlrs, part of the family of transmembrane proteins, which affect the transcription of genes involved in inflammatory and immune response-enhancing cellular activities such as phagocytosis, endocytosis, cytotoxic functions, and cytokine production ( , ) . the adjuvants most frequently used for the induction of th and th responses are tlr agonists, such as as , cpg/ dna, and others. menps seems to have capacity to induce the expression of toll-like receptors, such as tio nps and zirconium oxide nps that have been described to enhance tlr , tlr , and tlr expression in macrophages ( ) and tlr and tlr in mouse liver cells ( ) . zinc oxide nps (plus ova) generated an inflammatory response in balb/c mice and also improve tlr- , - and - expression, followed by activation of src family kinases ( ) . consequently, tio nps and ionps were shown to induce dc upregulation of co-stimulatory molecules (mhc ii, cd ) ( , , ) , which can also be related to tlr stimuli pathways. however, none of these works demonstrate the direct interaction of nps with tlr (using knock-out mice, agonists, or antagonist molecules) thus, this interaction must be further studied. the next step in the generation of adaptive responses is the tailoring of cytokine secretion by apcs at immunological synapses, which will guide the development of the response. several nps have been reported to trigger cytokine and chemokine production, which may be used as biomarkers for immunotoxicity ( ). among those described, tio nps were used in mimetic systems composed of blood vein endothelial component (including pbmc) and was reported to trigger pro-inflammatory cytokines (il- , ifn-γ, and tnfα) ( ); zinc oxide nps were shown to be preferentially associated with monocytes and, when used in pbmc, induced ifn-γ, tnf-α, and il- cytokine production ( ); aunp-stimulated bone marrow-derived dc produced il- , tnf-α, and ifn-γ ( ) ; and ionps were shown to induce the activation of apcs with an increase of il- , tnf-α, ifn-γ, and il- , as well as chemokines. the response generated by ionps, however, was weaker than that generated by the positive control lps which may be beneficial in controlling possible side effects ( ) . the generation of a cellular response associated with protection against intracellular pathogens is the ultimate goal of vaccination. however, the direct effects of nps on cellular responses have been evaluated in only a few studies. tio nps were shown to activate and induce proliferation of naïve cd + t cells and to generate a pronounced th response with ifn-γ and tnf-α production, associated with pro-inflammatory cytokine production (il- , il- a, il- b) and dc maturation (cd + and cd + expressions increase). schanen et al. ( ) hypothesized that the oxidative capacity of an np could impact the response and trigger pro-inflammatory (oxidant capacity) or anti-inflammatory (antioxidant capacity) responses. this oxidant effect could control ros generation and thus control downstream pro-inflammatory effects while antioxidants prevent the initiation of the innate immunity in lps-stimulated macrophages ( ) . this study was, however, conducted with mitogens (non-specific stimuli) and not with vaccine stimuli, but nevertheless serves as a warning about the direct action of nps, not only on the innate immune system but specifically on t cells. there is enough evidence to suggest that menps are not only particulate formulations but also immunostimulatory molecules with several studies demonstrating their capacity to generate humoral and cytotoxic responses. menps clearly have figure | metallic nanoparticles adjuvanticity and its prediction capacity to generate t-helper (th ) and th responses. to generate a cellular immune response, the np must be able to be recognized by the host innate immune response and stimulate a sequence of events that will lead to the release of a specific milieu of cytokines and better antigen presentation (bottom arrow). in the top arrow is the immune response elicited by metallic nanoparticles to aid th and th generation. nf-κb, nuclear factor kappa b; ccl, chemokine ligand; cxcl, chemokine (c-x-c motif) ligand; gm-csf, granulocyte macrophage colonystimulating factor; ifn, interferon; il, interleukin; m-csf, macrophage colony-stimulating factor; myd, myeloid differentiation factor; tcr, t cell receptor; th, t-helper cell; tlr, toll-like receptor; tnf, tumor necrosis factor. immunostimulatory capacity and can induce several reactions in all phases of vaccine development. these capabilities correlated with np physicochemical characteristics such as size, charge, and hydrophobicity, but there are several gaps in our understanding of their mechanism of actions and how they may lead to adjuvanticity, immunomodulation, or tolerance to the ag formulated with nps. there are also evidence of menp being capable of help in the generation of th and th ; figure presents an overview of the generation of these cells subsets and the possible role of menp in this induction. ln designed the review and wrote the first draft. aj-k edited the first draft and critically reviewed the manuscript. ak edited the first draft and critically reviewed the manuscript. all authors read and approved the final version of the manuscript and agreed to submission. this work is part of ln phd thesis at biotechnology and biodiversity graduate program from capes. this work was funded by fapeg (grant number - ) and cnpq (grant number: / - ). ln received a phd fellow from capes, and apjk (# / - ) and ak (# - - ) received a productivity research fellow from cnpq. key roles of adjuvants in modern vaccines novel adjuvant formulations for delivery of anti-tuberculosis vaccine candidates landmark 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tio( ) nanoparticles the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -g fwk authors: hachim, mahmood yaseen; al heialy, saba; hachim, ibrahim yaseen; halwani, rabih; senok, abiola c.; maghazachi, azzam a.; hamid, qutayba title: interferon-induced transmembrane protein (ifitm ) is upregulated explicitly in sars-cov- infected lung epithelial cells date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: g fwk current guidelines for covid- management recommend the utilization of various repurposed drugs. despite ongoing research toward the development of a vaccine against sars-cov- , such a vaccine will not be available in time to contribute to the containment of the ongoing pandemic. therefore, there is an urgent need to develop a framework for the rapid identification of novel targets for diagnostic and therapeutic interventions. we analyzed publicly available transcriptomic datasets of sars-cov infected humans and mammals to identify consistent differentially expressed genes then validated in sars-cov- infected epithelial cells transcriptomic datasets. comprehensive toxicogenomic analysis of the identified genes to identify possible interactions with clinically proven drugs was carried out. we identified ifitm as an early upregulated gene, and valproic acid was found to enhance its mrna expression as well as induce its antiviral action. these findings indicate that analysis of publicly available transcriptomic and toxicogenomic data represents a rapid approach for the identification of novel targets and molecules that can modify the action of such targets during the early phases of emerging infections like covid- . coronaviruses are a large family of viruses that were first described over years ago ( ) . since the turn of the millennium, there have been two major global outbreaks caused by coronaviruses, namely sars-cov in and mers-cov in ( ) . the ongoing covid- pandemic caused by sars-cov- represents the third and most devastating of these outbreaks. these outbreaks, notably the covid- pandemic, are harsh reminders of the challenges posed by emerging infectious diseases. the global impact of the covid- pandemic has brought to the forefront the need to rapidly develop and deploy an effective vaccine. however, despite ongoing concerted research efforts, it is accepted that such a vaccine will not be available in time to contribute to the containment of the ongoing pandemic. current management guidelines include the use of repurposed drugs such as chloroquine and its analog hydroxychloroquine as well as antiviral agents ( ) . however, the need for well-designed clinical trials to validate their efficacy continues to be highlighted. to effectively address the ongoing covid- pandemic, there is a recognized need for a framework for rapid identification of novel targets for diagnostic and therapeutic interventions as well as determine clinically approved drugs with high potential for repurposed use against sars-cov- . publicly available transcriptomic datasets generated from sars-cov infected humans, and mammalian cells represent a wealth of data that could be used to identify consistent differentially expressed genes, which could then be validated against sars-cov- infected epithelial cells transcriptomic datasets. a comprehensive toxicogenomic analysis of the identified genes could potentially identify possible interactions with clinically proven drugs. this simple approach can be used for the rapid identification of novel targets and drugs for further validation. in this study, we have applied this approach, and our findings have identified ifitm as an early upregulated gene and indicate that valproic acid enhances ifitm mrna expression and antiviral action. publicly available transcriptomic datasets were retrieved from gene expression omnibus (geo) mouse lung tissue transcriptome response to a mouse-adapted strain of sars-cov in wild type c bl /nj mice and tlr -/-mice c bl /nj ma gse ( ) (https://www.ncbi.nlm.nih.gov/geo/). only microarray gene expression datasets with the word "sars-cov, " virus, or modified strain infected vs. mock-infected and no more than h after the infection. twelve datasets fulfilled the criteria, as detailed in table . we used geoquery and limma r packages through the geo r tool for each dataset ( ) . after sorting the genes according to the false discovery rate (fdr), the top , differentially expressed probes with fdr < . were selected from each dataset. the annotated genes of the , probes in each dataset were intersected with differentially expressed genes (degs) from all other datasets. the degs that were common in at least out of the ( %) datasets were identified as shared genes that are consistently deg in the first h of sars-cov infection. enriched ontology clustering for the identified genes was performed to explore using the metascape (http://metascape.org/gp/index.html#/main/step ). the shortlisted genes expression was then explored in another dataset (gse ), where rna-sequencing of primary human lung epithelium (nhbe) mock-treated or infected with sars-cov- was done to examine whether there is a difference in the response of sars-cov- from other strains in terms of degs ( ) . in total, , genes were differentially expressed genes (degs) between mock-infected and virally infected models in the studies. thirty-eight genes that were degs in at least out of studies ( %) were considered common degs due to sars-cov infection of the lungs in the first h post-infection. these genes are listed in table . in order to identify deg in sars-cov infected lung tissuespecific to each of the models used and those which are shared, we intersected the degs from datasets that use the same model. human epithelial cells datasets (gse , gse , gse , gse , and gse ), mice datasets (gse , gse , gse , gse , and gse ), ferret (gse ) and cynomolgus macaques (gse ) were all intersected with the covid- infected epithelial cells dataset as shown in figure . the number of deg intersected between different species is listed in the table . epithelial cells infected with sars-cov- shared degs (mx , oas , xaf , ifi , mx , irf , stat , ifit , and ifit ) with human lung cells, mice, and cynomolgus maca. the identified genes are involved in the immune response against rna viruses as expected, the top genes identified are involved in innate immune responses against rna viruses. these include the cytosolic dna-sensing pathway, toll-like receptor signaling pathway, and negative regulation of binding. interferon (ifn) response to viral infections such as type i interferon signaling pathway, defense response to the virus, the antiviral mechanism by ifn-stimulated genes, regulation of type i interferon production, response to interferon-alpha, and regulation of defense response to virus and influenza a, were also upregulated. genes that play significant roles in activating immune systems such as regulation of response to cytokine stimulus, negative regulation of immune response, myeloid cell homeostasis, and positive regulation of the multi-organism process are also upregulated (figure and table ). the identified genes expression levels were higher in human bronchial epithelium infected with sars-cov- compared to those mock-infected (figure ) . however, only ifitm showed a significant difference (p < . ), while two other genes oas and mx showed a trend of enhancement, although it was not statistically significant (p = . using the two-stage linear stepup procedure of benjamini, krieger, and yekutieli). ifitm mrna levels were one of the highly expressed genes compared to the other identified genes at baseline in mock-infected hbe and were further induced by the virus, which results in overall high mrna levels. valproic acid can upregulate the ifitm mrna expression next, we searched the comparative toxicogenomics database (http://ctdbase.org/) to identify drugs/chemicals that might affect the mrna expression of ifitm in at least two reference studies ( ) . interestingly valproic acid, carbon nanotubes, nickel, and tert-butylhydroperoxide were shown to upregulate ifitm expression while pirinixic acid, acetaminophen, and ethinyl estradiol decreased such an expression ( table ) . in order to examine the effect of valproic acid therapy on the ifitm mrna expression in immune cells of the blood, a publicly available transcriptomics dataset (gse ) was extracted and reanalyzed. healthy controls were compared to responders and non-responders patients on valproic acid therapy. we found upregulation of the mrna expression of ifitm in patients, and the difference was significant in the responder group only (p < . ) compared to healthy controls (figure ) . in response to viral rnas, like in the case of sars-cov- , the innate immune system will unleash interferon (ifn), to activate antiviral mechanisms and effector cells like natural killers ( ) . in mice infected with sars-cov, a delayed and prolonged type i interferon (ifn-i) signaling leads to lung immunopathology as it promotes the accumulation of pathogenic inflammatory cells with increased lung cytokine/chemokine levels and vascular leakage ( ) . this prolonged ifn-i and virally induced il- set the scene for secondary bacterial infection, which can add a strong il β and tnfα-mediated inflammatory response to magnify lung damage ( ) . understanding how sars-cov- can manipulate ifn is vital in deciphering the battle of the body against the viral spread and consequence. our reanalysis of transcriptomic data showed that although the ifn pathway is upregulated consistently in sars-cov related infection, sars-cov- showed specific upregulation of the gene for a unique interferon-induced protein, namely ifitm . ifitm is a -kda protein that localizes to endosomes and lysosomes and is possibly acquired by mammalian ancestral cells via horizontal gene transfer ( ) . interferon-induced transmembrane proteins (ifitms , , and ) are innate immune responders to virus infections as they regulate the fusion of invading virus and endocytic vesicles and direct it to the lysosomes ( , ) . ifitm can further alter membrane rigidity and curvature to inhibit virus membrane fusion ( ) . such action is important to prevent the release of viral particles into the cytoplasm, which controls viral spread ( ) . during influenza a infection of human airway epithelial cells, ifitm was shown to clusters on viruscontaining endosomes and lysosomes within few hours postinfection, indicating its role in the early phase of viral entry ( ) . even platelets and megakaryocytes were shown to remarkably upregulate ifitm to prevent viral progression during influenza infection ( ) . bst , ifi , ifit , ifit , ifit , irf , isg , mx , mx , oas , oas , sp , stat , isg , ifitm , usp , xaf , zbp , rsad , trim , ddx , il , cxcl , cxcl the epithelial cell and resident leukocytes in lung upper and lower airways that constitutively express ifitm can withstand viral infections, and this is vital to decide viral tropism as viruses favor cells with low ifitm expression ( ) . ifitm enhances the accumulation of cd + t cells in airways to promote mucosal immune cell persistence ( ) . lung and circulating immune cells were reported to express less ifitm than other tissues, and this was a suggestive reason for covid- severity and cytokine release syndrome ( ) . interestingly, ifitm -rs -c/c snp prevalence in the chinese population is . %, and recent research confirmed that snps in ifitm could change the severity of influenza infection, as was shown in one case with covid- ( ) . ifitm polymorphisms have been linked with hospitalization and mortality during influenza virus infection ( ) . expressing the gene is not the only prerequisite to the antiviral action of ifitm , as it was found recently that within the protein, an amphipathic helix is critical for its blocking effect of viral fusion of similar pathogenic viruses like influenza a virus and zika virus ( ). another factor that regulates the ifitm trafficking specificity to such viruses is that it requires s-palmitoylation ( , ) . s-palmitoylation (s-palm) is the reversible process of linking a fatty acid chain to cysteine residues of the substrate protein ( ) . multiple zinc finger dhhc domain-containing palmitoyltransferases (zdhhcs) can palmitoylate ifitm to make it a fully functional antiviral protein ( ) . it seems that bats (order chiroptera), which act as natural hosts for many viral infections, use ifitm as an antiviral mechanism if there is s-palmitoylation of the protein; however, if this modification is disturbed, the bat can develop viral infection ( ) . based on that, we can suggest that severe covid- cases might be due to either non-functional ifitm by snp, failure of lung cells to upregulate ifitm in response to interferon, a mutation in amphipathic helix sequence or modification in s-palmitoylation. further examination and screening for the ifitm dynamics in covid- might explain the possible therapeutic and diagnostic options. our toxicogenomic analysis showed that valproic acid increased the mrna expression of ifitm , supporting a new report that the sars-cov- -human protein-protein interaction map showed that valproic acid might be a potential repurposing drug for covid- ( ) . virtual screening, docking, binding energy calculation, and simulation show that valproic acid forms stable interaction with nsp of cov and can inhibit its function ( ) . valproic acid is currently used for the treatment of epilepsy and known to target histone deacetylases (hdacs) that modify the gene expression epigenetically ( ) . valproic acid was shown to inhibit mature and fully infectious enveloped viruses release as it alters cellular membrane composition ( ) . the modest and broad antiviral activity of valproic acid made the drug an attractive possibility due to its availability, and limited side effects for a short term of use during acute viral disease ( ) . the reported side effects like hepatotoxicity and teratogenicity are mainly associated with the parental compound valproate and can be avoided by the use of its derivatives like valpromide (vpd) and valnoctamide (vcd) . a recent open-label proof-of-concept trial of days intravenous valproic acid for severe covid- showed a % reduction in the case fatality rate and length of stay ( ) . more studies are needed to explore the promising potential of valproic acid in the treatment of covid- . one limitation of the study is that it is based on the publicly available transcriptome dataset, which is limited in number, partly because this is a novel disease, but also because ongoing lockdowns have made it challenging for scientists to carry out the extensive laboratory work required. our evaluation showed that the analysis of publicly available transcriptomic data could be a reasonable approach to identify the novel target and suggest drugs that can modify the action of such targets during the early phases of emerging infections like covid- until a complete understanding of the disease become clear. this can justify the experimental use of clinically approved drugs and guide the clinicians in their limited options against such lethal disease. all datasets presented in this study are included in the article/supplementary material. coronaviruses in animals and humans three emerging coronaviruses in two decades covid- -navigating the uncharted lack of innate interferon responses during sars coronavirus infection in a vaccination and reinfection ferret model dynamic innate immune responses of human bronchial epithelial cells to severe acute respiratory syndrome-associated coronavirus infection comparative 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pharmacological strategies to fight enveloped viruses trends in antiviral strategies methods of an openlabel proof-of-concept trial of intravenous valproic acid for severe covid- . medrxiv all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. key: cord- -jcvrqeew authors: gelain, maria elena; bonsembiante, federico title: acute phase proteins in marine mammals: state of art, perspectives and challenges date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: jcvrqeew the term “acute phase response” (apr) is referred to a nonspecific and complex reaction of an organism that occurs shortly after any tissue damage, such as infection, trauma, neoplasia, inflammation, and stress. the apr can be identified and monitored with some laboratory tests, such as the concentration of several plasma proteins, the acute phase proteins (apps). the apps are components of the non-specific innate immune response, and their plasma concentration is proportional to the severity and/or the extent of tissue damage. the evaluation of health status of marine mammals is difficult because the classical clinical signs of illness used for human and domestic animals are difficult to recognize and understand. for this reason, in the past years, several efforts were done to identify laboratory markers of disease in these animals. the apps have demonstrated their role as early markers of inflammation in veterinary medicine, thus several apps were tested in marine mammals, such as c-reactive protein (crp), serum amyloid-a (saa), and haptoglobin (hp). however, the difficulty to extrapolate the knowledge about apps in one species to another, the lack of specie-specific reagents, the absence of data about negative apps have hampered their extent use in marine mammals. herein, the state of art of apps in marine mammals is reviewed, with particular attention to pre-analytical and analytical factors that should be taken into account in validation and interpretation of apps assays. moreover, the current application, potential utility and the future developments of apps in marine mammals is highlighted and discussed. the mammalian immune system includes innate or nonspecific immunity as well as adaptive or specific immunity. the responses of these two different pathways are distinct, but highly interconnected. the first reaction of an organism to different pathological conditions is an innate, non-specific response ( ), a more conserved response during evolution which aim is the immediate reaction against pathological stimuli ( ) . after the initial recognition of pathogens or tissue damages by the tissue-resident macrophages, which express the pattern recognition receptors (prrs), a variety of different inflammatory mediators are produced by leukocytes, endothelial cells, tissue cells or are derived from plasma proteins. these mediators include different chemokine, cytokine, vasoactive amines and products of the arachidonic acid: their primary effect is to elicit inflammatory response locally and to recruit leukocytes and plasma proteins in the site of injury ( ) . glycoprotein (agp) ( ) , in ruminants is haptoglobin (hp) ( ) ; in horse is serum amyloid a (saa) ( ) and in pigs are hp, saa, and major acute phase protein (map) ( ) . recently, in veterinary medicine, studies on the role of apps as markers of infectious, inflammatory and neoplastic diseases have proliferated ( ) and at least different plasma proteins have been identified as apps ( ) . their use as marker of homeostasis perturbation provides some advantages compared with traditional parameters like the white blood cell (wbc) counts. compared to wbc count, the diagnostic sensitivity of apps is higher and the change in concentration is faster ( ) . moreover, their stability in serum/plasma is high, so it is possible to measure apps in frozen samples ( ) . one limitation of the apps is the poor diagnostic specificity, for this reason they cannot be used as primary diagnostic test for a specific disease, but they were successfully used to detect subclinical diseases and to monitor clinical evolution and to assess the response to treatment ( ) . additionally, the combined measurement of several apps provides more information than the evaluation of a single protein: in the "app value index, " proposed by gruys et al. ( ) , sensitivity and specificity are improved by combining response of both positive and negative major and minor apps. marine mammals are a group of around mammalian species which depend on water environment for most of their needs. they include orders: carnivora (pinnipeds, otters, and polar bear), cetacea (dolphins, whale, and porpoise) and sirenia (manatee and dugogons). marine mammals are differently adapted to life in water, with some species, which are fully aquatic (cetaceans and sirenia), and others that spend part of their life on land (pinniped and polar bears) ( ) . from an immunological point of view, aquatic adaptation caused few differences in distribution and function of immune system between marine and terrestrial mammals ( ) . however, nowadays the marine mammal' immune system is deeply exposed to environmental pollution because they are a long-lived animals placed at the top of food chain, thus they are exposed to a progressive bioaccumulation of fat-soluble pollutants, such as pcbs, which affect both innate and adaptive immune function ( ) . for these reasons, increasing knowledge in cellular and humoral immune response is continuously required to understand their immune system and in particular its relationship with infectious pathologies and the environmental pollution ( ) . furthermore, marine mammals live also in controlled environment like aquaria, rehabilitation facilities and research center where health assessment is fundamental to evaluate the correct management of animals and to monitor the response to therapy during rehabilitation. from this perspective, the availability of new markers to asses immune functions is fundamental both for medical care and research purpose ( ) . innate immune response represent the first line of response against pathological stimuli, it's very fast and it's based primarily on effector cells (e.g., mast cells, macrophages, neutrophils) and antimicrobial substances (e.g., complement, reactive oxygen, and nitrogen species). on the other hand, adaptive immune system is antigen-specific, it takes more days to be effective and it's based on different t-cells response and on b-lymphocytes which are responsible of humoral response mediated by the different subclass of immunoglobulin (igg, igm, iga) ( ) . several assays were proposed to evaluate both immune response in marine mammals, generally based on isolated leucocytes with the aim to evaluate the leukocytes response against in vitro stimuli ( ) . to assess the response pattern of cetaceans' cellular innate immune system, the phagocytosis and the generation of reactive oxygen species of polymorphonuclear leukocytes were investigated. in particular, in vitro ingestion of latex beads and hydrogen peroxide production have been evaluated in beluga whales (delphinapterus leucas) and in bottlenose dolphins (tursiops truncatus) ( , ) whereas phagocytosis and respiratory burst assay, using whole blood from bottlenose dolphins, were used to assess the antimicrobial activity ( ) . in addition, the investigation of apr by analyzing the cytokine expression gives important information on the functionality of lymphoid cells. the production of specie-specific antibodies allows the development of immunological assays for the quantification of cytokine expression useful to investigate the inflammatory response in whales and dolphins. the coding regions of il- , il- β, il- , and tnf-α gene of the beluga whale have been sequenced, and a cytokine-specific rabbit antisera have been produced ( ) ( ) ( ) . in harbor porpoise (phocoena phocoena), the quantification of mrna of il- β, il- , il- , il- , il- , and tnf-α have been performed by rt-pcr ( ) , and the increase of il- was seen in harbor porpoises suffering from long lasting infectious ( ) . also in bottlenose dolphins, pacific white-sided dolphins (lagenorhynchus obliquidens), and beluga whales, il- , il- , il- , il- , il- , il- , tnf-α, tgf-β, and interferon (ifn)-γ quantification was performed using rt-pcr ( ) . an il- receptor expression assay and an il- elisa were developed in bottlenose dolphins and killer whale (orcinus orca), respectively ( , ) . similarly, both innate and the cell-mediated response were studied in pinnipeds. to better understand the innate response, phagocytic activity of isolated peripheral blood leukocytes was evaluated in harbor seal (phoca vitulina), gray seal (halichoerus grypus), and harp seal (phoca groenlandica) pups, in harbor seal female during lactation and in harbor seal pups admitted to rescue center ( , ) . the authors found an age-related variation in both pups and adults: phagocytosis increased with age in gray and harbor seal pups, while in female harbor seals decreased from sub-adult to adulthood. at the same time, pups after rehabilitation showed a decreased phagocytic activity, probably due to the decreased stimulation of innate response after therapy. also cytokine response was evaluate in harbor seal. pro-inflammatory cytokine mrna (il- β, il- , il- , and il- ) in pups in a rehabilitation center were higher at admission whilst il- was higher before the release ( ), demonstrating the recovery from inflammation. recently, a multiplex canine cytokine assay was validate in harbor, gray and harp seal to measure proteins levels in cell culture supernatant of peripheral blood mononuclear cells (pbmc) ( ) . however, all these techniques are not generally applicable in a clinical setting in which the primary goal is a sensitive diagnostic tool with a rapid turnaround, even if give us important information on factors affecting cetaceans' immune system. for this reason, in the past years, several efforts were made to identify laboratory markers of disease in these animals. first parameters tested were wbc and erythrocyte sedimentation rate ( ) . however, even if they are inexpensive and rapid, they lack specificity and sensitivity. moreover, changes in wbc occur after several hours after inflammatory stimuli. thus, efforts were directed to identify inflammation at earlier stage ( ) . to examine the humoral response, species-specific antibodies against igg were produced and used to evaluate serum igg levels in killer whale by radial immunodiffusion assay ( ) and by competitive elisa in bottlenose dolphins ( , ) . the determination of igg baseline values in free-ranging and in managed dolphins revealed higher levels of immunoglobulin in the first group with several values over the accurate range of the assay, probably due to the higher parasitic load in free-ranging dolphins ( ) . serum total protein, albumin, globulin and albumin:globulin ratio (a:g) are undoubtedly among the most measured markers in basic health assessment in domestic animals as well as in marine mammals. serum protein electrophoresis is also broadly applied in veterinary medicine and it has the advantage to produce an accurate measurement of albumin and the visualization of globulin fractions ( ) . the interpretation of total proteins values and electrophoretic pattern of serum proteins is receiving increased attention also in marine mammals in which a typical pathologic pattern could be identified in inflammatory diseases ( ) . reference intervals for these markers are available for free-ranging bottlenose dolphins ( ) and, compared to these, recently data on managed dolphins showed slightly lower values of tp, α-globulins, and γ-globulins and higher albumin and albumin/globulins ratio ( ) . it's interesting to note that hp, α -antitrypsin, α antichymotripsin, and α -macroglobulin migrate in the α-globulins fraction, while the igg and crp migrate in the γ-globulins fraction. albumin acts as a negative acute phase protein since the synthesis of this protein is decreased during an inflammation ( ) . thus, the lower concentration of positive apps associated to a higher concentration of albumin and the consequent higher albumin/globulins ratio could reflect lower antigenic stimuli in managed population compared to the free-ranging populations ( ) . serum total protein analysis were used to assess health status in several cetaceans species such as pantropical spotted dolphins (stenella attenuata) ( ), beluga ( ) , minke whales (balaenoptera acutorostrata) ( ) and killer whales ( ) as well as in other marine mammals, like harbor seals (phoca vitulina) ( ) and walruses (odobenus rosmarus) ( ) . in all these species, serum total protein analysis was demonstrated to be one of the most used and commonly accepted marker of inflammation. however, specific apps have demonstrated their superior role as early markers of inflammation, so based on the results obtained in humans and companion animals, several positive apps were tested in marine mammals ( table ) . published works had the primary aims to evaluate the feasibility of the assays to measure the apps, to validate the antibody-based assay and to determine the ris. in bottlenose dolphins three apps (crp, saa, and hp) were tested, even if not always complete validation studies were performed ( , ) . for these apps, the authors established the ris in free-ranging and managed dolphins using automated assays ( ) and they found significantly lower saa and higher hp levels in free ranging animals. the only clinical significance of these alteration was a higher ability to detect chronic inflammation for hp. regarding hp, segawa and colleagues validated commercially available hp-elisa and hp-hemoglobin binding assay in bottlenose dolphins with ''acceptable" intra-and inter-assay imprecision (cv: . % healthy dolphins and . % inflamed dolphins; cv: . % healthy dolphins and . % inflamed dolphins) and demonstrated that hp levels in the serum increase under inflammatory conditions ( ) . positive apps were tested also in florida manatees (trichechus manatus latirostris) to define the more accurate marker of inflammation. five different apps were tested: agp, crp, hp, fibrinogen, and saa. saa showed the highest diagnostic sensitivity and specificity ( % for both sensitivity and specificity) in the detection of inflammatory diseases, the diagnostic specificity of hp and fibrinogen were and %, respectively, while their diagnostic sensitivity were and %, respectively, ( ) . when used in stranded manatee suffering from cold stress and trauma, saa showed % of sensitivity and % of specificity in detecting diseased animals ( ) . by contrast, the abs used for the determination of agp and crp did not cross-react in this species ( ) . in harbor seal, an ab anti-crp and a competitive immunoassay was produced ( ), but hp is probably the app most used in pinnipeds. a multispecies assay based on hemoglobin binding capacity was used to demonstrate as hp is a sensitive marker of the health vs. disease status in harbor seal ( ) . in seal pups admitted in a rescue center. hp, total protein, igg and globulin values correlated positively, but hp levels increased during the hospitalization, probably reflecting age-related changes ( ) . hp is considered a health marker also in steller sea lions (eumetopias jubatus): significantly higher levels of hp were found in declining population compared to more stable ones ( ) . however, also genetic differences between distant and isolated population of wild animals could be the causes of this difference, not only a pathological condition. if some data on marine mammals positive apps are available in literature, quite surprising are the lack of data available on negative apps. for these reasons, the possibility to evaluate the usefulness of an "app value index" is still far from being applied. the availability of sensitive markers of inflammation both for free-ranging and managed marine mammals is nowadays considered fundamental to evaluate the health status and, in rehabilitation setting, to monitor the response to therapy and to define the prognosis. as serum markers, the apps have several advantages: they have longer stability compared to other blood component such as wbc; they can be performed on frozen serum, thus the samples can be shipped to references laboratories; some assays can be automated to obtain results in an excellent turnaround time. however, is important to consider that the knowledge about apps in one species cannot be readily generalized to another species, in which healthy levels, response to inflammation or infection, and prognostic significance may be different ( ) . moreover, the evolution of marine mammals and their adaptation throughout the millennia to an aquatic environment had led to a different physiology and metabolism compared to terrestrial mammals. thus, the understanding of the genetic, phenotypical and biochemical properties of marine mammals apps are essential prior to using them as a new biomarker. an example of how the biochemical properties influence the analytic method is paroxonase- (pon ), a hdl-bound esterase which protects against organophosphate compounds, acts as negative app and as oxidative stress marker. pon is usually assessed by enzymatic method and, based on the different pon functions, several substrates have been identified to evaluate serum pon activities. nevertheless, both in humans as in some terrestrial mammals, pon gene polymorphisms highly influence the enzymatic activity toward different substrate: the single-nucleotide polymorphisms (snps) leu met and gln arg increase the paraoxonase activity ( ) in humans and different pon genotypes influence activities toward paraoxon and phenyl-acetate in rabbit ( ) . also in cows, some snps in the promotor region of pon gene are associated with serum pon activity ( ) . recently, a phylogenetic study on convergent functional losses across marine mammals, has identified a pon functional loss in marine mammals, probably related to their different lipid metabolism and fatty acid oxidation due to adaptation to the marine environment and a high concentration of ω- fatty acids on their diet. as a consequence, in several marine mammals species paroxonase activity is very low, while enzymatic activity against other pon substrates is still present, such as arylesterase activity ( ) . for these reasons, the use of classical enzymatic assays is hampered in these animals and further studies are needed to elucidate the role of pon as possible negative app, oxidative stress marker and the consequences of its inability to detoxify organophosphates compounds. from an analytical point of view, another challenge in the evaluation of apps in marine mammals is the need of speciesspecific assays, especially for the immunological assay, such as elisa or immunoturbidimetry. this means the development of a de-novo method, often a time-consuming and expensive approach, or the validation of a commercial available assay used in other species ( ) . the latter approach is surely the most used in veterinary medicine, in which some human assays were validated for dogs, cats and horses ( ) . however, even if some apps appeared highly conserved among species, an accurate validation of antibody cross reactivity is needed as well as species specific standards and control material ( ) . among positive apps, saa is the most used across different species: it appeared as the most conserved app in mammals even if some difference in circulating isoforms were reported ( ) and it's considered a major app in all the mammals in which it was investigated ( ) . some commercial saa assays showed good results also in marine mammals, such as bottlenose dolphin, manatee and striped dolphin (stenella ceoreloualba) ( , , ) and its use as diagnostic and prognostic marker appears nowadays the most promising. to obtain accurate data, all the pre-analytical factors that could influence the results should be taken into consideration. the effect of storage, temperature and different anticoagulant had to be evaluated in a correct validation process as well as the interference of hemolysis and lipemia, as done in other species ( ) . the application of a novel biomarker required a full evaluation of all the analytical performances and the clinical value. this process is usually divided in steps: the assessment of analytical features (precision, accuracy, detection limits), the overlap performance (the ability to detect difference between healthy and diseased animals), the assessment of diagnostic capacity (sensitivity, specificity, accuracy, positive, and negative predictive values) and, at the end, the evaluation of the outcome of the new methods (which is the advantage of the test and its influence in the patient management) ( ) . in veterinary medicine, the validation studies do not always follow all these steps, mainly due to the lack of resources or technical limitation ( , ) . also in marine mammals, the majority of studies had performed only some steps ( , , , , ) . this is mainly due to the limitation in species-specific reagents, the number of samples from animal with known health status and, last but not least, the capability to generate appropriate reference intervals, hampered the possibility to perform complete validation studies. population-based reference intervals derived from an appropriate group of reference individuals are usually required for diagnostic purpose ( ) . however, a number of biological factors have to be taken in consideration to select the appropriate reference population. surely, age, sex and pregnancy could be used for partitioning ( , ) , but in marine mammals greater attention should be given to the difference between wild and managed animals. serum protein electrophoresis values obtained in managed bottlenose dolphins showed lower total proteins and higher albumin levels compared to reference intervals derived from free-ranging ( ) while wild-caught manatees, apparently clinically healthy, had saa level above reference limit ( ) . these data could indicate a trend to an inflammatory status or the presence of subclinical inflammation in free-ranging animals which are more exposed to immunological stimuli. in any case, this highlights the need to define appropriate reference intervals for animals living in different environment to have an accurate toll for the evaluation of clinical condition. compared to human and companion animals, the use of apps in marine mammals is just getting started. the increasing need of knowledge on immune system and its response against infectious diseases or chemical pollutants and the request of more sensitive inflammation markers have increased the effort of researchers to study the apr and apps. even if apps are considered a sensitive, but non-specific marker of inflammation, some studies revealed that, in some infectious diseases, apps showed a specific behavior and biochemical features. one example is the modification of the glycan moiety of agp in feline infectious peritonitis, fiv and felv, influencing the host-pathogens interaction and the immune response ( ) ( ) ( ) . currently, some of the greatest threats for wild marine mammals is pathogens, like morbillivirus, herpesvirus, brucella ceti, and toxoplasma gondii ( ): the evaluation of apr and apps patterns during these infectious diseases could lead to the identification of a distinctive response of the immune system and increase the understanding of hostpathogen interaction. secondly, for managed or rescued animals, the forthcoming needs are the increase of automated assays, the standardization of procedures across laboratories and the discovery of new markers, for example negative apps, to generate an app index also in marine mammals. these new tools will certainly increase the diagnostic and prognostic skills for health assessment and, especially for stranded animals, the development of new "health status" markers will provide valuable resources in evaluating the response to treatment and rehabilitation prior to the release into the wild. mg and fb analyzed the literature review, designed, and wrote the review. monitoring health by values of acute phase proteins inflammation and factors that may regulate inflammatory response origin and physiological roles of inflammation inflammatory mechanisms: the molecular basis of inflammation and disease acute phase proteins in dogs and cats: current knowledge and future perspectives initiation of acute phase response and synthesis of cytokines acute phase proteins: biomarkers of infection and inflammation in veterinary medicine current research on acute phase proteins in veterinary diagnosis: an overview the feline acute phase reaction application of acute phase protein measurements in veterinary clinical chemistry acute phase proteins as promising biomarkers: perspectives and limitations for human and veterinary medicine applications of acute phase reactants in infectious diseases an immunoturbidimetric assay for canine c-reactive protein bovine acute phase response following turpentine injection serum amyloid a protein (saa) in horses: objective measurement of the acute phase response the final hurdles for acute phase protein analysis in small animal practice study on biological variability of five acute-phase reactants in dogs haptoglobin and ceruloplasmin as determinants of inflammation in dogs chapter -introduction marine mammal immunology immunotoxic effects of environmental pollutants in marine mammals cetacean host-pathogen interaction(s): critical knowledge gaps immunology of whales and dolphins evaluation of the polymorphonuclear cell functions of bottlenose dolphins immune functions in beluga whales (delphinapterus leucas): evaluation of mitogen-induced blastic transformation of lymphocytes from peripheral blood, spleen and thymus simultaneous measurement of phagocytosis and respiratory burst of leukocytes in whole blood from bottlenose dolphins (tursiops truncatus) utilizing flow cytometry molecular cloning and characterization of beluga whale (delphinapterus leucas) interleukin- beta and tumor necrosis factoralpha molecular cloning, phylogenetic analysis and expression of beluga whale (delphinapterus leucas) interleukin molecular cloning and phylogenetic analysis of beluga whale (delphinapterus leucas) and grey seal (halichoerus grypus) interleukin development of a lymphocyte-transformation-assay for peripheral blood lymphocytes of the harbor porpoise and detection of cytokines using the reverse-transcription polymerase chain reaction increased blood interleukin- mrna levels in diseased free-ranging harbor porpoises (phocoena phocoena) quantitation of leukocyte gene expression in cetaceans development of an interleukin- receptor expression assay and its use in evaluation of cellular immune responses in bottlenose dolphin (tursiops truncatus) expression and functional characterization of killer whale (orcinus orca) interleukin- (il- ) and development of a competitive immunoassay immune status and function in harbor seal pups during the course of rehabilitation phagocytosis in pup and adult harbour, grey and harp seals cytokine and acute phase protein expression in blood samples of harbour seal pups validation of a commercial canine assay kit to measure pinniped cytokines clinical pathology crc handbook of marine mammal medicine measurement of serum immunoglobulin concentration in killer whales and sea otters by radial immunodiffusion development and validation of monoclonal and polyclonal antibodies for the detection of immunoglobulin g of bottlenose dolphins (tursiops truncatus) baseline circulating immunoglobulin g levels in managed collection and freeranging bottlenose dolphins (tursiops truncatus) acute phase response in animals: a review hematologic and serum biochemical reference intervals for freeranging common bottlenose dolphins (tursiops truncatus) and variation in the distributions of clinicopathologic values related to geographic sampling site detection of hereditary bisalbuminemia in bottlenose dolphins (tursiops truncatus fundamentals of veterinary clinical pathology hematological, serum, and plasma chemical constituents in pantropical spotted dolphins (stenella attenuata) following chase, encirclement, and tagging serum chemistry of freeranging white whales (delphinapterus leucas) in svalbard serum chemistry of the minke whale from the northeastern atlantic hematological and serum biochemical analytes reflect physiological challenges during gestation and lactation in killer whales (orcinus orca) hematology and serum chemistry in stranded and wild-caught harbor seals in central california: reference intervals, predictors of survival, and parameters affecting blood variables serum chemistry reference values in free-ranging north atlantic male walruses (odobenus rosmarus rosmarus) from the svalbard archipelago acute phase protein quantitation in serum samples from healthy atlantic bottlenose dolphins (tursiops truncatus) evaluation of immune and stress status in harbour porpoises (phocoena phocoena): can hormones and mrna expression levels serve as indicators to assess stress? comparison of methods used to diagnose generalized inflammatory disease in manatees (trichechus manatus latirostris) assessement of serum amyloid a levels in the rehabilitation setting in the florida manatee (trichechus manatus latirostris) plasma haptoglobin levels in threatened alaskan pinniped populations characterization of haptoglobin in the blood plasma of harbor seals (phoca vitulina) fibrinogen concentrations in captive bottlenose dolphins during pregnancy characterization of the circulating serum amyloid a in bottlenose dolphins molecular characterization and validation of commercially available methods for haptoglobin measurement in bottlenose dolphin harbor seal (phoca vitulina) c-reactive protein (c-rp): purification, characterization of specific monoclonal antibodies and development of an immuno-assay to measure serum c-rp concentrations acute phase protein haptoglobin in blood plasma samples of harbour seals (phoca vitulina) of the wadden sea and of the isle helgoland assay validation and diagnostic applications of major acute-phase protein testing in companion animals human paraoxonase- (pon ): gene structure and expression, promiscuous activities and multiple physiological roles rabbits possess a serum paraoxonase polymorphism similar to the human q r characterization of single nucleotide polymorphisms in the promoter region of the bovine paraoxonase (pon ) gene affecting serum enzyme activity in dairy cows ancient convergent losses of paraoxonase yield potential risks for modern marine mammals clinico-pathological findings in a striped dolphin (stenella coeruleoalba) affected by rhabdomyolysis and myoglobinuric nephrosis effects of age and sex on clinicopathologic reference ranges in a healthy managed atlantic bottlenose dolphin population association between faecal shedding of feline coronavirus and serum alpha -acid glycoprotein sialylation hyposialylated α -acid glycoprotein inhibits phagocytosis of feline neutrophils glycan moiety modifications of feline alpha -acid glycoprotein in retrovirus (fiv, felv) affected cats emerging infectious diseases in cetaceans worldwide and the possible role of environmental stressors clinico-pathological investigation of serum proteins in odontocetes the literature review presented in the manuscript is partially included in ph.d. thesis of bonsembiante ( ), supported by a ph.d. grant from the university of padova. key: cord- -tpjxt w authors: mandl, judith n.; schneider, caitlin; schneider, david s.; baker, michelle l. title: going to bat(s) for studies of disease tolerance date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: tpjxt w a majority of viruses that have caused recent epidemics with high lethality rates in people, are zoonoses originating from wildlife. among them are filoviruses (e.g., marburg, ebola), coronaviruses (e.g., sars, mers), henipaviruses (e.g., hendra, nipah) which share the common features that they are all rna viruses, and that a dysregulated immune response is an important contributor to the tissue damage and hence pathogenicity that results from infection in humans. intriguingly, these viruses also all originate from bat reservoirs. bats have been shown to have a greater mean viral richness than predicted by their phylogenetic distance from humans, their geographic range, or their presence in urban areas, suggesting other traits must explain why bats harbor a greater number of zoonotic viruses than other mammals. bats are highly unusual among mammals in other ways as well. not only are they the only mammals capable of powered flight, they have extraordinarily long life spans, with little detectable increases in mortality or senescence until high ages. their physiology likely impacted their history of pathogen exposure and necessitated adaptations that may have also affected immune signaling pathways. do our life history traits make us susceptible to generating damaging immune responses to rna viruses or does the physiology of bats make them particularly tolerant or resistant? understanding what immune mechanisms enable bats to coexist with rna viruses may provide critical fundamental insights into how to achieve greater resilience in humans. an estimated ∼ % of emerging infectious diseases are caused by pathogens which originate from a non-human animal source, referred to as zoonoses ( ) ( ) ( ) . moreover, the frequency of outbreaks caused by zoonotic pathogens has been increasing over time in the human population, with viruses being the most successful at crossing the species barrier ( ) ( ) ( ) . given the impact of viral zoonoses on global public health, considerable resources have been invested into better understanding patterns in their emergence to improve predictions of where they might arise. one key variable in such predictions is to determine the animal reservoir populations within which these novel viruses can be maintained indefinitely (with or without disease) and which therefore act as sources for transmission to humans ( ) . in some instances, epidemiological associations may provide clues to identifying a reservoir host species, and the detection of natural infection through seroconversion or the virus itself provides further evidence. recently, phylogenetic analyses have also been used to investigate viral origins-with a presence of greater diversity and of strains ancestral to those in humans being indicative of a virus circulating within a particular natural host population ( ) . once identified, viral reservoirs have historically been critical levers through which to reduce human cases ( ) . however, reservoir hosts may also provide us with fundamental insights into host-pathogen interactions and are a rich opportunity to examine the immunological processes that contribute to patterns governing which pathogens cross into humans, cause disease and why ( , ) . this can be particularly informative as in many instances, the zoonotic viruses that are so pathogenic in humans do not cause disease in the reservoirs with which they coexist. bats have been confirmed as reservoir hosts for many viruses, several of which are associated with fatality rates as high as % among diagnosed human cases. it has long been appreciated that rabies and other lyssaviruses causing lethal encephalitis can be transmitted from numerous bat species ( , ) . live marburg virus (marv) has been isolated from rousettus aegyptiacus fruit bats which, jointly with epidemiologic evidence and detection of viral rna, strongly suggests that r. aegyptiacus is a reservoir host of this filovirus ( ) . the related ebolavirus (ebov) likely also circulates in african fruit bats, with a few species having been implicated so far-the mobility of which accounts for the sudden appearance of ebola in west africa during the outbreak, a region where ebolavirus had not previously been detected ( , ) . the highly pathogenic henipaviruses, of which hendra virus emerged in australia and nipah virus in south-east asia via horse and pig intermediate hosts respectively, have been shown to be transmitted from pteropus bats ( , ) . in china, horseshoe rhinolophus bats have been identified as the reservoirs for sars coronavirus via palm civet intermediate hosts, the cause of a large outbreak of atypical pneumonia across several countries that began in in china ( ) ( ) ( ) . more recently, mers coronavirus that has caused lethal respiratory infections mostly in saudi arabia, likely transmitted via dromedary camels, was shown to be closely related to several bat coronaviruses, including those sequenced from neoromicia capensis, pipistrellus abramus, and vespertilio superans bats ( , ) . moreover, additional viruses may continue to emerge from bats, as in the single case of sosuga virus infection in a wildlife biologist collecting bats in south sudan ( ) . in addition to these emerging zoonotic viruses, bats may be the source of a number of viruses with which humans have older evolutionary associations. for instance, bats harbor viruses closely related to both mumps (rubula virus) and measles (morbilli virus) and have likely been donors of these viruses to other mammalian groups, possibly including humans ( , ) . furthermore, both old and new world bats carry diverse hepadnaviruses, some of which are related to hepatitis b virus and can infect human hepatocytes ( ) . hepaciviruses that are related to hepatitis c virus and pegiviruses that are related to human gb viruses were detected in the sera of many different bat species, and given the basal position of these bat viruses in phylogenetic trees, may also represent strains ancestral to those found in humans ( , ) . the preponderance of links between bat and human pathogens has led to a debate about whether bats disproportionately contribute to emerging viral infections crossing the species barrier into humans ( ) ( ) ( ) ( ) ( ) . given the diversity of the chiroptera order (figure ) , we may simply see more bat viruses because there are so many (> , ) species of bats ( ) . however, even when accounting for the fact that they make up ∼ % of extant terrestrial mammals, bats are overrepresented as reservoir hosts of pathogens with a high potential for spilling into human populations ( , ) . in fact, no known predictors that have been described to impact the likelihood of crossing the species barrier, including reservoir host ecology, phylogenetic relatedness to humans or frequency of reservoir-human contact, explain this pattern ( ) . thus, why bats are such a frequent source of pathogenic human viruses remains a tantalizing mystery. among viruses, those that have genomes encoded by rna generally jump across species boundaries more frequently, presumably due to their inherently greater mutation rates that facilitate the rapid adaptation to replicating within new hosts ( ) . interestingly, all pathogenic viruses that have made the jump to humans for which bat species may be reservoirs share the common feature that they have single-stranded rna genomes (with the exception of hepadnaviruses which have a dna genome but replicate via an rna intermediate). so far, available evidence suggests that bats remain disease-free when infected with the rna viruses they carry-even those highly pathogenic to humans-and are able to coexist with them without detectable fitness costs using measures such as changes in temperature, loss of body weight, or overt signs of inflammation ( ) . indeed, so far only one rna virus studied which circulates in a bat population has been shown to consistently cause significant morbidity and mortality: tacaribe virus in the jamaican fruit bat (artibeus jamaicensis), which recent evidence suggests is not a reservoir host for this virus ( ) . data from experimental rabies and lyssavirus infections suggests that rhabdoviruses may also cause disease in bats, although experimental infection outcome is very dependent on the infection route. intracerebral infection with different strains and in different bat species invariably led to death ( , ) . in contrast, intramuscular infection led to muscle weakness, paralysis and visible histological cns lesions in % of experimentally infected flying foxes (pteropus poliocephalus) ( ) . similarly, a subset of vampire bats (desmodus rotundus) experimentally infected intramuscularly with a high dose of rabies virus remained healthy despite viral shedding in the saliva and survived ( ) . naturally infected bats are thought to either die or remain healthy and seroconvert, but transmission in freeranging populations remains incompletely understood ( ) . while bats seem to be frequent hosts for rna viruses, current available data indicates that primates and humans disproportionately harbor dna viruses such as herpesviruses ( ) . interestingly, it is these dna viruses that can persist in an individual which can also be found in isolated, small indigenous groups-perhaps suggestive of humans having a more ancient relationship with such dna viruses ( ) . it may even be the case that persistent dna viruses in humans impact immune responses specifically to rna viruses, but this has not yet been examined. it is likely that differences in evolutionary history of pathogen exposure between bats and humans have led to distinct adaptations in anti-viral immune responses and the ability to tolerate certain infections without disease while being susceptible to others. importantly, bats differ in many aspects of their physiology and behavior from humans that may have direct or indirect effects on immune function. bats are a monophyletic mammalian group traditionally divided by morphological data into two suborders, the megabats and microbats, which more recent molecular data has revised into the yinpterochiroptera and yangochiroptera suborders (figure ). bats possess a suite of traits that make them distinct from other mammals in a number of ways. these unique life history traits may play a role in understanding which pathogens bats have evolved to coexist with and why. in particular, such traits may explain the ability of bat populations to maintain particular viral pathogens indefinitely, and may have effects on immune function through specific energetic or evolutionary trade-offs we have yet to better define. despite the diversity of viruses carried by bats, they are not typically known to cause mass bat die-offs or reduce bats' remarkable longevity. in this respect, bats represent a potential opportunity for long-term persistence of viruses within a population and across generations. bats live significantly longer than similarly-sized terrestrial mammals and, despite their small size, are characterized as "slow" mammals in the slow-fast continuum ( , ) . although their weights range from grams to kilograms, with respect to longevity bats group with large mammals such as humans and non-human primates ( ) . aerial living has an obvious advantage in avoiding predation, but bats outlive even birds. for example, the brandt's bat (myotis brandtii) lives up to years, compared to selasphorus platycercus, a bird species of similar size that lives for ∼ years ( , ) . thus, flight can only partially account for their extraordinarily long lives. initially, the longevity of some bats was attributed to seasonal hibernation, as temperate-zone species enter continuous torpor of up to days, with a dramatic drop in metabolic rate such that small fat reserves can sustain them throughout the entire hibernating season ( ) . however, even non-hibernating bat species live three times longer, on average, than predicted by their size, and heterothermy is not an accurate predictor of lifespan in other mammalian orders, suggesting that the driving force behind their surprising longevity is intrinsic to bats as a group ( ) ( ) ( ) . like other "slow" mammals, bat females typically only have one offspring per year, perhaps because the volant lifestyles of bats make it difficult to rear more than one offspring, as pregnant females and those with recent births must navigate and forage with added weight; on average, neonatal bat pups are ¼ of their mother's weight ( ) . the physical and energetic constraints of rearing multiple offspring may necessitate small litters, which would in turn require prolonged reproductive capability and enhanced longevity to ensure maintenance of the population over generations. thus, in bats, the dependence of colony survival as a whole may depend upon enhanced individual survival and delayed senescence ( ) . genetic analyses of several bat species have shown differences in the growth hormone (gh)/insulin-like growth factor (igf ) axis which in humans is associated with aging, resistance to diabetes and cancer ( ) . the determinants of adult survival in bats have been historically difficult to identify, as this requires tracking individuals over many years, and until recently longitudinal studies of bat mortality were conducted using tagged bats, of which only a fraction were recovered ( ) . recently, a year study of a colony of bechstein's bats demonstrated that unlike terrestrial mammals, survival could not be predicted by common indicators such as season, age, and body size. instead, the only accurate predictor of mortality was a single cataclysmic weather event that affected multiple countries in north-central europe. additionally, even the oldest female bats were reproductively capable, indicating that bat survival is primarily affected by catastrophic natural events rather than factors that normally dictate an individual's fitness ( ) . molecular phylogenetic studies of bats suggest that there are massive gaps in bat fossil records. as bats are the second most diverse order of mammals, outnumbered only by rodents, the number of species unrepresented in the fossil records is staggering. over half of microbat and nearly all of megabat fossil histories are missing ( , ) . the enormous incompleteness of the fossil records has made it difficult to identify when specific morphological traits of bats arose. as molecular phylogeny groups two echolocation-reliant microbat species with megabats (also called old world bats or pteropodids), which do not rely on echolocation, there is some debate as to whether echolocation first arose in the common ancestor of bats and was subsequently lost in megabats, or whether it arose twice, independently ( ) . pteropodids have adaptations that enhance visual acuity at night ( ), and they do not require echolocation for foraging ( ) . there are multiple types of echolocation that can be partially delineated by species, but are more clearly categorized by the type of environment. divergent species that inhabit the same type of environment, such as those that hunt in large, open spaces, often use the same form of echolocation, suggesting that habitat has a greater influence on echolocation than phylogeny ( ) . importantly, echolocation can result in the production of droplets or small-particle aerosols of oropharyngeal fluids, mucus, or saliva, thus facilitating transmission of viruses between individuals in close proximity ( , ) . the unique navigation tactic of many bat species may inadvertently facilitate virus transmission among bats in the same habitat. bats are the only mammal capable of powered flight, which likely evolved ∼ million years ago alongside birds following radical ecological changes that resulted in the extinction of the dinosaurs ( , ) . during flight, bats consume approximately four times as much oxygen, and they have a markedly higher concentration of red blood cells compared to small terrestrial mammals ( ). bat flight is markedly different from that of birds and insects, whose wing surfaces are typically composed of inflexible material, such as feathers or chitin. bat wings are constructed from live skin stretched across elongated arm and finger bones, making them extraordinarily malleable and sensitive to environmental cues ( ) . the plasticity of bats' wings allows them to navigate and inhabit diverse ecospheres, contributing to their extensive speciation. moreover, the capability of powered flight can allow the efficient spread of viruses and thus the introduction of pathogens to which colonies may otherwise have remained naïve. as flight is extremely metabolically demanding, in addition to evolving the physical mechanisms required for flight, bats have also evolved necessary underlying molecular mechanisms. the mitochondrial respiratory chain accounts for nearly all atp required for mobility in eukaryotes, and genetic analysis of both micro-and megabat species revealed an enrichment of genes specific to the oxidative phosphorylation (oxphos) pathway. specifically, . % of nuclear-encoded and % of mitochondrial oxphos genes have evidence of positive selection in bats, which is markedly higher than the expected % of orthologous genes in previous genome-wide studies that show evidence of positive selection ( ) . genomic analysis of pteropus alecto and m. davidii suggests positive selection for the dna damage checkpoint pathway and changes in overlapping aspects of this pathway with the innate immune system, indicating that evolutionary adaptations important for flight may have secondarily affected bat immunity ( ). as a group, bats exhibit the greatest diversity of social systems in mammals. tropical species are primarily responsible for this diversity, as temperate species are more restricted in their social behavior. generally, however, bats are extremely social creatures that tend to form dense roosting colonies ( ) , and almost all temperate-zone species live in closed societies with very little infiltration of foreign bats into established roosts ( , ) . in particular, female bats form maternity colonies in which males do not take part. as bats are capable of longdistance flight, dispersal barriers cannot explain the philopatry of females. instead, benefits such as knowledge of foraging areas and social thermoregulation likely selected for these colony types. additionally, there is evidence that forming closed societies limits the potential invasion of new pathogens, thereby protecting colony members that would otherwise be vulnerable to infection. for example, pseudogymnaoscus destructans has decimated north american bat populations that do not live in the type of closed societies observed elsewhere ( ) . dna analysis of a closed society of bechstein's bats revealed extraordinarily high conservation of mitochondrial dna and relatively low conservation of nuclear dna, suggesting stable maternal populations within colonies and gene flow between colonies via promiscuous mating with males. it is possible that the mating patterns of temperate-zone species may allow transmission of pathogens between colonies via traveling males while the frontiers in immunology | www.frontiersin.org more insular females may allow viruses to persist throughout generations within a colony. an important commonality among pathogenic rna viruses in humans presenting with disease is that the host response is an important contributor to the disease process, with dysregulated and excessive innate immune responses being particularly important drivers of tissue damage during infection ( ) . given the general absence of clinical signs of disease in bats infected with the same viruses that are so lethal in humans or other non-natural hosts infected experimentally, a critical question has been to understand whether bats might establish effective disease tolerance, thus maintaining fitness despite pathogen replication, or whether bats are more resistant to infection through more successful control of pathogen replication and what the contribution of the immune response is ( , ) . the lack of many fundamental immunological tools enabling the probing of bat immune responses has meant that truly mechanistic studies of bat immunity have been very limited, although recently there has been some progress in establishing approaches such as flow cytometry to identify distinct bat immune cell populations ( , ) . so far, studies of bat immunity have primarily taken one of three approaches, whereby each comes with important strengths and weaknesses that have to be kept in mind: (i) comparative genome studies, (ii) in vitro cell culture assays, and (iii) experimental infections. comparative genome studies have confirmed that the critical components of the innate and adaptive immune system are conserved in bats at the gene level and that bats have the machinery for innate responses to pathogen-associated molecular patterns (pamps), the production of anti-viral effector molecules such as type i interferons (ifn), t cell responses (variable t cell receptors, mhci and mhcii), and b cell responses [reviewed in ( ) ]. interestingly, based on the bat genomes sequenced so far, the only family of genes lost entirely in all of them are pyhin genes ( ) . members of the pyhin family are dna sensors capable of recognizing foreign dna, including dna viruses and damaged self dna which can be generated by rna viral infection. recognition of dna results in production of ifn through interaction with stimulator of interferon genes (sting). the pyhin family also encode the only identified class of dna sensors capable of activating the inflammasome. it has been hypothesized that the absence of the pyhin family may allow bats to limit activation of the innate immune response to damaged self-dna generated by rna viral infection, thus avoiding excessive inflammation ( , ) . genome comparisons highlighting contractions or expansions of specific gene families, specific genes under positive selection, or nonconserved sequence differences in critical protein domains can thus provide the basis for hypotheses worth testing further. however, it is important to note that much can be missed in absence of data on gene regulation, especially during infection when gene expression kinetics can make a critical difference to the infection outcome. moreover, the absence of a gene or gene family does not rule out that other proteins have evolved to compensate for their loss of function. thus, while whole genome analyses can provide a context for specific questions or be hypothesis-generating, on their own they cannot distinguish tolerance from resistance mechanisms. the repeated identification of signatures of positive selection in innate immune genes in particular, does however lend credence to the idea that bats have specific adaptations as a result of a long co-evolutionary history with viruses. cell culture assays with bat cell lines, or, in some instances, primary bat cells, have been used to assess whether bats are permissive for viral replication and to determine whether particular immune receptor signaling pathways are intact. as discussed below, such studies have probed the type i ifn pathway in particular, revealing some possible species-specific differences among bats ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . however, it is important to note that in some instances immortalized cells can behave differently from primary cells and that such cultures may miss additional differences imposed by changes in cell localization, cell recruitment or cell-cell interactions in a whole animal. careful experiments measuring the quality, magnitude, and kinetics of immune responses in bats during infection and upon administration with defined stimuli for which we have comparative information from humans remain to be done to provide additional evidence that specific innate immune pathways are wired differently. experimental infections come with the enormous challenge of having to house and/or breed colonies of bats and to have biosafety-level facilities in place to perform infections with viruses lethal to humans. moreover, some trial and error is involved in determining which route and dose leads to viral replication, establishing a source of the virus (humanadapted strains tend to replicate less well in bats than strains obtained from naturally infected bats), and amplifying this viral stock without extensive tissue culture passaging. studies to date have examined the kinetics of viral replication by quantifying the extent of viremia and dissemination to other tissues, and assessing changes in white blood cell counts, body mass, and temperature. given the generally low levels of viral shedding and short infectious periods observed so far it remains poorly understood how transmission occurs in the wild to sufficient levels that cross-species jumps occur. some infection experiments have also provided evidence that a particular bat species is unlikely to be a reservoir despite epidemiological evidence, for example for r. aegyptiacus and ebolavirus. certainly, once good experimental infection models are established, such studies have the potential to be hugely informative with regard to anti-viral immune responses elicited using, for instance, comparative transcriptome analyses. one drawback may be that experimental infections do not mimic the impact of chronic stress arising from the disruption of wildlife populations, which bats are particularly sensitive to jones et al. ( ) . comparison of either cave-roosting or foliage-roosting species in areas of malaysian borneo designated as actively logged forest, recovering forest, or fragmented forest revealed varying impacts of habitat disturbance on stress and circulating white blood cells ( ) . overall, the limited studies of bat immunity that have been done have focused largely on species: p. alecto and r. aegyptiacus. we summarize this work below, but comparisons of observations made across species suggest that although a number of species appear to be capable of avoiding the pathological effects of rna virus infection, each bat species may have achieved this through distinct pathways, possibly involving changes to both increase pathogen replication control and to mitigate any immunopathology through decreased inflammatory responses and hence increased disease tolerance. the most well studied bat species with regard to antiviral immune responses is the australian black flying fox (p. alecto). this interest has stemmed from the fact that pteropid bats have been identified as the natural reservoirs for the deadly hendra and nipah viruses ( ) , which continue to cause outbreaks [such as most recently in india in may ( )]. to date, several studies have examined the kinetics of viral infection in pteropus bats and the nature of transmission and replication in other susceptible species ( ) ( ) ( ) ( ) . in australia, all four species of pteropid bats (p. alecto, p. poliocephalus, p. scapulatus, and p. conspicillatus) have antibodies to hendra virus but only p. alecto and p. conspicillatus are considered to be the primary reservoir hosts ( , , ) . in south east asia, both pteropus spp. occurring in malaysia have been found to be seropositive for nipah virus neutralizing antibodies, and the virus has been isolated from p. hypomelanus and p. vampyrus ( , ) . experimental infections of pteroid bats with hendra or nipah virus result in sub-clinical infection with short periods of virus replication and shedding, and low antibody titres ( ) ( ) ( ) ( ) . upon subcutaneous infection of p. poliocephalus with hendra virus, viral antigen was detected by immunohistochemistry at dpi in blood vessels of spleen, kidney and placenta ( ) . similarly, oronasal hendra virus infection of p. alecto led to the presence of viral genome in lung, spleen, liver and kidney weeks later, but virus isolation was unsuccessful at this timepoint ( , ) . the malaysian flying fox, p. vampyrus and the australian species, p. poliocephalus demonstrate similarly short periods of viremia upon infection with nipah virus. in subcutaneously infected p. poliocephalus, virus was isolated from the kidney and uterus of bats euthanized at dpi, but no virus was isolated at any of the other timepoints examined ( , , , , or dpi) and there was no evidence of antigen in any tissue by immunohistochemistry, including tissues collected at dpi. in this study, low neutralizing antibodies were detected in all bats with the exception of one individual that developed a significant neutralizing antibody titre -possibly reflecting the fact that p. poliocephalus is not the natural host for nipah virus ( ) . in p. vampyrus challenged by oronasal nipah inoculation, viral genome was detected in a throat swab at dpi and a rectal swab of the same individual at dpi but virus was undetectable in tissues collected at postmortem from all individuals ( , , or dpi), consistent with a short period of viremia. similar to previous studies, antibody titres were low in all p. vampyrus bats ( ) . overall, these results are consistent with bats controlling replication rapidly, at least following experimental infections which involve higher doses of virus compared to what bats would likely be naturally exposed to in the wild. the absence of a robust antibody response also appears to be typical of all experimental hendra and nipah virus infections performed to date. since antibody responses are the only immune parameter that has been measured during experimental infections of bats so far, it is difficult to speculate on the mechanisms responsible for control of viral infections in vivo. pteropus alecto was among the first bat species to have its genome described in detail. genomic studies provided initial clues for possible differences in the innate immune system of bats, with evidence for selection of key innate immune genes and the expansion or contraction of specific immune gene families ( , , ) . the mhci region is contracted ( ) , as is the type i ifn locus, which in p. alecto contains fewer ifn genes than any other mammalian species sequenced, with only three functional ifn-α loci ( ) . in contrast, pteropid bats have the largest and most diverse family of apobec (apolipoprotein b mrna editing enzyme, catalytic polypeptide-like) proteins identified in any mammal ( ) . apobecs interfere with the replication of retroviruses by deaminating cytosine residues in nascent retroviral dna. this is notable, as bats are an important source of mammalian retroviruses, many of which have been transmitted to other mammals ( , ) . apobec diversification may therefore have occurred to counteract the effect of retroviruses and possibly other viruses, as apobecs have been shown to restrict the replication of other virus families including hepadnaviruses, and parvoviruses ( , ) . members of the apobeca protein family exhibit direct antiviral activity through dna cytosine deamination which results in hypermutation of the nascent retroviral dna which is then degraded or rendered non-functional ( ) . the mechanism of antiviral activity against non-retroviruses remains largely unknown. for parvovirus adeno-associated virus, apobec meditated inhibition has been speculated to involve direct interaction with the viral dna or the replication machinery ( ) . whether the expanded family of abobecs in bats have evolved other mechanisms to control dna and rna viruses remains to be determined. as apobecs can be induced by even low levels of type i ifn ( ) , one hypothesis to be tested is that bats, through their multiple apobecs, are able to restrict viral replication without causing inflammation. pteropus alecto is the only bat species to date in which apobec genes have been mapped, and whether the expansion of this gene family extends to other bat species remains to be determined. in addition to the identification of putative immune pathways distinct in p. alecto through genome studies, differences have been identified in the activation of innate immune effectors in p. alecto from studies performed in vitro, primarily using cell lines derived from tissues including the kidney and lung. ifns are the first line of defense following viral infection and unsurprisingly, because of this, they have been the most extensively studied group of genes in bats. both type i (ifna and ifnb) and iii (ifnl) ifns are detectable in bat cells. curiously, a unique characteristic of pteropid bats is the constitutive expression of mrna for ifna and the signaling molecule, ifn regulatory factor (irf ) in unstimulated tissues and cells [ , a] . constitutively expressed ifna and irf may allow bats to respond more rapidly to infection, thus avoiding the lag time between pathogen detection and response. furthermore, viral infection or stimulation with synthetic ligands result in little ifna induction in pteropid bat cells ( ) . the constitutive expression of ifna has been described in two species of pteropid bats (p. alecto and cynopterus brachyotis) and is a first for any species. ifnb and ifnl are activated following stimulation of cells from p. alecto and p. vampyrus with synthetic ligands such as polyic ( ) ( ) ( ) ( ) . moreover, bat ifns demonstrate antiviral activity ( , ( ) ( ) ( ) ( ) ) . however, viral infection of p. alecto splenocytes results in induction of ifnl but not ifnb, hinting at differences in the function of type i and iii ifns ( ) . in humans and mice, ifnl has recently been demonstrated to have a role not only in controlling virus replication, but also in dampening damage-inducing neutrophil functions and in modulating tissue-damaging, transcriptionindependent responses such as production of ros ( , ) . a hypothesis yet to be tested is whether upregulation of ifnl rather than ifnb has a similar function in bats. the endoplasmic reticulum (er) membrane protein, sting, is involved in induction of type i ifn by cytosolic dna ( ) . stimulation of bat splenocytes with gmp-amp, which is produced following sensing of cytosolic dna by cgas, results in little induction of ifn compared to responses observed in mouse splenocytes ( ) . bat sting contains an amino acid substitution of the highly conserved and functionally important serine residue s which may be responsible for dampening sting-dependent ifn activation in bat cells in response to dna. however, comparable levels of ifn induction in mouse and bat cells in response to the rna viral mimic polyic indicate that sting-associated inhibition of the ifn response does not extend to rna viruses ( ) , thus the relevance to rna viruses in bats remains unknown. downstream of the induction of ifns, novel subsets of ifn stimulated genes (isgs) have been detected in unstimulated and stimulated pteropid bat cells indicative of a response that is less damaging to the host. furthermore, the isg response is elevated for a shorter period of time in p. alecto compared to human cell lines which again may be a strategy to avoid tissue damage ( , ) . the less inflammatory profile of isgs may be the key to the ability of bats to tolerate higher ifn expression without adverse consequences. the balance between resistance and tolerance may therefore be achieved through careful selection of the pathways that are activated and shorter periods of activation or limited activation to prevent inflammation. in this regard, studies of the regulation of ifn signaling in bats is likely to provide important additional insights. a second bat species whose host responses to viral infections has been studied more recently is the egyptian fruit bat (r. aegyptiacus). marburg virus (marv) has been repeatedly isolated from this species with demonstrated seasonal pulses of active marv replication in juvenile bats living in caves in uganda ( , ) . moreover, r. aegyptiacus were a suspected reservoir for ebolavirus (ebov) based on epidemiological evidence and detected seroreactivity to ebov, but no infectious virus has been isolated thus far from wild rousettus bats ( ) . indeed, while cell lines from r. aegyptiacus are equally susceptible to marv and ebov ( , ) , experimental infections of r. aegyptiacus seem to confirm that it is a reservoir for marv, but is unlikely to be the source of ebov spillover to humans. subcutaneous ebov infection results in very low viral replication, no viremia, little dissemination to other tissues, and no viral shedding, although some animals seroconvert, suggesting that r. aegyptiacus are unlikely to perpetuate ebov in the wild ( , ) . in contrast, experimental marv infection of r. aegyptiacus resulted in acute viremia that peaked on days - post-infection (although generally at lower levels than in humans), oral shedding that peaked on days - postinfection, and dissemination to other tissues including spleen, liver, kidney and salivary glands ( , ( ) ( ) ( ) . interestingly, viral replication was not associated with increases in white blood cell counts, any clinical signs of infection such as changes in body temperature or body weight, and infected tissues showed little evidence of inflammatory infiltrates ( ) . in all experiments, viremia was cleared by day and oral shedding ceased by day . intriguingly, a cohousing experiment resulted in marv transmissions to uninfected bats - months after experimental infection, raising the question of whether persistent infection with intermittent shedding is possible or whether very long latent periods without detectable viral replication could follow exposure ( ) . upon secondary challenge of previously marv-infected bats, none showed any detectable viral replication or shedding, providing evidence that protective immunity is established ( ) . unlike for pteropus bats, no constitutive expression of type i ifns has been detected in r. aegyptiacus ( ) , but type i ifns are induced in r. aegyptiacus cell lines upon stimulation with sendai virus as seen in other mammals ( ) . furthermore, in r. aegyptiacus the type i ifn genes are expanded, again in contrast to p. alecto ( ), but like for p. alecto a number of genes in the type i ifn pathway or involved in innate immune recognition of pamps show signs of having been under positive selection ( ) . whether positive selection of genes in either bat species is associated with tolerance remains to be determined, especially given that innate immune genes in humans have also been under positive selection ( ) . a transcriptome study which generated rna sequencing libraries from tissues taken from female and male r. aegyptiacus found a reduced coverage of nk cell related genes compared to other mammals, but confirmed that in these bats the predominant t cells had an αβ t cell receptor, and showed that ige, igg, igm, and iga, as well as a number of pro-and anti-inflammatory cytokines, were all detectable ( ) . the recently sequenced r. aegyptiacus genome revealed substantial differences in the repertoire of nk cell receptors, with this bat species entirely lacking functional killer cell immunoglobulin receptors (kirs) and with all killer lectinlike receptors (klrs) encoding either activating and inhibitory interaction motifs, or inhibitory interaction motifs only ( ) . nk cells are important immune cell players in an antiviral response but without assessment of the consequences of these genomic differences it is difficult to draw any specific conclusions with regard to viral control or the magnitude of inflammation elicited upon infection with viruses like marv. nonetheless, these genomic data provide some interesting hypotheses to be tested in the future. some additional studies probing the induction of cytokines upon stimulation of bat cells with defined innate immune stimuli provides some evidence that innate immune recognition of viruses may be altered, leading to a reduction in proinflammatory responses. stimulation of kidney and myeloid cells from the big brown bat (eptesicus fuscus) with polyinosinicpolycytidylic acid (polyi:c) resulted in only limited activation of the inflammatory cytokine, tumor necrosis factor alpha (tnfα) compared to human cells which display a robust tnfα response. induction of tnfα is controlled by transcription factors, including the nf-kappa b (nf-κb) family which consists of five members, [rela (p ), relb, c-rel, nfκb- (p ), and nfκb- (p )] which form homo-or hetero-dimers that are bound by molecules of the inhibitor of nfκb (iκb) family and retained in the cytoplasm of the cell in an inactivated state ( ) . in e. fuscus, a potential repressor (c-rel) binding motif was identified in the tnfα promoter region which may explain the difference in induction of tnfα in e. fuscus cells. consistent with this hypothesis, partial knockdown of c-rel transcripts significantly increased basal levels of tnfα transcripts in e. fuscus cells ( ) . the transcription factor, c-rel has also undergone positive selection in the bat ancestor which may indicate that this mechanism is common to other species of bats ( ) . of note, low levels of tnfα induction have also been associated with tolerance in european bank voles which are a natural reservoir for puumala hantavirus (puuv) ( ) . stimulation of macrophages from the greater mouse eared bat (myotis myotis) suggested that this species may have also evolved mechanisms to avoid excessive inflammation caused by cytokines. while high levels of tnfα, il β, and ifnβ were produced in response to in vitro challenge with lipopolysaccharides (lps) and polyi:c, there was also a sustained, high-level transcription of the anti-inflammatory cytokine il- , which was not observed in mouse macrophages ( ) . furthermore, unlike in the mouse, m. myotis macrophages did not produce the proinflammatory and cytotoxic mediator, nitric oxide, in response to lps. the same study also showed evidence of bat specific adaptations in genes involved in antiviral and proinflammatory signaling pathways through comparison with other mammalian taxa, including rig-i, il b, il- , nlrp , sting, and casp , further supporting the evolution of adaptations associated with reducing inflammatory responses in bats ( ). even less is known about immune responses of bats to nonviral pathogens than to viral pathogens, but it is clear that while anti-inflammatory responses may be characteristic of antiviral responses in bats, they are susceptible to disease upon infection with particular pathogens-in some instances due to dysregulated and damaging immune responses. one particular example of this is the emerging infectious disease, white nose syndrome (wns), that has decimated north american bat populations beginning in , in what will likely rank as one of the most devastating wildlife diseases in history ( ) ( ) ( ) . for reasons that remain poorly understood, the psychrophilic fungus pseudogymnoascus destructans (formerly geomyces destructans) causes no mass mortality in european bats despite being abundantly detected ( , ) . indeed, evidence suggests that a single p. destructans genotype was introduced to north american bat species from europe ( ) . in north america, p. destructans infection is not specific to a particular bat genus, replicating in many different bat species during hibernation and targeting the furless skin of the wings, ears, and muzzle ( ) . distinct hypotheses have been proposed for why p. destructans is so deadly in north american bats, ascribing the impaired tolerance to infection compared to european bat counterparts to either physiological or immunological factors. on the one hand, more frequent arousal, electrolyte depletion, and dehydration are thought to contribute to mortality following infection ( , ) . the destruction of wing tissue in wns results in a marked electrolyte imbalance, as the wings play a critical role in maintaining water levels, especially during hibernation, during which bats are particularly vulnerable to dehydration ( , ) . dehydration catalyzes arousal in hibernating bats, which is extraordinarily metabolically costly and rapidly depletes the fat reserves necessary to survive until spring ( ) . an alternative hypothesis posits that the restoration of the immune system following emergence from hibernation induces the fatal pathology of wns. during hibernation, destruction of cutaneous tissue is limited and infiltrating immune cells are entirely absent, yet in the weeks following arousal, infected bats exhibit overt wing damage and corresponding neutrophilic and lymphocytic infiltration ( ) . hibernation does not preclude a localized immune response to p. destructans at the site of infection and transcriptomic analysis of infected tissue showed upregulation of some acute inflammatory genes in infected tissue ( , ) . however, the observed immune responses likely occur during arousal periods, which are more common in infected bats. ultimately, immunosuppression during torpor allows p. destructans to colonize infected bats relatively unchecked ( ) , and upon emergence from hibernation, the exuberant immune response may result in deadly immunopathology during wns ( ) . in addition to general studies of immune cell recruitment and transcriptional responses during wns, body mass and white blood cell counts were examined following lps administration in four bat species ( ) ( ) ( ) ( ) . subcutaneous lps challenge in of pallas's mastiff bats (molossus molossus) led to a loss of body mass of ∼ % within the first day, but did not result in changes in circulating white blood cell counts or body temperature ( ) . seba's short-tailed fruit bat (carollia perspicillata) also showed a decrease in body mass following lps challenge, but this was associated with increases in white blood cell counts as well as increases in derivatives of reactive oxidative metabolites (drom) ( ) . subdermal lps challenge of fish-eating myotis (myotis vivesi) led to body mass decreases, increased resting metabolic rate and skin temperature ( ) , while intraperitoneal lps challenge of wrinkle-lipped bats (chaerephon plicatus) caused an increase in circulating leukocytes, but did not result in a reduction in body mass compared to controls ( ) . the differential responses to lps challenge suggest that the immune response to bacterial infection varies across species. of note, postmortem examinations of ∼ dead bats comprising species from germany revealed inflammatory lesions, many of which had evidence of underlying bacterial or parasitic infections, particularly in the lung ( ) . bats have an array of unique life history characteristics that not only allow them to be particularly good reservoirs for viruses that are highly pathogenic in other species, but also appear to have shaped their immune systems. although research on bat antiviral immunity has focused on only a few species to date, at the genomic level, selection on genes is concentrated on the innate immune system across both suborders of bats. however, while these studies have provided a rich source of hypotheses, the majority remain to be tested at the functional level and many questions remain that cannot be answered from comparative genome studies. experimental studies to date have demonstrated some functional differences between bat species, with the common emerging theme that the overall antiviral response appears to converge on a lower inflammatory profile, with tight regulation of the cytokine and inflammatory response key to clearing viral infection without the pathological outcomes typically associated with infection. however, whether this is due to specific tolerance mechanisms that are at play or increased resistance to rna virus replication still remains unclear. fewer studies have examined the adaptive immune system than those probing innate immune pathways, but experimental infections with bat borne viruses have demonstrated that bats generate low or absent antibody responses which often wane rapidly. this is reminiscent of the response of another reservoir host, the sooty mangabey which is the natural reservoir for simian immunodeficiency virus (siv) and for yellow fever virus. sooty mangabeys given an attenuated yellow fever virus vaccine strain generate much lower, transient antibody responses as compared to humans or rhesus macaques. changes to innate immune responses are also evident in sooty mangabeys ( ) . thus, intriguingly, different reservoir hosts may have arrived at similar solutions to avoid the pathological 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circadian fluctuation of immune cells in wrinkle-lipped bats (chaerephon plicatus) diseases in free-ranging bats from germany distinctive tlr signaling, type i ifn production, and attenuated innate and adaptive immune responses to yellow fever virus in a primate reservoir host all authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.the handling editor declared a shared affiliation, though no other collaboration, with the authors jm and cs.copyright © mandl, schneider, schneider and baker. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -joiajgs authors: shah, vibhuti kumar; firmal, priyanka; alam, aftab; ganguly, dipyaman; chattopadhyay, samit title: overview of immune response during sars-cov- infection: lessons from the past date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: joiajgs after the flu pandemic, the world is again facing a similar situation. however, the advancement in medical science has made it possible to identify that the novel infectious agent is from the coronavirus family. rapid genome sequencing by various groups helped in identifying the structure and function of the virus, its immunogenicity in diverse populations, and potential preventive measures. coronavirus attacks the respiratory system, causing pneumonia and lymphopenia in infected individuals. viral components like spike and nucleocapsid proteins trigger an immune response in the host to eliminate the virus. these viral antigens can be either recognized by the b cells or presented by mhc complexes to the t cells, resulting in antibody production, increased cytokine secretion, and cytolytic activity in the acute phase of infection. genetic polymorphism in mhc enables it to present some of the t cell epitopes very well over the other mhc alleles. the association of mhc alleles and its downregulated expression has been correlated with disease severity against influenza and coronaviruses. studies have reported that infected individuals can, after recovery, induce strong protective responses by generating a memory t-cell pool against sars-cov and mers-cov. these memory t cells were not persistent in the long term and, upon reactivation, caused local damage due to cross-reactivity. so far, the reports suggest that sars-cov- , which is highly contagious, shows related symptoms in three different stages and develops an exhaustive t-cell pool at higher loads of viral infection. as there are no specific treatments available for this novel coronavirus, numerous small molecular drugs that are being used for the treatment of diseases like sars, mers, hiv, ebola, malaria, and tuberculosis are being given to covid- patients, and clinical trials for many such drugs have already begun. a classical immunotherapy of convalescent plasma transfusion from recovered patients has also been initiated for the neutralization of viremia in terminally ill covid- patients. due to the limitations of plasma transfusion, researchers are now focusing on developing neutralizing antibodies against virus particles along with immuno-modulation of cytokines like il- , type i interferons (ifns), and tnf-α that could help in combating the infection. this review highlights the similarities of the coronaviruses that caused sars and mers to the novel sars-cov- in relation to their pathogenicity and immunogenicity and also focuses on various treatment strategies that could be employed for curing covid- . the whole world is currently confronting a crisis situation that first appeared in late december as merely a few cases of pneumonia in wuhan, china. the patients were exhibiting common symptoms like fever, dry cough, sore throat, breathlessness, and fatigue. sample swabs from the oral cavity and anal region were collected along with the blood and bronchoalveolar lavage fluid (balf) from all seven of the patients, irrespective of their age and gender, which were then sent to the wuhan institute of virology for further examination. as the outbreak initiated at the seafood market with the onset of winter, similar to that of the previous severe acute respiratory syndrome (sars) infection, the scientists first screened the samples using pan-cov qpcr primers. surprisingly, five samples were reported positive for coronavirus. thorough investigation employing next-generation sequencing and phylogenetic analysis led to the identification of the causative agent of this respiratory disease, a novel coronavirus ( -ncov) ( ) . as more cases started to appear around the world, on february , , the world health organization assigned a name, corona virus disease or covid- , to the disease and declared it a pandemic on march , . the virus was renamed from -ncov to sars-cov- by the international committee on taxonomy of viruses on the basis of its genetic similarity to a previously known coronavirus, severe acute respiratory syndrome coronavirus (sars-cov) ( ) . transmission of sars-cov- occurs when a healthy individual inhales or comes into contact with respiratory droplets from an infected person. the average incubation period before patients exhibit disease symptoms ranges from to days ( ) . before the spread of covid- , sars emerged as an epidemic in , followed by middle east respiratory syndrome (mers) in , both caused by a novel coronavirus of zoonotic origin and assigned to the genus betacoronavirus ( ) . the worldwide outbreak of sars-cov- has put life on hold, having a major impact on the world's economy, and has claimed ∼ , lives globally as of june , ( , ) . unlike previous episodes of coronavirus spread, where it took months to identify the cause of infection and perform genome sequencing ( ) , advancement in science and technology made it possible to identify the causative organism swiftly. within a few weeks of the outbreak, different laboratories across the world had sequenced the whole viral genome and had also provided structural and functional insights into the essential proteins required by the virus for its survival. these immediate scientific inputs helped with developing diagnostic kits and defining treatment strategies for effective prognosis and prevention ( ) ( ) ( ) . in this review, we are emphasizing the immunological aspect of sars-cov- pathogenesis by taking into consideration the previous experimental and clinical knowledge obtained from the coronaviruses that were responsible for causing sars and mers. this approach will assist in utilizing immunotherapies, repurposing the previously approved antiviral drugs, and developing therapeutic vaccines specific to novel coronavirus more effectively. initial genome sequencing and phylogenetic analysis of novel coronavirus sars-cov- has shown that it is genetically similar to previously known coronavirus sars-cov and hence is placed under the family coronaviridae. coronavirus contains positivesense single-stranded rna (+ve ssrna) as its genetic material, which can be about kb in length and is mostly protected by an outer fatty layer of an envelope that also helps the virus to evade host immune response and assists its entry inside the host cell ( , ) . the subfamily coronavirinae is further subdivided into four genera, namely alpha-, beta-, gamma-, and delta-coronavirus (α-cov, β-cov, γ-cov, and δ-cov). viruses having the potential to infect humans are placed under the genus α-cov and β-cov (sars-cov & mers-cov), whereas viruses of γ-cov and δ-cov genera are mostly known to infect avians and pigs ( ) . the novel coronavirus, sars-cov- falls under the genus β-cov, as it shares % sequence identity with sars-cov-like coronaviruses (derived from bat) but is only % identical to sars-cov and % identical to mers-cov ( ) . thus, it can be deduced by its genome identity that the immediate host of this virus could be a bat, which then transmitted it to some unknown intermediate host that acted as a source for the transmission of the virus to humans. like those of sars-cov and mers-cov, the sars-cov- genome comprises of open reading frames (orfs) in number. at the ′ end of the viral genome, overlapping orfs a and b are present that encode the rna polymerase and other nonstructural proteins of the virus and occupy approximately twothirds of the genome. genes encoding structural proteins such as spike (s), membrane (m), envelope (e), and nucleocapsid (n), are present in the remaining one-third of its genome spanning from the ′ to the ′ terminal, along with several genes encoding non-structural proteins (nsps) and accessory proteins scattered in between, as shown in figure . despite being in the same serogroup, there is a slight difference in the nucleotide number, sequence, gene order, and expression method among previously known coronaviruses and the novel sars-cov- ( , , ) . recent reports highlight that a few amino acid substitutions have occurred in the novel coronavirus genes encoding the s protein, nsp , nsp , and receptor-binding domain (rbd). these mutations in the nsp & nsp are also believed to impart the enhanced infection abilities of the novel coronavirus ( , ) . rna viruses are prone to acquiring genetic mutations that eventually help them to escape the host immune system and develop drug resistance. researchers have also found minor mutations in sars-cov- genotype in different covid- patients ( ) . one such hotspot of mutation in the sars-cov- genome is the rna-dependent rna polymerase gene. on analyzing sequences across the globe, eight repetitive novel point mutations were observed. viral genetic sequences accessed from europe exhibited five mutation hotspots, whereas the remaining three point mutations were solely present in the sequences from north america. these unique mutations suggest that the viral strains are continuously evolving across the globe figure | schematic representation of the coronavirus structure and genomic comparison of coronaviruses. (a) representation of coronavirus showing different components of the particle, which is - nm in diameter. the single-stranded rna (ssrna) genome, covered with the envelope and membrane proteins, gains access into the host cell and hijacks the replication machinery. (b) the ssrna of sars-cov- is about kb and has similarities with the genomes of sars-cov and mers-cov. translation of this ssrna results in the formation of two polyproteins, namely pp a and pp ab, that are further sliced to generate numerous non-structural proteins (nsps). the remaining orfs encode for various structural and accessory proteins that help in assembly of the viral particle and evading immune response. and that the strains from europe, north america, and asia might have co-existed the whole time ( ) . another similar report analyzed , global viral genomic sequences and found unique mutation sites on sars-cov- genome that encodes nsps and s protein, suggesting that the virus is trying to adapt to its new host ( ) . as numerous drugs are currently being designed to target the proteins that are essential for the survival of the virus, rapid genetic mutation occurring in these proteins might not prove to be a potential candidate for drug design. therefore, the invariable region of the virus could be a better target to avoid drug failures. interestingly, sars-cov- , similar to sars-cov, exploits the angiotensin-converting enzyme (ace ) receptor to gain access inside human cells, whereas mers-cov binds specifically to dipeptidyl peptidase (dpp ) receptor ( , ) . binding of the virus particle to the specific receptor on the host cell plays a key role in governing its pathogenicity. functional evaluation was carried out to reveal the potential receptors for different betacoronaviruses (β-cov) including sars-cov- , and it was found out that the entry of the virus particle was enhanced in human cells expressing ace receptor instead of dpp or aminopeptidase n (apn) in the case of the novel coronavirus ( ) . recent structural insights provided by cryo-em studies of s protein in prefusion conformation highlighted that the binding efficiency of ace and s protein of sars-cov- is - times greater than for the previously known sars-cov ( , ) . the latest reports suggest that the trimeric s protein of sars-cov- is sliced by transmembrane protease serine (tmprss ), similar to sars-cov ( , ) . hence, profound knowledge of the potential receptors to which the virus particle can bind and its associated proteases will help us in designing specific antiviral drugs and neutralizing antibodies and will lead us to foresee whether particular coronaviruses of zoonotic origin could be able to adapt and infect humans. all coronaviruses initiate entry inside the target cell by engaging the host receptor with the s glycoprotein present on their surface so as to gain entry inside the target cell. the region of s protein containing the rbd is present on the s subunit. in a few coronaviruses, rbd is present at the n-terminus region of s , whereas in sars-cov, it is situated at the c-terminus region ( , ) . the fusogenic activity of virus-cell membrane is governed by two tandem domains, heptad repeats (hr , ) that are present on the s region of s protein ( , ) . initially, it was believed that sars-cov enters the target cell merely by virtue of cell membrane integration of virus particle and host cell membrane ( ) . later, it was discovered that an essential proteolytic cleavage event takes place in the s protein at the s position of sars-cov that results in membrane fusion and facilitates virus entry inside the cell ( ) . once the coronavirus is inside the host cell via membrane fusion, it releases its +ve ssrna genome into the cytoplasmic compartment, where the translation of orf- a and orf- b begins resulting in the formation of two large polyproteins (pp a and pp ab). three functional proteases then cleave the polyproteins into non-structural proteins (nsp - ), which eventually create the viral rna polymerase and other accessory proteins for virus assembly ( ) ( ) ( ) . an uninterrupted replication-transcription event results in the formation of various nested sets of subgenomic (sg) mrnas that eventually translate into numerous structural and accessory proteins ( ) . the e glycoproteins after synthesis are incorporated into the rough endoplasmic reticulum or golgi membrane. the +ve ssrna combines with capsid protein to form the nucleocapsid, followed by budding of assembled virus particles in the er-golgi intermediate compartment (ergic) ( ) . lastly, the virus particle-loaded vesicles are fused with the cell membrane for effective shedding of the virus ( ). these new virions are now accessible to infect the neighboring healthy cells and are also released into the surrounding environment via respiratory droplets that are highly contagious and hence potentially spread the disease to healthy individuals. the path followed by sars-cov- to reach the lungs is via the naso-oral cavity. once the virus is inhaled, it enters the epithelial cells of the nasal cavity by engagement of ace receptor with the viral rbd and initiates its replication ( , , ) . this initial asymptomatic phase lasts for about - days, during which the virus multiplies in the upper respiratory tract, where no major hindrance is caused by the innate immune cells. within - days of initial encounter, the common symptoms of covid- start to appear, which are similar to those of sars and mers, i.e., fever, dry cough, pharyngitis, shortness of breath, joint pain, and tiredness. numerous problems arise during this phase of the disease, including nosocomial and fomite transmission of infection, which enhances the chances of community spread ( ) . soon, the virus begins to move toward the lower respiratory tract via airways, and this triggers a strong innate immune response. patients at this stage start exhibiting enhanced pro-inflammatory response that leads to viral sepsis accompanied by other complications, including pulmonary edema, acute respiratory distress syndrome (ards), different organ failures, and death in the worst scenarios ( ) . the infected individuals rarely show the intestinal symptoms like diarrhea that were evident in other coronavirus infections. patients are recommended to be quarantined to prevent community spread of this pandemic virus ( ) . the severity of covid- has been found to be greater in aged individuals and in people with a health history, such as those immune-compromised by hiv infection or by chemotherapy for cancer. diabetic and asthma patients, along with individuals with hypertension, obesity, or heart, kidney, or liver disorders, are also at higher risk if they acquire the disease ( ) . autopsy reports of individuals who died due to sars show multi-organ dysfunction, with the highest viral titers in the lungs and immune cells in circulation, thus damaging the pulmonary and immune system ( , ) . as opposed to adults, only a very small population of children has been infected with sars-cov- . in one study, the symptoms displayed by children above years were found to be milder as compared to those of younger children, who showed severe symptoms but with rare deaths and better prognosis ( ) . the study speculated two major possibilities related to covid- severity in children among different age groups. one of these rests on the finding that ace activity is higher in children aged - years; after this age, it starts to decline until adolescence. this could be one of the reasons why lung fibrosis is observed mainly in younger children. secondly, differential cd + and cd + t cell populations have been seen in children as compared to adults ( , ) . a large number of clinical and epidemiological criteria were defined to assess probable pediatric cases of covid- ( ) . a preliminary report from a cross-sectional study of children admitted to us and canadian pediatric intensive care units (picus) during march -april , , revealed that the children were admitted in the usa whereas no covid- cases were reported in canadian picus. the study revealed that there are fewer covid- cases in children as compared to adults and that there is a median picu time of days ( ). a recent preprint from paris reports that children (age . - . ) were admitted experiencing symptoms similar to kawasaki disease (kd) along with gastrointestinal issues and elevated inflammatory markers. further investigation suggested that they were also sars-cov- -positive, speculating that this could be the reason for kd shock syndrome ( ) . similar cases have been observed in new york, where four otherwise healthy sars-cov- -positive children started displaying symptoms similar to kd and toxic shock syndrome, thereby needing intensive care ( ) . therefore, medical practitioners should be prepared to tackle such sudden post-infection complications to avoid the associated risks. once the virus gains access inside the target cell, the host immune system recognizes the whole virus or its surface epitopes, eliciting the innate or adaptive immune response (figure ). pathogen recognition receptors (prrs) present on immune cells, mainly toll-like receptors , , and , are the first to identify the virus, which leads to enhanced interferon (ifn) production. the function of host innate immune cells is impaired during sars-cov and mers-cov infection by their non-structural proteins, which affects the overall cytokine production ( ) ( ) ( ) . humoral response against sars-cov- has been found to be similar to that against other coronavirus infections, involving the characteristic igg and igm production. at the onset of sars-cov infection, b cells elicit an early response against the n protein, while antibodies against s protein could be detected after - days from the appearance of initial symptoms ( , ) . although n protein is smaller than s protein, it is highly immunogenic, and the absence of glycosylation sites on it results in n-specific neutralizing antibody production at an early stage of acute infection ( ) . sars-cov-specific iga, igg, and igm antibodies were detected after the onset of symptoms at different time points in infected patients. a persistent level of igg was detected for a longer period, whereas igm levels started to decline after months ( , ) . in an observational case study of sars-cov- patients, anti-s-rbd igg was detected in all of the subjects, whereas anti-n igg and anti-s-rbd igm were detected in patients and anti-n igm in patients ( ) . an elisa-based time kinetics study to detect the covid- specific humoral immune response showed that the patients produced igm and igg antibodies that did not cross-react with other human coronaviruses except sars-cov. igm and iga antibodies were detected days after the onset of initial symptoms, whereas igg was detected after days ( ) . another kinetic study of viral shedding and antibody detection was published in a preprint and reported the presence of higher igg and igm antibody titers in severe patients. they also observed that weak responders for igg antibody had higher viral clearance than strong responders. this observation suggests that robust antibody response leads to disease severity while feeble response is associated with the elimination of virus ( ) . a case study on pediatric patients reports that out of children showed a protective humoral response, with neutralizing igg and igm antibodies targeting the n and s-rbd proteins of sars-cov- ( ) . these studies propose that igm-based elisa can be used for early diagnosis of patients along with qpcr techniques to improve the sensitivity and specificity of the technique. in addition to neutralizing antibodies, which are defensive and useful, there are numerous non-neutralizing antibodies in the system that aid the infection of immune cells and apcs. previously existing sars-cov antibodies may promote the viral infection in fcr-expressing cells ( ) . this ace -independent pathway of viral entry does not result in viral replication; rather, viral shedding by macrophages enhances inflammation and tissue injury by myeloid cell activation. this mechanism of viral entry through non-neutralizing antibody that results in aberrant activation of immune cells is called ade (antibody-dependent enhancement) ( , ) . ade has been observed in a number of viral infections, including sars and mers. in the case of sars, anti-s antibodies were observed to be involved in ade to gain entry into fcr-expressing cells ( ) , while in mers, a neutralizing mab (mersmab ) targeting rbd aided in mers pseudo-virus entry via the dpp pathway ( ) . although there is no clear evidence regarding ade in sars-cov- infection, it is still necessary to consider all of the odds in the pursuit of developing vaccines and treatment regimens involving antibodies ( ) . during viral infection, t cells also recognize the viral antigens presented by mhc class i [mhc; human leukocyte antigen (hla) in humans], which in turn promotes the cytokine release and cytotoxic activity of cd + t cells ( ) . but in some other cases, mhc class ii is also found to present sars-cov peptides to cd + t cells. due to the genetic polymorphism of hla, some haplotypes, like hla-b * , hla-b * , hla-drb * ( ) , and hla-cw * ( ) , are found to be more susceptible to coronavirus infection, whereas the hla-drb * , hla-a * , and hla-cw * haplotypes are protected from sars-cov infection ( ) . similarly, hla-drb * and hla-dqb * were found to be vulnerable to mers-cov infection ( ) . additionally, mhc expression is also found to be reduced during the infection due to epigenetic modifications of downstream molecules ( , ) . so far, hla association is not very wellidentified for sars-cov- infection, and this could be crucial for the prevention and treatment of covid- . however, in a recent report, blood plasma from covid- patients was able to block the expression of hla-dr on cd + monocytes, which was restored effectively on inhibiting il- , suggesting that decreased hla-dr expression in sars-cov- patients is due to the buildup of hyper-inflammatory conditions ( ) . decrease in mhc expression is also evident in cancer cells, which is a mechanism by which they evade the immune response by epigenetically modifying calnexin promoter. but infection with influenza virus in these cancer cells results in enhanced mhc-i presentation due to the increased expression of chromatin remodeling proteins, which stabilizes p expression and hence augments the immune surveillance of cancer cells ( ) . therefore, molecules that can upregulate chromatin regulators and increase the mhc-i expression could potentially be used for covid- . most of the t-cell epitopes presented by mhc complex are derived from structural proteins such as the s and n proteins of the coronavirus in both humans and animal models, while the nsps have regulatory effects on the signaling cascade ( , ) . t cells can be stimulated by epitopes, most of which are observed to be located on orf and the s protein in sars patients ( ) . in a large cohort study during sars-cov infection, s protein was the only immuno-dominant epitope for cd + t-cell activation ( ) , whereas, in mers, cd + response was against the s and n proteins along with some of the m/e epitopes ( ) . these t-cell epitopes have been tested in animal models by assessing the lung pathology and t-cell response upon infection in balb/c and c bl/ mice ( , ). the sequence of sars-cov- being more similar to sars-cov than to mers-cov, with no mutation in epitopes, provides a prospective subunit vaccine for stimulating a strong t-cell response in covid patients ( ) . in a recent study, samples from convalescing covid- patients were analyzed to check the development of adaptive immune response during infection. the results highlighted that helper t cells were eliciting a robust immune response against s, m, and n protein. the effect of adaptive immune response on humoral immunity was also compared, where a strong cd + t-cell response against sars-cov- eventually resulted in an increase in anti-s-rbdspecific igg and iga antibody titer. along with cd + t cells, immunogenic epitopes on s, m, and n proteins were also able to activate cd + t cells. however, such t-cell response was not specific to recovered patients only but was also present in - % of the individuals who were not exposed to sars-cov- . further analysis showed that they had pre-existing cross-reactive cd + t cells, which might have been generated in response to some previous coronavirus infection. hence, these t-cells could impart protective immunity in such individuals against sars-cov- to some extent ( ) . these epitopes could be a promising factor in developing immunotherapy by small molecules that can increase the presentation of viral epitopes. a rapid and coordinated immune response during viral infection leads to enhanced secretion of various cytokines, which acts as a defense mechanism against the virus. numerous reports suggest that individuals affected with sars-cov or mers-cov have dysregulated cytokine production from both innate and adaptive immune cells. in the case of sars, infected hematopoietic cell, monocyte-macrophages, and other immune cells trigger enhanced secretion of pro-inflammatory cytokines like tnf-α, il- , and ifn-α/-γ, with reduced anti-inflammatory cytokines ( ) ( ) ( ) . similarly, mers-cov infection leads to delayed but increased production of ifn-α and pro-inflammatory cytokines like il- , il- , and il- β ( ) ( ) ( ) . such elevated levels of cytokines were associated with multi-organ dysfunctional syndrome (mods) and ards due to the accumulation of numerous immune cells like macrophages, neutrophils, and dendritic cells in the lungs causing alveolar damage and edema ( , , ) . similarly, in covid- patients, secretion of cytokines and chemokines, which attract the immune cells to the lungs, was increased, hence causing ards, which is fatal to critically ill individuals ( , ) . signature cytokines in severely ill covid- patients were consistent with those in sars and mers, i.e., enhanced expression of il- , tnf-α, macrophage inflammatory protein -α (mip- α), mcp , gm-csf, il- , and ip- along with elevated chemokines (ip- , ccl /mcp , cxcl , cxcl ) were also detected in sars-cov- infection ( ) ( ) ( ) ( ) . in children, the increased inflammatory markers include il- , il- , and c-reactive protein along with procalcitonin in serum ( ) . in a case study, a -year-old child with cytokine storm was treated with anakinra (il- receptor antagonist) in order to stabilize the respiratory illness and other clinical symptoms ( ) . transcriptomic analysis of pbmc and balf showed that a number of immune regulators were upregulated, particularly cxcl , with respect to balf. this study also reported that several apoptotic genes and p signaling molecules were upregulated, suggesting a possible reason for lymphopenia in these patients ( ) . therapeutic measures to control such cytokines involve neutralizing antibodies or small molecular drugs that can stop the signaling cascade for cytokine production. the most potent antiviral machinery acquired by immune cells is the secretion of interferons that act as secondary messengers stimulating the neighboring cells. most innate immune cells are efficient in producing ifns that are involved in obstructing cell proliferation, apoptosis, and immunomodulation ( , ) . as an escape mechanism, sars-cov or mers-cov uses several ways to overcome the host immune response, one of which is by severe leukopenia and lymphopenia ( ) ( ) ( ) . after gaining entry to the cell, these viruses encode different proteins that interact with downstream signaling molecules of tlrs and the jak-stat pathway. mers-cov encoded matrix protein, accessory proteins from orf a, b, and , which directly inhibits the ifn promoter and nuclear localization of irf ( ) . plpro, encoded by sars-cov and mers-cov, prevents the dissociation of nf-κb from iκbα, whereas nonstructural proteins of sars-cov, i.e., plpro and orf b, inhibit irf phosphorylation and hence its translocation to the nucleus ( , , ). these viral accessory proteins also inhibit the jak-stat pathway, resulting in inhibition of genes by isre promoters ( ) ( ) ( ) (figure ) . a new investigation revealed that sars-cov- infection leads to an overall decrease in the transcription of antiviral genes because of the lower production of type i and iii interferons with sufficient isg expression, along with elevated chemokine secretion. results obtained from in-vivo and ex-vivo covid- experiments were in tune with the in-vitro findings. therefore, a decrease in the innate antiviral response, along with hyper-inflammation, could be one of the causes of covid- severity ( ) . in addition to reduction in t cells, sars-cov- infection also enhances the exhaustion of effector t cells, decreasing the immune response against the virus ( , ) . exhaustion and loss in function of effector t cells is the result of increased expression of inhibitory receptors like pd- , tim- , and tigit on its surface as a result of cytokines like il- , il- , and tnf-α or by decreasing the regulatory t-cell population ( , ) . following viral/antigen clearance, most of the effector t cell undergoes apoptosis in the contraction phase. subsequently, a pool of memory t cells are generated that are programmed to fight against re-infection. cd + memory t cells, upon restimulation, trigger b cells and other immune cells by cytokine production, while cytotoxic memory t cells help in destroying the infected cells during subsequent infection ( , ). case studies in recovered sars patients showed that both cd + and cd + memory t cells were efficient in eliciting immune response from months to years without the presence of any antigens ( ) . in a case study of recovered sars-cov patients, the patients showed very low frequencies of memory b cells, while memory t cells elicited a response against the s protein in % of recovered individuals ( ) . considering the memory t-cell subset, n-specific helper t cells had more of central memory markers (cd ra − , ccr + , cd l − ) while the cd + t cell population had the effector memory (cd ra + , ccr − , cd l − ) phenotype in a steady-state manner ( ) . the study suggests that an effective vaccine or t cell epitopes could be used to target a particular population for rapid viral clearance. in recent reports, covid- subjects have shown reduced regulatory t cell populations and memory t cells, which may aggravate the inflammatory response leading to cytokine storm and hence enhance the tissue damage and organ failure ( ) . in a mouse model, the use of cd + memory t cells as a vaccine by the intranasal, but not the subcutaneous, route imparted a protective response against the human coronavirus. the infused cd + memory t cell, upon re-stimulation, produces ifn-γ and recruits cd + t cells for rapid clearance in response to sars-s peptide ( ) . recently, a human ace- -expressing mouse model has been developed by crispr/cas technology that recapitulates the human symptoms upon infection with sars-cov- through the intra-nasal route. this tool will be beneficial for evaluating the efficacy of vaccines for covid- and also to study its transmission and pathogenesis ( ) . just like sars and mers, there are no specific clinically approved drugs available for covid- as of june , ( ) . currently, the treatment regime focuses mainly on providing intensive care in order to alleviate the symptoms and discomfort associated with covid- . conservative fluid therapy accompanied by broad-spectrum antibiotics are also given to the patients as a protective measure to avoid opportunistic bacterial infections. however, ventilator support for respiration is provided to the patient under extreme conditions ( ) . numerous fda-approved antiviral drugs, vaccines, and immunotherapies that are already being used to treat other diseases have also been considered as a possible approach for treating covid- ( table ) . but this approach may reduce the availability of these drugs and vaccines for the intended diseases and for the patients with the greatest need. the molecular, structural, and functional relationships of lopinavir viral protease involved in immature, noninfectious hiv virus particle, and inhibits plpro or clpro in sars-cov- . favilavir viral rna polymerase purine analog blocking viral rna synthesis. remdesivir ( ) ribavirin guanosine nucleoside binds to nucleoside binding pocket of the enzyme. ( , , ) galidesivir adenosine analog, effective against ebola, zika, and other rna viruses. chloroquine/hydroxychloroquine heme polymerase and ace increases endosomal ph and terminal glycosylation of ace , inhibiting sars-cov- entry. ( , ) nitazoxanide glutathione-s-transferase alters ph and inhibits viral maturation. reported against tb, helminthic, and protozoan infection. umifenovir/arbidol n/a interacts with aromatic residues of viral glycoproteins. is being trialed for prophylactic action against covid- . sars-cov- with sars-cov might define the use of existing anti-viral drugs against covid- ( , ) , considering the total time it takes to perform clinical trials and get fda approval for the use of novel drugs and vaccines. the increasing knowledge of the genetic, immunological, and molecular mechanisms behind its enhanced pathogenicity might help in developing specific treatment approaches for covid- in the future. considering the studies on the molecular mechanism of coronavirus infection ( ) , several antiviral drugs could be repurposed for the treatment of covid- . remdesivir is a nucleotide analog that acts as an antiviral agent for a wide variety of viruses and has been tested widely against previous epidemics of coronavirus infections in both in-vitro and in-vivo models ( , ( ) ( ) ( ) . this adenosine analog gets incorporated into the newly synthesized viral rna, which inhibits the addition of further nucleotides by viral rnadependent rna polymerase and hence terminates the ongoing transcription. administration of intravenous remdesivir was found to be effective in treating the first known patient of covid- in the usa ( ) . a randomized double-blinded clinical trial on , adult hospitalized covid- patients was sponsored by the national institute of allergy and infectious diseases, usa, to further test the potency of intravenously administered remdesivir. the preliminary outcomes of the trial reported that remdesivir treatment decreased the median recovery time in the treatment group ( days) as compared to the placebo group ( days). the mortality rate was also less in the treatment group ( . %) in contrast to the placebo group ( . %) ( ) . numerous clinical studies, similar to this, are required so as to validate the proposed drugs for covid- . favipiravir, ribavirin, and galidesivir are also potential nucleoside analogs that might be useful against novel coronavirus infection ( ) . the combinatorial therapy approach of using remdesivir along with chloroquine, a well-known anti-malarial drug, has also been tested in vitro so as to study its effectiveness against sars-cov- ( , ) . it has been reported that chloroquine immuno-modulates the host microenvironment and also interferes with the replication of the virus and its interaction with the receptor ( , ) . in a randomized clinical trial (nct ) involving asymptomatic individuals across the us and canada who had come into close contact with potential covid- patients, the individuals were given either hydroxychloroquine or placebo as a prophylactic measure. the results revealed that hydroxychloroquine treatment had the same effect as did the placebo group. the usage of hydroxychloroquine resulted in minor side effects ( . %) as compared to the placebo treatment ( . %). however, no cardiovascular disorder or treatment-related major complications were observed ( ) . based on the putative function of hydroxychloroquine on the endosomal acidification, whereby it is presumed to hinder viral uncapping, it can be observed that it has a great potential for prophylaxis, not to prevent infection but to reduce effective viral load in patients and thus lead to milder disease. numerous clinical trials to further explore the usage of hydroxychloroquine in different combinations are in the pipeline and will finally provide a better understanding of the efficacy of this drug for covid- . a few anti-hiv drugs, such as lopinavir/ritonavir in combination with interferon beta (ifn-β), have been tested in vivo for treating coronavirus infections (sars-cov, mers-cov) and have also been used in the case of covid- ( , , ) . various complementary therapies could also be employed as a preventive measure against viral infections. many essential proteases, such as chymotrypsin ( c-like protease) and plpro, which are required by coronavirus for completing the replication process, can also be targeted using drugs. cinanserin, flavonoids, and some small molecules are known to inhibit clpro, whereas diarylheptanoids are used to inhibit plpro ( ) ( ) ( ) . in a recent study, potential anti-hcov drugs were identified through a systems biology-based approach, such as melatonin, mercaptopurine, sirolimus, dactiomycin, and toremifene, which are to be tested further for their potency ( ) . in the absence of any dependable vaccine or drugs with tested efficacy and when the pandemic onslaught is ongoing, a worthy therapeutic approach is passive immunization using purified antibodies. the source of such antibodies could be the sera of convalescing individuals, mabs, or genetically modified antibodies from an animal host, which can efficiently neutralize the virus. this is an age-old practice, with pioneering work having been done by the nobel laureate, emil behring, who applied this approach for diphtheria, and has been used whenever there are sudden outbreaks of viral diseases like sars, mers, h n , h n , ebola, and many others ( , , ) . as opposed to active vaccination, plasma therapy is the only means to provide immediate immunity for viral clearance, as in the case of sars-cov- . as in other epidemic diseases, convalescent sera are currently being employed for covid- in a number of countries ( , ) . although a randomized controlled trial is yet to be reported, limited studies in patients have been documented with no remission of severe respiratory afflictions on receiving neutralizing antibodies from convalesced donors with antibody titers of : , along with drugs and oxygen support ( ) . a report from hong kong suggested that this therapy had poor outcome in sars patients, with a number of limitations in their study ( ) . as with transfusion of any blood products, precautionary screening of infectious agent is warranted in plasma transfusion. recently, the fda in the usa has approved trials of convalescent plasma therapy in covid- under specific guidelines; plasma donation is advised weeks after a patient becomes virus-negative on pcr. the major challenge in this therapy is obtaining donors with similar blood antigens with a high antibody titer of sars-cov- ( ) . another potential adverse effect of this approach is ade of infection, which is common in so many other viruses. but, to date, the incidence of ade has not been reported in the case of sars-cov- . another major point of contention is the selection of patients for this therapeutic approach. in most clinical trials, patients with severe diseases are being recruited, while the presumed mechanism of action of convalescent plasma, based on its content of virus-neutralizing antibodies, rather points to plausible favorable outcomes in earlier phases of the disease because in the later, more severe phases, the hyperimmune response, rather than the viral load, becomes the more critical pathology. finally, there are no available data on the heterogeneity of response to convalescent plasma transfusion, which may further illustrate the importance of careful evidencebased patient selection, as heterogeneity of response may result from both virus and host-intrinsic factors which are, to date, not revealed. researchers around the world are working hard to develop a potential vaccine candidate so as to stop the deadly pandemic caused by sars-cov- . however, vaccine development is not an easy task, as a number of successful clinical trials are required before approval for patients. different approaches are being utilized for designing a specific vaccine targeting either the structural proteins or viral replication process, which eventually results in the inhibition of viral growth and its further transmission. the common strategies involve the use of live attenuated vaccine (lav), inactivated virus, subunit vaccines, monoclonal antibody vaccine, virus vectors, protein vaccines, and dna/rna-based vaccines ( ) ( ) ( ) ( ) . there are numerous subunit vaccines targeting all or a part of s protein that have already been tested for sars and mers in animal models ( ) and could be potential candidates for testing against sars-cov- . a recent pilot study with a purified inactivated sars-cov- virus vaccine displayed very promising outcomes in different animal models. the neutralizing antibodies generated after vaccination were able to effectively target different strains of sars-cov- without developing any ade of infection ( ) . various randomized controlled trials (nct , nct ) are also underway to evaluate the effectiveness of the bcg vaccine against sars-cov- for healthcare professionals. an adenovirus vector-based vaccine candidate, chadox (presently azd ), developed by oxford university (licensed to astrazeneca) for use against sars-cov- has been reported to activate both the humoral and cell-mediated immune response when tested in rhesus monkey ( ) . the phase i clinical trial to confirm its potency is also in progress (nct ). another group has followed a similar approach by using a recombinant adenovirus type (ad -ncov) vectorbased vaccine for covid- . the full report from the phase i clinical trial (nct ) of ad -ncov shows that it is very effective in generating both humoral and rapid t-cell response post immunization. the group is now ready for the next clinical trial phase to further strengthen the effectiveness of the ad -ncov vaccine ( ) . it should be noted that there are potential risks associated with the usage of live attenuated viruses, for example, complications resulting in lung damage by infiltrating eosinophils, as seen in in vivo models ( , ) . however, eosinophil immunopathology due to sars-cov vaccine could be reduced by using tlr agonist as an adjuvant ( ) . viral neutralizing antibodies specifically targeting various regions of s, i.e., s -rbd, s -ntd, or the s region, and blocking the interaction of virus with the receptor are well-known for sars and mers ( ) . these neutralizing antibodies could prove to be the best and potential candidate for cross-neutralization of sars-cov- . despite being structurally related, some of the sars-cov neutralizing monoclonal antibodies failed to interact with the s-protein of sars-cov- , which could be attributable to the substantial differences in their rbd ( ) . a recent study reported the presence of high titres of neutralizing anti-s-rbd igg antibodies, but no antibodies were detected against the n protein in recovered covid- patients, suggesting that anti-s igg persists longer than does anti-n igg. along with the humoral immune response, they also observed an s protein-specific t cell-population producing ifn-γ, which further contributes to conferring protective immunity against sars-cov- infection ( ). recently, a monoclonal antibody ( d ) has been identified from sars-spike hybridomas that targets the conserved s-rbd region (residue - ) and therefore can very effectively neutralize sars-cov- along with sars-cov ( ). on similar lines, a group has isolated a single-domain antibody from a phage display library targeting the s-rbd region of sars-cov- . the fully humanized singledomain antibody was able to neutralize the virus by interacting with a cryptic epitope in s protein ( ) . these mab and singledomain antibodies could be used to treat as well as to design quick diagnostic kits for covid- . the new technology of the microneedle array (mna) has been employed for delivering sars-cov- s subunit vaccine, which could be really helpful in the treatment of the emerging covid- outbreak ( ) . the transfer of s subunit by mna elicited a strong virus specific-antibody response in sars-cov- ( ) . a novel encapsulated mrna vaccine candidate developed by modernatx, inc. that encodes full length s protein of sars-cov- , is also under clinical trial (nct ). there is an urgent need to develop more such specific vaccines that could neutralize the novel coronavirus effectively ( ) . the host innate immune system encounters upcoming infections, and this results in elevated production of various cytokines and type i interferons (ifns). in the case of prolonged infection, hyperactivation of the immune system may also result in the development of a pro-inflammatory microenvironment, leading to adverse outcomes and even death. the induction of numerous lymphokines, such as il- , il- β, tnf-α, and ccl , that are pro-inflammatory in nature has also been observed in the case of covid- ( ) ( ) ( ) . a previous study in a mers animal model showed that treatment with recombinant type- ifn (rifn) decreased the viral rna level in lungs with a decrease in ifn-stimulating gene expression. early treatment with rifn resulted in a dampening of cytokine and chemokine release that lowered the migration of neutrophils and other cells in lung ( ) . an allogenic mesenchymal stem cell-based (remestemcel-l) therapy developed by mesoblast, which has been previously used for inflammatory conditions and graft vs. host disease in children and adults, is now being assessed for covid- ( ) ( ) ( ) . in this therapy, bone marrow-derived mscs from the donor are grown in vitro and are then transfused to the recipient patients. upon infusion, these cells exhibit antiinflammatory activity by reducing pro-inflammatory cytokine production via the recruitment of anti-inflammatory cells in the affected tissue ( ) . currently, a randomized placebo-controlled trial (nct ) with patients is ongoing for treating ards caused by covid- . treatment with rifn, inhibitors of the pro-inflammatory pathway, cytokine inhibitors such as tocilizumab, lenzilumab, and many others are still to be used in combination with other drugs for treating covid- . so far, there is not much evidence from clinical trials of such inhibitors with which to predict the outcome of these anticytokine therapies. considering the current situation of more than million people being infected, with ∼ , deaths as of june , , there is an urgent need to control the sars-cov- pandemic. the fatality rate of sars-cov- in lower than those of other coronaviruses that caused catastrophes in the past, but the higher infectivity rate makes it worse. raising awareness of this contagious virus is one of the many ways by which its spread can be prevented. the governing authorities concerned in every country have approved guidelines and taken necessary action to quarantine infected people and break the chain of community spread. antibodies, vaccines, and drugs developed for previously emerged coronaviruses could potentially be used for treating sars-cov- . the combination of various neutralizing antibodies against s protein could enhance the effectiveness of viral clearance. among various antivirals and other small molecules that are fda approved, chloroquine/hydroxychloroquine has shown better positive outcome in covid- patients. in clinical trials, some of the combinational antiviral drugs like lopinavir + ritonavir and blockers like angiotensin receptor blocker that were thought to be effective, have failed in curing the disease ( , ) . cytokine storm being one of the symptoms of infected individuals, anticytokine therapy for tnf and il- should be attempted to determine the efficacy of these antibodies in the treatment of sars-cov- infection. clinical trial chictr with tocilizumab, a monoclonal humanized antibody against il- receptor, has shown some efficacy, but this still needs to be tested in a larger cohort. with the increasing number of deaths, there is an immense need to accelerate the development of rapid and sensitive diagnostic kits and to commence clinical trials of the readily available and safe drugs to reduce the rising infections and covid- -related deaths so as to bring life back on track. vs and pf contributed equally in writing the review. conception of 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therapy for crohn's disease transplantation of ace -mesenchymal stem cells improves the outcome of patients with covid- pneumonia mesoblast to evaluate anti-inflammatory cell therapy remestemcel-l for treatment of covid- lung disease are patients with hypertension and diabetes mellitus at increased risk for covid- infection? key: cord- - h l authors: di lullo, giulia; calabresi, valentina; mariotti, feliciana; zambruno, giovanna; lanzavecchia, antonio; di zenzo, giovanni title: identification of a novel non-desmoglein autoantigen in pemphigus vulgaris date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: h l pemphigus vulgaris (pv) is an autoimmune bullous disease of the skin and mucous membranes characterized by the presence of circulating and tissue-bound autoantibodies against keratinocyte cell surface antigens, specifically desmoglein (dsg) and . the pathogenic role of anti-dsg antibodies is well-established, while the mechanism of blister formation is only partly defined. we have applied a previously developed method for the efficient immortalization of igg+ memory b cells to identify novel target antigens in pv. a human monoclonal antibody reactive with a hitherto unreported non-dsg antigen was isolated. immunoprecipitation and immunoblotting studies with keratinocyte extracts indicated α-catenin as the putative antigen, then confirmed by immunoblotting on the recombinant protein. four of ten pv sera reacted with recombinant α-catenin. although the isolated human monoclonal antibody was per se unable to dissociate keratinocyte monolayers and also to synergize with a pathogenic antibody in vitro, further studies are warranted to assess its possible in vivo contribution in the multifactorial pathogenesis and heterogeneous manifestations of pv disease. pemphigus vulgaris (pv) is a rare but highly disabling and, if untreated, almost always fatal immunobullous disease of the skin and mucous membranes. pv is characterized histologically by loss of cell-cell adhesion between suprabasal keratinocytes, leading to acantholysis, and immunopathologically by the presence of circulating and tissue-bound autoantibodies (autoabs) against keratinocyte cell surface antigens, specifically desmoglein (dsg) and . pv is considered as a paradigmatic organ-specific autoimmune disease in view of (i) present knowledge of disease autoantigens and pathogenesis and (ii) reproducibility of major clinical and pathogenic features in animal models ( ) . the existence of both pathogenic and non-pathogenic anti-dsg autoabs has recently been underscored by isolation of human monoclonal antibodies (hmabs) from pemphigus patients. anti-dsg hmabs characterization has shown that their pathogenic potential primarily depends on the targeted epitopes ( ) . we have been interested in characterizing the repertoire of naturally occurring autoreactive epithelium-specific memory b cells in pemphigus vulgaris patients. in a first work, we focused on autoantibodies targeting dsg ( ) . however, (i) the lack of tight correlation between anti-dsg autoab titers and disease activity in some patients and (ii) the significant degree of disease heterogeneity point at the importance of non-dsg autoabs, that have been progressively, even though not exhaustively, described ( , ) . in fact, besides dsg and dsg , other non-desmoglein autoabs, either pathogenic or nonpathogenic, have been identified in pemphigus patients. autoabs endowed with an acantholytic potential target desmocollin , α-acetylcholine receptor, pemphaxin, and keratinocyte mitochondria ( ) ( ) ( ) ( ) . on the other hand, the pathogenic role of autoabs recognizing other autoantigens, such as atp c , desmocollin , bp , periplakin, e-cadherin, desmoglein , desmoplakin , and desmoplakin , remains to be demonstrated ( ) . in line with this interest, our current work aimed to identify autoabs targeting non-dsg membrane-bound or membrane-associated intracellular antigens. in the present study, we report on the characterization of a hmab isolated from a pv patient and directed to a novel non-dsg antigen. the hmab reacts with α-catenin that is recognized by almost half of pv sera analyzed. peripheral blood was obtained from patients (pvc and pvf) suffering from active mucocutaneous pv. the patients showed typical clinical, histological, and immunopathological features and had high-titer anti-dsg circulating autoantibodies (pvc: dsg , u/ml, dsg , u/ml; pvf: dsg , u/ml, dsg , u/ml), as assessed by elisa kits based on ectodomain of dsg and dsg (mbl, nagoya, japan). hmabs were isolated by a highly-efficient protocol for the immortalization of igg+ memory b cell with epstein barr virus (ebv) in the presence of a toll-like receptor agonist, as previously described ( ) . in detail, igg+ memory b cells were isolated from cryopreserved peripheral blood mononuclear cells using anti-cd microbeads (miltenyi biotec, bo, italy) followed by depletion of cells carrying igm, igd, and iga by cell sorting. multiple replicate microcultures of - igg+ memory b cells/well (for a total of to × purified cells) were infected with ebv and cpg as previously described ( ) . culture supernatants were tested for binding to dsg -and dsg -coated elisa plates and for binding to hacat keratinocyte cell line monolayers (both on live cells and on fixed and permeabilized cells) by immunofluorescence (if) assay using an automated fluorescence microscope (pathway , bd). the specificity of positive polyclonal cultures was further assessed by if on primary human keratinocytes. positive reactivities were confirmed by the propagation of oligoclonal cultures. positive cultures were cloned by limiting dilution and expanded; antibodies were purified using protein g columns. serum samples were collected from pv and bullous pemphigoid (bp) patients and healthy donors. this study was carried out in conformity with the helsinki guidelines and with approval of the idi-irccs ethics committee. all the biological samples were obtained after patient's informed consent. if studies were performed according to the procedure described in di zenzo et al. with minor modifications ( ) . briefly, supernatants from immortalized human memory b cells were screened on monolayers of live and fixed/permeabilized hacat cells. after washing with phosphate-buffered saline, cells were stained with alexa fluor -conjugated goat anti-human igg (invitrogen, carlsbad, ca, usa). the isolated monoclonal antibodies were further tested on permeabilized hacat cells and on primary keratinocytes. non-keratinocyte cell lines, i.e., mrc , hela, and skmel cells, were used as controls for if analyses on cell monolayers. human antibodies of irrelevant specificity were used as negative controls. serial images of stained keratinocyte monolayers were acquired by the bd pathway automated fluorescence microscope. staining was also performed on cryo sections of normal human skin, guinea pig and monkey esophagus and revealed with fluorescein isothiocyanateconjugated anti-human igg antibody (agilent dako, santa clara, ca, usa). immunoprecipitation (ip) of s-labeled keratinocyte extracts by hmab pvf was carried out as previously described ( ) . the precipitated proteins, separated on sodium dodecyl sulfatepolyacrylamide gel electrophoresis on % gels under reducing conditions, were detected by autoradiography. immunoblotting (ib) experiments on keratinocyte extracts were performed as previously reported using both horseradish peroxidase (hrp)conjugated and alkaline phosphatase-conjugated secondary antibodies ( ) . bands were quantified using imagej software (national institutes of health, bethesda, md, usa) and their intensities were normalized respect to the positive control signal obtained by anti-α-catenin antibody. the cutoff value was set as the medium value + standard deviations obtained measuring signals obtained with healthy donors. commercial primary antibodies were purchased from progen biotechnik gmbh (heidelberg, germany) (anti-desmocollin ), bd biosciences (san jose, ca, usa; anti-α-catenin), and santa cruz biotechnology, inc (dallas, tx, usa; anti-γ-catenin, antiβ-catenin, anti-p , and anti-e-cadherin). recombinant tagged human α -catenin was purchased from abcam (cambridge, uk). to rule out that pv sera were mainly reacting against the fused glutathione s-transferase (gst) moiety of the α-catenin recombinant protein, ib experiments by using commercial tagged protein and equimolar gst were performed (data not shown). briefly, recombinant dsg and dsg ectodomains were produced in baculovirus and used for coating of elisa plates. the plates were, then, blocked with % bovine serum albumin and incubated with antibodies followed by hrp-conjugated antihuman igg (jackson immunoresearch, baltimore, pa, usa) ( ). the assay was performed as previously reported ( ) . briefly, primary human keratinocyte cells were seeded onto -well plates and h post-confluence treated with monoclonal antibodies. after adding exfoliative toxin a to cleave dsg protein, the cell monolayers were detached with dispase i (merck, kgaa, darmstadt, germany) and subjected to mechanical stress by pipetting. the monolayer fragments, fixed by adding a % formaldehyde solution, were subsequently stained using crystal violet. to investigate a possible synergistic effect pvf was applied to the monolayer together with a pathogenic antibody (pvb ) ( ) at optimal ( µg/ml) and suboptimal concentrations ( µg/ml and . µg/ml; figure ) and a non-pathogenic antibody ( , pvb in a germline version that is not able to dissociate the keratinocyte monolayer; data not shown). in order to identify hmabs targeting non-dsg membranebound or membrane-associated intracellular antigens, we took advantage of the same strategy by which we had previously isolated and finely characterized several dsg-reactive pv patient-derived monoclonal autoantibodies ( ) . in detail, peripheral blood samples were collected from patients with mucocutaneous pv: one with long-lasting steroid-resistant disease (pvc) and the other prior to treatment initiation (pvf). igg+ memory b cells were isolated by magnetic and fluorescence-activated cell sorting, seeded in -well microplates, and immortalized with ebv in the presence of irradiated mononuclear cells and oligodeoxynucleotides containing cpg motifs, as previously described ( ) . the reactivity of antibodies secreted in the supernatants of growing polyclonal cultures was screened by if staining both on live and on fixed and permeabilized cells from the human keratinocyte hacat cell line. the polyclonal antibodies produced by the vast majority of isolated cultures bound to surface antigens on the keratinocyte membrane and were reactive with dsg and/or dsg ectodomains by elisa, as previously reported ( , and data not shown). besides such prevalent pattern of reactivity, supernatants of rare cultures from both patients showed a distinctive membrane-associated fishnet reactivity, detected only on permeabilized keratinocytes, and were negative in elisa on dsg and dsg ectodomains. the human mabs pvf (igg -isotype), pvc (igg -isotype), and pvc (isotype igg isotype) were cloned by limiting dilution and showed the same if pattern as the original polyclonal cultures also on permeabilized hacat keratinocytes (figure a) , suggesting their specificity for a membrane-associated intracellular (i.e., non-exposed) autoantigen. in addition, they showed an intercellular staining pattern by if on human skin, guinea pig, and monkey esophagus (figures b-d) , very similar to if staining pattern of anti-dsg antibodies ( , and data not shown). supernatants from the selected clones were tested on non-keratinocyte cell lines, i.e., mrc- (fibroblast), skmel (melanoma), and hela (epithelial) cells: they failed to stain both live and permeabilized cells, apart from a reactivity on permeabilized hela cells (data not shown), hinting at a specificity for membrane-associated intracellular antigens expressed on keratinocytes and other epithelial cells. ip studies with the selected clones on radiolabelled keratinocyte extracts to identify the target antigen showed reactivity to a kda antigen for pvf (figure a) and to - kda antigens for pvc and pvc , respectively (data not shown). as the ip results from the latter two clones pointed at members of the plakin-family as candidate target antigens and autoreactivity of pv sera to plakin family members has been already documented ( , ) , we chose to further characterize pvf with the aim to identify a possible novel membrane-associated pv autoantigen. subsequent ib experiments using commercial antibodies reacting with keratinocyte membrane-associated proteins with a molecular weight of ∼ kda (desmocollin - kda; α-catenin- kda; e-cadherin- kda; β-catenin- kda; γ-catenin- kda; p -catenin - kda) indicated α-catenin as the putative antigen ( figure b) . sequential ip and ib experiments further supported α-catenin as the target antigen of pvf . specifically, a protein immunoprecipitated from keratinocyte extracts with pvf was recognized by a commercial anti-α-catenin antibody, and an anti-α-catenin antibody was in turn able to immunoprecipitate a protein recognized by pvf in ib ( figure c) . finally, ib experiments with the recombinant tagged protein unequivocally confirmed the specific binding of pvf to α-catenin ( figure d ). to determine whether autoabs of the same specificity as pvf were present in the sera of pv patients, we performed ib experiments with pv sera on recombinant tagged α-catenin figure | pvf binds a membrane-associated epithelial antigen: α-catenin. pvf immunoprecipitates (ip) an unknown antigen of kda from radiolabeled normal human keratinocyte extracts (a). immunoblotting (ib) experiments on keratinocyte extracts by using pvf and commercial antibodies suggest that α-catenin could be the putative antigen of kda: pvf and a monoclonal murine anti-α-catenin antibody (ab) react with a keratinocyte antigen of similar molecular weight ( kda) (b). anti-α-catenin commercial antibody reacts to α-catenin from keratinocyte extracts by ib (lane ); pvf immunoprecipitates α-catenin recognized by the commercial anti-α-catenin antibody by ib (lane ) and, viceversa, anti-α-catenin antibody immunoprecipitates α-catenin recognized by pvf by ib (lane ). the faint reactivity observed in lane could be related to the epitope recognized (c). ib performed with a recombinant gst-tagged human α-catenin ( kda) confirms that the target of pvf is α-catenin. the lower bands are likely degradation products (d). (figure ) . four out of pv patients ( %) showed the same reactivity of pvf to α-catenin, whereas the other pv sera and normal human control sera showed only a faint signal, indicating that this autoreactivity was well-represented in pv patients, even though not shared by all patients. in order to evaluate whether this reactivity is disease-specific, bp patient sera were analyzed on recombinant α-catenin by ib. only one of bp ( representative bp sera shown and other not shown) reacted to α-catenin (supplementary figure ) underlining the specificity of this autoantigen. the weak signals found in the other remaining pv, pb, and healthy donor sera might be ascribed to reactivity to the tag protein (gst). the presence of anti-α-catenin autoabs in several pv sera raised the question as to their pathogenic potential. previous studies showed the ability of intact autoabs to enter the cytosol or nucleus of living cells ( , ) . more recently, marchenko et al. reported that pv autoabs could penetrate keratinocytes and react with intracellular mitochondrial proteins ( ) . these findings suggested that a hmab to an intracellular antigen could exert its pathogenic ability on live keratinocytes. thus, an in vitro dissociation assay was used to evaluate the pathogenic activity of the hmab pvf . this approach allows to measure the ability of a specific antibody or a mixture of antibodies, such as a serum, to fragment a monolayer of primary human keratinocytes seeded to confluence. the keratinocyte monolayers, incubated for h with pvf , remained intact, similarly to monolayers incubated with an unrelated hmab used as negative control (figure ) . as expected, human (pvb ) and murine (ak ) pathogenic anti-dsg antibodies were able to dissociate the monolayers ( , ) (figure ) . in addition, pvf failed to synergize with a pathogenic antibody (figure ) . these findings exclude a primary pathogenic function of anti-α-catenin autoabs in pv, nevertheless a potential secondary role in the immunobiology of the disease cannot be excluded and warrants future studies. in the present work we went forward in the characterization of the diverse targets of epithelium-specific autoreactive b cells from pv patients. to this purpose, we took advantage of the figure | almost half of pv sera specifically react with recombinant α-catenin. immunoblotting with sera obtained from pemphigus vulgaris (pv) patients shows that of sera ( , , , ) react with the novel epithelial antigen, while healthy donor sera ( , , ) , representative of sera analyzed, show only a background signal. the positive control (c+) is the commercial anti-α-catenin antibody. the background signal, could be also due to reactivity to the tag protein (gst). quantification and normalization of bands using imagej analysis (data not shown) have confirmed the reported results (see materials and methods section). high-efficiency immortalization protocol of igg+ memory b cells we had previously developed ( ) and applied to isolate and molecularly characterize a number of anti-dsg hmabs ( ). our present focus was to detect plasma-membrane-associated antigens, either expressed on the cell surface or intracellularly, therefore we chose to screen the isolated polyclonal cultures on both live and permeabilized keratinocytes. our choice was based on the hypothesis that antigens associated to the cell membrane, even those with intracellular localization, could have a higher chance to contribute to pemphigus pathogenesis, which is due to the loss of cell-cell adhesion. as expected, the vast majority of cultures resulted specific for dsg and/or dsg , further confirming the major role of these antigens in pemphigus pathogenesis. among polyclonal cultures that reacted only with permeabilized keratinocytes, we selected and cloned those giving a membrane-associated fishnet-like staining pattern on stratified epithelia. among the cloned cultures, we further characterized clone pvf and demonstrated by if, ip and ib studies that α-catenin is its target. α-catenin is a component of adherens junctions (ajs), i.e., cellcell anchoring structures that, together with desmosomes, allow keratinocytes to adhere to one another and maintain epithelial integrity. α-catenin binds to β-catenin in ajs and is required for their formation and maintenance ( , ) . in addition, α-catenin was reported to be necessary for the organization of desmosomes in epithelial cells ( ) . a previous study reported a reactivity of pemphigus sera to another aj component, i.e., e-cadherin ( ) . likewise, we showed that anti-α-catenin autoreactivity was: (i) wellrepresented in pv patient sera, as α-catenin was recognized by almost half of the pv sera tested; (ii) more frequently associated with pemphigus than with other autoimmune bullous diseases, as α-catenin was very rarely recognized by the bp sera analyzed. then, considering that previous studies demonstrated the ability of intact autoabs to enter living cells ( , , ) , we addressed the potential pathogenicity of α-catenin-specific mab pvf by evaluating its acantholytic activity in an in vitro keratinocyte dissociation assay. in this regard, several observations suggest that ajs, and in particular e-cadherin, may be involved in pemphigus pathogenesis ( , ) . of note, with the exception of anti-desmocollin autoabs, all known non-dsg-reactive autoabs with reported pathogenicity do not possess acantholytic potential on their own but may act synergistically with anti-dsg antibodies ( , , ) . accordingly, marchenko et al. described a pathogenic role for intracellular anti-mitochondrial autoabs, even though not on their own ( ) . in our hands, the anti-α-catenin mab pvf was not able to dissociate the keratinocyte monolayer either alone or in combination with a suboptimal dose of a pathogenic anti-dsg antibody. nevertheless, we cannot exclude that regions of α-catenin different from that recognized by pvf could be recognized by pv sera and contribute to acantholysis. in addition, a possible role of anti-α-catenin hmabs as cofactors in disease initiation or maintenance could not be exhaustively addressed by our approach. in previous studies, several antibodies against intracellular antigens have been considered as triggers for autoimmunity. mabs specific for the cytoplasmic precursor form of dsg (predsg ) have been cloned from pemphigus patients and from healthy individuals ( , ) . yamagami et al. postulated that the presence of anti-predsg b cells is involved in the initiation of the autoimmune response in pemphigus patients. in particular, in the context of tissue damage they could present peptides which are part of mature dsg (i.e., the extracellular autoantigen recognized by most pathogenetic autoabs) derived from the processing of predsg . this intramolecular epitope spreading phenomenon could lead to the production of pathogenic autoabs targeting mature dsg and to the initiation of disease pathogenesis ( , ). moreover, natural autoabs (naas), i.e., antibodies to intracellular autoantigens that naturally occur in the healthy population and are per se unable to cause immune phatology, have been theorized as cofactors in the onset of autoimmunity, possibly by participating in the mechanisms of chronic tissue injury at the basis of intermolecular epitope spreading ( ) ( ) ( ) . in conclusion, while our previous study ( ) demonstrated that pathogenic pv antibodies primarily target the dsg- cisinterface, thus leading to desmosome disruption, our current results reveal novel antibodies targeting intracellular keratinocyte proteins. we are tempted to speculate that the cellular damage figure | pvf is not able to dissociate a keratinocyte monolayer even in the presence of suboptimal concentrations of pvb . a representative keratinocyte dissociation experiment. primary human keratinocytes, seeded to confluence, were incubated with pvf , an unrelated human mab (negative control) and, as positive controls, the human pathogenic mab pvb ( ) and the murine pathogenic mab ak ( ) , both dsg -specific (a). to investigate synergistic potential of cloned antibody we have employed pvf ( µg/ml) together with optimal ( µg/ml) and suboptimal concentrations ( µg/ml and . µg/ml) of the pathogenic anti-dsg antibody pvb without obtaining any difference in ability of pvb , with or without pvf , to dissociate the keratinocyte monolayer (b). induced by pathogenic anti-dsg antibodies may trigger an intermolecular epitope-spreading phenomenon resulting in an antibody response against intracellular antigens, among which α-catenin. further studies are needed: (i) to evaluate whether pvf may act synergistically with anti-dsg antibodies using more informative approaches, such as in vitro organ culture or in vivo models; (ii) to assess the possible role of anti-α-catenin autoabs in pemphigus initiation and evolution in vivo; and (iii) to characterize this novel antigen as a possible target of epitope spreading phenomena in pv. the datasets generated for this study are available on request to the corresponding author. this study was carried out in accordance with the recommendations of helsinki guidelines, idi-irccs ethics committee. the protocol was approved by the idi-irccs ethics committee. all subjects gave written informed consent in accordance with the declaration of helsinki. gdz and gdl have written the manuscript. gdz, gz, and al have designed the study. gdz, gdl, vc, and fm performed the experiments. all the authors have revised the manuscript and given the final approval for submission. the present work has been supported by the italian ministry of health (ricerca corrente - to gdz). immune response in pemphigus and beyond: progresses and emerging concepts pemphigus autoantibodies generated through somatic mutations target the desmoglein- cis-interface non-desmoglein antibodies in patients with pemphigus vulgaris the evolving story of autoantibodies in pemphigus vulgaris: development of the 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pathogenicity of antidesmoglein igg autoantidodies in pemphigus vulgaris envoplakin and periplakin are components of the paraneoplastic pemphigus antigen complex mucosal dominant pemphigus vulgaris with antidesmoplakin autoantibodies the penetrating potential of autoantibodies into live cells in vitro coincides with the in vivo staining of epidermal nuclei proceedings of the second international conference on the penetration of autoantibodies into living cells induction of pemphigus phenotype by a mouse monoclonal antibody against the aminoterminal adhesive interface of desmoglein identification of a neural alpha-catenin as a key regulator of cadherin function and multicellular organization alpha-catenin: at the junction of intercellular adhesion and actin dynamics identification of regions of alpha-catenin required for desmosome organization in epithelial cells e-cadherin is an additional immunological target for pemphigus autoantibodies cadherins in cutaneous biology a pathogenic autoantibody, pemphigus vulgaris-igg, induces phosphorylation of desmoglein , and its dissociation from plakoglobin in cultured keratinocytes critical role of the neonatal fc receptor (fcrn) in the pathogenic action of antimitochondrial autoantibodies synergizing with anti-desmoglein autoantibodies in pemphigus vulgaris synergy among non-desmoglein antibodies contributes to the immunopathology of desmoglein antibody-negative pemphigus vulgaris isolation of pathogenic monoclonal anti-desmoglein human antibodies by phage display of pemphigus foliaceus autoantidodies antibodies to the desmoglein precursor proprotein but not to the mature cell surface protein cloned from individuals without pemphigus humoral epitope spreading in autoimmune bullous diseases cellular and genetic mechanisms of self tolerance and autoimmunity predominant autoantibody production by early human b cell precursors conflict of interest statement: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest we thank naomi de luca for the excellent technical assistance. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material key: cord- - ksgl t authors: li, liang; xue, mei; fu, fang; yin, lingdan; feng, li; liu, pinghuang title: ifn-lambda mediates antiviral protection against porcine epidemic diarrhea virus by inducing a distinct antiviral transcript profile in porcine intestinal epithelia date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: ksgl t type iii interferon-lambda (ifn-λ) plays a critical role against infection, particularly in mucosal infection in the respiratory and gastrointestinal tract. our study and other previous studies have shown that porcine ifn-λ more efficiently curtails the infection of porcine epidemic diarrhea virus (pedv) in the intestine epithelia than type i ifn, whereas ifn-λ exerts a more potent effect than ifn-λ . however, the underlying mechanism remains elusive, and in particular, the transcriptional profile induced by ifn-λ has not been reported. here, to resolve the mechanism responsible for the disparity between ifn-λ and type i ifn in anti-mucosal virus infection, we compared the transcription profiles induced by the two ifns in porcine intestinal epithelial (ipec-j ) cells by rna-seq. our results showed that the pretreatment of ipec-j cells with ifn-λ resulted in the differential expression of genes. in contrast, ifn-α only modified the expression of genes, and of these genes were also observed in the response to ifn-λ . a transcriptional enrichment analysis indicated that ifn-λ or ifn-α regulates multiple cellular processes and that ifn-λ activates more robust signaling pathways, particularly the antiviral jak-stat signaling pathway, than ifn-α. furthermore, we verified the rna-seq results through an rt-qpcr analysis of ipec-j cells and porcine enteroids. moreover, transient expression of the porcine rsad and mx genes among the top genes induced by ifn-λ significantly inhibited pedv infection. collectively, the data showed that ifn-λ induces a unique transcriptional profile that does not completely overlap with that induced by ifn-α and strongly elicits a set of genes responsible for the antiviral activity of ifn-λ . these findings provide important knowledge regarding the elicited isgs of type i and iii ifns in restricting porcine intestinal viral infection. type iii interferon-lambda (ifn-λ) plays a critical role against infection, particularly in mucosal infection in the respiratory and gastrointestinal tract. our study and other previous studies have shown that porcine ifn-λ more efficiently curtails the infection of porcine epidemic diarrhea virus (pedv) in the intestine epithelia than type i ifn, whereas ifn-λ exerts a more potent effect than ifn-λ . however, the underlying mechanism remains elusive, and in particular, the transcriptional profile induced by ifn-λ has not been reported. here, to resolve the mechanism responsible for the disparity between ifn-λ and type i ifn in anti-mucosal virus infection, we compared the transcription profiles induced by the two ifns in porcine intestinal epithelial (ipec-j ) cells by rna-seq. our results showed that the pretreatment of ipec-j cells with ifn-λ resulted in the differential expression of genes. in contrast, ifn-α only modified the expression of genes, and of these genes were also observed in the response to ifn-λ . a transcriptional enrichment analysis indicated that ifn-λ or ifn-α regulates multiple cellular processes and that ifn-λ activates more robust signaling pathways, particularly the antiviral jak-stat signaling pathway, than ifn-α. furthermore, we verified the rna-seq results through an rt-qpcr analysis of ipec-j cells and porcine enteroids. moreover, transient expression of the porcine rsad and mx genes among the top genes induced by ifn-λ significantly inhibited pedv infection. collectively, the data showed that ifn-λ induces a unique transcriptional profile that does not completely overlap with that induced by ifn-α and strongly elicits a set of genes responsible for the antiviral activity of ifn-λ . these findings provide important knowledge regarding the elicited isgs of type i and iii ifns in restricting porcine intestinal viral infection. the surface epithelia of the mucosa are the major entry site of most pathogens in the host and serve as a first line of defense against invading pathogens. one of the most important antiviral cytokines in the host is interferons (ifns), which perform key roles in inhibiting viral infection ( , ) . the ifn family is categorized into three different types: type i ifn (ifn-α/β), type ii ifn (ifn-γ), and type iii ifn (ifn-λ). type ii ifn, which is primarily produced by t cells and natural killer cells, exerts limited direct antiviral activity and plays a key role in modulating the host immune response ( ) , whereas type i ifns (α/β) and the more recently discovered type iii ifns induce a strong antiviral state in responsive cells and play crucial roles in controlling viral infection ( ) ( ) ( ) ( ) ( ) . although type i ifns have generally been thought to be a key element against systemic infections, recent research has shown that ifn-λ plays a critical role in mucosal infections, such as enteric infection ( , ) . unlike type i ifns that are secreted by a wide range of different cell types upon stimulation, type iii ifns are primarily produced by epithelial cells, nk cells, and dendritic cells (dcs) ( , ( ) ( ) ( ) . ifn-λ acts primarily on the mucosal epithelium, which might result in fewer side effects compared with type i ifn treatment ( ) . these features make ifn-λ a potentially superior antiviral therapeutic candidate against local mucosal infection ( ) . although the receptors for type i and iii ifns are different, the binding of both type i and iii ifns to their corresponding receptors stimulates a janus kinase (jak)-signal transducer of transcription (stat) pathway, and the stimulation of this pathways subsequently drives the transcription of ifnstimulated genes (isgs) and prompts cells toward an antiviral status ( ) . consistent with the similarity of the induced signaling pathways, the spectrum of genes elicited by the two types of ifns show a high overlap ( ) . however, recent studies have demonstrated that type iii ifns are critical non-redundant antiviral mediators of type i ifns in the gi tract ( ) . to date, numerous studies in humans or mice have taken advantage of rna-seq or chip assays to show that ifn-λ and ifn-α elicit distinct downstream signaling events, even though many genes are induced by both type i and iii ifns ( , ) . mice with type i ifn or iii ifn receptor knockout experience more severe viral intestinal infections, but ifnl −/− mice show higher viral loads and more serious clinical symptoms than ifnar −/− mice ( , ) . studies conducted by pott et al. showed that intestinal epithelial cells exhibit stronger responses to ifn-λ compared with ifn-α/β in vivo ( , ) . a comprehensive understanding of the unique signaling profiles of type i and iii ifns has become increasingly important for understanding host-virus interactions and the development of ifn-λ therapeutics. however, thus far, no direct comparative analyses of the transcriptional profiles induced by porcine type i vs. type iii ifns in swine intestinal epithelia have been performed. the piglet diarrhea caused by enteric coronavirus porcine epidemic diarrhea virus (pedv) is a highly contagious disease characterized by watery diarrhea, dehydration, and causes up to % mortality in neonatal piglets. we and other research groups previously reported that porcine ifn-λ results in better suppression against pedv infection compared with ifn-α and that ifn-λ more efficiently inhibits pedv than ifn-λ ( ) ( ) ( ) . however, the mechanisms underlying the difference among ifn-λ , ifn-λ , and ifn-α in inhibiting enteric coronavirus remain less clear. previous studies have largely focused on the gene profiles induced by human or mouse ifn-λ and ifn-α, but the ifn-λ -and ifn-α-elicited genes have not been compared. in this study, we comprehensively compared the transcriptional profiling of ifn-λ -and ifn-α-induced genes in a porcine intestinal epithelial cell line (ipec-j ) and verified the rna-seq results by reverse transcriptase quantitative pcr (rt-qpcr) in vitro, and further confirmed the transcriptional profile difference in crypt-derived porcine enteroids. the intestinal porcine epithelial cell line j (ipec-j ; kindly provided by dr. anthony blikslager, north carolina state university, raleigh, nc, usa) was maintained in dulbecco's modified eagle's medium nutrient mixture f- (dmem/f ) supplemented with antibiotics ( units/ml penicillin and µg/ml streptomycin), . mm hepes (gibco, usa), and % heat-inactivated fetal bovine serum (fbs) (gibco). african green monkey kidney cells (vero e ) were grown and maintained in dmem supplemented with antibiotics ( units/ml penicillin and µg/ml streptomycin) and % heat-inactivated fbs (gibco). pedv strain cv of genotype (genbank accession no. kt ) was maintained at the harbin veterinary research institute of the chinese academy of agricultural sciences, harbin. the biological antiviral activity of e. coli-derived recombinant porcine ifn-lambda was prepared in our laboratory and evaluated in mdbk cells using a recombinant vesicular stomatitis virus (vsv) with a gfp reporter as described previously ( , ) . the weight-activity unit (u/ml) of samples was calculated using porcine prokaryotic-derived ifn-α ( . × u/mg) (prosit sole biotechnology, co., ltd., beijing, china) as a reference. porcine intestinal crypts were prepared from specific pathogenfree piglets using previously described protocols ( ) . in brief, the intestine was flushed with cold pbs with antibiotics ( units/ml penicillin and µg/ml streptomycin), cut into mm segments, and washed with cold pbs with antibiotics until the supernatant was clear. the washed intestinal pieces were suspended in ml of gentle cell dissociation reagent (stemcell, canada) and shaken at rpm for min to disassociate the crypts at room temperature (rt). the pellets of the intestinal pieces were suspended in ml of cold pbs with . % bovine serum albumin (bsa) and antibiotics (pen-strep) and passed through a -µm cell mesh. the crypt pellets were harvested by centrifugation at × g at • c for min and resuspended in ml of cold dmem/f . after counting, the intestine crypts were resuspended in µl of intesticult organoid growth medium (stemcell, canada) and µl of matrigel (bd biosciences, usa) per crypts and seeded into a -well plate at crypts per well. the plate was incubated at • c for min until the matrigel solidified. the plate was filled with complete intesticult organoid growth medium and then incubated at • c in a % co incubator. the culture medium was exchanged every - days. the institutional animal care and use committee of the harbin veterinary research institute approved all the protocols related to the animal experiments performed in this study. expanded d enteroids were recovered from the matrigel after - days of growth by the addition of ice-cold dmem/f medium, transferred into -ml tubes, and centrifuged at × g at • c for min. the pellet of enteroids was incubated in . % trypsin (gibco) for min at • c and dissociated by repeated pipetting to obtain a single-cell suspension. dmem-f with % (v/v) fbs was added into the single-cell suspension, and the mixture was centrifuged at × g for min. the cell pellets were resuspended in complete intesticult organoid growth medium at rt and seeded at enteroids per well in a matrigel-precoated -well plate. after differentiation for about - days, planar monolayers of d enteroids were ready for use in experiments. total cellular rna was extracted using the simply p total rna extraction kit (bioflux, china) according to the manufacturer's instructions. total rna ( µg) was reverse-transcribed to cdna using the primescript tm ii first-strand cdna synthesis kit (takara, china). the synthesized cdna was subjected to qpcr performed in triplicate using a lightcycler r ii real-time pcr instrument (roche, switzerland) and sybr green pcr mix (life technologies, usa) according to the manufacturer's instructions. all the data were acquired and analyzed using lightcycler r ii software . based on the cycle threshold ( ct) method ( ) . gapdh served as the internal control. the amplification efficiency of qpcr primers ranged from . to . %. the primers used in this assay were designed using primer premier software and are listed in table . three biological replicates of each of the three groups, namely, untreated ipec-j cells (mock control), ipec-j cells treated with ifn-λ ( , ng/ml) for h, or ipec-j cells treated with ifn-α ( , ng/ml) for h, were prepared for rna sequencing. total rna was purified using the trizol reagent according to the manufacturer's instructions (thermo fisher scientific, usa). the total rna from each sample was quantified and qualified using an agilent bioanalyzer (agilent technologies, usa), a nanodrop instrument (thermo fisher scientific, inc.), and a % agarose gel. one microgram of total rna with a rin value > was used for subsequent library construction. next-generation enzyme mix to repair both ends and add a da tail through one reaction, and the product was subjected to t-a ligation to add adaptors to both ends. size selection of adaptor-ligated dna was then performed using an axyprep mag pcr clean-up kit (axygen), and fragments of ∼ bp (with an approximate insert size of bp) were recovered. each sample was then amplified by pcr for cycles using the p and p primers carrying sequences that can anneal during bridge pcr with a flowcell and the p primer carrying a six-base index to allow multiplexing. the pcr products were cleaned up using the axyprep mag pcr clean-up kit (axygen), validated using an agilent bioanalyzer (agilent technologies, usa), and quantified with a qubit . fluorometer (invitrogen, usa). libraries with different indices were then multiplexed and loaded on an illumina hiseq instrument (illumina, usa) according to the manufacturer's instructions. sequencing was performed using a × -bp paired-end (pe) configuration and image analysis and base calling were conducted using hiseq control software (hcs) + olb + gapipeline- . (illumina) provided with the hiseq instrument. rna-seq was performed and analyzed using genewiz (suzhou, china). the analysis of the microarray data to identify differentially expressed genes was performed using edger software. the analysis starts with the count, and the data are standardized by tmm and then subjected to differential expression analysis. we selected | log (fold change) (logfc) | > and fdr < . as the screening criteria for the identification of differentially expressed genes. ipec-j or vero e cells were fixed with % paraformaldehyde for min at rt. the fixed cells were permeabilized with . % triton x- for min at rt and then blocked with blocking buffer (pbs with % fbs and % skim milk) for min at • c. the cells were then incubated with pedv n protein antibody at • c for h and then labeled with alexa fluor goat anti-mouse igg antibody (thermo fisher scientific, usa) at • c for h. dapi (sigma, usa) was used for the staining of cellular nuclei. the stained cells were visualized using an amg evos f fluorescence microscope (thermo fisher scientific, usa). the porcine rsad or mx coding region was amplified by rt-pcr, and cdna was prepared using the primescript tm ii st strand cdna synthesis kit (takara, china). rsad or mx was then amplified using a pair of primers specific for porcine rsad or mx (table ) , respectively. the pcr products were purified, digested, and cloned into pcaggs-ha through ecor i and kpn i. construction of the prsad /pmx -ha expression plasmid was confirmed by sequencing. vero e cells were grown in -well plates to - % confluence and then transfected with the prsad /pmx -ha expression plasmid using the attractene transfection reagent (qiagen), and the expression of prsad /pmx was verified by anti-ha ifa. graphpad prism software version was used to analyze the experimental results. the results are expressed as scatter plots in which the mean is indicated by a line. the differences between groups were compared using an unpaired mann-whitney test. p < . was considered significant, and the p-values are expressed as follows: * p < . , * * p < . , * * * p < . , and * * * * p < . . the inhibition of pedv in ipec-j cells by exogenous ifn-λ is superior to that achieved with ifn-α according to our study and other previous studies ( ) ( ) ( ) , ifn-λ , ifn-λ , and ifn-α all significantly inhibit pedv infection, but ifn-λ shows the strongest antiviral activity against pedv. to confirm this disparity in the antiviral activities of ifn-λ and ifn-α against pedv, ipec-j cells were primed with ifn-α ( , ng/ml) or ifn-λ ( , ng/ml) for h and then infected with pedv (moi = ). consistent with our previous results, both ifn-α and ifn-λ substantially suppressed pedv infection in ipec-j cells, as demonstrated by measurements of the viral genomes and titers by rt-qpcr ( figure a ) and tcid titration (figure b) , respectively. ifn-α reduced the number of pedv genomes by -fold, whereas ifn-λ decreased the number of pedv genomes by approximately -fold. the virus titers were consistent with results of pedv genomes: pedv titers decreased and -fold after pretreatment with ifn-α or ifn-λ , respectively. the inhibition of pedv infection by both ifnα and ifn-λ was further verified by pedv n protein ifa ( figure c) . the virus-infected cells were significantly decreased after pretreatment with ifn-α or ifn-λ , whereas the number of pedv-infected cells in the ifn-λ -pretreated group was significantly decreased, as demonstrated by only a few sporadically distributed cells, compared with those in the ifnα-pretreated group. thus, ifn-λ restricted pedv infection in ipec-j cells more effectively than ifn-α, regardless of the quantification of viral rna, infectivity, or viral protein (figure ). to assess the underlying mechanisms of the disparity between the anti-pedv responses induced by ifn-α or ifn-λ , we performed an rna-sequencing (rna-seq) analysis of total cellular rna isolated from ipec-j cell cultures stimulated with ifn-α or ifn-λ for h and ifn untreated ipec-j mock control. this duration of stimulation ( h) was selected based on the efficacy of viral inhibition after exposure to ifn-λ and ifn-α (figure ) ( ) . each of the triple replicate yielded more than . × clean reads and has more than % q (%), indicating the reliability of the rna-seq data. the ifn-λ stimulated cells showed a total of differentially expressed genes, including upregulated genes and downregulated genes, compared with the mock control, whereas ifn-α only upregulated the expression of genes among the total of differentially expressed genes and reduced the expression of four genes (figure a) . the number of ifn-λ -modified genes was approximately -fold higher than that of ifn-αmodified genes. these results indicate that the intestine epithelia respond better to ifn-λ than to ifn-α. we further grouped the corresponding genes through supervised partitioning clustering and combined the differentially expressed genes induced by ifn-λ and ifn-α to obtain the heat map ( figure b ) and the venn map ( figure c ). ifn-λ yielded different gene expression profiles compared with that induced by ifn-α, even though these showed substantial overlap ( figure b) . one hundred ten genes were upregulated in both the ifn-λ -and ifn-α-treated cells, whereas and genes were uniquely upregulated in the presence of ifn-λ and ifn-α, respectively ( figure c) . none of the coexpression genes were downregulated in both the ifn-λ -and ifn-α-treated cells, whereas and genes were only downregulated in the presence of ifn-λ and ifnα, respectively. these results demonstrated that ifn-λ induces a unique gene transcriptional profile in the intestine epithelia compared with ifn-α. to further clarify the functional consequences of the gene profiles elicited by either ifn-λ or ifn-α, we performed a gene ontology (go) enrichment analysis using a database established by the gene ontology consortium, which aims to define and describe the functions of genes and proteins in various species (figure ) ( ) . among the genes upregulated by ifn-λ , and genes ( . and . % of the total genes, respectively) were associated with a binding function and catalysis, respectively, and these two functions account for the main molecular functional changes. the dominant functions of the ifn-α-regulated genes are cellular transporter activity and nucleic acid-binding transcription factor activity, and these functions were associated with genes ( . % of total genes) and genes ( . % of total genes), respectively. the analysis of cellular components revealed that differentially expressed genes in the ifn-λ -treated group affected the cell part, and genes associated with this cellular component were differentially expressed in the ifn-α-treated group. with respect to biological processes, the ifn-λ -treated group included , , , , and differentially expressed genes associated with the cellular process, the single-organism process, biological regulation, the metabolic process, and response to stimulus, respectively. the pattern obtained for the ifn-α-treated group was similar to that of the ifn-λ -treated group, and the differentially expressed genes were also concentrated on the following five functions: the cellular process, the single-organism process, the biological regulation, the metabolic process, and the response to stimulus. however, the numbers of differentially expressed genes after ifn-α treatment were notably lower than those obtained after ifn-λ treatment. these results indicated that both ifn-λ and ifn-α are involved in the regulation of multiple cellular processes in ipec-j cells, such as cellular components and molecular functions, but ifn-λ exerts more potent effects than ifn-α. to further investigate the function of genes specifically induced by ifn-λ , we extracted the transcriptional profiles of ifn-λ and ifn-α regarding biological processes (p < . ) and combined them based on the -log (p) values to obtain a heat map that showed the enrichment of biological processes ( figure s ). the data demonstrated that in ipec-j cells, ifn-λ stimulation triggered more biological reaction processes than ifn-α, and these processes mainly involved the modulation of metabolic processes, including cellular metabolic processes, macromolecular metabolic processes, and primary metabolic regulation. taken together, these results indicate that the differentially expressed genes induced by ifn-λ are involved in more intracellular biological processes than those induced by ifn-α. to explore the clustering of the ifn-induced differential expression genes in anti-viral signaling pathways, we performed a kegg enrichment analysis of the differentially expressed genes using kobas . . the kegg enrichment analysis revealed that the differentially expressed genes induced by ifn-λ involved in much broader signal pathways compared with ifn-α though they both primarily modified the nf-κb signaling pathway, the jak-stat signaling pathway, the phosphoinositide- -kinase-akt (pi k-akt) signaling pathway, the mitogen-activated protein kinase (mapk) signaling pathway, and the cgmp-pkg signaling pathway ( figure a) . to determine the signaling pathways involving the differentially expressed genes induced by ifn-λ or ifn-α, we combined the differentially expressed genes and analyzed their expression patterns ( figure b) . the results showed that the differentially expressed genes were most abundantly involved in the jak-stat signaling pathway, followed by the pi k-akt signaling pathway and the mapk signaling pathway, which is consistent with the finding that the jak-stat signaling pathway primarily mediates ifn-induced antiviral responses. among the proteins encoded by the differentially expressed genes, sos , pik ca, jak , il- , socs , il- b, stat , il ra , and socs play a major role in the jak-stat signaling pathway, and kras, pik ap , prkaa , enssscg , enssscg , and map k play a major role in the pi k-akt signaling pathway. to predict the protein interactions between differentially expressed genes, we extracted the union of differentially expressed genes and introduced these into the web-based tool string (http:// www.string-db.org/) to generate protein-protein interaction networks ( figure c) . the results of the string analysis indicated that jak , stat , pten, pik ca, irs , kras, and il st are closely related to other proteins and displayed significant differential expression compared with the ifn-αinduced proteins, which play critical roles in the innate immune response induced by ifn. collectively, the results showed that the ifn-λ -induced differentially expressed genes are involved in more signaling pathways, particularly those associated with innate immunity, than those induced by ifn-α, which indicates that ifn-λ exhibits stronger antiviral activity in intestinal epithelial cells. to further elucidate the mechanisms underlying the antiviral activity discrepancy between ifn-λ and ifn-α, we focused on the top ifn-λ -induced genes (fold change compared with the mock control) and divided them into three subgroups (classical isgs, weakly ifn-λ -induced genes, and strongly ifn-λ -induced genes) as in a previous study ( ) . the expression of the top genes in all three subgroups induced by ifn-λ was significantly higher compared with that induced by ifn-α ( figure ) . the isgs in the classic isg subgroup are primarily the classical isgs induced by ifn reported in the literature ( , ) . the levels of isgs in this subgroup induced by ifn-λ were from -to -fold higher than those induced by ifnα. the weakly ifn-λ -induced gene subgroup contained genes, including three unknown genes (being denoted un , un , and un ). the analysis of this subgroup showed that both ifn-λ and ifn-α induced a more than -fold increase in the expression of isgs compared with mock control. the ifn-λ -induced expression levels of ifit , oasl, oas , and gbp , which are important innate immunity factors, were fold higher than those induced by ifn-α. the expression of of the genes in the strongly ifn-λ -induced gene subgroup was strongly induced by ifn-λ , whereas ifn-α did not induce a substantial increase in expression (< -fold compared with the mock control). interestingly, radical s-adenosyl methionine domain containing (rsad ), a multifunctional protein with broad antiviral activity that can inhibit both dna and rna viruses, is the top gene upregulated by ifn-λ ( ) . in contrast, ifn-α did not substantially upregulate rsad expression. protein interaction prediction analysis of differentially expressed genes by string database. the relevant genes were input into string (http://www.string-db. org/), the active interaction sources were selected as homology, and the experimentally determined interaction and database annotated were used as predictive conditions for protein interaction prediction. the thickness of the line represents combined score, which is the combined score of the prediction results of the three prediction conditions, indicating the strength of data support. several other genes (ifit , parp , and gbp ) in this subgroup are also important innate viral restriction factors ( , ( ) ( ) ( ) . thus, ifn-λ induces a stronger antiviral innate immune response compared with ifn-α. to verify the unique antiviral gene expression profile induced by ifn-λ that was detected by rna-seq, as shown in figure , we randomly selected three genes from each group to be verified by rt-qpcr. the analysis of the three subgroups confirmed that ifn-λ most substantially upregulated the expression of isgs (figure ) . specifically, ifn-λ upregulated the expression of large distinct classes of isgs in the classical isg subgroup, such as mx , isg , and ifit , the isg fold changes induced by ifn-λ were up to nearly -fold higher than those induced by ifn-α ( figure a ). the analysis of the weakly ifn-λ induced gene subgroup showed that the expression of oasl, oas , and isg (a) was induced by both ifn-λ and ifnα to significantly different levels. the oasl and oas fold changes induced by ifn-λ were up to -fold greater than those induced by ifn-α ( figure b ). more substantial fold changes in the expression of isgs in the strongly ifn-λ -induced gene subgroup were obtained with ifn-λ compared with ifn-α, which resulted in only slight changes in expression ( figure c ). the differences between groups were compared using an unpaired mann-whitney test. p < . was considered significant, and the p values are expressed as follows: *p < . , **p < . , ***p < . , and ****p < . . rsad , plac , and ifit were more substantially upregulated by ifn-λ than by ifn-α. collectively, the in vitro rt-qpcr results confirmed that ifn-λ induces stronger isg expression compared with ifn-α. enteroids derived from intestinal crypt stem cells, which mimic the diverse cellular nature and physiological activity of the small intestine while also maintaining the genetic identity of the host, constitute a unique ex vivo model for studying the intestine ( , ) . to further confirm whether the ifn-λ induced gene expression profile in enteroids was the same as that in ipec-j cells, we stimulated enteroids with ifn-λ or ifn-α under the same conditions as ipec-j cells and evaluated their gene expression by rt-qpcr. in all three groups, the expression pattern of the genes induced by ifn-λ and ifn-α in porcine enteroids was consistent with that found in the ipec-j cells (figure ) . in the classical isg subgroup, the upregulated levels of mx and ifit elicited by ifn-λ were ∼ -to -fold higher than those induced by ifn-α ( figure a ). in contrast, the upregulated levels of oasl and oas , which belong to the weakly ifn-λ -induced gene subgroup, induced by ifn-λ were -fold higher than those induced by ifn-α, and the fold change difference was moderate ( figure b) . for the strongly ifn-λ -induced gene rsad and plac , just as we observed in the other subgroups, enteroids were or were not primed with ifn-λ ( , ng/ml) or ifn-α ( , ng/ml) for h, and the total rna from the enteroids was extracted and used for rt-qpcr. the differences between groups were compared using an unpaired mann-whitney test. p < . was considered significant, and the p values are expressed as follows: *p < . , **p < . , ***p < . , and ****p < . . ifn-λ significantly induced higher expression than ifn-α does ( figure c ). in summary, ifn-λ induces higher expression of isgs than ifn-α in porcine enteroids, which suggests that ifn-λ exerts a greater effect in gastrointestinal epithelial cells than ifn-α. pmx and prsad inhibit pedv infection in vero e cells we selected mx (classical isg) and rsad (strongly ifn-λ induced gene) among the top genes induced by ifn-λ to evaluate the antiviral effect of the ifn-λ -induced expression of isgs against pedv infection. we cloned porcine mx or rsad into the eukaryotic expression vector pcaggs-ha, and the transient expression of pmx or prsad in vero e cells was verified by ifa (data not shown). as expected, pmx or prsad transient overexpression significantly inhibited pedv infection in vero e cells, as demonstrated by measuring the viral rna (figures a,c) and pedv n protein expression by ifa (figures b,d) . thus, these data indicate that the differentially upregulated pmx or prsad serves as an important ifn-λ elicited antiviral host factor against pedv. type i and type iii ifns, which establish a cellular antiviral state and restrict viral infection in the host, are key players at the earliest stages of innate immunity against viral infection. despite the similarities between the effects of the two types of ifns, increasing evidence demonstrates that each class of ifn is essential for antiviral host defense and is not functionally redundant ( ) . a previous study in mice demonstrated that the role of ifn-λ in functionally redundant intestinal viral infections cannot be compensated by ifn-α/β ( ) . therefore, clarification of the induction of cell-specific ifn signaling profiles is essential for understanding the non-redundant roles of ifn-λ and ifn-α in viral infection at the tissue and organism levels. in this study, we found that the transcriptional profile induced by type iii ifn in the intestinal epithelia the differences between groups were compared using an unpaired mann-whitney test. p < . was considered significant, and the p values are expressed as follows: *p < . , **p < . , ***p < . , and ****p < . . was unique compared with that induced by type i ifn. compared with ifn-α, stimulation with ifn-λ not only resulted in higher levels of most isgs but also substantially increased diversity in the gene profiles involved in various cellular functions. type i and iii ifns each signal through different heterodimeric complex receptors to initiate multiple downstream signaling pathways. in addition to activation of the jak-stat signaling pathway, ifns also activate the pi k and mapk signaling cascades ( , ) . the combined use of these signaling pathways by ifns and many other cytokines might help explain the different roles of ifns in regulating the antiviral and immune responses in a variety of environments and locations. we have very limited knowledge on the pathways activated by ifn-λ, which are important for understanding the immune-modulating activities of ifn-λ ( ) . in this study, we found that ifn-λ not only activates the classical antiviral response jak-stat signaling pathway but also primarily activated the nf-κb signaling pathway, the camp signal pathway, the pi k-akt signaling pathway, and mapk signaling pathway (figure ) . ifn-λ activated much more signaling pathways in epithelia than ifn-α, and this finding might provide new insights into the mechanism through which ifn-λ modulates other cellular functions beyond its direct antiviral activity. ifn-λ largely stimulated wnt signaling pathway in iec compared with ifn-α, which plays critical roles in maintaining the homeostasis of intestinal epithelia in vivo ( ) . further studies of different signaling pathways induced by ifn-λ or ifn-α will help dissect the complex interaction of ifn-induced signaling pathways with similar or overlapping intracellular signaling pathways stimulated by other cytokines. most somatic cells can induce and respond to type i ifns, but certain specialized cells, such as those in the mucosal epithelia, selectively produce and respond to type iii ifns during various virus infections. we demonstrated that the porcine intestinal epithelia respond to both type i and iii ifns, even though these ifns induced different isg expression levels. type iii ifns comprise four functional and highly homologous subtypes, namely, ifn-λ , ifn-λ , ifn-λ , and ifn-λ , in humans. our study and other studies have revealed differences in antiviral activity among different ifn-λ subtypes ( , , ) . ifn-λ is superior or equal to ifn-λ in terms of its antiviral activity in the intestinal epithelia ( ) and the immortalized liver cell line hepg in vitro ( ) . the different kinetics and magnitude of isg induction after stimulation might account for the variation in the antiviral activity among different types or subtypes ifns. previous studies that attempted to identify the gene profiles induced by type iii ifns primarily focused on the comparison of ifn-λ with type i ifn ( ) , and most of these studies were performed in immortalized human or mouse cell lines. the gene transcription profile induced by ifn-λ , particularly the gene transcription profile induced by ifn-λ in porcine intestinal epithelial cells, has not been reported. here, we performed comparative analyses of the transcriptional profiles induced by ifn-λ and ifn-α in porcine intestinal epithelia cells. more importantly, in this study, we verified the rna-seq results in primary swine crypt-derived enteroids, an in vitro system that well recapitulates the complicated cellularity of the gi tract, which exhibits a potent response to both type i and iii ifns ( , , ) . the differential expression in ipec-j or enteroids detected by qpcr is not related to the selection of housekeeping gene gapdh since we observed the same pattern when using another housekeeping gene actin (data not shown). therefore, studying the differences among ifn-λ -and ifn-α-induced gene expression in enteroids is more realistic and clinically significant. the different antiviral activities of ifn-λ and ifn-α are likely due to the different degrees of isg induction by these two cytokines, and ifn-λ induced increased isg expression. the functions of some of the genes that were strongly induced by ifn-λ have been reported, but the functions and mechanisms of more genes remain unclear. rsad , a host viral restrictor gene, showed the highest upregulated levels after ifn-λ rather than ifn-α stimulation, whereas mx was the most highly upregulated gene after ifn-α stimulation. a previous study demonstrated that porcine rsad effectively inhibited csfv replication in vitro, and this effect might occur via the interaction of rsad with csfv e protein in the cytoplasm ( ) . our study found that rsad transient expression also substantially curtailed pedv infection in vero e cells in vitro, but the mechanism is unclear. mx , an ifn-induced gtpbinding protein ( , ) , reportedly inhibits the replication of lentivirus hiv- , simian immunodeficiency virus (siv), and equine infectious anemia virus (eiav) in vitro ( , ) . the function of porcine mx has been poorly studied. we confirmed that porcine mx inhibits pedv infection in vero e cells. we also discovered some unknown genes that were differentially expressed in response to ifn-λ or ifn-α stimulation, and this finding might provide a foundation for further elucidation of the different mechanisms of action of ifn-λ and ifn-α. in summary, we compared the transcriptional profiles of ifn-λ and ifn-α in ipec-j cells and identified a unique set of genes that were strongly induced by ifn-λ compared with ifn-α. the transcriptional enrichment analysis indicated that ifn-λ or ifn-α is involved in the regulation of cellular processes, such as cellular components and molecular functions, in ipec-j cells, and that ifn-λ activates more robust signaling pathways, particularly the antiviral jak-stat signaling pathway, compared with ifn-α. ifn-λ preferentially upregulates antiviral genes in epithelial cells compared with ifn-α. these results indicate that ifn-λ selectively targets small intestinal epithelial cells and induces a non-redundant antiviral host response at the enteric mucosal site. all datasets generated in this study are included in the manuscript/supplementary material. the animal study was reviewed and approved by the harbin veterinary research institute of the chinese academy of agricultural sciences, harbin. written informed consent was obtained from the owners for the participation of their animals in this study. pl, ll, and lf designed the research studies and analyzed and interpreted the data. ll, mx, ff, and ly conducted experiments and acquired data. ll and pl drafted the manuscript and all authors contributed revisions. funding support for this work was provided by grants from the national key r&d program of china ( yfd ) and the national natural science fund ( ). the content is solely the responsibility of the authors and does not necessarily represent the official views of the funding resources. ifn-lambda decreases murid herpesvirus- infection of the olfactory epithelium but fails to prevent virus reactivation in the vaginal mucosa type iii interferons in antiviral defenses at barrier surfaces cell type-specific signaling in response to interferon-gamma interferon-lambdas: frontline guardians of immunity and homeostasis in the respiratory tract interferon lambda genetics and biology in regulation of viral control. front immunol lambda interferon is the predominant interferon induced by influenza a virus infection in vivo interferon-lambda: a potent regulator of intestinal viral infections. front immunol type iii interferons in viral infection and antiviral immunity interferon lambda protects the female reproductive tract against zika virus infection induction and function of type i and iii interferon in response to viral infection interferon induction and function at the mucosal surface ifn-alpha receptor- upregulation in pbmc from hcv naive patients carrying cc genotype possible role of ifn-lambda interferon-lambda: immune functions at barrier surfaces and beyond type iii interferon (ifn) induces a type i ifn-like response in a restricted subset of cells through signaling pathways involving both the jak-stat pathway and the mitogen-activated protein kinases distinct and overlapping genomic profiles and antiviral effects of interferon-lambda andalpha on hcv-infected and noninfected hepatoma cells identification of a predominantly interferon-lambda-induced transcriptional profile in murine intestinal epithelial cells commensal microbes and interferon-lambda determine persistence of enteric murine norovirus infection type iii interferon-mediated signaling is critical for controlling live attenuated yellow fever virus infection in vivo diverse intracellular pathogens activate type iii interferon expression from peroxisomes ifn-lambda determines the intestinal epithelial antiviral host defense porcine intestinal enteroids: a new model for studying enteric coronavirus porcine epidemic diarrhea virus infection and the host innate response ifn-lambda preferably inhibits pedv infection of porcine intestinal epithelial cells compared with ifn-alpha type iii interferon restriction by porcine epidemic diarrhea virus and the role of viral protein nsp in irf signaling establishment of a stable cho cell line with high level expression of recombinant porcine ifn-beta analyzing real-time pcr data by the comparative c(t) method gene ontology: tool for the unification of biology. the gene ontology consortium a family of ifn-gamma-inducible -kd gtpases protects against bacterial infection porcine viperin protein inhibits the replication of classical swine fever virus (csfv) in vitro decreased ifit expression promotes gastric cancer progression and predicts poor prognosis of the patients ifit is a restriction factor in rabies virus pathogenicity structure, function and inhibition of poly(adp-ribose)polymerase, member (parp ) regulation of type i interferon responses sustained in vitro intestinal epithelial culture within a wnt-dependent stem cell niche human interferon-lambda is a potent member of the type iii interferon family expression of ifnlr on intestinal epithelial cells is critical to the antiviral effects of interferon lambda against norovirus and reovirus cdna structures and regulation of two interferon-induced human mx proteins human mxb protein, an interferon-alpha-inducible gtpase, contains a nuclear targeting signal and is localized in the heterochromatin region beneath the nuclear envelope equine myxovirus resistance protein restricts lentiviral replication by blocking nuclear uptake of capsid protein the interferon-inducible mxb protein inhibits hiv- infection the authors thank dr. xiangxi pei (northeast agricultural university, harbin, china) for assistance and advice in rna-seq data analysis. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material figure s | the heat map of ifn-λ and ifn-α transcriptional profile biological process enrichment analysis. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © li, xue, fu, yin, feng and liu. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -or in m authors: nguyen, khue g.; vrabel, maura r.; mantooth, siena m.; hopkins, jared j.; wagner, ethan s.; gabaldon, taylor a.; zaharoff, david a. title: localized interleukin- for cancer immunotherapy date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: or in m interleukin- (il- ) is a potent, pro-inflammatory type cytokine that has long been studied as a potential immunotherapy for cancer. unfortunately, il- 's remarkable antitumor efficacy in preclinical models has yet to be replicated in humans. early clinical trials in the mid- 's showed that systemic delivery of il- incurred dose-limiting toxicities. nevertheless, il- 's pleiotropic activity, i.e., its ability to engage multiple effector mechanisms and reverse tumor-induced immunosuppression, continues to entice cancer researchers. the development of strategies which maximize il- delivery to the tumor microenvironment while minimizing systemic exposure are of increasing interest. diverse il- delivery systems, from immunocytokine fusions to polymeric nanoparticles, have demonstrated robust antitumor immunity with reduced adverse events in preclinical studies. several localized il- delivery approaches have recently reached the clinical stage with several more at the precipice of translation. taken together, localized delivery systems are supporting an il- renaissance which may finally allow this potent cytokine to fulfill its considerable clinical potential. this review begins with a brief historical account of cytokine monotherapies and describes how il- went from promising new cure to ostracized black sheep following multiple on-study deaths. the bulk of this comprehensive review focuses on developments in diverse localized delivery strategies for il- -based cancer immunotherapies. advantages and limitations of different delivery technologies are highlighted. finally, perspectives on how il- -based immunotherapies may be utilized for widespread clinical application in the very near future are offered. interleukin- (il- ) is a potent, pro-inflammatory type cytokine that has long been studied as a potential immunotherapy for cancer. unfortunately, il- 's remarkable antitumor efficacy in preclinical models has yet to be replicated in humans. early clinical trials in the mid- 's showed that systemic delivery of il- incurred dose-limiting toxicities. nevertheless, il- 's pleiotropic activity, i.e., its ability to engage multiple effector mechanisms and reverse tumor-induced immunosuppression, continues to entice cancer researchers. the development of strategies which maximize il- delivery to the tumor microenvironment while minimizing systemic exposure are of increasing interest. diverse il- delivery systems, from immunocytokine fusions to polymeric nanoparticles, have demonstrated robust antitumor immunity with reduced adverse events in preclinical studies. several localized il- delivery approaches have recently reached the clinical stage with several more at the precipice of translation. taken together, localized delivery systems are supporting an il- renaissance which may finally allow this potent cytokine to fulfill its considerable clinical potential. this review begins with a brief historical account of cytokine monotherapies and describes how il- went from promising new cure to ostracized black sheep following multiple on-study deaths. the bulk of this comprehensive review focuses on developments in diverse localized delivery strategies for il- -based cancer immunotherapies. advantages and limitations of different delivery technologies are highlighted. finally, perspectives on how il- -based immunotherapies may be utilized for widespread clinical application in the very near future are offered. keywords: interleukin- (il- ), cancer immunotherapy, cancer vaccine, cytokine delivery system, localized delivery, intratumoral administration overview of il- -based immunotherapies a brief history of cytokine and il- immunotherapies since the discovery of an "endogenous pyrogen, " now known as il- , in , scientists have anticipated the use of exogenous cytokines to manipulate a patient's immune system in an effort to control malignant neoplasms ( ) . early obstacles to cytokine-based immunotherapy centered on difficulties achieving reproducible manufacture of a sufficient and pure supply of cytokines for clinical trials. in the early s, recombinant dna technology and advances in the biochemical characterization of proteins combined to overcome this hurdle. finally, in , ifn-α broke through as the first cytokine to win fda approval as a single agent cytokine therapy for cancer ( ) . since then, hundreds, if not thousands, of studies have evaluated more than cytokines against a range of preclinical tumor models. a number of promising cytokines, including gm-csf, il- , tnfα, ifn-γ, and il- , subsequently entered clinical trials as single agents but failed to provide clinical benefit. currently, only of + identified cytokines are approved as single agent immunotherapies for a limited number of indications ( table ) . another fda approved cancer immunotherapy, talimogene laherparepvec (t-vec; imlygic tm ) is an oncolytic herpes simplex virus that uses gm-csf expression as an immune enhancer ( ) . perhaps the greatest disappointment in cytokine immunotherapy development thus far is il- . il- is a potent, pro-inflammatory cytokine produced by antigen presenting cells typically in response to microbial pathogens. it is comprised of two subunits, p and p , that are linked by three disulfide bridges to form a p heterodimer ( ) ( ) ( ) ( ) . il- is chiefly responsible for the induction and enhancement of cell-mediated immunity. among its diverse functions, il- has been shown to: (i) induce t h cell differentiation; (ii) increase activation and cytotoxic capacities of t and nk cells; and (iii) inhibit or reprogram immunosuppressive cells, such as tumor associated macrophages (tams) and myeloid-derived suppressor cells (mdscs) ( ) ( ) ( ) ( ) ( ) ( ) . il- also induces the production of large amounts of ifnγ which itself is cytostatic/cytotoxic ( , ) , anti-angiogenic ( , ) and can upregulate mhc i and ii expression on tumor cells for enhanced recognition and lysis ( ) . not surprisingly then, il- has demonstrated remarkable antitumor effects against a range of malignancies in preclinical studies ( ) ( ) ( ) ( ) . these effects are largely dependent on cd + t cells, nk cells, and nk t cells ( , , ) . in clinical studies, il- has been evaluated as an experimental treatment for numerous malignancies ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . unfortunately, the efficacy of il- at tolerated doses has been minimal ( , , ) . atkins and colleagues were the first to employ il- immunotherapy in a clinical trial ( ) . this phase i study enrolled patients, including with renal cancer and with melanoma, to investigate intravenous administration of recombinant hil- (rhil- ). one melanoma patient experienced a transient complete response and one renal cancer patient had a partial response ( ) . subcutaneous rhil- was employed in a separate pilot study that enrolled advanced melanoma patients ( ) . in this study, a fixed dose of rhil- ( . µg/kg) was given to patients on days , , and for two sequential cycles of days. no partial or complete responses were reported. minor tumor shrinkages involving some subcutaneous metastases and hepatic metastases were observed ( ) . in yet another early melanoma study, the administration of il- was found to induce a striking peripheral burst of hla-restricted ctl precursors directed to autologous tumors and to multiple immunogenic tumor-associated antigens ( ) . significantly, the infiltration of cd + t cells with an effector-memory phenotype was identified in posttreatment metastatic lesions, but not in pretreatment metastatic lesions of three patients ( ) . il- has also been shown to induce productive antitumor responses against cutaneous t cell lymphoma variants ( ) , aids-related kaposi sarcoma ( ) , and non-hodgkin's lymphoma ( ) . although il- has demonstrated robust antitumor activity in preclinical studies and potent immune-stimulating potential in humans, systemic administrations of il- have been shown to be exceedingly toxic. in one phase ii trial, a maximal dose of . µg/kg per day resulted in severe side effects in out of enrolled patients and the deaths of two patients ( ) . interestingly, the same dose of . µg/kg il- per day was found to be well-tolerated in patients that were enrolled in a previous phase i study. a change in dosing schedule accounted for the differences in toxicity between the phase i and phase ii trials. in the phase i trial, a single tester dose of il- was administered week before a multiple-dose regimen. the tester dose was found to blunt the toxicity induced by subsequent doses ( ) . overall, severe toxicities in early clinical trials, including on-study deaths ( ) due to frequent systemic injections of il- , together with disappointing clinical responses in large phase studies ( , ) , dampened enthusiasm for il- -based immunotherapy. the disappointing antitumor responses in clinical trials raised the possibility that il- is simply less active in humans. however, the severe toxicities outlined above indicate that il- has potent biological activity in humans. another possibility for the limited clinical efficacy is insufficient delivery of il- to the tumor microenvironment in humans. il- , like most cytokines, functions locally through paracrine and autocrine mechanisms. the ideal targets of il- immunotherapy are not lymphocytes in circulation, but rather immune cells within the tumor and nearby lymph nodes, including activated but exhausted t cells, nk cells, tams, and mdscs. therefore, maximizing the amount of il- that reaches the tumor seems critical for a robust antitumor response. we and others have noted that il- immunotherapeutics would be more effective and less toxic if delivered and maintained in the tumor through the use of novel delivery technologies. there are five benefits of local, persistent il- delivery. the first is enhanced spatiotemporal distribution of il- compared to systemic delivery. the failure of il- -based immunotherapies to achieve widespread clinical success may be at least partially attributed to the inability of a tolerated dose of systemically administered il- to reach therapeutic concentrations within human tumors. in mice, implanted or induced tumors are disproportionately large. for example, a . -g tumor (∼ . cm ) comprises % of the body weight of a -g mouse. a comparably sized tumor in a kg ( lbs) human would weigh . kg ( . lbs). furthermore, rapidly growing murine tumors are highly vascularized relative to their human counterparts ( , ) . taken together, a significantly larger fraction of a systemically administered il- dose can be expected to reach the tumor in a mouse compared to a human. localized delivery strategies, on the other hand, are capable of enhancing il- concentrations in the tumor microenvironment by one or more orders of magnitude ( ) ( ) ( ) ( ) . a second benefit of localized il- delivery is the ability to generate systemic antitumor immunity from a locally initiated intravenous injection ( , ) talimogene laherparepvec (t-vec) unresectable advanced melanoma intratumoral injection ( ) * subcutaneous and intramuscular injections are local deliveries. however, they result in systemic cytokine distribution and are utilized in the clinic as systemic therapies. immune response. as cancer metastasizes and becomes a "systemic" disease, conventional wisdom has suggested that metastases must be treated with systemic therapies such as i.v. administered chemotherapies or immune checkpoint inhibitors. however, systemic delivery increases the frequency of adverse events through off-target interactions. for instance, systemic il- therapy has the potential to cause activation and/or differentiation of all circulating t cells whereas activation/differentiation of only tumor-specific or tumor antigen-experienced t cells is preferred. fortunately, a growing mountain of evidence demonstrates that localized il- can generate systemic, adaptive immunological memory capable of controlling anestic tumors, inhibiting metastases, and preventing tumor recurrences ( ) ( ) ( ) . in particular, local administration of il- has been shown to activate or reactivate tumor infiltrating cd + t cells, improve antigen presenting machinery and subsequently cause the expansion of tumor-specific cd + effector t cells. this often leads to enhanced infiltration of contralateral untreated tumors ( ) . research from our own lab demonstrated that local/intravesical administration of il- eliminated untreated flank tumors only when a primary orthotopic bladder tumor was treated. these data indicated that t cells must be educated with antigens from a primary tumor in order to find and eliminate abscopal tumors. similarly, intratumoral injections of il- neoadjuvant to resection have been shown to inhibit metastases by multiple groups in a t cell, nk cell, or ifn-γ dependent manner ( , ) . taken together, the convincing evidence demonstrating that localized il- can induce abscopal immunity renders systemic il- delivery unnecessary even for the treatment of metastatic disease. third, as mentioned above, il- is a pleiotropic cytokine with context dependent consequences. when il- is administered systemically, it induces rapid increases in pro-inflammatory cytokines, such as ifn-γ, tnf-α, and il- ( ). this "cytokine storm" combined with rapid decreases in peripheral blood lymphocytes, monocytes, and neutrophils can be lethal ( ) . however, when controlled locally, pleiotropic cytokines have the potential to engage multiple antitumor effector mechanisms. for instance, il- increases the activation and cytolytic capacity of cd + t cells and nk cells and induces the production of ifn-γ. ifn-γ, in turn, may kill tumor cells directly, inhibit angiogenesis ( ) ( ) ( ) ( ) , and stimulate nk cells, ctls ( , ) , and macrophages ( ) while upregulating mhc i and ii molecules ( ) on the surfaces of tumor cells. fourth, high levels of locally administered il- can reverse tumor-supporting immunosuppression. the immunosuppressive tumor microenvironment is a major hindrance to the clinical efficacy of all cancer immunotherapies. in fact, the cancer vaccine literature teaches that the majority of patients in clinical studies are able to mount significant antigen-specific t cell responses, yet few patients experience clinical benefit ( ) ( ) ( ) . similarly, the extraordinary activity of car t cells against hematologic malignancies becomes less than ordinary against solid tumors. many solid tumors lack the chemokines and inflammation necessary to recruit cytotoxic t cells ( ) . moreover, dense tumor stroma prevents t cell penetration while immunosuppressive factors released by tumor cells, suppressor t cells and tams can cause t cell anergy. regarding the latter, many tumors bathe in a cocktail of immunosuppressive factors such as tgfβ, il- , ido, and larginase. fortunately, high intratumoral concentrations of il- can cause apoptosis and elimination of cd + cd + foxp + suppressor t cells in tumors ( ) . in addition, the tumor suppressive phenotype of tams can be converted to a cytotoxic, antitumor phenotype in the presence of localized il- ( ) . finally, il- has been shown to modulate and alter the suppressive activities of tumor-associated mdscs ( ) . lastly, and perhaps most importantly, activation of t cells in the presence of il- can not only enhance ctl function, but also reduce negative regulatory mechanisms such as pd- /pd-l signaling and autocrine ifnγ-induced apoptosis. this "protective" effect has been observed mostly in the cellular immunotherapy literature. standard protocols for ex vivo expansion of tumor infiltrating lymphocytes for adoptive cell therapy (act) traditionally used high dose il- to facilitate t cell proliferation ( ) . the inclusion of il- in conditioning/expansion media has been explored recently because it had been shown previously to result in optimal t cell priming ( ) . indeed, adoptive transfer of tumor-specific cd + t cells primed ex vivo in the presence of il- resulted in enhanced antitumor responses ( , ) , increased persistence of infused t cells ( , ) , as well as increased expression of il- rα (cd ], icos, ox , granzyme b, and ifnγ ( ) . importantly, cytotoxic t lymphocytes (ctls) stimulated with il- were more effective in controlling tumors following adoptive transfer than ctls stimulated with ifnα ( ) . il- -stimulated t cells expressed lower levels of pd- and higher levels of ifnγ and il- compared to ifnα-stimulated t cells ( ) . il- conditioning caused downregulation of ifnγr with a concomitant decrease in susceptibility to ifnγ-induced apoptosis of tumor-infiltrating cd + t cells ( , ) . il- delivery strategies can be divided into three general approaches. the first involves fusion of a targeting moiety to il- in order to facilitate accumulation in a tumor following a systemic injection. the most common of class of fusion molecules are immunocytokines, which involve linking a tumor binding antibody fragment to a cytokine. the second approach involves delivery of genetic material encoding il- directly to the tumor or a tissue of interest. this category can be further divided based on the type or method of gene delivery. plasmids, mrna, viruses, and transduced cells are all capable of expressing and delivering il- after a local injection. the third major approach involves controlled release of recombinant il- protein from a sustained delivery system. here, the cytokine delivery system is injected or implanted directly in a tumor or tissue of interest. the remainder of this section will present and discuss the most relevant preclinical and clinical data pertaining to each il- delivery strategy. a summary of current clinical trials utilizing localized il- delivery is presented in table . as mentioned above, immunocytokines are part or whole cytokines that have been engineered to contain antibody fragments or other targeting moieties. these "targeted" cytokines are administered systemically but are expected to accumulate within tumors at higher levels compared to non-targeted cytokines. various tumor-related features have been targeted by immunocytokines including: ( ) tumor antigens which are overexpressed or uniquely expressed by tumor cells; ( ) cryptic extracellular matrix epitopes found only in tumors; and ( ) neovasculature markers as tumors require angiogenesis for growth. developments in immunocytokines are discussed below. the pan-carcinoma antigen, epithelial cell adhesion molecule (epcam), is highly expressed by cancer cells of epithelial origin such as colon, prostate, breast, and lung carcinomas. huks-il- is an immunocytokine of il- fused to the fc fragment of a humanized antibody that recognizes epcam. in a murine prostate cancer model, huks-il- was found to suppress experimental metastases in scid mice reconstituted with activated human t and nk cells lymphocytes ( ) . another immunocytokine, hu . -il- , which is irrelevant in this system due to its targeting of ganglioside gd , was found to be somewhat less effective than huks-il- , although differences in antitumor activity were not statistically significant ( ) . dual immunocytokines in which both il- and il- were fused to the huks / antibody fragment were found to eliminate epcam-expressing llc flank tumors following intratumoral (i.t.) injection ( ) . interestingly, a mixture of huks-il- and huks-il- was less effective than the dual immunocytokine if delivered i.t., but exhibited similar in antitumor activity if administered i.v. ( ) . the epidermal growth factor receptor her /neu is overexpressed in roughly a third of breast and ovarian cancers, with high expression correlating with poor prognosis. trastuzumab, a monoclonal antibody targeting her , has been approved for the treatment of certain breast cancers for more than years. a mouse single chain il- fused to an anti-her /neu igg (mscil- .her .igg ) retarded the growth of ct -her /neu tumors in immunocompetent mice ( ) . a direct comparison demonstrated that mscil- .her .igg and free il- induced similar activities against ct -her /neu tumors ( ) . follow up studies revealed that mscil- .her .igg also displayed robust antitumor activity against mc /her /neu and d f /e tumors ( , ) . a more recent study revealed that disruption of the heparin binding domain in the mscil- .her .igg immunocytokine, reduced il- bioactivity ( ) . this result was consistent with recent studies showing that heparin and heparan sulfate bind to and enhance the activity of il- ( ) ( ) ( ) ( ) ( ) . while eliminating heparin binding reduces il- activity, we speculate that this reduction could be counterbalanced by an enhancement in tumor targeting as the il- immunocytokine may no longer bind to ubiquitous sulfated glycosaminoglycans in non-targeted tissues. mesothelin is a differentiation antigen that is highly expressed in a number of human cancers including mesotheliomas, pancreatic and lung adenocarcinomas, and ovarian and breast carcinomas. to direct il- to mesothelin expressing cancer cells, a scfv, called ss , that specifically binds to mesothelin was fused to the p subunit of a single-chain il- ( ) . human peritoneal mesotheliomas established in nude mice were significantly inhibited by i.p. injections of il -ss ( ) . that these studies were successful in nude mice seems to imply a prominent role for nk cells in this model. ca - is a cancer antigen that is expressed in about half of human ovarian cancers ( ) . a scfv of the b monoclonal antibody that binds to ca - was fused to mil- ( ) . systemically (i.v.) administered b scfv-mil- was found to inhibit the growth of subcutaneously implanted id ovarian tumors more effectively than non-targeted mil- ( ) . cd is expressed by activated lymphocytes and thus serves as a useful target for several types of lymphoma. a cd -targeted il- fusion protein was developed for cd + hodgkin's lymphoma therapy ( ) . the immunocytokine was found to induce activation of t and nk cells and secretion of proinflammatory cytokines resulting in enhanced cytotoxicity of cd + mc cells. interestingly, a cd -targeted il -il fusion protein outperformed targeted il- or il- alone in all in vitro measures. the dual cytokine construct induced regression of cd + but not cd -mc tumors in vivo ( ) . whether the dual cytokine fusion protein was better than the single cytokine constructs is not known as the latter were not evaluated in vivo. many solid tumors overexpress extracellular matrix (ecm) which serves as transport barrier to the penetration of therapeutics and immune cells. in ecm-rich solid tumors, it may frontiers in immunology | www.frontiersin.org targeting ecm proteins instead of cancer cells, therefore, is a promising strategy to encourage immunocytokine accumulation in tumors. there are two immunocytokines, hubc -il and il- -l , that have been developed to target the splice variant extra domain b (ed-b) of fibronectin, which is highly expressed in tumor tissues but undetectable in normal adult tissues with the exception of endometrium ( ) . bc- is a monoclonal antibody that recognizes the ed-b isoform, thus a hubc -il immunocytokine has been constructed from two molecules of il- fused to each of the igg heavy chains of humanized bc- . systemic administration of hubc -il was found to eliminate experimental pc metastases and suppress the growth of multiple human tumor lines in immunocompromised mice more effectively than il- alone ( ) . a phase i trial evaluated the safety of weekly infusions of as (hubc -il ) in renal carcinoma and malignant melanoma patients ( ) . the maximum tolerated dose (mtd) was found to be µg/kg. in contrast, the mtd of twice weekly i.v. il- was previously found to be . µg/kg ( ) . dose limiting toxicities, including fever, fatigue, and elevated transaminase levels, were consistent with known toxicities of il- ( ) . the second ed-b targeted immunocytokine, il- -l , is comprised of the ed-b-binding l scfv and il- ( ) . l -targeted cytokines have been shown to selectively accumulate in tumors following i.v. administration ( , ) . intravenous administration of il- -l every h was found to control the growth of primary c colon adenocarcinomas, f teratocarcinomas as well as experimental pulmonary c metastasis ( ) . biodistribution studies confirmed that a greater percentage of the injected dose of il- -l was found in tumors as compared to an il- -fusion negative control. il- -l also demonstrated synergistic antitumor activity when combined with l -tnfα ( ) . most recently, il- was fused to the collagen-binding proteoglycan lumican and mouse serum albumin (msa), to create il -msa-lumican ( ) . lumican binds to collagen types i and iv, components of the thick fibrotic capsule surrounding tumors and perivascular basement membrane, respectively. in mice bearing established subcutaneous flank b f tumors, treatment with il -msa-lumican resulted in prolonged tumor control and longer survival. significant weight loss was observed following il -msa compared to il -msa-lumican treatment, indicating that collagen targeting may reduce systemic toxicities of il- . finally, the combined treatment of lumican-msa-il and il -msa-lumican potentiated anti-pd- increasing survival in multiple models and completely protecting cured mice from live tumor rechallenge ( ) . another collagen-binding immunocytokine comprised of the a cbd of von willebrand factor fused to both subunits of il- was also recently developed ( ) . systemic (i.v.) administration of this cbd-il was found to accumulate in emt mammary carcinomas at significantly higher levels and induce higher rates of complete tumor regression against -week old b f and emt tumors compared to il- ( ) . inclusion of the cbd resulted in a - -fold decrease in plasma half-life despite the larger size of cbd-il . the distributions of cbd-il and il- in normal tissues following i.v. injection were surprisingly similar although typical sites of collagen targeted drugs, e.g., bone and skin, were not examined. most importantly, although elevated liver enzymes were observed, levels following cbd-il at an impressive dose of µg/mouse were similar to µg/mouse of il- ( ) . in general, the key advantage of systemically administered immunocytokines is their ability to preferentially accumulate within a site of disease, e.g., a tumor. however, immunocytokines retain complete cytokine activity in circulation which allows them to interact with circulating lymphocytes and induce similar cytokine-induced toxicities as parental cytokines ( ) . one clever strategy has been developed to reduce adverse effects associated with systemic il- by separating the targeted delivery of the p and p subunits. this split-immunocytokine approach involves first delivering a bivalent p -based antibody fusion protein (f -p s-f ). f binds to the alternatively spliced extra domain a (ed-a) domain which is present on the subendothelial extracellular matrix of tumor neovasculature ( ) . after allowing time for binding and clearance of unbound f -p s-f , a subsequent administration of p , which has no activity by itself, interacts with p to recover il- activity. quantitative biodistribution investigation in f teratocarcinomas bearing mice showed that both targeted subunits accumulated in the tumor ( ) . furthermore, the recombined subunits displayed robust il- activity in terms of ifnγ production and stat phosphorylation ( ) . tumor necrosis is a common feature of most advanced solid tumors. approaches to target dna strands that become uniquely exposed in necrotic foci are under investigation. the monoclonal antibody, chtnt- , recognizes single-stranded dna ( ) . a fusion between the variable heavy chain of chtnt- and hil- forms the necrosis-targeting immunocytokine, chtnt- /hil- ( ) . chtnt- /hil- was retained in a subcutaneous tumor after i.v. injection and resulted in a significant inhibition of du prostate tumors in human pbl-engrafted scid mice ( ) . another necrosis-targeting il- , capitalizes on the specificity of the nhs antibody for ssdna and dsdna ( , ) . nhs-il is comprised of the full length nhs antibody fused to single-chain il- molecules. systemic administration of a murine analog, nhs-muil , has been shown to delay the growth of mc -cea+ colorectal carcinomas in cea.tg mice ( ) . furthermore, tumorbearing mice treated with nhs-muil developed cd + t cell responses against an endogenous tumor antigen, p e. in vivo imaging studies have shown that nhs-muil accumulated in flank tumors following a s.c. injection ( ) . subcutaneous administrations of nhs-muil were also recently shown to provide significant reductions in orthotopic mb luc bladder tumors ( ) . tumor control was associated with a noticeable reduction in markers of immunosuppression, e.g., mdscs, macrophages and tumor-associated tgf-β ( ) . the combination of nhs-muil with avelumab, an anti-pd-l antibody, resulted in improved control of both mc and mb flank tumors with higher frequencies of cd + t cells and enhanced t cell activation compared to either agent alone ( ) . against orthotopic emt- mammary tumors (∼ mm ) the combination of nhs-muil and avelumab induced complete regression in of mice ( ) . the same treatment was shown to delay, but not completely regress, the growth of - mm established emt- tumors. importantly, nhs-muil plus avelumab was shown to induce protective immunity as all cured mice resist an emt- tumor challenge but not a t challenge. furthermore, treatments enhanced cytotoxic nk and cd + tcell proliferation, t-bet expression, plasma cytokine levels, and innate and adaptive immune genes ( ) . combining nhs-il with fcil- or il- mab resulted in improved antitumor immunity, increased survival, and longterm remission in sarcoma-bearing mice ( ) . fcil- is a fusion of interleukin- and an fc fragment while il- mab is a fusion of il- and a monoclonal antibody against il- , mab . separately, the combination of nhs-il with local tumor irradiation was shown to increase treatment efficacy ( , ) . in preparation for first-in-human clinical trials, a comparative oncology study in client-owned dogs with melanoma revealed that s.c. injections of nhs-il- induced transient increases in serum ifnγ and il- . two of dogs in a dose escalation cohort experienced a partial response while of dogs had increased levels of tumor-infiltrating cd + t cells ( ) . nhs-il is currently in phase i clinical studies either as a monotherapy (nct ) or in combination with avelumab (nct ). in the former study, nhs-il induced transient lymphopenia and elevated liver transaminases, but was otherwise well-tolerated with a mtd of . µg/kg ( ) . no objective tumor responses were observed, however, of patients experienced stable disease. immune assays revealed that nhs-il treatment increased nk cell frequencies and broadened the tcr diversity of tumor-infiltrating t cells ( ) . systemically administered immunocytokines can significantly reduce but are unlikely to completely avoid il- -related toxicities. as mentioned above, in circulation, immunocytokines will interact with immune cells and induce signaling outside of the tumor. in addition, all targeting moieties are susceptible to non-specific binding and distribution in normal, untargeted tissues. for example, radiolabeled nhs has been found in all major tissues in mice for - days after i.v. administration ( ) . furthermore, substantial amounts of il- -l were found in the livers of treated animals, likely leading to hepatotoxicity ( ) . on-target/off-tissue specific binding may create additional concerns. in the case of nhs-targeting moieties, cancer patients have high levels of circulating cell-free dna that is shed from tumors ( ). it is not clear how circulating dna impacts nhs targeting. in the case of neovasculature targeting moieties, angiogenesis is a normal process of wound healing and promotes collateral circulation for atherosclerotic blood vessels. disrupting non-cancerous angiogenesis could induce hypertension and cardiac ischemia which are among the adverse events associated with anti-angiogenic agents, such as bevacizumab. moreover, the potential immunogenicity of a nonendogenous immunocytokine is another factor that may limit therapeutic potential. as non-native proteins, immunocytokines could contain immunogenic epitopes against which an immune response, likely an antibody response, could be raised. antiimmunocytokine antibodies could induce pharmacological abrogation, therapeutic alteration, or hypersensitivity reactions ( ) . because of the potential for anti-immunocytokine antibodies, novel immunocytokines should be engineered to minimize the presence of immunogenic epitopes. overall, although immunocytokines remain capable of inducing il- -related adverse events, the use of targeting moieties may improve biodistribution enough to expand the therapeutic window of il- -based immunotherapies. intratumoral (i.t.) injections of dna and rna encoding il- have the potential to localize and sustain the production of il- in the tumor microenvironment. nucleic acids are much easier to produce, purify and manipulate than recombinant cytokines. however, mammalian host cells are not easy to transfect and typically require chemical, physical, or electrical assistance to achieve reasonable transfection rates. this section will highlight progress in nucleic acid-based approaches both preclinically and clinically. around the same time that recombinant il- was failing in clinical trials, a limited number of preclinical and clinical studies explored i.t. injection of plasmid dna encoding il- (pil- ) as a potentially less toxic approach. preclinically, i.t. pil- inhibited but did not eliminate b melanomas ( ) . in this study, il- was not detected in the serum following i.t. injection. in another study involving gray horses with metastatic melanoma, i.t. pil- resulted in detectable levels of pil- in the serum for up to h ( ). however, it is not clear if systemic dissemination of pil- resulted in significant systemic increases in serum il- or ifnγ as these were not measured ( ) . in a phase i/ii trial of intralesional injections with pil- , of and of patients with stage iv malignant melanoma experienced clinical and local responses, respectively ( ) . in a phase i/ib study, i.t. pil- was found to reduce the size of treated lesions by at least % in of malignant melanomas and renal cell carcinomas ( ) . pil- injections were welltolerated as no patient in either study experienced a significant treatment-related adverse event. despite successful safety studies, the use of naked pil- for cancer immunotherapy has not progressed, mostly likely due to poor transfection efficiency. the application of pulsed, high electric fields to facilitate cellular uptake and expression of genes has been a part of the molecular biologist's toolbox for decades. intratumoral injection of pil- immediately followed by electroporation, referred to here as pil- +ep, has been explored in several murine tumor models ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . as expected, the benefit of adding electroporation was immediately apparent as one early study showed pil- alone had no effect on b f tumor growth while pil- +ep significantly inhibited tumors and extended survival ( ) . importantly, the increase in antitumor efficacy was not associated with an increase in systemic il- levels ( ) . among the more notable responses in other early preclinical studies, nearly half of mice bearing established b f melanomas experienced complete tumor regression following weekly treatments with pil- +ep ( ) . in a follow up study, pil- +ep induced tumor regression in up to % of mice, whereas i.t. injections of pil- alone delayed but could not eliminate b f primary tumors ( ) . cured mice displayed protective immunity as of rejected a b f challenge ( ) . in the sccvii squamous cell carcinoma (scc) model, complete regressions were observed in % of mice following pil- +ep ( ). furthermore, of cured mice resisted a tumor challenge containing five times the original dose of tumor cells ( , ) . against bjmc murine mammary adenocarcinomas, ct murine colon adenocarcinomas and renca renal cell carcinomas, pil- +ep significantly suppressed, but did not eliminate implanted tumors ( , ) . against murine sa- fibrosarcomas, pil- +ep suppressed tumor growth and induced complete regression in % of treated mice with of becoming resistant to tumor rechallenge ( ) . in this study, il- and ifnγ were detected in the serum of treated mice, however, no side effects were observed ( ) . abscopal responses have been documented in several studies. against bilateral sa- tumors, pil- +ep treatment consistently eliminated primary, treated tumors, while slowing the growth of secondary, untreated tumors ( ) . similarly, pil- +ep treatment of mh hepatocellular carcinomas, inhibited both treated and untreated tumors while preventing spontaneous pulmonary metastases ( ) . a recent study using bilateral b tumors demonstrated that an optimized pil- +ep protocol ( ) was capable of regressing treated lesions while inhibiting the growth of contralateral untreated tumors ( ) . injecting pil- directly into tumors is important as multiple studies have confirmed that i.t. pil- +ep treatments were significantly more effective than either peritumoral or intramuscular (i.m.) routes ( , , , ) . the i.m. route also resulted in significantly more il- and ifn-γ in the serum ( , ) . in terms of mechanism, multiple studies agreed that pil- +ep treatment was associated with increased t cell infiltration, increased ifnγ expression and decreased angiogenesis ( , , , , , ) . these findings are consistent with known antitumor mechanisms of il- . more recent mechanistic studies have focused on changes in immune cell phenotype and function. for example, b f tumor regression following pil- +ep was mediated via the perforin/granzyme lytic pathway while antigen-specific cd + t cell responses were directed against tyrosinase-related protein epitope trp [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ( ) . in another study, pil- +ep-induced elimination of b f tumors was associated with increased tumor infiltration and polarization of macrophages toward an m phenotype ( ) . another group found that pil- +epinduced antitumor responses against b f tumors were correlated with a reduction in pd- expression on cd + and cd + t cells ( ) . yet, another group found that the treatment of bilateral b f tumors induced a unique population of cd + effector t cells with low pd- expression in both untreated tumors and systemically ( ) . this finding suggested that a subset of cd + effectors generated by pil- +ep may be protected or "armored" against checkpoint-mediated exhaustion ( ) . a unique feature of pil- +ep immunotherapy is the potential to manipulate electric field parameters to enhance transfection, and therefore, efficacy. an exploration of electric field parameters demonstrated that pil- +ep-induced cures ranged from to % in b f tumor-bearing mice depending on pulsing conditions ( ) . about half of these cured mice resisted a b f rechallenge ( ) . by further enhancing electric field intensity and/or pulse length, it is possible to directly kill tumor cells and release tumor antigens via irreversible electroporation. in one recent study, partial-irreversible electropermeabilization (pire) administered after peritumoral electrotransfection with pil- caused complete regression of about - % of treated b f tumors ( ) . two of cured mice completely resisted tumor rechallenge while the remaining two experienced delayed tumor growth from the rechallenge. this pil- plus pire approach was found to delay, but not eliminate distant, untreated tumors in about half of the mice ( ) . there have been several attempts to enhance antitumor activity through the incorporation of additional cytokineencoding plasmids. of note, two studies have demonstrated that ep using a combination of il- and il- plasmids outperformed il- alone in terms of antitumor activity ( , ) . the rationale to combine these two cytokines is wellsupported given that il- and il- synergize to enhance th responses and ifn-γ production. however, adverse events are also enhanced as systemic co-administration of recombinant il- and il- proteins leads to lethal toxicity in mice ( ) . in one study, addition of pil- to pil- increased serum il- and ifn-γ levels for at least days after ep although no inflammation was observed in liver, lung, and intestine samples ( ) . in a second study, pil- did not increase il- -induced serum ifn-γ, but intratumoral ifn-γ was significantly higher ( ) . several notable canine clinical studies have explored pil- +ep in dogs with naturally occurring tumors. in one such study, pil- +ep resulted in a - % reduction in mast cell tumor volume ( ) . treated nodules displayed increases in leukocytic inflammation and decreases in the number of malignant mast cells ( ) . in beagles with canine transmissible venereal tumors (ctvts), pil- +ep induced complete regression of all treated lesions ( ) . contralateral untreated tumors were also significantly inhibited. serum il- levels peaked days after treatment; however, relevant blood chemistries, i.e., liver and kidney enzymes, as well as cell counts were not different from those of control dogs ( ) . another study investigated pil- +ep for the treatment of canine oral malignant melanoma (omm). there were no differences in the percentages of helper cd + and cd + cells before and after treatment, while t reg frequencies declined from . to . %. one month post treatment, the objective response rate was % ( / ) but by the end of the observation period, all but one of the dogs developed progressive disease ( ) . a more recent study in dogs with a range of spontaneous cancers, demonstrated that pil- +ep induced immunostimulatory and anti-angiogenic effects ( ) . administration of three pil- +ep treatments every other day caused significant systemic toxicities, including anemia and thrombocytopenia. after switching to a weekly schedule, treatments were well-tolerated, however, all treated tumors continued to progress ( ) . several human clinical trials, mainly against advanced melanoma, have investigated the safety and efficacy of pil- +ep. in a phase i study, patients with stage iii or iv melanoma received i.t. pil- +ep (six µs, , v/cm pulses) on days , , and during a single -day cycle. fifty three percent of patients experienced a systemic response, defined as either stable disease or regression of untreated lesions, following pil- +ep ( ) . most notably, of patients showed complete regression of all metastases. no grade or higher adverse events were observed and neither il- nor ifnγ was detectable in serum samples. in a follow up phase ii study, patients with in-transit or m a melanoma were treated with up to four -week cycles of pil- +ep as described above ( ) . intermediate results revealed an objective response rate of % with % complete responses. the treatment was found to increase nk cell levels both intratumorally and systemically ( ) . no grade / drug-related adverse events were noted. a subsequent analysis of clinical samples revealed that responses were associated with increased intratumoral infiltration of cd + t cells ( ) . in addition, t cell receptor beta chain (tcrβ) sequencing revealed a focusing of the tcr repertoire following treatment. however, there were no differences in t cell clonality between responders and non-responders to pil- +ep ( ) . a second study from the same trial, nct , found a complete response rate of up to . % and a best overall response rate of . % in patients with stage iii/iv melanoma ( ) . nearly half of these patients experienced regression of at least one anenestic lesion. an analysis of transcripts in melanoma biopsies found increases in t cell trafficking, immune activation, and antigen presentation ( ) . genes associated with adaptive resistance, e.g., pd-l , tgfβ, and trail, were also increased ( ) . recent results from a safety study in patients with locoregional merkel cell carcinoma (mcc) and patients with metastatic mcc received or up to cycles of pil- +ep, respectively ( ) . the overall response rate in the metastatic mcc cohort was % ( / ). of patients with measurable untreated lesions, experienced abscopal regressions. in addition, patients experienced clinical responses lasting and + months, respectively. two of the locoregional mcc patients, all of whom were treated with definitive surgery after pil- +ep, were recurrence-free at + and + months, respectively ( ) . serum il- levels were not measured, but treatments were well-tolerated, and no serious adverse events were observed. lipoplexes, polyplexes, and lipopolyplexes are complexes of lipids, polymers, and lipids plus polymers, respectively, with dna. these complexes are under investigation to enhance the delivery and transfection efficiency of plasmids encoding genes of interest, including pil- . numerous studies have explored a range of different materials to create novel pil- complexes ( ) ( ) ( ) ( ) ( ) . studies demonstrating antitumor efficacy following local or targeted delivery of pil- complexes are highlighted below. polyethyleneimine (pei), a highly cationic polymer that readily complexes with negatively charged dna, has been widely used to enhance gene delivery. pei protects dna from degradation in vivo, encourages interaction with negatively charged cell membranes, and enhances release from lysosomes by acting as proton sponge ( ) . pei:il- complexes were shown to transfect lung tissue following delivery via nebulization ( ) . this approach led to production of il- in the lungs which was not detectable in the plasma of treated mice ( , ) . weekly or twice weekly administration of aerosolized pei:il- was found to suppress or eliminate experimental pulmonary metastases of saos- human osteosarcomas in athymic nude mice ( ) . recent attempts to enhance uptake and il- production have focused on modification of pei with tetraiodothyroacetic acid (tetrac) which binds the α v β integrin receptor that is overexpressed in some tumors ( ) or diethylene triamine penta-acetic acid (dpta) which can reduce the surface charge of pei:il- complexes ( , ) . as of this writing, no in vivo data using either modification have been published. polyvinylpyrridilone (pvp) is another cationic polymer that readily complexes with dna. twice weekly i.t. injections of pil- /pvp complexes were found to eliminate and % of - mm renca and ct tumors, respectively ( ) . most mice that were cured of a primary tumor rejected a subsequent rechallenge ( ) . in a follow-up study, pil- /pvp was found to be more effective than pifnα/pvp in controlling preclinical tumors, while the combination of pil- /pvp and pifnα/pvp synergized to eliminate % of renca and % of ct tumors ( ) . in both studies, cd + t cells but not cd + t cells were identified as primary effectors. complexation with poly-a-( -aminobutyl)-l-glycolic acid (paga), a biodegradable polyester, enhanced transfection efficiency of pil- and expression of il- in vitro and in vivo ( , ) . however, t cell infiltration of injected ct colon adenocarcinomas and antitumor activities following repeated injections of paga/pil- and naked pil- were similar ( ) . encapsulation of pil- in nanoparticles comprised of poly-(d,l-lactic-co-glycolic acid) (plga) and , -dioleoyl- -(trimethylammonium) propane (dotap) demonstrated complete regression of up to % of established heterotopic bnl hepatocarcinomas following a single i.t. injection ( ) . importantly, treatment with encapsulated pil- was more effective than treatment with nanoparticles with pil- adsorbed to the surface ( ) . long term expression of inflammatory cytokines could be a concern as both il- and ifnγ were detected in the serum for up to days after treatment ( ) . a similar, so-called dmp nanoparticle, comprised of dotap and methoxy-poly(ethylene glycol)-poly(lactide) (mpeg-pla), has been developed to facilitate gene delivery ( ) . complexation with pil- resulted in inhibition of ct tumors with no signs of systemic toxicity, as determined by appearance, body weight, fecal output, and urinary excretion ( ) . a slightly different dmp nanoparticle that uses polycaprolactone (pcl) instead of pla, inhibited the growth of intraperitoneal c colon carcinomas and ll/ lewis lung carcinomas ( ) . in addition to delaying tumor growth, dmp/il- particles resulted in high il- gene expression and t cell infiltration although the treatment regimen consisted of daily or every other day treatments ( ) . plasmids complexed with mannosylated chitosan (mc) are under development as a method to target mannose receptors on i.t. dcs. chitosan is a linear co-polymer of β-linked dglucosamine and n-acetyl-d-glucosamine. it is primarily derived from the structural polysaccharide, chitin, found in shells of crustaceans. i.t. injection of mc/pil- complexes elicited modest growth delay of ct tumors, which was associated with increased tumor cell apoptosis and decreased angiogenesis ( ) . lipopolymers, which incorporate a lipid tail on a polymer backbone, are also under exploration for non-viral gene delivery. water soluble lipopolymers (wslp) comprised of cholesterol conjugated to pei have been developed to increase cell membrane permeability and reduce pei-mediated toxicity ( ) . wslp/p cmvmil- dna complexes inhibited the growth of ct tumors and improved survival following a single i.t. injection ( ) . however, the antitumor efficacies of wslp/pil- complexes and naked pil- appeared similar. in a later study, wslp/p cmvmil- complexes injected every days outperformed single injections and multiple injections with naked pil- or pei/pil- complexes ( ) . the vast majority of injected wslp/p cmvmil- was found in the tumor for up to h with small but increasing accumulation in the liver and blood ( ) . subsequent studies demonstrated that i.t. wslp/p cmvmil- significantly suppressed the growth of primary and metastatic t , tsa, and emt- mammary carcinomas ( , ) . polytraxane (prx) is a composite molecule made of polyethylene glycol (peg) and cationic cyclodextrin (cd) that self assembles with dna into a spherical particle ( ) . a -arm configuration, rather than a linear configuration, had a higher accumulation in mc -luc tumors and lower accumulation in the lungs following systemic delivery. the -arm prx/pil- also produced higher levels of il- and significantly slowed mc -luc tumor growth after five i.v. injections of the complex starting days after tumor implantation. ( ) systemic injections of prx/pil- complexes induced moderate lymphopenia, but no elevation of liver enzymes ( ) . pil- complexed with a polyethyleneglycolpolyethylenimine-cholesterol (ppc) lipopolymer was shown to inhibit t and sccvii tumors ( ) and increase survival in mice with intracranial gl gliomas ( ) following localized injections. of note, although the i.p. route is frequently used to deliver drugs systemically, i.p. injections of pmil- /ppc for treatment of id ovarian carcinomas resulted in high levels of il- and ifn-γ in ascites but low levels in serum ( ) . in this study, i.p. pmil- /ppc was well-tolerated with no significant changes in serum chemistries ( ) . in a phase i study, phil- /ppc was administered i.p. to women with chemo-resistant recurrent ovarian cancer ( ) . escalating doses of phil- /ppc were well-tolerated with no dose-limiting toxicities. five of the treated patients reported a serious adverse event, however, only one was possibly related to the phil- /ppc ( ) . similar to preclinical studies, no detectable increase in serum il- was found following phil- /ppc treatment ( ) . a subsequent phase ii study in patients with platinum-resistant recurrent ovarian cancer demonstrated similar safety following weekly i.p. phil- /ppc, however, with no objective clinical responses observed ( ) . a different phase i trial evaluated the safety of phil- /ppc in ovarian cancer patients when combined with carboplatin and docetaxel chemotherapy ( ) . while there were no dose limiting toxicities, grade adverse events included manageable abdominal pain and cytokine release syndrome. two of patients experienced complete response while of experienced a partial response ( ) . a more recent phase i trial combined weekly i.p. pil- /ppc with i.v. pegylated liposomal doxorubicin in patients with persistent or recurrent platinum-resistant ovarian or peritoneal cancers ( ) . although increased levels of il- , ifn-γ, and tnf-α were found in peritoneal fluid, no dose limiting toxicities were observed and a maximum tolerated dose was not reached. the best partial response ( . %) and stable disease ( . %) rates were found at the highest dose ( mg/m ) of pil- /ppc ( ) . pil- complexed with a cationic lipid (+/-)-n-( -hydroxyethyl)-n,ndimethyl- , -bis(tetradecyloxy)- propanaminium bromide/dioleoylphosphatidylethanolamine (dmrie/dope), and injected i.t. was found to inhibit and eliminate ct and renca tumors while protecting up to and %, respectively, of mice from rechallenge ( ) . interestingly, a direct comparison between naked pil- and dmrie/dope/pil- revealed no difference in antitumor activity ( ) . polyphosphazene particles were modified with hydrophobic n,n-diisopropylethylenediamine (dpa) and hydrophilic monomethoxy poly-(ethylene glycol) (mpeg) to create weakly cationic particles to complex with pil- ( ) . mpeg/pil- polymersomes delayed ct tumor growth when administered i.v. body weights were unaffected by mpeg/pmil- treatments. tumor il- levels steadily increased, reaching about pg/g tumor on day , while serum il- concentration remained on average about pg/ml after day and continued to day . the concentration of ifn-γ in the tumor reached a maximum of pg/g tumor on day , while serum levels of ifn-γ slightly increased from day to day , reaching a concentration of only pg/ml. while mpeg/pmil- polymersomes did not affect cd + cd + cells in the tumor, there was a -fold increase of cd + cd + cells and significant increases of cd − nk . + and cd + nk . + cells in the tumor. in another study, all-trans-retinoic acid (atra) was incorporated in cationic liposomes and complexed with pil- ( ) . atra was previously found to increase the expression of tnf receptor and mediate apoptosis of lung cancer cells via tnfα ( , ) . i.v. injections of atra-cationic liposome/pil- reduced lung nodules and extended survival compared to cationic liposome/pil- treatment in an experimental pulmonary metastasis model using c cells expressing luciferase ( ) . concentration of il- in the lungs reached a maximum of pg/mg protein at h post injection, while levels of il- in the spleen and liver were significantly lower and nearly eliminated by h. interestingly, the incorporation of atra reduced liver enzymes levels and thus hepatic toxicity suggesting a possible anti-inflammatory role ( ) . recently, mrna delivery platforms have received tremendous attention, most notably as front running vaccines against sars-cov- . mrna, like dna, can encode an unlimited number of proteins and polypeptides. although mrna-based platforms are less stable than dna-based platforms, mrna can be protected from digestion through encapsulation in polymeric or lipidbased micro-or nanoparticles. a key advantage of mrna is their ability to directly translate encoded proteins in the cytoplasm. in contrast, dna must first translocate to the nucleus to be transcribed to mrna before translation in the cytoplasm. regarding the use of mrna to deliver il- locally, a recent study demonstrated that weekly i.v. delivery of lipid nanoparticles (lnp) loaded with mrna encoding il- reduced tumor burden in a myc-driven transgenic mouse model of hepatocellular carcinoma (hcc) ( ) . the tumor inhibition and extended survival were attributed to increased infiltration of cd + cd + cd + immune cells and not suppression of myc ( ) . similarly, moderna/astrazeneca has developed, medi , an lnp formulation with il- mrna. medi is currently in phase i clinical trials for intratumoral injection of advanced solid tumors in combination with durvalumab ( table ) ( ) . in preclinical studies, a single intratumoral injection of mrna encoding murine il- (mil- ) increased ifnγ expression and genes associated with a th response in mc tumor-bearing mice ( ) . when combined with anti-pd-l , enhanced t cell infiltration and expanded tumor-specific t cell subsets were observed ( ) . in another phase i clinical trial, sanofi and biontech are testing sar (bnt ), an mrna platform encoding a cocktail of il- sc, il- sushi, ifnα, and gm-csf for intratumoral injection as a monotherapy and in combination with cemiplimab ( ) . to our knowledge, no preclinical data with sar have been disclosed. in general, nucleic acid-based il- delivery approaches are limited by variable transfection rates as well as unregulated production of gene products. in other words, it is easy to control the amount of pil- or il- mrna delivered but it is not easy to control the dose of recombinant il- that each subject receives. variable transfection rates may be responsible for conflicting reports on whether pil- +ep does ( - , , , ) or does not ( , , ) produce significant increases in serum il- and ifn-γ. fortunately, lethal il- -related toxicities have not been reported in the any of the preclinical or clinical studies detailed above. nevertheless, the potential for severe il- -related adverse events caused by continued and/or unregulated production of il- remains. strategies to enhance safety and efficacy, either by localizing gene-based il- though incorporation of an anchoring or binding domain or by incorporation of an inducible safety switch to turn off il- production, will help improve the therapeutic window of promising nucleic-acid-based il- delivery technologies. another potential limitation that must be considered, is any type of adverse reaction against components of the delivery vehicle or against the nucleic acid vector itself. regarding the former, foreign delivery components have the potential to induce immune responses which could influence il- delivery. most notably, it has been reported that about in humans have circulating anti-peg antibodies ( ) . the high prevalence of anti-peg antibodies could limit the efficacy of any peg-based delivery vehicle. regarding the immune responses against il- vectors, nucleic acids, particularly dna that is found in the cytoplasm have the potential to stimulate the cgas-sting pathway, which may induce its own inflammatory response. thus, studies utilizing nucleic acid-based delivery must take care to decouple the effects of il- from sting activation. lastly, although an exceedingly rare event, dna vectors could become integrated within a cell's genome. depending on the site, such integration could have deleterious or even transforming effects. given that only transient il- expression is desirable, strategies capable for preventing integration, like the use of circular instead of linearized plasmids, should be preferred. adenoviruses, herpes simplex viruses, semliki forest viruses, poxviruses, and other viral vectors have been engineered to express biologically active il- . these engineered viruses injected directly into a tumor are able to infect cancer cells and induce expression of il- within the tumor microenvironment. furthermore, many viruses have the unique ability to selectively lyse cancers cells after infection. such oncolytic viruses take advantage of defective cell cycle and interferon signaling pathways that are hallmarks of cancer cells but not normal cells ( ) . oncolytic viruses can kill compromised cancer cells in a variety of ways from direct virus-mediated cytotoxicity to indirect destruction of tumor-feeding blood vessels ( ) . the following sections discuss the progress and limitations of il- encoding viral vectors. adenoviruses are the most well-studied among the il- expressing vectors ( , ( ) ( ) ( ) ( ) ( ) ( ) ( ) . in preclinical studies, i.t. injections of adenoviruses encoding il- (ad-il- ) have mediated regressions of murine colorectal carcinomas ( ) ( ) ( ) , breast carcinomas ( , , ) , prostate carcinomas ( , ) gliomas ( , ) , bladder carcinomas ( ) , fibrosarcomas ( , ) , laryngeal squamous cell carcinoma ( ) , hepatomas ( ) and hepatocellular carcinomas ( , ) medullary thyroid carcinomas ( ) , thyroid follicular cancer ( ) , and ewing's sarcoma ( ) . among the more robust responses, ad-il- induced complete regression of subcutaneous neuro- a neuroblastomas in nearly half of mice receiving a single i.t. injection ( ) . mice becoming tumor-free also rejected a subsequent tumor rechallenge ( ) . similar results were found against ct colon adenocarcinomas with more than three-fourths of mice completely eliminating their tumors and all cured mice rejecting a tumor rechallenge ( ) . the antitumor immune response was mediated primarily by cd + t cells. impressively, of mice with bilateral tumors experienced complete regression of an untreated tumor ( ) . against - rat medullary thyroid carcinomas, i.t. injection of adtcpmil- caused complete regression of more than % of treated tumors ( ) . all cured rats rejected a tumor rechallenge while separate experiments showed that treatment of a single tumor resulted in inhibition of a distant untreated tumor ( ) . while liver infection following i.t. adtcpmil- injection was documented, no toxicity was observed ( ) . a follow up study found similar antitumor and abscopal responses against rat thyroid follicular cancer ( ) . in the pymt-derived transplanted mammary carcinoma model, adenoviral vectors encoding il- induced complete regression in % and partial regression in % of mice ( ) . ten of tumor-free mice completely rejected a tumor rechallenge. in contrast with similar studies using ad vectors expressing il- , no obvious toxic side effects due to admil- were noted ( ) . in another difficult model, a single i.t. injection of ad. / .crgd-mil p resulted in > % long term survival of mice with intracranial gl gliomas ( ) . in a murine model of ewing's sarcoma (tc ), twice weekly i.t. injections of ad.mil- significantly delayed treated tumors as well as untreated tumors ( ) . ad.mil- also induced complete regression in all treated mice bearing heterotopic mb bladder carcinomas ( ) . although body weights were not affected, serum ifn-γ levels due to i.t. ad.mil- were maximal from (∼ , pg/ml) to days (∼ , pg/ml) post injection ( ) . in a useful comparison against other cytokines, one study demonstrated that ad-ifn-γ had no greater antitumor activity than an empty ad vector, whereas admil- induced complete regressions of p mastocytomas in > % of treated mice ( ) . similarly, ad-gm-csf inhibited the growth of frtl-tc rat thyroid tumors, however, adil- was found to be much more effective ( ) . adil- also generated systemic immunity capable of inhibiting the growth of distant tumors ( ) . an adenoviral vector expressing a single chain il- (scil- ) was developed to enhance bioactivity over the native heterodimeric form ( ) . long-term tumor-free survival was observed in up to % of rats with established mh- a hepatocellular carcinomas following i.t. infections with ad.scil- ( ) . all tumor-free mice were protected from tumor rechallenge. however, despite i.t. injections, il- and ifn-γ were detected in the serum of treated mice ( ) . an oncolytic adenovirus expressing single-chain il- (ad-dhscil ) was more effective than non-replicating (nononcolytic) adenoviruses expressing il- at controlling liver metastases of pancreatic cancer in hamsters ( ) . however, il- levels in serum and non-tumor tissues were similar ( ) . in clinical studies, i.t. ad.il- was well-tolerated with no dose-limiting toxicities in a phase i trial in patients with advanced pancreatic, colorectal, or primary liver malignancies ( ) . serum ifnγ levels peaked day after ad.il- administration and was likely responsible for the grade adverse events observed. one patient had a partial response and % of patients experienced stable disease. four of assessable patients experienced increases in tumor infiltrating cd + and cd + cells. delayed-type hypersensitivity tests with inactivated adenovirus indicated that all patients developed an immune response against the adenovirus ( ) . despite robust antitumor immune responses, interest in ad-il- waned in the mid- s due to the aforementioned lethal toxicities associated with systemic administration of ril- and the inability of ad-il- , even if administered intratumorally, to prevent systemic dissemination of the cytokine. in recent efforts to mitigate systemic il- dissemination and associated toxicities, two strategies have been developed. the first involves engineering il- to prevent its dissemination. this has been accomplished either by anchoring il- to the surface of tumor cells via fusing a transmembrane domain or glycosylphosphatidylinositol (gpi)-anchored signal sequence to the cytokine ( , ) or by deleting the n-terminal signal peptide and thus preventing il- secretion ( ) . using the former technology, i.t. injection of an adenoviral vector encoding membrane-anchored il- (ad/scil- -b tm) eliminated the majority of primary ct tumors and suppressed the growth of untreated contralateral tumors ( out of mice), in which complete regression occurred in out of mice ( ). importantly, negligible il- was found in the circulation of mice treated with ad/scil- -b tm. the second technology deletes the signal peptide from the p subunit of il- to prohibit il- secretion ( ) . newly designed oncolytic adenoviral vectors with three genes deleted, i.e., triple deletion (td), and encoding either wild-type il- (ad-td-il- ) or a non-secreting il- (ad-td-nsil- ) were evaluated in syrian hamster models of pancreatic cancer. six i.t. injections of either ad-td-il- or ad-td-nsil- were found to eliminate subcutaneous hpd nr tumors in all mice ( ) . against peritoneally disseminated shpc tumors and orthotopic hap-t pancreatic tumors, ad-td-nsil- outperformed ad-td-il- in terms of overall survival ( ) . most importantly, ad-td-nsil- resulted in significantly lower serum il- levels and reduced systemic inflammatory cytokine expression ( ) . the second strategy to mitigate systemic il- dissemination involves conditional expression of il- . the rheoswitch therapeutic system r (rts) is an ecdysone receptor-based gene regulation platform in which a transcription factor becomes activated only in the presence of a synthetic small molecule ligand ( ) . an adenoviral vector encoding the rts switch and mil- (ad-rts-mil- ) and controlled by the oral activator, veledimex (vdx) was recently shown to extend survival in mice bearing intracranial gl gliomas ( ) . intratumoral ad-rts-mil- plus oral vdx was found to induce il- expression in a dose-dependent manner. local il- expression correlated with increases in tumor-infiltrating lymphocytes. il- and ifnγ were detected in the sera of treated animals, albeit at an order of magnitude lower than levels found in tumors ( ) . several clinical studies utilizing the regulatable ad-rts-il- platform are underway ( table ) . recent results from a phase i study in patients undergoing resection of recurrent highgrade glioma demonstrated that vdx induced il- expression in a dose-dependent manner ( ) . likewise, the frequency and severity of adverse events, including grade cytokine release syndrome, also increased with vdx dose. demonstrating the advantage of the inducible system, all serious adverse events were reversible with vdx discontinuation. interestingly, the use of corticosteroids negatively impacted survival ( ) . at the optimal dose, and in the absence of corticosteroids, the median overall survival of . months was encouraging ( ) . while the localized injection of ad-rts-hil- in the resected tumor bed induced il- expression in a recurrent tumor microenvironment, systemic dissemination of il- and its resultant toxicities could not be avoided. herpes simplex viruses (hsvs) are another family of viruses that have been widely explored for localized il- delivery. wild-type hsv are cytolytic and thus must be significantly attenuated or rendered replication-incompetent to avoid systemic infection. injection of replication-incompetent hsv-il- into established hepatomas prior to partial hepatectomy inhibited the engraftment of an intraportal tumor cell challenge in preclinical studies ( ) . importantly, no changes in serum il- were detected in treated buffalo rats ( ) . in order to capitalize on their lytic potential, hsvs have been engineered, through deletion or mutation of genes responsible for viral replication such that only cancer cells are lysed. not surprisingly, these oncolytic hsvs (ohsvs) have been shown to exhibit greater antitumor potential than their replicationincompetent, parental counterparts. for instance, treatment of established hepatomas with a non-cytokine encoding ohsv exhibited significant antitumor activity which was further increased with an il- insert ( ). the ohsv-il- treatment was also more effective than ohsv at protecting animals from a tumor rechallenge ( ) . similar antitumor responses to ohsv and increased efficacy with ohsv-il- were observed against flank ct and sccvii tumor models ( ) ( ) ( ) . a single i.t. injection was able to delay the growth of established sccvii tumors whereas multiple injections induced complete regressions in of treated mice ( ) . in contrast, multiple injections of ohsv-gm-csf cured only half of treated mice ( ) . in the ct model, ohsv-il- inhibited or eliminated injected mm tumors while inhibiting non-injected, contralateral tumors ( , ) . when low dose mil- was added to i.t. ohsv-il- , both treated and untreated ct tumors were eliminated in more than two-thirds of mice ( ) . the importance of t cells in this model was established as ohsv-il- had no antitumor effect in ct -bearing nude mice ( , ) . intraperitoneal administration of ohsv-il- also increased survival in misiir-tag mice bearing ovarian carcinomas ( ) . treatment was associated with tumor antigen-specific cd + t cell infiltration ( ) . against flank sarc- and sarc- sarcomas, ohsv-il- extended survival compared to saline injections ( ) . although there was no difference is survival between control and il- encoding ohsv, the ohsv-il- was found to increase tumor-infiltrating effector t cells while decreasing immunosuppressive mdsc and t reg populations ( ) . in an interesting comparison of cytokines, ohsv-il- was more effective at inhibiting flank prostate tumors, tramp-c and pr - , than ohsv encoding gm-csf (ohsv-gm-csf) which was no more effective than non-cytokine encoding ohsv ( ) . only of mice treated with ohsv-il- exhibited an increase in serum il- days after treatment ( ) . ohsv-il- also significantly outperformed ohsv-gm-csf in a model of ct metastasis ( ) . in addition to localizing il- , i.t. injections may be key in assuring safety as intrasplenic injections of ohsv-il- induced concerning increases in il- and ifnγ in both serum and liver specimens ( ) . frequent and worthwhile targets of ohsv-il- immunotherapy are the various forms of brain cancer. in one early study, ohsv-il- was found to extend survival and cure approximately one-fourth of mice bearing -day-old intracranial neuro- a neuroblastomas ( ) . treatment was associated with an influx of cd + and cd + t cells as well as macrophages. the inclusion of il- was critical, as median survivals of mice treated with the parental, non-cytokine encoding ohsv or saline were similar ( ) . likewise, ohsv-il- but not non-cytokine ohsv was effective at extending survival against intracerebral mouse glioblastomas ( ) . in an intracranial c glioma model, ohsv-il- was significantly more effective than ohsv at extending survival and curing mice ( ) . in a model of breast cancer metastasizing to the brain, i.t. ohsv-il- modestly extended the survival of mice bearing intracranial sck tumors ( ) . intracerebral injections of ohsv-il- in owl monkeys resulted in neither histopathological changes in brain tissue nor clinical evidence of toxicity, as assessed by changes in temperature, neurologic performance, feeding or social behavior, or weight ( ) . in addition to encoding cytokines, hsvs can be engineered to target cancer-associated antigens. four i.t. injections of a her -targeted ohsv-il- was significantly more effective at inhibiting both day and day tumors than the non-cytokine encoding parental hsv (day : / ohsv-il- vs. / hsv becoming tumor free; day : / ohsv-il- vs. / hsv becoming tumor free) ( ) . immune responses to ohsv-il- included elevated levels of ifnγ, il- , granzyme b, tbet, and tnfα as well as th polarization and nk activation ( ) . this her- targeted ohsv-il- was also found to induce complete remission in more than one-fourth of treated mice bearing orthotopic high grade gliomas (hgg) expressing her ( ) . cured mice were protected from hgg rechallenge regardless of her expression ( ) . this is an important finding given the heterogeneity of her expression among and within tumors. the clinical precedence for hsv has been established with talimogene laherparepvec, which encodes for gm-csf and is approved for intratumoral injection in patients with advanced, non-resectable melanoma. a clinical grade preparation of hsv- encoding hil- (m ) induced no adverse clinical signs after intracerebral injection in non-human primates ( ) . a phase clinical trial exploring the safety of m in patients with recurrent or progressive glioblastoma multiforme, anaplastic astrocytoma, or gliosarcoma is currently recruiting [nct ]. semliki forest virus (sfv) is an alphavirus that was first isolated from mosquitos in uganda and has a broad range of hosts, making it ideal for translation ( ) . in addition, modified sfv can produce higher levels of recombinant protein than retroviral vectors, and expresses protein more stably than adenoviral vectors ( ) . sfv is also less pathogenic in humans, and induces apoptosis of tumor cells at the end stage of virulence ( ) . sfv encoding il- (sfv-il- ) was found to extend the survival of mice with established orthotopic gliomas ( ) . because sfv infection induces apoptosis, uptake of infected tumor cells by dendritic cells in the presence of il- is posited as a potential mechanism of enhanced antitumor activity. in a b brain tumor model, the same group demonstrated a prolonged median survival by days when immunizing with sfv-il- pulsed dendritic cells, compared to a retroviral vector encoding il- ( ) . in a woodchuck hcc model, induced by chronic infection with woodchuck hepatitis virus (whv), using a single intratumoral treatment with sfv-enhil- , which included separate injections at different sites of one tumor, partial tumor regressions of up to % occurred in out of animals ( ) . although all tumors regrew after treatment, injections of sfv-enhil- resulted in a favorable safety profile with only transient reductions in body weight ( ) . in a transgenic mouse model of spontaneous hcc, i.t. sfv-il- treatments achieved % survival for at least days ( ) . interestingly, sfv-il- , which induces transient infection and expression of il- , was found to be more effective and less toxic than long-term il- expression induced by a plasmid encoding il- which was delivered hydrodynamically ( ) . i.t. sfv-il- inhibited tumor growth and extended survival in mice bearing orthotopic t mammary carcinomas ( ) . when administered neoadjuvant to resection and combined with attenuated salmonella (lvr ) as a post-surgery adjuvant, an impressive % long-term tumor free survival was achieved ( ) . while there is consensus on its benefits, the mechanism of sfv-il- -induced tumor regression is debated. initial studies performed in a subcutaneous b melanoma model did not find a significant decrease in tumor regression when using t cell-deficient nude or nk cell-deficient beige mice. rather, this group suggested that inhibition of angiogenesis mediated by ifnγ production causes massive tumor necrosis ( ) . a decade later, the same group further supported this conclusion using inos deficient mice that express high levels of vegf ( ) . the effect of il- was even more pronounced, with fewer tumor vessels in inos −/− mice than in wild-type mice given the same treatment with sfv-il- ( ) . however, they did find immunohistochemical evidence that nk cell activation and recruitment is correlated with murine endothelial cell death ( ) . more recently, the efficacy of sfv-il- was found to be dependent on type i interferons produced by macrophages and dendritic cells ( ) . furthermore, the type i interferon receptor, ifnar, is necessary for il- -dependent cd + t cell expansion ( ) . different routes of administration were compared using a subcutaneous p model and demonstrated that i.t. injection of sfv-il- was superior in producing ifnγ compared to either s.c. or i.v. routes ( ) . importantly, none of the sfv-il- injections resulted in increased serum levels of ifnγ ( ) . the combination of sfv-il- with monoclonal antibodies or adjuvants is another strategy that has been studied. by administering sfv-il- and anti-cd to provide costimulation to t cells, survival was improved in both s.c. b -ova and s.c. tc- models with a maximum longterm survival of % in both models ( ) . in the bilateral b -ova model, % of treated and % of untreated tumors experienced complete regression with sfv-il- viral particles and anti-cd ( ) . cd + t cells were crucial for this tumor regression and sfv-il- increased the ratio of cd t/t regs compared to anti-cd alone. a subsequent paper further demonstrated that sfv-il- induces pd-l expression on b -ova cells and therefore combined pd- /pd-l blockade with the viral construct ( ) . this combination significantly enhanced survival compared to individual components using b -ova and mc models, with long-term survival > % ( ) . modifications to improve the performance of sfv-il- vectors have also been explored. an enhanced (sfv-enhil- ) vector with separate promoters for each subunit of il- increased il- expression -fold over the single promoter construct ( ) . one i.t. injection of viral particles using either the original or enhanced constructs resulted in > % longterm tumor free survival of mc tumor-bearing mice ( ) . lower doses of sfv-enhil- induced tumor regression more efficiently than sfv-il- , although the enhanced vector induced higher levels of serum il- ( ) . a separate enhanced sfv vector with x higher gene expression, called psfv -e-il , has also been developed ( ) . this vector also included separate promoters for p and p with an additional capsid enhancer to drive enhanced expression of the recombinant protein. two helper vectors with instructions for structural proteins were cotransfected with the enhanced sfv to produce virus-like particles (vlps). intratumoral injections in subcutaneous k-balb and ct tumors caused complete regression and furthermore inhibited primary tumor growth and reduced metastases in a heterotopic t model ( ) . in an effort to limit undesirable virus-mediated cytotoxicity for intracranial applications, sfv vlps expressing il- have been developed ( ) . low dose ( × vlps) treatment decreased orthotopic rg rat glioma volumes by % and extended survival by a little more than % over vehicle controls. high dose ( × vlps) treatment resulted in enhanced tumor reduction but also caused cns inflammation, necrosis, and treatment-related death ( ) . the broad infectivity of sfv-based vectors is a major limitation to their use in cns applications. vaccinia virus (vv), which was used extensively during the eradication of smallpox, has a large capacity for gene insertion, a wide host range and high gene expression efficiency. vv expressing il- (vv-il- ) has been found to inhibit, but not eliminate, c gliomas in nude mice following i.t. injection ( ) . low vv-il- doses, - pfu, and high vv-il- doses, - pfu, resulted in similar levels of tumor inhibition ( ) . all doses above pfu resulted in cytokine-associated toxicities punctuated with a % mortality rate in the high dose group ( ) . similar tumor inhibition and mortality were found in a subsequent publication which also correlated high plasma levels of ifnγ and tnfα with toxicity ( ) . vv-il- and il- expressing vv (vv-il- ) exhibited similar inhibition of c gliomas ( , ) however, against ae mesotheliomas, vv-il- cured % of mice compared to only % of mice treated with vv-il ( ) . recently, an oncolytic vv-il- was shown to increase lymphocytic infiltration and inhibit llc and b f tumors with - % complete responses ( ) . antitumor efficacy was enhanced by the addition of an il- expressing vv (vv-il ) as well as immune checkpoint inhibitors ( ) . the combination of vv-il- and vv-il did not affect mouse body weight ( ) and appears to be better tolerated than previous vv-il- treatments which utilized different vv strains ( , ) . a recombinant canarypox virus (alvac) encoding il- has been shown to induce complete regression of ts/a mammary carcinomas after six i.t. injections, whereas a single i.t. injection of alvac-il- had no effect on tumor growth ( ) . of the mice that were rendered tumor-free, % were protected from a contralateral rechallenge. antitumor activity was driven by il- as the alvac vector itself displayed very limited tumor inhibition. in a phase i study in patients with unresectable metastatic melanoma, i.t. injections of alvac-il- resulted in increased intratumoral il- mrna expression in only of patients ( ) . three patients experienced modest increases in serum il- and ifnγ levels and no dose-limiting toxicities were observed. one patient had a complete response of an injected lesion and an uninjected in-transit metastases ( ) . in a similar phase i study performed by the same group, only of evaluable patients exhibited an increase in intratumoral il- mrna after alvac-il- administration ( ) . attenuated measles viruses (mevac) have been used safely as a vaccine to prevent measles in billions of people. mevac is another type of oncolytic virus which makes it an attractive potential cancer therapy. mevac encoding il- (mevac fmil- ) achieved complete regression of carcinoembryonic antigen (cea) expressing mc tumors in % of mice treated i.t. ( ) . mev encoding anti-pd-l or igg-fc induced complete regressions in and % of treated mice, respectively. antitumor activity was cd + t cell dependent and correlated with high levels of intratumoral ifnγ. all mice experiencing complete regressions following mevac fmil- also complete rejected tumor rechallenge ( ) . a follow up study demonstrated that mevac fmil- was more effective at controlling tumors than mev encoding il- in two murine tumor models ( ) . against mc -cea tumors, mevac fmil- eliminated tumors in all treated mice whereas mev-il induced complete tumor regressions in % of mice ( ) . effects appear to be model dependent as treatment of b tumors expressing human cd with mev-il- and mev-il only modestly extended survival ( ) . vesicular stomatitis virus (vsv) is another potent oncolytic virus under exploration for cancer therapy. vsvs engineered to express il- (vsv-il- ) were recently evaluated in an orthotopic floor of mouth model of sccvii murine squamous cell carcinoma ( ) . multiple i.t. injections of vsv-il- were more effective at eliminating tumors and induced higher longterm survival rates ( vs. %) compared to non-cytokine encoding vsv ( ) . moloney murine leukemia virus (momlv) is a positive sense, single-stranded rna (ssrna) retrovirus. a momlv expressing il- was engineered to display an anti-erbb scfv against the her receptor ( ) . eight daily i.t. injections of -day-old flank mbt tumors was found to result in inhibition and complete regression in about one-fifth of mice treated with the her targeted momlv-il- ( ) . a hybrid viral vector which pairs the high infectivity of adenoviruses with the high gene expression of sfv has been engineered to specifically infect and express il- in hepatocellular carcinoma cells ( ) . in buffalo rats bearing orthotopic mca-rh , the adv-sfv hybrid vector expressing il- induced complete regression in of treated animals. while the non-hybrid sfv-il- induced complete regression in % of treated rats, treatment was accompanied by elevated liver enzymes indicating hepatotoxicity ( ) . in contrast, liver enzyme levels following administration with the tissue-specific, hybrid vector were no different than levels in saline-treated control rats ( ) . newcastle disease virus (ndv) is an oncolytic, enveloped, negative-sense, single-stranded rna virus. ndvs have been engineered to express checkpoint inhibitor molecules and checkpoint inhibitor-cytokine conjugates ( ) . although the antitumor activity of i.t. ndv expressing il- alone was not tested, i.t. injections of ndv-anti-cd -mil- plus i.p. anti-ctla- or ndv-antipdl -mil- plus i.p. anti-ctla- resulting in complete responses in or %, respectively of mice bearing day-old b f flank tumors ( ) . the same treatments of one of two bilateral tumors resulted in and % complete responses, respectively, of untreated tumors ( ) . a major benefit of virus-based il- delivery is high transfection rates, particularly when compared to non-viral vectors. however, virus-based delivery is limited in several aspects. first, neutralizing immune responses including anti-viral antibody production ( , , ) and dth responses ( ) may preclude repeated injections with the same vector. however, the extent to which anti-viral immunity limits repeated injections is unclear. mice previously exposed to a viral vector demonstrated a . -fold reduction in intratumoral transgene expression. this level of reduction did not affect antitumor activity; however, the potential exists for reduced antitumor immune responses when lower doses of virus are used ( ) . on the positive side, viral dissemination and transgene expression in non-targeted liver tissue was reduced by more than , -fold in mice pre-exposed to the viral vector ( ) . secondly, viral il- delivery requires transfection of host cells, which has been shown to be highly heterogeneous due to inherent susceptibility or previous exposure to related vectors ( ) . in an extreme example of susceptibility, a dose of adcmvmil- that was found to be lethal in % of treated c bl/ mice did not result in a single death in the balb/c strain ( ) . livers of c bl/ mice exhibited much higher levels of adenovirus transduction ( ) . the current coronavirus outbreak further illustrates the point that some individuals appear to be more susceptible than others to viral infections. third, although clinical trials have shown that recombinant viruses are tolerated, viral dissemination, and infection in liver cells following i.t. injection were reported at an alarming degree ( ) ( ) ( ) . even when small volumes ( - µl) of recombinant adenoviruses were injected i.t., transgene expression was found in the liver, intestine, spleen, kidney, and brain. as much as % of the injected adv infected non-targeted normal tissues ( ) . similar levels of transgene expression was found in the liver and tumor days after an i.t. injection ( ) . fourth, some viral vectors, such as momlv and mev, can insert their genetic information into an infected cell's genome causing undesirable mutations and uncontrolled longterm expression of il- . the benefits of using such integrating vectors must be weighed against these risks as other viruses, such as adv, hsv, vsv, and ndv, are non-integrating and induce transient expression ( ) . lastly, and most relevant to the topic of localizing il- , even local injections of viruses encoding il- are not able to prevent il- dissemination ( , , , , ) . consequently, antitumor responses and il- -mediated adverse events are strongly correlated with virus dose. similar to plasmidbased il- delivery, novel strategies that anchor il- to an injection site or prevent its secretion appear best suited to break this correlation and widen the therapeutic window of virusencoding il- . dendritic cells ( , ) , fibroblasts ( , ) , macrophages ( , ) , tumor cells ( , ) , and mesenchymal stem cells (mscs) ( ) ( ) ( ) have all been engineered to express il- . transduced dcs, fibroblasts, tumor cells, and macrophages have been directly injected into tumors, while mscs have been injected systemically for migration to tumor beds ( , ) . a much larger number of preclinical and clinical studies have used il- -transduced tumor cells for s.c. immunization. these studies are not covered in this review as il- in this context is functioning as a vaccine adjuvant following injection of transduced cells in healthy, non-cancerous s.c. tissue rather than a strategy to localize il- to a tumor. dendritic cells and macrophages have been engineered to express a variety of cytokines including il- . in addition to supplying the tumor microenvironment with il- , transduction of these antigen presenting cells increases their ability to induce robust antitumor immune responses. following i.t. injection, dcs and macrophages are capable of capturing tumor antigen, migrating to draining lymph nodes, presenting antigen and priming tumorspecific t cell responses. transfection of antigen presenting cells with il- helps polarize t cells to a th phenotype and facilitate robust tumor-specific ctl responses ( ) . in an early preclinical study, bone marrow-derived dendritic cells (bmdcs) expressing il- were more effective than dcs expressing il- in controlling the growth of b f melanomas and generating tumor-specific ctl activity ( ) . il- -transduced bmdcs were also found to significantly suppress established mca fibrosarcomas, b melanomas, d adenocarcinomas, and - bma prostate carcinomas following a single i.t. injection ( , , ) . in one study, % of treated mca tumors were completely rejected. tumor-free mice also rejected a subsequent mca rechallenge ( ) . the growth of contralateral, non-treated tumors was also suppressed, implying that systemic tumor-specific immunity can be induced following i.t. injection. il- -transduced bmdcs were substantially more effective than il- -transduced fibroblasts or non-transduced bmdcs at inducing tumor-specific ctl activity and ifnγ production by draining lymph node cells and splenocytes ( ) . enhanced trafficking of dcs to regional lymph nodes provides a major advantage over non-migratory cells such as il- transduced tumor cells or fibroblasts. dcs transfected with adenovirus to express il- (dc-adcmvil- ) were found to eliminate ct and mc colon adenocarcinomas in - % and % of mice, respectively ( , ) . mice experiencing complete regression exhibited tumor-specific ctl activity and rejected tumor rechallenge. i.t. injections of adcmvil- -infected fibroblasts or allogeneic dc-adcmvil- were not effective ( ) . in a similar study, dc-adcmvil- reversed immune suppression mediated by tbj neuroblastomas and induced complete tumor regression ( ) . dcs engineered to co-express both il- and il- displayed synergistic antitumor activity that was greater than either dcencoding cytokine alone ( ) . all mice experienced complete regression of cms tumors while treated metha tumors were substantially reduced ( ) . while serum ifn-γ levels reached pg/ml days after dc administration, no toxicities were noted ( ) . bmdcs transfected by simian virus (sv ) to express il- or il- demonstrated complete regression of ct colon adenocarcinomas in or % of mice, respectively ( ) . there was no synergy between il- and il- when delivered together. interestingly, maturation of dcs with lps prior to i.t. injection impaired antitumor activity, presumably due to reduced antigen loading in the tumor. however, this hypothesis was not evaluated ( ) . transfection of bmdcs isolated from cms sarcoma-bearing mice with an adenoviral vector expressing il- was shown to restore antigen-presenting and allostimulatory functions ( ) . direct injections of the rescued bmdcs were shown to eliminate intrahepatic cms tumors and induce protective immunity ( ) . dcs can also be isolated from mouse spleens prior to transduction with an il- encoding vector. a recent study demonstrated that splenic dcs overexpressing il- can inhibit the growth of melanomas in mice ( ) . il- -transduced macrophages are less well-studied, however, like dcs, macrophages have antigen presenting capabilities that can be enhanced by il- . a single i.t. injection of peritoneal exudate-derived macrophages transduced by admil- induced suppression of primary and metastatic - bma orthotopic prostate tumors ( ) . serum il- levels peaked days after injection and remained elevated for at least weeks. antitumor activity was correlated with increased leukocytic infiltrate and cytolytic activity ( ) . the admil- -transduced macrophages were found to migrate to draining lymph nodes following i.t. injection. similar to some viral constructs, to reduce the potential for toxicity, il- expression can be placed under the control of an inducible promoter. one inducible system consists of cassettes including gal -ecr;vp -rxr and il- . first, gal -ecr and vp -rxr are expressed under the constitutive ubiquitin c promoter. the heterodimerization of gal -ecr and vp -rxr are triggered by addition of small-molecule ligand, e.g., pharmacologically inert diacylhydrazine. gal -ecr-vp -rxr heterodimers bind to a synthetic promoter containing gal binding site and induce the expression of il- ( ) . dcs with inducible il- demonstrated inhibition of renca and metha tumors following i.t. administration ( ) . combining il- expressing dcs with either il- -or ifnα expressing dcs did not improve antitumor efficacy ( ) . the ability to turn cytokine expression "on" and "off " is an attractive feature which may potentially allow for better control over cytokine delivery. to date, only one clinical study has been published. in a phase i study, autologous dendritic cells transfected to express il- were administered i.t. to patients with metastatic gastrointestinal carcinomas ( ) . of the treated patients, one experienced a partial response and two experienced stable disease. serum ifnγ, but not il- , levels were elevated following treatment. four instances of transient grade lymphopenia were observed, but no grade toxicities were observed. in vitro il- production was highly variable, most likely due to differences in susceptibility of dendritic cell infection with adenoviruses encoding il- ( ) . fibroblasts expressing il- caused the elimination of -day mca tumors in - % of mice after a single peritumoral injection ( ) . in addition, local il- -mediated regression was also able to control tumors on the contralateral flank. as has been shown in other systems, i.t. injections were more effective than distant s.c. injections at controlling tumor growth. repeated injections of il- -engineered fibroblasts were welltolerated, with no abnormalities detected in liver and renal function tests ( ) . a similar peritumoral implantation of alginate encapsulated nih t fibroblasts expressing il- was found to inhibit ct tumor growth ( ) . in a phase i study, peritumoral injections of il- -transduced autologous fibroblasts induced transient tumor reductions in four of nine patients with disseminated cancers ( ) . treatment with fibroblasts capable of secreting up to , ng of il- per day was well-tolerated ( ) . mesenchymal stem cells (mscs) are multipotent adult stem cells that divide and differentiate in order to repair skeletal tissues, such as bone, cartilage, and muscle. as such, mscs are predisposed to traffic from bone marrow to a wound site based on expression of soluble factors and unique receptors in damaged tissues. tumors can express many of the same factors and receptors which results in mscs trafficking to, and infiltrating tumors following systemic injections ( ) . thus, using il- transduced mscs as a "trojan horse" to target tumors and deliver il- is a promising approach. in one preclinical study, ad.il- -transduced mscs inhibited the growth of tc human ewing sarcomas in mice ( ) . one potential concern about il- -expressing mscs (msc/il- ) is their tropism for some normal tissues. in the above study, msc/il- were found in tumor, liver, lung and spleen tissues but not kidney, heart, or brain tissues days after i.v. injection ( ) . in a different study, high levels of msc/il- were found in the tumor but not in the liver, lung, and spleen days after administration ( ) . earlier time points were not presented. il- levels were high in the tumor; however, significant serum il- levels were found to persist for at least weeks after msc/il- administration ( ) . these high levels of intratumoral il- resulted in the inhibition of - renal cell carcinomas in nude mice ( ) . in a study that illustrates the importance of tropism, mscs that were non-virally transfected with pil- significantly inhibited b f lung metastases but not subcutaneous b f tumors following i.v. administration ( ) . only when administered i.t., were msc/il- cells able to inhibit s.c. b f tumors ( ) . despite the tumor-homing potential of mscs, published data suggest that msc/il- may be most effective at treating tumors in tissues where mscs have a natural tropism, e.g., lung, liver and bone ( ) . several other studies have favored local over systemic administration of msc/il- to treat various tumors. intracranial gl glioma-bearing mice treated with i.t. ucb-msc-il m generated from human umbilical cord blood experienced significantly prolonged survival ( ) . specifically, % of ucb-msc-il m treated mice survived more than days post tumor implantation whereas all mice treated with mscs expressing gfp succumbed within days ( ) . cured mice were completely protected from ipsilateral and contralateral tumor rechallenge ( ) . in another study, bone marrow derived mscs were transfected with a lentivirus to express mil- ( ) . these lenti-mil- -mscs reduced the volume of h and metha ascites and increased survival when administered i.p. and days after tumor inoculation ( ) . no pathological abnormalities were noted on biopsies taken from internal organs of lenti-mil- -msc treated mice ( ) . human mscs transduced with a retroviral vector expressing a modified il- (mscs/il- m) inhibited b f melanomas following five i.t. injections over weeks ( ) . safety was implied as no il- was detected in the serum following treatment. interestingly, the antitumor activities elicited by i.t. injections of mscs/il- m and il- plasmid dna were similar ( ) . in a rare comparison of cytokine delivery technologies, i.t. injections of mscs transfected with rad/il- m inhibited b f primary and metastatic tumors more effectively than i.t. injections of rad/il- m alone ( ) . despite the homing feature tsa mammary carcinoma cells transduced to secrete il- were found to cure of mice bearing -day established tsa tumors following local injection ( ) . when tumors were allowed to establish for seven days prior to treatment, antitumor efficacy dropped, with only of mice experiencing tumor regression. local delivery of l gliosarcoma cells engineered to express il- ( l-il ) significantly prolonged the survival of rats challenged intracranially with wild-type l tumors ( ) . similar results were observed whether the l-il treatment was given at the same time or days later than the tumor inoculation. the authors noted that only mice receiving the delayed treatment were protected from tumor rechallenge ( ) . natural killer (nk) cells and chimeric antigen receptor (car) t cells have also been transduced to produce il- . in one recent study, primary nk cells isolated from c bl/ mice and transduced to express il- (nk il− ) increased mhc class i expression in ova-transfected melanoma cells (b m ) compared to mock plasmid transduced nk cells ( ) . combination treatment studies involving tumor-specific cytotoxic t lymphocytes (ctls), pegylated il- , and activated nk il− revealed that only groups containing activated nk il− demonstrated statistically significant prolonged survival over mock constructs, untreated, and ctl alone controls in b -ova tumor bearing mouse models. in particular, nk il− plus il- treatment increased survival by . days, while nk il− plus ctl and il- prolonged survival for . days. nevertheless, all mice eventually succumbed to the disease and no stable tumor regressions were reported ( ) . car t cells have shown remarkable efficacy against hematologic malignancies but have yet to achieve clinical benefit against solid tumors. engineering car-t cells to express il- is under investigation to improve antitumor efficacy against solid tumors. in one study, i.v. infusion of il- expressing anti-vegfr car t cells induced curative regressions in - % of mice in five different tumor models including - -day-old b f melanomas, mca- sarcomas, mc colorectal adenocarcinomas, mc - sarcomas or - days old ct colon adenocarcinomas ( ) . t cells singularly transduced with either anti-vegfr- or il- were markedly less effective ( ) . because constitutive systemic expression of il- can be toxic, inducible il- expression strategies have been developed. by using an nfat-responsive promoter, il- could be expressed only after tcr engagement ( ) ( ) ( ) . this modification reduced toxicity but required lymphodepletion to achieve durable tumor regressions ( ) . the efficacy of car t cell therapy in general is dependent upon lymphodepletion via chemotherapy or radiotherapy. in an attempt to eliminate the need for lymphodepleting preconditioning, cd car t cells expressing il- (cd /il- ) have been investigated ( , ) . indeed, treatment with cd /il- car t cells significantly enhanced the survival of cd -el tumor-bearing mice vs. treatment with cd car t cells ( vs . % survival at day after tumor inoculation) ( ) . similarly, second generation cd /il- car t cells produced a % long-term survival rate in mice with established a b cell lymphomas. without the il- insert, all mice treated with cd car t cells succumbed to disease ( ) . h - z/il- car t cells, which are specific for the muc- ecto antigen and secrete il- , controlled skov human ovarian carcinomas in scid-beige mice following i.p. infusion or weeks after tumor implantation ( ) . specifically, ∼ or % of mice treated or weeks, respectively, after tumor implantation with h - z/il- car t cells survived for days. in contrast, ∼ % and % of mice treated or weeks, respectively, after tumor implantation with h - z car t cells, not containing the il- gene, survived for days ( ). glypican- (gpc )-specific car t cells with inducible expression of il- (gpc - z-nfat-il- car t) significantly inhibited or eliminated established plc/prf/ and huh- human hccs ( ) . the il- secreting car t cells resulted in decreased t reg infiltration. serum ifnγ and il- levels were elevated although neither pathological changes to critical organs nor significant loss in body weight were observed ( ) . it should be noted, however, that these studies were performed in immunocompromised mice which is not a reliable model for immune-related adverse events. from a clinical standpoint, administrations of il- -transduced cells have been well-tolerated, with patients experiencing only transient symptoms of lymphopenia, fever, and malaise; however, significant, durable clinical responses have been elusive. from a manufacturing perspective, the transduction of allogeneic and xenogeneic cells is easier and more reproducible. however, these cells are rapidly recognized and rejected by the host immune system. thankfully, recent advances in autologous cell transduction due to the emergence of car t cell immunotherapy has eliminated many concerns related to technical feasibility and patient heterogeneity ( ) . local administration of recombinant il- protein is the most direct and most quantifiable strategy in terms of ensuring the accuracy and reproducibility of a delivered dose. however, recombinant cytokines disseminate rapidly from local injection sites into the systemic circulation ( ) . thus, in order to maintain high levels of recombinant il- in the tumor microenvironment while minimizing systemic exposure, sustained delivery technologies, capable of locally releasing proteins and cytokines over extended periods of time following direct injection, are under development ( ) . the encapsulation of pharmaceuticals in polymeric microspheres for sustained delivery has been explored for several decades ( , ) . the use of microspheres to deliver cytokines is a more recent trend. synthetic polymers, such as poly-lactic coglycolic acid (plga), poly-caprolactone (pcl), and poly-lactic acid (pla), that have been widely explored for drug delivery, are being adapted to encapsulate and deliver various cytokines including il- ( , ( ) ( ) ( ) ( ) ( ) . human il- -loaded pcl:pla microspheres, when co-delivered with human peripheral blood lymphocytes (pbls), were shown to prevent engraftment of human tumors in scid mice ( ) . peg-il- also suppressed tumor engraftment and growth, but not as effectively as il- -loaded microspheres. although high levels of il- and ifn-γ were found in sera after administration of il- -loaded microspheres, this localized delivery strategy was more effective at preventing tumor engraftment than repeated i.p. injections of il- ( ) . similarly, a single i.t. injection of il- -loaded pcl:pla microspheres outperformed bolus injections of il- in the inhibition of human head and neck tumor tissue xenografts containing human pbls ( ) . il- -loaded microspheres completely suppressed engraftment in % of mice, whereas il- alone slowed but could not eliminate tumor growth ( ). in the above xenograft studies, antitumor activity was mediated by cd + t cells and cd + natural killer cells ( , ) . in yet another study, lung tumor xenografts using non-disrupted human lung tumor tissue were completely suppressed when injected with il- -loaded pla microspheres week after implantation ( ) . mechanistically, quiescent cd + effector memory t cells present within the tumor microenvironment were reactivated upon exposure to high local levels of il- ( ) . the proliferation of reactivated cd + cells and their production of ifn-γ facilitated tumor destruction ( ) . in murine tumor models, i.t. injections of il- -loaded pla microspheres eliminated up to % of line- lung alveolar carcinomas ( ) . in contrast, i.t. injections of free il- induced a complete response in only one of five treated mice. interestingly, mice experiencing complete tumor regression following microsphere administration were more resistant to tumor rechallenge than mice vaccinated with either live or irradiated line- cells co-injected with il- -loaded microspheres ( ) . in other studies, i.t. injections of il- loaded pla microspheres significantly reduced the incidence and number of spontaneous pulmonary tumor nodules ( , ) . when administered neoadjuvant to tumor resection, il- -loaded microspheres inhibited local recurrence, reduced pulmonary metastases, and improved disease-free survival as compared to resection alone ( ) . a similar study using more advanced line- tumors, which were , - , mm at study onset, demonstrated that neoadjuvant i.t. injections of il- -loaded microspheres prevented both local recurrence and pulmonary metastases better than surgery alone ( ) . the addition of gm-csf-loaded pla microspheres enhanced antitumor activity and survival. however, more cytokines were not always better, as the addition of low dose il- to neoadjuvant immunotherapy with il- -and gm-csf-loaded microspheres abrogated antitumor immunity ( ) . a theme common to nearly all il- -based immunotherapies was that il- -loaded microspheres induced cd + t cell and nk cell activation, including increases in cytolytic function and ifn-γ expression. in-depth effector mechanisms are elegantly described in a series of papers by egilmez and kilinc et al. ( , , ( ) ( ) ( ) ( ) . in addition to inducing a strong effector response, a potentially more important feature of i.t. injected il- -loaded microspheres is their potential to reverse immunosuppression in the tumor microenvironment. for example, a single i.t. injection of il- -loaded microspheres with and without gm-csf-loaded microspheres induced apoptosis and elimination of tumor-infiltrating cd + cd + foxp + t suppressor cells ( , ) . il- -loaded microspheres were also shown to reprogram immunosuppressive tumor-infiltrating macrophages (tims) and tumor-associated macrophages (tams) to a proinflammatory, antitumor phenotype ( , ) . microsphere platforms are well-suited to explore codelivery of multiple cytokines. the combination of il- -and tnfα-loaded pla microspheres was shown to be more effective than il- and gm-csf-loaded microspheres at eliminating mt- mammary carcinomas ( ) and b melanomas ( ) . nearly and % of mt- tumor-bearing mice were rendered tumor-free following i.t. injections with dual cytokine loaded microspheres of il- /tnfα and il- /gm-csf, respectively ( ) . dual cytokine loaded microspheres of il- /tnfα also induced increases in tumor-specific cytokine release from effector cells as compared to microspheres containing either cytokine alone ( ) or the combination of il- and gm-csf ( , ) . a single i.t. injection of il- -and il- -loaded pla microspheres outperformed injections of either cytokine-loaded microsphere alone in terms of delaying the growth of b melanomas ( ) . unlike previous studies demonstrating the elimination of % of line- tumors ( ) and impressive reductions in metastatic lesions ( , ) , il- -loaded microspheres had no impact on either primary b tumor growth or pulmonary metastases ( ) . interestingly, the triple combination of il- /il- /tnfα loaded in microspheres was found to reduce antitumor activity in the t model ( ) . in her- /neu transgenic mice, which develop spontaneous, multifocal mammary carcinomas, il- -and gm-csf-loaded microspheres injected intratumorally caused complete regression of primary tumors in up to % of mice ( ) . unfortunately, responses were temporary, and all tumors recurred. interestingly, chronic immunotherapy with cytokine-loaded microspheres could not control tumor burden due to a progressive decline in the intensity of antitumor t cells and an increase in tumor-infiltrating cd + cd + foxp + t suppressor cells with repeated injections ( , ) . subsequent studies have shown that administration of cyclophosphamide can block the postimmunotherapy increase in t suppressor cells ( , ) . recently, intratumoral delivery of il- microspheres (il- ms) in combination with stereotactic body radiation (sbrt) resulted in remarkable tumor regression in several types of preclinical pancreatic ductal adenocarcinoma mouse models ( ). this study showed that intratumoral levels of ifnγ were enhanced following the treatment of sbrt/il- ms, which in turn increased cd + t cell activation. sbrt/il- ms treatments also established systemic tumor immunity that was confirmed by antitumor effects on liver metastases ( ). chitosan (cs) is a bioadhesive polysaccharide derived primarily from the exoskeletons of crustaceans. it is nontoxic, biodegradable, and non-immunoreactive ( ) with an established safety profile in humans. co-formulation of il- in cs (cs/il- ) has been shown to increase the i.t. residence of il- from - days to - weeks ( ) . cs is believed to hinder il- diffusion through viscous and electrostatic interactions. the sustained presence of cs/il- induced complete regression of - % of mice bearing mc , panc , ct , e , and mb tumors ( , ) . administration of cs/il- neoadjuvant to t tumor resection resulted in complete clearance of lung metastases and long-term survival in about two-thirds of mice. in contrast, all mice treated with surgery alone died within days and mice receiving il- without cs neoadjuvant to resection had a median survival of days ( ). cured mice rejected a subsequent challenge with live t cells, but not live renca cells illustrating the specificity of the antitumor immune response. in another demonstration of systemic tumorspecific immunity from localized il- , % of mice bearing both flank and orthotopic mb bladder tumors were able to clear the flank tumor following intravesical administration of cs/il- ( ) . in contrast, mice with only a flank tumor, did not benefit from intravesical cs/il- in a naïve bladder. these data indicate that intravesical cs/il- induced systemic tumor-specific immunity only when il- was localized in or near a tumor. in a subsequent study, orthotopic bladder tumor regression following intravesical cs/il- immunotherapy was associated with a rapid infiltration of macrophages and granulocytes followed by increases in cd + and cd + effector t cells along with a reduction of immunosuppressive t regs and mdscs ( ) . importantly, local administrations of cs/il- did not result in significant toxicity, such as elevated liver enzymes, commonly observed following regular, free il- injections ( ) . a polysaccharide gel matrix, designated f gel, is comprised of % deacetylated poly-n-acetyl glucosamine (p-glcnac) coformulated with lactate salt. p-glcnac gels were evaluated as slow-release cytokine depots for gm-csf, il- , and il- ( ) ( ) ( ) ( ) ( ) . in particular, i.t. injections of il- in p-glcnac gel delayed the growth of ova-transfected ae malignant mesotheliomas ( ) . interestingly, gm-csf and il- depots had no impact on mesothelioma growth, although only a single i.t. injection was given. the p-glcnac gel by itself induced a strong, transient inflammatory response which was thought to be due to a foreign body reaction or toxic species that may have been contained within the gel ( , ) . human il- has been encapsulated in multilamellar liposomes for sustained release after i.t. injection ( ) . antitumor activity on xenografts of human lung tissues indicated that liposomal encapsulation is a promising approach capable of eliminating tumor cells and inducing lymphocyte infiltration weeks after i.t. injection. liposomal leakage and/or il- liposome dissemination could be problematic following delivery of large doses ( µg/mouse) as significant sustained levels of il- and ifn-γ were found in sera of treated mice ( ) . capitalizing on the tumor disruption caused by cationic liposomes, il- -and tlr ligand monophosphoryl lipid a (mpl)-loaded cationic liposomes were injected i.t. to treat t tumors. this combination produced an abscopal effect which arrested growth in both treated and distal tumors but did not result in tumor elimination ( ) . to date, sustained release technologies for recombinant il- have not been evaluated in clinical studies. while sustained release technologies are designed to maximize il- concentrations in the tumor microenvironment, some amount of delivered il- can be expected to reach the systemic circulation. however, due to the infrequency of administration for these long-acting platforms, they are unlikely to induce the same lethal toxicities that were observed following the frequent systemic il- administrations in early clinical trials ( ) . nevertheless, it will be essential to perform rigorous pharmacokinetic and toxicology studies prior to clinical translation of any sustained recombinant il- platform. a possible limitation of the microsphere encapsulation technology is the use of organic solvents during the encapsulation process. organic solvents can denature cytokines immediately or adversely affect long term storage. one study indicated that cytokines lose about half of their bioactivity during microencapsulation ( ) . loss of bioactivity appears to be cytokine dependent ( ) . specific activity of il- after encapsulation in pla microspheres was %. however, more than % of the bioactivity of il- was lost when pla/il- microspheres were stored for days ( ) . in addition, because many commonly used biodegradable polymers are comprise of acidic monomers, polymer degradation often forms highly acidic microenvironments that may inactivate cytokines ( ) . a number of techniques are under investigation to either raise ph ( ) or stabilize encapsulated cytokines ( ) . a common limitation of liposomal carriers is their inherent leakiness, and therefore the choice of lipid and modifications to stabilize the bilayer are critical for sustained release. high temperature phase-transition lipids, such as dspc, increase the retention of cargo and enhance drug circulation time. incorporation of cholesterol into the lipid bilayer increases the rigidity and further decreases leakage of interior cargo ( ) . another major limitation of liposomes is opsonization by serum proteins and clearance by phagocytes of the reticuloendothelial system (res). pegylation can reduced res clearance, however, peg can sterically inhibit ligand-cell interaction ( ) . if administered i.t., loss of tumor targeting is likely not detrimental. while some localized il- immunotherapies have shown limited efficacy in clinical studies, a long-awaited breakthrough leading to widespread application of localized il- as a monotherapy does not appear imminent. however, there are a number of near-term opportunities to combine localized il- with traditional or experimental cancer management approaches. and in fact, most contemporary experimental therapies for aggressive or advanced cancers involve combination approaches. thus, near future opportunities involving translatable combination approaches are explored in the following sections. the majority of cancer patients with solid tumors undergo tumor resection as part of their treatment. surgery, by itself, is unable to prevent recurrence and/or metastasis which is responsible for out of cancer-related deaths. patients at high risk of recurrence receive adjuvant chemotherapy and/or radiotherapy following resection. while chemotherapy and radiotherapy may help reduce the risk of recurrence, they induce life-altering side effects and fail in a significant fraction of patients. as a result, most metastatic cancers arise from previously treated non-metastatic disease ( ) ( ) ( ) . administration of localized il- to the tumor microenvironment prior to surgery has the potential to reduce recurrence rates and/or eliminate occult metastases through the induction of systemic, tumor-specific immunity. as mentioned above, our own investigation demonstrated that neoadjuvant i.t. injections of cs/il- prior to resection can improve overall survival from to % in a highly metastatic t breast carcinoma model ( ) . cured mice demonstrated tumor-specific immunity via tumor rechallenge, ctl activity, and elispot assays. if proven to be safe, localized il- can be administered neoadjuvant to any solid tumor resection, not just in high risk patients. in fact, at the time of surgery, it is likely that a significant fraction of patients already has occult metastases that cannot be detected via radiographic imaging. the median primary breast tumor size for which % of patients had micrometastases was found to be only . cm ( ) . while most immune-oncology trials focus on developing therapies for metastatic disease, an approach, such as neoadjuvant localized il- , capable of preventing metastasis in the first place, may be a more effective strategy to reducing cancer mortality. the combination of il- -based immunotherapy and chemotherapy ("biochemotherapy") is also under exploration. several clinical studies have shown significant clinical benefit when certain cytotoxic drugs are combined with il- ( , , ) . there are three distinct advantages for combining chemotherapy with il- immunotherapy. first, chemotherapy provides a debulking benefit with the elimination of some or most of the tumor. second, some chemotherapeutics can deplete immunosuppressive cells. cyclophosphamide has been shown to deplete t regs while -fluorouricil and gemcitabine have been shown to deplete mdscs. third, some chemotherapies cause upregulation of t cell-attracting chemokines in the tumor ( ) . for example, melanoma-bearing mice treated with temozolomide, cisplatin, or dacarbazine increase expression of ccl , cxcl , and cxcl ( ) . the timing of chemotherapy relative to local il- immunotherapy is expected to be critical. unlike the neoadjuvant resection approach mentioned above, chemotherapy should be administered prior to il- therapy to avoid elimination of beneficial proliferating immune cells. chemotherapy may synergize with il- by reducing tumor burden and creating a source of tumor antigen for in situ vaccination. in fact, certain chemotherapies can induce an immunogenic cell death that is capable of priming an antitumor immune response ( ) . to date, several examples demonstrate the promise of chemotherapy plus local il- delivery. antitumor efficacy of pil- /ppc is improved with paclitaxel, cyclophosphamide, or taxol/paraplatin ( , , ) . the antitumor effect of il- lipoplexes against intracranial glioma was substantially improved when combined with fda-approved carmustine (bcnu) wafers ( ) . finally, a phase- clinical trial found that intraperitoneal il- gene therapy in combination with intravenous pegylated liposomal doxorubicin resulted in greater clinical benefit relative to the chemotherapeutic alone ( . % vs. - . %) in patients (n = ) with platinum-resistant epithelial ovarian cancer ( ) . standard-of-care radiotherapy of inoperable tumors can be administered prior to local il- delivery. radiation-induced tumor cell death can supply a source of tumor antigens, while radiation has been shown to upregulate co-stimulatory molecules on the surface of tumor cells which makes them more susceptible to immune-mediated killing ( ) . the addition of tumor irradiation after pil- +ep was found to improve antitumor activity ( ) . similarly, radiotherapy improved the antitumor activity of i.t. oncolytic adenoviruses expressing il- and gm-csf ( ) . it has also been found that radiotherapy can induce infiltration of mscs in the tumor site. through a combination of radiation and il- -expressing mscs, both primary tumor growth and metastases were reduced in hca-i and hepa - hepatoma models ( ) . in another study, radiation along with an i.t. injection of an il- -encoding adenoviral vector greatly reduced tumor growth and metastasis compared to either treatment alone in a bnl-p hepatocellular carcinoma model, while a near-complete abscopal response occurred in a ct colorectal cancer model ( ) . an abscopal response was also induced in a fully humanized rhabdomyosarcoma xenograft model. mice with bilateral tumors were treated with a combination of single-tumor radiation and systemic nhs-il , which resulted in significantly lower tumor burdens than either individual treatment ( ) . cryoablation, radiofrequency ablation (rfa), microwave ablation (mwa), embolic microspheres and high intensity focused ultrasound (hifu) are routinely used in the clinic to destroy some operable and many inoperable solid tumors. irreversible electroporation is another ablative technology in clinical and preclinical development ( ) ( ) ( ) ( ) . all of these strategies have the potential to induce in situ vaccinations through the release of tumor antigen, although abscopal responses or robust anti-tumor immune responses are rare. among the ablation strategies, cryoablation appears distinct from most other ablation approaches that kill via high temperatures and result in coagulation, enzyme inactivation, and antigen denaturation. cryoablation is thought to mostly preserve tumor antigen structure ( , ) and has been shown to induce greater levels of tumor antigen uptake by dendritic cells compared to rfa ( ) . in addition, cryoablation also induces higher levels of inflammatory cytokines, such as il , il , and tnfα, compared to rfa and mwa ( , ) . perhaps most importantly, cryoablation was recently shown to induce expansion of oligoclonal t cells both in tumor tissues and in peripheral blood of kidney cancer patients ( ) . the addition of localized il- to the above ablation strategies may enhance tumor control as well as antitumor immunity. our recent preclinical data, demonstrated that the combination of cryoablation and cs/il- could eliminate multiple tumor types and induce abscopal immunity in a bilateral flank model ( ) . ongoing studies are aimed at determining mechanisms of systemic tumor control. immune checkpoint inhibitors are ineffective against noninflamed, "cold" tumors. localized il- induces profound intratumoral infiltration that can be expected to synergize with checkpoint inhibitors. indeed, the addition of anti-pd- or anti-ctla- improved the antitumor activity of l -il- in multiple murine tumor models ( ) . similarly, systemic anti-ctla- in combination with intratumoral il- induced synergistic antitumor responses in gl- glioblastoma and sma- astrocytoma models ( ) . also, as mentioned in a previous section, i.t. injections of vv-il- and vv-il enhanced the responsiveness of poorly immunogenic tumors to checkpoint inhibition ( ) . finally, a recent phase ii study of i.t. pil- +ep and systemic pembrolizumab in patients with non-infiltrated melanomas resulted in a % objective response rate with % complete responses ( ) . correlative studies demonstrated increased antigen cross-presentation along with enhance t cell infiltration and activity leading to higher than expected clinical responses ( ) . il- enhances cytotoxic t and nk cell activity while reversing tumor-induced immunosuppression, inhibiting angiogenesis, increasing lymphocyte trafficking and antigen presentation either directly or through induction of ifnγ ( ) . based on these diverse mechanisms as well as its profound antitumor activity in numerous preclinical studies, il- is arguably one of, if not, the most potent antitumor cytokine evaluated to date. unfortunately, the much-anticipated clinical translation of il- -based immunotherapies suffered a tremendous setback in the late s/early s due to severe clinical toxicities associated with systemic il- injections. through the development of several clever approaches to localize il- to the tumor microenvironment while limiting systemic exposure, il- -based immunotherapies are making a comeback. several clinical studies evaluating localized il- have been initiated ( table ) with more on the way. while each delivery strategy has limitations, the approaches reviewed above may retain enough il- in the tumor while eliminating the need for frequent systemic injections. by reducing the potential for il- -mediated toxicities associated with systemic injections, localized il- can expand the therapeutic window and finally allow il- to fulfill its considerable potential. as exemplified by the current immune-oncology clinical trial landscape, combination approaches appear to be most effective for accelerating clinical impact. several promising preclinical combination approaches with localized il- are likely to be translated in the near future. there is unprecedented interest in finding immunomodulators that can enhance lymphocytic infiltration in order to improve the efficacy of checkpoint inhibitors. localized il- , based on its ability to drive th responses, enhance cytolytic activity, and protect t cells from pd- /pd-l exhaustion and ifnγ-induced apoptosis may be an ideal partner for checkpoint inhibitors. if safety concerns are assuaged, localized il- could be used in earlier stage cancer patients as a neoadjuvant to resection. for tumors that are inoperable, combining localized il- with ablation may help increase local as well as distant tumor control. with the interesting combination approaches, as well as the uptick in il- -based immunotherapies in clinical trials, there is reason to believe that localized il- may play a major role in cancer immunotherapy in the near future. studies on the pathogenesis of fever. ii. characterization of fever-producing substances from polymorphonuclear leukocytes and from the fluid of sterile exudates anticancer cytokines: biology and clinical effects of interferon-alpha , interleukin (il)- , il- , il- , and il- melanoma: therapeutic options with recombinant interferons results of treatment of patients with metastatic renal cell carcinoma who received high-dose recombinant interleukin- therapy high-dose recombinant interleukin therapy for patients with metastatic melanoma: analysis of patients treated between and ( ) talimogene laherparepvec improves durable response rate in patients with advanced melanoma coexpression of two distinct genes is required to generate secreted bioactive cytotoxic lymphocyte maturation factor identification and purification of natural killer cell stimulatory factor (nksf), a cytokine with multiple biologic effects on human lymphocytes cloning of cdna for natural killer cell stimulatory factor, a heterodimeric cytokine with multiple biologic effects on t and natural killer cells cloning and expression of murine il- il- rapidly alters the functional profile of tumor-associated and tumor-infiltrating macrophages in vitro and in vivo the role of interleukin- on modulating myeloid-derived suppressor cells, increasing overall survival and reducing metastasis interleukin- : a master regulator immunological mechanisms of intravesical chitosan/interleukin- immunotherapy against murine bladder cancer. oncoimmunology differential effects of il- on tregs and non-treg t cells: roles of ifn-gamma, il- and il- r il- triggers a programmatic change in dysfunctional myeloidderived cells within mouse tumors antiproliferative effects of four different cytokines on renal carcinoma cell lines ifn-gamma induces apoptosis in ovarian cancer cells in vivo and in vitro inhibition of angiogenesis in vivo by interleukin inhibition of tumor-induced angiogenesis by the administration of recombinant interferon-gamma followed by a synthetic lipid-a subunit analogue (gla- ) cellular responses to interferon-gamma interleukin- in anti-tumor immunity and immunotherapy intratumoral immunotherapy of established solid tumors with chitosan/il- effect of interleukin on tumor induction by -methylcholanthrene therapeutic effect of interleukin on mouse haemangiosarcomas is not associated with an increased antitumour cytotoxic t-lymphocyte activity the anti-tumor activity of il- : mechanisms of innate immunity that are model and dose dependent antitumor and antimetastatic activity of interleukin against murine tumors phase i evaluation of intravenous recombinant human interleukin in patients with advanced malignancies pilot study of subcutaneous recombinant human interleukin in metastatic melanoma phase i trial of subcutaneous recombinant human interleukin- in patients with advanced renal cell carcinoma interleukin- therapy of cutaneous t-cell lymphoma induces lesion regression and cytotoxic t-cell responses phase i study of intraperitoneal recombinant human interleukin in patients with mullerian carcinoma, gastrointestinal primary malignancies, and mesothelioma phase study of the intravesical administration of recombinant human interleukin- in patients with recurrent superficial transitional cell carcinoma of the bladder a phase ii trial of interleukin- in patients with advanced cervical cancer: clinical and immunologic correlates. eastern cooperative oncology group study e e nk and cd + t cell-mediated eradication of poorly immunogenic b -f melanoma by the combined action of il- gene therapy and - bb costimulation intratumoral recombinant human interleukin- administration in head and neck squamous cell carcinoma patients modifies locoregional lymph node architecture and induces natural killer cell infiltration in the primary tumor activity of subcutaneous interleukin- in aids-related kaposi sarcoma phase ii clinical trial of interleukin- in patients with relapsed and refractory non-hodgkin's lymphoma and hodgkin's disease peripheral burst of tumor-specific cytotoxic t lymphocytes and infiltration of metastatic lesions by memory cd + t cells in melanoma patients receiving interleukin after initial setback, il- regaining popularity interleukin : still a promising candidate for tumor immunotherapy? effects of single-dose interleukin- exposure on interleukin- -associated toxicity and interferon-gamma production evaluation of recombinant human interleukin- in patients with recurrent or refractory ovarian cancer: a gynecologic oncology group study randomized multicenter phase ii trial of subcutaneous recombinant human interleukin- versus interferon-alpha a for patients with advanced renal cell carcinoma selective ablation of immature blood vessels in established human tumors follows vascular endothelial growth factor withdrawal patterns of vasculature in mouse models of lung cancer are dependent on location direct intratumoral injection of an adenovirus expressing interleukin- induces regression and long-lasting immunity that is associated with highly localized expression of interleukin- co-delivery of antigen and il- by venezuelan equine encephalitis virus replicon particles enhances antigen-specific immune responses and antitumor effects right time and place for il : targeted delivery stimulates immune therapy new insights into il- -mediated tumor suppression gene therapy of cancer based on interleukin neoadjuvant immunotherapy with chitosan and interleukin- to control breast cancer metastasis intravesical chitosan/interleukin- immunotherapy induces tumorspecific systemic immunity against murine bladder cancer characterization of abscopal effects of intratumoral electroporation-mediated il- gene therapy neoadjuvant intratumoral cytokine-loaded microspheres are superior to postoperative autologous cellular vaccines in generating systemic anti-tumor immunity repeated administrations of interleukin (il)- are associated with persistently elevated plasma levels of il- and declining ifn-gamma, tumor necrosis factor-alpha, il- , and il- responses transcriptionally active stat is required for the antiproliferative effects of both interferon alpha and interferon gamma ifn-gamma regulates donor cd t cell expansion, migration, and leads to apoptosis of cells of a solid tumor ifn-gamma-mediated upmodulation of mhc class i expression activates tumor-specific immune response in a mouse model of prostate cancer ifn-gamma-mediated inhibition of tumor angiogenesis by natural killer t-cell ligand, alpha-galactosylceramide interferon: a cytotoxic t lymphocyte differentiation signal il- and ifn-gamma are two necessary lymphokines in the development of cytolytic t cells gamma interferon enhances macrophage transcription of the tumor necrosis factor/cachectin, interleukin , and urokinase genes, which are controlled by short-lived repressors interferon and major histocompatibility complex genes: a model to analyse eukaryotic gene regulation? placebo-controlled phase iii trial of patient-specific immunotherapy with mitumprotimut-t and granulocyte-macrophage colony-stimulating factor after rituximab in patients with follicular lymphoma evaluation of ipilimumab in combination with allogeneic pancreatic tumor cells transfected with a gm-csf gene in previously treated pancreatic cancer cancer immunotherapy comes of age cancer immunotherapy strategies based on overcoming barriers within the tumor microenvironment reversing tumor immune suppression with intratumoral il- : activation of tumor-associated t effector/memory cells, induction of t suppressor apoptosis, and infiltration of cd + t effectors use of tumor-infiltrating lymphocytes and interleukin- in the immunotherapy of patients with metastatic melanoma. a preliminary report il- priming during in vitro antigenic stimulation changes properties of cd t cells and increases generation of effector and memory cells synergy of brief activation of cd t-cells in the presence of il- and adoptive transfer into lymphopenic hosts promotes tumor clearance and anti-tumor memory ex vivo interleukin- -priming during cd + t cell activation dramatically improves adoptive t cell transfer antitumor efficacy in a lymphodepleted host ex vivo conditioning with il- protects tumor-infiltrating cd + t cells from negative regulation by local ifn-gamma cutting edge: il- and type i ifn differentially program cd t cells for programmed death re-expression levels and tumor control switching on of the proliferation or apoptosis of activated human t lymphocytes by ifn-gamma is correlated with the differential expression of the alpha-and beta-chains of its receptor clinical trial listed in this table meet the following four criteria: ) involve a delivery technology; ) be administered locally, i.e. intratumoral or peritumoral; ) use il- as an effector and ) demonstrate antitumor activity of il- . clinical trials listed in this table include: ) nhs-il for solid tumors ) a first-in-human dose escalation and expansion study to evaluate intratumoral administration of sar as monotherapy and in combination with cemiplimab in patients with advanced solid tumors antibody-il- fusion proteins are effective in scid mouse models of prostate and colon carcinoma metastases bifunctional cytokine fusion proteins for gene therapy and antibody-targeted treatment of cancer a single-chain il- igg antibody fusion protein retains antibody specificity and il- bioactivity and demonstrates antitumor activity mechanism of antitumor activity of a single-chain interleukin- igg antibody fusion protein (mscil- .her .igg ) cytokines fused to antibodies and their combinations as therapeutic agents against different peritoneal her /neu expressing tumors long-term immunity elicited by antibody-cytokine fusion proteins protects against sequential challenge with murine mammary and colon malignancies amino acid residues involved in the heparin-binding activity of murine il- in the context of an antibody-cytokine fusion protein modulation of interleukin- activity in the presence of heparin molecular mechanisms of heparin-induced modulation of human interleukin bioactivity species-specific differences in heparin-induced modulation of il- family cytokines efficient production and purification of recombinant human interleukin- (il- ) overexpressed in mammalian cells without affinity tag novel immunocytokine il -ss (fv) inhibits mesothelioma tumor growth in nude mice prognostic value and characterization of the ovarian cancer-specific antigen ca - construction, expression, and function of b scfv-mil- , a fusion protein that attacks human ovarian carcinoma an il -il -antibody fusion protein targeting hodgkin's lymphoma cells potentiates activation of nk and t cells for an anti-tumor attack expression of the extra domain b of fibronectin, a marker of angiogenesis, in head and neck tumors hubc -il , an immunocytokine which targets edb-containing oncofetal fibronectin in tumors and tumor vasculature, shows potent anti-tumor activity in human tumor models a phase study of as , a novel antibody-cytokine fusion protein, in patients with malignant melanoma or renal cell carcinoma phase i trial of twice-weekly intravenous interleukin in patients with metastatic renal cell cancer or malignant melanoma: ability to maintain ifn-gamma induction is associated with clinical response an engineered antibody-interleukin- fusion protein with enhanced tumor vascular targeting properties enhancement of the antitumor properties of interleukin- by its targeted delivery to the tumor blood vessel extracellular matrix engineered vascular-targeting antibody-interferon-gamma fusion protein for cancer therapy enhancement of the antitumor activity of interleukin- by targeted delivery to neovasculature synergistic therapeutic effects of a tumor targeting antibody fragment, fused to interleukin and to tumor necrosis factor alpha anchoring of intratumorally administered cytokines to collagen safely potentiates systemic cancer immunotherapy collagen-binding il- enhances tumour inflammation and drives the complete remission of established immunologically cold mouse tumours a dose-escalation and signal-generating study of the immunocytokine l -il in combination with dacarbazine for the therapy of patients with metastatic melanoma targeted reconstitution of cytokine activity upon antigen binding using split cytokine antibody fusion proteins a new chemically modified chimeric tnt- monoclonal antibody directed against dna for the radioimmunotherapy of solid tumors the immunocytokine nhs-il as a potential cancer therapeutic characterization of a phage display-derived human monoclonal antibody (nhs ) counterpart to chimeric tnt- directed against necrotic regions of solid tumors temporal changes within the (bladder) tumor microenvironment that accompany the therapeutic effects of the immunocytokine nhs-il enhanced antitumor effects by combining an il- /anti-dna fusion protein with avelumab, an anti-pd-l antibody combination therapy with nhs-muil and avelumab (anti-pd-l ) enhances antitumor efficacy in preclinical cancer models cancer-targeted il- controls human rhabdomyosarcoma by senescence induction and myogenic differentiation tumortargeted il- combined with local irradiation leads to systemic tumor control via abscopal effects in vivo. oncoimmunology enhanced binding of necrosis-targeting immunocytokine nhs-il after local tumour irradiation in murine xenograft models defining the pharmacodynamic profile and therapeutic index of nhs-il immunocytokine in dogs with malignant melanoma first-in-human phase i trial of a tumor-targeted cytokine (nhs-il ) in subjects with metastatic solid tumors immunogenicity to biotherapeutics -the role of anti-drug immune complexes tumor regression of human and murine melanoma after intratumoral injection of il- -encoding plasmid dna in mice in vivo induction of interferon gamma expression in grey horses with metastatic melanoma resulting from direct injection of plasmid dna coding for equine interleukin intratumoral injection of dna encoding human interleukin into patients with metastatic melanoma: clinical efficacy intratumoral injection of il- plasmid dna-results of a phase i/ib clinical trial effective tumor therapy with plasmid-encoded cytokines combined with in vivo electroporation il- plasmid delivery by in vivo electroporation for the successful treatment of established subcutaneous b .f melanoma il- gene therapy using an electrically mediated nonviral approach reduces metastatic growth of melanoma evaluation of toxicity following electrically mediated interleukin- gene delivery in a b mouse melanoma model regression of tumor growth and induction of long-term antitumor memory by interleukin electro-gene therapy administration route-and immune cell activation-dependent tumor eradication by il electrotransfer intratumoral delivery of interleukin expression plasmids with in vivo electroporation is effective for colon and renal cancer radiosensitizing effect of intratumoral interleukin- gene electrotransfer in murine sarcoma in vivo electrogene transfer of interleukin- inhibits tumor growth and lymph node and lung metastases in mouse mammary carcinomas local and systemic antitumor effect of intratumoral and peritumoral il- electrogene therapy on murine sarcoma electroporation-mediated interleukin- gene therapy for hepatocellular carcinoma in the mice model improving therapeutic efficacy of il- intratumoral gene electrotransfer through novel plasmid design and modified parameters systemic administration of interleukin- can restore the antitumor potential of b melanoma-draining lymph node cells impaired at a late tumor-bearing state intratumoral electroporation of il- cdna eradicates established melanomas by trp ( - )-specific cd + ctls in a perforin/granzyme-mediated and ifn-gamma-dependent manner: application of trp ( - ) peptides gene electrotransfer of plasmid-encoding il- recruits the m macrophages and antigen-presenting cells inducing the eradication of aggressive b f murine melanoma il- gene electrotransfer triggers a change in immune response within mouse tumors pre-clinical investigation of the synergy effect of interleukin- gene-electro-transfer during partially irreversible electropermeabilization against melanoma in vivo electroporation-mediated transfer of interleukin- and interleukin- genes induces significant antitumor effects against melanoma in mice combination of il- and il- of electro-gene therapy synergistically inhibits tumor growth coadministration of interleukin- and interleukin- induces a fatal inflammatory response in mice: critical role of natural killer cell interferongamma production and stat-mediated signal transduction electrogene therapy with interleukin- in canine mast cell tumors electroporation-mediated il- gene therapy in a transplantable canine cancer model a combination of electrochemotherapy, gene electrotransfer of plasmid encoding canine il- and cytoreductive surgery in the treatment of canine oral malignant melanoma intratumoural interleukin gene therapy stimulates the immune system and decreases angiogenesis in dogs with spontaneous cancer phase i trial of interleukin- plasmid electroporation in patients with metastatic melanoma intratumoral plasmid il electroporation therapy in patients with advanced melanoma induces systemic and intratumoral t-cell responses intratumoral delivery of tavokinogene telseplasmid yields systemic immune responses in metastatic melanoma patients intratumoral delivery of plasmid il via electroporation leads to regression of injected and noninjected tumors in merkel cell carcinoma integrin-targeted nanocomplexes for tumour specific delivery and therapy by systemic administration conjugation of poly(amidoamine) dendrimers with various acrylates for improved delivery of plasmid encoding interleukin- gene preparation and characterization of gelatin nanoparticles containing pdna encoding il- and their expression in ct- carcinoma cells preparation and characterization of chitosan/beta-cyclodextrin nanoparticles containing plasmid dna encoding interleukin- efficient gene delivery by egf-lipoplexes in vitro and in vivo polyethylenimine: a versatile, multifunctional nonviral vector for nucleic acid delivery aerosol gene therapy with pei: il- eradicates osteosarcoma lung metastases eradication of osteosarcoma lung metastases following intranasal interleukin- gene therapy using a nonviral polyethylenimine vector tetraiodothyroacetic acid-conjugated polyethylenimine for integrin receptor mediated delivery of the plasmid encoding il- gene enhanced delivery of plasmid encoding interleukin- gene by diethylene triamine penta-acetic acid (dtpa)-conjugated pei nanoparticles delivery of plasmid encoding interleukin- gene into hepatocytes by conjugated polyethylenimine-based nanoparticles intratumoral delivery of il- gene by polyvinyl polymeric vector system to murine renal and colon carcinoma results in potent antitumor immunity combination of interleukin and interferon alpha gene therapy induces a synergistic antitumor response against colon and renal cell carcinoma biodegradable polymer-based interleukin- gene delivery: role of induced cytokines, tumor infiltrating cells and nitric oxide in anti-tumor activity soluble biodegradable polymer-based cytokine gene delivery for cancer treatment in vivo targeted gene delivery by cationic nanoparticles for treatment of hepatocellular carcinoma modified nanoparticle mediated il- immunogene therapy for colon cancer local and systemic delivery of interleukin- gene by cationic micelles for cancer immunogene therapy mannosylated chitosan nanoparticle-based cytokine gene therapy suppressed cancer growth in balb/c mice bearing ct- carcinoma cells intratumoral delivery of p cmvmil- using water-soluble lipopolymers tumor regression by repeated intratumoral delivery of water soluble lipopolymers/p cmvmil- complexes combination of local, nonviral il gene therapy and systemic paclitaxel treatment in a metastatic breast cancer model local, non-viral il- gene therapy using a water soluble lipopolymer as carrier system combined with systemic paclitaxel for cancer treatment development of self-assembled multi-arm polyrotaxanes nanocarriers for systemic plasmid delivery in vivo synthesis and application of a non-viral gene delivery system for immunogene therapy of cancer a safety and efficacy study of local delivery of interleukin- transgene by ppc polymer in a model of experimental glioma treatment of disseminated ovarian cancer using nonviral interleukin- gene therapy delivered intraperitoneally phase-i clinical trial of il- plasmid/lipopolymer complexes for the treatment of recurrent ovarian cancer a phase ii trial of intraperitoneal egen- , an il- plasmid formulated with peg-pei-cholesterol lipopolymer in the treatment of persistent or recurrent epithelial ovarian, fallopian tube or primary peritoneal cancer: a gynecologic oncology group study phase i trial of a formulated il- plasmid in combination with carboplatin and docetaxel chemotherapy in the treatment of platinum-sensitive recurrent ovarian cancer a phase i trial of intraperitoneal gen- , an il- plasmid formulated with peg-pei-cholesterol lipopolymer, administered with pegylated liposomal doxorubicin in patients with recurrent or persistent epithelial ovarian, fallopian tube or primary peritoneal cancers: an nrg oncology/gynecologic oncology group study intratumoral injection of interleukin- plasmid dna, either naked or in complex with cationic lipid, results in similar tumor regression in a murine model cationic polyphosphazene vesicles for cancer immunotherapy by efficient in vivo cytokine il- plasmid delivery enhanced growth inhibition of metastatic lung tumors by intravenous injection of atra-cationic liposome/il- pdna complexes in mice retinoic acid receptors and cancers all-trans-retinoic acid upregulates tnf receptors and potentiates tnf-induced activation of nuclear factors-kappab, activated protein- and apoptosis in human lung cancer cells lipid nanoparticles that deliver il- messenger rna suppress tumorigenesis in myc oncogene-driven hepatocellular carcinoma visual evidence of acidic environment within degrading poly(lactic-co-glycolic acid) (plga) microspheres a study of medi in sequential and concurrent combination with durvalumab in subjects with advanced solid tumors abstract : medi , a novel il- mrna therapy for intratumoral injection to promote t<sub>h</sub> transformation of the patient tumor microenvironment a first-in-human dose escalation and expansion study to evaluate intratumoral administration of sar as monotherapy and in combination with cemiplimab in patients with advanced solid tumors analysis of pre-existing igg and igm antibodies against polyethylene glycol (peg) in the general population viruses as anticancer drugs oncolytic virotherapy adenovirus-mediated interleukin- gene therapy for metastatic colon carcinoma construction of a double recombinant adenovirus vector expressing a heterodimeric cytokine: in vitro and in vivo production of biologically active interleukin- adenovirus-mediated expression of interleukin- induces natural killer cell activity and complements adenovirus-directed gp treatment of melanoma lung metastases construction and characterization of a recombinant adenoviral vector expressing human interleukin- intranasal therapy with an adenoviral vector containing the murine interleukin- gene eradicates osteosarcoma lung metastases antitumor mechanism of intratumoral injection with il- -expressing adenoviral vector against il- -unresponsive tumor the antitumor effects of adenoviralmediated, intratumoral delivery of interleukin require endogenous il- induction of antitumor immunity by direct intratumoral injection of a recombinant adenovirus vector expressing interleukin- regression of colon cancer and induction of antitumor immunity by intratumoral injection of adenovirus expressing interleukin- role of nk and t cells in il- -induced anti-tumor response against hepatic colon carcinoma adenovirusmediated interleukin- gene therapy for prostate cancer: suppression of orthotopic tumor growth and pre-established lung metastases in an orthotopic model attenuation of the glucocorticoid response during ad il- adenovirus vector treatment enhances natural killer cell-mediated killing of mhc class i-negative lncap prostate tumors depletion of myeloid-derived suppressor cells during interleukin- immunogene therapy does not confer a survival advantage in experimental malignant glioma in situ adenoviral interleukin gene transfer confers potent and longlasting cytotoxic immunity in glioma eradication of murine bladder carcinoma by intratumor injection of a bicistronic adenoviral vector carrying cdnas for the il- heterodimer and its inhibition by the il- p subunit homodimer a single intratumoral injection of a fiber-mutant adenoviral vector encoding interleukin induces remarkable anti-tumor and anti-metastatic activity in mice with meth-a fibrosarcoma growth suppression of human laryngeal squamous cell carcinoma by adenoviral-mediated interleukin- enhanced antitumor effect of combined replicative adenovirus and nonreplicative adenovirus expressing interleukin- in an immunocompetent mouse model adenovirus-interleukin- -mediated tumor regression in a murine hepatocellular carcinoma model is not dependent on cd -restricted natural killer t cells gene therapy of orthotopic hepatocellular carcinoma in rats using adenovirus coding for interleukin effective gene therapy for medullary thyroid carcinoma using recombinant adenovirus inducing tumor-specific expression of interleukin- gene therapy of a rat follicular thyroid carcinoma model with adenoviral vectors transducing murine interleukin- intratumor murine interleukin- gene therapy suppressed the growth of local and distant ewing's sarcoma neuroblastoma regression and immunity induced by transgenic expression of interleukin- high frequency of specific cd + t cells in the tumor and blood is associated with efficient local il- gene therapy of cancer thyroid cancer immuno-therapy with retroviral and adenoviral vectors expressing granulocyte macrophage colony stimulating factor and interleukin- in a rat model low-dose adenoviral immunotherapy of rat hepatocellular carcinoma using single-chain interleukin- treatment of pancreatic cancer with an oncolytic adenovirus expressing interleukin- in syrian hamsters phase i trial of intratumoral injection of an adenovirus encoding interleukin- for advanced digestive tumors membrane-tethered proteins for basic research, imaging, and therapy tumor therapy applying membrane-bound form of cytokines re-designing interleukin- to enhance its safety and potential as an anti-tumor immunotherapeutic agent cancer immunotherapy using a membrane-bound interleukin- with b - transmembrane and cytoplasmic domains the rheoswitch system for inducible up-and down-regulation of gene expression regulated intratumoral expression of il- using a rheoswitch therapeutic system((r)) (rts((r))) gene switch as gene therapy for the treatment of glioma regulatable interleukin- gene therapy in patients with recurrent highgrade glioma: results of a phase trial neoadjuvant interleukin- immunogene therapy protects against cancer recurrence after liver resection in an animal model neoadjuvant treatment of hepatic malignancy: an oncolytic herpes simplex virus expressing il- effectively treats the parent tumor and protects against recurrence-after resection interleukin secretion enhances antitumor efficacy of oncolytic herpes simplex viral therapy for colorectal cancer angiogenesis inhibition by an oncolytic herpes virus expressing interleukin cytokine gene transfer enhances herpes oncolytic therapy in murine squamous cell carcinoma in situ cancer vaccination: an il- defective vector/replication-competent herpes simplex virus combination induces local and systemic antitumor activity augmentation of antitumor immune responses by multiple intratumoral inoculations of replicationconditional hsv and interleukin- il- expressing oncolytic herpes simplex virus promotes anti-tumor activity and immunologic control of metastatic ovarian cancer in mice newly characterized murine undifferentiated sarcoma models sensitive to virotherapy with oncolytic hsv- m enhanced therapeutic efficacy of il- , but not gm-csf, expressing oncolytic herpes simplex virus for transgenic mouse derived prostate cancers cytokine-secreting herpes viral mutants effectively treat tumor in a murine metastatic colorectal liver model by oncolytic and t-cell-dependent mechanisms engineered herpes simplex virus expressing il- in the treatment of experimental murine brain tumors multifaceted oncolytic virus therapy for glioblastoma in an immunocompetent cancer stem cell model increased efficacy of an interleukin- -secreting herpes simplex virus in a syngeneic intracranial murine glioma model preclinical evaluation of oncolytic deltagamma( ) . herpes simplex virus expressing interleukin- for therapy of breast cancer brain metastases preclinical evaluation of a genetically engineered herpes simplex virus expressing interleukin- a fully-virulent retargeted oncolytic hsv armed with il- elicits local immunity and vaccine therapy towards distant tumors eradication of glioblastoma by immuno-virotherapy with a retargeted oncolytic hsv in a preclinical model evaluation of the safety and biodistribution of m , an attenuated herpes simplex virus type expressing hil- , after intracerebral administration to aotus nonhuman primates the molecular pathogenesis of semliki forest virus: a model virus made useful? semliki forest virus vectors engineered to express higher il- levels induce efficient elimination of murine colon adenocarcinomas induction of a therapeutic antitumor immunological response by intratumoral injection of genetically engineered semliki forest virus to produce interleukin- marked enhancement of antitumor immune responses in mouse brain tumor models by genetically modified dendritic cells producing semliki forest virus-mediated interleukin- semliki forest virus expressing interleukin- induces antiviral and antitumoral responses in woodchucks with chronic viral hepatitis and hepatocellular carcinoma short-term intratumoral interleukin- expressed from an alphaviral vector is sufficient to induce an efficient antitumoral response against spontaneous hepatocellular carcinomas neoadjuvant administration of semliki forest virus expressing interleukin- combined with attenuated salmonella eradicates breast cancer metastasis and achieves long-term survival in immunocompetent mice transfer of the murine interleukin- gene in vivo by a semliki forest virus vector induces b tumor regression through inhibition of tumor blood vessel formation monitored by doppler ultrasonography the anti-angiogenic activity of il- is increased in inos-/-mice and involves nk cells strict requirement for vector-induced type i interferon in efficacious antitumor responses to virally encoded il immunotherapy with recombinant sfv-replicons expressing the p a tumor antigen or il- induces tumor regression immunotherapeutic synergy between anti-cd mab and intratumoral administration of a cytopathic semliki forest virus encoding il- virotherapy with a semliki forest virus-based vector encoding il synergizes with pd- /pd-l blockade regression of mouse tumours and inhibition of metastases following administration of a semliki forest virus vector with enhanced expression of il- semliki forest virus-mediated gene therapy of the rg rat glioma low-dose vaccinia virus-mediated cytokine gene therapy of glioma evaluation of cytokine toxicity induced by vaccinia virus-mediated il- and il- antitumour immunotherapy cytokine-armed vaccinia virus infects the mesothelioma tumor microenvironment to overcome immune tolerance and mediate tumor resolution intratumoral expression of il- and il- using an oncolytic virus increases systemic sensitivity to immune checkpoint blockade canarypox virus-mediated interleukin gene transfer into murine mammary adenocarcinoma induces tumor suppression and long-term antitumoral immunity intratumoral administration of a recombinant canarypox virus expressing interleukin in patients with metastatic melanoma phase i study of the intratumoral administration of recombinant canarypox viruses expressing b . and interleukin in patients with metastatic melanoma oncolytic measles virus encoding interleukin- mediates potent antitumor effects through t cell activation. oncoimmunology immunological effects and viral gene expression determine the efficacy of oncolytic measles vaccines encoding il- or il- agonists interleukin- expression enhances vesicular stomatitis virus oncolytic therapy in murine squamous cell carcinoma enhancement of antitumor activity of gammaretrovirus carrying il- gene through genetic modification of envelope targeting her receptor: a promising strategy for bladder cancer therapy increased efficacy and safety in the treatment of experimental liver cancer with a novel adenovirus-alphavirus hybrid vector engineering newcastle disease virus as an oncolytic vector for intratumoral delivery of immune checkpoint inhibitors and immunocytokines immunotherapy of established tumors in mice by intratumoral injection of an adenovirus vector harboring the human il- cdna: induction of cd + t-cell immunity and nk activity effect of viral dose on neutralizing antibody response and transgene expression after aav vector re-administration in mice pre-existing immunity to adenovirus does not prevent tumor regression following intratumoral administration of a vector expressing il- but inhibits virus dissemination genetic heterogeneity in the toxicity to systemic adenoviral gene transfer of interleukin- systemic vector leakage and transgene expression by intratumorally injected recombinant adenovirus vectors systemic dissemination of viral vectors during intratumoral injection in vivo cancer gene therapy with a recombinant interleukin- adenovirus vector gene therapy leaves a vicious cycle intratumoral delivery of adenovirus-mediated interleukin- gene in mice with metastatic cancer in the liver preclinical toxicology of oncolytic adenovirus-mediated cytotoxic and interleukin- gene therapy for prostate cancer induction of systemic and therapeutic antitumor immunity using intratumoral injection of dendritic cells genetically modified to express interleukin intratumoral injection of bone-marrow derived dendritic cells engineered to produce interleukin- induces complete regression of established murine transplantable colon adenocarcinomas fibroblasts genetically engineered to secrete interleukin can suppress tumor growth and induce antitumor immunity to a murine melanoma in vivo cancer immunotherapy of established tumors with il- . effective delivery by genetically engineered fibroblasts therapeutic effects of gelatin matrix-embedded il- gene-modified macrophages in a mouse model of residual prostate cancer macrophages transduced with an adenoviral vector expressing interleukin suppress tumor growth and metastasis in a preclinical metastatic prostate cancer model antitumor efficacy of adenocarcinoma cells engineered to produce interleukin (il- ) or other cytokines compared with exogenous il- paracrine delivery of il- against intracranial l gliosarcoma in rats expression of interleukin- by adipose-derived mesenchymal stem cells for treatment of lung adenocarcinoma. thorac cancer therapeutic potential of human mesenchymal stem cells producing il- in a mouse xenograft model of renal cell carcinoma anti-tumor activity of mesenchymal stem cells producing il- in a mouse melanoma model enhancement of antitumor immunity against b melanoma tumor using genetically modified dendritic cells to produce cytokines route of administration influences the antitumor effects of bone marrowderived dendritic cells engineered to produce interleukin- in a metastatic mouse prostate cancer model augmented anti-tumor effect of dendritic cells genetically engineered by interleukin- plasmid dna upregulation of natural killer cells functions underlies the efficacy of intratumorally injected dendritic cells engineered to produce interleukin- interleukin- transduced dendritic cells induce regression of established murine neuroblastoma intratumoral delivery of dendritic cells engineered to secrete both interleukin (il)- and il- effectively treats local and distant disease in association with broadly reactive tc -type immunity intratumoral injection of dendritic cells transduced by an sv -based vector expressing interleukin- induces curative immunity mediated by cd + t lymphocytes and nk cells injection of il- gene-transduced dendritic cells into mouse liver tumor lesions activates both innate and acquired immunity intratumoral injection of dendritic cells overexpressing interleukin inhibits melanoma growth conditional interleukin- gene therapy promotes safe and effective antitumor immunity therapeutic effect of intratumoral administration of dcs with conditional expression of combination of different cytokines intratumoral injection of dendritic cells engineered to secrete interleukin- by recombinant adenovirus in patients with metastatic gastrointestinal carcinomas continuous release of interleukin from microencapsulated engineered cells for colon cancer therapy interleukin gene therapy of cancer by peritumoral injection of transduced autologous fibroblasts: outcome of a phase i study concise review: mesenchymal stem cell tumorhoming: detection methods in disease model systems murine bone marrow-derived mesenchymal stem cells as vehicles for interleukin- gene delivery into ewing sarcoma tumors reversal of tumor growth by gene modification of mesenchymal stem cells using spermine-pullulan/dna nanoparticles gene therapy of intracranial glioma using interleukin -secreting human umbilical cord blood-derived mesenchymal stem cells mesenchymal stem cells genetically modified by lentivirus-mediated interleukin- inhibit malignant ascites in mice the effects of mesenchymal stem cells injected via different routes on modified il- -mediated antitumor activity adoptive transfer of natural killer cells promotes the anti-tumor efficacy of t cells local delivery of interleukin- using t cells targeting vegf receptor- eradicates multiple vascularized tumors in mice il- release by engineered t cells expressing chimeric antigen receptors can effectively muster an antigen-independent macrophage response on tumor cells that have shut down tumor antigen expression improving adoptive t cell therapy by targeting and controlling il- expression to the tumor environment tumor-targeted t cells modified to secrete il- eradicate systemic tumors without need for prior conditioning cd car t cells expressing il- eradicate lymphoma in fully lymphoreplete mice through induction of host immunity il- secreting tumor-targeted chimeric antigen receptor t cells eradicate ovarian tumors in vivo armored inducible expression of il- enhances antitumor activity of glypican- -targeted chimeric antigen receptor-engineered t cells in hepatocellular carcinoma chitosan solution enhances the immunoadjuvant properties of gm-csf sustained release technology and its application in environmental remediation: a review designing hydrogels for controlled drug delivery recent advances in hydrogel based drug delivery systems for the human body neoadjuvant therapy with interleukin- -loaded polylactic acid microspheres reduces local recurrence and distant metastases in situ tumor vaccination with interleukin- -encapsulated biodegradable microspheres: induction of tumor regression and potent antitumor immunity cytokines delivered by biodegradable microspheres promote effective suppression of human tumors by human peripheral blood lymphocytes in the scid-winn model synergistic effect of intratumoral il- and tnf-alpha microspheres: systemic antitumor immunity is mediated by both cd + ctl and nk cells intratumoral delivery of encapsulated il- , il- and tnf-alpha in a model of metastatic breast cancer antitumor immune response of human peripheral blood lymphocytes coengrafted with tumor into severe combined immunodeficient mice interleukin- delivered by biodegradable microspheres promotes the antitumor activity of human peripheral blood lymphocytes in a human head and neck tumor xenograft/scid mouse model human cd + t cells present within the microenvironment of human lung tumors are mobilized by the local and sustained release of il- to kill tumors in situ by indirect effects of ifn-gamma human cd + effector memory t cells persisting in the microenvironment of lung cancer xenografts are activated by local delivery of il- to proliferate, produce ifn-gamma, and eradicate tumor cells transient activation of tumor-associated t-effector/memory cells promotes tumor eradication via nk-cell recruitment: minimal role for long-term t-cell immunity in cure of metastatic disease cancer immunotherapy with interleukin and granulocyte-macrophage colony-stimulating factor-encapsulated microspheres: coinduction of innate and adaptive antitumor immunity and cure of disseminated disease dichotomous effects of ifn-gamma on dendritic cell function determine the extent of il- -driven antitumor t cell immunity central role of tumorassociated cd + t effector/memory cells in restoring systemic antitumor immunity activated cd + t-effector/memory cells eliminate cd + cd + foxp + tsuppressor cells from tumors via fasl mediated apoptosis central role of ifngamma-indoleamine , -dioxygenase axis in regulation of interleukin- -mediated antitumor immunity rapid release of cytoplasmic il- from tumor-associated macrophages is an initial and critical event in il- -initiated tumor regression intratumoral il- and tnf-alpha-loaded microspheres lead to regression of breast cancer and systemic antitumor immunity generation of a tumor-specific systemic response after intratumoral injection of il- and il- -loaded polylactic acid microspheres il- + gm-csf microsphere therapy induces eradication of advanced spontaneous tumors in her- /neu transgenic mice but fails to achieve long-term cure due to the inability to maintain effector t-cell activity chronic immune therapy induces a progressive increase in intratumoral t suppressor activity and a concurrent loss of tumor-specific cd + t effectors in her- /neu transgenic mice bearing advanced spontaneous tumors chronic chemoimmunotherapy achieves cure of spontaneous murine mammary tumors via persistent blockade of posttherapy counterregulation effect of chitosan properties on immunoreactivity intravesical immunotherapy of superficial bladder cancer with chitosan/interleukin- characterization of a sustained-release delivery system for combined cytokine/peptide vaccination using a poly-n-acetyl glucosamine-based polymer matrix sustained release of granulocyte-macrophage colony-stimulating factor from a modular peptide-based cancer vaccine alters vaccine microenvironment and enhances the antigen-specific t-cell response paracrine release of il- stimulates ifn-gamma production and dramatically enhances the antigen-specific t cell response after vaccination with a novel peptide-based cancer vaccine intratumoral poly-n-acetyl glucosamine-based polymer matrix provokes a prolonged local inflammatory response that, when combined with il- , induces regression of malignant mesothelioma in a murine model using poly-n-acetyl glucosamine gel matrix to deliver il- with anti-schistosomasis vaccination evaluation of the biocompatibility of a chitosan scaffold in mice biocompatibility evaluation of crosslinked chitosan hydrogels after subcutaneous and intraperitoneal implantation in the rat il- delivered intratumorally by multilamellar liposomes reactivates memory t cells in human tumor microenvironments adjuvant cationic liposomes presenting mpl and il- induce cell death, suppress tumor growth, and alter the cellular phenotype of tumors in a murine model of breast cancer cytokine immunotherapy of cancer with controlled release biodegradable microspheres in a human tumor xenograft/scid mouse model characterization of cytokine-encapsulated controlled-release microsphere adjuvants stabilization of proteins encapsulated in injectable poly (lactide-co-glycolide) biodegradable microand nanoparticles as long-term delivery vehicles for interferon-alpha challenges in development of targeted liposomal therapeutics advances and challenges of liposome assisted drug delivery surgery for cancer: a trigger for metastases chemotherapy-induced metastasis in breast cancer radiotherapy targeting cancer stem cells "awakens" them to induce tumour relapse and metastasis in oral cancer biological behavior of human breast cancer micrometastases a novel synergistic combination of cyclophosphamide and gene transfer of interleukin- eradicates colorectal carcinoma in mice a phase i clinical trial of adoptive t cell therapy using il- secreting muc- (ecto) directed chimeric antigen receptors for recurrent ovarian cancer the next challenge in cancer immunotherapy: controlling t-cell traffic to the tumor chemotherapy induces intratumoral expression of chemokines in cutaneous melanoma, favoring t-cell infiltration and tumor control immunogenic effects of chemotherapy-induced tumor cell death radiation-induced effects and the immune system in cancer a novel combination treatment of armed oncolytic adenovirus expressing il- and gm-csf with radiotherapy in murine hepatocarcinoma irradiation-induced localization of il- -expressing mesenchymal stem cells to enhance the curative effect in murine metastatic hepatoma combination of radiation and interleukin eradicates large orthotopic hepatocellular carcinoma through immunomodulation of tumor microenvironment the role of irreversible electroporation (ire) for locally advanced pancreatic cancer: a systematic review of safety and efficacy irreversible electroporation reverses resistance to immune checkpoint blockade in pancreatic cancer systematic review of surgical and percutaneous irreversible electroporation in the treatment of locally advanced pancreatic cancer high-frequency irreversible electroporation is an effective tumor ablation strategy that induces immunologic cell death and promotes systemic anti-tumor immunity immunologic response to cryoablation of breast cancer thermal ablation of tumours: biological mechanisms and advances in therapy synergy between in situ cryoablation and tlr stimulation results in a highly effective in vivo dendritic cell vaccine changes in interleukin- beta and after hepatic microwave tissue ablation compared with radiofrequency, cryotherapy and surgical resections image-guided thermal ablation of tumors increases the plasma level of interleukin- and interleukin- characterization of the cryoablation-induced immune response in kidney cancer patients abscopal immunity achieved via in situ vaccination using a novel combination of cryoablation and interleukin- the antibody-based delivery of interleukin- to solid tumors boosts nk and cd + t cell activity and synergizes with immune checkpoint inhibitors intratumoral il- combined with ctla- blockade elicits t cell-mediated glioma rejection phase ii trial of il- plasmid transfection and pd- blockade in immunologically quiescent melanoma revisiting interleukin- as a cancer immunotherapy agent abbreviations a-nk il− or activated nk il− , activated primary natural killer (nk) cells transduced to express il- adcmvil- or adcmvmil- , recombinant defective adenovirus expressing il- or mil- under control of a cmv promoter adenoviral vector expressing murinedil- protein under the control of the hcmv promoter and sv polyadenylation sequences; ad-dhscil , adenovirus with replication dependent on hypoxia-inducible factor (hif) activity expressing single-chain il- ; ad-il- or adil- or ad.il- or admil- or ad.mil- inducible adenoviral vector engineered to express il- under the control of the rheoswitch therapeutic system r (rts) gene switch adtcpmil- , recombinant replication-defective adenoviral vector expressing murine interleukin- (mil- ), driven by a modified calc-i promoter oncolytic triple-deletion (td) adenoviral vector encoding wild-type il- oncolytic triple-deletion (td) adenoviral vector encoding non-secreting il- antibody-dependent cellular cytotoxicity; ad.scil- , adenoviral vector expressing single-chain il- ad -ycd/muttksr rep-hil , a replication-competent oncolytic adenovirus encoding the murine pro-inflammatory cytokine interleukin- (il- ) gene and two suicide fusion genes, a yeast cytosine deaminase (ycd) and a mutant form of herpes simplex virus type thymidine kinase / .crgd-mil p , a replication-deficient double targeted ad backbone-based vector carrying a chimeric ad / fiber with integrin-binding rgd motif incorporated in its ad knob domain expressing murine il- p adenoviral vector encoding membrane-anchored murine single-chain il- with b - transmembrane and cytoplasmic domains atra-cationic liposome/il- pdna, pil- complexed with cationic liposomes incorporating all-trans-retinoic acid chimeric antigen receptor; cbd, collagen binding domain; cbd-il- , collagen-binding immunocytokine comprised of a cbd of von willebrand factor fused to both subunits of il- chtnt- /huil- , necrosis-targeting immunocytokine comprised of huil- fused to the variable heavy chain of chtnt- antibody dendritic cells transfected with adenovirus expressing il- under control of the cmv promoter enzyme-linked immune absorbent spot fda, food and drug administration; gm-csf, granulocyte-macrophage colony-stimulating factor glypican- -specific car t cells with nfat-inducible expression of il- herpes simplex viruses (hsv) encoding il- ; hubc -il , immunocytokine comprised of two molecules of il- fused to each of the igg heavy chains of humanized bc- antibody immunocytokine comprised of il- fused to the fc fragment of humanized ks antibody; hu- . -il- , immunocytokine comprised of il- fused to gd targeting hu dmp/il- , pil- complexed with dmp cationic micelles -dimyristyloxypropyl)-n,n-dimethyl-( -hydroxyethyl)ammonium bromide/dioleoyl phosphatidylethanolamine (dmrie/dope) cationic lipids; il- , interleukin- ; il- m, modified il- ; an n-glycosylation mutant of il- at asn ; il -il , immunocytokine comprised of il- and il- fused to anti-cd scfv antibody immunocytokine comprised of il- fused to l scfv antibody; il -msa, il- fused to mouse serum albumin (msa) il- fused to mouse serum albumin (msa) and lumican, a collagen-binding proteoglycan immunocytokine comprised of p subunit of single-chain il- fused to ss , a mesothelin-binding single-chain variable fragment lenti-mil- -mscs, bone-marrow derived mscs transfected with lentivirus to express il- mc/pmil- , pmil- complexed with mannosylated chitosan (mc) myeloid-derived suppressor cells mevac fmil- , attenuated measles viruses (mevac) encoding il- histocompatibility complex; momlv-mil- , moloney murine leukemia virus (momlv) expressing il- ; mpeg/pmil- , polymersomes comprised of pmil- complexed with polyphosphazene particles modified with dpa and mpeg; mscs, mesenchymal stem cells; mscil- .her .igg , mouse single-chain il- fused mesenchymal stem cells (mscs) expressing il- ; l -mil , immunocytokine comprised of murine il- fused to l scfv antibody; nhs-il , necrosis-targeting immunocytokine comprised of two single-chain il- molecules fused to fulllength nhs antibody; nhs-muil , murine analog of nhs-il ; nk cells a biodegradable polyester; pbl, peripheral blood lymphocytes; pd- , programmed cell death protein ; pd-l , programmed death-ligand ; pei:il- , il- complexed with polyethyleneimine (pei); pil- enhanced sfv vector with x higher gene expression of recombinant il- protein; rad/il- m, recombinant adenovirus expressing il- m; rndv-anti-cd -mil- , newcastle disease virus (ndv) expressing immunocytokine comprised of anti-cd antibody fused to mil- ; rndvanti-pdl -mil- , newcastle disease virus (ndv) expressing immunocytokine comprised of anti-pdl antibody fused to mil- ; rsfv/il , recombinant semliki forest viruses (sfv) encoding il- ; rvsv-il , recombinant vesicular stomatitis virus (vsv) encoding il- ; rvv-mil- severe combined immunodeficient; scil- , single-chain il- ; sfv-il , semliki forest viruses encoding il- sfv-il- , semliki forest viruses encoding il- tumor associated macrophages; tlr, toll-like receptor; tnf, tumor necrosis factor; ucb-msc-il m, human umbilical cord blood-derived mesenchymal stem cells (ucb-mscs) expressing il- m vv-il- , vaccinia virus (vv) expressing il- il- expression plasmid complexed with water-soluble lipopolymers car t cells which are specific for the muc- ecto antigen and secrete il- ; b scfv-mil- , immunocytokine comprised of mil- fused to scfv of b antibody key: cord- -o k ae authors: he, bing; wang, jun; wang, yudie; zhao, juan; huang, juan; tian, yu; yang, cheng; zhang, heng; zhang, mingxia; gu, lixing; zhou, xiaocui; zhou, jingjiao title: the metabolic changes and immune profiles in patients with covid- date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: o k ae to explore the metabolic changes and immune profiles in patients with covid- , we analyzed the data of patients with mild and severe covid- as well as young children with covid- . of the leukocytes, % (iqr, – ) were lymphocytes [ . × ( )/l (iqr, . – . )], and monocytes were . × ( )/l (iqr, . – . ) in young children with covid- . in mild covid- patients, circulating monocytes were . × ( )/l (iqr, . – . ). twenty-one severe patients had low po( ) [ mmhg (iqr, – )] and so( ) [ % (iqr, – )] and high lactate dehydrogenase [ u/l (iqr, – )], cardiac troponin i [ . ng/ml (iqr, . – . )], and pro-bnp [ pg/ml (iqr, – , )]. serum d-dimer and fdp were . mg/l (iqr, . – . ) and . mg/l (iqr, . – . ), and a large number of rbc ( /μl (iqr, – ) was presented in urine, a cue of disseminated intravascular coagulation (dic) in severe patients. three patients had comorbidity with diabetes, and patients without diabetes also presented high blood glucose [ . mmol/l (iqr, . – . )]. fifteen of ( %) severe cases had urine glucose +, and nine of ( %) had urine ketone body +. the increased glucose was partially caused by reduced glucose consumption of cells. severe cases had extraordinarily low serum uric acid [ μmol/l (iqr, – )]. in the late stage of covid- , severe cases had extremely low cd (+) t cells and cd (+) t cells, but unusually high neutrophils [ . × ( )/l (iqr, . – . )], procalcitonin [ . ng/ml (iqr, . – . )], c-reactive protein [ mg/l (iqr, – )] and an extremely high level of interleukin- . four of ( %) severe cases had co-infection with fungi, and two of ( %) severe cases had bacterial infection. our findings suggest that, severe cases had acute respiratory distress syndrome (ards) i–iii, and metabolic disorders of glucose, lipid, uric acid, etc., even multiple organ dysfunction (mods) and dic. increased neutrophils and severe inflammatory responses were involved in ards, mods, and dic. with the dramatical decrease of t-lymphocytes, severe cases were susceptible to co-infect with bacteria and fungi in the late stage of covid- . in young children, extremely high lymphocytes and monocytes might be associated with the low morbidity of covid- . the significantly increased monocytes might play an important role in the recovery of patients with mild covid- . in december , an unknown viral pneumonia emerged in wuhan, china, and it then escalated into an unprecedented outbreak ( ) . chinese authorities have identified a new type of coronavirus named severe acute respiratory syndrome coronavirus (sars-cov- ) ( ). on february , , the infectious disease caused by this viral strain was officially named covid- (coronavirus disease ) by the world health organization (who) ( ) . by march , covid- had swept into at least countries and killed more than , people, and who officially announced a pandemic of covid- viral disease ( ) . as of june , , covid- cases have been confirmed in countries and territories, and the total was up to , , , including , deaths ( ) . so far, according to reported patients' data, some remarkable phenomena have been observed. first, only % of patients with covid- were infants and young children, and very few young patients have developed severe covid- ( ) . leukocytes are the main immune cells to fight against pathogens, and the total leukocyte count is higher in young children than in adults ( ) . moreover, the thymus gland of an infant is large and continues to grow throughout childhood. thus, the thymus produces more than enough matures tlymphocytes throughout the child's life ( ) . we explored whether the count and differential of leukocytes in infants and young children are associated with very low morbidity rates of covid- . second, from the epidemiology and clinical characteristics of covid- , % of patients were diagnosed as mild cases, and most mild cases can recover from covid- infection ( ) . so, it could be that specific leukocytes contributed to the recovery of patients with mild covid- . monocytes are important immune sentinel cells critical in the defense against viral infection in the blood. they achieve this via diverse mechanisms that include the detection of viruses, migration into infected tissues, differentiation into macrophages and dendritic cells, and pathogen clearance by phagocytosis and intracellular killing ( , ) . besides monocytes, the effect of lymphocytes on mild covid- cases is still unclear. in this study, mild patients have been examined to explore the potential roles of monocytes and lymphocytes in the recovery of patients with mild covid- . third, according to an analysis of nearly , confirmed cases, % of patients with covid- have been identified as severe cases and critically ill cases, involving severe pneumonia and metabolic disorders, developing into acute respiratory distress syndrome (ards), multiple organ dysfunctions (mods), and even septic shock and death ( , ) . some studies suggested that the immunopathogenesis after viral infection has been linked to the development of the disease into severe cases ( , ) . to explore the potential roles of immunopathogenesis in the progress of covid- infection, severe covid- patients have been investigated to explore how the immunopathogenesis was involved in ards and metabolic disorders, even mods, disseminated intravascular coagulation (dic), and death. in this study, we investigated mild cases and severe cases infected with sars-cov- , as well as healthy young children and adults. our multiple comparative analysis showed that not only is leukocyte composition different in healthy groups, these differences can also be found during various stages of sars-cov- infection. our study suggests that monocytes, neutrophils, and t-lymphocytes are associated with the onset and progress of covid- infection, and immunopathogenesis was involved in ards, metabolic disorders, and mods in severe cases. this study increases our understanding of the immune responses during covid- infection and provides support to develop novel, feasible, and effective treatments for covid- infection. research sources: covid- patients and healthy individuals covid- infection was rapidly endemic in wuhan, china, in january, . renmin hospital of wuhan university is at the very forefront of the fight against covid- . we collected the data of patients with covid- , including the clinical records, laboratory results and chest computed tomography (ct) scan images of mild and severe cases in the hospital. for comparison with covid- cases, the data of healthy adults and young children have been collected from the physical examination center of the hospitals. these healthy individuals have no significant medical condition and were in stable physical condition at that time. the data of patients with covid- and healthy persons have been all reviewed by a group of professional doctors from the hospitals, including basic features, nucleic acid tests, clinical data, laboratory results, co-infection with other pathogens, ct images, and other primary data. the study design has been approved by the ethics committee of the hospital. nasopharyngeal swab samples were collected from patients, and tested as soon as possible to increase the detection rate of sars-cov- . reverse transcription polymerase chain reaction (rt-pcr) kit (daan gene, shenzhen, china) was used to detect the conserved genes of sars-cov- , such as orf ab gene, n gene, and e gene with lightcycler system (roche, switzerland). if two or more of these three targeted genes has been detected as positive or one gene has been detected positive in two different samples from the same patient, the result is considered as positive for sars-cov- . meanwhile, the results can also be analyzed in conjunction with the patient's chest ct images. blood samples were collected from patients for laboratory tests. serum biochemical tests, including aspartate aminotransferase (ast), alanine aminotransferase (alt), creatine kinase (ck) and lactate dehydrogenase (ldh) were determined with cobas c testing system (roche, germany). procalcitonin (pct) and cardiac troponin i (ctn i) were analyzed by cl- i chemiluminescence immunoassay system (mindray, shenzhen, china). coagulation indicators were detecting with acl top hemostasis testing systems (werfen, usa). all the blood samples from healthy persons were used for comparison. to study the count and differential of lymphocytes, the blood samples from covid- patients were stained with cd , cd , cd , cd , cd , and cd antibodies (bd multi-test imk kit, usa) and were analyzed by bd facscanto ii flow cytometer (bd, usa). th /th kit (bd, usa) was used to quantitatively measure il- , il- , il- , tnf, and ifn-γ protein levels. to examine the effect of sars-cov- on the patients' humoral immune function, immunoglobulins (igm, igg, iga, and ige), complement (c ) and complement (c ) were tested (siemens healthineers, usa). c-reactive protein (crp) and interleukin (il- ) were measured for covid- patients (mindray, shenzhen, china). serum samples of patients were collected and tested for the igm of respiratory tract pathogens with pneumoslide igm kit (vircell, spain), including human respiratory syncytial virus, influenza a virus (subtypes h n and h n ), influenza b virus, parainfluenza virus / / , metapneumovirus, common coronavirus, epstein-barr virus, cytomegalovirus, rhinovirus, adenovirus, and bocavirus, as well as legionella pneumophila serum type i, mycoplasma pneumonia, and chlamydia pneumoniae. nasopharyngeal secretions were tested for nucleic acids of respiratory pathogens (health gene technologies, ningbo, china). sputum culture was performed to identify bacterial and fungal co-infection. the fungal examination was performed with fungus ( - )-β-d-glucan kit (dynamiker biotechnology, tianjin, china) and platelia aspergillus ag kit (bio-rad, usa). continuous measurements have been presented as median and interquartile range (iqr) and categorical variables as percentages. for assessing laboratory results, we also assessed whether the measurements were outside the normal range. unpaired t-test with welch's correction was used for comparison, and p < . and < . were considered statistically significant and highly statistically significant, respectively. graphpad prism . . (san diego, ca, usa) and spss . (ibm, armonk, ny, usa) were used for all analyses. patients with fever and/or cough were admitted to hospital after february , . chest ct images indicated multiple patchy, ground-glass opacity in the lungs ( figure a) . thirty-two patients were further diagnosed as infected with sars-cov- by real-time rt-pcr. there were men and women, and the median age of these mild cases was . the clinical characteristics of mild patients were presented in supplementary table s . compared with healthy adults, the count of leukocytes and neutrophils in mild covid- patients did not increase, but the median percentage and count of lymphocytes were % (iqr, - ) and . × /l (iqr, . - . ), respectively, which were significantly less than those of healthy adults, % (iqr, - ) and . × /l (iqr, . - . ), respectively (p < . ). interestingly, the median percentage and count of monocytes were . % (iqr, . - . ) and . × /l (iqr, . - . ), which were significantly higher than those of healthy adults . % (iqr, . - . ) and . × /l (iqr, . - . ) (p ≤ . ) ( table ). the significantly increased number of monocytes could play an important role in the recovery of patients with mild covid- . to investigate why infants and young children have low morbidity of covid- , we analyzed the clinical characteristics of young children with covid- , and collected the data of circulating leukocytes of young children with/without covid- . comparative analyses showed that young children have much higher leukocyte counts [ . × /l (iqr, . - . )] than adults. of note, % (iqr, - ) of leukocytes are lymphocytes [ . × /l, (iqr, . - . )] in young children. the median count of monocytes in young children is . × /l (iqr, . - . ), which is much higher than that of adults [ . × /l (iqr, . - . )] (p = . ). lymphocytes of young children with covid- was a little lower than those of healthy children, but remained at a high level [ . × /l (iqr, . - . )]. young children with covid- had a high level of monocytes [ . × /l (iqr, . - . )] as well ( table ) . such a high number of lymphocytes and monocytes has benefit to fight against sars-cov- , which might be associated with the low morbidity of covid- in young children. we collected and compared the data of severe cases and mild cases. chest ct images of severe cases indicated that there was critically diffuse ground-glass opacity in both lungs. the critically ill patient's bedside chest x-ray showed the lung texture enhanced and the translucency decreased, and multiple patchy shadows in both lungs. (d) serum il- concentration between mild patients (n = ) and severe patients (n = ). the normal range of il- is ≤ pg/ml. **p < . . (e) the analysis of th /th cytokine panel between mild patients (n = ) and severe patients (n = ). the normal range of il- , ifn-γ, il- , and il- are ≤ . pg/ml, pg/ml, . pg/ml, and . pg/ml, respectively. *p < . . a representative ct image is presented in figure b . in bedside chest x-ray results of the critically ill patients, the translucency of both lungs was diffusely decreased, and a large area of patchy shadow appeared with uneven density. tracheal intubation can be observed in the trachea and the heart shadow outline ( figure c) . the clinical characteristics of severe patients were presented in supplementary table s . these ct and x-ray images showed that the primary and most significant changes were in the lower respiratory tract of patients with severe covid- . among the respiratory indicators we measured, severe cases had lower partial pressure of oxygen (po ) and oxygen saturation (so ), mmhg (iqr, - ) and % (iqr, - ), respectively, and suffered from different degrees of ards, i to iii ( table ) . table ) . increased glucose and low uric acid in blood should be noted here. the level of blood glucose was . table ) . the total of leukocytes was . × /l (iqr, . - . ) in the peripheral blood of severe cases, which were much more than those in mild cases. compared with mild cases, severe cases had low levels of monocytes [ . × /l (iqr, . - . )]. however, the percentage and count of lymphocytes in severe cases were only % (iqr, - ) and . × /l (iqr, . - . ) respectively, which were significantly lower than those in mild cases ( table ) . the subsets of lymphocytes were examined by flow cytometry, including natural killer (nk) cells (cd + cd + ), b cells (cd + ), and t cells (cd + ). the results showed that severe cases had nk cells [ /µl (iqr, - )] and b cells [ /µl (iqr, - )], which was not a significant difference from the mild cases (p > . ). in addition, the functions of b cells and complements were tested, including igm, igg, iga, ige, c , and c , for both mild and severe covid- cases. for severe cases, the values of igm, c , and c were slightly lower than those in mild cases, but these values were still within the normal range. however, compared with mild cases, severe cases had much lower levels of cd + t cells and cd + t cells, /µl [iqr, - ] and /µl (iqr, - ), respectively. the decrease of cd + t cells was much more than that of cd + t cells, and the ratio of cd + t cells/cd + t cells increased by . (iqr, . - . ) ( table ). further examination of th /th cytokines also indicated that severe patients had normal levels of il- , and ifnγ, as well as il- in peripheral blood, but the level of il- in severe patients was times higher than normal (figure e ). in this study, the clinical course of severe cases was over weeks, and severe cases had a potentially high risk of co-infection with other pathogens due to critical exhaustion of cd + and cd + t cells. the respiratory tract pathogens were tested in severe cases, including viruses, legionella pneumophila, mycoplasma pneumoniae, and chlamydia pneumoniae, which were all negative. the fungal examinations, g assay and gm assay, were also performed in severe cases. the results of bacterial and fungal examinations indicated that four of ( %) severe cases had co-infection with fungi, and two of ( %) severe cases had co-infection with bacteria. a high number of white blood cells (wbc) [ /µl (iqr, ] was found in the urine of severe cases ( table ). further examinations showed that the median pct was . ng/ml (iqr, . - . ) in severe cases, a cue of potential sepsis/septic shock. among the inflammatory factors tested in severe cases, the median of crp was mg/l (iqr, - ), which was much higher than those in mild cases ( table ) . il- slightly increased in mild cases, but exceptionally high level of il- presented in severe cases, even times higher than normal in some critically ill cases (figure d) . the release of the inflammatory factors triggered by sars-cov- replication and/or co-infection with bacteria and fungi, played important roles in the progress of covid- infection. in the late stage of the disease in severe covid- cases, % (iqr, - ) of leukocytes were neutrophils [ . × /l (iqr, . - . )] ( table ) . previous studies showed that largely number of neutrophils triggered inflammatory responses and caused excessive organ injury in acute inflammatory disease, such as sepsis ( , ) . exceptionally high neutrophil numbers might be involved in severe inflammatory responses and might be associated with ards, mods, and even sepsis/septic shock, dic, and death during the late stage of severe covid- infection. in this study, we first analyzed the clinical features and leukocyte differential of mild covid- patients admitted to the hospital after february , . thirty-two mild cases, with a median age of years, had recovered from covid- infection. our data showed that compared with healthy adults, patients with mild covid- had lower lymphocytes in the acute stage, which was consistent with previous studies ( ) . however, mild covid- cases had high counts of circulating monocytes [ . × /l (iqr, . - . )]. in addition, mild patients had normal level of il- and il- in peripheral blood, but they had a - -fold increase of il- . monocytes/macrophages play very important roles in fighting against invading foreign viruses. literature from the past years has emphasized links among il- and innate immune response, such as mononuclear phagocytes ( , , ) . for patients with mild covid- , a high monocyte count and slight increase of il- might be helpful for eradicating the sars-cov- infection and were associated with recovery from covid- . based on the epidemiology and clinical characteristics of covid- , young children under six have the lowest morbidity rate, and very few young children with covid- develop severe cases ( , ) . according to our comparative analysis, young children under six have highly circulating monocytes, and % (iqr, - ) of leukocytes are lymphocytes had a high level of monocytes [ . × /l ( . - . )] as well. the intricate process of t-lymphocyte development in the thymus is essential in the formation and maintenance of the peripheral t-lymphocytes. the thymus of a young child is big, and has the function of maintaining the large amounts of t-lymphocytes in the peripheral blood ( , ) . extremely high levels of circulating lymphocytes and monocytes would benefit to fight against sars-cov- infection, which might be associated with the low morbidity of covid- in young children. to explore the metabolic changes and immune responses in the progress of covid- cases, we investigated patients with severe covid- infection. the median age of these patients was , and the clinical course was more than weeks. ct scan images showed multiple patchy ground-glass shadows in the left and right lungs. bedside chest radiography of critically ill patients indicated that the brightness of both lungs was decreased and multiple patchy shadows were observed. these clinical characteristics of severe cases are very similar to those reported in previous studies ( , ) . the severe covid- cases had ards i to iii, and had extremely high levels of ctni, ldh, and pro-bnp, a marker of severe cardiac dysfunction and even heart failure. besides that, an extraordinarily low level of serum uric acid [ µmol/l ( - )] was found in severe cases. uric acid is synthesized mainly in the liver and other tissues, which usually dissolves in the blood, and is removed from the body through urine. the extraordinarily low level of serum uric acid might indicate that potential liver and/or rental metabolism dysregulated in severe patients. among severe cases, three patients had the comorbidity of diabetes, and other patients also had very high blood glucose we further investigated immune responses in patients with severe covid- . first, different subpopulations of lymphocytes were investigated. the percentage and count of b cells and nk cells did not obviously change, which is consistent with the results from a previous report ( ) . the results of igm/igg/iga/ige, c and c also indicated that b cells and complements held normal functions. however, compared with mild cases, severe covid- cases had lower levels of cd + t cells [ /µl (iqr, - )] and an even more significant reduction in cd + t cells [only /µl (iqr, - )], which has a sharper drop than cd + t cell. we further analyzed th /th panel, in severe patients, th cytokines (il- and ifn-γ) were in the normal range, but il- , one of th cytokines, was about four times higher than normal. previous studies presented that in severe patients, cd + t cells and cd + t cells highly expressed the exhaustion markers, including nkg a, pd- , and tim- ( , ) . the dramatical decrease and functional exhaustion of cd + t cells and cd + t cells represents an important immunological characteristic of severe covid- infection. following the exhaustion of t cells, severe cases had high potential for co-infection with other pathogens. in this study, of ( %) severe patients had coinfection with fungi, and two of ( %) severe patients had bacterial co-infection. twenty-one severe cases had a high level of pct and crp, . ng/ml (iqr, . - . ) and mg/l (iqr, - ), respectively. il- was much higher than normal in severe cases, even times higher than normal in some critically ill cases. with sars-cov- replication and/or coinfection with bacteria and fungi, severe inflammatory responses played important roles in the progress of severe covid- infection. in the late stage of severe covid- , % (iqr, - ) of leukocytes were neutrophils [ . × /l (iqr, . - . ). a high number of wbc [ /µl (iqr, ] was presented in urine of severe patients. previous studies suggest that, in sepsis, a large number of neutrophil and the formation of neutrophil extracellular traps (net) triggered severe inflammatory responses and excessive tissue damage ( , , ) . the significant increase in neutrophils might be involved in severe inflammatory responses and mods, even dic and death in severe covid- patients. additionally, uric acid is the predominant anti-oxidant molecule in the plasma and respiratory tract, and is necessary for induction of type immune responses. uric acid plays a pivotal role in protecting against pathogen infections and autoimmune diseases ( , ) . whether the decrease of serum uric acid is associated with the inflammatory responses in severe covid- cases need to be explored. there are several limitations to this study. first, we investigated young children with covid- and adult cases, including mild cases and severe cases. more cases will need to be collected for comparative analysis of the difference between severe and critically ill patients. second, more inflammatory cytokines and chemokines will be analyzed for severe and critically ill patients and will be further evaluated for inflammatory storm mediated ards, dic, mods, and coagulation disorders. third, the mechanisms by which sars-cov- infection causes the reduction and functional exhaustion of cd + t cells and cd + t cells are still unclear. in-vitro and in-vivo experiments need to be performed to explore the mechanisms of t cell exhaustion. in summary, our findings suggest that extremely high level of lymphocytes and monocytes could help hamper sars-cov- replication, which might be associated with the low morbidity of covid- in infants and young children. a high number of monocytes would be helpful for removing sars-cov- and play an important role in the recovery of patients with mild covid- . in the late stage of the disease, severe cases suffered from ards, metabolic disorders, mods and coagulation disorders. with dramatical decrease of cd + t cells and cd + t cells, extraordinarily increased neutrophils and severe inflammatory responses are involved in ards, mods, and coagulation disorders and can even lead to dic and death in severe cases. whether the decrease of serum uric acid is associated with the inflammatory responses in severe covid- cases needs to be further explored. these findings can not only greatly improve our understanding of metabolic and immunological characteristics, but also provide a mechanistic basis for the prevention and treatment of covid- infection. all datasets generated for this study are included in the article/supplementary material. the studies involving human participants were reviewed and approved by renmin 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of hospitalized patients with novel coronavirus-infected pneumonia in wuhan longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of sars-cov- infected patients. medrxiv functional exhaustion of antiviral lymphocytes in covid- patients reduction and functional exhaustion of t cells in patients with coronavirus disease (covid- ) neutrophil extracellular traps in immunity and disease physiological functions and pathogenic potential of uric acid: a review barrier epithelial cells and the control of type immunity the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © he, wang, wang, zhao, huang, tian, yang, zhang, zhang, gu, zhou and zhou. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - f xbq authors: kaneko, kazunari; akagawa, shohei; akagawa, yuko; kimata, takahisa; tsuji, shoji title: our evolving understanding of kawasaki disease pathogenesis: role of the gut microbiota date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: f xbq kawasaki disease (kd) was first described by dr. tomisaku kawasaki in . the etiology of kd has been studied comprehensively but remains largely unknown. the disease seems to result from the interplay of genetic and environmental susceptibility factors with infectious triggers, followed by a subsequent abnormal immune response characterized by increased levels of inflammatory cytokines and chemokines during the acute phase. evidence has mounted to suggest that an imbalance between t helper cells (th s) and regulatory t cells (tregs) is associated with aberrant immune responses in kd. recent advances in culture-independent techniques for detection and identification of intestinal commensal bacteria enabled the discovery that th and treg differentiation are regulated by short chain fatty acids (scfas), in particular butyrate, produced by the gut microbiota. this finding provided a mechanistic link between dysbiosis, defined as changes in the composition of the gut microbiota, and various inflammatory diseases. on this basis, we propose that dysbiosis, with reduced production of scfas leading to imbalances of th s/tregs, could be involved in the etiology of kd. a pilot study supported this hypothesis, as only fecal concentrations of butyrate were significantly reduced in kd patients among scfas. this evolving perspective prompted us to undertake metagenomic analyses of bacterial dna from the feces of kd patients who were antibiotic-naïve at diagnosis. simultaneous measurements of th s/tregs in peripheral blood and scfa concentrations in feces would provide valuable information regarding the association between dysbiosis and dysregulated immune responses in kd. kawasaki disease (kd), named after dr. tomisaku kawasaki deceased june th, , mainly affects young children between the ages of months and years ( ). kd is characterized by persistent fever, bilateral conjunctival congestion, changes of the lips and oral cavity, polymorphous exanthema, changes of peripheral extremities, and acute non-purulent cervical lymphadenopathy ( , ) . although kd was originally reported to be self-limiting and benign ( ) , it is now recognized as a systemic vasculitis with a specific predilection for forming coronary artery lesions. these develop in up to % of children with kd who are not treated with intravenous immunoglobulin ( ) . coronary artery lesions associated with kd are the most common causes of pediatric heart disease in developed countries. the incidence of kd in the japanese population continues to increase and reached cases per , children aged ≤ years per year in ( ) . despite extensive ongoing research into the etiology of kd, the underlying mechanisms of this enigmatic vasculitis are not fully understood ( , ) . the current paradigm of kd pathogenesis is that the disease results from a pathologically amplified immune response against infectious agent(s) in a genetically and environmentally susceptible child ( ) . this paradigm is based on the following observations. first, there is clinical overlap between kd and infectious diseases such as adenovirus and streptococcosis. second, seasonal clustering of kd in the winter and spring mimics that of several viral diseases ( ) . third, temporal clusters of epidemics have been reported in japan, the us, canada, and finland ( ) . moreover, an outbreak in japan began in tokyo and spread throughout the country over a period of months ( ). finally, low incidence in the first months of life suggests at least partial protection from trans-placental antibodies ( ) . the low incidence of kd in schoolchildren indicates a potential role of common antigen(s) that most children encounter uneventfully in early childhood and against which they mount an appropriate and protective immune response ( ) . however, efforts to find a single unifying microbiological cause of kd have been, to date, unsuccessful. standard microbiological techniques, molecular methods and serological investigations have all failed to identify an etiological agent. it has long been held that infection by one or more widely distributed microorganism(s) might elicit dysregulated immune responses in genetically susceptible children resulting in kd. candidate pathogens include epstein-barr virus ( , ) , human herpes virus ( ), human immunodeficiency virus ( ) , human adenovirus ( ) ( ) , and candida spp. ( ) . candida albicans (c. albicans) has recently drawn attention, as administration of caws (water-soluble extracellular polysaccharide from culture supernatants of c. albicans) induces coronary arteritis similar to kd in mice ( ) . several reports suggested that c. albicans plays an important role as an infectious trigger of kd ( , , ) . considering the recent pandemic of coronavirus disease (covid- ), potential links between kd and covid- are also deserving of mention ( ) ( ) ( ) ( ) . clusters of children presenting with kd-like symptoms have been documented in the uk, us, france, and italy, and some of them were confirmed to have covid- . it appears that hyperinflammation associated with covid- could act as primer for kd development in individuals having genetically or environmentally determined predisposition. however, the specific mechanism is not yet defined ( ) . the higher incidence of kd in asian countries suggests a genetic predisposition for acquiring the disease ( ) . increased risk in family members of kd patients in japanese populations ( ) also hint at genetically determined susceptibility. recent advances have been made in identifying disease-susceptibility genes from genome-wide association studies. candidate genes contributing to kd susceptibility include b-lymphoid kinase (blk) ( ) epidemiologic studies have extensively searched for environmental factors that may explain variation and seasonality in kd incidence. surveys from the us and japan have demonstrated that higher precipitation and lower temperatures were associated with higher incidence of kd ( , ) . an investigation of the role of the early social environment in kd susceptibility in a japanese population found that higher household income, smaller family size, and urbanization were associated with increased kd incidence ( ) . this study, however, did not find a significant association between absence of infectious exposures during early life and kd. the prominent role played by the immune system in kd has been confirmed by many studies demonstrating activation of neutrophils and other immune cells as well as overproduction of inflammatory cytokines and chemokines such as tumor necrosis factor-α, interleukin (il)- , il- , il- , and il- , and monocyte chemotactic protein- ( ). levels of inflammatory cytokines and chemokines are reported to be elevated during the acute phase of kd. however, the mechanisms responsible for abnormal immune responses and overexpression of inflammatory cytokines remain unclear. several lines of evidence have revealed decreased numbers of regulatory t cells (tregs) and imbalances between t helper cells (th s) and tregs in acute kd ( ) ( ) ( ) . jia et al. elegantly demonstrated that th proportions and cytokine (il- , il- , and il- ) levels were significantly increased, while treg proportions and expression of treg transcription factors (e.g., foxp ) were significantly decreased in patients with acute kd ( ) . they concluded that th expansion and treg depletion were characteristic of acute kd. furthermore, recent studies suggested that treatment efficacy was associated with decreased th proportions and increased treg proportions, with both returning closer to a normal range ( , ) . thus, th /treg imbalances may contribute to exaggerated immune responses in kd patients. growing evidence suggests that disturbances within intestinal communities of commensal bacteria may lead to illness through aberrant immune system development. while the study of gut microbiology related to human health has a centennial history, recent technological advances have enabled us to explore this field in a more sophisticated manner. current approaches rely primarily on culture-independent methods such as amplification of conserved regions of the s rrna gene present in all bacteria ( ) . studies using these techniques have demonstrated that an adult humans harbor trillion gut bacteria comprising more than , different species and approximately species per person per fecal sample ( ) . development of the gut microbiota, defined as its colonization by microorganisms, might begin not at birth but in utero. however, the existence of viable bacteria in the womb was recently questioned ( ) ( ) ( ) . the maternal microbiota provides the first microbial inoculum, and from birth, microbial diversity increases and converges toward an adultlike microbiome within the first - years of life ( ) . the composition of the microbiota in childhood depends on various factors including sanitation, mode of delivery, maturity at birth, infant diet, antibiotic use during infancy, immunizations, and environmental factors such as geography or diet ( , , , ) . ethnic and genetic factors may also give rise to dysbiosis ( , ) . the factors that can alter the microbiome are being studied as potential drivers of changing trends in non-communicable diseases. dysbiosis, defined as changes in the composition of the gut microbiota, may be associated with several clinical conditions including obesity and metabolic diseases ( ), cancer ( ), autoimmune diseases ( ), allergy ( ), chronic inflammatory bowel diseases ( , ) , chronic kidney diseases ( ) , and autisticspectrum disorders ( ) . we also found that dysbiosis was present in children with idiopathic nephrotic syndrome ( ) . although research on the mechanism(s) through which dysbiosis impairs human health has just begun, regulation of the gut immune system by microbiota is believed to be involved. experiments with germ-free animals (deficient in commensal microbiota including gut microbiota) have demonstrated that microbial colonization promotes anatomical development of the intestinal epithelium, increases epithelial cell turnover rates, and initiates the maturation of gut-associated lymphoid tissue ( ) . both tregs and th s have received much attention in terms of their role in the gut immune system and its regulation by microbiota ( ) . tregs were originally identified as cd -positive, cd -positive and foxp -positive t cells that exerted inhibitory control over immune responses ( ) , maintained tolerance to self-antigens, and prevented autoimmune disease ( ) . there are several immunosuppressive mechanisms mediated by tregs, including secretion of immunosuppressive cytokines such as il- and tgf-β, functional modification or killing of antigenpresenting cells, and cell contact-dependent suppression via cytotoxic t-lymphocyte-associated protein ( ) . tregs mainly arise from naïve t cell precursors following stimulation by short chain fatty acids (scfas) such as acetate, propionate or butyrate produced by the gut microbiota ( , ) . previous studies demonstrated that members of the genus clostridium were potent inducers of treg differentiation through butyrate production ( , ) and that reduced luminal concentrations of scfas resulted in impaired development of intestinal tregs in germ-free mice ( , ) . in these mice, reconstitution with commensal bacteria or administration of scfas, especially butyrate, restored treg frequency ( ) , supporting the role of bacterial metabolites in treg development. therefore, it has been proposed that a decrease in the relative abundance of butyrateproducing microbes may disrupt mucosal immune homeostasis ( ) . the gut microbiota also plays a crucial role in the induction of effector t cell responses in the intestine. th s are a subtype of cd -positive t cells specialized for mounting immune responses against fungi and some extracellular bacteria. in addition to il- a, th s produce il- f, il- , il- , and il- ( ) . because il- is a potent proinflammatory cytokine that amplifies ongoing inflammation, aberrant regulation of th s contributes to development of inflammatory and autoimmune disorders. in germ-free mice, the number of th s was markedly decreased. however, the th compartment was restored by reconstitution with conventional microbiota ( ), thus indicating a crucial role for gut microbes in th development. among commensal bacteria, segmented filamentous bacteria (sfb) are one of the most potent inducers of th s. colonization of mice by sfb causes abundant accumulation of th s in the small intestine via enhanced production of serum amyloid a ( , ) . the existence of human commensal bacteria equivalent to sfb in rodents is probable because mixtures of bacterial strains isolated from fecal samples ulcerative colitis patients could induce th development ( ) . the gut, the largest interface between microbial factors and the host, contains the largest proportion of bacteria and the largest amount of lymphoid tissue in the body. thus, it was hypothesized that the intestinal environment might be reshaped in patients with kd. indeed, kd patients frequently exhibit gastrointestinal symptoms and complications ( ) . the contribution of the gut microbiota to kd has been evaluated in limited numbers of small cohorts using culture-based methods. several studies have been performed to identify the causative microbial agent(s) of kd at disease onset. takeshita et al. showed that the gut microbiomes of kd patients were distinguished by a lack of lactobacilli during the acute phase ( ), while nagata et al. isolated both hsp -producing gram-negative bacteria and gram-positive cocci capable of inducing vβ t cell expansion from kd patients ( ) . these results indicated that distinctive gut microbes might be involved in the pathophysiology of kd. however, because these studies of the microbiomes of kd patients were carried out using culture-based methods, microorganisms that cannot be cultured, which constitute more than half of the human gut microbiome, would have been overlooked. unlike culture methods, metagenomic analyses can reveal the composition of the intestinal microbiota irrespective of the ability to culture microbes. kinumaki of a metagenomic analysis of feces using culture-independent methods ( ) . they collected paired fecal samples from children with kd during the acute and non-acute phases and demonstrated that streptococcus spp., including s. pneumoniae, s. pseudopneumoniae, s. mitis, s. oralis, s. gordonii, and s. sanguinis, were more abundant during the acute phase while the genera ruminococcus, roseburia and faecalibacterium were less abundant. unfortunately, more than half of subjects were treated empirically with antibiotics during the early phase of kd when the fecal samples were collected because clinical and laboratory findings often do not fulfill the diagnostic criteria of kd ( ), but are instead suggestive of bacterial infections ( ) . as antibiotic administration rapidly perturbs the gut microbiota ( ), these results may reflect the effects of antibiotic therapy on the gut microbiota and not dysbiosis associated with kd. here, we would like to focus on a novel viewpoint on the role of the gut microbiota in kd: dysbiosis, defined as changes in the composition of the gut microbiota and caused by various prenatal and postnatal factors that are not necessarily infectious agent(s), might contribute to genetically and environmentally determined predilection for kd. this perspective is illustrated in figure and can be explained as follows: [ ] various factors during the in utero and postnatal period drive dysbiosis in young children; [ ] dysbiosis results in reduced intestinal production of scfas including butyrate; [ ] reduced levels of scfas in the gut cause an imbalance of th s/tregs; and [ ] individuals with th /treg imbalances develop hypercytokinemia triggered by ubiquitous infectious agents(s), followed by kd (figure ) . recent observations revealed that viral respiratory infections may alter microbial growth in the gut leading to dysbiosis ( ) . in addition, the gut microbiota has been shown to play an important role in regulating the generation of virus-specific cd -positive and cd -positive t cells and antibody responses following influenza virus infection ( ) . therefore, we hypothesize that young children with dysbiosis are prone to a vicious cycle of hypercytokinemia following infection by viruses. this paradigm for the pathogenesis of kd contrasts with previous hypotheses, which focused on specific microorganisms, toxins, or pathogen-associated molecular patterns ( ) ( ) ( ) . as butyrate has been reported to limit th differentiation and promote treg development ( ) ( ) ( ) , we hypothesize that kd-associated dysbiosis might be characterized by lower abundance of butyrate-producing bacteria. interestingly, the genera roseburia and faecalibacterium, which were reported to be less abundant in patients with acute kd by kinumaki et al. ( ) , are butyrate-producing bacteria ( , ) . furthermore, the strong association between kd and allergic diseases ( ) ( ) ( ) in which dysbiosis plays an important role ( ) also supports this perspective. recent observations of a potential association between previous antibiotic therapy and development of kd also supports our hypothesis ( ) . in this study, the median interval between the final dose of antibiotics and the onset of kd was . months, which was insufficient time for restoration of the gut microbiota and complete resolution of dysbiosis caused by antibiotic use ( ) . to confirm our hypothesis, further investigations involving metagenomic analysis of bacterial dna from the feces of a larger number of antibiotic-naïve patients with kd is clearly needed. it would be worthwhile to compare the proportions of specific microbial species such as butyrate-producing bacteria. simultaneous analysis of th /treg ratios in peripheral blood and measurement of fecal butyrate concentrations, reflecting the intestinal production of scfas, would also be helpful. we have just begun a project to test our hypothesis with approval from our institutional ethics committee (approval no. ) and parental informed consent. fecal samples will be collected not only from kd patients and healthy children but also from controls with viral infections. this will allow us to specifically characterize dysbiosis in kd because recent observations suggested that viral infection itself may cause dysbiosis ( ) . the results of our pilot study of four acute kd patients (median age . years, range . - . years; boys and girl) and four healthy children (median age . years, range . - . years, boys and girl) supports our perspective as fecal butyrate concentrations were significantly lower in kd patients (p < . , mann-whitney u test). in contrast, fecal concentrations of acetate, lactate, and propionate did not differ between kd patients and healthy children (figure ) . the kd patients studied had a median body mass index of . (range: . - . ), median maximal body temperatures of . • c (range: . - . • c), and median maximal c-reactive protein levels of mg/l (range: - mg/l). all kd patients presented with typical clinical features and fulfilled the diagnostic criteria ( ). none reported diarrhea or constipation and none received antibiotics. fecal samples were provided before intravenous immunoglobulin administration. no cases were complicated by coronary artery lesions. what factors perturb the gut microbiota and cause dysbiosis in young children? as shown in figure , both prenatal and postnatal conditions affect the establishment of the intestinal microbiota in infancy. factors that influence the initial colonization of the gut by microbes include maternal factors such as the maternal gut microbiota, vaginal infection or periodontitis as well as postnatal factors such as cesarean delivery, formula feeding, excessive antibiotic use, host genetics, and the environment ( ). interestingly, formula feeding ( ) and social environment factors such as higher household income, smaller family size, and urbanization were associated with both increased risk of dysbiosis and increased kd incidence ( ) . in addition, the peak age of kd onset ranging from months to years corresponds to the critical period for establishment of the gut microbiota during the first , days of life ( ) . we believe that dysbiosis underlies kd and could contribute to genetically and environmentally determined predilections for kd. therefore, kd might be included in the growing list of dysbiosis-associated conditions. if our perspective is confirmed, it would be valuable to investigate whether supplying probiotics starting at birth could reduce the risk of kd in infancy. the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. the studies involving human participants were reviewed and approved by ethics committee of kansai medical university. written informed 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previous antibiotic use and the development of kawasaki disease: a matched-pair casecontrol study the pervasive effects of an antibiotic on the human gut microbiota, as revealed by deep s rrna sequencing the microbiome in early life: implications for health outcomes breastfeeding and risk of kawasaki disease: a nationwide longitudinal survey in japan shaping microbiota during the first days of life we thank edanz group (https://en-author-services.edanzgroup. com/) for editing a draft of this manuscript. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © kaneko, akagawa, akagawa, kimata and tsuji. this is an openaccess article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -dx cqqt authors: kunkl, martina; amormino, carola; frascolla, simone; sambucci, manolo; de bardi, marco; caristi, silvana; arcieri, stefano; battistini, luca; tuosto, loretta title: cd autonomous signaling orchestrates il- expression and il- -regulated epithelial barrier functions in human t lymphocytes date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: dx cqqt il- is a member of the il- cytokine family involved in host protection against extracellular pathogens, by promoting epithelial cell regeneration and barrier functions. dysregulation of il- production has also frequently been observed in acute respiratory distress syndrome (ards) and several chronic inflammatory and autoimmune diseases. we have previously described that human cd , a crucial co-stimulatory receptor necessary for full t cell activation, is also able to act as a tcr independent signaling receptor and to induce the expression of il- a and inflammatory cytokines related to th cells, which together with th cells represent the main cellular source of il- . here we characterized the role of cd autonomous signaling in regulating il- expression in human cd (+) t cells. we show that cd stimulation in the absence of tcr strongly up-regulates il- gene expression and secretion. as recently observed for il- a, we also found that cd -mediated regulation of il- transcription requires the cooperative activities of both il- -activated stat and rela/nf-κb transcription factors. cd -mediated il- production also promotes the barrier functions of epithelial cells by inducing mucin and metalloproteases expression. finally, by using specific inhibitory drugs, we also identified cd -associated class a phosphatidylinositol -kinase (pi k) as a pivotal mediator of cd -mediated il- expression and il- –dependent epithelial cell barrier functions. il- has been discovered in as a member of the il- family that affects the functions of several non-hematopoietic cells, such as epithelial cells, keratinocytes, and hepatocytes ( ) . il- is mainly produced by th cells, a subset of cd + t helper (th) cells that regulate efficient immune defense against extracellular pathogens ( ) and by a unique cd + th cell subset, named th ( ) ( ) ( ) . other cell types have been described to produce il- such as natural killer (nk) cells, ilc subset of innate lymphoid cells and at lower extent macrophages ( ) ( ) ( ) . il- elicits its main effect on epithelial cells by stimulating their regeneration and proliferation, and by promoting epithelial barrier functions essential for the host defense against extracellular microbes at mucosal surfaces ( ) ( ) ( ) ( ) ( ) ( ) . however, high levels of il- and il- were detected in plasma of patients with sepsis-induced acute respiratory distress syndrome (ards) and were correlated with poor prognosis ( ) . more recently, a novel severe acute respiratory syndrome-related coronavirus (sars-cov ) has been identified as the etiological agent of covid- ( ) , a respiratory pandemic disease that, in several patients, causes ards associated with high levels of pro-inflammatory cytokines ( ) including th cytokines such as il- , il- , and il- ( , ) . moreover, an excessive production of il- together with other cytokines of the th signature has also been involved in maintaining and amplifying the chronic state of several inflammatory and autoimmune diseases ( ) ( ) ( ) ( ) ( ) ( ) ( ) . therefore, the identification of stimulatory molecules and associated signaling pathways regulating and/or amplifying il- production may provide new insight into immune defense against pathogens and novel therapeutic targets for inflammatory and autoimmune diseases. cd is one of the most important costimulatory receptor required for full t cell activation and differentiation ( ) . cd interacts with b . /cd or b . /cd on the surface of professional antigen presenting cells (apcs) and lowers the threshold of the t cell receptor (tcr), thus enhancing the early signaling events necessary for optimal cytokine production, cell cycle progression and survival ( , ) . human cd is also able to deliver tcr-independent signals, which leads to the production of pro-inflammatory cytokines and chemokines ( , ) in cd + t cells. we also evidenced that cd autonomous stimulation of peripheral blood cd + t cells from either healthy donors (hd) or type diabetes (t d) or multiple sclerosis (ms) patients promotes the production of inflammatory cytokines and chemokines related to the th cell phenotype ( ) ( ) ( ) , including il- ( ) . in this work, we elucidated the role of cd autonomous stimulation in the regulation of il- expression and il- mediated effector functions. we found that il- gene expression and secretion was strongly up-regulated by cd in human cd + t cells. similar to il- a, cd -induced il- gene expression was regulated by both rela/nf-kb and il- -regulated stat transcription factors. co-culture experiments of cd -activated t cells with caco- epithelial cell lines evidenced a critical role of il- in regulating the expression of mucins and metalloproteases. finally, we also evidence a pivotal role of cd -associated class a phosphatidylinositol -kinase (pi k) in regulating both il- expression and il- -dependent epithelial barrier functions. human primary cd + t cells were enriched from pbmc by negative selection using an easyseptm isolation kit (# , stemcell technology) and cultured in rpmi supplemented with % human serum (euroclone, uk), lglutamine, penicillin and streptomycin. the purity of the sorted population was % to %, as evidenced by staining with anti-cd plus anti-cd abs. human naïve cd + cd ra + and effector/memory cd + cd ro + t cells were sorted using a high speed cell sorter (moflo astrios eq, beckman coulter). purity of sorted cells was consistently > %, and was acquired on a cytometer (cytoflex, beckman coulter). pbmcs were derived from buffy coats or anonymous healthy blood (hd) donors provided by the policlinico umberto i (sapienza university of rome, italy). written informed consent was obtained from blood donors and both the informed consent form and procedure was approved by the ethics committee of policlinico umberto i (ethical code n. bis/ , / / ). caco- epithelial cell line from human colon was provided by atcc (maryland, usa) and cultured in dmem supplemented with % fbs (euroclone, uk), l-glutamine, penicillin and streptomycin. the following antibodies were used: anti-human cd (cd . , # , mg ml − ), anti-human cd (ucht , # , mg ml − ), goat anti-mouse (gam, # , mg ml − ), anti-human cd -pe (# ), anti-human cd ra bv (# ), mouse anti-human stat (# ), antihuman cd ro pe (# ) (bd biosciences); rabbit antihuman lck (#sc- , mg/chip), rabbit anti-human rna polymerase ii (n- , #sc- , mg/chip), anti-human relb (c- , #sc- , mg/chip), anti-human gapdh (#sc- ), antihuman mucin (muc ) (#sc- ) (santa cruz biotechnology, usa), rabbit anti-human rela (# s) (cell signaling technology, usa), mouse anti-human il- (#mab , mg ml − ), mouse anti-human il- (#mab , mg ml − ) (r&d systems, usa), anti-human cd fitc (# - - , miltenyi biotec, italy). human recombinant il- (ril- ) was from miltenyi biotec (# - - , ng ml − ). the following inhibitory drugs were used: ps (#p , sigma aldrich), s - (#sc- , santa cruz biotechnology), as (#a , sigma aldrich). caco- cells were plated at . × ml − and co-cultured with cd + t cells ( × ml − ) in trans-well plates as indicated at °c. at the end of incubation, total cell extracts were obtained by lysing cells for min on ice in % nonidet p- lysis buffer ( mm nacl, mm tris-hcl (ph , ), mm egta, mm mgcl , mm naf, mm na p o ) in the presence of inhibitors of proteases and phosphatases ( mg ml − leupeptin, mg ml − aprotinin, mm navo , mm pefablock-sc). proteins were resolved by sds-page and blotted onto nitrocellulose membranes. blots were incubated with the anti-muc or anti-gapdh ( : dilution), extensively washed and after incubation with horseradish peroxidase (hrp)-labeled goat anti-rabbit (#na v, : dilution) or hrp-labeled goat anti-mouse (#na v, : dilution) developed with the enhanced chemiluminescence's detection system (ge healthcare life sciences, italy). protein levels were quantified by densitometric analysis using the imagej . i program (national institute of health, usa). cd + t cells were plated at × ml − in -well plate or well plate inserts in the experiments of co-culture with caco- cells ( . × ml − ) and stimulated for the indicated times with control isotype abs (ig) or crosslinked anti-cd ( mg ml − ), or anti-cd ( mg ml − ) or anti-cd plus anti-cd abs ( mg ml − ). secretion of human il- , il- , and mmp was measured from the supernatants of cd + t cells cultured alone or with caco- cells and stimulated with control isotype abs or crosslinked anti-cd . ( mg ml − ), by using human il- (#dy ), il- (#dy ), and mmp (#dy ) elisa kits (r&d systems). data were analyzed on a bio-plex (bio-rad, hercules, ca, usa). the assays were performed in duplicate. the sensitivity of the assay was . pg ml − for il- and . pg ml − for il- and mmp . cd + t cells were plated at × ml − in -well plate or well plate inserts in the experiments of co-culture with caco- cells and stimulated for the indicated times with control isotype matched abs (ig) or crosslinked anti-cd ( mg ml − ), or anti-cd ( mg ml − ) or anti-cd plus anti-cd abs ( mg ml − ). total rna was extracted by either cd + t cells or caco- cells using trizol according to the manufacturer's instructions and was reverse-transcribed into cdna by using moloney murine leukemia virus reverse transcriptase (thermo fisher scientific ca, usa). taqman universal pcr master mix and human il- , il- , mmp , muc , and gapdh primer/probe sets were from thermo fisher scientific. the relative quantification was performed using the comparative c t method. the results were expressed as arbitrary units (au) by using the mean value of cells stimulated with control ig as c t calibrator or fold induction (f.i.) over control ig-stimulated cells after normalization to gapdh. the pil- (- )-gfp construct containing the gfp under the control of the − bp region upstream of the transcriptional start site and the + region upstream the translational start site was generated as previously described ( ) . briefly, human il- promoter fragment (− /+ ) was amplified from genomic dna isolated from human pbmcs by using amplitaq (applied biosystem). the primers used (excluding additional flanking cloning/restriction sites) were as follow: forward ′-ggtatttgcattttgatacttgcatttgct- ′ and ′-tgcagacaattctaactcgag- ′. by using an internal asei restriction site located at position − of the human il- promoter, the pil- (- )-gfp construct was obtained by subcloning the pcr fragment into asei-hind iii sites within the cmv promoter of pegfp-n vector (clontech, uk). the sequence of human il- promoter fragment (− /+ ) was verified by dna sequencing and was as follow: ′-attaata plasmid vectors expressing ha-tagged human rela, ikka, ikkb and nik were previously described ( , ) . cd -positive jurkat t cells were electroporated (at v, mf) in . ml rpmi- supplemented with % fbs with mg of pil- (- )-gfp together with pcdna control vector mg ha-rela, or ha-ikka or ha-ikkb or mg ha-nik. twenty-four hours after transfection, the cells were analyzed by using a bd facscan flow cytometer (bd biosciences, italy). mean fluorescence intensity was calculated on a total of × gfp-positive events by gating for ssc and fsc. chromatin immunoprecipitation cd + t cells were stimulated as indicated and chromatin immunoprecipitation (chip) assays were performed as previously described ( ) . briefly, after fixing in % formaldehyde, t cells were lysed for min in mm tris, ph . , mm edta, . % np- , % glycerol supplemented with proteases inhibitors. nuclei were suspended in mm tris, ph . , % sds, and mm edta. chromatin was sheared by sonication, centrifuged and diluted times in mm tris, ph . , . % np- , . m nacl, . mm edta. after pre-clearing with a % suspension salmon sperm-saturated protein-a or protein g sepharose beads (amersham), lysates were incubated at °c overnight with anti-rela ( : dilution), anti-relb ( mg), anti phoshotyr stat (pstat , : dilution), anti-rna-polymerase ii (pol ii, mg), or control anti-lck abs ( mg). immune complexes were collected with sperm-saturated protein-a or protein g sepharose beads, washed three times with high salt buffer ( mm tris, ph . , . % sds, % np- , mm edta, mm nacl), and five times with × tris/edta (te). immune complexes were extracted in × te containing % sds, and protein-dna cross-links were reverted by heating at °c overnight. dna was extracted by phenol-chloroform and about / of the immunoprecipitated dna was analyzed by real-time pcr. quantitative real-time pcr with sensimix ™ sybr ® hi-rox kit (bioline, uk) was performed for the human il- promoter. specific enrichment was calculated as previously described ( ) by using the cycle threshold (ct): (ct of control chip-ct of control input)/ (ct of specific chip -ct of specific input) . the human il- promoter primers used for each specific chip were as follow: rela i, relb i and pstat i, ′-gcttactcagcc actataggagatca- ′ and ′-ccggagggtatttta c a g a c a a a t c c - ′ ; rela ii, ′ -a c c c t c c g g g ctctaatagtgac- ′ and ′-agaacgacctaaccacca cagga- ′; pstat ii and pol ii ′-cctgtggtggtta ggtcgttct- ′ and ′-gctgcttttatagcccccag gat- ′. the cytotoxicity of as on caco- cells was evaluated by propidium iodide (pi) staining ( mg ml − ). caco- cells were plated at . × cells ml − in -well plates and treated with mm as or dmso, as vehicle control, for h. cytotoxicity was analyzed by a bd biosciences facscalibur (mountain view, ca) by quantifying the percentage of pi positive cells. results were calculated from at least three independent experiments. the sample size was chosen based on previous studies to ensure adequate power. parametrical statistical analysis (mean and sem) was performed to evaluate differences between continuous variables through prism . (graph pad software, san diego, ca) using student t test. for multiple group comparisons, significant differences were calculated using nonparametric mann-whitney u or wilcoxon tests, and linear regression analyses were performed using the pearson chi-squared test. for all tests, p values < . were considered significant. we have recently found that cd stimulation induces the expression of th related cytokines in cd + t cells from hd, relapsing-remitting ms (rrms) and t d patients ( ) ( ) ( ) . more recent preliminary data also evidenced that cd stimulation up-regulated il- gene expression in peripheral cd + t cells from stable rrms patients ( ) . in order to verify whether cd -induced il- expression was also effective in hd and clarify the molecular basis of this activity, we performed a kinetic analysis of il- gene expression by stimulating human cd + t cells from hd with an agonistic anti-cd ab (cd . ) binding the same epitope recognized by b molecules ( ) or anti-cd (ucht ) or anti-cd plus anti-cd abs. cd stimulation of cd + t cells from one representative hd induced a significant increase (p < . ) of il- gene expression within h that further increased to h and decreased h after stimulation ( figure a) . no significant differences in neither il- mrna levels nor kinetics were observed between cd and cd plus cd stimulation, whereas cd stimulation alone induced only a slight increase of il- gene expression after h ( figure a) . similar results were obtained by analyzing il- gene expression in cd -stimulated cd + t cells from a larger sample size (n = ) (figures b, c) . cd -induced il- gene expression was also associated with a strong increase of il- cytokine secretion after to h from stimulation ( figures d, e) . in the human system, il- gene expression has been described to be induced by il- ( ). consistently with our previous data ( ) , cd stimulation induced a strong il- secretion ( figure f ). il- -mediated signaling alone was not sufficient for inducing il- gene expression ( figure g) . however, the blockade of il- -mediated signaling by a neutralizing anti-il- ab strongly inhibited cd -induced il- gene expression ( figure h ) and secretion ( figure i ) in cd + t cells from hd. as we previously observed for il- a ( ) and other pro-inflammatory cytokines ( ) , the analysis of cd -induced il- secretion in purified naïve cd + cd ra + (figure a ) or effector/memory cd + cd ro + ( figure b ) evidenced no significant differences between the two population ( figure c) . these data evidence that cd intrinsic signaling regulates il- gene expression and secretion in both naïve and effector/ memory cd + t cells in a il- -dependent manner. the human il- gene promoter contains two putative stat responsive elements (stat i and ii) and two nf-kb binding sites (nf-kb i and ii) upstream of the transcription start site that have been involved in il- promoter trans-activation ( , ) ( figure a) . we have previously demonstrated that human cd individual stimulation induces the activation and nuclear translocation of rela ( , ) and, more recently, we evidenced that cd induces the phosphorylation on tyr of stat (pstat ) and its nuclear translocation in a il- dependent manner. pstat and rela/nf-kb in turn cooperate for inducing the promoter trans-activation and transcription of il- a ( ) . to assess if pstat and rela were also involved in cd -mediated up-regulation of il- , we performed chromatin immunoprecipitation (chip) assays in peripheral cd + t cells stimulated with agonistic anti-cd . abs. to do that, three distinct oligonucleotide probes were used; probe i (− to − ) for both stat i and nf-kb i binding sites, probe ii (− to − ) for nf-kb ii binding site and probe iii (− to − ) for both stat ii binding site and tata box ( figure a) . the kinetic analysis of the specific recruitment to the il- promoter evidenced that rela efficiently bound nf-kb i but not nf-kb ii consensus sequences within h from stimulation and persisted over h, whereas no significant recruitment of relb was observed ( figure b ). pstat was significantly recruited on both stat i and stat ii consensus sequences on the il- promoter with similar kinetics ( figure c ). the specific recruitment of both rela on the nf-kb i and pstat on both i and ii binding sites was confirmed in cd + t cells from a larger sample size (n = ) and was associated to cd -induced transcriptional activation of il- promoter, as evidenced by rna polymerase ii (pol ii) promoter occupancy ( figure d ). the significant inhibition of il- gene expression exerted by the stat inhibitor s - ( , ) and the nf-kb inhibitor ps ( ) (figure e ), confirmed the mutual cooperation of both transcription factors in regulating cd induced il- expression. the main functional effects of il- are exerted on epithelial cells, such as intestinal cells, where il- promotes the production of anti-microbial peptides, mucins and matrix metalloproteases (mmps) required for epithelial barrier functions and protection against extracellular pathogens ( , ) . we next analyzed the functional role of cd -mediated activation of il- -producing t cells on intestinal epithelial cells. to do that, cd + t cells were stimulated with control isotype matched abs (ig), or anti-cd , or anti-cd or anti-cd plus anti-cd abs and co-cultured in trans-well plates with caco- cells for different times. the gene expressions of mucin (muc ), mmp and s a anti-microbial peptide were then analyzed. the kinetic analysis evidenced that both mmp ( figure a ) and muc ( figure b ) gene expressions were strongly up-regulated in caco- cells after h of coculture with cd -stimulated cd + t cells, whereas no significant increase in s a was observed ( figure c ). similar results were obtained by analyzing mmp ( figure d ) and muc ( figure e ) gene expression in caco- cells cocultured with cd -stimulated cd + t cells from a larger sample size (n = ). moreover, the up-regulation and muc gene expression induced by co-culturing caco- cells with cd -stimulated cd + t cells was also associated with a strong increase of muc protein content ( figure f ) after h from stimulation. in contrast to the gene expression data ( figure d ), when we looked at mmp secretion we found high levels of mmp in culture supernatants from caco- cells cocultured with cd + t cells stimulated with control ig that further increased following cd stimulation (supplementary figure s a) , thus suggesting that cd + t cells also produced mmp , as previously reported ( ) . cd + t cells secreted, indeed, high levels of mmp in culture supernatant and no significant differences were observed following cd stimulation (supplementary figure s b) . figure h ) and secretion ( figure i ), muc gene expression was strongly impaired by anti-il- neutralizing abs in caco- cells cocultured with cd -stimulated cd + t cells ( figure h ). cd pro-inflammatory functions depend on the ability of its intracytoplasmic tail to recruit and activate class a pi k ( ) ( ) ( ) . we have recently demonstrated that class a pi k activity is crucial for cd -induced up-regulation of pro-inflammatory cytokines related to th cell phenotype in both hd and rrms patients ( , ) . consistently, a non-cytotoxic dose of the class a pi k inhibitor as ( ) (figures a, b) , significantly impaired mmp and muc gene expression in caco- cells co-cultured with cd -stimulated cd + t cells ( figure c ) as well as cd induced il il- ( figure d ) and il- secretion ( figure e) . moreover, the inability of recombinant il- to restore impaired il- gene expression in as -treated cd -stimulated cd + t cells, strongly support a role of class a pi k downstream cd . altogether these data support a crucial role of cd associated pi k in il- -regulated epithelial barrier functions. frontiers in immunology | www.frontiersin.org october | volume | article discussion il- is an important cytokine that strengthen epithelial barrier functions against extracellular pathogen and regulates tissue homeostasis and repair. however, excessive production of il- has been also associated with severe complications in several pathogen infections ( ) , including the recently identified sars-cov ( ) as well as with inflammatory bowel diseases such as crohn's disease ( ) , neurological disorders such as ms and guillain-barrè syndrome ( , ) , and rheumatoid arthritis ( , ) . therefore, the identification of receptors and associated signaling mediators regulating il- expression could represent an important goal of the ongoing research in inflammation and autoimmunity. in mouse t cells, il- is mainly regarded as a th cytokine ( , , ) , whereas in human cd + t cells il- expression often does not correlate with the expression of il- or the master transcriptional regulator of th cells retinoic acid receptor-related orphan nuclear receptor gt (rorgt) ( , ) and is produced by a unique memory th cell subset ( , ) . herewith, we evidence that cd stimulation induced il- expression and production in the absence of tcr engagement (figure ) . the up-regulation of il- transcription by cd autonomous stimulation of peripheral cd + t cell followed a kinetic ( figures a, b ) similar to il- a ( ) and no significant differences were observed between naïve (cd ra) and effector/ memory (cd ro) cd + t cells ( figure ) . moreover, we found that cd -induced up-regulation of il- depended on cd -induced il- ( figure f) , as demonstrated by the strong inhibitory effects exerted by neutralizing anti-il- abs ( figures h, i) . however, although il- itself is able to prime il- production in activated human cd + t cells ( ) , stimulation of cd + t cells with il- alone is not sufficient for up-regulating il- gene expression ( figure g ) but requires a second signal delivered by cd . in this contest, among the most promising therapeutic strategies for dampening excessive inflammatory responses and ards in sars-cov infection, tocilizumab, an anti-il- r mab inhibitor that interferes with il- -mediated signaling and has already approved for ra treatment ( , ) resulted effective in preventing the cytokine storm, ards and to reduce mortality in covid- patients ( ) ( ) ( ) . il- regulates the expression of both il- a and il- through il- receptor-associated stat ( , , ( ) ( ) ( ) ( ) . we have recently demonstrated that cd stimulation induced a strong and persistent stat phosphorylation on tyr and its nuclear translocation in cd + t cells. we also found that activated stat was also recruited to the proximal human il- a promoter and induced its trans-activation in response to cd stimulation ( ) . in t cells from stat deficient mice, il- expression is impaired ( , ) , whereas the overexpression of constitutively active stat up-regulates il- expression ( ) . for instance, two functional stat binding sites have been identified upstream of the transcription start site within the mouse il- promoter ( ) . the human il- promoter contains similar high consensus sequences ( figure a ) and we found that cd stimulation promoted the recruitment of stat on both binding sites in human cd + t cells ( figures c, d) . these data together with the strong inhibition of cd induced il- expression by the stat inhibitor s - ( figure e) , support a direct role of stat in regulating il- gene expression in cd -stimulated cd + t cells. in the human il- promoter, two putative nf-kb responsive elements ( ) , nf-kb i and ii (figure a) , containing the consensus binding sequence gggrnnyycc (where r represents a or g, n represents any nucleotide, and y represents c or t) has been also identified ( ) . the nf-kb family comprises five members: p /nf-kb , p /nf-kb , rela/p , relb and c-rel. rela, c-rel and relb contain a transactivation domain and form transcriptionally active heterodimers in association with p and/or p ( ) . we have demonstrated that cd autonomous stimulation by either b . /cd or anti-cd . agonistic abs activates a non-canonical nf-kb -like cascade ( ) by recruiting and activating nik and ikka ( , ) , thus leading to the nuclear translocation of rela-containing nf-kb dimers and to the transactivation of pro-inflammatory target cytokines, including il- a ( ) ( ) ( ) ) . consistently, rela overexpression strongly upregulated human il- promoter-gfp construct [hil- prom (- )-gfp], containing the gfp construct under the control of the il- ′-flanking region (− ) upstream of the transcriptional start point (supplementary figure s ) . moreover, as observed in ilc cells ( ) , rela/nf-kb binds the high homology consensus sequence ii at position − of the human il- promoter ( figure a) and cooperates with stat for inducing il- transcription in cd -stimulated cd + t cells (figures b, d) . although both ikka and ikkb induced a significant (p < . ) trans-activation of hil- promoter at similar levels ( . fold inductions) when overexpressed in jurkat cells, a strongest up-regulation ( fold inductions) of hil- promoter was induced by nik overexpression (supplementary figures s d, e) . these data are consistent with those from rudloff et al., who identified ikka as an important regulator of il- gene expression ( ) . il- regulates essential functions in host defense against pathogens by mediating the crosstalk between the immune system and epithelial cells. in fact, il- regulates the expression of several genes that maintain the barrier functions of intact epithelium ( ) . these include molecules involved in tight and gap junctions such as claudins that keep epithelial cells together and regulate the permeability of intestinal epithelial barrier to water, nutrients and ions ( ) , anti-microbial proteins such as s a ( , ) and mucins. muc , a membrane-bound mucin, is the main component of the intestinal mucus layer that provides a physiological barrier to prevent close contacts between epithelial cells and microbes ( ) . in addition to its physiological barrier functions, muc has been described to modulate the functions of inflammatory t cells. in muc -deficient mice, th cells and il- -producing ilc cells were expanded and exacerbated colitis ( ) . similarly, a reduced glycosylation of muc increased both susceptibility to colitis and progression to colon cancers ( , ) . in both human and mouse colonic epithelial cell lines, il- has been reported to promote muc expression ( , ) . we extend these data by evidencing an important role of cd in regulating epithelial barrier functions. during cd stimulation, rela/nf-kb and il- -associated stat cooperate for inducing il- expression and secretion ( figure ) that in turn acts on epithelial cells by promoting the up-regulation of muc ( figure ) . moreover, il- secreted by cd -activated cd + t cells also stimulate mmp expression in caco- cells (figure ) . interestingly, pujada et al. recently showed that mmp expression in colonic epithelium was associated with a more efficient epithelial barrier functions by maintaining epithelial integrity, microbiota and immune homeostasis ( ) . however, mmp is also up-regulated in inflamed intestine of ibd patients and has been recently suggested as a predictor marker of disease exacerbation in cd ( , ) . moreover, high levels of mmp were also found in the serum and cerebrospinal fluid of ms patients and increase during relapses ( , ) . thus, anti-inflammatory compounds able to dampen excessive production of mmps might represent potential therapeutic strategies for inflammatory diseases. our findings on the ability of class a pi k inhibitor to impair both cd -induced il- and il- production as well as il- -dependent mmp and muc expression in caco- cells ( figure ) are consistent with data from yan et al. who identified the pi k/akt/mtorc as a crucial pathway in regulating both th cell-mediated inflammation and lung injury in ards ( ) . our results provide novel insights on the role of cd and associated signaling mediators in il- regulation in human cd + t cells and may provide the biological bases for the development of new therapeutic strategies to dampen inflammatory and autoimmune disorders. the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. the studies involving human participants were reviewed and approved by ethical committee of the policlinico umberto i (ethical code n. bis/ , / / ). the patients/ participants provided their written informed consent to participate in this study. mk performed most of the experiments, analyzed the data, interpreted the results, and helped in writing the manuscript. ca, sf, sc, ms, and mb performed parts of the experiments and data analyses. sa contributed with human samples for the study. mk, ca, sf, ms, and lb were involved in the discussion about the data. lt designed the study, coordinated the work, and wrote the manuscript. all authors contributed to the article and approved the submitted version. il-tif/il- : genomic organization and mapping of the human and mouse genes differentiation and function of th t cells production of interleukin but not interleukin by a subset of human skinhoming memory t cells identification of a human helper t cell population that has abundant production of interleukin and is distinct from t(h)- , t(h) and t(h) cells th cells represent a distinct human t cell subset involved in epidermal immunity and remodeling interleukin- : a cytokine produced by t, nk and nkt cell subsets, with importance in the innate immune defense and tissue protection a human natural killer cell subset provides an innate source of il- for mucosal immunity interleukin- : immunobiology and pathology signaling at the epithelial-microbiota interface il- -producing cd + t cells: middle-men between the immune system and its environment il- and il- : siblings, not twins il- : there is a gap in our knowledge directing traffic: il- and il- coordinate pulmonary immune defense il- mediates mucosal host defense against gram-negative bacterial pneumonia rapamycin attenuates acute lung injury induced by lps through inhibition of th cell proliferation in mice a novel coronavirus from patients with pneumonia in china clinical features of patients infected with novel coronavirus in wuhan th responses in cytokine storm of covid- : an emerging target of jak inhibitor fedratinib marked t cell activation, senescence, exhaustion and skewing towards th in patients with covid- pneumonia emerging role of interleukin- in autoimmune diseases clinical importance of il- cascade in ibd interleukin- in human inflammatory diseases and viral infections the interleukin- cytokines in intestinal diseases exploring the role of interleukin- in neurological and autoimmune disorders the interleukin- family of cytokines and their role in the cns the modulators of inflammation in multiple sclerosis. cells cd costimulatory signals in t lymphocyte activation: emerging functions beyond a qualitative and quantitative support to tcr signalling cd and ctla- coreceptor expression and signal transduction signaling amplification at the immunological synapse nf-kappab family of transcription factors: biochemical players of cd co-stimulation a non-conserved amino acid variant regulates differential signalling between human and mouse cd cd ligation in the absence of tcr stimulation up-regulates il- a and pro-inflammatory cytokines in relapsing-remitting multiple sclerosis t lymphocytes cd individual signaling up-regulates human il- a expression by promoting the recruitment of rela/nf-kb and stat transcription factors on the proximal promoter isa- b, a phosphatidylinositol -phosphate -kinase alpha inhibitor, impairs cd -dependent costimulatory and pro-inflammatory signals in human t lymphocytes cd autonomous signaling up-regulates c-myc expression and promotes glycolysis enabling inflammatory t cell responses in multiple sclerosis mechanisms of rapid induction of interleukin- in activated t cells and its modulation by cyclosporin a rela/nf-kappab recruitment on the bax gene promoter antagonizes p -dependent apoptosis in costimulated t cells mitogen-activated kinase kinase kinase regulates t cell receptor-and cd -mediated signaling events which lead to nf-kappab activation epigenetic remodeling of the il- and ifn-gamma loci in memory cd t cells is influenced by cd t cells differential recognition by cd of its cognate counter receptors cd (b . ) and b (b . ): analysis by site directed mutagenesis interleukin- , a t(h) cytokine, mediates il- -induced dermal inflammation and acanthosis il- induces il- production in cd + t cells il- drives ilc proliferation and promotes il- production via nf-kappab cd delivers a unique signal leading to the selective recruitment of rela and p nf-kappab subunits on il- and bcl-xl gene promoters isoform selective inhibition of stat or stat homo-dimerization via peptidomimetic probes: structural recognition of stat sh domains selective chemical probe inhibitor of stat , identified through structurebased virtual screening, induces antitumor activity effects of ikk inhibitor ps on nf-kappab function, proliferation, apoptosis and invasion activity in prostate carcinoma cells human t lymphocytes synthesize the kda type iv collagenase (gelatinase b) phosphatidylinositol -phosphate -kinase alpha activation critically contributes to cd -dependent signaling responses phosphatidylinositol -phosphate -kinase alpha and vav mutual cooperation in cd -mediated actin remodeling and signaling functions cutting edge: cd -mediated transcriptional and posttranscriptional regulation of il- expression are controlled through different signaling pathways functional redundancy of class i phosphoinositide -kinase (pi k) isoforms in signaling growth factor-mediated human neutrophil survival emerging cell and cytokine targets in rheumatoid arthritis the il- cytokine family in rheumatoid arthritis and spondyloarthritis interleukin (il)- and il- are coexpressed by th cells and cooperatively enhance expression of antimicrobial peptides expression and regulation of il- in the il- -producing cd + t lymphocytes cd -mediated regulation of il- frontiers in immunology | www.frontiersin.org october a critical function for transforming growth factor-beta, interleukin and proinflammatory cytokines in driving and modulating human t(h)- responses multiparametric analysis of cytokine-driven human th differentiation reveals a differential regulation of il- and il- production tocilizumab monotherapy versus adalimumab monotherapy for treatment of rheumatoid arthritis (adacta): a randomised, double-blind, controlled phase trial a new era for the treatment of inflammatory autoimmune diseases by interleukin- blockade strategy tocilizumab treatment in covid- : a single center experience tocilizumab, an anti-il receptor antibody, to treat covid- -related respiratory failure: a case report tocilizumab in patients with severe covid- : a retrospective cohort study il- programs t (h)- cell differentiation by promoting sequential engagement of the il- and il- pathways il- is produced by th cells and drives il- production in a stat -dependent manner stat regulates cytokine-mediated generation of inflammatory helper t cells diverse targets of the transcription factor stat contribute to t cell pathogenicity and homeostasis essential autocrine regulation by il- in the generation of inflammatory t cells interactions of nf-kappab with chromatin: the art of being at the right place at the right time shared principles in nf-kappab signaling a novel association between filamin a and nf-kappab inducing kinase couples cd to inhibitor of nf-kappab kinase alpha and nf-kappab activation vav- and the ikk alpha subunit of i kappa b kinase functionally associate to induce nf-kappa b activation in response to cd engagement il- increases permeability of intestinal epithelial tight junctions by enhancing claudin- expression il- regulates the expression of genes responsible for antimicrobial defense, cellular differentiation, and mobility in keratinocytes: a potential role in psoriasis structure and function of the cell surface (tethered) mucins the membrane-bound mucin muc regulates t helper -cell responses and colitis in mice cutting edge: transgenic expression of human muc in il- -/-mice accelerates inflammatory bowel disease and progression to colon cancer increased susceptibility to colitis and colorectal tumors in mice lacking core -derived o-glycans il- ameliorates intestinal inflammation in a mouse model of ulcerative colitis expression of il- , an activator of the jak /stat /socs cascade, is enhanced in inflammatory bowel disease matrix metalloproteinase mmp maintains epithelial barrier function and preserves mucosal lining in colitis associated cancer ecm remodelling in ibd: innocent bystander or partner in crime? the emerging role of extracellular molecular events in sustaining intestinal inflammation serum mmp- : a novel biomarker for prediction of clinical relapse in patients with quiescent crohn's disease, a post hoc analysis interplay between matrix metalloproteinase- , matrix metalloproteinase- , and interleukins in multiple sclerosis patients imaging matrix metalloproteinase activity in multiple sclerosis as a specific marker of leukocyte penetration of the blood-brain barrier the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . / full#supplementary-material key: cord- -js nr d authors: yu, junyang; wu, yuzhang; wang, jingxue title: activation and role of nacht, lrr, and pyd domains-containing protein inflammasome in rna viral infection date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: js nr d nacht, lrr, and pyd domains-containing protein (nlrp ) inflammasome activation and effects during ribonucleic acid (rna) viral infection are the focus of a wide range of research currently. both the pathogen-associated molecule pattern derived from virions and intracellular stress molecules involved in the process of viral infection lead to activation of the nlrp inflammasome, which in turn triggers inflammatory responses for antiviral defense and tissue healing. however, aberrant activation of the nlrp inflammasome can instead support viral pathogenesis and promote disease progression. here, we summarize and expound upon the recent literature describing the molecular mechanisms underlying the activation and effects of the nlrp inflammasome in rna viral infection to highlight how it provides protection against rna viral infection. the ribonucleic acid (rna) virus has single-stranded (ss)rna or double-stranded (ds)rna as its genetic material. infections with rna viruses are responsible for a variety of diseases are significant threats to public health, including influenza, hepatitis, viral encephalitis, and autoimmune deficiency syndrome, to name a few of the most prominent. host antiviral defense mechanisms are critical for clearing rna viral infections and controlling the manifested diseases. the initiation of antiviral defense in response to rna viruses involves interferon (ifn) response and inflammasome activation. the rna viral infection usually leads to activation of the nacht, lrr, and pyd domainscontaining protein (nlrp ) inflammasome, which is the intracellular multiprotein complex formed upon sensing pathogen-associated molecule patterns and/or damage-associated molecule patterns ( ) . the inflammasome response is critical for the innate immune response's ability to initiate innate and adaptive immunity pathways during rna viral infections ( ) . the nlrp inflammasome has been extensively investigated. its major components include pro-caspase- and apoptosis-associated speck-like protein containing a card (asc), in addition to the nlrp molecules. upon sensing activating signals, the nlrp oligomerize to generate a caspase- -activating scaffold, resulting in autocleavage, and activation of pro-caspase- . the activated caspase- then shears pro-interleukin (il)- β and pro-il- into il- β and il- , leading to production of their functional forms and release from cells to mediate protective (or sometimes detrimental) inflammatory responses. the activated caspase- , however, can also promote caspase- -dependent pyroptosis, a type of inflammatory programmed cell death; although, the consequence of pyroptosis induced by the nlrp inflammasome remains to be fully elucidated in rna viral infection ( ) . table . this review will focus on recent advances in our knowledge of the activation mechanisms and functions of the nlrp inflammasome in response to such rna viral infections, discussing the related protective, and/or detrimental mechanisms that may represent manipulable targets for prevention and/or treatment. priming mechanisms of the nlrp inflammasome in rna viral infections nacht, lrr, and pyd domains-containing protein inflammasome activation requires two types of signals. signal primes the nlrp inflammasome and signal initiates the sequence of its assembly, activation of the pro-caspase- , and cleavage of pro-il- β and pro-il- . during rna viral infections, signal is usually derived from the signals evoked upon pathogen recognition by toll-like receptors (tlrs) ( ) and retinoic acid-inducible gene-i (rig-i)-like receptors ( ) . recent research has shown that signal triggering of the nlrp inflammasome involves transcriptiondependent and post-translation-dependent priming. the transcription-dependent priming is executed by activation of nuclear factor (nf)-κb and ifn-regulatory factor transcription factors, and subsequent up-regulation of transcription of genes encoding the major inflammasome components through the classical tlr signaling pathway: the myd -mediated and trif-mediated pathway. the specific role of tlr was shown by the reduced pro-il- β transcription in bone marrow-derived macrophages from tlr −/− mice in response to h of iav infection ( ) . another in vivo study of the iav infection model provided evidence that commensal bacteria lipopolysaccharide can provide priming signal ( ) . investigations of vsv infection showed that rig-i promotes the synthesis of pro-il- β through engagement by the viral ′-triphosphate rna ( prna). moreover, bone marrow-derived cells lacking rig-i, as well as the downstream molecules mavs and card , showed reduced synthesis of pro-il- β but normal caspase- p subunits when stimulated with vsv and prna ( ) , suggesting that the rig-i/mavs/card signaling pathway provides the signal in vsv infection. post-translation-dependent priming occurs upon the simultaneous engagement of tlrs and nod-like receptors with their ligands, even causing rapid activation of the nlrp inflammasome (within min) ( , ) . viral rna (vrna) moleculeinduced nlrp inflammasome activation also comes about by post-translational priming, but involving the rip /caspase /rip signaling pathway ( , ) , which may ultimately result in the deubiquitination of nlrp protein by brcc ( , ) . rip -deficient mice showed reduced caspase- activation and il- β production following infection with vsv, sendai virus, or iav ( ) . bone marrow-derived macrophages and mice lacking rip both displayed substantial reduction in caspase- cleavage and levels of il- β and il- following challenge with iav ( , ) . in various rna viral infection studies, the rip / rip -mediated signaling pathway has been demonstrated to provide the endogenous signal for the nlrp inflammasome, being derived from reactive oxygen species (ros) production by mitochondrial fission initiated by the rip /rip /drp pathway ( , , ) . signal is responsive to a wide range of stimuli [i.e., nlrp agonists, such as adenosine triphosphate (atp), silica, alum, and ultraviolet radiation]. however, the spectrum of different structures represented by these varied and distinctive agonists indicates that nlrp does not simply bind directly to each but instead relies on a common intracellular signal transmission pathway, triggered by the agonists themselves, for activation of the nlrp inflammasome. to date, five major signal mechanisms have been proposed as those responsible for or contributing to the nlrp inflammasome activation ( , ) . in rna viral infections, in particular, viroporins and some viral functional proteins act as the stimuli for signal (as shown in table ) . viroporins are a group of virus-encoded proteins that enhance permeability of host cell membrane (through interactions with the lipid bilayer) to promote release of viral particles from cells ( ) , making them a critical component of virus replication and virion release in the host system. the viroporin effect on permeability also modifies the cell's ability to regulate ion passage, disrupting ion homeostasis ( ) . several viroporins, such as the iav m protein ( ), rsv sh protein ( ) , hcv p protein ( ), sars-cov e protein ( ), emcv b protein ( ) , and rhinovirus b protein ( ) , have been reported to activate the nlrp inflammasome by disturbing intracellular ionic concentrations, particularly through potassium efflux, calcium flux, and ph alteration. in the viroporin-induced nlrp activation itself, the host-encoded nek protein may contribute to the nlrp inflammasome assembly and activation via formation of the nlrp -nek complex ( ) . among the other viral proteins involved in nlrp inflammasome activation are iav pb -f (a small protein encoded by an alternate + open reading frame in the viral pb gene) ( ) and ev d protein (an rna-dependent rna polymerase) ( ) . pb -f activates the nlrp inflammasome through mitochondrial ros production, which could be a major mechanism of pathogenic inflammation induced by highly pathogenic iav strains ( ) . the zbp protein can interact with rip upon sensing the viral protein pb , and contributes to nlrp inflammasome activation in iav but not in vsv infection through post-translational regulation ( ) . in contrast, the ev d protein can directly bind to nlrp and asc, and promotes nlrp inflammasome assembly and activation ( ) . plasma vrna, another common agonist for nlrp , may bind with nlrp and act to subsequently activate it. although nlrp contains a nucleotide-binding domain ( ), rna-sensing molecules have been reported to participate in the process by which vrna activates the nlrp inflammasome; these include the dexd/h-box helicase (dhx) family members ( , , , ) and the ′, ′-oligoadenylate synthetase (oas, or - a)/rnase l system ( ) . however, some debate exists as to the exact role of dhx , in particular, in vsv-induced nlrp inflammasome activation ( ) . the dhx family members contain some domains required for atp binding, hydrolysis, and nucleic acid binding and unwinding, which usually acts as a cytosolic rna sensor and triggers type i ifn response. dhx , ddx a, and ddx have been reported to regulate nlrp inflammasome activation. dhx was shown to combine directly with vrna to bind to nlrp through the dead domain of dhx and the nacht domain of nlrp , forming a dhx /nlrp /asc inflammasome complex to promote nlrp inflammasome activation ( ) . in that same study, down-regulation of dhx expression led to a substantial reduction in cleavage of caspase- and secretion of il- and il- β from the human monocyte cell line, thp- , upon stimulation with viral dsrna purified from reovirus, and produced by rsv during the replication process ( ) . dhx has also been demonstrated to contribute to nlrp inflammasome activation and il- β secretion during vsv and iav infections ( ) , the process of which was found to be dependent upon the oas/rnase l system induced by ifn. oass recognize cytosolic viral dsrna and subsequently trigger synthesis of - a in the presence of three atp molecules. then, rnase l is activated by - a and cleaves intracellular vrna. these rnase l-mediated rna cleavage products will directly interact with dhx and induce a dhx /mavs/nlrp complex to trigger assembly of the nlrp inflammasome. another study demonstrated that ddx a binds porcine reproductive and respiration syndrome virus (prrsv) genomic rna through its atp-binding domain and c-terminal domain and directly interacts with the nlrp inflammasome in highly pathogenic-prrsv-infected primary porcine alveolar macrophages, dependent on the ddx a atp-binding domain, and figure | activation and effects of nacht, lrr, and pyd domainscontaining protein (nlrp ) inflammasome in response to ribonucleic acid (rna) virus infections. viroporin, viral nucleic acids, and some virus-encoded functional proteins are able to activate the nlrp inflammasome. viroporin-mediated ion concentration change, virus replication-produced reactive oxygen species, and even viral protein direct binding promote assembly of the nlrp inflammasome. the cytokines interleukin (il)- and il- contain extensive downstream effector molecules and effector cells, which ultimately orchestrate innate and adaptive immunity, leading to antiviral defense, and/or inflammatory injury during rna virus infections. the nlrp nacht and lrr domains. knockdown of ddx a expression led to reductions in both il- β secretion and virus titers in the prrsv-infected porcine alveolar macrophages ( ) . thus, ddx a appears to be required for both antiviral defense and nlrp inflammasome activation. rig-i (ddx ) is a key intracellular sensor for cytosol ssrna and provides the transcriptional priming signal for nlrp inflammasome during infections with vsv ( ) and iav ( ) , upon recognition of the vrna. however, in primary respiratory epithelial cells, iav infection promotes rig-i binding with asc and caspase- , implying that rig-i contributes to inflammasome assembly ( ) . during the process of rna viral infection, nlrp inflammasome activation contributes to either enhancing antiviral defense and tissue healing, or inflammatory pathological injury, which depends mainly on the time point and extent of nlrp activation. the appropriate and early phase activation of the nlrp inflammasome in some rna viral infection systems, such those of iav, encephalomyocarditis virus, and west nile virus (wnv) (shown in table ), usually provide protection, thereby decreasing mortality and viral load, as shown in virus-infected mouse models ( , , , ) . in rhesus monkeys, neither nlrp inflammasome activation nor ifn response can be established successfully within h following challenge with the simian immunodeficiency virus, which may be the key reason underlying the observation of viral dissemination occurring rapidly from the inoculation site to a distal site (within the first day of infection) ( ) . reciprocally, when using hiv- -specific broadly neutralizing antibodies, pgt is capable of inducing innate immune responses such as nlrp inflammasome activation in vrna-positive tissues within h; in addition, viral replication is reduced in distal tissues significantly ( ) . these findings imply the early nlrp inflammasome activation as well as ifn response play critical roles in establishment of effective antiviral defense. similarly, the administration of mc (a specific nlrp inhibitor) on days , , and but not days - following iav challenge also resulted in accelerated weight loss and mortality in iav-infected mice ( ) . at weeks after challenge with iav, the mortality rates among nlrp −/− , caspase- −/− ( , ), and il- ri −/− ( ) mice are significantly higher (by - %) than for the wild-type counterparts. thus, proper nlrp inflammasome activation in the early phase of rna viral infection protects the infected host. in contrast, if activated at a later phase or over-activated, the nlrp inflammasome response to rna virus results in inflammatory injury. production of il- β and il- , and pyroptosis are the critical and fundamental reactions directly induced by activated nlrp inflammasome (figure ) . il- β and il- serve to activate myriad downstream cell responses, and orchestrate innate and adaptive immunity through myd /irak /traf -mediated nf-κb signaling and the jnk/p mitogen-activated protein kinase pathways ( - ), which may represent key events for the nlrp inflammasome-dependent antiviral defense. pyroptosis is a pro-inflammatory caspase-dependent programed cell death ( ). nlrp inflammasome-dependent pyroptosis has been described in hcv-infected hepatoma cells ( ) , dengue virus (dv)-infected monocytes ( ) , and hiv-infected podocytes ( ) . the data to date suggest that pyroptosis in viral infection leads to host injury and pathogenesis. in mice, iav-induced nlrp inflammasome activation initiates protective inflammation by recruitment of monocytes and neutrophils to the lung (as noted in the bronchoalveolar lavage fluid), which occurs within - days of the iav infection, and the subsequent promotion of secretion of various cytokines, such as tumor necrosis factor (tnf)-α, ifn-α, and mip- α ( , ) . furthermore, the impaired il- response by il- ri deficiency results in impaired production of immunoglobulin m, reduced recruitment of cd + t cells in the bronchoalveolar lavage fluid ( ) , and impaired priming of cd + t cells in the lung and draining mesenteric lymph nodes through the intervention of cd b lo cd + dendritic cell migration and maturation ( ) . in a recent study, il- was confirmed to be required for ifn-γ production in cd + vα . + mucosal-associated invariant t cells, which has been observed clinically to be increased in recovered but not succumbed patients hospitalized with avian h n influenza pneumonia and which implies a protective role in influenza virus infection ( ) . in wnv infected mice, the il- β produced by cd + cd b + infiltrating t cells promotes the wnv-specific cd + t cell entry to the central neuronal system nlrp inflammasome induced by rna virus frontiers in immunology | www.frontiersin.org october | volume | article via regulation of cxcl -mediated t cell adhesion, eventually controlling the viral infection ( , ) . in bv- mouse microglia cells infected by japanese encephalitis virus, the nlrp inflammasome induces production of il- β and il- rapidly (within h of exposure) and of tnf-α, ccl , and il- later (within h after exposure) ( ) ; the findings suggest that the nlrp dependent protective inflammatory response is a very early phase innate immune response against rna viral infection. in addition to initiating protective inflammation to regulate and orchestrate innate and adaptive immunity, the nlrp inflammasome also promotes tissue healing and reduces respiratory epithelial necrosis, which contributes to enhancing host disease tolerance ( , ) . thus, the nlrp inflammasome provides protective roles through enhancing host tolerance capacity and orchestrating innate immunity and adaptive immunity during rna viral infections. aberrant nlrp inflammasome activation usually results in progression of viral pathogenesis and the manifested disease in host, such as the robust nlrp activation induced by pb -f during the iav infection ( ). the major pathogenic mechanism associated with pb -f -induced aberrant nlrp inflammasome activation involves excessive neutrophil influx ( , ) . the nlrp activation induced by hcv promotes chronic intrahepatic inflammation in macrophages ( ) and interrupts cellular lipid metabolism ( ) in hcv-infected hepatoma cells, both of which contribute to chronic liver injury and liver disease progression. in hiv-infected microglia, nlrp inflammasome activation is involved in the occurrence of chronic neuroinflammation ( ) . in addition, nlrp -dependent pyroptosis during rna viral infection also supports the pathogenesis of virus infection diseases. hiv-induced podocyte pyroptosis could be associated with hiv-associated nephropathy ( ) . nlrp inflammasome-mediated pyroptosis is also observed in dv-infected monocytes ( ) and in hcv-infected and bystander hepatoma cells ( ) , and could be associated with viral pathogenesis ( table ) . unlike rna viruses, the dna viruses, which have a large dna genome, can encode viral homologs of some complex intracellular inflammasome-regulating molecules to regulate inflammasome activity; an example of this is the pyrin-only proteins and bcl- encoded by poxvirus ( , ) . the rna viruses, in contrast, have evolved some simple yet effective strategies to inhibit activation of the nlrp inflammasome and evade host immunity. specifically, the proteases c and a of ev are able to cleave nlrp at the g -l or q -g junction ( ) . furthermore, the protease c of ev is capable of degrading gasdermin d, a protein critical to the induction of pyroptosis ( ) , and host failure to inhibit ev replication ( ) . the ns protein of influenza virus can bind with nlrp directly, thereby inhibiting assembly of the nlrp -asc-caspase- complex and secretion of il- β; this inhibition is dependent on rna and the trim- domain of the ns protein ( ) . the measles virus v protein also targets nlrp , probably serving to interfere with the nlrp inflammasome assembly and ultimately facilitating escape from the host immune response ( ) . finally, the non-structural protein encoded by prrsv also antagonizes the nlrp inflammasome effect in the macrophages, through decreasing expression of pro-il- β ( ). nacht, lrr, and pyd domains-containing protein inflammasome activation in response to rna virus is a fundamental innate immune response as well as an ifn response, which usually contributes to antivirus defense, although aberrant and inappropriate nlrp inflammasome response still results in the immunopathology and exaggeration of infectious disease, especially in some persistent virus infections. the activation itself is induced by different signals and probably occurs in different phases of the viral infection, triggering inflammatory response (appropriate/protective or inappropriate/detrimental), and affecting outcome of the viral infection substantially. thus, the proper regulation of nlrp inflammasome activation with specific nlrp inhibitors may have therapeutic benefit in controlling virus-related diseases and in clearance of the infecting virus. jy collecting literatures and drafting manuscript. jw design of the work, collecting literatures, drafting, and revising manuscript. yw discussing literatures, design of the work, and revising manuscript. this work was supported by grants from the national natural science foundation of china (grant no. and ) and the natural science foundation of chongqing (grant no. cstc, bb ). the inflammasomes inflammasome control of viral infection viruses and the diversity of cell death the nlrp inflammasome mediates in vivo innate immunity to influenza a virus through recognition of viral rna the intracellular sensor nlrp mediates key innate and healing responses to influenza a virus via the regulation of caspase- frontiers in immunology | www.frontiersin.org october influenza virus activates inflammasomes via its intracellular m ion channel activation of the nlrp inflammasome by iav virulence protein pb -f contributes to severe pathophysiology and disease zbp /dai is an innate sensor of influenza virus triggering the nlrp inflammasome and programmed cell death pathways reassessing the role of the nlrp inflammasome during pathogenic influenza a virus infection via temporal inhibition hiv promotes nlrp inflammasome complex activation in murine hiv-associated nephropathy hiv- tat primes and activates microglial nlrp inflammasome-mediated neuroinflammation hiv- viral protein r activates nlrp inflammasome in microglia: implications for hiv- associated neuroinflammation interleukin coexpression during respiratory syncytial virus infection results in enhanced disease mediated by natural killer cells the dhx rna helicase senses cytosolic rna and activates the nlrp inflammasome human respiratory syncytial virus viroporin sh: a viral recognition pathway used by the host to signal inflammasome activation il- beta signaling promotes cns-intrinsic immune control of west nile virus infection il- r signaling regulates cxcl -mediated t cell localization and fate within the central nervous system during west nile virus encephalitis il- beta production through the nlrp inflammasome by hepatic macrophages links hepatitis c virus infection with liver inflammation and disease hcv genomic rna activates the nlrp inflammasome in human myeloid cells the hepatitis c virus-induced nlrp inflammasome activates the sterol regulatory element-binding protein (srebp) and regulates lipid metabolism the p viroporin of the hepatitis c virus contributes to liver inflammation by stimulating production of interleukin- beta platelets mediate increased endothelium permeability in dengue through nlrp -inflammasome activation resistance of interleukin- beta-deficient mice to fatal sindbis virus encephalitis the inflammatory cytokine, interleukin- beta, mediates loss of astroglial glutamate transport and drives excitotoxic motor neuron injury in the spinal cord during acute viral encephalomyelitis effect of interleukin- on viral myocarditis: enhancement of interferon-gamma and natural killer cell activity encephalomyocarditis virus viroporin b activates nlrp inflammasome the nlrp inflammasome detects encephalomyocarditis virus and vesicular stomatitis virus infection rna viruses promote activation of the nlrp inflammasome through a rip -rip -drp signaling pathway rnase l activates the nlrp inflammasome during viral infections rhinovirus infection of primary cultures of human tracheal epithelium: role of icam- and il- beta rhinovirus-induced calcium flux triggers nlrp and nlrc activation in bronchial cells reciprocal regulation between enterovirus and the nlrp inflammasome ev d protein binds with nlrp and enhances the assembly of inflammasome complex stimulation of host-defense mechanism with synthetic adjuvants and recombinant cytokines against viral infection in mice critical role for cryopyrin/nalp in activation of caspase- in response to viral infection and double-stranded rna zika virus induces inflammasome activation in the glial cell line u -mg involvement of nlrp inflammasome in cvb -induced viral myocarditis japanese encephalitis virus differentially modulates the induction of multiple pro-inflammatory mediators in human astrocytoma and astroglioma cell-lines nlrp inflammasome: key mediator of neuroinflammation in murine japanese encephalitis recognition of rna virus by rig-i results in activation of card and inflammasome signaling for interleukin beta production microbiota regulates immune defense against respiratory tract influenza a virus infection cutting edge: tlr signaling licenses irak for rapid activation of the nlrp inflammasome irak- bypasses priming and directly links tlrs to rapid nlrp inflammasome activation caspase- scaffolding function and mlkl regulate nlrp inflammasome activation downstream of tlr non-transcriptional priming and deubiquitination regulate nlrp inflammasome activation deubiquitination of nlrp by brcc critically regulates inflammasome activity the rip -rip complex initiates mitochondrial fission to fuel nlrp regulation of inflammasome activation viroporins: structure and biological functions nek is an essential mediator of nlrp activation downstream of potassium efflux pb -f derived from avian influenza a virus h n induces inflammation via activation of the nlrp inflammasome cryopyrin/nalp binds atp/datp, is an atpase, and requires atp binding to mediate inflammatory signaling type i ifn triggers rig-i/tlr /nlrp -dependent inflammasome activation in influenza a virus infected cells ddx a senses viral rna and mediates nlrp -dependent inflammasome activation interleukin- is responsible for acute lung immunopathology but increases survival of respiratory influenza virus infection rapid inflammasome activation following mucosal siv infection of rhesus monkeys antibodymediated protection against shiv challenge includes systemic clearance of distal virus the role of il- in innate immunity the il- family: regulators of immunity interleukin- (il- ) pathway molecular mechanisms and functions of pyroptosis, inflammatory caspases and inflammasomes in infectious diseases hepatitis c virus infection of cultured human hepatoma cells causes apoptosis and pyroptosis in both infected and bystander cells dengue virus-infected human monocytes trigger late activation of caspase- , which mediates pro-inflammatory il- beta secretion and pyroptosis il- r signaling in dendritic cells replaces pattern-recognition receptors in promoting cd (+) t cell responses to influenza a virus human mucosal-associated invariant t cells contribute to antiviral influenza immunity via il- -dependent activation mx reveals innate pathways to antiviral resistance and lethal influenza disease pyrin-and card-only proteins as regulators of nlr functions inflammasomes and its importance in viral infections cleavage of gsdmd by inflammatory caspases determines pyroptotic cell death enterovirus inhibits pyroptosis through cleavage of gasdermin d the rna-and trim -binding domains of influenza virus ns protein are essential for suppression of nlrp inflammasome-mediated interleukin- beta secretion measles virus v protein inhibits nlrp inflammasome-mediated interleukin- beta secretion the endoribonuclease activity essential for the nonstructural protein of porcine reproductive and respiratory syndrome virus to inhibit nlrp inflammasome-mediated il- beta induction key: cord- -x xo t authors: theresine, maud; patil, neha d.; zimmer, jacques title: airway natural killer cells and bacteria in health and disease date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: x xo t natural killer (nk) cells are innate lymphoid cells at the interface between innate and adaptive immunity and mostly studied for their important roles in viral infections and malignant tumors. they can kill diseased cells and produce cytokines and chemokines, thereby shaping the adaptive immune response. nowadays, nk cells are considered as a strong weapon for cancer immunotherapy and can for example be transduced to express tumor-specific chimeric antigen receptors or harnessed with therapeutic antibodies such as the so-called nk engagers. whereas a large body of literature exists about the antiviral and antitumoral properties of nk cells, their potential role in bacterial infections is not that well delineated. furthermore, nk cells are much more heterogeneous than previously thought and have tissue-characteristic features and phenotypes. this review gives an overview of airway nk cells and their position within the immunological army dressed against bacterial infections in the upper and predominantly the lower respiratory tracts. whereas it appears that in several infections, nk cells play a non-redundant and protective role, they can likewise act as rather detrimental. the use of mouse models and the difficulty of access to human airway tissues for ethical reasons might partly explain the divergent results. however, new methods are appearing that are likely to reduce the heterogeneity between studies and to give a more coherent picture in this field. historically, human natural killer (nk) cells have mostly been harvested from and studied in peripheral blood (pb), which is an easy way to access them, and where they usually represent - % of all lymphocytes ( ) ( ) ( ) . two different subsets have been initially described, called cd bright cd − (up to % of pb nk cells) and cd dim cd bright (at least % of pb nk cells). phenotypic and functional (cytokine production for the former and cytotoxic activity for the latter) characteristics distinguish both populations ( ) ( ) ( ) . however, things are not that simple, as four additional subpopulations have been identified, which are (i) cd bright cd dim , (ii) cd dim cd − , (iii) cd − cd bright and finally (iv) cd dim cd dim ( ) , the latter still being almost systematically overlooked in the literature ( ) . human nk cell functions are governed by a balance between the messages transmitted through inhibitory receptors (ir), such as kir, cd /nkg a, ilt , tigit, and activating receptors (ar), such as particularly nkg d and the natural cytotoxicity receptors (ncr) nkp , nkp , and nkp ( ). when stimulated, nk cells exert natural cytotoxic activity against tumor cells and virally infected cells, antibody-dependent cellular cytotoxicity (adcc) toward antibody-coated target cells via the fcγ receptor cd , and cytokine and growth factor production ( , ) . most of the ligands of the ir are represented by human leukocyte antigen (hla) class i molecules, so that target cells lacking those molecules in part or in total, become killed by the nk cells. the ir nevertheless have another important function, as they are responsible for nk cell education. indeed, before a developing nk cells becomes functional, its self-specific ir must interact with their ligands expressed by cells in their micro-environment ( , ) . nk cells without such ir, which can represent up to % of all pb nk cells, remain uneducated, and hyporesponsive ( , ) . however, they can be activated under certain conditions, such as some viral infections ( ) . a hot topic in the nk cell field is of course their potential use as immunotherapeutic anticancer agents. to reach this aim, several approaches are studied, and for example the chimeric antigen receptor (car) nk cells, which allow the specific targeting of a tumor antigen ( ) , or the use of multi-specific antibody constructs directed simultaneously at several nk cell ar and tumor surface molecules ( ), appear as particularly promising. it has also been discovered that nk cells, which had been previously considered as exclusively innate immune cells, can develop a memory-like behavior ( ) . finally, nk cell metabolism, which appears to be different between educated and uneducated cells, is extensively studied ( , ) . another aspect that has changed our view on nk cells in recent years is the observation of a broad heterogeneity of this population. not only are there many subsets in pb based on the clonal distribution of several ir, immature, partly mature and completely mature fractions based on the relative expression of cd , cd and the ir nkg a and kir ( ) , conventional and adaptive nk cells ( , ) , but there are also heterogeneous aspects between pb and various tissues ( , ) . very recent data by dogra et al. ( ) suggests a model in which the phenotype, the degree of maturity and the functions of nk cells are closely dependent on the anatomic location, with no influence of age and gender. it is quite difficult to find a substantial amount of references regarding upper airway nk cells. in human, the articles were mostly reporting on the investigation of nk cells in chronic rhinosinusitis, an inflammatory state of the mucosa of the nose and the sinuses ( ) with a significant impact on quality of life. two different forms, one with nasal polyps and one without nasal polyps, are distinguished ( , ) . bacterial pathogens are considered as one of the etiological factors in this disease ( ) . however, as the bacteriology of ethmoidal biopsies was the same regardless of the presence or absence of polyps, niederfuhr et al. questioned the bacterial role in the pathogenesis of the polyps as well as a systematic antibiotic treatment ( ) . in a study of patients, further subdivided into those responding and those resistant to treatment, and healthy controls, kim et al. investigated exclusively pb nk cells. the authors demonstrated that the pb nk cells from the patients had decreased effector functions compared to the healthy controls, with the treatmentresistant individuals being most severely affected ( ) . the recent manuscript by kaczmarek et al. ( ) reported not only on pb nk cells, but also on those from nasal mucosa and from nasal polyps. however, the exploitation of the material was limited to cd − cd + cd + events, which excluded the population of cd bright cd − nk cells that might be numerically well represented in these tissues. the phenotypic investigations of this subset in the nose revealed a predominance of relatively immature, cd + nk cells. furthermore, the ar nkg d was expressed at lower frequencies (lower percentages of nkg d + cells) and lower density of expression in the nasal mucosa and the polyps compared to pb (populations negative for nkg d were identified in the tissues). finally, the percentage of nk cells among lymphocytes (mean: %) was significantly higher in the polyps than in pb ( ) . okada et al. published a paper about nk cells in the nasal mucosa of the mouse on the c bl/ background ( ) , in which they showed that these nk cells were more immature (according to the relative levels of expression of cd and cd b) and phenotypically more activated (reduced expression of cd l, higher percentage of cd + cells) than those from spleen and lung. around % expressed the tissue residency and activation marker cd and one third of those also cd . the pattern of expression of the ly receptor family was different between the three tissues. functionally [cd a staining and interferon (ifn)-γ production], nasal nk cells appeared to be hyporesponsive compared to their spleen, but not their lung counterparts ( ) , which might be related to the possibility that the fraction of cd + nk cells was not sufficient to significantly activate the global nk cell population in the chosen experimental readouts. although this dataset is interesting per se, it should not be ignored that casadei and salinas ( ) , in a review about different animal models of nasal infections and immunity, cited several anatomic (functional vomeronasal organ in contrast to human, no waldeyer's ring) and physiologic (macrosmatic, no coughingsneezing reflex, lower sensitivity to human viruses) limitations of the mouse in this context, so that such results should always be considered with care before extrapolating to the human situation. lung nk cells have recently been extensively reviewed in this journal ( , ) , so that a summary of their most important features might be sufficient. lungs are constantly exposed to microparticles from the environment. particularly, as the mucosal lung epithelium is at the interface between the outside world and the organism, it can become the entry site for infectious pathogens, be they bacterial, fungal, or viral in nature. therefore, an extensive and sophisticated local immune response is waiting to be triggered at this anatomic location, and human nk cells, which represent around - % of lung lymphocytes ( ) , are a part of it. the work of marquardt et al. has established that most human lung nk cells represented the circulating subset and had the mature cd dim cd bright phenotype ( ) . they expressed more frequently the differentiation marker cd as well as educating kir than blood nk cells from the same donors but were relatively hyporesponsive upon stimulation with hla class i-negative target cell lines. in addition, however, a putative tissue-resident subset (around % of all lung nk cells), further subdivided into relatively immature cd bright cd − and cd dim cd − cells ( , ) , expressed the tissue residency markers cd , cd a, and cd . these cells were characterized in detail again by marquardt et al. ( ) , who showed that they were functional, especially after stimulation with the cytokine interleukin (il)- and displayed a unique transcriptional profile. several subpopulations could be distinguished based on the relative expression of cd a and cd ( , ) . natural killer cells have likewise been investigated in mouse lungs, particularly by wang et al. ( ) and michel et al. ( ) . both groups found that lung nk cells were more mature than those from the spleen ( ) or other organs ( ) according to the relative expression of cd and cd b. whereas the former authors described a higher expression level of the ir cd /nkg a and a lower level of the ar nkg d, the second paper could confirm this data only regarding nkg d in terms of mean fluorescence intensities. lung nk cells proliferated less, degranulated less (cd a assay) and were less cytotoxic than splenic nk cells ( ) , but these functions were rapidly up-regulated upon bacterial lung infection ( ) . this suggests that at homeostasis, lung nk cells are inhibited to avoid damage to normal autologous cells, but that they can quickly intervene in case of an infectious insult ( ) . michel et al. showed in in vitro co-culture systems that both spleen and lung macrophages could significantly up-regulate the cytotoxic activity of lung nk cells through a contact-dependent mechanism ( ) . regarding the homeostatic situation, research in recent years has revealed that in contrast to the older view of the lungs as sterile organs, a lung microbiota is present in the lower airways which exerts significant effects in health and disease, although it is not as abundant as in the gut ( ) ( ) ( ) ( ) . the term "microbiota" refers to all the microorganisms present, namely bacteria, fungi, protozoans, and viruses ( ) , but here we will only consider the role of bacteria. six phyla are predominantly represented in the lower airways: firmicutes, proteobacteria, bacteroidetes, actinobacteria ( , ) , acidobacteria, and fusobacteria ( ) . this microbiota is supposed to be transient in healthy donors and to be established from micro-aspiration and inhalation ( ) and its composition at any given time point submitted to the parameters of bacterial arrival, bacterial removal, and local immune responses ( , ) . in this way, an equilibrium state is reached that depends also strongly on the gut microbiota through various bacterial metabolites and contributes to the maintenance of homeostasis in the lower airways (gut -lung axis) ( ) ( ) ( ) . everything that disturbs this balance, such as some medications and particularly antibiotics, increases in nutrients (high fat diet, low fiber diet), cigarette smoke, infectious agents, chronic inflammation, can disturb the gut as well as the lung microbiota and lead to a state of dysbiosis, characterized by an increased number of airway bacteria and a change in its composition. the dysbiosis is profoundly linked to several severe lung diseases [asthma, chronic obstructive pulmonary disease (copd), infections, cancer] ( ) ( ) ( ) ( ) ( ) ( ) ( ) . natural killer cells have, to our knowledge at least, not been investigated in detail in the context of a normal lung microbiota to date. as most lung nk cells are not activated nor tissueresident (as illustrated by their negativity for cd ), they might not react very strongly to a normal microbiota. however, as they are expressing several bacteria-specific toll-like receptors (tlrs) that signal in the presence of bacterial pathogens ( ) , it might be conceivable that they could also mount an immune response toward microbiota components and that this would contribute to homeostasis. the overall immunosuppressed state of lung nk cells at baseline would help to avoid aggression of harmless and useful bacteria and of uninfected autologous cells ( ) . yang et al. ( ) , as well as fuchs and colonna ( ) , discuss data claiming that at steady state, alveolar macrophages secrete immunosuppressive cytokines which keep nk cells in respect. this is in contrast with the results of michel et al. ( ) , discussed above. however, the macrophages and dendritic cells (dc) switch to pro-inflammatory cytokine production in case of a bacterial or viral infection and thereby activate the nk cells. this entity is the third cause of mortality in the united states of america ( ) and worldwide ( ) and is in most cases the consequence of prolonged cigarette smoking ( ) . it is characterized by airflow obstruction, emphysema, recurrent infections ( , ) , chronic inflammation, and overproduction of mucus ( ) . acute exacerbations significantly limit the quality of life of the patients ( , ) . the exacerbations are in principle caused by viral or bacterial infections, the latter most frequently due to haemophilus influenzae, streptococcus pneumoniae, and moraxella catarrhalis ( ) . pseudomonas aeruginosa is another bacterium frequently involved and one of the most dangerous ones, based on its highly pathogenic properties ( ) , and its remarkable level of resistance to antibiotics. natural killer cells have been investigated in human copd as well as in animal models of this disease. motz et al. demonstrated that exposure of pulmonary leukocytes to viral pathogenassociated molecular patterns (pamp) induced higher functional properties (degranulation measured with the cd a assay, and ifn-γ production) ex vivo in chronic cigarette smoke exposed than in non-exposed c bl/ mice ( ) . interestingly, bacterial pamp appeared to be less efficient in this model, as among five molecules tested, only fsl- (bacterial lipopeptide, tlr / agonist) and lipopolysaccharide (lps, tlr ligand) increased the percentage of ifn-γ + nk cells above the one of the non-exposed mice. in contrast, other papers reported that nk cell functions are diminished in copd ( ) . it has been further repeatedly demonstrated that in copd or relevant animal models, nk cell cytotoxic activity is increased relative to non-copd smokers and healthy individuals ( , ) . based on the model of lung nk cell hypo-responsiveness at baseline, cigarette smoke and even more the inflammatory state of the lower airways in copd would activate the alveolar macrophages and induce their production of pro-inflammatory cytokines. these would, in turn, unleash the nk cells and increase their cytotoxic activity, cytokine and chemokine expression, leading to a further aggravation of the inflammation and the clinical status of the patients. indeed, in accordance with this concept, freeman et al. ( ) showed that cd + cells (in fact a mixture of nk cells and cd + t lymphocytes) isolated from lung parenchymal samples of non-copd smokers and copd patients with a smoking history, although similar in terms of frequencies between the cohorts, had a different cytotoxic activity toward autologous lung epithelial cells. the cd + lymphocytes from the copd patients were more cytotoxic than the cells from the non-copd smokers, in an experimental setup without additional stimulation. the target cells were supposed to be mostly epithelial cells based on their positivity for cd , their size, and their negativity for the hematopoietic cell marker cd . the cytotoxicity was measured as the percentage of annexin v + target cells after the co-culture with the effectors and was around % in most samples. this was not a lot, but the nk cells and cd + t cells were not otherwise activated. most of the parenchymal lung nk cells were cd + cd + and the minor rest cd + cd − ( ). another study was provided by the same group in ( ) . it showed that isolated, purified lung nk cells induced apoptosis in autologous epithelial cells. this time, the mean level of cytotoxicity was rather high compared to the previous paper, and it was very significantly stronger in copd patients than in non-copd smokers. the nk cells, but not the target cells, determined this increased cytotoxic activity, because k , a hla class i-negative myeloid leukemia cell line used as the standard nk cell target, was also lysed more efficiently by copd nk cells than by their non-copd counterparts. the authors confirmed their data in a mouse model and then showed that the nk cells were primed by dc via trans-presentation of il- ( ), a phenomenon first described in by andreas diefenbach and his group ( ) . this would nicely explain the higher level of nk cell cytotoxicity observed in copd. along the same line, okamoto et al. administered il- and il- to normal mice and observed a lethal effect within days, selectively involving the lungs, with a profound interstitial infiltration of lymphocytes dominated by nk cells ( ) . high levels of various cytokines and chemokines were found in serum and lungs. depletion of the nk cells by antibodies completely abrogated the lethal injury, which is a convincing demonstration of the potentially destructive power of nk cellactivating cytokines and nk cells themselves ( ) . this work was intended as a contribution to the elucidation of the pathogenesis of interstitial pneumonia, but similar mechanisms, in the presence of high levels of pro-inflammatory cytokines in bacterial infections, might contribute to copd. in human cancer patients, administration of high dose il- induced a vascular leakage syndrome where the so-called lymphokine activated killer cells (equivalent to highly activated nk cells) destroyed endothelial cells, causing a generalized edema, and damaging several organs ( ) . hodge et al. demonstrated a higher number of nk cells in the bronchoalveolar lavage fluid (balf) of copd patients (the cohort was composed of current smokers and of ex-smokers) than in healthy smokers ( ), a higher content of the cytolytic molecule granzyme b and, most importantly, a significantly increased cytotoxic activity against k . they also found a reduction in the percentage of balf nk cells expressing cd (which they consider as ir, although it is more a chaperone molecule for the true ir nkg a). nevertheless, this indirect measure of a down-regulation of nkg a could indicate that it contributes to the higher nk cell cytotoxic activity observed in copd ( ) . recently, osterburg et al. presented a multiparameter flow cytometry study of pb nk cells from copd patients compared with smokers and never smokers ( ) . in contrast to those, copd patients and smokers highly expressed the maturation marker cd as well as the ar nkp and nkp (normally only present on activated but not on baseline nk cells), but lower levels of cd . certain nk cell subpopulations were indicative of prior exacerbations ( ) . the ar nkg c, which is significantly present only on adaptive nk cells from human cytomegalovirus (cmv)-infected individuals, was not differentially expressed in pb of copd patients with a smoking history and healthy volunteers, but present on a higher percentage of nk cells in the patients with the most frequent exacerbations and the most reduced lean mass ( ). a relationship with the bacterial burden cannot be excluded in this context, as there might be a correlation between the viral reactivations and the bacterial colonization, contributing together to the higher number of exacerbations. most of the papers discussed above investigated the nk cell cytotoxicity toward autologous cells or conventional nk target cells, but what about a potential direct bacterial killing? nk cells, upon appropriate stimulation, release cytolytic molecules called perforin, granzymes and, in human but not in mice, granulysin, which have an additive or synergistic cytolytic effect toward bacteria ( ) . they can form pores in the target cell walls and thereby eliminate the microorganisms, but in addition they are able to eliminate some types of eukaryote cells infected by bacteria ( , , ) . furthermore, in addition to the direct effect, nk cells are embedded in the immunological network and react (through an increased cytotoxic activity and cytokine production) to the immune cells and the cytokines/chemokines in their environment ( ) , which is strongly shaped in case of a bacterial infection ["cellular crosstalk" ( ) ]. data about chronic rhinosinusitis, nasal polyposis and copd are summarized in table . as mentioned above, this ubiquitous gram-negative pathogen is part of those colonizing the lower airways in copd, but it is also a major problem in cystic fibrosis and in nosocomial infections, with a high morbidity and mortality ( ) . the role of nk cells in the host defense against this bacterium has been quite extensively studied by the team of michael t. borchers ( , ) in consequences of the indicated diseases on nk cell phenotype and functions. copd, chronic obstructive pulmonary disease; balf, bronchoalveolar lavage fluid; and ifn-γ , interferon-gamma. mouse models. in the chronologically first work, outbred cd- mice were intranasally infected with the p. aeruginosa laboratory strain pao ( ) and evaluated h later. the findings can be summarized as follows: (i) the infection increased the expression of ligands for the ar nkg d, present on almost all nk cells but also on a subpopulation of cd + t lymphocytes, in vivo; (ii) similarly, these ligands increased in an infected human lung epithelial cell line in vitro; (iii) the inhibition of the ar nkg d with a monoclonal antibody significantly reduced the clearance of p. aeruginosa from the lungs; (iv) antibody-mediated nkg d blockade down-regulated the amount of the cytokines il- β, ifn-γ and tumor necrosis factor (tnf)-α and in addition of nitric oxide; and finally, (v) the same experiment also revealed a threefold reduction of epithelial cell damage, measured as shedding of these cells into the balf ( ) . the latter point brings us again to the recurrent theme of lung cell damage that can be induced by activated nk cells, whereby it would have to be determined if this is beneficial (elimination of infected cells by nk cells) or deleterious (exaggerated damage to the epithelium). the follow-up paper ( ) then presented a conditional mouse model with an inducible expression of nkg d ligands on lung epithelial cells. here, the bacterial clearance was significantly higher in those mice that overexpressed the nkg d ligands. moreover, the survival up to h post-infection and the level of phagocytosis were significantly increased in the latter group. similarly, in in vitro experiments, where the nk cells were stimulated with lps, the percentage of nk cells producing ifnγ was much higher in the mice with the increased expression of nkg d ligands. as expected, this percentage dropped (but was not completely abolished) in nk cells from infected mice treated with an anti-nkg d antibody ( ) . however, the p. aeruginosa-derived exotoxin a, which in combination with il- α may induce a dangerous inflammatory state with tissue damage in the host, has also been shown to inhibit nk cell cytotoxic activity against k , even in the presence of usually stimulating cytokines such as il- ( ). the inhibition was almost complete with a high dose of the toxin and still partial with a low dose ( ) . the effector cells were not purified nk cells but peripheral blood mononuclear cells (pbmc), so that an indirect effect on the nk lymphocytes might play a role in this readout. furthermore, pedersen and kharamzi described already in that the p. aeruginosa-derived alkaline protease and elastase inhibited nk cell cytotoxic activity against k , presumably due to a reduction in the effector-target conjugate formation ( ) . in addition, these molecules strongly reduced the binding of an anti-cd (called leu- at that time) antibody ( ) . this group of pathogenic gram-negative bacteria is composed of several species, of whom some are dangerous for cystic fibrosis patients, as they are highly resistant to multiple antibiotics ( ). li et al. ( ) investigated the interaction between burkholderia cenocepacia and nk cells, and first demonstrated that the nklike leukemia cell line yt ( ) , as well as primary purified human nk cells, significantly reduced the number of living bacteria (measured as cfu) after a co-incubation of to h. the results were confirmed with life cell imaging techniques and bacterial uptake of propidium iodide (pi). the authors then wanted to know if the killing activity was contact-dependent or not, and first showed that yt cells bound the fluorochromelabeled bacteria. then, they could demonstrate that a direct contact was needed for the killing activity, as nothing happened to the bacteria when they were separated from the nk cells by a porous membrane, allowing passage of soluble molecules but not of cells ( ) . most bacteria remained extracellular and were not taken up by the yt cells. killing was almost completely abrogated after treatment with strontium chloride (srcl ), which is known to deplete nk cells from their cytotoxic granules ( ) . finally, it was established that src family kinases were activated in yt cells after the contact with b. cenocepacia ( ) . this is a very nice demonstration that nk cells are able to directly kill certain extracellular bacterial species through nk cell -bacteria contact, although the precise mechanism is still unknown. other possible mechanisms of nk cell-mediated elimination of bacteria are the lysis of intracellular pathogens within the infected cells and the activation of other immune cells, and particularly of macrophages, via nk cell-derived cytokines (such as ifn-γ) ( ) , and most likely also the killing of bacteria-infected cells expressing ligands for nk cell ar. this is another gram-negative pathogen which poses a major problem due to its frequent causative involvement in nosocomial infections (particularly in pneumonia) and the steady increase of strains multi-resistant to antibiotics ( ). chalifour et al. ( ) demonstrated that the outer membrane protein a (kpompa) from this microorganism, known to signal via tlr , induced ifn-γ and α-defensin (an antimicrobial peptide) synthesis and release in human nk cells. in the mouse, both nk cell-derived ifn-γ ( ) and il- ( ) have been described to be necessary for bacterial clearance ( , ) . in the paper from xu et al., it was nicely shown with genetic controls and depletion experiments that the immune defense against this pathogen indeed deeply involved nk cells and that a subset of them produced il- . the nk cells had a conventional and mature phenotype (less cd + , more klrg + ) distinct from other innate lymphoid cells (ilc) ( ). ivin et al. focused on the fact that ifn-γ production by nk cells, likewise necessary for the elimination of the bacteria through a network with alveolar macrophages, was dependent on the nk cell-intrinsic stimulation by type i ifn, in turn induced by k. pneumoniae ( ) . in contrast to the crucial role of nk cell-derived cytokines, their granzymes (a and b) , one of the constituents of the lytic granules, did not seem to play a major role in this model ( ) . however, this does not rule out that in human, granulysin and perforin together might have a cytotoxic effect on these bacteria. in the case of helicobacter pylori, responsible for chronic gastric inflammation with the potential to lead to ulcers or cancer, preincubation with fixed bacteria increased the cytotoxic activity of nk cell-enriched pbmc toward k and other tumor target cells, as well as the release of ifn-γ ( ). furthermore, rudnicka et al. showed that the bacterial glycine acid extract induced nk cell expansion and ifn-γ production, whereas the lps from the same bacteria inhibited these parameters, and instead favorized the apparition of il- -producing nk cells ( ) . although this might just marginally be relevant for nk cells in the lungs, it nevertheless shows to which extent these cells can react to bacteria and how the latter try to manipulate them. legionella pneumophila, the agent of legionnaires' disease, is replicating intracellularly in macrophages. here again, nk cell production of ifn-γ, induced probably through direct tlr messages ( ) one of the most frequent culprits in community-acquired pneumonia is s. pneumoniae. regarding the role of nk cells in this infection, their beneficial or detrimental action depended on the pathogen's serotype ( ). thus, the control of serotype depended on nk cells, as demonstrated by baranek et al. in a mouse model ( ). these authors investigated the consequences of a defect in the transcriptional cofactor four-and-a-half lim-only protein (fhl- ) on nk cells in general and on pneumococcal infection particularly. it had been previously established that ifn-γ was, once more, the crucial factor in host defense in this context, and that nk cells were one of its major producers ( ) . in the spleen and the lungs of fhl- knockout (ko) mice, the number of nk cells and their expression of the ar nkg d and nk . (cd c) were down-regulated and a negative effect of the deficiency on nk cell maturation was observed. mortality to s. pneumoniae lung infection was strongly increased in the ko mice but could be rescued by the adoptive transfer of wildtype nk cells. finally, the authors showed that ifn-γ production by nk cells was severely reduced and that less neutrophils were recruited to the lungs of the ko animals ( ). the role of the mostly immunosuppressive cytokine il- in dampening the immune response to pneumococcal infection was shown in , when van der poll et al. administered the pathogen intranasally together with il- and observed early mortality and reduced levels of the pro-inflammatory factors ifn-γ and tnf. conversely, all this was restored when the mice were pre-treated with an anti-il- antibody ( ). these results were very recently confirmed by clark et al. ( ) , who worked with il- reporter and il- -ko mice to observe that s. pneumoniae induced il- production by nk cells (around % of total lung nk cells) with a negative effect on animal survival, and that the bacterial burden was diminished in the lungs of the ko mice compared to wildtype animals. nk cell depletion in the latter induced a strong reduction in the bacterial lung counts and in il- . furthermore, il- -deficient mice had significantly more neutrophils and monocytes in the infected lungs. finally, the virulence protein spr from s. pneumoniae was identified as the il- -inducing factor ( ) . none of these papers investigated the potential balance between the pro-inflammatory and anti-inflammatory effects of ifn-γ and il- , respectively, on the outcome of this infection, which would anyhow have been technically challenging. one might suppose that il- is there to down-regulate an overwhelming immune response that would damage lung tissues, but on the other hand, it might also be counterproductive to dampen it too much and thus to lose control over the pathogens ( ) . other groups have described that human as well as mouse nk cells could produce and release il- ( , ), although, according to perona-wright et al., this only occurred in the case of a systemic, but not a localized, pulmonary infection (with the gram-negative bacterium yersinia pestis) ( ) . in the case of systemic infections with listeria monocytogenes and y. pestis, approximately % of blood nk cells became il- + , and the cytokine was produced by a nk cell subset circulating in blood prior to the infection ( ) . before studying s. pneumoniae ( ), clark et al. had already shown that l. monocytogenes elicits il- production by nk cells via the virulence factor p (with, as a consequence, an inhibition of the recruitment and the activation of myeloid cells) in a mouse model of systemic infection, where the lungs were not further investigated ( ) . another frequently encountered nosocomial and multiresistant infectious agent is staphylococcus aureus. small et al. could demonstrate the fundamental role of nk cells in the response to these bacteria in the case of mouse lung infections ( ) , as (i) nk cell numbers in the airways increased; (ii) in vitro contact with products from the pathogens activated nk cells; (iii) co-culture of nk cells with alveolar macrophages increased the phagocytic activity of the latter, (iv) il- -ko mice were much more susceptible to the infection than wildtype mice, whereas they had much more neutrophils and macrophages in the lungs; and (v) nk cell depletion rendered even wildtype mice highly sensitive, despite a conserved il- production ( ). these findings demonstrate indeed once again the important role of nk cells in immune defense against extracellular bacteria. in accordance with this model, zhao et al. showed that particular matter, associated epidemiologically with enhanced numbers of lung infections, diminished the amount of nk cells migrating to rat lungs in case of infection with s. aureus, whereas adoptive nk cell transfer restored a vigorous nk cell response ( ) . in ex vivo experiments, nk cells improved, as in the previous study, the phagocytosis of the pathogens by alveolar macrophages. it is well known that after influenza, recovering patients are very susceptible to bacterial superinfection, notably by s. pneumoniae and s. aureus ( ) . the contribution of nk cells to this phenomenon was demonstrated in a mouse model of h n influenza virus infection followed by intratracheal instillation of s. aureus. the sequentially double-infected mice were much more susceptible to the infection (weight loss, survival rate) than those receiving pbs or bacteria only. this went hand in hand with severe changes in the histopathological aspect of the lungs and a marked reduction of local nk cell numbers and tnfα + nk cells. furthermore, the concentrations of tnf-α and of the chemokines ip- and mip- α were diminished in the balf. adoptive transfer of naive nk cells could restore the immune response. the nk cells needed tnf-α to perform their antibacterial effect and this was organized via an interaction with alveolar macrophages and increased phagocytosis ( ) . the conclusion that might be drawn from all these papers is that nk cells are very important, at least in mouse models, for the immune response to and the defense against pulmonary infections due to gram-positive bacteria, with, on the other hand, a detrimental influence of these lymphocytes in case they produce too much immunosuppressive factors [the same old story ( ) ]. mycobacterium tuberculosis is the agent of tuberculosis (tb), an infectious disease that puts a high burden on the populations in developing but also in developed countries and increasingly shows resistance to conventional antibiotics. it latently infects about % of the total population and becomes clinically apparent in ten million patients per year, according to estimations from the world health organization (who) ( , ) , rendering it a major public health issue. as recently reviewed by cong and wei ( ) , nk cells could interact with this intracellular pathogen through the ar nkp , nkp , and nkg d, as well as tlr . although they became activated under these conditions, they seemed to play only a negligible protective role, according to junqueira-kipnis et al. ( ) . these authors showed in a mouse model that lung nk cells augmented in number within the first weeks after exposure to aerosols containing the mycobacteria and up-regulated cd , ifn-γ and perforin. however, their depletion did not at all change the kinetics of the infection. human pb nk cells likewise up-regulate ifn-γ after contact with m. tuberculosis ( ) . barcelos et al. ( ) compared pb nk cells in cohorts of patients with active tb, isolated tuberculin + skin tests, and tuberculin − healthy donors. they found a different subset distribution according to the cohorts, with putative tb-exposed but-resistant individuals (defined as those with a positive tuberculin test) having overall less nk cells but an increased percentage of cd − cd + , cd + cd − and especially cd bright cd −/+ nk cell subsets compared to the other two donor groups. in contrast, tb patients displayed lower frequencies of cd + cd + cells. the authors speculated that this different subset distribution might have been related to the resistance or sensitivity to active tb, but of course, as the cells stem from pb, this dataset might have to be interpreted with some caution. surprisingly, however, roy chowdhury et al. ( ) , by following a cohort of adolescents from an endemic region in south africa, could demonstrate with mass cytometry and functional experiments, that latent tb was associated with increased responses of pb nk cells, with a particular role for the ar cd . indeed, the percentages of nk cells among total living cells, of total cd + cells, of granzyme b + and of perforin + cells were significantly higher in patients with latent tb than in healthy, non-infected donors. in addition, adcc (mediated via cd ) against p target cells was also higher in latent tb. by following further cohorts, the authors found that the percentage of pb nk cells was dynamically regulated during latency, progression of the disease and responses to antibiotic medication. this level of nk cells in pb even correlated inversely with inflammation in the lungs of patients with active tb ( ) . such observations push nk cells again at the forefront of immune defenses in tb and at a possible role in the maintenance of latency. with a similar cohort-based approach, harris et al. ( ) evaluated nk cell phenotype and functions in individuals with latent tb compared with healthy controls. furthermore, participants were separated in infected and non-infected in a tb-endemic region in kenya and a healthy volunteer cohort from the united states. among the three groups, the persons from the united states had the significantly lowest percentage of cd − cells, which are known to expand in chronic human immunodeficiency virus (hiv) infection and other viral diseases and to be dysfunctional ( ) . the kenyan volunteers displayed, among cd dim nk cells, a higher expression of granzyme b and of the non-mhc class i-specific ir tigit, with the highest levels found in the healthy cohort. furthermore, these individuals had an increased expression of the ar nkp . within the cd bright subpopulation, the kenyan participants showed increased expression of nkg d but again decreased levels of nkp , compared to the cohort from the united states. functionally, degranulation (cd a assay), ifnγ production (intracellular flow cytometry) and cd expression were compared between the three cohorts after co-culture with k cells (evaluation of natural cytotoxicity) and p mouse cells plus anti-mouse antibody (evaluation of adcc). whereas for the latter parameter, no significant differences were observed, frequencies of cd + , cd a + , and ifn-γ + nk cells turned out to be significantly higher in the united states study population, such as if an environment endemic for tb would impact the "missing self "-recognition capacities of nk cells ( ) . the same regional discrepancies were observed after stimulation of total pbmc with three different antigen extracts from m. tuberculosis, and the reactivity to these antigens was shown to be at least partially dependent on the presence of il- and il- , supposed to be derived from accessory cells. conradie et al. ( ) described that the level of activation of pb nk cells (frequency of cd + and cd + hla-dr + events) allowed, among other parameters, to discriminate between m. tuberculosis-induced immune reconstitution syndrome, hiv infection and co-infection with both pathogens. although these three papers suggest some influence of m. tuberculosis on pb nk cells, it is not clear yet to which extent nk cells really intervene in the immune defense against this pathogen that persists in the lungs. an investigation on tuberculous pleurisy ( ) revealed a large predominance of cd bright cd − nk cells in the pleural fluid, and an apoptotic effect of soluble factors from this environment predominantly on cd + nk cells. m. tuberculosis induced ifn-γ production from cd bright nk cells in the absence of monocytes, t cells and b cells, leaving open the possibility of a direct productive interaction between the bacteria and the nk cells. lai et al. ( ) presented a work on nontuberculous mycobacterial lung infections, which means due to other mycobacterial species, such as mycobacterium abscessus and mycobacterium kansasii. as the latter become more and more prevalent in developed countries, these authors performed a study in c bl/ mice that were infected intratracheally with m. kansasii. they found that nk cell depletion increased bacterial burden, mortality, and pathogenetic postinfectious changes (macrophage phagocytosis, dc activation, cytokine production, and development of granuloma). the same observations were made in ifn-γ-ko animals and restored after transfer of wildtype nk cells. these cells were also the most important producer of ifn-γ in this model ( ) . lai et al. further cited papers that had demonstrated a similar protective effect of ifn-γ produced by nk cells in the infections with bordetella pertussis, francisella tularensis, and chlamydia muridarum in mouse models of respiratory infection. previous publications by the same group had shown that nk cells can directly lyse m. tuberculosis and m. kansasii via the cytotoxic proteins granulysin and perforin in a contactdependent manner disrupting mycobacteria cell wall integrity ( ) , and that in some patients with mycobacterial infections, anti-ifn-γ autoantibodies were detected ( ) . the killing process involved signaling through nkg d and ncr as well as map kinases, suggesting that similar mechanisms are involved for the killing of bacteria and of eukaryotic target cells ( ) . this is potentially a very important observation, as it strongly suggests that both conventional cytotoxic mechanisms and cytokine production might be relevant in anti-mycobacterial defense. some studies were also performed on mycobacterium bovis bacillus calmette-guérin (bcg), an attenuated mycobacterial strain used as an anti-tuberculous vaccine ( ) . for example, it was demonstrated in vitro that cd bright nk cells reacted to this microorganism by proliferation and ifn-γ production, whereas their cd dim counterparts better up-regulated the cytolytic proteins perforin and granzyme a ( ), all of which was largely expected based on what is known about the functional specialization of these two nk cell subsets ( ) ( ) ( ) . in a mouse in vivo model, where bcg was directly administered (intratracheally) into the lungs, nk cell-mediated production of ifn-γ rapidly increased in the first days after infection, similarly to the number of lung nk cells ( ) . after nk cell depletion, the reduction of body weight was less pronounced compared to non-depleted mice, whereas the bacterial load remained identical. importantly, inflammation and injury of the pulmonary structures was much less pronounced in the nk cell-depleted animals, suggesting a pathogenic role for these lymphocytes. indeed, the level of pro-inflammatory cytokines and chemokines was also reduced in the absence of nk cells, and the percentages of ifn-γ + cd + and ifn-γ + cd + t cells was significantly increased in these mice. bacillus calmette-guérin-infected macrophages up-regulated nkg d ligands, which induced their lysis via this receptor-ligand interaction. finally, the blocking of nkg d with a monoclonal antibody restored the survival of the macrophages and the t cell-mediated immune response ( ) . it is difficult to make a coherent synthesis of all these observations on nk cells and mycobacteria, but it is nevertheless quite appealing that again positive, negative and neutral aspects are described, which may vary according to the models and the experimental setup. this shows that nk cells still hide a lot of secrets regarding their function in anti-mycobacterial infections as well as in bacterial pathogenesis overall. a summary of the relationships between the bacteria discussed and nk cells is presented in table . these are obligate intracellular pathogenic bacteria that are responsible for several types of human and mouse diseases. various studies dedicated to this type of microorganisms illustrated the concept that nk cells usually do not respond as a pure population as may be the case for in vitro experiments, but that in vivo they are part of a tightly controlled immune network composed of cells, cytokines, chemokines and exosomes. thus, in mouse models, nk cells influenced the interaction between dc, t helper (h) and th t lymphocytes in c. muridarum lung infection ( ) , modulated the balance between th and th t cells and t regulatory cells (treg) in the same type of infection ( ) , and again positively regulated the interactions between dc and t lymphocytes against chlamydophila pneumoniae ( ) . in all these situations, nk cells exerted a protective and disease-controlling effect via their influence on the bridge between innate and adaptive immune responses. with the ambitious aim to experimentally investigate the famous "hygiene hypothesis, " han et al. ( ) studied mice infected with c. muridarum and rendered allergic to ovalbumin (ova). they observed that prior infection could inhibit at least certain parameters of allergy. however, nk cell depletion partly suppressed the "beneficial" effect of the lung infection. adoptive transfer of nk cells from infected mice inhibited partially the development of an allergic response in non-infected recipients. nk cell-devoid mice coherently produced more th type cytokines ("pro-allergic" th cytokines, il- , and il- ) than ifn-γ ("anti-allergic" th cytokine). a detrimental effect of nk cells had been shown for the immune response to the respiratory rodent pathogen mycoplasma pulmonis, related to the human infectious agent mycoplasma pneumoniae. indeed, in a quite complicated experimental setup, bodhankar et al. demonstrated that nk cell depletion interfered positively with the development of a protective adaptive immunity after nasal-pulmonary immunization with bacterial antigens ( ) . this could be explained because nk cells shaped the t cell cytokine response toward more il- , il- , and il- but away from ifnγ production. wurzel et al. presented large cohort studies of children with protracted bacterial bronchitis (pbb) and mild bronchiectasis, associated or not with human adenovirus co-infection ( , ) . besides typical socio-economic and clinical factors, an elevated nk cell number relative to the values of healthy children of the same age was observed in the pb of diseased children in general and with adenovirus species c particularly. nk cell phenotype and function were not further investigated. recently, sim et al. ( ) made the important discovery that the hla-c-specific activating kir ds did recognize a conserved bacterial peptide presented by hla-c, and more precisely by hla-c * : . the sequence of the peptide required for this recognition was a "rare" self-peptide, but the epitope of interest is conserved in the recombinase a (reca) of many bacterial species (more than according to the authors' claims), most of them belonging to serious human pathogens, such as helicobacter pylori, brucella, campylobacter jejuni, and chlamydia trachomatis ( ) . interestingly, activation of resting nk cells via kir ds alone was sufficient to induce degranulation and cytokine production, whereas all other known ar, except cd , need at least one co-activating molecule engaged at the same time ( ) . there was, furthermore, an inverse correlation between the frequency of the kir ds full length gene and the hla-c * : allele. thus, it appears that the kir family is not only involved in nk cell licensing and in multiple disease associations, but also, most likely, in antibacterial defense. this paper received an accompanying commentary by peter parham, which places the findings in the broader context of kir and hla class i molecules ( ) . to sum up, nk cells might be directly activated by various bacteria via contact-dependent mechanisms whose modes of functioning are still unknown, via tlr, via kir ds , or more indirectly via the up-regulation of ligands for their ar, such as nkg d, by infected cells. they might also react to cytokines released into the microenvironment by antigen-presenting cells (macrophages, dc). dietert et al. published a plea for the histopathological evaluation of the consequences of different infectious lung diseases in mouse models and described the pathogen-specific features characteristic for each of them ( ) . indeed, many variations were observed between the infecting microorganisms, be they bacterial or viral in nature. the authors emphasized that histopathology remains the "most conclusive and practical read out" for the evaluation of the effects of the various infectious models on mouse lungs. although this is true, more "modern" and state-of-theart methods are being developed and are about to enter the laboratories, as a consequence of general scientific and technical progress but also of the " r" approach regarding experiments with animals. in , the team of hans clevers described the generation of human airway organoids derived from surgical material or from balf ( ) . these were long-term proliferating structures that recapitulated a normal airway with different types of cells that are physiologically present in vivo. the beauty of the system per se was already an accomplishment, but it could be used for the study of various lung diseases, such as cystic fibrosis, cancer, or viral infections ( ) . therefore, it is likely that bacterial infections could similarly be investigated in this system, and the data obtained would probably be more relevant to human pathology than the mere mouse models (and save the life of many mice by the way). the same year, ross et al. ( ) published a review on the "ex vivo human lung." they worked with donor lungs not retained for transplantation, extracted primary cells from them and developed an "ex vivo-perfused single human lung" that would allow the investigation of different lung diseases. the system seems at first sight less elegant than the lung organoids and is maybe also more limited in the spectrum of possible pathologies that can be investigated. the advantage would be that an entire, complete organ is available and not just an organoid. yet another option is the "alveolus-on-a-chip, " developed by deinhardt-emmer et al. ( ) . it was a three-dimensional structure with an air phase and a liquid phase, where endothelial cells, epithelial cells and macrophages could be co-cultured. in the presented work, a primary influenza virus infection, followed by a s. aureus superinfection, were investigated, and it was shown that the endothelium was seriously damaged under these conditions. likewise, single cell transcriptomics is a powerful tool that can reveal huge amounts of details about all kinds of immune cells, and among them nk cells, as exemplified by lung cancerinfiltrating immunocytes in human and mouse ( ) . one aspect of nk cells is their putative potential for a dual role as "pro-inflammatory" and "regulatory" effectors, which might be mediated by different subsets ( ) . our group has previously touched the problem that nk cells are in fact a double-edged sword, meaning that they might have sometimes beneficial but sometimes rather deleterious effects ( ) . this has again become clear throughout this review, although the models and studies presented and discussed were all but homogeneous. it might be expected that this will change in the coming years if more and more teams will use the organoid and organ on-a-chip technologies and go into various "omics." overall, given the current and justified hype for nk cells as efficient agents for cancer immunotherapy, it would be difficult to convince the nk community that their favorite cells might also have a dark side. we emphasize that several methods to improve nk cell antitumoral efficiency, such as particularly car-nk cells ( ) and nk cell engagers, recently described by the vivier group ( ) , should be sufficient to stand up for the use of nk cells in this indication. however, what about the therapeutic indication of nk cells in infectious diseases in general and in the lungs particularly? due to the current covid- pandemic, this question has gained increased interest ( , ) , and in addition, most of the papers discussed here that describe an influence of nk cells on the disease course in the airways conclude with the statement that nk cells should be targeted in respiratory infections. but it has clearly been shown that these lymphocytes can have detrimental side effects and cause significant damage to the airways, at least in viral diseases ( , ) . available literature does not give clear indications regarding bacterial pathogenesis, but the issue was already discussed in , with the question if nk cells are angels or devils in bacterial infectious diseases ( ) . this problem is, in our opinion, not yet resolved and a lot of research work will be necessary in the field, keeping in mind that the number of multi-resistant bacterial strains is increasing at a terrifying rate and that alternatives to antibiotics must be discovered and developed. finally, a general problem in the field and a caveat to many of the presented studies is the difficulty of distinguishing nk cells reliably form ilc , which also produce ifn-γ as a signature cytokine and have a partially overlapping phenotype. a high plasticity within the ilc family renders even possible the conversion of nk cells, in certain microenvironments, into ilc like cells ( ) ( ) ( ) . however, whereas both nk cells and ilc require the transcription factor t-bet for their development and function, nk cells need and express eomes in addition. ilc are preferentially located in tissues and are very rare in peripheral blood, in contrast to nk cells. thus, one can be confident that the studies discussed here that worked with blood (human) and blood or spleen (mouse) nk cells really investigated nk cells and not ilc . for tissue-based studies, the differences might be more blunted, although ilc are considered as non-cytotoxic cells ( ) ( ) ( ) . these difficulties are in line with the increasing number of "new" cell types that are currently discovered [for example mr t cells ( ) ], as a consequence of the ever growing diversification and performance 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model single-cell transcriptomics of human and mouse lung cancers reveals conserved myeloid populations across individuals and species regulatory nk cells suppress antigen-specific t cell responses multifunctional natural killer cell engagers targeting nkp trigger protective tumor immunity flattening the covid- curve with natural killer cell based immunotherapies the promise and peril of natural killer cell therapies in pulmonary infection innate lymphoid cells: diversity, plasticity, and unique functions in immunity nk cells and ilcs in tumor immunotherapy plasticity of innate lymphoid cell subsets mr -restricted t cells are unprecedented cancer fighters the authors would like to thank prof. dr. markus ollert, md, the director of the department of infection and immunity of the luxembourg institute of health, for his continuous support. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © theresine, patil and zimmer. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -m fsrwvw authors: elbahesh, husni; gerlach, thomas; saletti, giulietta; rimmelzwaan, guus f. title: response modifiers: tweaking the immune response against influenza a virus date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: m fsrwvw despite causing pandemics and yearly epidemics that result in significant morbidity and mortality, our arsenal of options to treat influenza a virus (iav) infections remains limited and is challenged by the virus itself. while vaccination is the preferred intervention strategy against influenza, its efficacy is reduced in the elderly and infants who are most susceptible to severe and/or fatal infections. in addition, antigenic variation of iav complicates the production of efficacious vaccines. similarly, effectiveness of currently used antiviral drugs is jeopardized by the development of resistance to these drugs. like many viruses, iav is reliant on host factors and signaling-pathways for its replication, which could potentially offer alternative options to treat infections. while host-factors have long been recognized as attractive therapeutic candidates against other viruses, only recently they have been targeted for development as iav antivirals. future strategies to combat iav infections will most likely include approaches that alter host-virus interactions on the one hand or dampen harmful host immune responses on the other, with the use of biological response modifiers (brms). in principle, brms are biologically active agents including antibodies, small peptides, and/or other (small) molecules that can influence the immune response. brms are already being used in the clinic to treat malignancies and autoimmune diseases. repurposing such agents would allow for accelerated use against severe and potentially fatal iav infections. in this review, we will address the potential therapeutic use of different brm classes to modulate the immune response induced after iav infections. influenza viruses (ivs) are responsible for significant morbidity and mortality in the human population with ∼ , annual deaths worldwide. ivs can cause severe acute respiratory disease especially in high-risk populations like children, the elderly and the immunocompromised. while both influenza a and b viruses (iav and ibv, respectively) cause annual epidemics, the majority of severe human infections are caused by iav. ivs have segmented negative-sense single-stranded rna genomes. the lack of proof-reading activity of the viral rna-dependent rna polymerase (rdrp) and successive replication can lead to the accumulation of nucleotide mutations which drive antigenic drift. in addition, the segmented nature of their genome allows genetic reassortment between iv's to take place, which can produce novel strains that have acquired alternative antigenically distinct hemagglutinin, also known as antigenic shift. both antigenic drift and antigenic shift contribute to the iv's ability to evade pre-existing host immunity induced by previous infections. early recognition and responses to iv infection are largely mediated by innate immune sensors expressed by its primary target, the alveolar epithelial cells ( , ). recognition of ivs is mediated by pattern recognition receptors (prrs) that include toll like receptors (tlrs), retinoinc acid inducible gene-i (rig-i), and nucleotide oligomerization domain (nod)-like receptor family pyrin domain containing (nlrp ); all of which can recognize viral rnas during various stages of the infection cycle ( - ). activation of these sensors triggers signaling cascades that lead to the production of interferons as well as pro-inflammatory cytokines and chemokines ultimately resulting in an antiviral state within the surrounding cells/tissue ( ). accordingly, ivs have multiple mechanisms to evade these responses mediated by the viral nonstructural protein (ns ), polymerase basic protein (pb ), polymerase basic protein (pb ), polymerase acidic (pa) and nucleoprotein (np) [ in otherwise healthy individuals, iav infections are mild and the ensuing pro-and anti-inflammatory responses are balanced. in contrast, a "cytokine storm" is typically associated with severe infections including those caused by highly pathogenic iv strains. during a cytokine storm, chemokine and cytokine responses are dysregulated in both intensity and kinetics resulting in excessive damage to the host due to infiltration of inflammatory immune cells. acute lung injury (ali) caused by this inflammatory response is typically characterized by significant damage or destruction of the respiratory epithelium leading to acute respiratory distress syndrome (ards) ( , ). clinical treatment options for severe influenza virus infections remain limited and relying heavily on the administration of antiviral neuraminidase inhibitors (nais) and supportive critical care ( ). however, nais have not been effective in patients with severe h n or h n infections and there is evidence that fatal outcomes are associated with development of antiviral resistance in patients ( - ). while virus-targeted therapies remain the standard approach, iv's mutability and adaptation to current antivirals has highlighted the need for new therapeutic options that target host factors that regulate iv infections and resulting immune responses. in either approach, the focus is to prevent or limit damage to the lung epithelium due to exaggerated or dysregulated immune cell responses. biological response modifiers (brms) can alter the immune response thereby offering an additional therapeutic approach to treating severe infections. in this review, we highlight several studies that have shown the viability of brms as potential treatment options. for clarity, brms are categorized based on the type of biological agent (table ) . therapeutic antibodies iav infections and some vaccines elicit broadly-neutralizing antibodies (abs) that target the viral ha-stem. however, their abundance and immune-subdominance is overshadowed by abs targeting the ha-head domain. the effectiveness of these hastem abs against a broad range of iav subtypes, makes them an attractive target not only for vaccine development but also as antivirals. indeed, several ha-stem specific human monoclonal abs are now being evaluated in clinical trials [reviewed in davidson ( ) ]. mhaa a, medi , and vis are human monoclonal abs that have been shown to control viral replication and improve symptoms of human patients in phase clinical trials ( - ). while virus-specific abs aim to reduce antigenic load, abs to host targets aim at limiting the secondary wave of cytokines and reduce prolonged damaging cellular infiltration during severe infections. host-target directed antibodies have been utilized to target key regulators of this inflammatory wave and could potentially be used to dampen these overt responses. angiopoietin-like (angptl ) is a soluble angiogenicregulating protein. following proteolytic cleavage, the cterminal portion (cangptl ) is involved in integrin-dependent wound repair and can regulate vascular permeability ( , ) . angptl was significantly elevated in lung biopsies from iav-induced pneumonia patients ( ). in mouse studies, neutralizing anti-angptl abs reduced pulmonary tissue leakiness significantly accelerating lung recovery and improved lung tissue integrity ( ). neutrophil infiltration into the alveolar space occurs within day following iav infections ( ) . neutrophil extracellular traps (nets) released during iav-induced pneumonia into the alveolar space caused alveolar damage ( ) . the complement protein c a was shown to induce nets release and administration of anti-c a abs (ifx- ) reduced h n -induced ali due to reduced infiltration of lung macrophages and neutrophils as well as reduction of viral load in african green monkeys ( , ). tumor necrosis factor alpha (tnfα) is a key cytokine for controlling severe iav infections. it regulates two main antiviral functions: the induction of (i) the nfkb pathway, which ultimately controls expression of several inflammatory cytokines and (ii) apoptosis through multiple signaling cascades ( , ) . tnf upregulation during iav infections correlates with infection severity, especially following highly pathogenic iav-infections ( ) ( ) ( ) . mice treated with anti-tnf abs showed reduced disease burden; however, the authors of that study reported no effect on viral replication ( ). tnf-related apoptosis inducing ligand (trail) can trigger apoptosis in iav-infected cells. iav-infected human epithelial cells are sensitized to trail-mediated apoptosis while peripheral blood mononuclear cells upregulate trail expression. moreover, administration of monoclonal abs against trail increases survival rate following iav infections in mouse studies ( , ). antimicrobial peptides (amps) are host proteins that have direct antibacterial and antiviral activities and can modulate immune responses to infections. while the literature is largely focused on the antibacterial aspects of amps, several studies have highlighted the antiviral potential of amps against several viruses including ivs [reviewed in hsieh and hartshorn ( ) anti-angptl -reduced pulmonary tissue leakiness, significantly accelerated lung recovery and improved lung tissue integrity in mice. -mouse-adapted laboratory iav (h n ) c a ifx- antibody -reduced viral load and virus-induced ali due to reduced infiltration of lung macrophages and neutrophils in iav-infected african green monkeys. -highly-pathogenic avian iav (h n ) trail anti-trail -increased survival rate following iav infections in mouse studies. -improved symptoms and increased survival of iav infected mice. ed survival after h n or h n mouse infections. - pandemic iav (h n ); mouse-adapted laboratory iav (h n ); pandemic iav (h n ) ( ) ( ) ( ) and albericio and kruger ( )]. ll- is a human cathelicidin derived amp that is found predominantly in neutrophils and its expression can also be induced in epithelial cells and macrophages ( ) . aerosol administration of either human ll- or its mouse counterpart mcramp led to reduced morbidity and mortality to similar levels as the neuraminidase inhibitor zanamivir that is used for the treatment of human influenza patients ( ). both cellular and viral fadd-like il- β-converting enzymeinhibitory protein (cflip and vflip, respectively) protect cells from death receptor mediated apoptosis. kα is a vflip-derived peptide that consists of amino acids from the α helix of the kaposi's sarcoma herpes virus (kshv) death effector domain protein. a synthetic version of this peptide, tat-kα , was generated by fusing kα to a portion of the hiv tat protein ( , ) . in mouse challenge studies, intranasal administration of tat-kα at the time of infection with highly pathogenic avian h n virus resulted in protection of the treated mice. no replicating virus was detected in the lungs at either or days after infection suggesting complete protection from infection ( ). it should be noted that this effect is largely due to direct destabilization of the virions by the tat-kα peptide and it is likely that infection in treated mice was not established; the efficacy of this amp has not been determined during an established infection and warrants further investigation. host kinases regulate not only iav entry and replication but also initiate antiviral signaling cascades that regulate expression of pro-inflammatory chemokines and cytokines during infections and present viable targets for intervention ( , ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . iav infection has been shown to upregulate c-jun n-terminal kinases and (jnk /jnk ). these kinases directly regulate the induction of pro-inflammatory responses. iav-induced jnk /jnk activation mediates production of chemokines and cytokines including tnf-α, interferon β (ifn-β), and interleukin (il- ) ( ) . in vivo inhibition of jnk /jnk resulted in reduced levels of pro-inflammatory cytokines and reduced viral titers ( , ). the mitogen activated protein kinase (mapk), p , regulates viral entry and replication ( , ) . furthermore, p regulates ifn stimulated gene (isg) gene expression and ultimately cytokine production via stat phosphorylation ( ) . using either of two specific p inhibitors (sb or sb ), mice were protected from lethal h n infection exhibiting reduced mortality and pro-inflammatory responses ( ) . activation of another mapk, mek, is required for efficient iav replication and its inhibition results in viral ribonucleoprotein (vrnp) retention and reduced titers of progeny virus ( , , ) . importantly, treatment of mice with the clinically approved mek inhibitor (ci- ) showed reduced lung viral load and mortality of mice following infection with a lethal dose of pandemic h n iav; interestingly, this inhibitor significantly out-performed the clinically recommended oseltamivir in these studies ( ) . another central regulator of immune responses at the epithelium as well as immune cells is the nf-κb signaling pathway. accordingly, iav has evolved several mechanisms to modulate this pathway to counteract antiviral responses including directly targeting the ikb kinase (ikk) ( , ) . sc is a potent nfkb inhibitor that functions by reducing the ability of the p subunit of the nfkb complex to bind dna; thereby limiting its transcription-regulating functions ( , ) . in vivo administration of sc at days after lethal infection with either h n or h n avian viruses resulted in significant protection with most mice surviving and showing little to no clinical symptoms; similar results were obtained by prophylactic administration ( ) . g-protein coupled receptor kinase (grk ) is best known for its phosphorylation of gpcrs in cardiac tissue resulting in recruitment of β-arrestin to facilitate rapid receptor internalization and lysozomal degradation ( ) . recent phosphoproteomic studies identified grk as a potentially proviral host protein for iav that plays a major role in virion uncoating ( ) . although in vivo inhibition of grk using paroxetine led to a significant reduction in upper respiratory tract viral load and to a modest reduction in lower respiratory tract titers at days post infection, this inhibition was not protective from lethal infections ( ) . however, it is possible that the route of administration (intraperitoneal vs. intranasal) and dosing regimen influenced the results. sphingosin kinases (sphk) are lipid kinases that mediate conversion of sphingosine to bioactive lipid sphingosine phosphate (s p) ( ), a known modulator of central apoptotic pathways ( ) . iav infections leads to increased expression and activation of sphk and sphk ( ) and in vitro inhibition of sphk was shown to decrease iav rna synthesis via suppression of nfkb activation ( ) . treatment of mice with specific inhibitors to either sphk or sphk or a pan-sphk inhibitor led to prolonged survival of mice following lethal iav infection ( ) . peroxisome proliferator-activated receptors (pparα, pparβ, and pparγ) regulate metabolic homeostasis and are important mediators of the inflammatory response. several ppar agonists have been investigated for efficacy during iav infections with varying results. gemfibrozil (pparα agonist) not only improved symptoms when administered days after infections with an h n virus, but also increased survival of iav infected mice ( ) . prophylactic treatment of h n -infected mice with pioglitazone (pparγ agonist) resulted in increased survival ( ) . combined activation of pparγ and its downstream target ampk improved survival of mice infected with pandemic iav strains ( ) . protease activated receptor (pars) link protease activity to inflammatory cellular responses ( ) . par expression is upregulated in the mouse airways following iav infections ( ) . intranasal administration of a par antagonist (sch ) at the time of infection with various iav strains including highly pathogenic avian h n and pandemic h n viruses led to increased survival and a decrease in inflammatory responses. moreover, this effect was also observed when sch was administered - h after infection ( ) . the use of statins, angiotensin ii receptor blockers (arbs) and angiotensin converting enzyme inhibitors (acei) has been proposed to regulate the iav-induced cytokine storm in severe infections ( , ) . retrospective studies conducted separately in mexico, netherlands, uk and usa reported an association of reduced iav-related pneumonia and lower case fatality due to lower respiratory tract iav infections with statin treatment ( ) ( ) ( ) ( ) . however, this association was contested in two additional studies that found no benefit of statin treatment on iavinduced disease burden ( , ) . this uncertainty regarding the iav therapeutic potential of these widely used compounds warrants further investigations at the basic science level and in clinical trials. the continuous accumulation of adaptive mutations and the introduction of novel viruses in the human population continue to pose a threat to public health, especially to individuals at high risk to influenza. the emergence of strains resistant to existing classes of antiviral drugs and reduced vaccine effectiveness highlights the need for the development of alternative intervention strategies. therefore, therapeutic approaches that can diminish the potential for drug-resistance while being effective against multiple iav subtypes/strains are highly desirable. targeting host cell factors meets these criteria and is more likely to avoid overtly robust immune responses thereby reducing disease severity and improve patient outcome (figure ) . a large effort has been made in recent years to identify host proteins to serve as intervention targets against iv infections. several genetic and proteomic screens have identified several promising hits with potential roles in the iv replication cycle ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . in addition to these genome-wide screens, viral and host protein interactions can be mapped into networks that can also be used to identify host factors critical for iv replication ( , ) . interestingly, meta-analysis of some these studies shows limited overlap in the genes/proteins identified as required host factors ( , ( ) ( ) ( ) . this is likely due to study-specific variations in iv types/strains and cell-lines used, inclusion/exclusion criteria, limited hit-validations and methods used to "knock-down/out" these genes. local microenvironment within a given tissue can dictate the quality and intensity of an immune response. inhibition or activation of critical signaling pathways expressed in both respiratory tract epithelial and immune cells by brms can have opposite and unintended consequences. as discussed above, trail regulates immune cell-mediated apoptosis of infected cells and several studies have shown that blocking trail signaling by genomic deletion or depletion by monoclonal antibody administration can improve infection outcome in iavinfected mice. indeed inhibition of trail signaling in alveolar macrophages and other monocytes limits their ability to induce apoptosis in alveolar cells, prevents lung tissue damage and promotes survival ( , , ) . however, cd + t cells from trail−/− mice are less able to protect mice from severe infections, consistent with impaired trail-mediated effector functions of cd + t cells ( ). similarly, opposing beneficial and detrimental outcomes have also been observed in studies using bcl- inhibitors to treat iav infections ( , ) . brm delivery should be guided by immune system "compartmentalization" to ensure they elicit balanced immune responses. ideally, mucosal delivery deposits brms that reduce viral titers at the site of iav replication; however, systemic delivery of certain brms might be required to dampen dysregulated responses. this not only depends on the brms used but also on the timing of their administration. moreover, the duration of treatment with brms must be considered because sustained inhibition of certain inflammatory responses can result in an immune status that increases susceptibility to secondary opportunistic infections. repurposing of clinically approved drugs could potentially be used as brms for the treatment of severe iav infectious and should be explored ( , , ) . considering that susceptibility to severe iav infections is influenced by host genetics and hostspecific immune responses, selection of therapeutic brms should be carried out using in vivo model systems that are representative of the immune status spectrum and underlying conditions of high-risk influenza patients (young, immunocompromised, nonnaive, obese, pregnant, or aged). using these model systems will increase the likelihood of identifying brms with clinically relevant antiviral and immunomodulatory potentials. he, tg, gs, and gr conceptualized and composed the manuscript. gr and he oversaw all aspects of the manuscript preparation. this work was funded by the alexander von humboldt foundation in the framework of the alexander von humboldt professorship endowed by the german federal ministry of education and research. we apologize to any investigators whose relevant work was not included due to space limitations. regulatory roles of c-jun in h n influenza virus replication and host inflammation inhibition of p mitogen-activated protein kinase impairs 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lombardo, gloria; pinna, debora; strati, francesco; morone, diego; seehusen, frauke; hu, yue; bajoria, sakshi; xiong, jian; kumru, ozan selahattin; joshi, sangeeta bagai; volkin, david bernard; piantanida, renato; benigni, fabio; grassi, fabio; corti, davide; pizzuto, matteo samuele title: prophylactic activity of orally administered flid-reactive monoclonal siga against campylobacter infection date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: hlre i campylobacter infection is one of the most common causes of bacterial gastroenteritis worldwide and a major global health threat due to the rapid development of antibiotic resistance. currently, there are no vaccines approved to prevent campylobacteriosis, and rehydration is the main form of therapy. secretory immunoglobulin a (siga) is the main antibody class found in mucous secretions, including human milk, and serves as the first line of defense for the gastrointestinal epithelium against enteric pathogens. in this study, we describe the prophylactic activity of orally delivered recombinant siga generated from two human monoclonal antibodies (caa and ccg ) isolated for their reactivity against the flagellar-capping protein flid, which is essential for bacteria motility and highly conserved across campylobacter species associated with severe enteritis. in an immunocompetent weaned mouse model, a single oral administration of flid-reactive siga caa or ccg at h before infection significantly enhances campylobacter clearance at early stages post-infection, reducing the levels of inflammation markers associated with epithelial damage and polymorphonuclear (pmn) cells infiltration in the cecum lamina propria. our data indicate that the prophylactic activity of caa and ccg is not only dependent on the specificity to flid but also on the use of the siga format, as the immunoglobulin g (igg) versions of the same antibodies did not confer a comparable protective effect. our work emphasizes the potential of flid as a target for the development of vaccines and supports the concept that orally administered flid-reactive siga can be developed to prevent or mitigate the severity of campylobacter infections as well as the development of post-infection syndromes. campylobacter infection is one of the most common causes of bacterial gastroenteritis worldwide and a major global health threat due to the rapid development of antibiotic resistance. currently, there are no vaccines approved to prevent campylobacteriosis, and rehydration is the main form of therapy. secretory immunoglobulin a (siga) is the main antibody class found in mucous secretions, including human milk, and serves as the first line of defense for the gastrointestinal epithelium against enteric pathogens. in this study, we describe the prophylactic activity of orally delivered recombinant siga generated from two human monoclonal antibodies (caa and ccg ) isolated for their reactivity against the flagellar-capping protein flid, which is essential for bacteria motility and highly conserved across campylobacter species associated with severe enteritis. in an immunocompetent weaned mouse model, a single oral administration of flid-reactive siga caa or ccg at h before infection significantly enhances campylobacter clearance at early stages post-infection, reducing the levels of inflammation markers associated with epithelial damage and polymorphonuclear (pmn) cells infiltration in the cecum lamina propria. our data indicate that the prophylactic activity of caa and ccg is not only dependent on the specificity to flid but also on the use of the siga format, as the immunoglobulin g (igg) versions of the same antibodies did not confer a comparable protective effect. our work emphasizes the potential of flid as a target for the development of vaccines and supports the concept that orally administered flid-reactive siga can be developed to prevent or mitigate the severity of campylobacter infections as well as the development of post-infection syndromes. keywords: secretory iga, monoclonal antibodies, prophylaxis, campylobacter, flagellar-capping protein, flid campylobacter is an established cause of diarrhea worldwide and has recently been added to the who list of bacteria whose antibiotic resistance might pose a global threat to human health ( ) . campylobacter's epidemiology differs between high-income countries, where infections are sporadic, and low-and middle-income countries, in which the infection is common in early childhood and a major cause of lifethreatening acute watery diarrhea in infants ( ) . considered as a leading zoonosis, campylobacter infection is mainly associated with the consumption of contaminated undercooked animal meat (poultry being the primary bacteria reservoir), water, or unpasteurized milk ( ). campylobacter jejuni and campylobacter coli are the major causes of campylobacter enteritis in humans, and despite the clinical relevance of other emerging species ( ), they will unlikely be eclipsed in terms of prevalence and magnitude of the disease. campylobacteriosis typically results in an acute, gastrointestinal illness characterized by watery or bloody diarrhea, fever, weight loss, and cramps that last on average days ( , ). although considered a self-limiting disease, the severe dehydration associated with campylobacter enteritis represents a significant cause of death among newborns and children particularly in developing countries ( ) . furthermore, c. jejuni infections has been consistently linked with the onset of autoimmune conditions such as guillain-barré syndrome (gbs) ( , ) and inflammatory bowel disease (ibd) ( ) . despite a yet incomplete understanding of campylobacter pathogenesis, flagellum-mediated motility is widely presumed pivotal for virulence and pathogenicity, as demonstrated in both experimental animal models and in human healthy volunteer studies ( ) . nevertheless, flagellin (flaa), the major constituent of the flagellum, is not highly conserved even within the same c. jejuni species, and its heavy glycosylation pattern varies greatly depending on the strain and growth phase ( , ) . in addition, a recombinant non-glycosylated form of c. jejuni flagellin was shown to be poorly immunogenic in clinical trials ( ) . moreover, the possibility to use c. jejuni as vaccines has been limited by the risk of gbs development associated with ganglioside mimicry of bacterial lipo-oligosaccharide (los) ( ) . due to these shortcomings, there are currently no vaccines approved to prevent campylobacter infection. rehydration is the main form of therapy, and albeit antibiotics have been shown to be beneficial in severe infections, they are often not recommended due to the rapid development of antibiotic resistance ( ) . despite recovering from infection, continuous exposure of infants in low-income countries to intestinal pathogens, including campylobacter, has been linked to environmental enteropathy (ee)/environmental enteric dysfunction (eed), a subclinical chronic inflammation of the small intestine. this inflammation is associated with malabsorption of nutrients, growth faltering, impaired cognitive development, changes in microbiota, and reduced responsiveness to oral vaccination ( ) . secretory immunoglobulin a (siga) represents the most abundant class of antibodies produced in humans and serves as the first line of defense at the level of intestinal mucosa against enteric toxins and pathogens ( ) . typically, a siga consists of two monomeric iga linked tail to tail by disulfide bonds via a -kda protein called joining (j) chain and further complexed with a heavily glycosylated secretory component (sc) ( ) . while iga monomers and the j chain are synthesized, assembled, and secreted by plasma cells in the lamina propria, the sc consists of the ectodomain of the polymeric immunoglobulin receptor (pigr) that remains bound to polymeric iga (piga) following transcytosis across the enterocyte ( ) . the sc protects the antibody from proteolytic degradation, confers innate recognition functions, and contributes to appropriate tissue localization by anchoring the immunoglobulin to the mucus lining the epithelial surface ( , ) . siga acts primarily by preventing adhesion of pathogens to epithelial cells through receptor blockade, steric hindrance, and/or immune exclusion, eventually facilitating their clearance from the intestinal lumen via peristaltic and mucociliary activities ( , ) . furthermore, antigen-bound siga can be transported back into peyer's patches (pps) subepithelial dome (sed) by microfold (m) cells in a process known as reverse transcytosis, which promotes migration and activation of follicular dcs, thus conditioning the inflammatory response associated to infection by enteric pathogens ( , ) . siga is also the main antibody class found in mucous secretions, including human milk, in which the ig portion of the molecule is produced by mammary gland plasma cells that originate in the small intestine (entero-mammary pathway) ( ) . the provision of siga by breastfeeding has been reported to be critical for the protection against enteric pathogens especially in early infancy, when the contribution of endogenous production is limited ( ) ( ) ( ) . in this study, an immunocompetent mouse model of campylobacter infection was used to evaluated the prophylactic activity of orally delivered recombinant siga generated from human monoclonal antibodies isolated and selected for their reactivity against the flagellar-capping protein flid, which is pivotal for bacteria motility ( ), and highly conserved across campylobacter species frequently associated with severe neonatal enteritis ( ) . we demonstrated that, regardless of the iga isotype, prophylactically administered flid-reactive siga can enhance campylobacter clearance at early stages post-infection, dramatically reducing the levels of inflammation markers associated with epithelial damage and polymorphonuclear (pmn) cells infiltration. our data indicate that the prophylactic activity of these antibodies is not only dependent on the specificity to flid but also on features strictly associated with the siga format as the igg versions of the same antibodies did not produce a comparable beneficial effect. flid amino acid sequences from c. jejuni (n = ) and c. coli (n = ) isolates were retrieved from genbank and aligned using mega software to obtain a consensus sequence. condon optimization and synthesis of the corresponding nucleotide sequence was outsourced (genscript), and the construct was subcloned in pet a in frame with the c-term his tag. the plasmid was transformed to escherichia coli rosetta strain. gene expression was induced by the addition of mm isopropyl-beta-d-thiogalactopyranoside (iptg) to log-phase grown bacteria. after h of induction at • c, bacteria were collected by centrifugation and stored at − • c for h before being resuspended in sonication buffer ( mm nah po , mm nacl, mm imidazole, and ph . ) and being sonicated × s in ice. lysed bacteria were spun ( , × g, h, and • c), and the supernatants were filtered and then loaded on a nickel column (hitrap chelating hp, ge healthcare). column was washed with washing buffer ( mm nah po , mm nacl, mm imidazole, and ph . ) followed by washing buffer + . % triton-x and washing buffer again. bound protein was eluted from the column with elution buffer ( mm nah po , mm nacl, mm imidazole, and ph . ) and dialyzed for h at • c in dulbecco's phosphate-buffered saline (dpbs). protein size was measure on size exclusion column (hiload / superdex pg; ge healthcare), and the concentration was measured before storage at − • c. flid-reactive monoclonal antibodies caa and ccg were isolated, as previously described ( , ) , from iga + and igg + memory b cells derived from tonsillar donors who had given written informed consent, following approval by the cantonal ethical committee of cantone ticino, switzerland (ce - ). the sequences for the variable regions of the mabs were obtained from memory b cell clone messenger rnas (mrnas) following reverse transcription pcr (rt-pcr) and sequencing. the vh region of each mab was cloned into a plasmid encoding iga , iga allotype m( ) ( ), or igg heavy chains. the constructs were transiently transfected in expi cells together with those for the corresponding light chains and, in the case of siga, also with those encoding for the joining chain (j) and the ectodomain of pigr (sc) ( ) . after h, cells were supplemented with supplement medium and further incubated for days under shaking. next, cells were spun down, and the supernatant was filtered before being purified using captureselect tm iga affinity matrix (thermo fisher). siga assembly was appraised by ultrahigh performance liquid (uhpl) chromatography using acquity beh sec guard column (waters) and by denaturing nonreducing gel using siga purified from human milk (mp biomedicals) as positive control. the purified immunoglobulins were quantified with pierce tm rapid gold bca protein assay kit (life technologies) and then aliquoted and store at − • c. n-glycan oligosaccharide analysis was performed as previously described ( ) . briefly, a glycoworks rapifluor-ms n-glycan kit (waters corporation, milford, ma) was used to identify and quantify n-linked glycans following the manufacturer's instructions. fluor-ms n-glycan analysis was performed using an agilent , infinity ii hplc system equipped with a , fld detector (agilent, santa clara, ca) and an agilent , electrospray ionization time-of-flight mass spectrometer (agilent, santa clara, ca). a hilic advancebio glycan mapping column ( Å, . × mm, . µm), operated at • c, was used to separate various n-glycans. fifty microliters of prepared samples was injected into liquid chromatographymass spectrometry (lc-ms) system, with a flowrate of . mg/ml and a gradient run time of min. fluorescence was obtained using excitation and emission wavelengths of and nm, respectively. ms was acquired simultaneously from to , m/z at a constant scan rate of one spectrum per second. n-glycans were assigned based on m/z values using the n-glycan glycobase database, and nglycan quantification was calculated on integration of the fluorescence chromatogram. total carbohydrate analysis was performed as previously described ( ) . briefly, a glycoprotein carbohydrate estimation kit (thermo-fisher # ) was used to determine the total carbohydrate content (both n-and o-linked oligosaccharides) in mab samples as a percentage of total protein mass. lysozyme, bovine serum albumin (bsa), ovalbumin, apo-transferrin, fetuin, and α -acid glycoprotein were used as glycoprotein standards to construct a standard curve. size exclusion chromatography (sec) was performed as previously described ( ) . briefly, a shimadzu prominence ultrafast liquid chromatography hplc system equipped with a diode array detector (with absorbance detection at nm) was equilibrated at . ml/min flowrate in . m sodium phosphate buffer, ph . for at least h. ten microliters of each mab ( µg total protein) was injected and separated by a tosoh tskgel g swxl column ( µm particle size, . mm id × cm) with the corresponding guard column operated at ambient temperature (tosoh biosciences) using a -min run time. gel filtration molecular weight standards (bio-rad, hercules, ca) were injected before and after the mab samples to ensure integrity of the column and hplc system. potential presence of larger aggregates was determined by running mab samples with and without the sec column (i.e., protein percentage recovery). data were analyzed using lc-solution software (shimadzu, kyoto, japan). sedimentation velocity analytical ultracentrifugation (sv-auc) was performed as described previously ( ) using a proteome lab xl-i (beckman coulter) analytical ultracentrifuge equipped with a scanning ultraviolet-visible optical system. all experiments were performed at • c after at least h of equilibration after the rotor reached • c. sv-auc was performed at a rotor speed of , rpm and with detection at nm. the data were analyzed using sedfit software (dr. peter schuck, nih). intrinsic tryptophan fluorescence spectroscopy measurements as a function of temperature and polyethylene glycol (peg) relative solubility measurements were both performed as described previously ( ) . for fluorescence spectroscopy measurements, µl of each mab sample was diluted to . mg/ml in pbs, ph . and loaded into a -well plate (hard-shell -well pcr plates) and overlaid with µl of silicon oil (thermo fisher scientific, waltham, ma). samples were excited at nm, and the emission spectra were recorded from to nm with an integration time of ms using a fluorescence plate reader equipped with a charge-coupled device (ccd) detector (fluorescence innovations, minneapolis, mn). temperature ramps were programmed from to • c with an increment of . • c per step. the mean center of spectra mass peak algorithm was used to analyze the data to determine the shift in fluorescence peak position as a function of temperature. with respect to relative solubility measurements, various concentrations of peg , solutions ranging from to % w/v were prepared with a final mab concentration of . mg/ml. samples were incubated overnight at room temperature in the dark in -well plates with µl in each well. the next day, the plates were centrifuged for min at ∼ , ×g, and the supernatant was transferred to a clean -well plate. relative protein concentration in each well was then measured at nm using a spectramax m plate reader (molecular devices). thermal melting temperature values (t m ) and % peg midpoint calculations were performed using origin v . software (originlab, northampton, ma). quantification of recombinant siga and igg by elisa was performed by coating -well plates with goat anti-human iga or igg (southernbiotech). two-fold serial dilutions of the mabs in duplicates were incubated for h at room temperature. in the case of siga, detection was performed using human pigr biotinylated antibody biotinylated anti-human sc antibody (r&d system) followed by incubation with streptavidin-ap, while goat anti-human igg-ap (southernbiotech) was used for igg. siga purified from human milk (mp biomedicals) and rituximab (mabthera) were used as quantification standards for siga and igg, respectively. binding of caa , ccg siga, and igg counterparts to the recombinant antigen was measured by elisa using flidcoated -well-plates following the same detection protocol described above. epitope binning was tested by biolayer interferometry (bli) using octet red (fortebio) immobilizing recombinant flid antigen ( µg/ml) onto aps biosensor (pall fortebio) for min. after incubation with blocking buffer (bb), the sensor was incubated with caa siga ( . µg/ml) for min before being further incubated with ccg siga , caa siga , or bb for additional min. binding of siga to flid antigen in denaturing and reducing conditions was tested by western blot. briefly, µg of recombinant flid was loaded on sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page) nupage % bis-tris protein gel (thermo fisher scientific) in the presence of a reducing agent. blotting was performed using iblot system (invitrogen), and the membrane was blocked with % milk (biorad) in tris-buffered saline with tween (tbst) for h at room temperature (rt). the mabs were biotinylated by incubation with mm biotin using the ez-link nhs-peo solid phase biotinylation kit followed by desalting with zeba spin desalting columns (thermo scientific). the membrane was incubated with a % blocking grade blocker (biorad) in tbst containing µg/ml of biotinylated mabs followed by washes in tbst and by incubation with streptavidin-horseradish peroxidase (hrp). detection was performed using ecl detection kit (ge healthcare) on fusion fx (witec ag). sensitivity to igase was evaluate by incubating caa siga and siga with recombinant iga protease from neisseria gonorrhoeae (igase pro-pro-y-pro-mo bi tec). briefly, µg of caa siga and siga were incubated at • c for h with . µg of igase or with reaction buffer alone. the post-incubation products were visualized in a denaturing non-reducing gel with himark prestained protein standard (thermo fisher). c. jejuni - and c. coli nctc strains were purchased from the american type culture collection (atcc) and public health england bacteria collection, respectively. the campylobacter species were grown from glycerol stocks in mueller-hinton agar (sigma aldrich) ( g/l) supplemented with vancomycin and trimethoprim ( µg/ml) (sigma-aldrich) for h at • c in microaerophilic jar with oxoid tm campygen tm . l sachet (fisher scientific). in vitro binding of the different flid-reactive siga to a pure culture of c. jejuni was measured by flow cytometry. fifty microliters of each mabs ( µg/ml) was incubated with µl of c. jejuni - ( cfu/ml) for h at • c. after washing with pbs % fetal bovine serum (fbs), bacteria were incubated for min with biotin-conjugated anti-human iga (southern biotech, cat. no. - ). immunoglobulin-bound bacteria were then stained with streptavidin-pb at • c for min. samples were washed with pbs % fbs, resuspended in % paraformaldehyde and acquired on lsr fortessa flow cytometer (bd biosciences, franklin lakes nj, usa) using forward scatter (fcs) and side scatter (ssc) parameters in logarithmic mode. data were analyzed using the flowjo software (treestar, ashland, or, usa) or facs diva software (bd biosciences, franklin lakes nj, usa). binding of caa and ccg siga on the poles of c. jejuni was also confirmed by microscopy. briefly, µg of caa , ccg , and hgn siga were incubated with cfu of c. jejuni - for h at • c in microaerophilic conditions. next, bacteria-mabs complexes were washed and further incubated with antihuman iga-af conjugated ( : , jackson immunoresearch, cat. no. - - ) and sytobc ( : , , thermo fisher scientific, cat. no. s ) for additional min before being poured in microscope slides. images were acquired with a leica sp laser scanning confocal microscope with a -and -nm excitation. the resulting fluorescence emission was collected using - -nm (for sytox green) and - nm (for alexa ) windows. samples were imaged with a × objective (n.a. . ) in xy optical sections of . µm ( , × , pixels) with a pixel size of . nm. to improve lateral resolution, the pinhole was set to . au. animal experiments were performed in accordance with the swiss federal veterinary office guidelines and authorized by the cantonal veterinary office (ti- - ). twentyone-day-old c bl/ female mice were purchased from charles river laboratories. mice were housed, five per cage, under standardized conditions ( ± • c, ± % relative humidity, h light/dark cycle), at the institute for research in biomedicine. food and water were available ad-libitum, and mice were monitored daily. in order to measure c. jejuni specific antibody binding to the mouse microbiota, stools were collected and homogenized in sterile pbs ( . g of stools in µl pbs). samples were centrifuged for min at g to pellet large debris, and then, the supernatant were centrifuged at , g for min to pellet bacteria. the supernatant was removed, and the bacterial pellet was resuspended in µl ( µg/ml) of the different anti-campylo mabs and incubated at • c for min. after washing, bacteria were incubated at • c for min with biotin-conjugated anti-human iga (southern biotech, cat. no. - ) and syto bc ( : , , thermo fisher scientific, cat. no. s ). bacteria were washed once and then incubated with streptavidin-pb and, at the end, resuspended in % paraformaldehyde in pbs for flowcytometry analysis. all the samples were acquired on an lsr fortessa flow cytometer (bd biosciences, franklin lakes nj, usa) using fcs and ssc parameters in logarithmic mode. data were analyzed using the flowjo software (treestar, ashland, or, usa) or facs diva software (bd biosciences, franklin lakes nj, usa). differences in localization and persistence of siga vs. igg in the gastrointestinal (gi) tract of -day-old c bl/ mice were evaluated by testing the pk of the two species in the three gi subcompartments: small intestine, cecum, and colon. weaned mice were administered with a pbs solution containing µg of campylobacter-irrelevant antibody hgn , which targets hiv gp and presents no cross-reactivity with the murine microbiota, in the form of siga m( ) or igg. a control group was administered only with pbs. at , , and h postoral administration, five animals per group were euthanized, and the content of the three different intestinal tracts was collected and resuspended in . ml pbs. the samples were then homogenized, and after centrifugation, the supernatants were collected and stored at − • c. the detection of human iga or igg in the different parts of the gi tract was performed by elisa. briefly, -well plates were coated with goat antihuman iga or igg antibodies (southern biotechnology: - and - ) and blocked with % bsa blocking buffer. plates were then incubated with -fold serial dilutions ( : to : ) of each intestinal sample for h at room temperature. after four washes with pbs + . % tween , the presence of the antibodies was detected by incubation with goat anti-human lambda antibody ap conjugated (southern biotechnology: - ) for h at room temperature followed by min incubation with p-nitrophenylphosphate (pnpp) and detection at nm. taking into account the different numbers of lambda light chains possessed by the polymeric immunoglobulin, to quantify the amount of siga or igg in the samples, two distinct standard curves were built using hgn siga and hgn igg as standards, respectively. no signals were detected testing the samples from the control animals administered only with pbs. the inflammation status of mice was evaluated by measuring the levels of lipocalin- (lcn- ) in fecal supernatants via elisa assay (r&d systems, duoset elisa mouse lipocalin- /ngal) , , and h post-infection. briefly, . g feces was resuspended in µl in pbs, centrifuged for min at maximum speed, and diluted before performing the elisa assay, according to manufacturer's instructions. ceca from all animals were examined at necropsy, fixed in % neutral buffered formalin for at least h prior to embedding in paraffin, and stained with hematoxylin and eosin (h&e). pathological scores were determined in a blinded manner using a previously described scoring scheme ( ) . briefly, each tissue section was assessed for ( ) submucosal edema ( , no change; , mild; , moderate; , severe); ( ) crypt hyperplasia ( , no change; , - %; , - %; , > %); ( ) goblet cell depletion ( , no change; , mild depletion; , severe depletion; , absence of goblet cells); ( ) epithelial integrity [ , no pathological changes detectable; , epithelial desquamation (few cells sloughed, surface rippled); , erosion of epithelial surface (epithelial surface rippled, damaged); , epithelial surface severely disrupted/damaged, large amounts of cell sloughing; , ulceration [with an additional score of added for each % fraction of tissue in the cross-section affected up to a maximum score of ( + ) for a tissue section that had entirely lost its crypt structure due to epithelial cell loss and immune cell infiltration]; ( ) mucosal mononuclear cell infiltration (per × magnification field) ( , no change; , < ; , - ; , > cells/field); ( ) submucosal pmn and mononuclear cell infiltration (per × magnification field) ( , < ; , - ; , - ; , > cells/field). a maximum score under this scale is . statistical analyses were performed using graphpad prism v . (graphpad software, la jolla, ca, usa). mann-whitney u test was used to determine the statistical significance of the results. a p < . was considered significant in all cases. different campylobacter's proteins were evaluated as potential targets to prevent infection on the basis of (i) the presence of surface exposed epitopes, (ii) role in the bacteria pathogenesis, (iii) ability to stimulate an immune response in the context of natural infection, and (iv) level of amino acid sequence conservation within but not limited to the c. jejuni species. the flagellar-capping protein flid, also known as hookassociated protein (hap ), is a -kda protein with high sequence conservation across the c. jejuni species ( ) (supplemental figures a,b ). based on structures and selfoligomerization mechanisms characterized for other flagellated pathogens ( , ), campylobacter flid oligomers form the cap protein complex located at the tip of the flagellum, which controls the distal growth of the filament by regulating the assembly of the flagellin molecules. due to its functional role in filament elongation, flid-deficient mutants exhibit defects in bacterial motility ( ). the potential of flid as a promising target is strengthened by the proposed involvement in cell adherence ( ) and the immunogenicity in chickens during natural infection. accordingly, it has been suggested as a vaccine candidate for the prevention c. jejuni in broilers ( , ) . flid was therefore selected as a candidate target antigen for monoclonal antibody (mab) development. the frequencies of igg + and iga + flid-reactive memory b cells in tonsillar samples of swiss origin were evaluated using the antigen-specific memory b cell repertoire analysis (ambra) ( ) (supplemental figure ) . igg + and iga + memory b cells from selected tonsils were then immortalized under clonal conditions ( ) , and culture supernatants were screened using a -well plate-based high-throughput platform to identify b-cell clones expressing flid-reactive antibodies. using this approach, memory b cell clones producing two human monoclonal antibodies, namely caa and ccg , were isolated and selected based on their specificity and affinity for recombinant flid. caa was originally isolated as an iga encoded by v h - /d - /j h with a -amino acid hcdr and v k - /j k , whereas ccg was isolated as an igg encoded by v h - /d - /j h bearing a shorter -amino-acid hcdr and v l - /j l . humans present two different iga subclasses that differ mainly for the length and glycosylation of the hinge region. iga possesses a -amino-acid longer hinge than iga containing up to five o-linked glycans at serine and threonine residues. the longer hinge not only confers to iga antibodies higher flexibility and a longer fab reach but is also linked to the sensitivity to a specific class of bacterial proteases known as iga proteases. bearing a shorter hinge region lacking proline-serine and/or proline-threonine peptide bonds, iga is instead resistant to these proteases ( ) . in addition, when compared to igg, igm, and iga , the iga isotype has the unique characteristic to undergo reverse transcytosis by contacting dectin-i receptor on the surface of pps m cells ( ) . both the cα region and the glycosylation pattern appear pivotal for the interaction at the basis of this mechanism, which has been suggested as a strategy to boost adaptive immunity against pathogens ( ) . the recombinant siga , siga , and diga versions of the flid-reactive mabs were generated by transient transfection of expi cells and were then purified by affinity chromatography. various physicochemical measurements were performed to monitor key structural attributes of recombinant flid-reactive polymeric igas as summarized in supplemental figure a . the conformational stability of the mabs was determined by monitoring their overall tertiary structure as a function of temperature (intrinsic tryptophan fluorescence spectroscopy), and a - • c lower thermal melting temperature (tm) value was observed for siga when compared to siga and diga (supplemental figure a) . the difference in conformational stability could be due to structural differences in the hinge region between the two subtypes. the relative solubility of the mabs was evaluated by a peg precipitation assay, and diga was found to be relatively more soluble compared to siga and siga , a result of which suggests that the presence of the secretory component protein may affect mab solubility at neutral ph. molecular size analysis by sedimentation velocity analytical ultracentrifugation (sv-auc) revealed a heterogeneous distribution of species for each mab (supplemental figure a) . we observed that siga had a considerably higher relative percent composition of larger molecular weight species than the other two mabs (as compared to the percent main peak). as expected, diga had a later elution time when compared to siga and siga in size exclusion chromatography (supplemental figure b) . each of the caa iga molecules were heavily glycosylated with carbohydrates ranging from ∼ - % of the total mass of each mab. as expected by sharing the same iga backbone, the nlinked oligosaccharide structural types were overall very similar in siga and diga antibodies (supplemental figure c ). differently to these antibodies, caa siga lacked g f + gn, g f -gn, gif + gn and g f + nana, while it contained g f and man g + nana (supplemental figure c) . finally, incubation of caa siga and siga with recombinant igaase from n. gonorrhoeae confirmed the differences in sensitivity to bacterial iga proteases of the two isotypes, which have been associated to the length and sequence of the hinge regions (supplemental figure d) . based on its resistance to bacterial iga proteases and the ability to undergo reverse transcytosis, the iga scaffold was initially selected over the iga for our studies, and both caa and ccg siga were further characterized in vitro. to include a control antibody that was not reactive with the antigen in our experiments of campylobacter infection, we also generated the siga version of the hgn mab, which targets an epitope on the v loop of hiv- env glycoprotein ( ) . caa and ccg siga molecules maintained the original flid binding activity, displaying similar ec (caa = . ; ccg = . µg/ml) for the flagellar protein ( figure a) . epitope binning studies by bli indicated that the binding of one antibody to the antigen did not prevent the binding of the other mab (figure b) , pointing to the recognition of two distinct epitopes. in addition, caa , but not ccg , recognized flid by western blot analysis under denaturing and reducing conditions, confirming the binding to structurally different antigenic determinants (figure c) . accessibility of the two mabs to these epitopes in the flagellum was assessed by flow cytometry using two flid-divergent historical isolates c. jejuni - and c. coli nctc . caa and ccg stained both pathogens, thus confirming epitope accessibility and breadth ( figure d) . consistent with flid localization, confocal imaging on c. jejuni - confirmed that the binding of the two mabs occurred at the cell poles ( figure e ). the murine intestine is highly resistant to campylobacter due to intense competition exerted by the rich gut microbiota and to a certain level of tolerance, which limits inflammation ( ) ( ) ( ) . to overcome the challenge represented by the resident microbiota, pre-treatment via oral gavage with vancomycin, for which campylobacter species are inherently resistant ( ) , is largely adopted. although the pretreatment allows robust bacterial colonization in the cecum, it does not appear to enhance the pathology in adult wild-type mice since minimal signs of inflammation were previously observed ( ) . higher susceptibility to c. jejuni infection of infant wild-type mice in comparison to adult animals has been previously reported and linked to significant differences in the microbiota composition ( ) . to set up a model that would allow us to evaluate the prophylactic activity of our mabs under immunocompetent conditions, we evaluated the sensitivity to c. jejuni - infection of pups ( days old), weaned ( days old), and adult ( days old) c bl/ mice. animals were pre-treated with vancomycin via oral gavage to deplete the murine microbiota before infection with c. jejuni - at cfu/mouse. campylobacter isolation from stools at days post-infection revealed a ∼ log higher increase in shedding from -day-old animals than from -and -day-old mice (figure a) . in line with these results, weaned mice displayed significantly higher intestinal inflammation as measured by the levels of lipocalin- in the stools and histological score values in the cecum in comparison to pups and adult mice (figures b,c) . since antibiotic pretreatment should provide comparable ecological niches for infection in the different animals, other factors could be accountable for the different sensitivity to c. jejuni infection displayed by the three groups of mice. analysis of murine iga in the stools of -, -, and -day-old animals revealed different concentrations among the groups (figure d ). a phylogenetic tree built using neighbor-joining method with jukes-cantor distance measurement is shown to provide the amino acid distance of flid between the two historical isolates tested (in red). one representative experiment out of three is represented. (e) binding of caa siga to c. jejuni as observed in confocal microscopy. bacteria were stained using syto bc (green), whereas caa was detected using anti-human iga af conjugated (red). interestingly, weaned mice presented negligible levels of iga in the stools in comparison to both pups and adult mice. this observation is consistent with the transition between the exogenous supply of maternal antibodies provided along with the milk ( -day-old mice) and the beginning of the endogenous production that is established in adult animals (i.e., -day-old mice). therefore, a likely important factor in the susceptibility of weaned mice to c. jejuni infection could be the lower concentration of secretory iga due to the relative immaturity of intestinal immune system and the depletion of maternal antibodies in these animals. based on these observations, weaned mice were selected as an immune competent animal model to study the prophylactic activity of flid-reactive mabs. since off-target binding of caa and ccg to the murine microbiota would result in reduced bioavailability and thus activity against pathogens in a prophylactic setting, we evaluated potential cross-reactivity of the recombinant siga with the microbiota of weaned mice. stools from animals orally infected with c. jejuni, c. coli, or pbs (mock infected) were collected h post-infection and incubated with the two flid-reactive mabs and the control hgn . ccg displayed limited levels of cross-reactivity with the murine microbiota ( . %), which were comparable with those observed for the control antibody ( . %) (supplemental figure a) . conversely, caa was binding the microbiota to a higher extent ( %), suggesting a possible fabmediated recognition of antigens in the commensal bacteria that present structures similar to flid (supplemental figure a) . nevertheless, for both flid-reactive mabs, the percentage of igacoated bacteria dramatically increased in the stools of infected animals confirming the prompt recognition of the c. jejuni and c. coli species (caa , . and . %; ccg , . and . %). interestingly, we also observed an increase in hgn binding to the bacteria in the stools of these animals ( . and . %) that could be consistent with the innate binding activity of iga and secretory component against various pathogens ( , ) . finally, to examine the conditions for testing the prophylactic efficacy of the mabs, we evaluated the pharmacokinetics of orally administered siga in different gastrointestinal tracts of weaned mice. the campylobacter-irrelevant hgn siga , which displayed negligible cross-reactivity with the murine microbiota (supplemental figure a) , was administered as a single oral gavage of µg in pbs (≈ mg/kg), and its concentration in the different intestinal subcompartments was measured at , , and h post-administration (supplemental figure b) . hgn siga concentration in the cecum was maintained almost constant within the first h post-administration and then tended to rapidly decrease by h. the human antibody was not detectable at or h post-administration (data not shown). we next evaluated the prophylactic activity of orally administered flid-reactive recombinant siga in immunocompetent-weaned mice infected with c. jejuni - . as such, vancomycin pretreated animals were administered with a single oral gavage of µg/mouse of caa , ccg , hgn siga , or pbs h before oral infection with cfu/mouse of c. jejuni - . treated animals and the corresponding control groups were monitored for h, during which the severity of infection and degree of inflammation were recorded. of note, analysis of the stools from treated mice revealed a trend characterized by higher campylobacter shedding at h post-infection followed by a significant decrease over time. conversely, untreated and hgn -treated groups presented lower shedding at early time points followed by a consistent cfu increase at h post-infection ( figure a) . these results suggest that that caa and ccg could prevent or reduce the ability of the pathogen to adhere to the surface of the mucosal epithelium, thus facilitating the clearance of bacteria via peristalsis or mucociliary activity at early stages postinfection. this hypothesis is further supported by the significant lower levels of lipocalin- , a marker of intestinal inflammation linked to epithelial damage and neutrophil infiltration, recorded at h post-infection in the stools of caa -and ccg -treated animals in comparison to the control groups (infected/nontreated and infected/hgn -treated groups) ( figure b ). in addition, animals treated with a single administration of flidreactive mabs presented levels of pmn cells infiltration and histological score values in the cecum significantly lower than the hgn -treated group (figures c,d) , which indicates that the activity exerted by caa and ccg is largely fab mediated. similar findings were observed in animals administered h before infection with higher or lower campylobacter infection doses ( or cfu/mouse). indeed, in both cases caa -administered animals presented significantly higher bacterial clearance than the untreated group (supplemental figures a,b) and levels of intestinal inflammation similar to those of noninfected mice (supplemental figures c,d) . furthermore, no significant differences were observed in the reduction in bacterial shedding, pmn infiltration in the cecum lamina propria, and lipocalin- levels in the stools when caa siga was provided h before infection or when it was premixed with c. jejuni - before oral administration to the animals (supplemental figures e-g) . taken together, these results indicate that prophylactic administration of flid-specific caa and ccg siga increase protection from campylobacter infection by accelerating bacterial clearance at early stages after infection, thus significantly reducing inflammation. since longer fab reach, higher flexibility, and different glycosylation may affect the cross-linking ability and/or persistence of the polymeric ig in the intestine, we next investigated whether the two iga isotypes may exert different prophylactic activities in our mouse model of campylobacter infection. caa siga and siga displayed comparable binding to flid (supplemental figure a) , and no significant differences in reactivity to campylobacter species in vitro or with murine microbiota ex vivo were observed between the two formats (supplemental figures b,c) . the prophylactic activity of the two isotypes was then tested in the murine model of campylobacter infection. in line with our previous findings, animals administered with the flid-reactive mabs displayed higher campylobacter shedding at early time postinfection followed by decrease over time, while infected non-treated animals produced an opposite trend ( figure a) . although caa siga accelerated shedding faster than siga as confirmed also by the percentage of human iga-coated bacteria in the stools (supplemental figure d) , both subclasses were equally capable to limit inflammation in infected animals as shown by the levels of lipocalin- in the stools, pmn infiltration, and the corresponding histological score in the cecum at h postinfection (figures b-d) . overall, our results indicate that differences in flexibility and glycosylation associated to the two human iga subclasses do not have an impact in the prophylactic activity exerted by caa in the in vivo model. although siga are the most abundant antibodies expressed in association with the intestinal mucosa and the first line of defense against enteric pathogens, they are characterized by a complex structure, and their development as drugs might result challenging in comparison to igg-based monoclonal antibodies. because the activity of the campylobacter-reactive mabs was previously shown to be dependent on the specificity for flid, we generated caa , ccg , and the campylobacterirrelevant antibody hgn as human igg and evaluated their prophylactic activity in comparison to their corresponding siga counterparts. since glycosylation might affect the ability of mabs to interact with mucin on the mucosal surface, the localization and persistence of hgn igg in the murine intestinal tract were appraised by administering the antibody with a single oral gavage to weaned mice and then by measuring the its concentration in the different intestinal subcompartments after , , and h (supplemental figure c) . as in the case of hgn siga , at every time point, the highest concentration of the igg was detected in the cecum; however, in this intestinal subcompartment, the igg concentration tended to decrease faster than for siga , with a significant reduction already at h post-administration (supplemental figures b,c) . next, we evaluated the prophylactic activity of the flidreactive mabs caa and ccg as igg or siga both administered to weaned mice h before infection with c. jejuni - . interestingly, while animals treated with siga antibodies displayed the previously observed pattern characterized by higher shedding at h postinfection followed by a significant decrease at and h, the groups treated with the igg version of the same antibodies revealed trends similar to the non-treated groups ( figure a and supplemental figure a ). the importance of the siga format for the in vivo efficacy was further confirmed by the lower ability of caa and ccg igg to limit inflammation in comparison to their polymeric counterparts, as shown by pmn cells infiltration in the cecum and lipocalin- levels in the stools of the infected animals at h post-infection (figures b,c and supplemental figures b,c) . overall, no significant differences in the histological scores were observed between the mice treated with the flid-reactive igg antibodies and the non-treated animals ( figure d and supplemental figure d) . conversely, the siga versions of the antibodies were able to replicate the beneficial effect previously observed, maintaining the histological score in the cecum to values significantly lower than both non-treated and igg-treated mice (figure d and supplemental figure d) . taken together, these data indicate that the caa and ccg igg have limited prophylactic activity when orally administered prior to campylobacter infection. the reduced activity of caa and ccg igg might be dependent on both the lower stability and persistence in the gastrointestinal tract and/or on the decreased "bonus effect of multivalency" at the basis of the cross-linking activity associated with the polymeric format. the aim of this study was to evaluate the ability of orally administered recombinant siga, derived from selected flidreactive monoclonal antibodies, to prevent infections by campylobacter species frequently associated with severe neonatal gastroenteritis. despite its pivotal role in bacteria motility, epithelial cells adherence ( , ) , and the high level of conservation among c. jejuni (supplemental figure ) , flid has never been assessed to date as a potential target for therapeutic mabs. consistent with the immunogenicity previously reported for this antigen ( , ) , our results indicate that flid-reactive mabs can be isolated from the human memory b cell repertoire. further development of these antibodies, namely, caa and ccg , resulted in recombinant siga that preserved the ability to bind flid on the campylobacter's flagellum of both c. jejuni and c. coli species (figure d) . genetically manipulated animals characterized by an exacerbated inflammatory response to bacteria, such as sigirr or il − / − , have been proposed as models to study campylobacter pathogenesis ( , ) . these mutations, however, dramatically alter the murine immune system to an extent that even the presence of commensal microbes can potentially result in spontaneous enterocolitis ( ) . the sensitivity of wild-type mice to campylobacter infection has consistently been linked to age-dependent variations in the composition of the resident microbiota ( ) . of note, our findings indicate that different levels of iga in the murine intestine also play a role in campylobacter pathogenesis in mice. in particular, we observed that weaned mice represent the most permissive wild-type age group and that this is likely due to the transition between the exogenous iga supply provided by the maternal milk and iga endogenous production ( figure d) . moreover, giallourou et al. described a weaned mouse model of c. jejuni that can recapitulate enteropathy and diarrhea ( ) . in the current study, we established a weaned mouse model of campylobacter infection to evaluate the prophylactic activity of the mabs based on bacterial shedding and inflammatory biomarkers (figures a-c) . in this animal model, we documented a consistent effect following a single prophylactic administration of flid-reactive siga h before c. jejuni infection, which is characterized by significantly higher campylobacter shedding at the early stage of infection followed by a rapid decline at later time points (figure a) . our hypothesis is that by binding flid, the siga can limit bacteria motility and ability to adhere to the surface of the mucosal epithelium, thus accelerating their clearance via peristalsis. this idea is further strengthened by the fact that animals administered with flid-reactive mabs display considerably lower levels of inflammatory biomarkers in the cecum and stools than the control groups, which present an opposite shedding trend. flid-reactive mabs efficacy relies on the specific recognition of the bacterial surface antigen, as an irrelevant antibody in the same format does not produce similar beneficial effects (figures a-d) . further studies will elucidate whether these antibodies may act primarily by limiting bacteria motility (cross-linkage), by limiting attachment to cell receptors (steric hindrance), by limiting secretion via the t ss of proteins that contribute to host cell invasion (e.g., ciab) or via a combination of all these activities. interestingly, similar prophylactic activity was observed when caa was premixed with c. jejuni or administered h before infection, thus confirming siga stability during the passage through the small intestine and their persistence in the cecum (supplemental figures e-g) . despite the fact that siga showed a slightly higher ability to accelerate bacterial shedding at early stage of infection in comparison to the siga counterpart (figure a) , differences in length and glycosylation of the hinge region between the two human iga isotypes do not appear to significantly undermine the ability of the polymeric immunoglobulins to prevent inflammation (figures b-d) . however, iga might represent the most promising backbone for development of orally deliverable mabs due to resistance to iga proteases expressed by different pathogens (supplemental figure d ) and to the unique ability to undergo reverse transcytosis ( ) , which in turn could contribute to boost a potential vaccinal effect ( ) . the prophylactic activity of flid-reactive caa and ccg was significantly decreased when these mabs were administered as igg . indeed, in the igg format, the mabs were neither able to boost campylobacter shedding at early time postinfection nor able to limit inflammation to an extent comparable with their corresponding siga (figure and supplemental figure ) . the superior efficacy of caa and ccg as siga might be related to higher resistance to proteases in the small intestine ( , ) , higher localization and better persistence in the cecum ( , ) , and/or to the mechanisms of action (i.e., cross-linking) associated with the polymeric immunoglobulin structure ( , ) . in summary, our results indicate that the flagellar-capping protein flid represents a promising target for the development of campylobacter-reactive mabs capable to exert a prophylactic activity dependent on both their affinity to the antigen and on features strictly related to the siga format. although improvement in water sanitation and healthcare conditions are pivotal to reduce campylobacter's impact in infants in middle-and low-income countries, these findings suggest that the development of orally deliverable recombinant siga targeting flid might represent a novel strategy to prevent or mitigate both disease severity and the development of postinfection syndromes. the datasets generated for this study are available on request to the corresponding author. the animal study was reviewed and approved by swiss federal veterinary office guidelines and the cantonal veterinary office (ti- - ). who publishes list of bacteria for which new antibiotics are urgently needed status of vaccine research and development for campylobacter jejuni the clinical importance of emerging campylobacter species the global view of campylobacteriosis: report of an expert consultation pathogen-specific burdens of community diarrhoea in developing countries: a multisite birth cohort study (mal-ed) guillain-barre syndrome-related 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transmembrane forms the polymeric immunoglobulin receptor: bridging innate and adaptive immune responses at mucosal surfaces high-avidity iga protects the intestine by enchaining growing bacteria dectin- is essential for reverse transcytosis of glycosylated sigaantigen complexes by intestinal m cells breastfeeding: maintaining an irreplaceable immunological resource protection of breast-fed infants against campylobacter diarrhea by antibodies in human milk protection against campylobacter diarrhea: role of milk iga antibodies against bacterial surface antigens self-oligomerizing structure of the flagellar cap protein flid and its implication in filament assembly evaluation of flagellum-related proteins flid and fspa as subunit vaccines against campylobacter jejuni colonisation in chickens rapid development of broadly influenza neutralizing antibodies through redundant mutations an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus mucosal immune defense: immunoglobulin a preformulation characterization and stability assessments of secretory iga monoclonal antibodies as potential candidates for passive immunization by oral administration a novel mouse model of campylobacter jejuni gastroenteritis reveals key pro-inflammatory and tissue protective roles for toll-like receptor signaling during infection bacterial flagellar capping proteins adopt diverse oligomeric states host cell binding of the flagellar tip protein of campylobacter jejuni characterization and antigenicity of recombinant campylobacter jejuni flagellar capping protein flid clonal dissection of the human memory b-cell repertoire following infection and vaccination the iga proteases of pathogenic bacteria secretory iga as a vaccine carrier for delivery of hiv antigen to m cells analysis of memory b cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from hiv- -infected individuals novel murine infection models provide deep insights into the "menage a trois" of campylobacter jejuni, microbiota and host innate immunity modification of intestinal microbiota and its consequences for innate immune response in the pathogenesis of campylobacteriosis campylobacter jejuni colonization of mice with limited enteric flora mechanisms of antibiotic resistance in campylobacter species campylobacter jejuni infection of infant mice: acute enterocolitis is followed by asymptomatic intestinal and extra-intestinal immune responses glycosylation of human iga directly inhibits influenza a and other sialic-acidbinding viruses binding of lactoferrin and free secretory component to enterotoxigenic escherichia coli the role of serine protease htra in acute ulcerative enterocolitis and extra-intestinal immune responses during campylobacter jejuni infection of gnotobiotic il- deficient mice c bl/ and congenic interleukin- -deficient mice can serve as models of campylobacter jejuni colonization and enteritis a novel mouse model of campylobacter jejuni enteropathy and diarrhea increased risistance of immunoglobulin a dimers to proteolytic degradation after binding of secretory component genomic characterization of the guillain-barre syndrome-associated campylobacter jejuni icdccj isolate campylobacter jejuni strain cg : a refined model for the study of campylobacteriosis and evaluation of campylobacter vaccines in human subjects campylobacter jejuni lipopolysaccharides in guillain-barre syndrome: molecular mimicry and host susceptibility the authors would like to thank dr. antonio pellanda, dr. elena scotti, dr. stefano ermanni (eoc-ospedale regionale di lugano, switzerland), and dr. nicola melik (eoc-ospedale regionale di locarno, switzerland) for providing tonsillar samples from patients undergoing tonsillectomy. we would also like to thank omar vandal and jeremy blum for helpful advice throughout the execution of the study. lp, fg, dc, and mp conceived the study. lp and mp initiated the study design. sj, gl, dp, fst, rp, and fb helped with implementation. dm and fse provided expertise in microscopy and immunohistochemistry, respectively. yh, sb, jx, ok, sbj, and dv performed the physiochemical characterization of the recombinant mabs. lp, fg, dc, and mp outlined and wrote the manuscript, whereas ok, sbj, and dv contributed to its finalization. all authors approved the final manuscript. the remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © perruzza, jaconi, lombardo, pinna, strati, morone, seehusen, hu, bajoria, xiong, kumru, joshi, volkin, piantanida, benigni, grassi, corti and pizzuto. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - i gk et authors: bachmann, maría consuelo; bellalta, sofía; basoalto, roque; gómez-valenzuela, fernán; jalil, yorschua; lépez, macarena; matamoros, anibal; von bernhardi, rommy title: the challenge by multiple environmental and biological factors induce inflammation in aging: their role in the promotion of chronic disease date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: i gk et the aging process is driven by multiple mechanisms that lead to changes in energy production, oxidative stress, homeostatic dysregulation and eventually to loss of functionality and increased disease susceptibility. most aged individuals develop chronic low-grade inflammation, which is an important risk factor for morbidity, physical and cognitive impairment, frailty, and death. at any age, chronic inflammatory diseases are major causes of morbimortality, affecting up to – % of the population of industrialized countries. several environmental factors can play an important role for modifying the inflammatory state. genetics accounts for only a small fraction of chronic-inflammatory diseases, whereas environmental factors appear to participate, either with a causative or a promotional role in % to % of patients. several of those changes depend on epigenetic changes that will further modify the individual response to additional stimuli. the interaction between inflammation and the environment offers important insights on aging and health. these conditions, often depending on the individual’s sex, appear to lead to decreased longevity and physical and cognitive decline. in addition to biological factors, the environment is also involved in the generation of psychological and social context leading to stress. poor psychological environments and other sources of stress also result in increased inflammation. however, the mechanisms underlying the role of environmental and psychosocial factors and nutrition on the regulation of inflammation, and how the response elicited for those factors interact among them, are poorly understood. whereas certain deleterious environmental factors result in the generation of oxidative stress driven by an increased production of reactive oxygen and nitrogen species, endoplasmic reticulum stress, and inflammation, other factors, including nutrition (polyunsaturated fatty acids) and behavioral factors (exercise) confer protection against inflammation, oxidative and endoplasmic reticulum stress, and thus ameliorate their deleterious effect. here, we discuss processes and mechanisms of inflammation associated with environmental factors and behavior, their links to sex and gender, and their overall impact on aging. the inflammatory response is different in men and women. adult females develop stronger innate and adaptive immune responses than males. these sex-related differences can determine the ability of immune cells to generate an effective inflammatory response, which translates into epidemiological differences on the prevalence of various pathologies, including allergies ( ), asthma ( , ), autoimmune diseases ( ), anaphylaxis ( ), neonatal sepsis ( ), and cancer ( ), among several pathologies. the immune response of women is polarized towards an increased production of th cells, t regulatory cells (treg), m macrophages, il , il , and gata- cytokines, and decreased th , th , tbet, and rorgt lymphocytes ( - ). on the contrary, men show an immune response that depends on th lymphocytes ( , ), high il production ( ) and low levels of reactive mast cells ( ) . men have also an increased response of microglia in the central nervous system (cns) and an increased presence of tnfa and prostaglandins in response to inflammatory stimuli ( ). differences in inflammatory response between men and women vary among specific tissues. in the cns inflammation, women show greater levels of b-cell (cd +, cd +, cd d hi b ) migration from the spleen to the site of injury than men, followed by an increase of macrophages/microglia (cd b+, cd ), which appears to generate a lower neuroinflammatory response in female compared with male mice ( ). in addition, women develop an increased immunoreactivity due to high numbers of ifn-producing dendritic cells ( , ). female mice tend to have m phenotype and activated eosinophils and mast cells show a higher reactivity than in male mice ( , ) . however, in response to an acute inflammatory stimulus, males produce higher amounts of inflammatory cytokines, cd a+ neutrophil and t cells infiltration of the injury site ( ). conversely, the inflammatory microenvironment in female mice is characterized by an increased production of antibodies ( , ) and a differential pattern migration of antibody-secreting cells ( ). the immune system responds differently in men and women not only because of the influence of sex hormones, but also differences in the patterns of autosomal methylation and x chromosome methylation, which determine distinctive profiles of gene expression ( , ). sex hormones exert antagonist effects on the immune system: both estradiol and testosterone have a suppressive effect on the immune response ( ). estrogen is the sex hormone with the greatest impact on the immune response, being described as one of the non-modifiable regulators of the immune system, due to its immunoregulatory and protective effects in many inflammatory models ( ). however, this is contradictory with the fact that women have a higher prevalence of autoimmune diseases than men, although estrogens should be a protective condition ( ). the sex-dependent difference in the immune response is time-, and estrogen dose-dependent ( ). variations on the estrogen concentration during the ovulatory cycle, puberty or menopause, can promote the development of immune-related diseases ( ). mice exposed to chronic estrogen-treatment generate hormone resistance, decreasing the clonal expansion of treg lymphocytes in autoimmune diseases ( , ) . estrogen regulates immune response primarily through aand b-estrogen receptors (era/b), mitogen-activated protein kinase (mapk) pathways, estrogen-dependent ′- ′-cyclic adenosine monophosphate (camp) response element-binding (creb), and modifications in the production of camp in immune cells ( ). in addition to estrogen receptors, the presence of il receptors influences the type of immune response; female macrophages express greater amounts of il receptors than males. il receptors favor the m phenotype when stimulated by estrogen. in agreement with that, estrogen induces an increased expression of il on naive cd + t cells ( , ) . for a better general view of estrogen´s mechanisms and effect on the innate immune system cells we recommend reviews that have extensively covered those topics ( - ). sex regulates gene expression in multiple human tissues, in fact, one third of the autosomal genes that are expressed in a sexbiased manner exhibit androgen or estrogen hormonal response elements ( , ). sex hormones play a strong role in sexually dimorphic gene networks ( ), inducing aberrant expression in immune response genes via differential methylation ccl cxcl il ( ) . there are changes in the methylation pattern of sex-dependent immune response genes during embryonic development, which are reinforced in puberty by the estrogenmediated induction of active forms of chromatins that are maintained during adulthood ( ) . immune response-related genes located in chromosomes and x are differently expressed in b lymphocytes depending on the sex of the individual ( ). among the differentially expressed genes that are relevant for the immune/inflammatory response, can be mentioned the toll-like signaling, cytokine receptors, jak-stat pathway and genes related to the activation of t-cell receptors ( ) . phenotypically, the different pattern of gene expression may explain the greater female t-cell expandable capacity when exposed to an antigen ( ) . female t cells present higher activation and division capacities than their male counterparts. however, male t cells can develop greater infiltration potential and a lower self-reactive phenotype than female ones ( , ) . these differences could be due to the high expression of peroxisome proliferator-activated receptors (ppars) ( ) , prostaglandins, and cyclooxygenase- (cox- ) in males ( ) . the influence of sex on the immune response is observed throughout life and is accentuated with aging. in the neonatal stage, women have a lower concentration of regulatory t lymphocytes than men ( ) . during childhood, men develop a more intense immune response and are more likely to develop infections by various pathogens compared with women ( , ) . with increasing age, the dynamics and proportion of lymphocytes and myeloid cells differ depending on the sex due to the differential expression of genes of the immune response in men and women ( ) . also, in aged individuals, epigenomic changes generate a more robust innate and pro-inflammatory response in men and an increased activity in the adaptive immune response in women ( , ) . in recent times, during the covid- pandemic, it has been observed that the infection by sars-cov- in older adults shows conspicuous differences; men have elevated plasma levels of il and il and a high amount of monocytes whereas women develop a robust activation of t lymphocytes ( ) . this differences in the immune response could explain the higher mortality of covid in men than in women ( , ) . to recapitulate, sex hormones and genetic expression patterns in men and women can generate distinct immune and inflammatory responses that determine singularities in the epidemiological distribution of immune diseases. research protocols in immune response and inflammation must be redefined to avoid results biased by sex. furthermore, research in women is urgently needed to define the efficacy for women of several therapies that were originally tested in men. the increase in noncommunicable diseases (ncds), such as obesity, hypertension and cancer as well as the low-grade chronic inflammation that characterizes most ncds ( ) can be affected by environmental factors that change the immune response. lifestyle factors like nutrition can modulate the immune system. it has been reported in mice that western diet-induced systemic inflammation and reprogramming of myeloid cell precursors is mediated by the activation of the nlrp inflammasome, which is a key sensor of the innate immune system for metabolic danger signals, such as uric acid and cholesterol ( ) . metabolic regulation appears to be very robust and long lasting, being reported that proper nutrition during pregnancy can reduce the risk for ncds in the offspring even at adult age ( , ) . the impact of the diet on the immune response and inflammation some diet types can result in metabolic and epigenetic changes that affect immune function ( ) , as reported in populations that consume a high-fat and low-fiber western diet, who show a prevalence of ncds higher than populations that consume a mediterranean diet or a diet based on bioactive compounds, like the hydroxytyrosol in olive oil ( ) ( ) ( ) . there is evidence supporting the anti-inflammatory activity of phenolic extracts from olive oil, such as their ability to reduce lipopolysaccharide (lps)-stimulated nitric oxide (no) production by the raw- . macrophage cell line. the hydroxytyrosol stearate and the hydroxytyrosol oleate decrease no production in a concentration-dependent manner ( ) . in addition, olive oil extracts increase total plasma glutathione concentration ( ) , increasing the antioxidative response of the individual. nordic diet has many similarities with the mediterranean diet, but its effects on low-grade chronic inflammation are less known. both diets include abundant fruits, vegetables, whole grain products, fish and vegetable oil, but restrict saturated fat and red and processed meats ( , ) . observational ( , ) and interventional ( , ) studies report an inverse association between the adherence to nordic diet and the concentration of high sensitivity c-reactive protein (hscrp). single intervention studies reported beneficial effects, reducing il receptor a (il ra) ( ) and cathepsin s ( ) , and downregulation of inflammatory mediators in the adipose tissue ( ) and peripheral blood mononuclear cells (pbmcs) ( ) . a key nutrient in fish are the n polyunsaturated fatty acids (pufas) ( ) . the greenland inuit population, which has a high dietary intake of n -pufas, have a lower incidence of myocardial infarction than the danish population ( ) . numerous studies associate the cardioprotective effects of n- pufas to their effect on immunomodulation ( ) ( ) ( ) , and control of inflammation, including neuroinflammation during aging ( ) . the mechanism of the anti-inflammatory effects of n -pufas n -pufas can regulate the transcription and expression of inflammatory mediators such as cytokines, chemokines and adhesion molecules in cardiomyocytes, fibroblasts, endothelial cells, and monocyte-macrophages ( ) ( ) ( ) ( ) . anti-inflammatory effect of eicosapentaenoic acid (epa), docosahexaenoic acid (dha) and their biologically active metabolites (d and e resolvinsmediators derived from omega- fatty acids, primarily epa and dha that block the production of proinflammatory mediators and regulate leukocyte trafficking to inflammatory sites) can be mediated through one of the mechanisms capable of reducing inflammation of raw- . cells and of primary intraperitoneal macrophages ( ) . one of the mechanisms is the activation of g-protein coupled receptors (gpr), ea. gpr inhibition of toll-like receptor (tlr )-mediated inflammatory response, which blocks nfkb activation. the other is mediated by nuclear receptors, particularly ppars-a/g. dha binds to ppars with high affinity resulting in the activation of anti-inflammatory cascades ( ) , which appears to be responsible for the beneficial health effects ( ) . the inhibition of nfkb-mediated pro-inflammatory activity ( ) is the common mechanism of immunomodulation by n -pufas, being dha more effective than epa in reducing lps-n -pufas induced inflammatory cytokine production by macrophages ( ) . n -pufas are incorporated into phospholipid bilayers and in human atherosclerotic plaques. their incorporation is associated with a reduction in the number of foam-and t cells, and a decrease in inflammation ( ) . the increased incorporation of n -pufas in membranes affects both the innate and adaptive immune responses, impairing the maturation of dendritic cells and the function of macrophages, as well as the polarization and activation of t and b cells ( ) ( ) ( ) . it is well known that n - pufas compete with n -pufas for being incorporated into cell membranes and for the active sites of cox- and lipoxygenase, resulting in the production of less potent pro-inflammatory or even anti-inflammatory mediators, such as the -series of prostaglandin and thromboxane ( ) . resolvins reduce also neutrophil-derived ros production, favoring neutrophil apoptosis and clearance by macrophages, and inhibit chemokine signaling ( ) . the partial agonist/antagonist activity of resolvin e (rve ) on the leukotriene b receptor on polymorphonuclear cells (pmns), inhibits nfkb activation, reduces release of pro-inflammatory cytokines and reduces infiltration by pmn ( ) . moreover, rve reduces tnfa and ifng presence in the aortic wall, decreases the levels of the inflammatory marker crp and reduces macrophage infiltration of the intima. thus, rve attenuates atherosclerosis and atherosclerotic plaque formation ( ) . aging is associated with the activation of inflammatory signaling pathways ( , ) , which can be targeted by specific nutrients with anti-inflammatory effects, such as n -pufas ( , ) . in the brain, the main n -pufa is dha, representing - % of total fatty acids ( ) . aging and neurological disorders are associated with decreased levels and turn-over rate of brain n -pufas ( ) ( ) ( ) ( ) . in aged mice, n -pufa supplementation and diets enriched in dha have been reported to revert age-induced spatial memory deficits and impairment on learning and memory ( ) ( ) ( ) . in older adults, a low consumption of n -pufas and decreased erythrocyte dha levels are associated with cognitive impairment ( , ) . dietary supplementation with dha is positively correlated with an improvement in declarative memory test performance, improved cognitive function ( , ) and a lower risk of developing neurological disorders ( ) . the probable mechanisms by which n -pufas mediate their effects in the resolution of age-related neuroinflammation are the increased synthesis of n -pufa-derived rvd and decreased n -pufaderived oxylipins, displaying an anti-inflammatory profile ( , ) . to recapitulate, the evidence indicates that n -pufas and their bioactive metabolites have immunomodulatory and antiinflammatory properties. potential cardioprotective lipid mediators, through multiple mechanisms, including changes in cell membranes composition, and modification of both cell signaling and gene expression, shift the pattern of lipid metabolites toward a more anti-inflammatory metabolite profile. dietary habits may be essential regulators of the inflammatory profile and promote healthy aging, reinforcing the recommendation of a n -pufa rich diet. the long term chronic psychological stress is increasing among the world's population ( ) . its circuit arises at high cortical centers through the limbic system to the hypothalamus, where corticotropin-releasing factor (crf) is produced, which is responsible for inducing the pituitary gland to liberate adrenocorticotropic hormone (acth) that signals the adrenal cortex to synthesize and secrete glucocorticoids (gcs) ( ) . stress also activates the sympathetic nervous system (sns), particularly the adrenal medulla, activating chromaffin cells to produce epinephrine (epi), a main stress hormone along with gcs. the latter plays a key regulation feature inhibiting the hypothalamic-pituitary-adrenal (hpa) axis through negative feedback at the pituitary gland, hypothalamus, and medial prefrontal cortex, reducing crf secretion [rewieved in ( ) ]. the interplay of social and environmental stressors induces inflammation through multiple biological mechanisms, including epigenetic factors ( ) . studies in rats show that the methylation patterns of genes involved in the stress response, such as the glucocorticoid receptor (nr c ) and crf, can be modified by psychosocial factors from early childhood ( ) . similarly, early life adversity induces acute and long-lasting epigenetic modifications in nr c genes, regulating hpa axis and cytokine production, reinforcing the importance of the activation inputs during critical periods of development ( , ) . acute short-term emotional stress, such as speaking in public, leads to a transient increase in circulating inflammatory biomarkers and natural killer (nk) cells by the sns catecholaminergic activity ( ) . on the contrary, chronic stress results in a reduction of cytotoxic nk activity, determining a poorer response to cytokines ( ) . therefore, stress appears to have short term beneficial immune effects, whereas chronic stress in the absence of immune challenge has the opposite effect ( , ) , activating constantly the hpa axis with the consequent persistent elevation of systemic gcs and reduction of nk cell responsiveness to cytokines ( ) , affecting the balance of the t helper cell type /type (th /th ) cytokine networks, predisposing to a wide range of diseases ( ) . the stress magnitude has been associated with il b mrna overexpression in peripheral pbmcs, providing a molecular mechanism by which psychological stress is translated into an immune system response ( , ) . when stress becomes chronic, such as in depression, there is a maintained overproduction of inflammatory cytokines, which have been associated with gcs resistance. immune cells become less sensitive to their anti-inflammatory effects because of their persistent secretion, leading to chronic low-grade inflammation ( , ) . activation of the innate and adaptive immune system by chronic mild stressors increases inflammatory cytokines gene expression, maturation and trafficking of dendritic cells (dc), increased macrophage number and t cells recruitment and activation. social stressors can induce an increase in inflammatory responses and a state of gcs resistance at different levels ( , ) . the acute repeated social defeat stress (rsds) and chronic restraint stress (crs) models induce an inflammatory response that results in neuroinflammation and depressive behavior ( ) . stress activates the hpa axis and the sympatho-adrenomedullar (sam) axis causing neuroinflammation by circulating cytokines that crossed the blood-brain barrier (bbb) at the circumventricular organs and by cytokine bbb transporters. an inflammatory response that promotes bbb permeability, allowing more inflammatory factors entering the brain, including crf, metalloproteinase- , il , and tnfa ( ) . additionally, microglia produce chemokines that attract monocytes into the brain ( ) . activation of sns and hpa axis through continuous psychological stress dysregulate cytokine production, and together with the stress hormones corticosteroids and catecholamines, can affect endothelial adhesion molecules, causing endothelial damage ( ) . corticosteroids could facilitate the infiltration of monocytes by increasing the expression of il and il receptors on endothelial cells. these monocytes and lymphocytes, after attaching to such sites, would commence the process of infiltration into the wall vessels, leading to foam cell formation and thrombotic events ( , ) . chronic unpredictable mild stress (cums) decreases body mass and impairs the metabolism of carbohydrates and lipids. a model for cums showed an increased liver and pancreas protein-lipid peroxidation and protein oxidation ( ) . high ros production in both organs could be a result of a response mechanism to stress at the cellular level. in the liver, protein oxidation can be due to the regulation of metabolic impairments by gcs and epi ( ). the antioxidant system of the liver is in general more efficient than the pancreas. however, it is insufficient to clear the reactive species increased as consequence of chronic stress, which could be due to alterations in the antioxidant enzymatic activity ( ) . altogether, stress appears to have short term beneficial effects on the immune function, whereas chronic stress ( , ) activates persistently the hpa, elevating systemic gcs, and impairing the cytokine balance. the overproduction of inflammatory cytokines lead to gcs resistance driven by immune cells that lose their sensitivity to gcs, leading to a state of chronic low-grade inflammation ( , ) . this gcs imbalance, shares common features with aging, mediating an enhanced neuroinflammatory priming ( ) . the presence of psychological stress potentiates the defective immune response observed in aging, which at the same time conditionate an exaggerated sickness response to immune challenges (such as chronic stress). thus, chronic stress contributes to the phenomenon of inflammaging, which promotes the development of several age-related pathologies, including atherosclerosis and diabetes among others [reviewed in ( ) ]. additionally, there is an impairment of the antioxidant defense system to manage ros production after chronic stress, resulting in the damage of various tissues ( ) . in addition, people exposed to chronic stress age rapidly, showing a faster telomere shortening in their cells ( ) ( ) ( ) . on the other hand, epigenetic changes acquired during critical developmental stages could shape chronic stress-response along the lifespan, either promoting or reducing pathological aging ( , ) . substance abuse, such as alcohol and drugs, are important triggers of chronic inflammatory processes ( , ) . the effects of alcohol on human health are complex and depend on multiple factors. however, many of those factors are associated with the generation of immunosuppression and increased morbimortality in heavy users. those effects, which have been previously reviewed by goral et al. ( ) will not be discussed in this review. here, we will describe the effect of cocaine and methamphetamine abuse. both drugs are potent psychostimulants that, when repeatedly consumed, significantly disrupt the functioning of the cns, and modify the regulation of the immune response, leading to a chronic neuroinflammatory state ( ) . in general, it is known that drug abuse, among other factors, increases nfkb transcription of multiple proinflammatory genes that spread across brain cell types further amplifying of nfkb transcription, as has been reviewed by crews et al. ( ) . cocaine (benzoylmethylecgonine according to the international common denomination) is a strong stimulant tropane alkaloid that acts by modulating the catecholaminergic neurotransmitter dopamine. studies of the striatum of mice after the administration of various drugs showed that h after administration of mg/kg cocaine, there is a significant increase in gene arrays for hypoxia-inducible factor (hif- ), transcription factors, and cytokine receptors (il r, tnfa). two hours after cocaine administration, there is an increased gene expression for various tnf receptors, inducible no synthase (inos) and adhesion molecules ( ) . in the nucleus accumbens of mice stimulated with cocaine, there is a significant increase in matrix metalloproteinase (mmp ), macrophage colony stimulating factor (mcsf) and major histocompatibility complex ii (mhc-ii) ( ) . the brain of human subjects consuming cocaine shows an increased density of macrophages and activated microglia ( ) . cocaine induces the activation of microglia through the endoplasmic reticulum stress and autophagy pathways ( ) . studies of human and rodent immune cell populations after cocaine administration show decreased numbers of t lymphocytes, modulation of nk activity and cytokine production ( ) . among brain glial cells, astrocytes are the most abundant, and perform critical functions, being involved in neurogenesis, promotion of neuronal survival, elimination of free radicals, and the production of no to maintain neuronal homeostasis ( ) . nevertheless, astrocytes can also be activated by toxic stimuli, leading to a new phenotype called "reactive astrocytes", similar with the changes observed after inflammatory activation. this phenomenon has been described in various neuropsychiatric disorders, such as alzheimer's and parkinson's disease, amyotrophic lateral sclerosis and multiple sclerosis ( ) . the reactivity of astrocytes to toxic stimuli, such as cocaine, infection or disease, potentiates the neuroinflammatory process ( ) . methamphetamine (desoxyephedrine; meth) is a synthetic adrenergic agonist with psychostimulatory effects, structurally related to the ephedrine alkaloid and adrenaline. studies on the effect of meth are limited. however, it has been determined that its abuse affects the immune response. animals exposed to both acute and chronic meth use show alkalization of normally acidic organelles in immune cells, inhibition of antigen presentation, and impairment of phagocytosis ( ) . meth also generates mitochondrial oxidative damage, dysfunction of t lymphocytes and decreased production of antibodies and cytokines ( ) . meth has effects in various tissues ( ) . in the lungs, the number of t lymphocytes decreases compared with that of untreated animals, indicating a reduction in circulating cd + cells, and levels of il and il increases. in the spleen, recruitment of pmn and the number of ly- g+ and f / + are increased, whereas cd + cells are significantly reduced. in addition, levels of tnfa, ifng, il , and il are higher than those of control mice. in the liver, there is an increase of t lymphocytes and macrophages, hepatocellular atrophy, and increased levels of ifng, tnfa, il b, - , - , - , and - in the group exposed to meth compared with control animals ( ) . in the cns, meth can induce the activation of calpains and caspases; the production of ros with the subsequent induction of oxidative stress, and the release of high amounts of glutamate, causing excitotoxicity ( ) . recently, raineri et al. reported that meth induces activation of astrocytes and microglia, increasing the levels of il and tnfa mrna and its receptor (tnfr ) in the mouse striatum and hippocampus ( , ) . medical advances have resulted in the increment of the average life expectancy in developed countries. the aging of the population is associated with an increase in the number of older people using drugs of abuse. from to , the number of cocaine users aged or older that required treatment for drug addiction in the us increased by % ( , ) . aging is associated with low-grade basal inflammation that can be compounded by substance use. as cocaine exposure is associated with elevated inflammation and altered immune functioning, the presence of cocaine use disorder might exacerbate inflammatory processes in aging adults ( ) . a recent report by soder et al, compared the levels of inflammation (through the neutrophil to lymphocyte ratio) in older adults with cocaine use disorder (cud) and in healthy older adults, finding that the group with cud had a significantly higher baseline level of inflammation ( ) . the use of illegal drugs such as cocaine or methamphetamine has not been shown to affect cognitive function in older adults at the clinical level. however, the evaluation of the cognitive function of young drug users reveals a decreased performance compared with healthy young people. in fact, the cognitive function of young drug users is similar to that of adults older than years of age ( , , ) . in summary, both cocaine and meth can directly impair the immune response, induce the activation of glial cells and stimulate the release of pro-inflammatory mediators in the cns. all those effects cause relevant changes in glial cell regulation and inflammatory activation, triggering chronic neuroinflammation and potentiating pathological aging. air pollution has become an important threat to public health. air pollutants consider a mixture of gases such as nitrogen oxides (nox), sulphur oxides (sox), tropospheric ozone (o ), volatile organic compounds (vocs), and particulate matter (pm) ( ) . pm can enter the respiratory tract leading to severe in situ damage as well as inducing additional systemic effects ( ) . the world health organization (who) suggests a maximum annual exposure of μg/m³ of pm . , however, the exposure of % of the world's population exceeds the proposed limit ( ) . exposure to air pollutants is associated with increased morbimortality associated with respiratory, cardiovascular, metabolic, neurological, carcinogenic and autoimmune diseases ( , ( ) ( ) ( ) . inflammation is the main pathophysiological mechanism induced by air pollutants. in terms of the molecular and cellular mechanism induced by pollutants, pm and sox can generate ros, inducing oxidative stress, together with mitochondrial dysfunction and the consequent energy deprivation ( ) ( ) ( ) . as a direct consequence, nfkb and mapk inflammatory pathways are activated, triggering an innate immune activation ( , ) . despite the attempts to resolve the inflammatory event, the outcome appears to be an imbalance in lymphocyte homeostasis and immune system dysregulation, with inhibition of th and treg lymphocytes ( ) . there is also an increase of th lymphocytes and recruitment of eosinophils, resulting in respiratory disorders such as asthma ( , , ) . in parallel, pm deactivates the nuclear factor erythroid pathway (nrf ), involved in antioxidant regulation and prevention of oxidative stress, a necessary process for the resolution of inflammation. therefore, to maintain oxidation-reduction reactions becomes impossible, becoming a breaking point towards increased ros production and the non-resolution of the inflammatory event ( ) . another mechanism of action of pollutants is the activation of the aryl hydrocarbon receptor (ahr) by toxic agents. the binding of pm to ahr increases circulating th and decreases treg lymphocytes. increase in th associates to the release of il , promoting an abrupt increase of th lymphocyte response. these changes promote the dysregulation of the immune response associated with the development of autoimmune processes ( ) . aberrant increases in th may result in increased inflammation, with consequences such as asthma and acute respiratory failure syndrome (ards), due to neutrophil infiltration and tissue damage ( ) . studies suggest the existence of a decline in treg levels and, therefore, an inability to suppress th , th and phagocyte responses ( , ) . in addition, exposure to pm has been associated with fibrotic events, where il increases synthesis and secretion of collagen in the lung parenchyma ( , ) . in addition, it has been described that pm also induces the expression of tgfb, directly promoting fibroblast differentiation, which could also induce collagen deposition followed by a lower antifibrotic process in the liver ( ) . pollutants may promote direct dna damage through oxidation of nitrogenous bases. hu and yu described in a paper different mechanisms and changes in mirna expression that comprise specific targets of dna methyltransferases, which can impair the methylation of tumor suppressor genes ( ) . furthermore, urban populations show increased levels of mitochondrial methylation genes due to pm exposure ( ) . there is evidence of the existence of methylation, acetylation and phosphorylation of histones h and h , markers found in genes involved in the activation of immune cells and cardiovascular diseases ( , ( ) ( ) ( ) ( ) . altogether, air pollutants can generate dna adducts promoting carcinogenesis and deteriorate telomerase activity, as reviewed by martens and nawrot ( ) , and contributing to continuous dna damage and premature aging ( , ) . in vivo studies suggest that the inflammatory activation is doseand time-dependent. mice exposed to pm show that both variables are determinant for the outcome. however, inflammatory effects and major genetic changes appear to be especially dependent on the exposure to high concentrations of pm. one possible explanation is that a prolonged exposure could induce an adaptive response of the inflammatory activation ( ) , which may be mediated by the inactivation of the nrf pathway, generating a loss of antioxidant capacity and deregulation of the immune system ( ) . the resolution appears to depend on the exposure context. acute exposure would result in high levels of ros and damage, whereas prolonged stimulation, even a low-grade one, generates a constant production of ros and chronic low-grade inflammation ( ) , consequent with the potentiation of disease risk and an epigenetic age acceleration ( ) , promoting pathological aging. direct causes of the deregulation of the inflammatory resolution process resulting from inhaled contaminants are still unknown, however, the burden of associated chronic diseases is expected to increase. it is mandatory to intensify environmental policies specifically in lower-middle-income countries in prevention of the development of inflammatory conditions and the subsequent chronic diseases. aging, characterized by a progressive loss of cellular functions, is an inevitable physiologic process inherent to all living beings ( ) . the number of older adults is increasing. during the next years, up to % of the world population will be older than years ( ) . this demographic change is accompanied by a higher incidence of ncds accumulated in the aging population ( ) . therefore, various strategies have been proposed to improve the health and quality of life of older adults ( ), along with recommendations for the development of public policies that support the fiscal expenditure resulting from ncds ( ) . one of the most studied events of aging is the impairment of the immune system, characterized by an aberrant-increased activation of the innate immunity ( , ) , and high levels of circulatory inflammatory mediators that establish an inflammatory environment, and a decrease of the adaptive immune response ( , ) and a decrease of the adaptive immune response ( ) due to this low-grade chronic inflammation ( , ) , which together would promote the inflammaging phenomenon ( ) . interestingly, it is proposed that age would not be the cause per se of these diseases associated with aging ( ) . thus, there is a deterioration of the immune system's response to external stimuli, which depends on the individual's history ( ) . also, several epigenetic mechanisms can modulate the immune response in aging, enhancing changes in intercellular communication that could perpetuate inflammatory events ( ) . on the other hand, it is described that epigenetic clocks would be useful to analyze mechanisms associated with this environmental influence ( ) . finally, they would be capable of modulating the immune response in aging, enhancing changes in intercellular communication that could perpetuate inflammatory events ( ). multiple age-dependent changes play important roles in the promotion of ncds, with increased oxidative stress standing out as one of the main mechanisms. over the last two decades, evidence has revealed that increased oxidative stress and inflammation are involved in various ncds such as alzheimer's disease ( ) , rheumatoid arthritis ( ) , cardiovascular diseases ( , ) , and cancer ( ), among others. also, recent studies propose that the activation of nfkb signaling pathways could be the main driver of these associations ( ) ( ) ( ) ( ) . interestingly, de almeida et al. showed different sources of low-grade chronic inflammation that promote cardiovascular disease ( ) . in the cns, high levels of ros lead to the activation of astrocytes and microglia, further increasing the overproduction of ros and proinflammatory cytokines that promote the development of neurodegenerative changes ( , , ) . in fact, several systemic biomarkers appear to be associated with neuroinflammation and the development of cns diseases associated with aging ( ) . these modifications trigger the phenotype of senescent or aged cells characterized as sasp ( , ) extensively studied in the context of the deleterious effects of aging. however, sasp is also essential for remodeling and promoting wound healing, which requires a strict control of the inflammatory response, thus avoiding the induction of cell aging phenotypes that contribute to the development of chronic inflammatory diseases ( ) . the immune imbalance in aging occurs due to various alterations in cellular behavior and phenotype, which cause functional deficiencies in immune cells ( ) . for example, this context induces polarization of macrophages towards an inflammatory phenotype characterized by strong activation of the inflammasome ( ) . thus, these events could induce il b and tnfa release, changes in the chemoattraction of neutrophils mediated by the reduction of the intercellular adhesion molecule (icam- ) expression, and the aberrant activation of the phosphoinositide lipid kinase- (pi k) ( ) . also, there is a decrease in the expression of pattern recognition receptors (prr), which leads to the activation of proinflammatory signaling promoting tissue damage ( , ) . finally, the reduced level of certain hormones due to the impaired hypothalamic function causes the loss of muscle mass and an increase in adipose tissue, further contributing to the release of inflammatory cytokines and changes in metabolism ( ) . despite the remarkable effort being made to understand the basis of the processes underlying the inflammatory imbalance during aging, it is not fully understood. in aging, there are cumulative epigenetic changes that promote low-grade inflammation ( , ) , including a decrease in the global genome methylation, with increased methylation in specific regions, as those with repressive histone marks of cd + and cd + t cells ( ) and bivalent chromatin domains ( ) and histone acetylation and methylation. however, the influence of genomic methylation during aging remains undetermined ( ) . several studies correlate the methylation of multiple sites on cpg islands with the increase of the low-grade inflammation marker, crp ( , , ) . nonetheless, stevenson et al. propose that the dna methylation could be better associated with the low-grade chronic inflammation than crp ( ) . in addition, the age-related mitochondrial dysfunction, with the resulting oxidative stress and decreased atp production ( ) , affect the expression and activity of dna methyltransferases, which are responsible for maintaining the methylation pattern of dna ( ) . the reduced methylation results in the demethylation of the tnfa promoter in leukocytes and macrophages ( ) and the adhesion of immune cells to the endothelium ( ) . also, many epigenetic events contribute to the differentiation of proinflammatory t cells, th ( ) , which can compromise immunocompetence, associated with repression of differentiation of immune cells, loss of treg function ( ) and the alteration of the hematopoietic stem cells differentiation ( ) . thus, epigenetic mechanisms appear to have a major role in the inflammatory imbalance, which are associated with the accumulation of damage in time that ultimately leads to the perpetuation of a constant inflammatory response. according to the who, % of the world population is sedentary, lacking the benefits of physical exercise ( ) . conditions such as sedentarism, unhealthy diet, overweight, obesity and aging induce chronic low-grade inflammation. physical exercise increases the anti-inflammatory potential and reduces the pro-inflammatory effect ( ) . this equilibrium is partly modulated through tlrs ( ) , which are fundamental for the recognition of prrs, including the damage-associated molecular patterns (damps) and the induction of an inflammatory response in the absence of pathogens. there is evidence that in young people, physical exercise decreases tlrs expression, co-stimulatory molecules cd / cd , and mhcii ( , ) in cd + monocytes. physical exercise also affects the adipose tissue. exercising reduces tlr mrna expression and tnfa production in adipocytes ( , ) in obese mice. chronic physical exercise decreases tnfa and tlr gene expression in the skeletal muscle ( ) . the evidence suggests that obesity-or cerebral ischemiainduced neuroinflammation, which are associated with the overexpression of tlr and tlr , may be reduced by physical exercise through the reduction of tlrs expression as well as their downstream signaling molecules (tnfa, il b, myd , traf , tak , and nfkb), together with the reduced microglial activation ( , ) . there is evidence that cigarette smoking induces inflammatory status [reviewed in ( ) ]. however, exercise training reduces smoke-induced inflammation. in that sense, training for min with endurance exercise for days in smoke-exposed mice demonstrated that therapeutic exercise training significantly reduces the expression of il b and tnfa mrna in rectus femoris ( ) . physical exercise has been used as a therapeutic tool in chronic pathological conditions. in that sense, obese older adults (body mass index ± kg/m ; ± years) undergoing an exercise program consisting in physical therapy, endurance, and resistance for min, days per week, show a reduced expression of tlr , il , and tnfa mrna in skeletal muscle ( ) . in older adults, -week physical exercise reduces the expression of tlr and tlr , as well as tlrs downstream mediators, such as myd , p , pp , trif, ikki/ikkϵ, irf , and pirf in pbmcs ( ) . similarly, dendritic cells from multiple sclerosis patients undergoing an exercise (endurance and resistance) program for weeks reduce tnfa and mmp secretion when stimulated with a tlr ligand (lps in combination with ifng, or a tlr ligand) ( ) , suggesting that long-term physical exercise decrease tlr responsiveness. on the other hand, high-intensity physical exercise in untrained individuals induces inflammation, resulting in the increased expression of tlr , ap , nfkb, and p in mice myocardium and in adipose tissue ( ) ( ) ( ) . physical exercise associated with eccentric contractions causes expression of tlr and nfkb in skeletal muscle and liver in rats ( , ) . furthermore, this phenomenon induces muscle damage, which can increase chemotaxis, attracting nk, cd + t cells, macrophages and neutrophils to the site of injury, promoting the production of cox , inos, monocyte chemotactic protein- (mcp- ), tnfa, il , and il b, in addition to the production of ros and the activation of nfkb ( , ) . in healthy young males, one session of intense endurance exercise ( h intense cycling immediately followed by h intense running), increases plasmatic concentrations of il and il , in addition to increased gene expression of proinflammatory il receptor (il r) and tlr signaling pathways. moreover, plasma myoglobin changes in correlation with neutrophil tlr gene expression (r= . ), suggesting that their transcriptional activity was particularly induced by damps ( ) . therefore, inflammation and muscle damage are mainly associated with the type and intensity of the exercise, with loads that exceed individual physical abilities. chronic physical exercise generates epigenetic modifications. the physical exercise associated with an energy expenditure > kilocalories per week, results in hypomethylation of the il gene and hypermethylation of the tnfa gene ( ) , with an inverse correlation between tnfa methylation and tnfa mrna expression ( ) . the methylation of the caspase recruitment domain (asc) of the apoptosis-associated specklike protein gene, the main regulator of inflammasome and promoter of the activation of il b and il , decreases with aging. however, older adults who maintain physical exercises regularly express higher levels of asc methylation than subjects not exercising, which would imply a decreased release of inflammatory cytokines ( ) ( ) ( ) ( ) . similarly, in review a -month walk training can induce hypermethylation of the nfkb- gene, suppressing inflammation through the inhibition of the nfkb pathway ( ) . as life expectancy increases, age-related diseases thrive. aging is a complex multifactorial process of molecular and cellular decline that renders individuals susceptible to disease and death. maintenance of cell integrity, cell metabolism and host-defense mechanisms are tightly regulated by the surrounding microenvironment. a growing body of evidence in different biological models has contributed towards identifying biological mechanisms that ward off structural and functional deterioration. these data offer us insights into healthy aging. molecular integrity of the genome, telomere length, epigenetic stability, and protein homeostasis are all features linked to more youthful stages (regardless of the age), associated with mitochondrial fitness, metabolic regulation, efficient intercellular communication, stem cell renewal, and regenerative capacity in tissues. a good understanding of the environmental and endogenous mechanisms that underlie agerelated normal and deleterious changes, and how these pathways interconnect, remains a major challenge for slowing pathological aging while extending older adults' healthy lifespan. the study of the environmental influence on the development of complex-chronic diseases shows that in addition to genetic predisposition, the pathogenesis is promoted by changes in metabolism and behavior, cellular environment, and epigenetic regulation patterns. the type of nutrient, or environmental cytokine milieu dramatically affects not only the homoeostasis of tissues but also of complete organs and even of the whole individual. thus, tissue stress, malfunction, and damage may induce inflammation alarm responses, which result either in resolution of tissue damage, restoration of normal cell function or development of chronic disease ( figure ). older adults often present inflammaging, characterized by increased levels of proinflammatory cytokines il , il , il , tnfa/crp ( ) . however, the cellular sources of these cytokines are partially unknown. the increased inflammatory cytokines have been proposed to be a driver of unsuccessful aging (increased morbidity, degenerative processes, or frailty) and shortened health-span. the inflammatory scenario is complex and occurs in response to various internal and environmental stimuli ( figure ) mediated mainly by a high level of proinflammatory cytokines. indeed, in healthy aging, increased production of the anti-inflammatory cytokines tgfb and il , can regulate the pro-inflammatory state ( , ) . research into the impact of environmental factors on inflammaging is at an early stage and the involved mechanisms are not completely understood. several hypotheses have been developed to explain the chronic inflammation: aging-related increase of stress ( ) and oxidative stress ( ) , dna damage in senescent cells [reviewed in ( ) ], and stem cell aging ( ) . the proposed mechanisms are likely interdependent, resulting in the generation of ros causing oxidative damage and amplification of the cytokines secretion, thus perpetuating a vicious circle of systemic inflammation where tissue injury and healing mechanisms proceed in parallel while damage slowly accumulates over the lifespan of the individuals. endocrine and metabolic alterations are linked to the shift towards a pro-inflammatory profile, which could explain some age-related pathologies, such as alzheimer's and parkinson's disease, osteoporosis, diabetes, cancer, and frailty ( , ) . regarding stress-induced immune modifications, new evidence suggests that cross talk signals between the cns, endocrine and immune system are required for optimal response to stress [discussed in ( ) ]. various stressors can affect the activity and regulation of immune cells via direct regulation by the autonomic and peptidergic system or through the release of neuroendocrine mediators. moreover, neuronal catecholamines modulate immune cell functions. these interactions are bidirectional, cytokines produced by immune cells, such as il , can modulate the production of corticotropin-releasing hormone (crh) by the hypothalamus. chronic diseases are favored by some modern living conditions, such as the intake of high-caloric foods and the low level of physical activity, or endogenous signals produced by the chronic stress of modern life. there are many challenges in conducting research on biosocial processes, which will define novel disease-trigger factors. tailor-made approaches will depend on genetics, epigenetics and a constellation of factors depending on the historical as well as the present exposure to the environment. although environmental factors also express themselves as epigenetic changes, the combinatorial effect of the multiple factors generates complex patterns of epigenetic regulation, and the concomitant exposure to environmental factors can further modify the individual response. all authors contributed equally on the conception of the work, the analysis of literature and preparing the content of the review. rbe drafted and organized the manuscript. all authors contributed to the article and approved the submitted version. endogenous and environmental factors can be mostly beneficial (in green) and deleterious (in red) or can have both beneficial and deleterious effects depending on the specific context. the interplay of lifespan endogenous and environmental factors regulates the aging phenotype depending on dna damage, epigenetic changes, and inflammation. these drivers can induce functional aging hallmarks: changes in endocrine and metabolic regulation, and defective immune regulation that will further determine the response of the individual. in yellow we show processes that can participate in both protection and damage. exposure to various alarm signals induce an acute inflammation that, when associated with deleterious environmental and biological factors, potentiates chronic inflammation, which can be further promoted by excess ros production and oxidative stress that results from mitochondrial dysfunction or nox activity, leading to inflammaging and eventually to age-related disease. on the contrary, in the presence of protective environmental and biological factors, the initial inflammatory activation will be resolved and lead to a healthy aging process. ros, reactive oxygen species. sarcopenic obesity and 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longevity emerged from studies in humans role of tgf b signaling in the pathogenesis of alzheimer's disease age-dependent changes in the activation and regulation of microglia stress responses and innate immunity: aging as a contributory factor oxidative stress, inflamm-aging and immunosenescence dna damage response (ddr) and senescence: shuttled inflamma-mirnas on the stage of inflamm-aging emerging models and paradigms for stem cell ageing from inflamm-aging to immune-paralysis: a slippery slope during aging for immune-adaptation aging and parkinson's disease: inflammaging, neuroinflammation and biological remodeling as key factors in pathogenesis impact of stress on aged immune system compartments: overview from fundamental to clinical data the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © bachmann, bellalta, basoalto, goḿez-valenzuela, jalil, lépez, matamoros and von bernhardi. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -g ibmo authors: valenti, piera; rosa, luigi; capobianco, daniela; lepanto, maria stefania; schiavi, elisa; cutone, antimo; paesano, rosalba; mastromarino, paola title: role of lactobacilli and lactoferrin in the mucosal cervicovaginal defense date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: g ibmo the innate defense system of the female mucosal genital tract involves a close and complex interaction among the healthy vaginal microbiota, different cells, and various proteins that protect the host from pathogens. vaginal lactobacilli and lactoferrin represent two essential actors in the vaginal environment. lactobacilli represent the dominant bacterial species able to prevent facultative and obligate anaerobes outnumber in vaginal microbiota maintaining healthy microbial homeostasis. several mechanisms underlie the protection exerted by lactobacilli: competition for nutrients and tissue adherence, reduction of the vaginal ph, modulation of immunity, and production of bioactive compounds. among bioactive factors of cervicovaginal mucosa, lactoferrin, an iron-binding cationic glycoprotein, is a multifunctional glycoprotein with antibacterial, antifungal, antiviral, and antiparasitic activities, recently emerging as an important modulator of inflammation. lactobacilli and lactoferrin are largely under the influence of female hormones and of paracrine production of various cytokines. lactoferrin is strongly increased in lower genital tract mucosal fluid of women affected by neisseria gonorrheae, chlamydia trachomatis, and trichomonas vaginalis infections promoting both innate and adaptive immune responses. in vaginal dysbiosis characterized by low amounts of vaginal lactobacilli and increased levels of endogenous anaerobic bacteria, the increase in lactoferrin could act as an immune modulator assuming the role normally played by the healthy microbiota in vaginal mucosa. then lactoferrin and lactobacilli may be considered as biomarkers of altered microbial homeostasis at vaginal level. considering the shortage of effective treatments to counteract recurrent and/or antibiotic-resistant bacterial infections, the intravaginal administration of lactobacilli and lactoferrin could be a novel efficient therapeutic strategy and a valuable tool to restore mucosal immune homeostasis. the innate defense system of the female mucosal genital tract involves a close and complex interaction among the healthy vaginal microbiota, different cells, and various proteins that protect the host from pathogens. vaginal lactobacilli and lactoferrin represent two essential actors in the vaginal environment. lactobacilli represent the dominant bacterial species able to prevent facultative and obligate anaerobes outnumber in vaginal microbiota maintaining healthy microbial homeostasis. several mechanisms underlie the protection exerted by lactobacilli: competition for nutrients and tissue adherence, reduction of the vaginal ph, modulation of immunity, and production of bioactive compounds. among bioactive factors of cervicovaginal mucosa, lactoferrin, an iron-binding cationic glycoprotein, is a multifunctional glycoprotein with antibacterial, antifungal, antiviral, and antiparasitic activities, recently emerging as an important modulator of inflammation. lactobacilli and lactoferrin are largely under the influence of female hormones and of paracrine production of various cytokines. lactoferrin is strongly increased in lower genital tract mucosal fluid of women affected by neisseria gonorrheae, chlamydia trachomatis, and trichomonas vaginalis infections promoting both innate and adaptive immune responses. in vaginal dysbiosis characterized by low amounts of vaginal lactobacilli and increased levels of endogenous anaerobic bacteria, the increase in lactoferrin could act as an immune modulator assuming the role normally played by the healthy microbiota in vaginal mucosa. then lactoferrin and lactobacilli may be considered as biomarkers of altered microbial homeostasis at vaginal level. considering the shortage of effective treatments to counteract recurrent and/or antibiotic-resistant bacterial infections, the intravaginal administration of lactobacilli and lactoferrin could be a novel efficient therapeutic strategy and a valuable tool to restore mucosal immune homeostasis. keywords: lactobacilli, lactoferrin, cervicovaginal defense, vaginal homeostasis, inflammation introduction the basic structures of the female reproductive system are ovaries, fallopian tubes, uterus, cervix, and vagina. ovaries are responsible for the production of the ovum and secrete both estrogen and progesterone. when an ovum is developing in an ovary, it is encapsulated in a sac known as an ovarian follicle. on maturity of the ovum, the follicle and the ovary's wall rupture, allowing the ovum to escape and enter the fallopian tube to reach uterus. the uterus is a muscular organ useful to accept a fertilized ovum, which becomes implanted into the endometrium. the cervix is the neck of the uterus, which protrudes through the upper anterior vaginal wall. the vagina is a fibromuscular canal that connects the upper part of female genital tract to the outside of the body and represents the portal of entry of pathogenic microorganisms. the epithelial mucosa of the lower genital tract is extensively colonized by commensal microorganisms, while the tissues of the upper genital tract are generally considered to be sterile ( ) . however, bacterial colonization of the upper genital tract of healthy asymptomatic women remains a somehow controversial issue ( ) . the vaginal tract is colonized by microorganisms, recognized as the vaginal microbiota (vm). these microorganisms, in addition to a complex synergism among secretion's proteins and peptides, epithelial, and immune cells, perform a pivotal role in the defense of female genital tract against infectious and inflammatory processes. in the state of mucosal health, the various components are in balance. the rupture of mucosal homeostasis determined by the alteration of one of the various actors often results in an increased host susceptibility to infections. healthy vm is dominated by lactobacillus spp., but other microorganisms can be present at lesser extent (gardnerella, prevotella, streptococcus, ureaplasma, peptostreptococcus, staphylococcus, corynebacterium, clostridium, mycoplasma, enterococcus, bacteroides, escherichia, bifidobac terium, veillonella, and candida) ( ) ( ) ( ) ( ) ( ) . over species of lactobacillus have been detected in the vagina. however, in the majority of women, the healthy vaginal microflora contains one or two lactobacillus species among lactobacillus crispatus, lactobacillus gasseri, lactobacillus jense nii, and lactobacillus iners ( , ) . currently, the role of l. iners in vaginal health is still unclear ( ) . indeed, l. iners has been recently detected in both dysbiotic and healthy women, and its presence and amount are inversely correlated with l. crispatus ( , , ) . lactobacilli are involved in maintaining the healthy vaginal environment by counteracting overgrowth of other resident microorganisms ( ) . lactobacilli can also colonize the human cervix. in different studies, a range of - % of women resulted colonized by lactobacilli in the cervix, and within a single subject, usually the same lactobacillus strains colonized both the cervix and the vaginal tract ( ) ( ) ( ) . lactobacilli exert their protective effects by several mechanisms: (i) microbial competition for the nutrients and for adherence to the vaginal epithelium; (ii) reduction of the vaginal ph by the production of organic acids, especially lactic acid, through the degradation of glycogen released by vaginal cells thus exerting selective antimicrobial activity against non-resident microbiota; (iii) production of antimicrobial substances, such as bacteriocins and hydrogen peroxide (h o ) able to suppress the growth of several microorganisms; and (iv) modulation of the local immune system ( ) . homeostasis of vaginal environment results from complex interactions and synergies among the host and different microorganisms that colonize the vaginal mucosa, and the maintenance of high numbers of resident lactobacilli is an effective hallmark of woman's health and a wellorganized protection against pathogens causing sexually tranmitted infections (stis). abnormal vm involving a strong reduction or disappearance of lactobacilli characterizes a pathologic condition known as bacterial vaginosis (bv) that afflicts fertile, premenopausal, and pregnant women with an incidence rate ranging from to % ( ) . bv is a polymicrobial clinical syndrome resulting from the replacement of the normal lactobacillus spp. with high number of anaerobic bacteria such as gardnerella vaginalis, prevotella spp., mobiluncus spp., ureaplasma, mycoplasma, and other fastidious or not culturable anaerobes ( , ) . in bv, the overgrowing anaerobes produce compounds such as polyamines and other molecules capable of inducing the release of proinflammatory cytokines such as il- β, il- , and il- ( , ) . bv represents an independent risk factor for severe reproductive tract sequelae associated with pelvic inflammatory disease and tubal factor infertility ( , ) . changes in the vm have been also associated with obstetrical complications such as late miscarriage and premature birth ( ), thus exerting a profound impact also on the health of newborns. moreover, women with lactobacillus poor flora show an increased susceptibility to sexually transmitted pathogens. several studies indicate that abnormal vm lacking lactobacilli is associated with the acquisition of infections by neisseria gonorrhoeae, chlamydia trachomatis, and trichomonas vaginalis ( ) ( ) ( ) ( ) ( ) . furthermore, the alterations in vm are associated with increased risk of acquiring viral sexually transmitted diseases (stds). indeed, longitudinal and cross-sectional studies demonstrated the association between altered vm and the increased prevalence/incidence of many viral stis such as human immunodeficiency virus (hiv), herpes simplex virus (hsv), human papillomavirus, and cytomegalovirus infection ( ) . in addition to lactobacilli, the cervicovaginal fluid (cvf) exerts a significant microbicidal activity against gram-positive and gram-negative bacteria, fungi, and certain viruses as well as an anti-inflammatory activity through several peptides and proteins, all characterized by common cationic features ( ) . the main antimicrobial peptides and proteins present in the cvf are shown in table ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . these peptides and proteins act through different mechanisms: (i) microbial lysis; (ii) depletion of environmental nutrients essential for microbial growth; (iii) competitive binding to host cells; (iv) degradation of negatively charged microbial surface components; (v) interference with host cell signaling pathways; and (vi) modulation of inflammation and other functions involved in host defense ( , ) . the bactericidal activity of many of these compounds is strictly associated with their cationic features. the concentration of some of these molecules in cvf is lower than that required for in vitro inhibition of pathogens; however, it is known that several antimicrobials display synergistic effects. indeed, human β defensin and cathelicidin antimicrobial peptide ll- ( ) , secretory leukocyte protease inhibitor (slpi) and lysozyme ( ) , and lactoferrin (lf) and lysozyme ( ) display synergistic effects that potentially increase innate immune protection in the female reproductive tract ( ) . many of these antimicrobial/immunomodulatory compounds appear to be under hormonal control ( ) . in cvf α and β defensins, slpi and lysozyme levels are high during the proliferative phase, greatly decrease at mid-cycle/ovulation, and increase again during the late secretory phase. lactoferrin, belonging to transferrin family, is a multifunctional glycoprotein of about amino acids and a mw of ( , , ) . lf, abundantly found in most biological fluids of mammals, is synthesized by exocrine glands, many mucosal epithelial cells and released by neutrophils during inflammation. the highest level of the human lf (hlf) is found in colostrum ( mg/ml), while decreases in mature milk ( . - . mg/ml). in the tears, hlf is detected at low concentration (about . mg/ml), while in saliva, small intestine, earwax, vaginal fluid, amniotic fluid, upper airway fluid, seminal plasma, and the cervical mucus at very low levels (< . mg/ml) ( ) . in particular, the concentration of hlf in human vaginal fluid corresponds to - µg/ml, while it is extremely high ( µg/ml) in the cervical mucus plug ( , , ) . of note, a total number of neutrophils release µg of hlf in sites of inflammation and infection ( ) . in human body fluids, the concentration of free available iron must not overcome - m to avoid microbial multiplication and to hinder the precipitation of insoluble ferric hydroxides as well as the formation of reactive oxygen species. hlf, by its iron-binding ability, guarantees that free available iron does not exceed - m. hlf and bovine milk derivative lf (blf) possess high homology of sequence. from three-dimensional structure, lf is folded into two homologous lobes, each structured in two domains (n and n , c and c ). one fe (iii) ion is chelated by each lobe. when lf is completely iron saturated, its conformation appears in a closed state, more resistant to proteolytic enzymes than the unsaturated open form ( ) . the low iron availability ( - m), hindering microbial growth, is a signal of health and wellness, while iron concentration higher than - m favors not only microbial replication but also the biofilm formation ( , ) and persistence ( ) . interestingly, wiesner and vilcinskas have reported that proteins and peptides of mucosal secretions possess several functions ( ) . accordingly, hlf and blf are multifunctional glycoproteins effective against bacteria, mycetes, viruses, and parasites, possessing also anti-inflammatory and immunomodulatory properties ( ) . in particular, blf, available in large quantities and recognized by food and drug administration (fda, usa) as a safe substance, is the main lf used in in vitro studies ( ) as well as in clinical trials ( , ( ) ( ) ( ) ( ) and in mice ( ) . the level of vaginal hlf and others antimicrobial peptides change in response to microbial infections. it has been demonstrated that hlf and defensin levels increase in genital secretion of women with c. trachomatis, n. gonorrhoeae, t. vaginalis, or candida spp. infection and bv in comparison to healthy condition ( , ) . lactobacilli the innate defense system of the female mucosal genital tract involves a complex interaction among the healthy vaginal flora, immune cells, and several proteins that defend the host from pathogens. it is broadly suggested that the crucial role of vaginal lactobacilli is to protect female genital tract through the production of lactic acid responsible for low vaginal ph that inhibits sexually transmitted pathogens. lactic acid is in equilibrium with lactate anion. the former is the predominant form in healthy vaginal conditions and low ph (< . ), thus exerting antimicrobial activity against pathogens. lactate anion predominates at higher ph (> . ) conditions in women with dysbiosis ( ) . in vitro experiments demonstrated that at physiological concentrations ( - mm) of lactic acid and at ph . , bv-associated bacteria such as g. vaginalis and atopobium vaginae were inactivated without effects on typical vaginal species of lactobacilli ( ) . furthermore, inactivation of bv-associated species was dependent on lactic acid itself rather than ph, since a ph value of . determined by other acids was significantly less microbicidal. recent in vitro studies, however, demonstrated that c. trachomatis multiplication is inhibited by different strains of vaginal lactobacilli, independently from ph alterations ( ) . c. trachomatis, an obligate intracellular pathogen, responsible for the most common bacterial std worldwide, causes acute and chronic infections. unlike acute infections, which can be cured with oral or topical administration of antibiotics, chronic infections are difficult to eradicate and need prolonged therapies, thus increasing the risk of developing antibiotic resistance ( ) . therefore, novel alternative therapies are needed. the difficulty in finding new agents against c. trachomatis infection resides in the complex life cycle of this peculiar pathogen. in fact, c. trachomatis has a unique biphasic developmental cycle, alternating between the extracellular infectious elementary bodies (ebs), metabolically inactive, and the intracellular non-infectious reticular bodies (rbs), which are metabolically active. it has been recently demonstrated that vaginal lactobacilli inhibit ebs adhesion to epithelial cells as well as the intracellular rbs replication ( ) . the effect on the early phases of infection was related both to co-aggregation between lactobacilli and c. trachomatis and to competition for epithelial cell adhesion. the inhibition of chlamydial infection by lactobacilli was strain and dose dependent, suggesting that the strains and the amount of lactobacilli in the vagina are responsible for the protection from chlamydial infection. lactobacilli have been demonstrated to protect lower female genital tract also from n. gonorrhoeae infection, the second most common bacterial sti. the interaction between bacteria and host cells determines the success of the pathogen mucosal colonization or its elimination through the continuous fluid flow. in this respect, vielfort et al. ( ) showed that lactobacilli compete with n. gonorrhoeae for adhesion to human cervical cells. it has been also demonstrated that l. jensenii atcc could reduce both adhesion and invasion of n. gonorrhoeae, whereas l. gasseri atcc could displace adherent gonococci from the cell surface ( ) . the protection exerted by healthy vm toward viral infections can be ascribed to a direct virucidal effect or to the maintenance of natural defense factors present in the vaginal milieu. some mechanisms have been suggested by results obtained from both in vitro experiments and clinical observations in infected women ( ) . lactobacillus metabolites possessing antimicrobial activity may be directly protective against viral infections. hydrogen peroxide (h o ) produced by lactobacilli plays an important role as a natural microbicide within the vaginal ecosystem due to its toxic activity against a number of microorganims and viruses, including hiv- ( ) and hsv- ( ) . it has been observed that a range of - % of lactobacilli present in the vaginal flora of healthy women produce h o . this percentage drops to % in women affected by vaginal infections ( ) . the physiological acid vaginal ph value (≤ . ) determined by lactobacilli inactivates hiv ( ) and hsv- ( ) . in addition, hsv- is inactivated by lactic acid concentrations leading to ph values similar to the ones detected in the healthy human vagina ( ) . several compounds released from lactobacilli can impair the efficiency of target cells in supporting viral replication. a nonprotein cell wall component extracted from a vaginal strain of lactobacillus brevis strongly reduces hsv- replication in cell culture ( ) , whereas acid lactobacillus metabolic products decrease activation of t lymphocytes, with a consequent lower lymphocyte susceptibility to hiv- infection ( ) . a healthy vm contributes to the maintenance of the natural defense mechanisms from invading pathogens. the gel layer coat of the vaginal and cervical mucosa represents a physical barrier that hinders viral binding to cell membrane receptors, thus protecting women from viral infections. indeed, in vitro studies demonstrated that hsv could be trapped into the viscous cervical mucus ( ) . bv-related microorganisms are able to produce higher levels of mucin-degrading enzymes, such as mucinase and sialidase, in comparison to lactobacilli-dominated healthy vaginal flora ( ) ( ) ( ) . therefore, an increased degradation of the protective mucus layer may promote binding of hsv- and other viruses to the underlying epithelial cell receptors. further studies have demonstrated that vaginal lactobacilli are able to inhibit the first steps of hsv- infection in cell culture ( , ) . the antiviral activity exerted by the presence of lactobacilli during hsv- binding to the cell membrane is strain dependent and appears directly related to the adhesion capacity of lactobacillus strains ( ) . in conclusion, several mechanisms may be involved in the antimicrobial effect of vaginal lactobacilli: interference with microorganisms in the process of adhesion or entry into host cells, production of metabolites with a direct antimicrobial effect, production of compounds able to inhibit obligate intracellular pathogen replication, and contribution to the maintenance of natural defense factors present in the vaginal milieu. as already discussed, hlf is one of the most important defense proteins of cvf. in fact, endogenous hlf has been found increased in the genital fluid of women affected by neisseria gonorrheae, c. trachomatis, and t. vaginalis infections and/or vaginal dysbiosis ( ) . in this respect, hlf released by neutrophils recruited in situ could represent a marker of non-healthy conditions and one of the mediators involved in counteracting the inflammatory mileu. the first function of hlf, recognized in vitro, was the bacteriostatic activity depending on its ability to sequester iron necessary for bacterial survival and growth ( ) . hlf and blf establish a battle for iron acquisition with pathogens, capable to counteract these iron-binding proteins by synthesizing siderophores, small high affinity iron-chelating molecules, or through iron acquisition from other sources ( ) . as a matter of fact, g. vaginalis, lacking of siderophores, acquires iron by the lysis of erythrocytes, using hemoglobin as iron source. this is consistent with the observation that g. vaginalis level increases during menses ( ) . moreover, independently from iron-binding ability, blf exerts several antibacterial activities: (i) bacterial lysis through its binding to lipopolysaccharide (lps); (ii) inhibition of bacterial adhesion to the epithelial cells; and (iii) inhibition of the entry into host cells by facultative or obligate intracellular bacteria through competitive binding to host cells and/or to microbial surface components [( ) and references therein ( , ) ]. of note, facultative intracellular pathogens require intracellular nutrients, including iron, for replication in mammalian cells, and obligate intracellular c. trachomatis is no exception ( ) . a preparation of blf, iron saturated at % to consent further iron chelation, was utilized in in vitro model to check its antichlamydial activity ( ) . similar to that observed using vaginal lactobacilli, the incubation of cell monolayers with blf before the infection or at the moment of the infection significantly inhibited the adhesion and entry of c. trachomatis into epithelial cells. therefore, the inhibition of c. trachomatis infectivity by blf was dependent on its interaction with the cell surface and especially with glycosaminoglycans and heparan sulfate proteoglycans ( ) , which are potential receptors for c. trachomatis adhesion ( ) . conversely, the preincubation of blf with c. trachomatis ebs did not influence its infectivity, supporting the idea that the specific interaction between blf and epithelial host cells could be the sole mechanism responsible for the inhibition of c. trachomatis invasion ( ) . recently, the inhibition of il- and il- synthesis by blf has been demonstrated in in vitro model, mimicking the in vivo chlamydial infection. blf, added to infected cells h postinfection, produced a significant decrease of il- and il- without any effect on the number of intracellular chlamydia. similarly, il- levels were reduced when blf was added h postinfection to epithelial monolayers infected with other facultative intracellular bacteria or to lps-stimulated macrophages ( ) ( ) ( ) . the anti-chlamydial activity of blf related to its anti-inflammatory function has been shown in vivo. in a pilot study, of pregnant women showing cervical specimens positive for c. trachomatis were treated with the intravaginal administration of blf ( mg) every h for days. interestingly, after month, six women resulted negative for c. trachomatis and showed significant decreased il- levels in their cvf ( ) . similar to what observed in in vitro model, intravaginal administration of blf seems to act by protecting mucosal host cells against the adhesion and entry of chlamydial ebs, which are released extracellularly after redifferentiation of rbs to ebs. the decrease of il- levels could be a marker for the inhibition of c. trachomatis ebs infection of host cells due to the presence of blf. in other words, blf protects host cells and prevents the early phase of infection by ebs. unlike lactobacilli ( ), blf does not affect the replication of rbs ( ) . the potential influence of exogenous blf on microbial communities populating vagina has been recently investigated. vaginal blf administration to women with bv has been shown to be able to modify vm composition. in fact, the treatment induced a reduction of bv-associated gardnerella, prevotella, and lachnospira genera as well as an increase of lactobacillus species ( ) . these data suggest the therapeutic potential of blf in counteracting female genital tract diseases. indeed, it would be relevant to unveil the molecular mechanisms as well as the immunological changes accompanying blf effects on microbiota. furthermore, blf antiviral activity, verified against both enveloped and naked viruses, is exerted in the early stage of infection, thus inhibiting viral binding and entry into the host cell. this activity is mainly due to blf binding to heparan sulfate glycosaminoglycan cell receptors or viral particles or both ( ) . similar to viral particles, the inhibition of plasmodium endocytosis is attributed to the interaction between blf and both cell surface heparan sulfate and lipoprotein receptor-related protein ( ) ( ) ( ) ( ) . in this respect, blf represents the most relevant protein symbolizing a brick in the wall of natural non-immune defenses of human mucosal fluids against microbial infections ( ) . it is well known that lactobacilli are endowed with healthpromoting and immunomodulatory properties. along with bifidobacteria, they have been proposed as candidates for prevention and/or treatment of allergy, colitis, infections, and other inflammatory conditions ( ) . some lactobacillus strains have been also proposed as vaginal microbicide candidates against sti (e.g., n. gonorrhoeae, candida albicans, and hiv). besides mechanisms related to the bacterium itself (e.g., enhancement of epithelial barrier function and competition with pathogens), the capability to redirect the immune response underlies many of the beneficial effects of lactobacilli. in vitro data demonstrate that l. crispatus ( ) ( ) ( ) and l. jensenii ( ) act not only through colonization of epithelial cells but also influencing the cytokine secretion pattern. in particular, upon recognition through tolllike receptor (tlr) / and / , nf-κb signaling is activated without induction of pro-inflammatory mediators (il- β, il- α, and tnf-α). furthermore, secretion of cytokines as il- is inhibited, while production of il- , il- , and defensins can be induced ensuring homeostasis of immune responses. although innate immunity is the first level to be influenced by the probiotic interaction with mucosal epithelium, other cells (e.g., dendritic cells) can be shaped by lactobacilli to skew adaptive responses. an example is represented by l. crispatus sj- c-us strain, which was shown to confer anti-inflammatory properties to dendritic cells by inducing upregulation of il- production and induction of regulatory t cells ( ). as mentioned above, a key metabolite produced by lactobacilli is lactic acid. besides its antimicrobial properties, many of the immune modulation mechanisms exerted by lactobacilli can be ascribed to this compound. in particular, lactic acid has been shown to induce an anti-inflammatory response from vaginal and cervical epithelial cells by inhibiting il- , il- , rantes, and tnf-α secretion stimulated by tlr agonists used to mimic pathogen-associated molecular patterns (pamps) from microbes ( ) . given that il- , il- , and tnf-α are known to promote replication of hiv through activation of nf-κb transcription in hiv target cells, lactic acid produced by lactobacilli in the vaginal environment could be relevant in the context of viral infection acquisition. furthermore, the anti-inflammatory cytokine il- ra was induced by lactic acid treatment of cervicovaginal epithelial cells. all these anti-inflammatory effects were mediated by both l-and d-lactic acid isomers and by the protonated form which predominates in healthy conditions with low values of vaginal ph ( , ) . very few data about the immunological changes associated with benefits induced by lactobacilli administration are available in the vaginal tract in vivo in both physiological and pathological conditions. lactobacillus salivarius crl and l. gasseri crl have been proposed as good candidates to keep a balanced microbiota and immune surveillance. indeed, in a murine model set up to evaluate the benefits of lactobacilli and their effects on the mouse vaginal mucosa and innate immune cells, lactobacilli inoculation did not modify the amounts of granulocytes and macrophages in vaginal washings ( ) . in humans, it has been shown that administration of probiotic lactobacillus vaginal tablets produces a significant reduction in the levels of vaginal il- β and il- cytokines demonstrating the capacity of lactobacilli to modulate the production of inflammatory cytokines in both women with bv and women with healthy vaginal flora ( , ) . a link between oral probiotic administration and vm/immune markers has been recently demonstrated by vitali et al. on pregnant women ( ) . the authors investigated the effects of dietary supplementation with vsl# probiotic mixture containing eight species of lactobacillus, bifidobacterium, and streptococcus on the vm during late pregnancy. interestingly, no changes in the bacterial counts of the most represented populations were revealed upon probiotic administration. however, the probiotic mixture was able to change the composition of less abundant vaginal microorganisms by avoiding the reduction of bifidobacterium and the increase of atopobium recorded in the last trimester of pregnancy in control healthy women. significant modifications of the local immune system were also associated with the consumption of the probiotic showing anti-inflammatory effects. in particular, vaginal levels of il- and il- were maintained in balance compared to the reduction observed in control group. furthermore, vaginal levels of eotaxin, a pro-inflammatory chemokine, were reduced upon probiotic dietary supplementation. similar to lactobacilli, also blf exhibits effects on the host immune system, ranging from inhibition of inflammation to promotion of both innate and adaptive immune responses ( ) . innate immunity is shaped by endogenous hlf through its interaction with pamps and/or pattern recognition receptors (prrs) expressed by host cells. in vitro studies demonstrated that carbohydrate chains of hlf make it able to interact directly with tlr resulting in moderate activation of tlr associated pathways ( ) . lps is a typical pamp, which is bound by hlf, resulting in the inhibition of cell activation and inflammatory responses ( , ) . it has been proposed that in vivo hlf inhibits lps-stimulated tlr signaling and depresses endotoxemia ( ) . in particular, upon lps binding, hlf acts in reducing tnf-α, il- , and il- production by immune cells (macrophages, neutrophils, and lymphocytes), as well as il- release by endothelial cells ( ) . it has been demonstrated that levels of hlf are increased in inflammatory diseases such as rheumatoid arthritis ( ) , severe acute respiratory syndrome ( ) , inflammatory bowel disease ( ) , and as mentioned above, some std and bv ( ) . these observations suggest that hlf could be used as a clinical marker of inflammatory conditions. besides prrs and pamps binding, hlf displaces proteases from heparin in mast cells thus playing antiallergic effects ( ) . furthermore, hlf competes with il- through the binding to proteoglycans on endothelium, thus interfering with neutrophils recruitment to the site of inflammation ( ) . hlf has been shown to bind also dna in the neutrophil extracellular traps (nets), and this capability plays a key role in the context of netosis, which is the nets production by neutrophils. hlf adheres to the dna structures released due to chromatin decondensation and spreading exerting its antimicrobial properties ( ) . in vitro experiments showed that recombinant hlf is able to induce maturation of antigen-presenting cells such as dendritic cells, thus suggesting that it can represent a link in shaping adaptive immunity ( ) . depending on the external stimulus (pathogens, allergen, tumor antigens, etc.) and host immune status, hlf can modulate il- , il- , il- , or ifn-γ levels, thus providing different outcomes: strong th polarization (infections, tumor), reduction of excessive th responses, and correction of th /th balance (allergy, autoimmunity). furthermore, lf is able to support proliferation of t cell precursors and their differentiation. besides its role in cellular-mediated immunity, hlf influences activation of b cells, thus playing a role also in humoral responses ( ) . the effects of exogenous lf on immune responses have been evaluated in different in vitro systems. different epithelial cell monolayers infected with various facultative or obligate intracellular pathogens produce pro-inflammatory cytokines and the addition of blf significantly decreased il- β, il- , il- , and nf-kb levels ( , , , ) . the results obtained in different in vitro models and in various clinical trials confirm the blf ability in downregulating pro-inflammatory cytokine synthesis. it has been demonstrated that exogenous blf localizes into cell nucleus, thus acting as transcriptional factor and inhibiting pro-inflammatory cytokines ( , ( ) ( ) ( ) . although the mechanisms by which blf exerts its anti-inflammatory activity are still under debate, recently, the blf ability to decrease the high levels of il- in cvf seems strictly related to its capacity to restore iron homeostasis disorders ( , ) . therefore, lf is not only a key element in the host defense system ( , , ) but also a pivotal component that is able to regulate the inflammatory response and iron homeostasis ( , ( ) ( ) ( ) . recently, blf is emerging as an attractive molecule for treating inflammation by ranging pro-inflammatory macrophagic phenotypes m to regulatory/ anti-inflammatory m phenotypes ( ). women life: estrogens, lactobacilli, and lf lactobacilli defenses of female mucosal genital tract are largely under the influence of hormones and paracrine production of various cytokines. vaginal environment undergoes overtime shifts in the representation and abundance of microbial key species that are influenced by factors that may include age, hormonal fluctuations, sexual activity, use of medication, and hygiene ( ) . the vagina is lined by stratified not keratinizing squamous epithelium, which is variable in thickness and structure depending on life stages. the vaginal epithelium consists of three cell layers: superficial, intermediate, and basal capable of storing glycogen under the influence of estrogen. in the pre-pubertal and the postmenopausal women, the epithelium is thin and characterized by a basal layer of cells and several layers of parabasal cells. this thin atrophic epithelium is susceptible to infection and frequently shows degenerative and inflammatory changes. vaginal epithelium reflects the hormonal changes of the menstrual cycle with increased mitosis of the basal layers. under the influence of estrogen in the proliferative phase of the cycle, the whole epithelium thickens and is multilayered. during the secretory phase of the cycle, the intermediate layers become thick and the cells stuffed with glycogen. therefore, the glycogen content of the vaginal epithelium co-variates with estrogen levels. breakdown of glycogen by resident healthy vm produces an acid ph in the vagina, which deters infections. indeed, reproductive-age women carry lactobacillus species (predominant lactic acid bacteria) and genera of streptococcus and atopobium, which conserved the ecological function of lactate production in the vaginal microbiome ( ) . it is well demonstrated that fluctuations in the vm occur not only based on intercourse and infections but also during menstrual cycle. in general, high levels of estradiol may favor a lactobacillidominant environment, especially l. crispatus, l. gasseri, and/or l. jensenii ( ) , which can be underrepresented in low estrogen conditions such as the beginning of a menstrual cycle or in postmenopausal women. in one study, l. crispatus appears to decline during menses, while g. vaginalis increases along with l. iners and subsequently the concentration of both species decreases after menses ( ) . in other studies, l. crispatus has also been reported to decline -fold during menses, while the numbers of l. iners strongly increase ( , , ) . recently, cultivation-independent methods have highlighted the complexity and temporal variability of the vm ( , ) . in particular, gajer et al. described temporal changes in the composition of vm in reproductive-age women within a -week period ( ) . in general, the highest variability of microbiota community was associated with menses. for example, a subject can show a community dominated by l. crispatus, which could be replaced by l. iners during menses or by streptococcus spp. in a different subject. the same community could then revert to a community dominated by l. crispatus at the end of menses. moreover, the results also showed how some bacterial communities changed greatly over short time periods, while others were more stable. hormone levels were combined with diversity data showing that an increase in estradiol and progesterone corresponded to decreased microbial variability. vaginal metabolome data added information about community function, which was maintained despite changes in its composition. indeed, shifts in community composition involved only changes in the relative dominance of a little number of different bacteria that are able to produce lactic acid ( ) . interestingly, a recent study performed to investigate timing and sequence of changes that occur in the vaginal and vulvar microbiota during puberty showed that vm of perimenarcheal girls resembles those of reproductive-age women ( ) . in fact, l. crispatus, l. iners, l. gasseri, l. jensenii, and, in some subjects, streptococcus spp. were dominant in the microbiota of girls before the onset of menarche in the early to middle stages of puberty. further studies should be performed to increase knowledge about the link between estrogen, vaginal glycogen levels, lactic acid bacteria abundance, and vaginal ph. other important fluctuations in the vaginal microbiome are recorded during pregnancy. aagaard et al. showed that microbiome was enriched in l. iners, l. crispatus, l. jensenii, and l. johnsonii ( ) . the increase in lactobacilli may be due to the increase in estrogen levels that occurs during pregnancy although further investigations are needed to better understand the relationship between specific species of lactobacillus and estrogen levels. however, another study shows that l. crispatus and l. iners dominate the vaginal flora as pregnancy progresses and maternal age seems to be important for the dominance of l. crispatus or l. iners, with l. iners being dominant in older gravidae ( ) . as mentioned above, menopause usually represents a phase in which lactobacilli levels are low, but it seems that their implication is even more pronounced involving other features. actually, inverse correlation has been found between lactobacillus levels and vaginal dryness, a common condition of postmenopausal period, which was shown to be associated with changes in vaginal epithelial cell integrity and inflammation ( ) . besides the endogenous hormonal fluctuations, clinical evidences demonstrate that the use of hormonal contraceptives is also able to induce changes in vm, thus influencing the susceptibility to sti and bv. therefore, sti and bv (which in turn predisposes to sti) depend, in part, on modification of vaginal bacterial communities induced by some contraception methods. clinical trials based on nugent score endpoint and questionnaires have revealed a reduced bv rate in women who use estrogen-containing contraceptives ( , ) . the effects of progestin-containing contraceptives such as depot medroxyprogesterone acetate (dmpa) and levonorgestrel are less clear. in a systematic review of eligible studies, authors have shown that combined oral contraceptives (cocs; combination of an estrogen and a progestin) and dmpa reduce bv by a range of - and - %, respectively ( ) . accordingly, in a recent retrospective study on fertile women, based on s rrna sequencing, authors found that women using coc or dmpa showed a reduced colonization by bv-associated bacteria compared to women using condoms. in the same study, women using progestinbased therapies have a significantly higher abundance of taxa associated with a dysbiotic vm. on the other hand, coc users have a lactobacilli-dominated vm and a higher proportion of h o -producing lactobacillus species (l. crispatus, l. gasseri, and l. jensenii) correlating with vaginal health compared to progestin-containing contraceptives ( ) differences in the results reported in other studies, which do not reveal any changes in vaginal bacterial communities associated with progestin use, could be due to the different study population characteristics (e.g., ethnicity of subjects) and/or the design of the study itself such as the specific bv status examination ( , ) . in women with bv, vm is not the only level that hormonal contraception acts on. in fact, changes in genital tract immunity by effect of hormonal contraceptives have been shown in terms of both suppression and activation of responses. in a study on women, cervical secretions contained lower levels of pro-inflammatory molecules (tnf, ifn-γ, and gm-csf) in subjects with bv using hormonal contraceptives compared to those not using them ( ) . on the other hand, an increase of inflammatory cytokines (mip- α, mip- β, il- , il- , ip- , and rantes) has been associated with hormonal contraception in a different cross-sectional analysis including african women ( ) . conflicting data existing in these studies on immunomodulatory potential of hormonal contraception in female genital tract are complicated by their variability in terms of in vitro versus in vivo models used and sample tested (plasma or blood versus cervical fluid). however, the investigation on these features may represent a key point in understanding the association between hormonal contraception and hiv acquisition. in fact, women using progestin dmpa, but not those who use coc have been found to be at significantly increased risk of hiv infection compared to women not using hormonal contraception ( ) . in a more recent case-control selection of specimens from a large, prospective, clinical study, the same authors have investigated on innate immunity mediators in cervical samples collected from women at their visit before hiv seroconversion and matched visits from women remaining hiv uninfected. higher levels of pro-inflammatory markers such as rantes have been found in hiv seroconversion and in dmpa users, suggesting a possible role of this cytokine in the association between the contraceptive and hiv risk acquisition ( ) . the possible explanation could be that the upregulation of rantes, observed in women using dmpa and women with bv, may simultaneously block the ccr cellular hiv receptor but also facilitate transmission through recruiment of target cells. also other sti can be influenced by hormonal contraception, such as candidiasis, which has been found increased in women using coc but not dmpa ( ) . taken together, these observations suggest that altered immune responses by effect of hormonal contraception may predispose to infections from pathogens. in cases of a pre-existing condition of dysbiosis or specific cervicovaginal infection, suppression or activation of immunity by pathogens could increase the risk of infections by cumulative action with exogenous hormones. similar to the estrogen-induced changes in the species and number of lactobacilli, the hlf concentration fluctuates accordingly to circulating estrogen levels ( ) ( ) ( ) . in addition to the hlf levels produced by uterine epithelium and released in cvf, the synthesis of iga and igg is also modulated by estrogen and progesterone, thus exerting an immune protection against sexually transmitted pathogens ( ) . figure shows a comparison among estrogen, progesterone, and lf levels during the proliferative, ovulatory, and secretory phase. the hlf levels increase in line with estrogen production, reaching the highest levels during ovulatory phase, while they are inversely correlated with progesterone levels. as a matter of fact, secretory phase accordingly to the increase of progesterone shows the lowest levels of hlf. at the end of the secretory phase, hlf returns to increase in parallel to the progesterone decrease ( ) ( ) ( ) ( ) ( ) . obviously, the use of oral contraceptives by decreasing estrogen synthesis suppresses the production of hlf as well as immunoglobulins for the duration of hormone exposure, thus possibly increasing the susceptibility of women to infections ( ) . in rats, pretreatment with progesterone prior to exposure to chlamydia thracomatis infection induced a persistent infection ( ) . furthermore, the rats treated with progesterone were also found to be more vulnerable to chlamydial intrauterine infection, whereas the treatment with estradiol, the major female estrogen sex hormone, reduced the susceptibility to infection ( ) . the sex steroid hormones are also important mediators of inflammation ( ) and may influence resistance or susceptibility to parasitic infections ( ) . sex steroid hormones are also involved in viral infections. it has been proposed that hiv- utilizes a window of vulnerability during the menstrual cycle. this crucial period overlaps with the mid-cycle when innate and adaptive immune responses are suppressed by estrogens and/or progesterone to facilitate reproductive processes. hiv- presumably exploits this time frame, during which antiviral factors are suppressed, to establish and propagate infection in the female mucosal genital tract ( , , ) . in menopausal women, the low concentration of hlf in the secretions, related to the low levels of sexual hormones ( ) , may lead to recurrent infections. it is important to underline that during menses the decrease of this important natural defense glycoprotein is balanced by the presence of neutrophils that, through the synthesis of granules, can restore, at least partially, hlf concentration and its antimicrobial activity when the epithelial barrier is disrupted. the recruitment of neutrophils occurs through high levels of inflammatory biomarkers as pro-inflammatory cytokine as il- , il- or c-reactive protein ( ) . as matter of fact, during menstruation, as well as in aging and menopause, the decrease of estrogens is related to the increase of inflammatory processes ( ) . interestingly, hlf expression in endometrium suggests that, during the gestational period, hlf produced by uterine epithelium and neutrophils and released in cvf is controlled by sex steroid hormones ( ) . it has been reported that high levels of progesterone parallel low levels of estrogens in normal pregnancy, while this ratio is inverted in pregnant women with the preterm delivery threat ( ) . determining the existence of a regulatory circuit linking hlf synthesis and sex steroid hormone fluctuations may unravel novel mechanisms leading to preterm birth. figure | lactobacillus spp. and lactoferrin interplay on infection and inflammation in female genital tract. a schematic representation of lactobacillus spp. and lactoferrin balance: a multitasking strategy to protect against pathogen challenge and maintain immune homeostasis. (a) healthy genital tract; (b) vaginosis: high levels of pro-inflammatory cytokines, decrease of lactobacilli and increase of gram-negative anaerobes, and increase of lactoferrin concentration released by neutrophils; (c) decrease of pro-inflammatory cytokines by lactoferrin and restoration of healthy microbiota. lactobacillus spp. and lf are pivotal components of first-line defense in the female mucosal genital tract involved in protection against a multitude of microbial infections and the most effective natural mechanism to dampen inflammatory processes. to inhibit cervicovaginal infections, an ideal drug should inhibit: • microbial growth; • microbial adhesion and entry into host cells; • microbial intracellular replication; and • infection of new host cells by microbes extracellularly released from the infected cells. in the vaginal environment of women of childbearing age, the inhibition of bacterial multiplication through the synthesis of antibacterial substances by lactobacilli or by competition between microbes and lf for iron acquisition represents an effective natural defense mechanism. both lactobacilli and lf can inhibit the adhesion and consequently the microbial entry inside the cells through an interaction with the cell surface components potential receptors for pathogens. lactobacilli and lf appear complementary since lactobacilli inhibit microbial intracellular replication and together with lf hinder the infection of still healthy cells by microbes extracellularly released. this close cooperation is also exerted through their anti-inflammatory function. in this scenario, the mucosal environment represents a good model of mutualism and reciprocity against the injury by microbes. a schematic representation of lactobacillus spp. and lf balance in protecting against pathogens and maintaining immune homeostasis in the vaginal tract is shown in figure . considering the shortage of effective treatments to counteract antibiotic-resistant bacterial infections, the intravaginal administration of lactobacilli and lf could be a novel efficient therapeutic strategy and a valuable tool to restore mucosal immune homeostasis. author contributions pm, rp, and pv conceived the topic concept and wrote and revised the final manuscript; lr, ac, ml, dc, and es provided figures and contributed to manuscript preparation and editing. all authors read and approved the final version. acknowledgments this work was granted by sapienza university of rome funds to pv. microbiota of the upper and lower genital tract emerging role of lactobacilli in the control and maintenance of the vaginal bacterial microflora understanding the bacterial flora of the female genital tract characterization of vaginal flora and bacterial vaginosis in women who have sex with women the human vaginal bacterial biota and bacterial vaginosis temporal variability of human vaginal bacteria and relationship with bacterial vaginosis the vaginal microbiome: new information about 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peter j.; granata, guido; la marra, fabiola; kuijpers, taco w.; quinti, isabella title: on the dark side of therapies with immunoglobulin concentrates: the adverse events date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: h therapy by human immunoglobulin g (igg) concentrates is a success story ongoing for decades with an ever increasing demand for this plasma product. the success of igg concentrates on a clinical level is documented by the slowly increasing number of registered indication and the more rapid increase of the off-label uses, a topic dealt with in another contribution to this special issue of frontiers in immunology. a part of the success is the adverse event (ae) profile of igg concentrates which is, even at life-long need for therapy, excellent. transmission of pathogens in the last decade could be entirely controlled through the antecedent introduction by authorities of a regulatory network and installing quality standards by the plasma fractionation industry. the cornerstone of the regulatory network is current good manufacturing practice. non-infectious aes occur rarely and mainly are mild to moderate. however, in recent times, the increase in frequency of hemolytic and thrombotic aes raised worrying questions on the possible background for these aes. below, we review elements of non-infectious aes, and particularly focus on hemolysis and thrombosis. we discuss how the introduction of plasma fractionation by ion-exchange chromatography and polishing by immunoaffinity chromatographic steps might alter repertoire of specificities and influence ae profiles and efficacy of igg concentrates. i.e., cold-ethanol or ion-exchange chromatography, contaminants, route of application, i.e., intra muscular (imig), intravenous (ivig), or subcutaneous (scig), the rate of increase of the exogenous igg in the circulation of the recipient over time and, last but not least an eventually existing risk factor from patients' side ( figure ) as well as incorrect handling of the concentrate are factors having a role in inducing non-infectious aes related to administration of igg concentrates ( table ) . igg concentrates represent a defined part of the adaptive immune system, are isolated from pooled human plasma of at least donors, which contribute to the repertoire diversity in the final product. therapies with igg concentrates manufactured according to regulators requirements are acknowledged to be safe in general. this does not exclude the occurrence of aes which in their majority are rare and clinically mild to moderate. below, we like to give a few insights into various aspects and possible mechanisms of aes. table . contemplating on the active ingredient of the concentrates, the administration of the igg molecules inevitably results in the interactions of the exogenous igg with the various parts of the immune system of the recipient and vice versa. this interaction in its principle generates an inflammatory condition. a key parameter deciding on the intensity of such a condition is the rate of increase over time of exogenous igg in the circulation of the recipient. insert to figure (shadowed): ∆igg in the circulation over ∆time is dependent on the combination of infusion rate (ir) and the strength of the solution applied. main figure: the mode of application, i.e., intravenous or subcutaneous, are additional factors being decisive for kinetics and the area under the curve of generation of pro-inflammatory mediators and for passing the individual threshold (short-dashed line) for a clinically noticeable ae. the threshold in turn depends on eventually exiting risk factors form the patient side. although the area under the curve might be the same for low (long-dashed line) and high (solid line) irs, at high ir, the system might not be able to cope with the extent of reactions. at low ir (long-dashed line), all the events might remain below the threshold of clinically observable ae. subcutaneously applied igg concentrate reaches the circulation slowly and systemic aes are less frequent than with ivig. in contrast, local mild to moderate aes are more frequent with scig. virus inactivation and virus elimination methods by validating an already performed step of the fractionation process or by introduction of dedicated steps (figure ) . a hallmark of virus elimination introduced in the late s in berne by the team of christoph kempf is the large-scale virus filtration technique (formerly also termed "nanofiltration") ( ) . meanwhile, virus filtration became a versatile tool to eliminate a variety of pathogens, the suspected agent of variant creutzfeldt-jakob disease included. thanks to the tightly implemented regulatory framework, pathogen safety of plasma products is at a level never reached before. this is well supported by the fact of reports missing in the last decade of transmission by igg concentrates of emerging viruses (sars coronavirus, west nile virus, mers coronavirus, and others), zoonotic pathogens, or the agent of variant creutzfeldt-jakob disease (vcjd). furthermore, the development of specific mass screening techniques might help to eradicate in any blood product the transmission of vcjd in the future ( ) . the human immune system is in charge of controlling invading organisms and mediates homeostasis. the immunoglobulin pools in mammals (igm, igg, and iga) to its smaller part provide defense and to the larger part homeostasis. human igg has a role in both. efficient host defense is supported by "immune antibodies." these have undergone somatic hypermutations and have in their vast majority narrow specificities and high affinities. antibodies, generated in absence of external stimuli are termed "natural antibodies" (nabs). they occasionally recognize self structures. in general, the v(d)j genes of nabs are in germ-line configuration or have undergone a few somatic hypermutations only. furthermore, they have broad specificity, are of low affinity (with exceptions) and high avidity ( ) . these nabs can participate in primary host defense, i.e., at a time point when an immunologic reaction has not provided the specific antibodies, react, e.g., with repetitive structures which can be found on bacteria or viruses ( ) . the self-reactive nabs, which we like to term physiologic autoantibodies, comprises various populations of antibodies such as (i) those able to interact through their complementary variable regions (v-regions) with the v-regions of circulating and membrane-bound (bcr) immunoglobulins and the t cell receptor (tcr) β-chain variable region, providing a peripheral immune network (v-connected network) ( ); (ii) the nabs reacting in a non-idiotypic manner with the hinge region of immunoglobulins ( , ) ; (iii) populations of nabs showing a wide variety of specificities toward growth factors, cytokines, or anaphylatoxin ( ); (iii) and the population reacting with the soluble or membrane-bound forms of cell surface molecules having immunological importance, the last described being the fc receptors cd and cd ( ) . antibodies reacting with docking structures for viruses or bacteria can have additional first-line defense potential ( ) . these populations of nabs were described having a peripheral immune network homeostatic and anti-inflammatory function ( , ) . although the primordial humoral proteins comprising the complement and lectin-like proteins in the plasma play a definite role, another population of self-reactive nabs reacting with, e.g., epitopes conserved over the evolution apparently has tissue homeostatic function and might support the efficient removal of roughly altered/senescent cells of the body per day (for references see below). the signal for research on nabs in ivig was the description of igg autoantibody-mediated immune thrombocytopenia (itp) being corrected by infusion of a polyclonal, polyspecific igg concentrate ( , ) . this research has expanded ever since. the populations of immune antibodies and nabs in igg concentrates upon infusion/injection inevitably react with occasional pathogens, toxins, or superantigens and concomitantly infusion/injection also results in recognition of a wide array of tissue antigens and v-regions of the recipient's immune system. reactions with tissue antigens and v-regions are conveyed by the self-reactive antibodies of the many donors in the igg concentrate. vice versa, the recipient's immune system reacts with the infused igg. a bewildering wide range of possible reactions can occur which primarily are dependent on the immune status of the recipient at the time of therapy and to a smaller part on the igg concentrate(s). the therapeutic effect achieved depends on the disease treated, and can depend on the concentration reached locally, i.e., can have agonistic or antagonistic effects ( , ( ) ( ) ( ) . in summary, it is our opinion that igg concentrates always provide more or less the same "bouquet" of igg specificities (similarity); however, it is the recipient's actual immune condition which decides from which igg specificities the patient's derailed immune system is profiting (diversity). parameters of igg-mediated aes are: (i) the content in the product of biologically highly active likely beneficial ingredients that have to be kept under control (e.g., content of "dimers" devoid of remarkable complement activation in vivo; see below), and the content in unwanted active ingredients that have to be discarded during manufacturing (alloantibodies); (ii) impurities such as iga (anaphylactoid reaction); (iii) activated coagulation and contact activation factors (thromboembolic events) and; (iv) excipients such as sucrose (osmotic nephrosis). below, we like to add and contemplate on how fully native igg molecules not harmed by the manufacturing process might add to aes. the above mentioned inevitable interaction of the exogenous igg with the immune system of the recipient and vice versa in its principle might evoke an inflammatory condition. the sum of the potentially beneficial reactions might overshoot and lead to aes (figure ) . the principle of induction of mild inflammatory www.frontiersin.org figure | staying within a regulatory network is mandatory for remaining at the sunny side of the moon. handling of blood/plasma products to obtain therapeutic goods has to be performed within a regulatory framework. manufacturing of stable blood product has to adhere strictly to current good manufacturing practice (cgmp). application of cgmp starts from the moment of collecting whole blood and isolation of plasma thereof (r = recovered plasma) or the machine-supported collection of plasma (s = source plasma, apheresis plasma). application of cgmp ends at delivery of blood/plasma products to health care professionals. not following cgmp can lead to withdrawal of a plasma product from the market from one to the other day. conditions upon each infusion of a well-tolerated ivig was confirmed when several dozen normogammaglobulinemic volunteers in all cases except one, showed a more or less moderate inflammatory reaction as indicated by the increase of tumor necrosis factor alpha (tnfα) at . h post initiation of infusion. the only person in the cohort not showing a measurable tnfα increase was a woman caring at home for her brother with full blown aids ( ) . subcutaneously applied igg concentrate reaches the circulation slowly and systemic aes are less frequent compared to ivig but they are not absent ( - ) (a case of unintentional i.v. application of scig is not considered). in contrast, local mild to moderate aes are more frequent with scig ( ) . in summary, the intensity of the resulting aes is depending on the immune status of the recipient, the infusion rate (ir), e.g., how rapidly the active ingredients (the various igg specificities), the impurities, and the excipients reach the circulation of the recipient. thus, the i.v. application has the highest chance for the occurrence of aes. in the early days of ivig therapy, complement-mediated "anaphylactoid" (i.e., immediate) and "phlogistic" (i.e., inflammatory) aes were distinguished ( , ) . the complement-mediated aes were considered to be caused by aggregates in the product ("spontaneous complement activation" or anti-complementary activity or aca) or by in vivo formation of immune complexes (ics, patient's condition related; e.g., subclinical infections or the unnoticed presence of anti-iga antibodies) and therefore only igg concentrates with low or absent aca is accepted by authorities for human use. below, we present one instructive case of each type of reaction. the first reports of rapid onset aes concerned either the application of complement-activating fractions in an igg concentrate or the in vivo formation of complement-activating ics ( ) ( ) ( ) . a very rare but potentially fatal condition is the formation of iga/anti-iga complexes in patients being initiated on replacement therapy and having serum igg antibodies against infused iga not recognized before the start of the ivig infusion ( ) . prerequisite for the presence of anti-iga antibodies is the most common primary immunoglobulin defect, i.e., selective iga deficiency (sigad) or igad associated with diminution of other immunoglobulin classes. igad is defined by serum levels of < . or < . g/l (depending on laboratories). a marked diminution of serum iga consistent with igad in various ethnic groups is estimated being : to : , ( ) . the mean frequency in caucasians is approximately : ( ) . up to % of patients with igad have been reported having anti-iga antibodies in the serum with titers ranging between : and : , . in approximately % of patients with common variable immunodeficiency (cvid), and occasionally in patients with other primary immunodeficiency diseases, measurable anti-iga can be detected ( , ). these antibodies are predominantly of the igg class, but anti-iga antibodies of other immunoglobulin classes have been described as well ( , ) . the reason for their emergence remains unknown. taken the above numbers, the infusion of human-derived products containing iga resulting in severe anaphylactoid type aes should be considerable. this is not the case ( ) . questions about the clinical relevance of above numbers emerge as soon as blood banks (i) estimate the theoretical risk of iga anaphylactic reactions ( ); (ii) assess the relation of severe igad with the presence or absence of anti-iga antibodies ( ) ; (iii) screen donors for very low iga levels in order to become able to provide blood and plasma-derived products free of iga and find a considerably lower frequency than expected ( ) . alternatively, the test systems may not reliably detect anti-iga antibodies being as yet insensitive and inaccurate or -at least -do not correspond to the clinically relevant fraction of antibodies. this comes to mind when a more close look to "anti-iga" gives "unexpected" results, including "anti-iga" in blood donors with normal serum iga level or "anti-iga" that cannot be neutralized with purified iga ( ); or when blood products containing proven anti-iga do not elicit severe aes ( ) . among patients on replacement therapy, those with cvid may rarely develop severe immediate aes ( ) . the discrepancy between anti-iga positive patients and frequency of aes raises the question about the nature of the many reported anti-iga antibodies and also raises the question about the immunologic condition which allows the formation of anaphylactoid anti-iga antibodies. there might be some logic in supposing that anaphylactoid anti-iga cannot evolve at iga levels otherwise fulfilling the definition of igad. such a condition would constantly generate ics which in turn could activate complement, react with immune cells, and be deposited in lung and kidney. indeed, horn et al. found anti-iga frontiers in immunology | primary immunodeficiencies antibodies in cvid patients missing iga + b cells and presenting with iga levels < . g/l, a level which is more than -to fold lower than the threshold for igad ( ) . however, a possibility for an iga-mediated anaphylactoid reaction at measurable iga serum levels might exist. serum iga contains approximately % subclass of iga (iga ) and only % subclass of iga (iga ). selective deficiency of iga and -although evidence is lackingthe presence of a highly specific anti-iga antibody theoretically could elicit a severe ae. the kinetics of anti-iga after infusion of blood products have been studied in a few cases. in these patients, a fall in anti-iga titers has been noticed followed by an increase during subsequent weeks or months. this suggests that at appropriate proportions, iga of the infused material and anti-iga present in the patients' serum combine with each other to form ics. in turn, ics activate complement that are bound and eliminated by macrophages most likely leading to cytokine release. the increase in anti-iga titers over time indicates that the infused iga-containing product has a booster effect ( , , ) . such boosting effect together with the presence of anti-iga before the application of an igg concentrate can be taken as the ultimate confirmation of a supposed iga/anti-iga reaction. figure depicts a well-documented case of iga/anti-iga reaction in a patient who progressed from sigad to cvid. the events during the first h at occasion of the first infusion of ivig were as follows (shadowed area in figure ): min after the start of the infusion, having received eight drops of an igg solution (iga < . g/l; % solution), she experienced a flush, back pain, rigors, difficulty in breathing, and hypotension. the infusion was immediately stopped. after approximately h, the reaction has weaned, and h later the patient felt well again, and the infusion of total g igg could be continued without further complications. although the patient fairly assured having never received any blood or plasma product in the past, the follow-up of her anti-iga titers from before infusion to year later confirmed a true anaphylactoid reaction mediated by anti-iga, as the anti-iga became undetectable immediately after the infusion and showed a boosting phenomenon during the following months. true anaphylactoid reaction was further confirmed by follow-up of total complement hemolytic activity (ch ) on the day of infusion. interestingly enough, the ch value reached its nadir at the end of the infusion when the patient had no complains. although a single case only, the events during the first infusion call for the following remarks: (i) severe aes most likely occur at concomitant complement and cell activation with cytokine release; (ii) infusion of minute to low amounts of ivig hours before the main infusion can "anergize" cells and stop release of pro-inflammatory www.frontiersin.org figure | an individual's slithering into the dark. a female patient has been suffering from recurrent airway infections since adolescence, occasionally complicated by pneumonia. at age , selective iga deficiency was diagnosed. ten years later, she was hospitalized with pneumonia. within these years, her serum igg had dropped from to . g/l (long-dashed line) and iga was undetectable. the diagnosis was corrected into cvid, and ivig replacement therapy was initiated (shadowed area). two minutes after the start of the infusion of a % igg solution (iga < . g/l; % solution), she experienced a flush, back pain, rigors, difficulty in breathing, and hypotension. the infusion was immediately stopped and later continued without further complications (see text). the confirmation of a true anaphylactoid reaction due to anti-iga in the serum of the patient was achieved by follow-up of anti-iga (solid line) and ch (short-dashed line). cytokines; (iii) "anergized" cells loose reactivity toward ongoing formation of ics and complement activation products. a non-complement-mediated anaphylactoid reaction was ascribed to the unforeseen release of elastase and other proinflammatory substances from neutrophils activated by the formation of in vivo iga/anti-iga complexes. complement activation or mast cell-dependent release of vasoactive substances was excluded as pathogenic mechanisms. although the iga/anti-iga complexes usually do not cause clinically relevant neutrophil degranulation within the circulation, the presence of a rare genotype encoding a novel gain-of-function igg receptor on neutrophils may provoke premature degranulation by these complexes. this phenomenon was only relevant in hypogammaglobulinemic patients in the presence of in vivo iga/anti-iga complexes ( ). the low prevalence of this genotype combined with an igad or cvid may add how to explain the rarity of serious anaphylactoid reactions in newly ivig-treated patients. authors share the opinion of janne björkander who at occasion of a discussion panel "dilemmas in diagnosis and management of antibody deficiencies: ask the experts" held at occasion of the th annual meeting of the american academy of allergy, asthma & immunology (aaaai), new york city, march - , came to the following conclusion: a clinician has to be aware of the risk, particularly at occasion of first infusions, but otherwise iga is not a major concern (from tape record). in the early days of ig-therapy, the nature of the "phlogistic" aes was obscure. however, it was already known that an ae can be prevented or its evolution halted when the patient receives a low dose of ivig first or the infusion is stopped early and is continued several hours later. hours later the infusion can be (re)started at high rates without further problems ( figure ) . one of the authors had a particular opportunity to get an insight into what a "phlogistic reaction" might be. at the occasion of a voluntary infusion of an investigational liquid ivig, he encountered a severe flu-like ae of more than h duration. before injection, the investigational liquid preparation had passed all release criteria for human use, including spontaneous complement activation assessed by aca and was free of prekallikrein activator (pka). in those days, assays for cytokines in biological samples just began to become available and were included into the parameters assessed in the study. infusion was stopped after h because of a drop of pulse rate and heavy discomfort provoking the laconic comment by the proband's technician who was taking samples:"you look green." the infusion was continued after another min when the heart rate had almost normalized. the infusion could be completed within an additional . h (a total of . g/kg b.w.) without further aggravation of malaise. the leukocyte count transiently had dropped to a nadir of % at h followed by a leukocytosis peak at h. complement activation, as assessed by generation of c a/c a[desarg] and the formation of the terminal complement complex c b- , apparently did not occur: the c a/c a[desarg] value reached a maximum of ng/ml (norm: < ng/ml) at h while the c b- value never moved outside the normal range. instead, a sequence of rapid transient massive increases of pro-inflammatory cytokines was observed: (i) tnfα started to increase min post initiation of infusion from a value of pg/ml to a peak value at h which was above the calibration range of the test kit of pg/ml; (ii) interleukin (il- ) increase started after h from pg/ml and peaked at . h with pg/ml post initiation of infusion; (iii) interleukin (il- ) secretion started after h with an undetectable level and peaked at h with pg/ml. all pro-inflammatory cytokines fell sharply while the second part of the infusion was still ongoing. the day after infusion, the pro-inflammatory cytokine profiles were back to normal and the flu-like syndrome was gone. in contrast, interleukin receptor antagonist (il- ra) values started increasing at . h ( pg/ml), peaked at h (> , pg/ml), and decreased slowly to reach a value of , pg/ml h after initiation of the infusion. soluble tnf receptor p level started at . ng/ml, reached a peak with . ng/ml at the same time as il- ra, and h after initiation of infusion was still at . ng/ml. thus, in this normogammaglobulinemic subject similar cytokine profiles and leukocyte number changes were observed as reported for hypogammagobulinemia under replacement therapy ( , ) . a series of further experiments with investigational and marketed ivigs was performed. all igg concentrates were analyzed for their molecular weight (mw) distribution. the most remarkable differences emerged in the mw range of dimers while the presence of minute amounts of higher oligomers could not be excluded with certainty. below, we will use the term "dimers" for that fraction of igg with higher mw. subsequent findings indicated that levels of "dimers" > % were responsible for complement-independent cell activation and cytokine release. the tnfα peaks assessed at . h post initiation of infusions correlated with "dimer" content of the ivigs and mirrored a clinical score of aes ( ) ( ) ( ) . frontiers in immunology | primary immunodeficiencies a few years before a complement-independent induction of a hypotensive factor by igg di-and oligomers was reported in animal experiments ( ) . a key role for macrophages in the generation of the hypotensive lipid factor was identified as plateletactivating factor, being induced by dimers and polymers ( , ) . several years later, the dimer-mediated aes in animal experiments were confirmed ( , ) . yet, at the same time, the dimer content of ivig apparently correlated with the clinical efficacy in a murine itp model ( , ) . variables such as "ir," "genetic background," "endogenous immunoglobulin levels," or "proportional fraction of polymers versus dimers" may impact on the balance between the phlogiston (being cytokines, active lipid substances, or a combination of factors) and the therapeutic efficacy (blocking igg receptors on liver/spleen macrophages to prevent clearance of "opsonized" material such as platelets in itp). as of today, reports on release of cytokines in humans in association with aes or tolerability toward dimers remain scarce and to the best of our knowledge studies in humans of causative factors/fraction in an igg concentrate has not been adequately addressed ( , , ( ) ( ) ( ) ( ) . in igg preparations, various forms of dimers might be present: formed through covalent binding ( ) by denaturation, hydrophobic interactions of the fc-parts, and by idiotype/anti-idiotype interactions, as part of the v-connected network of peripheral immune homeostasis ( ) . for a commercially viable fractionation process, pooling of donated plasma is mandatory in order to obtain a volume of starting material large enough to cover ever increasing costs for documentation, in-process, and batchrelease testing as it is required by cgmp. pooling also intends smoothing the batch-to-batch differences in antibody titers, a goal apparently difficult to achieve to levels as theory might imply ( ) . consequences of pooling are on the one hand the enrichment of public/common immune antibodies while diluting out individual specificities; on the other hand, the antibodies of the immune network of an individual donor are exposed to those of many other donors. the more individuals contribute to the pool, the more complex the possible "immune-network" interactions among iga, igg, and igm will become. the subsequent fractionation process has far-reaching effects on immunoglobulins from a given pool: only trace amounts of iga and igm are retained in the final product, i.e., igg is deprived of its counterparts of the v-connected immune network. the igg molecules of the homeostatic network "naked" at their v-regions can interact with each other at random combining site-interactions of single donor-derived monomeric igg ( ), otherwise not existing in vivo. this interaction is largely reversible. with increasing numbers of donors included into the pool, the immune network recognition among the "naked" igg molecules of the v-connected network becomes more and more complex, and the dimer and lower oligomer content in the resulting igg concentrate increases ( ) ( ) ( ) . in lyophilized igg concentrates, the dimer formation is "frozen" at a low level while in liquid preparations an equilibrium between monomers and dimers is achieved over time reaching a dimer content of % or more if not hampered by stabilizers. specificities, as far as they have been addressed, in the dimer fraction considerably differ from the monomeric fraction ( ) ( ) ( ) ( ) ( ) . in conclusion, the immunomodulatory efficacy of igg concentrates in part depends on the capacity and extent to form "dimer" fractions devoid of remarkable complement activation in vivo. the "art" of manufacturing a liquid igg concentrate is not to eliminate the monomeric igg having potential for "dimer" formation but to inhibit extensive"dimerization."in summary,aes might be associated with the induction of pro-inflammatory cytokines in absence of measurable complement activation in vivo where all regulatory mechanisms and removal processes of a body are at disposition. at reasonable irs in the open system of the human body, clinically relevant systemic complement activation apparently needs oligomers formed of three or more igg molecules. there are multiple reports of ig-induced hemolytic anemia (ha) in patients receiving high doses of ivig ( , - ) (table ; figure ; www.adrreports.eu). by spontaneous reporting, risk factors recognized for ig-induced hemolysis include beside high doses (more than g ivig over - days), female gender and histo-blood group type a, b, or ab of recipients. www.frontiersin.org a significant proportion of patients receiving ivig develop a positive direct antiglobulin test (dat) detectable after h for up to days after the ivig infusion ( , ) . however, it should be underlined that the dat positivity due to the factors mentioned above ( , ) is not sufficient per se to diagnose hemolysis and dat positivity does not necessarily imply the presence of active hemolysis. dat-positive mild hemolytic reactions can be easily missed and the true incidence of such reactions is difficult to document without careful clinical and laboratory follow-up. in the majority of reports on ha, intravascular red blood cell (rbc) destruction via complement activation or extravascular rbc sequestration and removal by the reticulo-endothelial system was proposed to result from igg alloantibodies with specificity for rbc antigens a, b, d, or c. hemolytic anemia induced by high-dose ivig has an average incidence of . % ( ) . low-dose igg replacement therapy is considered universally as safe, and only few cases of hemolysis following low-dose ivig or scig administration have been described ( , , ) . a baseline wbc and rbc count prior to ivig initiation and a close clinical and laboratory follow-up was suggested as a useful tool for early diagnosis and treatment. a possible work up might be to check hemoglobin (hb) level prior and - h after ig infusion. in case of a drop of hb, the presence of dat, an increase in unconjugated bilirubin, lactate dehydrogenase (ldh), and reduced haptoglobin level, followed by a rise in reticulocyte count should be assessed (figure ) . we systematically reviewed case reports related to ivig-induced hemolysis from to and identified articles containing reports of patients. baseline characteristics of the patients are shown in table . when available, blood group, dat, hb drop, and outcome are indicated. all reports showed positive dat, except for a case of yin et al. ( ) ; in this case, dat was performed days after ivig administration and the dat negativity might have been due to a rapid removal of sensitized rbcs. in the majority of patients, the outcome was positive: out of patients recovered with or without packed rbc transfusions; three patients died after ha, with the hemolytic episode representing a precipitating factor of a severe underlying condition. elution experiments were performed and the search for blood group antibodies revealed anti-a and anti-b specificity in the majority of cases; anti-d specificity was assessed in four reports, often associated with other specificities ( , , ) . a search for other specificities such as anti-band or anti-gal was not performed. only one report detected anti-c specificity in three patients; in one of them associated with anti-d irregular antibodies ( ) . although studies were restricted to blood group antibodies, this finding demonstrated that polyvalent igg preparations might contain clinically significant non-blood group antibodies, which are not part of the lot-release criteria in that their titration is not yet required by the european pharmacopeia. antibodies in ha, such as anti-c, may have unexpected hemolytic consequences ( ) ( ) ( ) ( ) ( ) . beside passive transfer of alloantibodies, igg administration also has been demonstrated to lead to unspecific enhanced erythrocyte sequestration, in particular, in patients with underlying inflammatory disorders ( , ) . in , the canadian ivig hemolysis pharmacovigilance group elaborated criteria to define an "ivig-induced hemolysis" ( ) . they included a reduction of hb levels ≥ g within days after ig administration, with frontiers in immunology | primary immunodeficiencies appearance of a positive dat and, at least, two of the following criteria: increase in the reticulocyte count, elevation of ldh and unconjugated bilirubin serum levels, low haptoglobin, hemoglobinuria, hemoglobinemia, presence of significant spherocytosis, in the absence of alternative causes of anemia. the passive transfer of igg alloantibodies through igg concentrates is difficult to explain as polyvalent igg is prepared from plasma of thousands of donors. since immunization to rbc alloantigens can occur because of past transfusions or pregnancy, the hypothetical numbers of alloimmunized plasma donors should be rather low. recently, other mechanisms underlying alloimmunization related to molecular mimicry have been demonstrated ( ) . the mechanism of high-dose ivig-induced ha is complex and it might vary from patient to patient. ivig cause hemolysis due to: (i) diseaseassociated pre-coating of rbcs; (ii) igg with hemolysis triggered by passive transfer of igg binding to blood group antigens; (iii) transfer of high levels of alloantibodies to rbc pre-coated at a low level only; or (iv) transfer of clinically tolerable levels of isoagglutinins plus transfer of additional rbc-reacting physiological autoantibodies. indeed, hemolytic reactions could not be related exclusively to transfer of alloantibodies. hence, antibodies other than histo-blood group alloantibodies (pre-)coated to rbcs might contribute to hemolysis in igg recipients need to be identified. in addition, hemolytic episodes may possibly be precipitated by some sort of complexed/denatured igg that co-purify with other igg in the product ( , , , ) . recently, a two hit mechanism for ivig-induced hemolysis has been proposed: the passive transfer of alloantibodies through ivig representing the first hit and the underlying inflammatory state representing the second hit ( ) . nowadays all commercial ig products have to undergo anti-a and anti-b testing and regulatory requirement ask for respective igg antibody titers of ≤ : at % solution strength (w/v) ( , ) . nevertheless, hemolysis might occur even in recipients of igg products that meet these specifications ( ) . consequently, it has been suggested that igg recipients should be monitored for clinical signs and symptoms of hemolysis ( ) . with the detection of the immunomodulatory potential of igg concentrates, their clinical use has continuously increased ( ) . to cover the need, at a first glance, an increase of the volume of plasma fractionated seems to be the most convenient option. however, this might economically not be viable because fractionation of plasma products is interconnected ( ) and before increasing output of one product (e.g., ivig), the market absorbance of the other products as well (e.g., albumin) must be ascertained. on a longer-run, a more viable option is to improve recovery. considering recovery, the cold-ethanol fractionation apparently has reached its limits. as of today, four manufactures have invested into a"modern"fractioning technique on the basis of ion-exchange chromatography. ion-exchange chromatography allows elevated recovery at high purity. as of today, five ivigs, one scig, and one anti-d concentrate are fractionated by ion-exchange chromatography. pharmacovigilance has shown that all chromatographically fractionated ivig and scig, more or less prominently, show a tendency for elevated frequencies of hemolytic aes. anti-a and anti-b alloantibody titers are now lot-release criteria (see above) as they constitute the major risk parameter for hemolytic reactions mediated by igg concentrates. to overcome the threat of end up on the dark side of the moon, two manufacturers have taken measures to reduce anti-a and anti-b titers in their igg products. one measure chosen was adsorption of the alloantibodies by affinity chromatography ( ) . reported reduction in both alloantibodies was significant and levels were similar to those in cold-ethanol fractionated immunoglobulins ( ) . the other measure chosen was reduction in anti-a using an automated indirect agglutination test for donor screening and exclusion of high-titer donations (approximately . %) from plasma pooling and fractionation ( ) . this measure reduced anti-a in the igg concentrate by one titer step. to ensure staying on the safe and sunny side, the manufacturer has announced the introduction of an alloanti-a and alloanti-b immune-affinity chromatography step into the manufacturing process ( ) . preliminary results indicate depletion in anti-a and anti-b by > % in investigational lots. subsequently, we want to discuss possible consequences of (extensive) removal of antibodies reacting with histo-blood group antigens a and b. three facts have initiated our thinking about possible consequences of removal of histo-blood group a and b reacting www.frontiersin.org antibodies from igg concentrates. (i) in collaboration with hans u. lutz, formerly biochemistry eth zurich, we have observed the non-intended removal of natural anti-c autoantibodies regulating complement activation by large-scale immune-affinity adsorption of iga from an igg concentrate ( ) . anti-c antibodies belong to the family of "nabs" and have a particular role in homeostasis: they control activation of complement, among others, in the frame of nab-mediated opsono-phagocytosis of altered or senescent cells, including rbcs ( ) ( ) ( ) . thus, the intention to target one particular antibody by affinity chromatography might reduce that antibody specificity but at the same time affect other specificities as well. (ii) it should be kept in mind that the blood groups a and b are in fact "histo-blood group" antigens, i.e., they are also found on white blood cells, t lymphocytes, and proteins and also can be found in soluble form ( ) . alloantibodies reacting with histo-blood group antigens a and b thus have much broader tissue recognition than rbcs only. in addition, alloanti-a and alloanti-b belong to the population of nabs recognizing non-self and most likely participate in primary host defense ( ) . (iii) in contrast to cold-ethanol fractionation, where low titers of alloanti-a and alloanti-b are achieved on basis of their isoelectric points (ieps), the (extensive) immune-affinity removal might affect a much wider iep range, thereby removing broadly reacting antibodies and impairing some desirable functions of the igg concentrate. thus, the struggle for staying on the sunny side of the moon might have consequences for the antibody repertoire in an igg concentrate. antibodies reacting with terminal di-, tri-, and tetrasaccharides belong to the large family of human anti-glycan nabs. histo-blood group a and b epitopes in terminal position are tetrasaccharides. alloantibodies to these tetra-saccharides are found in the plasma of healthy individuals depending on the blood group they have. a considerable body of research into the nature of these nabs has been performed so far, all using for isolation the corresponding terminal di-or tri-saccharides ( ) ( ) ( ) . recently, the repertoire and epitope specificity of such immunoglobulins was addressed in depth by including the tetra-saccharide as well ( , ) . it proved that serum of healthy individuals contain respectable amounts of di-or tri-saccharide-reacting nabs. these nabs proved to be pseudo-anti-a and pseudo-anti-b nabs as they are not reacting with the tetra-saccharide of histo-blood groups a and b. in contrast, alloanti-a and -b antibodies able to react with tetra-saccharides are reacting with the corresponding terminal di-and tri-saccharides. reasoning about the biological role of these "high-titer and population conservative" anti-di-and anti-tri-saccharide nabs and the consequence of their potential removal by immunoaffinity is outlined below. a population of the anti-glycan nabs are the anti-αgal nabs which recognize galα - gal and galα - (fucα - )gal epitopes. anti-αgal nabs have been described being xenoreactive, recognizing bacterial galα - gal ( ) and having tissue homeostatic function. the daily removal of altered/senescent cells of the body is~ . removal is mainly mediated by apoptosis (no inflammation, no necrosis). rbcs, when they do not encounter a pathological condition, over their life span of - days remain intact although they shrink, do not undergo apoptosis but progressively become senescent, mainly due to cumulative oxidative stress. removal of intact rbcs with a daily turnover of~ × , corresponding to~ g cell mass, is effectuated by increased exposure of otherwise cryptic structures such as spectrin, band , or αgal epitopes. these exposed structures are recognized by low affinity, high avidity, c -bearing nabs, which promote the efficient removal of intact senescent rbcs ( , , ) . immunoaffinity adsorption by tri-saccharides columns of di-and tri-saccharide reacting nabs from igg concentrates can eliminate anti-histoblood a and b alloantibodies while it also eliminates αgal and this might have a janus effect. the face directed to the sun tells that adsorbing αgal nabs reacting with altered and senescent self on rbc might prevent an increase in the igg load of rbcs over the threshold level of relevant hemolysis in individuals at risk. the face directed to the dark indicate that adsorption of tissue homeostatic antibodies might deprive an igg concentrate of potentially beneficial antibodies. although they are nabs, tri-saccharide reacting antibodies can be induced by feeding bacteria bearing the corresponding carbohydrate epitopes ( ) . these inducible nabs are considered to participate in primary host defense. other antibodies possibly involved in primary host defense are the anti-αgal nabs. they show a broad specificity and can react with a number of related αgal-terminated oligosaccharides, including those on bacteria ( ) . thus, the immunoadsorption of di-and trisaccharide reacting nabs might diminish the potential of an igg concentrate to mediate primary host defense. therefore, when choosing affinity resins for immunoadsorption, there might be some aspects worth to consider. in summary, the principles of avoiding co-fractionation through cold-ethanol fractionation ( ) versus immune-affinity removal of histo-blood group alloantibodies can have an impact on the presence of homeostatic and first-line defense antibodies. according to present knowledge, only resins coated with the corresponding tetra-saccharides can ascertain the selective removal of histo-blood group alloantibodies presumably involved in ha. resins coated with the corresponding di-and tri-saccharides also remove blood group alloantibodies, however not selectively. such resins in addition might remove a broad range of nabs present in igg concentrates at relative high amounts. in the literature, the use of tri-saccharide-coated resin was reported ( ) ( ) ( ) . we have found no information available in the public domain indicating which type of resin is/will be used for reduction of the histo-blood group alloantibodies in large-scale fractionation of igg. furthermore, we suggest that the effect of reduction of anti-a and ant-b reacting antibodies by immune-affinity on the antibody repertoire of igg concentrates can only be assessed by, e.g., using pathogens/commensals, which share the saccharide epitopes, that have been used to coat the affinity resins or alternatively by exposing senescent rbcs stripped off the iggs coated in vivo. finally, techniques are required, which allow detection of low affinity, high avidity nabs. ivig administration-related aes, including thrombosis, have been extensively described ( ) . thrombotic aes are severe aes and patients with risk factors require a special care. reported average incidence of ivig-induced thrombosis ranges from to % ( ) . recognized risk factors for ivig-induced thrombosis frontiers in immunology | primary immunodeficiencies include male gender; age > ; diabetes; renal insufficiency, dyslipidemia; hypertension; immobility; coronary disease; pre-existing vascular disease, family history of early thromboembolic disease; atrial fibrillation, high-dose and high-speed ivig infusions. ivig-induced thrombosis is reported both as venous events such as thrombosis stroke, pulmonary embolism (pe), deep venous thrombosis (dvt), and arterial ischemia events such as myocardial infarct and stroke. the mechanisms leading to ivigassociated thrombosis are still not completely clear; three main mechanisms have been proposed, emphasizing the role of an increased blood viscosity causing a hypercoagulable state ( ) , the role of anticardiolipin antibodies passively transferred through ivig ( ) , and the role of factor xia or other biologically highly active factors passively transferred via igg concentrates, such as pka. avoiding activated coagulation factors in igg concentrates starts with appropriate anticoagulation of donated blood/plasma, i.e., careful mixing of anticoagulant and sample over the whole donation process. alterations in an established manufacturing process neglecting appropriate controls can also lead to increased risk of transmission of activated coagulation factors. high mw proteins passively transferred by ivig are probably contributing to this phenomenon ( ) . in patients with other risk factors, such as vascular disease, the increase in blood viscosity can precipitate thromboembolic events. as elderly individuals are prone for such aes, we like to point to the possibility of elevated altered/senescent self-reacting with infused homeostatic nabs being a possible factor facilitating thrombotic events as well. a relationship between ivig administration and cerebral vasospasm has also been suggested by sztajzel et al. ( ) ; blood viscosity is a determinant for oxygen delivery to the tissues, and changes in viscosity can lead to a reduction in cerebral or myocardial perfusion. we systematically reviewed case reports related to iviginduced thrombosis from to (figure ) . literature search identified articles containing reports concerning patients ( , , , . when data were available, diagnosis, risk factors, the number of ivig infusion prior to thrombosis event, and outcome have been indicated. baseline characteristics of the patients are shown in table . high-dose ivig induced thromboembolic events in patients at low to medium ivig doses. marie et al. ( ) observed that the frequency and type of arterial events was inversely related to the time elapsed from ivig infusion; almost % ( versus reports) of arterial ischemic events occurred within h following ivig, while about % of venous thrombosis occurred after more than h. no correlation between number of infusions and occurrence of ae was observed. the main risk factors observed in this review were hypertension ( cases, % of prevalence), previous vascular disease ( %), and dyslipidemia ( %). average mortality for thrombotic events was % (arterial ischemia % versus venous thrombosis %, pe representing the main venous fatal event). predicting iviginduced thrombosis is difficult. risk factors should be assessed for each patient including instrumental exams when needed. doppler ultrasound can be useful as early diagnostic tool for thrombosis or to detect the presence of abnormal blood flow especially after prolonged immobility. ivig should be administered at low ir to reduce the risk. the administration of antiplatelet or anticoagulant prophylaxis was suggested in patients with several risk factors ( ) . however, thrombotic events have been reported even after several previous uncomplicated courses of treatment. www.frontiersin.org ( ), arrhythmia ( ) first ( ), several ( ) arterial ( ), venous ( ) days recovery ( ), death ( ) al-riyami et al. ( ) second ( ), third ( ), several ( ) arterial ( ), venous ( ) h ( ), days ( ), weeks ( ) recovery ( ) stamboulis et al. ( ) several ( ) arterial ( ) h ( ), days ( ) death ( ) clinical and immunological characteristics of patients described in case reports. numbers in parenthesis indicate the number of patients with the given condition. in such cases, patients should be examined for signs and symptoms of thrombosis during each courses of ivig. immunoglobulin g concentrates are widely acknowledged to offer a safe, high-dose, long-term therapy option for a variety of diseases. aes occur rarely and mainly are mild to moderate. deviations from this rule of thumb are addressed by authorities and the plasma fractionation industry to achieve corrections. above, we have reviewed two types of ae which have shown elevated frequency in the near past. we tried to give some insights which might help in reducing frequencies of aes bed side. authors are deeply grateful to hans-hartmut peter, freiburg, germany, for his careful reading of the manuscript, the valuable comments, and the correction of english. epidemic 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red cell antibody autoimmune hemolytic anemia in kawasaki disease: a case report haemolysis induced by intravenously-administered immunoglobulin massive intravascular hemolysis associated with intravenous immunoglobulin in bone marrow transplant recipients hemolytic anemia following intravenous gamma globulin administration hemolysis following intravenous immune globulin therapy transient haemoglobin drop following high dose intravenous immunoglobulin in vivo administration of intravenous immunoglobulin (ivig) can lead to enhanced erythrocyte sequestration hemolysis in patients with antibody deficiencies on immunoglobulin replacement treatment batches of intravenous immunoglobulin associated with adverse reactions in recipients contain atypically high anti-rh d activity hematologic toxicities associated with intravenous immunoglobulin therapy anti-a and anti-b activity in batches of different intravenous immunoglobulin products determined using a direct haemagglutination method international collaborative study to evaluate candidate reference reagents to standardize haemagglutination testing for anti-a and anti-b in normal intravenous immunoglobulin products european directorate for the quality of medicines and healthcare. anti-a and anti-b haemagglutinins european directorate for the quality of medicines and healthcare. test for anti-d antibodies in human immunoglobulin immune complex-like moieties in immunoglobulin for intravenous use (i.v.ig) bind complement and enhance phagocytosis of human erythrocytes regulation of primary alloantibody response through antecedent exposure to a microbial tcell epitope a follow-up study of adult patients with idiopathic thrombocytopenic purpura treated with high-dose immunoglobulins and anti-d immunoglobulins hemolysis upon intravenous immunoglobulin transfusion report of the fda meeting on strategies to address hemolytic complications of immune globulin infusions kreuth ig working group. european consensus proposal for immunoglobulin therapies the art of balanced production in vitro and in vivo properties differ among liquid intravenous immunoglobulin preparations patient safety through an ivig mastered manufacturing process. posters isoagglutinin reduction in human immunoglobulin products by donor screening igg product development isoagglutinin reduction measures. oral presentation naturally occurring antibodies/autoantibodies in polyclonal immunoglobulin concentrates. lutz, h. u. naturally occurring antibodies (nabs) naturally occurring anti-band- antibodies and complement together mediate phagocytosis of oxidatively stressed human erythrocytes high doses of immunoglobulin g attenuate immune aggregate-mediated complement activation by enhancing physiologic cleavage of c b in c bn-igg complexes intravenously applied igg stimulates complement attenuation in a complementdependent autoimmune disease at the amplifying c convertase level histoblood group antigens as allo-and autoantigens blood group isoantibody stimulation in man by feeding blood group-active bacteria normal human serum contains natural antibodies reactive with autologous ab blood group antigens a unique natural human igg antibody with anti-alpha-galactosyl specificity normal human serum contains high levels of anti-galα - glcnac antibodies natural anti-a and anti-b of the ab system: allo-and autoantibodies have different epitope specificity repertoire of human natural anti-glycan immunoglobulins. do we have autoantibodies? interaction between human natural anti-alpha-galactosyl immunoglobulin g and bacteria of the human flora innate immune and non-immune mediators of erythrocyte clearance comparison of serum anti-band and anti-gal antibody binding to density-separated human red blood cells specificity of human antibodies against galα - gal carbohydrate epitope and distinction from natural antibodies reacting with galα - gal or galα - gal anti-a and anti-b haemagglutinin trend analysis during manufacturing process 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events associated with intravenous immunoglobulin infusion in antibody-deficient patients transverse sinus thrombosis and ivig treatment: a case report and discussion of risk-benefit assessment for immunoglobulin treatment thrombotic complications after intravenous immunoglobulin therapy in two patients pulmonary embolism after intravenous immunoglobulin adverse effects of intravenous immunoglobulin therapy in patients with autoimmune diseases acute myocardial infarction associated with high dose intravenous immunoglobulin infusion for autoimmune disorders. a study of four cases deep venous thrombosis of the arm after intravenous immunoglobulin infusion: case report and literature review of intravenous immunoglobulin-related thrombotic complications cerebral infarction complicating intravenous immunoglobulin therapy in a patient with miller fisher syndrome central retinal vein occlusion complicating treatment with intravenous immunoglobulin acute myocardial infarction during treatment with intravenous immunoglobulin for idiopathic thrombocytopenic purpura (itp) myocardial infarction as a complication of immunoglobulin therapy iatrogenic central retinal vein occlusion and hyperviscosity associated with high-dose intravenous immunoglobulin administration the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord- -ousbar c authors: horman, william s. j.; nguyen, thi h. o.; kedzierska, katherine; butler, jeffrey; shan, songhua; layton, rachel; bingham, john; payne, jean; bean, andrew g. d.; layton, daniel s. title: the dynamics of the ferret immune response during h n influenza virus infection date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: ousbar c as the recent outbreak of sars-cov- has highlighted, the threat of a pandemic event from zoonotic viruses, such as the deadly influenza a/h n virus subtype, continues to be a major global health concern. h n virus strains appear to exhibit greater disease severity in mammalian hosts compared to natural avian hosts, though the exact mechanisms underlying this are somewhat unclear. knowledge of the h n host-pathogen interactions have mainly been constrained to natural sporadic human infections. to elucidate the cellular immune mechanisms associated with disease severity and progression, we used a ferret model to closely resemble disease outcomes in humans following influenza virus infection. intriguingly, we observed variable disease outcomes when ferrets were inoculated with the a/anhui/ / (h n ) strain. we observed relatively reduced antigen-presenting cell activation in lymphoid tissues which may be correlative with increased disease severity. additionally, depletions in cd (+) t cells were not apparent in sick animals. this study provides further insight into the ways that lymphocytes maturate and traffic in response to h n infection in the ferret model. in recent years cases of zoonotic strains of avian influenza (ai) causing severe disease in humans have caused significant global concern, with fears that these viruses may lead to devastating pandemic events in future ( , ) . one such group of viruses are strains of h n influenza virus, which have caused over , cases of infection (with a ∼ % mortality rate) in humans since their first detection in ( , ) . of most concern with the h n viruses has been the ability for this virus to present as a low pathogenicity virus in its native avian hosts and yet present with severe clinical symptoms and death in humans, without obtaining virulence factors such as multi-basic cleavage sites usually required for high pathogenicity infections. deciphering the immune cellular mechanisms associated with disease severity progression will provide a better understanding as to why h n infections can produce severe disease in humans. changes in leukocyte subsets have previously been shown to correlate with more severe outcomes during ai infection, with decreases in t cell populations commonly reported in both human (h n virus) and avian (h n virus) hosts ( , ) . decreases in t lymphocytes are often accompanied by upregulation of several pro-inflammatory cytokines such as interferons (ifn), most notably ifn-γ, as well as interleukins (il) such as il- ( ). overproduction of cytokines or hypercytokinemia has also been identified as a key contributing factor in severe ai pathogenesis in both chickens and macaques, where induction of pro-inflammatory cytokines is associated with cellular apoptosis and tissue damage ( , ) . furthermore, macrophages have been shown to be predominantly pro-inflammatory responders to h n strains in a mouse model ( ) , though highly pathogenic strains such as h n and h n display attenuated macrophage inflammation responses compared to seasonal strains such as h n and h n ( , ) . while hypercytokinemia is common amongst many ai virus infections, h n strains such as the human infecting a/anhui/ / virus have been associated with dampened ifn responses in humans ( , ) . furthermore, they have previously been associated with an attenuated humoral immune response in the mouse model ( ) . these studies demonstrate the unpredictable nature and wide spectrum of pathogenicity of ai viruses. investigations into the pathogenesis and transmission of many human-infecting influenza viruses have been conducted in ferrets, as the clinical presentation in these animals is considered a more robust representation of human illness when compared to mice ( ) . however, only a limited number of studies exist looking at influenza-specific immunity in ferrets, which have primarily focused on seasonal influenza strains ( ) ( ) ( ) . in this study, we aimed to examine the ferret immune response to h n influenza virus infection by analyzing leukocyte population variation associated with disease pathogenesis. this study found the ferret model may allow for increased knowledge of the outcomes of h n infections and help in boosting our understanding of both this model and of these viruses in readiness for potential future outbreaks. all procedures described here were reviewed and approved by the commonwealth scientific and industrial research organization (csiro), australian center for disease prevention (acdp) animal ethics committee (aec# ) and were performed in accordance with the australian code for the care and use of animals for scientific research ( th edition ). influenza virus a/anhui/ / (h n ) used in this study was propagated by allantoic cavity inoculation of - -days of embryogenesis specific-pathogen-free (spf) embryonated chicken eggs. the virus stock was titrated in chicken eggs and the % egg infectious dose (eid ) /ml was calculated according to reed and muench ( ) . all in vitro and in vivo work involving live virus was conducted within biosafety level three facilities at acdp. animal work was performed using personal protective equipment and powered air purifying respirators. fitch ferrets that were ∼ months of age (csiro werribee animal facility) and serologically negative by hemagglutination inhibition (hi) assay for h n were used in this study. prior to initiation of the study, all ferrets were free from signs of clinical disease. body temperatures were measured using an implantable subcutaneous microchip (destron fearing, delray beach, fl, usa). baseline body weights and temperatures were obtained for consecutive days before challenge (i.e., day − , − , and − ) and on the day just prior to the challenge (day ). six ferrets were infected intranasally with × eid of virus diluted in . ml of sterile pbs, and four ferrets were mockinfected with an equivalent dilution of allantoic fluid collected from spf chicken eggs in sterile pbs as non-infected controls. following viral challenge, ferrets were monitored daily for body weight, temperature, and clinical signs of illness (including sneezing, lethargy, nasal discharge, diarrhea, and neurological dysfunction) for the duration of the study. blood samples were collected every second day. animals were anesthetized with ketamine/xylazine, and blood samples of - µl were taken from the jugular or axillary vein on days , , , and from the heart at the time of euthanasia for the terminal bleed. for virus titration, nasal washes were collected on days , , , and upon euthanasia, as described previously ( , ) . all ferrets were euthanised on day (study endpoint) or earlier due to ethical endpoints (≥ % weight loss or escalation of clinical signs). histological analysis of ferret tissues following infection was performed as previously described ( ) . tissues were fixed in % neutral-buffered formalin for at least h, processed into paraffin wax, cut and stained using haematoxylin and eosin for examination for histopathological lesions. consecutive tissue sections were stained in an immunohistochemistry (ihc) test for influenza a virus nucleoprotein ( ) . lung, spleen, and mediastinal lymph nodes were harvested and processed, as previously described ( ) . briefly, lung samples were manually minced using a scalpel followed by enzymatic digestion ( u/ml collagenase i and u/ml dnase i) while single-cell suspensions of spleen and lymph node samples were prepared by passing the tissue though a µm strainer. peripheral blood mononuclear cells were isolated by hypotonic lysis of red blood cells using erythrocyte lysing solution ( . m nh cl, mm khco , and mm edta ph . ). viral titers in tissues were measured on mdck cells by standard tcid assay. relative expression of ferret immune genes was assessed using a steponeplus tm real-time pcr system and the comparative threshold cycle (ct) method according to manufacturer's instructions (applied biosystems, foster city, ca, usa). relative gene expression was calculated using mean values obtained from ct relative to the housekeeper gene (gapdh), with each ferret compared to the average of the control ferrets for each gene. primers for ferret cytokines, as well as relative gene expression calculations, were obtained from carolan et al. ( ) . cells processed from ferret tissues were stained with anti-cd (alexafluor , csiro acdp sourced from wehi ( ) , geelong, vic, australia), anti-cd (pe, clone okt , ebioscience, ca, usa), anti-gl (alexafluor , clone gl , bd phamingen, san diego, ca, usa), anti-mhc-ii (biotin, clone cat a, kingfisher, saint paul, mn, usa), and anti-cd b (alexafluor , clone m / , bd pharmingen, san diego, ca, usa). cells were stained for h at • c, washed in facs buffer (pbs, % fcs, . % sodium azide) and analyzed using the lsr ii (becton-dickinson, franklin lakes, nj, usa). flow cytometry data were analyzed using flowlogic software (version . . , inivai technologies, mentone, vic, australia). to assess antibody responses, serum was collected prior to infection and at the point of euthanasia. haemagglutinin inhibition assays were performed on rde treated sera by using homologous antigen (influenza a/anhui/ / ) as per the standard method. hi titers were expressed as the reciprocal of the highest serum dilution causing complete inhibition of hemagglutination. ferret lung and spleen cells were pelleted by centrifugation at , × g max for min and washed in pbs. cells ( . × / well) were cultured with or without live h n virus for h at • c/ % co . the ferret ifn-γ elisa development kit (alp) (mabtech, stockholm, sweden) was used to determine the quantity of ifnγ secreted by cells ex vivo at endpoint and after restimulation with live h n virus (moi . ). elisa plates were measured using a multiskan ascent plate reader with ascent software version . (thermofisher, waltham, ma, usa). ifn-γ-producing cells were detected using a ferret ifn-γ elispot assay, as per the manufacturer's instructions (mabtech, stockholm, sweden). for analyzing the infected ferrets compared to the non-infected controls, all six ferrets were grouped regardless of timepoint to give an overall view of the infection time course. for these analyses, student t-tests were conducted between the two groups. time course data was analyzed by -way anova. to assess the impact of h n virus in ferrets, viral pathogenicity was firstly assessed based on observation of clinical signs of infected ferrets throughout the -day study (figure ) . from the six h n -infected ferrets, three ferrets showed little to no clinical signs and survived until the study endpoint (day ). of the remaining ferrets, two showed mild clinical signs but severe weight loss, leading to euthanasia at day post-infection. one ferret, however, showed moderate to severe clinical signs consistent with those seen in highly pathogenic avian influenza infections ( ) , and was euthanised at day due to escalation of these clinical signs ( figure a ). this ferret's increase in clinical signs was identified by a play score of two, with play scores > signifying moderate-to-severe clinical signs ( figure b ). significant weight losses (> % compared to baseline, p < . ) were observed in all the infected ferrets when compared to the control ferrets from day until the study endpoint ( figure c) . similarly, infected ferrets also showed a significant increase (p < . ) in body temperature at day post-infection, with an average temperature of . ± . • c compared to the controls at . ± . • c ( figure d) . consequently, h n infection in ferrets resulted in variable clinical symptoms, but overall body weight losses and heightened body temperatures at h postinfection, which are within the typical timeframe for onset of influenza illness in humans. to further examine the clinical progression between the infected ferrets, viral titres from the nasal washes were determined to assess whether differences in viral replication or clearance were correlative with worsened disease progression. titres > . log tcid /ml were obtained for all infected ferrets at day postinfection and virus was still detected in all ferrets at day postinfection (> . log tcid /ml, figure a ). one ferret had cleared the virus by day post-infection, while the other two ferrets that survived until the end of the study showed viral clearance at day (figure a ). neither the day nor the day ferrets showed viral clearance by the point of euthanasia. only one ferret euthanised on day showed live virus in the lung (> . log /ml, figure b ). additionally, antibody titres were measured in the ferret sera, with animals euthanized on day showing the highest hi titres (all > ) and the ferret euthanized on day showed a hi titer > . the two ferrets euthanised at day had no detectable hi titer ( figure c ). infected ferrets showed a variety of pathological outcomes following viral challenge. challenged ferrets exhibited epithelial metaplasia in the nasal turbinates (figure a) , with ferrets euthanised at the earlier day time point exhibiting viral infection of the nasal epithelium (figure b) . the day ferret showed the most severe lung pathology, with the lungs of this ferret showing diffuse interstitial pneumonia, with severe alveolar oedema and inflammatory cell infiltration in the alveolar spaces and around the blood vessels (figure c) . other infected ferrets showed broncho-interstitial pneumonia and interstitial inflammation (figure d ) and bronchitis, with infected epithelial cells and early stage lesions observed in one of the day ferrets (figure e) . bronchial adenitis was also present in some of the infected ferrets, with necrosis of the bronchial glands observed along with viral antigen (figure f) . these changes in pathology contrast to what is seen in the healthy nasal turbinates (figure g) , and lung (figure h ) of uninfected ferrets where no lesions were present. the number of localized lesions and qualitative assessment of epithelial metaplasia was also recorded to support the histopathological findings (supplementary figure ) . here, we observed epithelial metaplasia in the turbinates of all ferrets sampled, as well as several occurrences of bronchio-interstitial pneumonia and bronchio-adenitis. we also observed viral antigen in a number of tissues sampled (supplementary figure ) , though interestingly, viral antigen was not observed in the diffuse interstitial pneumonia of lung lesions associated with the ferret euthanized due to clinical disease. changes in certain pro-inflammatory cytokines (including il- , tnfα, and ifnγ) have been associated with h n disease severity in humans and animal models ( ). we measured levels of mrna transcripts of several pro-inflammatory cytokines commonly associated with influenza infections. in the blood there were few differences between the groups, with mcp the only tested cytokine showing an average fold increase > fold compared to the controls at day post-infection (data not shown). however, intriguingly the ferret euthanised on day showed a large decrease in il- ( figure a , -fold) and ifn-γ (figure b , -fold) transcript levels on day . this also coincided with a lack of detectible ifn-γ in terminal serum by elisa, though this trend was consistent across all tested ferrets (data not shown). in the spleen at endpoint, there appeared to be variable outcomes for the day ferrets, with somewhat increased pro-inflammatory cytokine levels such as type i ifns (ifn-α and ifn-β) in the day ferrets, and decreased levels in the day ferret compared to the controls (figure c) . in the lung, tnfα was significantly reduced (figure d , p < . ) in the infected animals, though no other consistent trends were seen. the ability to produce ifn-γ ex vivo or after restimulation with live h n virus was also assessed in spleen ( figure e ) and lung cells (figure f ). there were no significant differences except for in the infected lungs, where ifn-γ-producing cells were significantly higher compared to the control ferrets ex vivo (p = . ). based on these findings, our ferret infection model showed observable changes in the cytokine profiles at a key site of infection in the lung compared to non-infected controls. depletions in t cell numbers in the spleen and lungs have previously been reported for highly pathogenic avian strains h n and h n in both chickens and mice ( , ) . similarly, patients that died from h n infection were found to have lower t cell numbers in the blood compared to those that survived ( ) . therefore, in order to assess lymphocyte involvement in disease progression we investigated the cellular responses associated with h n -infection both in the blood at several timepoints, and in the tissues at endpoint. here, we found that in the blood the numbers of cd + and cd + t cells in infected ferrets were generally lower than the control ferrets, particularly for the cd + t cell population at day (p < . ). nevertheless, these t cell populations remained low but relatively stable over the days of infection ( figure a) . the gl monoclonal antibody can be used as a marker activated t lymphocytes. however, gl expression was absent on the t cell subsets examined. interestingly we observed significantly higher numbers of cd + t cells in the spleen (p = . ) and in the lungs (p = . ) of infected ferrets, and a trend toward higher numbers of cd + t cells in the lungs (figures b,c) , indicating a t cell response to h n infection at a key site of infection. antigen presenting cells (apcs), such as macrophages residing in the lungs, have previously been recognized as key proinflammatory responders during h n , h n , and h n infections in mice ( ) . cells expressing higher levels of cd b + have been shown to directly kill virally-infected cells in humans, and act as important antigen-presenting cells by upregulating mhc class ii expression in the ferret model correlating to reduced pathology associated with influenza infection ( , ) . in general, h n -infected ferrets showed higher levels of cd b/mhc-ii dual-expressing cells (cd b + mhc-ii + ) in the spleen, lung and lymph nodes compared to the control ferrets (figures a-c) . at the site of infection in the lungs, infected ferrets showed significantly higher levels of mhc-ii single-positive cells (mhc-ii + , p = . ). conversely, cd b single-positive cells (cd b + ) from infected ferrets were found at elevated levels in the spleen and lymph nodes. individually, we did observe that the ferrets euthanised on days and had higher cd b + cells in the spleen, suggesting a less activated and immature phenotype, and a trend toward lower cd b + mhc-ii + cells in the lymph nodes when compared to the other ferrets. when analyzed by time point, there are statistically greater proportions of these cells in the ferrets that survived until trial endpoint compared to the controls (p = . ) and the earlier timepoint ferrets (p = . , figure d ). as such, as these markers are often attributed to antigen-presenting cell maturation in the mouse model ( ) , apc activation in response to h n infection may be associated with disease severity seen in the ferret model. the recent sars-cov- outbreak has brought a sharp focus onto zoonotic viruses causing severe global pandemics. while sustained human-to-human transmission of avian-origin h n viruses has yet to occur, if a zoonotic outbreak of these highconsequence viruses were to occur, the results could be a pandemic much like the sars-cov- outbreak but with a virus which has potential for much higher mortality rate. as such, avian influenza viruses continue to present as a major global health concern, with one of the key concerns being what makes these viruses cause such severe consequences in mammalian hosts. influenza-related symptoms of ferrets more closely resemble human clinical symptoms than that of mice ( , ) , with outbred ferret populations giving a more accurate representation of the genetic variability seen in human populations compared to clonally inbred mouse colonies. however, husbandry requirements and a general lack of reagents for ferrets limits the scope of experimental research using these animals to date. these issues are exacerbated by biosafety level (bsl ) requirements for pathogens such as h n , which make immunological experiments in ferrets difficult to undertake. as a result, ferret cellular immunology is still a developing field of research in the context of influenza viruses, with only a handful of studies investigating changes in leukocyte populations following influenza virus infection ( ) ( ) ( ) . for avian-origin influenza viruses such as h n , little work has been done to investigate how cellular subsets are affected by viral infection, with the bulk of experiments using ai in the ferret model focusing on classifying viruses through clinical outcome-based pathogenesis and transmission studies ( ) ( ) ( ) . thus, our study aimed to give new insight into how h n affects mammalian hosts in the ferret model with a focus on cellular subsets. the ferrets in our viral challenge presented clinical outcomes with different severities. five of the six ferrets showed little to no clinical signs, nevertheless only three survived until study endpoint without complications. two ferrets reached the ethical weight loss cut-off of ≥ % by day post-infection, giving us two different time points to assess the immune response. furthermore, one ferret showed a quite different disease outcome, in which the ferret presented an escalation of clinical signs and was euthanised for ethical reasons at day post-infection. other studies have previously shown clinical variation between strains of h n ( ) , however most studies with a /anhui/ / (h n ) show ferrets presenting only mild clinical signs ( , , ) . to our knowledge, this is the first reported result showing variable disease outcome in an animal study using a single low pathogenicity h n strain, in which one ferret showed severe clinical outcomes. while the findings of this study are novel for this model, we were limited by the number of animals that could be used in this study and thus limited by our statistical power. furthermore, there are limitations in directly comparing ferrets euthanised on different days as, particularly for our serological data, it is difficult to discern whether the results are a function of observed severity or differences in the sampling time. as such, we believe the results from our observational study can provide a rationale for developing future ferret studies with greater numbers. it is also worth noting that ferrets used for these experiments are outbred animals, which may suggest that other combinations of host factors may be contributing to the variability seen in clinical presentation. the airway pathology in these infected ferrets was variable, with different degrees of severity observed in the six animals. these findings were generally consistent with pathology observed in other studies in which ferrets were infected with lpai h n or pdm (h n ) viruses ( , , , ( ) ( ) ( ) . while the day ferret which showed worsened disease progression did exhibit the most severe lung pathology, the lack of viral antigen in and around these lesions (supplementary figure ) means they cannot conclusively be classified as being caused by the influenza infection, and thus we are hesitant to classify disease presentation in this ferret as like a high pathogenicity infection based on the histopathology findings regardless of the increase in clinical signs. hypercytokinemia has frequently been associated with worsened disease progression in cases of severe ai infections, therefore we aimed to assess the induction of pro-inflammatory cytokines in this ferret model of infection. upregulation of proinflammatory cytokines such as tnf-α and il- commonly observed following h n infection in both cell culture ( , ) and in severe human cases, which follow similar patterns of pathogenesis to infections with hpai h n viruses ( , , ) . whilst the localized pro-inflammatory cytokine response to influenza infection has been characterized for circulating influenza strains in ferrets ( ) , examination into these responses for avian influenza viruses remains limited. our study found limited induction of cytokine responses at the site of infection in the lungs and in lymphoid tissues, and while attenuated ifn responses have been previously reported in human bronchial epithelial cells infected with h n ( ), and in ferrets severely infected with seasonal influenza strains ( ) , the overall lack of cytokine induction was an unexpected finding. a limitation of this study was that these tissues could only be sampled at study endpoint, which may suggest the pro-inflammatory response had abated at the site of infection by the time of sampling given that these responses typically occur in short timeframes of - h post-infection ( ) , though a lack of an evident cytokine response in the day ferret still presenting clinical signs and shedding live virus was surprising. furthermore, levels of ifn-γ were below the threshold of detection by elisa in the serum of infected ferrets, and a previous study has shown ifn-γ detectable in the lungs of seasonal influenza-infected ferrets from day post-infection at low levels, with greater detection observed from days - ( ) . these results suggest both timing and sampling may be critical for future studies to best capture the overall ifn-γ response to h n , if there is such a response occurring. sampling in the blood of infected ferrets over the course of the study also found no large-scale upregulation of the cytokines tested, with only the early innate cytokine mcp showing an average fold increase > fold compared to the controls at day post-infection. however, in the day ferret, decreases in both ifn-γ ( -fold) and il- ( -fold) where observed at day post-infection, coinciding with an escalation in clinical signs in this animal. the decrease in il- in particular was unexpected, as previous studies have implicated higher levels of il- correlating to worsened disease progression ( ) . our findings here suggest an immune dysregulation, rather than an over-activation, for worsened disease progression, and may be attributed to other factors in the cellular response. immunological work to determine the innate and adaptive responses to these viruses has mostly been conducted from infected patients or in the mouse model. human cases are often marked by leukopenia, though these measurements are routinely made via hematology rather than flow cytometric analysis ( , ) . in other species however, we have previously described the lymphopenia for highly pathogenic strains of avian influenza, measuring splenic cd + t cells following infection with highly pathogenic h n in chickens via facs analysis ( ) . in this study, cd + t cells levels were relatively stable. a loss of lymphocytes detected via hematological analysis at day (data not shown) was reconciled with our facs data showing a significant difference between cd + t cells in the blood, in which control animals had much higher levels compared to the infected animals. however, this loss was short-lived, as no further significant differences were observed across the study time points in the blood. this data is suggestive of a potential early "transient lymphopenia" of circulating leukocytes, which has previously been identified in one study during influenza infection in ferrets, and is also a phenomenon seen in humans following influenza a infection ( , ) . our data appears to show cd + t cells levels in the blood of infected animals remaining relatively and consistently low across time points, whilst greater variation was observed in the uninfected control levels. furthermore, reagents for facs analysis in the ferret are still relatively novel, often not well-characterized, and heavily reliant on cross-reactivity from other species. while cd + and cd + t cells have previously been examined in influenzainfected ferrets ( ) , little work has been conducted for innate cell subsets. myeloid-derived and antigen-presenting cells (apcs) have only been identified and analyzed in a handful of ferret studies, with one such study speculating that cd b + cells are likely granulocytes such as neutrophils ( , ) . as such, we have used these studies, together with mouse and human studies ( ) ( ) ( ) ( ) , as a basis for possible classification of apcs, with cd b + mhc-ii + cells suggested as likely "mature" or "activated" apcs. however, further work is needed to classify these innate cell subsets in the ferret model. apcs such as alveolar macrophages have been recognized as key early responders against influenza virus infection, as they mount robust pro-inflammatory cytokine responses within h of infection ( ) . while our study found no significant differences in apc numbers in infected lungs compared to control numbers, there was a trend toward much greater levels of mhc-ii + apcs in the lung, especially in our cell counts. rather, it was in the peripheral tissues that we found variability in the apc subsets. cd b + mhc-ii + "mature" cells were found in the spleens of infected ferrets at significantly higher levels (p = . ), however the trend toward higher cd b + cells in the earlier time point ferrets suggests a higher level of immature myeloid cells, or the presence of pro-inflammatory granulocytes; however, the latter is less likely due given the lack of pro-inflammatory cytokines detected in the spleen. moreover, in the draining lymph node a significant increase in cd b + cells (p = . ) was observed. interestingly, in the lymph node the day seven ferrets showed noticeably higher levels of cd b + mhc-ii + "activated" cells, with statistically greater proportions of these cells compared to the controls (p = . ) and the earlier timepoint ferrets (p = . ), which may suggest that these apc responses do not occur as early as day . this study found increased pathology in the ferret model diverges from the commonly observed pathogenesis markers such as lymphopenia and hypercytokinemia, suggesting another varied pathway that h n viruses can cause severe disease in mammalian hosts. further investigation into the ferret model may allow for better characterization of these outcomes and assist in increasing our knowledge of these viruses in preparation for any potential pandemic events. all datasets generated for this study are included in the article/supplementary material. the animal study was reviewed and approved by australian animal health laboratories (aahl) animal ethics committee, csiro. characterization of h n influenza a viruses isolated from humans the drivers of pathology in zoonotic avian influenza: the interplay between 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associated with lethal outcome in mice highly pathogenic avian influenza viruses inhibit effective immune responses of human blood-derived macrophages infection with highly pathogenic h influenza viruses results in an attenuated proinflammatory cytokine and chemokine response early after infection human h n virus induces a more pronounced pro-inflammatory cytokine but an attenuated interferon response in human bronchial epithelial cells when compared with an epidemiologically-linked chicken h n virus suboptimal humoral immune response against influenza a(h n ) virus is related to its internal genes the ferret as a model organism to study influenza a virus infection peripheral leukocyte migration in ferrets in response to infection with seasonal influenza virus flow cytometric and cytokine elispot approaches to characterize the cellmediated immune response in ferrets following influenza virus infection cellular immune response to human influenza viruses differs between h n and h n subtypes in the ferret lung a simple method of estimating fifty per cent endpoints evidence for viral interference and cross-reactive protective immunity between influenza b virus lineages the pathobiology of two indonesian h n avian influenza viruses representing different clade . sublineages in chickens and ducks taqman real time rt-pcr assays for detecting ferret innate and adaptive immune responses development of an anti-ferret cd monoclonal antibody for the characterisation of ferret t lymphocytes predicting disease severity and viral spread of h n influenza virus in ferrets in the context of natural exposure routes lymphopenia associated with highly virulent h n virus infection due to plasmacytoid dendritic cell-mediated apoptosis of t cells innate immune response of human alveolar macrophages during influenza a infection differential response of respiratory dendritic cell subsets to influenza virus infection chapter -the ferret as an animal model of influenza virus infection pathogenesis of avian influenza a (h n ) viruses in ferrets novel avian-origin human influenza a (h n ) can be transmitted between ferrets via respiratory droplets infectivity, transmission, and pathology of human-isolated h n influenza virus in ferrets and pigs pathogenesis and transmission of avian influenza a (h n ) virus in ferrets and mice pathogenicity and transmissibility of north american triple reassortant swine influenza a viruses in ferrets severity of clinical disease and pathology in ferrets experimentally infected with influenza viruses is influenced by inoculum volume comparative pathology in ferrets infected with h n influenza a viruses isolated from different hosts pro-inflammatory cytokine dysregulation is associated with novel avian influenza a (h n ) virus in primary human macrophages cytokine and chemokine levels in patients infected with the novel avian influenza a (h n ) virus in china fatal outcome of human influenza a (h n ) is associated with high viral load and hypercytokinemia characterization of the localized immune response in the respiratory tract of ferrets following infection with influenza a and b viruses severe seasonal influenza in ferrets correlates with reduced interferon and increased il- induction timing and magnitude of type i interferon responses by distinct sensors impact cd t cell exhaustion and chronic viral infection human infections with the emerging avian influenza a h n virus from wet market poultry: clinical analysis and characterisation of viral genome comparison of patients hospitalized with influenza a subtypes h n , h n , and pandemic h n avian influenza virus a h n infects multiple mononuclear cell types in peripheral blood and induces dysregulated cytokine responses and apoptosis in infected monocytes identification of myeloid cell subsets in murine lungs using flow cytometry distinct macrophage subpopulations characterize acute infection and chronic inflammatory lung disease all authors agreed on the final draft of the manuscript prior to submission. wh wrote the first draft and revised the manuscript. tn, kk, jbu, ss, jbi, jp, ab, and dl reviewed and edited the manuscript. wh was supported by a joint csiro/university of melbourne onehealth scholarship. this project was supported by csiro strategic funding. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © horman, nguyen, kedzierska, butler, shan, layton, bingham, payne, bean and layton. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -tk vn v authors: brooks, wesley h. title: viral impact in autoimmune diseases: expanding the “x chromosome–nucleolus nexus” hypothesis date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: tk vn v viruses are suspected of significant roles in autoimmune diseases but the mechanisms are unclear. we get some insight by considering demands a virus places on host cells. viruses not only require production of their own proteins, rna and/or dna, but also production of additional cellular machinery, such as ribosomes, to handle the increased demands. since the nucleolus is a major site of rna processing and ribonucleoprotein assembly, nucleoli are targeted by viruses, directly when viral rna and proteins enter the nucleolus and indirectly when viruses induce increased expression of cellular polyamine genes. polyamines are at high levels in nucleoli to assist in rna folding. the size and activity of nucleoli increase directly with increases in polyamines. nucleolar expansion due to abnormal increases in polyamines could disrupt nearby chromatin, such as the inactive x chromosome, leading to expression of previously sequestered dna. sudden expression of a large concentration of alu elements from the disrupted inactive x can compete with rna transcripts containing intronic alu sequences that normally maintain nucleolar structural integrity. such disruption of nucleolar activity can lead to misfolded rnas, misassembled ribonucleoprotein complexes, and fragmentation of the nucleolus. many autoantigens in lupus are, at least transiently, components of the nucleolus. considering these effects of viruses, the “x chromosome–nucleolus nexus” hypothesis, which proposed disruption of the inactive x by the nucleolus during stress, is now expanded here to propose subsequent disruption of the nucleolus by previously sequestered alu elements, which can fragment the nucleolus, leading to generation of autoantigens. previously, we presented the "x chromosome-nucleolus nexus" hypothesis ( ) ( ) ( ) . in the hypothesis it was proposed that enlargement of the nucleolus in response to cellular stress could disrupt neighboring chromatin, such as the inactive x chromosome. as a result, sequestered alleles (e.g., polyamine genes on the inactive x), elements (e.g., alu elements), and viruses could be opened for transcription. this could lead to eventual creation of autoantigens due to overexpression of genes and elements from both the previously active chromatin and the, now, reactivated chromatin. here is presented new details to the hypothesis, explaining how the disrupted chromatin can lead to subsequent disruption of the nucleolus, even nucleolar fragmentation, which results in ineffective nucleolar functioning, misfolded rnas, misassembled or incompletely assembled ribonucleoprotein (rnp) complexes, and stabilization of nucleolar components in autoantigenic conformations. many of the major autoantigens in autoimmune diseases like systemic lupus erythematosus (sle) are, at least transiently, components of the nucleolus (e.g., splicosome subunits). among the factors that could cause extraordinary cellular stress, viruses are highly suspected of causing such disruption in autoimmune diseases. exposomics is the study of all environmental factors which a person may encounter during their lifetime, even including prenatal exposure. these environmental factors in the exposome can include components of the diet, gut microbiota, chemicals, air pollutants, heavy metals, and infectious agents. these factors can cause cellular stress and can have a cumulative effect in cells through accumulation of genetic damage and/or disruption of epigenetic control, especially in genetically predisposed individuals, that establishes the conditions for eventual manifestation and progression of an autoimmune disease. within the exposome is the infectome which is the collection of pathogens that may contribute to an individual's onset and progression of an autoimmune disease ( ) . this can be complicated by the latency of some pathogens and the synergistic effects of multiple pathogens. however, unless one is looking for pathogen antigens, the specific pathogen and its effect on the immune system may be masked by a larger response to more abundant autoantigens some of which the pathogen's antigens may mimic. in addition, it has been difficult to prove these associations since, in the case of viruses, many viruses can establish latent infections but the autoimmune disease may not manifest itself until several years after the initial infection, thus clouding the true extent of their association. for example, a mononucleosis infection, a.k.a. the "kissing disease, " which involves the epstein-barr virus (ebv), increases the risk for subsequent appearance of multiple sclerosis (ms) but manifestation of the ms might not occur until as long as years after the mononucleosis episode ( ) . add to this the fact that in the interim the individual has had other infections caused by other pathogens that complicate the situation, potentially triggering the actual autoimmune disease for which an initial ebv infection set the stage. a subsequent infection with another virus could allow activation of latent viruses giving a combined stressful impact on the cell. for example, a primary infection by cytomegalovirus can lead to reactivation of latent ebv which provokes an immune response ( ) . the induction of one latent virus by another virus shows the potential complexity underlying autoimmune diseases. the general population has had exposure to many of the viruses associated with autoimmune diseases but for most individuals there is no autoimmune disease development, suggesting that genetic susceptibility is also important as well as possible epigenetic and environmental factors. as an example, most adults have had exposure to ebv but few develop an autoimmune disease, suggesting other factors are involved rather than just ebv. a genetic possibility for these differing responses may be based on different hla types, for example, entry of ebv into a host cell via binding of the ebv's gp glycoprotein to human cd and lymphocytic antigen type hla-dr. other hla sub-types may have different expression levels or have different affinity for the gp and not be as compliant for ebv entry. in addition, the extracellular portion of the ebv bzlf protein can suppress antigen presentation by binding hla-dr delaying detection of the ebv ( ) . table lists many of the viruses that have shown associations with autoimmune diseases. we should note that there is variety in the route of transmission and entry among these viruses: ( ) respiratory and oral secretions (saliva, sputum, nasal mucus) (e.g., ebv, parvovirus); ( ) gut (e.g., enteroviruses); ( ) insect vector transmission (e.g., mosquitos for zika, west nile); ( ) sexual interactions (e.g., hpv, hiv); and ( ) transfusions (e.g., hiv). the tissue types in which viral sequestration occurs may vary, such as ebv behind the blood-brain barrier associated with ms or possible ebv in the synovium associated with ra. we should also note that there are both rna and dna viruses listed in table and most of these viruses can persist in a latent state in the host. appearance of viral antigens does not necessarily mean that the virus is the cause of the autoimmune disease episode since it may only be the result of stress from an autoimmune disease episode that leads to subsequent activation of a hidden virus. for most of these associations (table ) , it remains to be determined if the virus is the causative agent, one of several combined contributing agents, or simply appearing as a result of impaired host cell suppression of the virus. for example, the measles virus is suspected of involvement in ms due to the appearance of antibodies to measles virus antigens in cerebrospinal fluid of ms patients ( ) . whether the ms is a direct result of the measles virus or the appearance of measles antigens is a consequence of the ms or is simply coincidental is not known. there may, in fact, be another virus or another environmental agent that has disrupted the suppression of the latent measles virus. as it is, the situation is even more perplexing since the introduction of measles vaccination for the general population has not caused a significant change in the occurrence rate of ms ( ) . other viruses with an infrequent association with an autoimmune response can have reemergence in other forms, such as the varicella zoster virus which causes chicken pox and which can reemerge as shingles and is associated with ms ( ) . and we should bear in mind that human endogenous retroviruses are suspected of involvement in serious diseases, including autoimmune diseases ( , ) . among the viruses listed in table , ebv has received the most attention as a virus with links to autoimmune diseases ( , ) . an association with ebv infection has been observed in sle ( , ) , ms ( ) , ra, and sjögren's syndrome (sjs) ( ) and prior ebv infection, as indicated by sero-positivity for ebv antigens, is observed in . % of controls and . % of ms patients ( ) . in addition, ebv (human gammaherpesvirus , hhv- ) is representative of several herpes viruses that have shown associations with autoimmune diseases. therefore, based on current knowledge, ebv is most useful for describing possible viral involvement in autoimmune diseases in general. one way in which a viral infection could provoke an autoimmune reaction is by disrupting the host cell's epigenetic control during a particularly strong cellular stress response to the viral activity leading to expression of previously sequestered gene alleles. expression from those newly opened sites could lead to imbalance in the protein and rna products. the female predominance of many autoimmune diseases suggests that the x chromosome and possibly disruption of the inactive x chromosome, a major epigenetic structure in the cell, could be of significance in such a scenario of viral disruption of epigenetic control ( , ) . one point of concern is fragile sites, which are particularly susceptible to viral insertion and dna breaks. fragile sites can be hundreds of thousands, even millions of base pairs in length. the x chromosome has four major fragile sites (fraxa at xq ; fraxb at xp ; fraxc at xq ; and fraxd at xq ) ( , ) . reactivation of part or all of the inactive x chromosome could open these fragile sites for expression of hidden viruses in the fragile sites, adding to the cellular stress. once a virus becomes active in a cell, one of its prime targets in taking over the cell is the nucleolus. the virus is dependent on the host cell's machinery, including ribosomes and transfer rnas (trna), in order for viral proteins to be synthesized. and viral rnas need to be properly folded and assembled into ribonucleoprotein complexes (rnps). rna and rnp processing and assembly are major functions of the nucleolus. the virus puts additional demands on the nucleolar functions beyond the host cell's needs and, since the virus does not code for ribosomes and such, the virus needs to induce increased nucleolar capacity and activity. localization of viral rna and proteins to the nucleolus takes advantage of nuclear and nucleolar localization signals (nols) and chaperones. in the case of viral rna transcripts, rna pol iii transcribed rnas bind ssb/la and ssa/ro which assist in nucleolar entry and processing along with any required refolding. for viral proteins, nuclear localization signals (nls), which are sequences of basic amino acids, and nols are used but, since these signals are frequently closely positioned, it has been difficult to decipher the nols from the more recognizable nls ( ) . some progress has been made in determining nols, such as for the adeno-associated virus serotype assembly activating protein ( ) , and for nucleolar retention signals, such as found in coronavirus nucleocapsid proteins ( ) . the nucleolus-structure, function, and dynamics the nucleolus [reviewed in ref. ( ) ( ) ( ) ( ) ( ) ] is a prominent feature in the nucleus, appearing as a vacant area when imaging nuclear dna content. there is, in fact, some dna in the nucleolus. as far as active dna in the nucleolus, nucleoli associate primarily with repetitive copies of ribosomal dna (rdna) genes expressing ribosomal rnas from nucleolar organizing regions (nors) which are located on the acrocentric autosomes , , , , and . other dna sequences may be involved at the periphery of the nucleolus transiently, such as in repair of breaks in dna near the rdna genes. a role in dna repair in general is emerging for the nucleolus since many proteins involved in dna repair have associations with the nucleolus ( ) . further, centromeric dna is associated with nucleoli as part of the nucleolar regulation of the cell cycle ( ) . the nucleolus does not have a membrane defining its structure but nucleoli are typically surrounded by a shell of heterochromatin established in chromosomes containing nucleolar-associated chromatin domains (nads) ( ) . this then serves to define the boundaries of the nucleolus. the nads contain satellite dna, mostly from centromeric and pericentromeric regions of chromosomes. the nads also contain gene poor and silent chromatin. in addition, the inactive x chromosome (a.k.a. the barr body), a heterochromatic body found in most human female cells, is found in close proximity to nucleoli in one-third of cells throughout the cell cycle and % of cells in s phase suggesting a putative role for the nucleolus in maintaining x chromosome inactivation ( ) . the heterochromatin-nucleolar associations are facilitated, in general, by insulator proteins, ctcf (ccctc-binding factor) and nucleophosmin and additionally, in the case of the inactive x chromosome, by x inactivation specific transcript rna. ebv latency can be controlled by ctcf bound in the promoter region of the epstein-barr virus nuclear antigen (ebna- ) gene ( ) . disruption of chromatin-nucleolar interactions could lead to changes in ebv latency when ctcf interactions with inserted ebv genes are disrupted. in addition, another very important point to keep in mind is that nucleolin bound to rna polymerase ii (rna pol ii) transcripts that contain intronic alu elements appear to have a critical role in maintaining the integrity of the nucleolus ( ) . when caudron-herger and colleagues added rna pol iii transcribed alu element sequences, even as short as nucleotides, there was fragmentation of nucleoli into small nucleolar-like units that were very inefficient in carrying out nucleolar functions of rna and rnp processing and assembly (figure ) . the authors proposed that the nucleolar fragmentation was attributable to dicer-facilitated degradation of hybridized rna pol iii alu sequences with rna pol ii intronic alu sequences. the work of caudron-herger and colleagues demonstrates a close connection between nucleolar integrity and the complexes of nucleolin with rna pol ii transcripts containing intronic alu sequences. another possibility for nucleolar disruption by rna pol iii alu transcripts that caudron-herger and colleagues did not mention is possible competition for nucleolin between the rna pol ii intronic alu sequences and a sudden abundance of rna pol iii alu transcripts. we believe this could have a major role in generation of autoantigens as we will explain below. normally nucleoli contain three discernable sub-regions: the fibrillary centers (fcs), the dense fibrillary centers (dfcs), and the granular components (gcs). the fcs are the sites of rdna transcription by rna polymerase i (rna pol i) to generate the initial ribosomal rna transcript, the pre-rrna. only % of the ~ rdna repeats in the human diploid genome are transcriptionally active ( ) . processing of the pre-rrna occurs primarily in the dfcs assisted by small nucleolar rnas (snornas). assembly of the final ribosomal subunits occurs in the gc, which has a high concentration of proteins needed to complete the rnps ( ). other rnas and rnps processed and assembled in the nucleolus include: the signal recognition particle (srp) which controls translation and localization of extracellular proteins by transporting them to the endoplasmic reticulum (er) for eventual extracellular release; trnas which require extensive folding; small nuclear ribonucleoprotein complexes involved in splicing of messenger rnas (mrnas); and centromere components. therefore, the nucleolus is involved directly or indirectly in many cellular functions, such as regulation of mitosis; cell-cycle progression; cell proliferation; mrna processing via splicing; translation; protein localization; and various forms of stress response. the nucleolar proteome contains over , proteins according to the nucleolar proteome database, nopdb . ( ). about % of these proteins are involved in ribosome biogenesis. since the demands on nucleolar output can change rapidly, the nucleolar proteome is very dynamic. in addition, the size of the nucleolus can change dramatically depending on the needs. increased nucleolar size correlates directly with increases in polyamine synthesis ( ) . the polyamines, spermidine and spermine, are involved in many cellular functions but their highest concentrations are found in the nucleoli where the polyamines assist in folding of rna transcripts and assembly of rnps. the polyamines have a unique combination of length (spermidine ~ Å spermine ~ Å), flexibility (all single bonds) and high cationic charge at physiological ph (spermidine + ; spermine + ) which makes them ideal counter ions to assist in folding the negatively charged rna transcripts in the nucleolus. nucleoli are very dynamic structures in the cell and cell cycle. there can be more than one nucleolus in the nucleus and, figure | nucleolar integrity from rna pol ii intronic alu sequences. (a) caudron-herger and colleagues reported that the nucleolus has a high content of rna pol ii transcripts with intronic alu sequences (red) and these transcripts associate with nucleolin to maintain nucleolar integrity ( ) . (since the actual localization in the nucleolus and structure of the nucleolin-rna complexes are not known, they are shown simply as part of the nucleolar perimeter.) (b) addition of rna fragments from rna pol iii alu sequences, even as short as bases, leads to loss of nucleolar integrity which caudron-herger and colleagues attribute to dicer degradation of hybridized alu sequences. this leads to fragmentation of the nucleolus into subunits that are substantially less efficient in nucleolar functions of rna folding and ribonucleoprotein assembly. combined, they can occupy up to % of the nucleus. they can expand rapidly, facilitated by increased polyamines, in response to cellular stress since the cell may need to have more ribosomes and trnas to synthesize new proteins to recover from the stress. however, we should remember that the nucleolus surrounds itself with heterochromatin so there is the possibility of displacement or disruption of neighboring heterochromatin due to the nucleolar dynamics ( ) . with regards to the cell cycle, nucleoli disappear in mitosis and reappear in telophase and early g forming around nors with the rdna genes and pre-existing rrna and ribosomal complexes ( ) . in addition, cdk cyclin kinases have a key role in controlling nucleoli during cell cycling, and centromere complexes are generated in the nucleolus giving further importance to nucleoli in cell cycling. once in the host cell, the virus can be sequestered into the host chromatin or it can initiate viral replication. in the case of ebv, the multi-functional epstein-barr nuclear antigen (ebna- ) protein from the ebv genome can assist in the spread and attachment of viral dna to metaphase chromosomes ( ) . ebna- can also disrupt the host cell's usp -assisted stabilization of p / tp , thereby preventing the host cell from entering apoptosis, setting the stage for continual viral replication ( ) . however, viruses do need host cell machinery produced in the nucleolus, such as ribosomes, to facilitate viral replication. therefore, the virus will attempt to increase nucleolar activity and turn on viral gene expression. the ebv genome has a snorna, called v-snorna , which is found in the nucleolus in infected cells ( ) . the v-snorna appears to be involved in activation of the viral dna polymerase. another ebv early gene is epstein-barr nuclear antigen (ebna- ) which is a transactivator of viral and host genes. ebna- can associate with rna polymerase ii promoters to induce increased transcription and this includes the myc gene ( , ) . myc induces increased rna polymerase iii (rna pol iii) activity which creates viral rna transcripts and many of the rna transcripts for nucleolar assembled complexes ( ) . and myc induces increased transcription by rna pol i to create rrna transcripts ( ) . the myc interactome consists of approximately % of genes throughout the host genome ( , ) . included among these are genes involved in polyamine synthesis: ornithine decarboxylase (odc); spermidine synthase (sds); and spermine synthase (sms) ( ) ( ) ( ) . and so the virus induces polyamine synthesis which is directly associated with an increase in the size and activity of the nucleolus ( , ) . the cationic polyamines are ubiquitous and have many important interactions throughout the cell and local extracellular environment (e.g., spermine in neural synapses). the highest levels of polyamines are found in the nucleolus where the polyamines play a critical role in rna folding by neutralizing anionic charges in the rna sufficiently for intra-strand rna-rna interactions to form. polyamine availability directly correlates with increased rna expression and processing ( ) . and the polyamines can assist in rnp complex assembly. mostly these are transient interactions of the polyamines but, in some cases, (a) the inactive x chromosome (xi) is typically located at the nuclear periphery next to the nuclear membrane and is associated with a nucleolus in % of cells in s phase and one-third of cells throughout the cell cycle except during mitosis when nucleoli disappear ( ) . this places one of the most inactive structures in the cell, the inactive x, next to one of the most active, multi-functional, and dynamic structures, the nucleolus. (b) the nucleolus can rapidly expand during cellular stress, such as viral activation. trapped between the nucleolus and the nuclear membrane, the xi could be disrupted by the expanding nucleolus. the polyamines remain as part of the final rna complex, such as in trnas ( ). since viral genes need to be expressed and viral rnas need to be folded and some viral proteins localize to the nucleolus for folding and assembly into the final virion, it is understandable why a virus would want to increase the host cell's polyamine content to increase nucleolar capacity and activity ( ) . however, the relation between viral activation and subsequent rna synthesis is more complex and can vary among viral types. for example, poliovirus inhibits rna pol i activity by inducing sl cleavage and ubf posttranslational modification ( ) , whereas hepatitis c virus stimulates rna pol i activity which is involved in transcription of the rdna genes ( ) . viruses can influence the nucleolar proteome leading to abnormal redistribution of nucleolar components to the nucleus, cytoplasm, and even cell surface ( ) . for example, viruses induce cell surface exposure of ssb/la which normally facilitates termination of rna pol iii transcription in the nucleus and then, along with ssa/ro, acts as a chaperone for the rna transcript as it is processed in the nucleolus ( ) . viruses can also disrupt the cell-cycle related kinases in the nucleolus, suppressing normal cell cycling and, thereby, hijacking the nucleolus to focus on viral rna and protein synthesis and assembly of viral rnp complexes ( ) . the usual effect of cellular stress, such as viral activity, on the nucleolus is to cause enlargement of the nucleolus but on some occasions the nucleolus can actually decrease in size. inhibition of rna pol i by poliovirus, as mentioned above, could be such a situation since a drop in rrna transcripts would inhibit the major nucleolar function of ribosome synthesis. the "x chromosome-nucleolus nexus" hypothesis: disruption of the inactive x the stress that viral activity can put on the nucleolus can lead to extensive enlargement of the nucleolus. this could potentially disrupt the epigenetic silencing in heterochromatin neighboring the nucleolus. the inactive x chromosome, a.k.a. the barr body, would be especially vulnerable, as we proposed in the original version of the "x chromosome-nucleolus nexus" hypothesis ( ), since the barr body is frequently found in close proximity to a nucleolus ( ) and against the nuclear membrane ( ), as depicted in figure a . sandwiched between the nucleolus and the nuclear membrane, the barr body would not be able to avoid exposure and disruption due to the nucleolin and nucleophosmin that are involved in chromatin remodeling from an expanding nucleolus ( , ) . in addition, exposure of the chromatin to the high content of polyamines in the nucleolus could add to the disruption of chromatin since the cationic polyamines can compete with histones for dna binding. moreover, the polyamines have the potential to stabilize alternate dna conformations, such as z-dna, which is targeted by autoantibodies in some cases of sle and ra. negative supercoiling stress is stored in nucleosomes as the double-stranded right-hand coiled b-dna makes a left-handed supercoil over the surface of the nucleosome's histones. displacement of the histones during chromatin remodeling could release the negative supercoiling stress, allowing it to flux through the dna and potentially flipping into left-hand coiled z-dna which is also a form of negative supercoiling storage. z-dna appears only transiently in chromatin since most dna is wrapped up as b-dna in nucleosomes. z-dna is not flexible enough to bend around histones so it is excluded from the bp bound to the surface of the histones. since nucleosomes in human chromatin occur every bp on average, there is normally little opportunity for z-dna to form and persist. exposure to high levels of polyamines from the nucleolus concomitant with disruption of nucleosomes could increase the likelihood of z-dna persistence when there is a shift of negative supercoiling storage from nucleosomes to z-dna ( ) . in a similar manner, dna cruciforms are formed from negative supercoiling stress but their occurrence is also suppressed by positioned nucleosomes. the alu elements, of which there are more than one million throughout the human genome, contain sequences capable of cruciform formation ( ). since there are approximately million nucleosomes associated with the human genomic dna, there is ample stored negative supercoiling stress. this potential for rapid dynamic changes in chromatin, including disruption of higher order "stacked" nucleosomes, alternate dna conformations, displacement of bound proteins, and dna strand separation, is perhaps figure | establishment of the inactive x chromosome (xi). early in embryogenesis one of the two x chromosomes in female cells is inactivated by persistent expression of the x inactivation specific transcript rna (xist) from the x inactivation center (xic). xist rna does not code for protein but remains in the nucleus and binds contiguous chromatin (i.e., the xi, a.k.a. the barr body), recruiting epigenetic silencing effectors (e.g., dna methyltransferases). approximately % of genes from the long arm (xq) and % of genes from the short arm (xp) are silenced. silenced genes shown as dark blue, while genes that escape inactivation are shown as light blue [based on ref. ( ) ]. the result is the barr body which appears as a dense heterochromatic structure near the nuclear membrane. the bulk of the heterochromatic core contains xq genes with some xp genes, and the euchromatic-like surface layer has primarily xp genes that are: actively expressed; potentiated for expression; or silenced but adjacent to expressed genes. particularly interesting in the xp is the pseudo-autosomal region (par ) which has an abundance of alu elements ( ) . in addition, xp contains a "hot" line- sequence that can code for a fully functional reverse transcriptase. xp also contains a fragile site (fraxb). fragile sites are preferential locations for viral insertions. and xp on the xi contains epigenetically silenced genes for spermine synthase (sms) and spermidine/ spermine n acetyltransferase (sat ). overexpression of sms and/or sat that could occur with disruption of epigenetic silencing on the xi can impact cellular methylation and polyamine types and levels. this could also impact polyamine activity in the nucleoli. under-appreciated aspects of epigenetics and could come into play when the nucleolus encroaches on surrounding chromatin. normally males have only one x chromosome whereas females have two x chromosomes. most genes on the x are not sex-related so females only need one active x chromosome. therefore, early in embryogenesis, each female cell inactivates one x chromosome, either the maternally derived or the paternally derived x, and each daughter cell will inherit that inactivation pattern. the process of x chromosome inactivation [reviewed in ref. ( ) ], which results in the heterochromatic barr body, begins from the x inactivation center at xq of the x chromosome's long arm (xq) (figure ) . approximately % of genes on the xq and % of genes on the short arm (xp) are inactive and form the heterochromatic core of the barr body ( ) . the surface of the barr body would be more characteristic of euchromatin with genes that escape inactivation and some inactive genes that are surrounded by active genes or genes potentiated for activity. especially interesting are genes on the xp from xp to the xp telomere, including the pseudo-autosomal region (par ). these would be at the surface of the barr body and more readily disrupted by an expanding nucleolus under stress. sms and spermidine/spermine n acetyltransferase (sat ) are involved in polyamine synthesis and recycling, respectively, and normally sms and sat , located at xp . , are inactive on the barr body ( ) . however, disruption of the barr body by enlargement of the nucleolus, as shown in figure b , could lead to reactivation of sms and sat . this would result in a rapid increase in polyamine synthesis and recycling beyond what was already induced by the host cell and the viral activity. there would be an increase in acetylated polyamines by sat that could interfere with rna folding and, through oxidation, generate putrescine which, in turn, could allosterically increase s-adenosylmethionine (sam) decarboxylase activation reducing sam needed for dna and protein methylation ( ) . excess free polyamines can be conjugated to proteins by transglutaminases and acetylated polyamines, and the conjugated polyamines and putrescine can be oxidized to toxic acrolein. there is a close relationship between the intensity of sjs and the appearance of acrolein-conjugated proteins ( ) . the net effect of expression of sms and sat from the disrupted barr body is dysregulation of polyamine levels. add to this the fact that sat can undergo super induction, meaning there could be a several -fold increase in polyamine acetylation. in the nucleolus, there could be an increase in polyamines and now acetylated polyamines that interfere with normal rna folding and rnp assembly. once sat becomes active from both x chromosomes from nucleolar disruption of the barr body, going forward there could be a drop in polyamines during subsequent stress events as super induction of sat acetylates polyamines. it may follow that the nucleolus can no longer expand sufficiently to adapt to new stresses and the nucleolus can no longer work efficiently in proper folding and assembly of rnas and rnps, leading to creation of autoantigens. in other words, an initial stress-induced polyamine-driven expansion of the nucleolus could disrupt the barr body leading to rna pol iii alu transcript-driven fragmentation of the nucleolus. subsequently, with reactivation of sat from the barr body, it may reduce polyamines and reduce the ability of the nucleolus to function normally in folding and assembly of rnas and rnps and fail to react effectively to future stressful events since polyamines will be rapidly acetylated as they are synthesized. this scenario could result in ongoing generation of abnormal, potentially autoantigenic rnps due to the compromised nucleolus that lacks sufficient polyamines. another problem that could arise is reverse transcription. most line- elements have mutated sufficiently so that they no longer code for functional reverse transcriptases. a few, including one in xp , can still produce functional reverse transcriptases but are suppressed by positioned nucleosomes ( ) . reverse transcription of alu elements could be particularly consequential. alu elements are rich in g-c base pairs; therefore, reverse transcribed alu dna would require significant methylation since hypomethylated dna would be interpreted as exogenous. line- reverse transcriptases preferentially reverse transcribe line- rna at a rate of , × and alu rna at a rate of × in comparison to other rnas ( ) . the cell could quickly become inundated with hypomethylated alu dna. li and steinman reported a high content of alu dna (up to %) in the free dna in sera of lupus patients whereas alu elements only account for . % of the human genome ( ) . those authors suggested that reverse transcription could be a possible cause. we proposed that fully functional line- elements activated from disruption of the x chromosome could be involved in such reverse transcription. expanding the "x chromosome-nucleolus nexus" hypothesis: disruption of the nucleolus the earlier version of the "x chromosome-nucleolus nexus" hypothesis suggested that there could be consequences from expression of previously sequestered alu elements, particularly from the par of the x chromosome short arm where there is an exceptionally high content of alu elements ( ). we can now add detail to the hypothesis regarding what consequences could arise from rna pol iii expression of these alu elements. there are over , , alu elements spread throughout the human genome but most are suppressed by a positioned nucleosome. displacement of the nucleosome could open the alu element's internal rna pol iii transcription start site. rna pol iii can be quite prolific since it does not require energy (atp), does not require extensive assembly of transcription factors, can initiate from the intragenic promoter in alu elements, and can rapidly reinitiate to generate multiple transcripts. in addition, since alu elements average only bp and the intragenic promoter requires only about bp for transcription factor binding, displacement of only one nucleosome would be all the opening needed. the abundance of rna pol iii typically found near the nucleolus, particularly the perinucleolar compartment, could rapidly generate thousands of alu rnas if there were a disruptive event, such as encroachment of the nucleolus into the barr body. especially vulnerable is the dense cluster of alu elements in the par region near the surface of the barr body. whereas alu elements comprise . % of the human genome, they are at only % in the x chromosome. however, alu elements account for . % of the par region and % of the adjacent s region ( ) . since par has approximately . million base pairs, there are estimated to be more than , alu elements in par that could potentially flood the nucleus and nucleolus with alu rna transcripts ( figure a ). contrast this with the approximately active ribosomal rna genes in the nucleolus. the alu rnas could interfere with assembly of the srp which contains an alu domain that binds srp / heterodimers ( ) . free alu rna could compete in binding the srp / leaving incomplete srps that cannot halt ribosomal activity in the cytoplasm when needed during synthesis of extracellular targeted proteins. this could lead to improper modifications (e.g., transglutamination) and localization of proteins. and, opening of the alu elements, which have extensive intra-strand matching sequences, could also facilitate formation of cruciforms in the dna which could be stabilized by polyamines. these cruciforms could be interpreted as autoantigens by the immune system. perhaps, the greatest danger from expression of alu elements from the disrupted barr body is deduced from the work of caudron-herger and colleagues mentioned previously ( ) . an abundance of rna pol iii alu transcripts from the disrupted barr body could compete with or lead to degradation of the rna pol ii intronic alu rna that, along with nucleolin and nucleophosmin, provides structural integrity for the nucleolus. this would lead to fragmentation of the nucleolus into inefficient subunits (figure b) . these nucleolar-derived subunits could have abnormal levels of polyamines and acetylated polyamines that cannot properly fold rna and assemble rnps. in fact, the needed components for assembly of an rnp like the ribosome may be unequally distributed among the nucleolar fragments preventing complete assembly. and there could be viral proteins and rnas competing to join rnp assemblies. the rnps and partial assemblies could be stabilized in abnormal conformations and associations by the polyamines and become autoantigenic when released from the cell. such extracellular exposure could occur by blebbing and microparticle release as the cell enters apoptosis, netosis or other forms of termination ( ) . with a loss of integrity of the nucleolus and expression of viral components, some nucleolar material could be displayed on the cell surface, such as the la protein ( ) . in addition, nucleolar fragmentation could lead to loss of nucleolar control of cell cycling, such as assembly of centromeres. another problem that could arise is involvement of cyclic gmp-amp synthase (cgas) which detects cytosolic dna. this includes detection of micronuclei that contain dna from a disrupted nucleus or from dna damage ( ) . fragmentation of the nucleolus, as described above, could potentially generate such micronuclei, especially when centromere assembly and functioning are disrupted or when there are lagging chromosomes during mitotic segregation of chromosomes. the appearance of hypomethylated reverse transcribed alu dna in the cytosol, possibly originating from x-linked line- reverse transcription of par alu element rna, could also trigger the cgas-sting pathway. formation of cyclic gmp-amp (cgamp) can trigger activation of the stimulator of interferon genes (sting) protein which induces transcription of interferon β (ifnβ) as part of the innate immune response in antiviral, antibacterial, and anticancer activity and is suspected of involvement in autoinflammatory and autoimmune diseases ( ) . therefore, the original "x chromosome-nucleolus nexus" hypothesis, which explained how the nucleolus could disrupt the inactive x chromosome, can now be expanded to include disruption of the nucleolus by x-linked alu rna transcripts that lead to nucleolar fragmentation and generation of autoantigenic material. the "x chromosome-nucleolus nexus" hypothesis in relation to other diseases the "x chromosome-nucleolus nexus" hypothesis was developed primarily with sle in mind but it could be involved in many autoimmune diseases. the mechanism could have differing effects due to the cell types and locations involved. for example, ra and sle can have some of the same autoantigens targeted, figure | autoantigens generated by disruption of the nucleolus. (a) the original version of the "inactive x chromosome and nucleolus nexus" hypothesis proposed that there is disruption of the inactive x by the nucleolus due to an extraordinary expansion of the nucleolus under stress (figure b) . this disruption could open previously sequestered dna, especially alu sequences and genes in the short arm of the xi that are located in the euchromatic-like surface layer of the xi ( ). now, based on the work by caudron-herger and colleagues ( ), we can propose additions to the hypothesis, that x-linked alu transcripts generated by the abundant rna pol iii near the nucleolus can disrupt the nucleolin-rna pol ii intronic alu complexes, either by dicer degradation or by direct competition between the rna pol iii alu transcripts and the intronic alu sequences. (b) the subsequent fragmentation of the nucleolus could result in nucleolar fragments that contain conformationally abnormal autoantigenic structures due to improperly folded rnas or improperly assembled ribonucleoprotein complexes (rnps). for example, in some nucleolar fragments there may be insufficient quantities of ribosomal components (either rnas or proteins) and, therefore, complete functional ribosomes cannot be formed. there may also be incorporation of viral rna and/or viral proteins into the rnps. also, overexpression of x-linked spermine synthase and/or spermidine/spermine n acetyltransferase could result in abnormal types and levels of polyamines in the nucleolus and nucleolar fragments. for example, there may be putrescine, acetylated polyamines, and/or nuclear aggregates of polyamines in the nucleolus that interfere with rna folding. normally one would expect only spermine and spermidine to be present in large quantities. extracellular release (by apoptosis, necrosis, netosis) of these abnormal nucleolar products could provoke an autoimmune reaction that later targets the more abundant normal products due to epitope spreading. such as z-dna, but ra is primarily behind the synovial membrane reducing full exposure of antigenic and autoantigenic material to the immune system. however, continual attraction of neutrophils to the same confined inflammation site in ra where the neutrophils undergo netosis in an ineffective attempt at clearing abnormal material would produce chronic local exposure of cells and extracellular material to the neutrophil's active peptidyl arginine deiminases (pads) producing high levels of citrullinated proteins (e.g., collagen) that eventually provokes the adaptive immune system into producing autoantibodies targeting the modified collagen as a major autoantigen in ra ( ) . in a similar manner, ms is confined behind the bloodbrain barrier reducing access of autoantigenic material to the immune system but, again, neutrophils continually attracted to an ms lesion could be releasing pads that citrullinate myelin, eventually triggering autoantibodies to citrullinated myelin basic protein which is a major autoantigen in ms. sle is a systemic disease suggesting that the immune system can more readily react to the broad array of abnormal material seen in sle and generate autoantibodies. compared to ra or ms, the autoantigens, autoantibodies and complexes of the two in sle have easier access to the circulatory system allowing the reaction to spread to and deposit in different organs rather than being confined behind a membrane barrier. sjs can be a primary disease or it can be secondary to sle, ra or ms. this suggests that there could be similar mechanisms occurring in all four of these disorders. involvement of polyamines, possibly due to loss of epigenetic control of x-linked polyamine genes, is suspected in sjs since the appearance of acrolein conjugated proteins is related to the intensity of sjs and acrolein is an oxidation product of polyamines ( ) . the hypothesis may even play a role in alzheimer's disease (alz) since there is a female bias in the disease and there are autoantibodies involved in alz ( ) . in addition, polyamine levels are altered in alz ( , ) along with increased acrolein ( ) ; sam levels are greatly decreased ( ) ; polyamines are involved in plaque formation ( ) and nucleolar poly (adp-ribose) polymerase (parp ) is decreased in alz ( ) . cellular stress that leads to disruption of the inactive x chromosome and/or the nucleolus could play a role in the altered polyamine activity, decreases in sam, decreases in parp , and appearance of acrolein. the hypothesis as a whole or in parts (part : inactive x disruption and/or part : nucleolar disruption) could have a role in some cancers. the inactive x chromosome is often missing in tumor cells from breast and ovarian cancers ( ) . the inactive x may have reactivated from a decrease in methylation (possibly due to over activity of polyamine synthesis and recycling) or the inactive x was lost due to improper segregation of chromosomes to daughter cells (possibly due to centromere assembly in the nucleolus). viruses could be involved in the loss of x inactivation since viruses increase polyamine levels in order to increase nucleolar activity for their benefit, as exemplified by ebv induction of odc, sds, and sms via increased myc activity. the subsequent reduction in sam due to polyamine synthesis would make it difficult for the cell to maintain chromatin methylation required for epigenetic silencing in the x chromosome and other chromosomes leading to disruption of control of oncogenes and tumor suppressor genes and there is the possibility of opening previously sequestered viruses that then try to take control of the nucleoli. the inactive x has the greatest demands for methylation but it is the last chromosome to be replicated and repackaged in late s phase or even early g when sam levels would have already been impacted by methylation of other chromosomes. reactivation or loss of the inactive x chromosome could explain some of the cases of triple-negative breast cancer in which there is no overexpression of her , estrogen, or progesterone receptors ( ) . so this epigenetic scenario of barr body disruption could explain some of the enigmatic cases of cancers. also, keep in mind that viral disruption of nucleoli could interfere with nucleolar involvement in dna repair, nucleolar assembly of centromeres, and alter nucleolar control of cell-cycle kinases ( ) . fragmenting of nucleoli as centromeres are being formed could lead to abnormal distribution of chromosomes resulting in daughter cells of differing karyotypes, such as a parent ( ,xx) cell generating daughter cells of ( ,x ) and ( , xxx) . disruption of the nucleolus in tumor cells could also lead to appearance of autoantigens. autoantigens can arise in cancers but they differ from those normally seen in autoimmune diseases such as sle. the differences could arise from: the cell type involved (proliferating versus mature, differentiated); the nucleolar activity and content at the time of disruption; and the rapidity of the disruption (acute versus gradual accumulation). we can consider that nucleoli in proliferating cells would be heavily involved in cell cycling, such as generating centromere components, and so many of the autoantigens that arise would be expected to be related to cell cycling and suppression of apoptosis ( ) . autoimmune diseases, such as sle, give rise to autoantigens that are components more routinely found in abundance in nucleoli, such as ribosomal or splicosomal components. there is a slightly higher risk of cancers among autoimmune patients but the risk varies with regards to the type of cancer ( ) . therapeutics taken by the patient targeting the autoimmune disease could contribute to cancer development. hematological, thyroid, lung, and vulva cancers show an increased risk with non-hodgkin's lymphoma showing a × to × greater risk in lupus patients, while breast, endometrial, and ovarian cancers show a lower risk. for now there is no direct connection between viruses and the "x chromosome-nucleolus nexus" hypothesis to the increased risk of cancers among autoimmune disease patients but we can consider the induction by viruses of increased polyamine levels and the possible reactivation of x-linked polyamine genes as means by which competition for the cellular methyl donor, sam, could reduce dna methylation and open oncogenes for overexpression in proliferation competent cells. increased nucleolar size due to increased polyamines could add to the disruption of neighboring epigenetically silenced chromatin to expose alleles for expression. the focus of this discussion has been on viruses and the "x chromosome-nucleolus nexus" hypothesis since we now understand the effects a virus can have on the nucleolus and how it is to the benefit of the virus to influence the nucleolar activity. and ebv has been used as the primary example of viral involvement in autoimmune diseases since it is one of the viruses most suspected of having such a role and we can connect ebv actions (e.g., increased myc activity) to increases in polyamines that could directly impact the nucleolus and trigger the hypothesized mechanism. other factors besides viruses, such as bacteria or chemicals, can contribute to autoimmune diseases but the means is less clear and may not closely follow the mechanism proposed. bacteria, for example, produce putrescine and spermidine without the extensive controls on polyamine synthesis seen in eukaryotes but the bacteria can produce their own machinery and are not as dependent as viruses on the cell's nucleolus. in addition, disruption of heterochromatin, such as the inactive x chromosome, can open latent viruses. the x chromosome has four major fragile sites and fragile sites are frequently the locations chosen for viral insertion. the particulars of the fragile site and its vulnerability to viral insertion may add to the genetic susceptibility of an individual. the previous version of the "x chromosome-nucleolus nexus" hypothesis, or simply the "nucleolus" hypothesis, explained how an overly stressed nucleolus could disrupt neighboring heterochromatin, especially the barr body. as a result, there could be detrimental increases in synthesis and recycling of polyamines that could impact cellular methylation and potentially stabilize autoantigenic complexes of nucleolar and chromatin components. the point was made that many autoantigens in sle are, at least transiently, components of the nucleolus but the means by which such nucleolar components could become autoantigenic was not presented. now, with this work, the hypothesis has been extended to include disruption of the nucleolus as an additional step. a disrupted barr body could generate an abundance of polyamines and alu rna from x-linked genes and elements that further stress and damage the nucleolus, making it very inefficient in its functions, even fragmenting it and possibly leading to cell death. and there may be overexpression of sat that hampers the nucleolus in subsequent stress events since polyamines may be converted to a predominance of acetylated polyamines that are less effective at or even detrimental to proper nucleolar folding and assembly of rnas and rnps. meanwhile during nucleolar disruption autoantigens may be created, stabilized and released extracellularly. further work is needed to understand how the various autoantigens provoke the autoimmune response. is it, for example, a conformational alteration of the ribosomal subunits stabilized by polyamines, or incomplete assembly of an rnp? or could it be "guilt by association" such as ssa/ro bound to misfolded rnas stabilized with polyamines that prevent proper refolding? there may be incorporation of viral components in the rnps that make it autoantigenic. or could it be abnormal localization and/or modification of proteins that are misdirected due to alu rna interference with srp assembly ( ) . epitope spreading from the autoantigenic complex to the normal endogenous protein would seem to have a role in the autoimmune response with the greater abundance of the endogenous protein then providing more of the provocation than the original autoantigenic complex. and the fragmentation of nucleoli, as described here, could lead to extracellular signaling and extracellular exposure of autoantigens. testing of the hypothesis can use powerful approaches, such as computational molecular dynamics and single cell analysis, that have now reached sophistication that allow us to explore the interactions of nucleolar components as they are normally processed and the possibilities of how abnormalities could occur. for example, what is the effect of acetylated polyamines if they were to compete with spermidine and spermine in the nucleolar folding and assembly of rnps? what are the interactions and resulting structures of intronic alu rna with nucleolin? and what is the distribution and composition of rnas and proteins in nucleolar fragments compared to intact nucleoli? and, perhaps most important, what does this new hypothesis present as far as therapeutic targets? certainly suppressing viral activity, myc activity, polyamine synthesis, and polyamine recycling are important targets but also newer areas, such as the cgas-sting pathway are promising targets too. the importance of alu elements implied by this hypothesis calls into question the use of mouse models of autoimmune diseases since mice do not have the extensive amount of alu elements seen in humans (certainly not a cluster of . % seen in the par of the human x). in addition, the mouse x chromosome is telocentric (just one long arm) whereas the human x is submetacentric (a long arm and a short arm). the mouse x inactivation would be relatively consistent since it does not negotiate a centromere. x inactivation researchers complain that it is difficult to study partial x reactivation in mice due to the consistency of inactivation along the mouse x. this makes the murine x more robust under stress, at least with regards to this hypothesis ( ). we have previously discussed the short arm of the x chromosome (xp) and especially the portion from xp . to the terminus as having a major role in sle, particularly with regards to possible reactivation of the inactive x chromosome ( - , , , , ) . this section includes: a "hot" line- (codes for a fully functional reverse transcriptase); the polyamine genes spermidine/spermine n acetyltransferase and sms; and the par region with an abundance of alu elements. other groups are just now coming to the conclusion that the xp arm has a major role in autoimmune diseases ( ) although they have not mentioned the alu elements, line- and polyamine genes we have mentioned and they have not made the connection to the nucleolus. it is hoped 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international multi-centre cohort study systemic lupus erythematosus and related autoimmune diseases are antigen-driven epigenetic diseases autoimmune disorders result from loss of epigenetic control following chromosome damage rare x chromosome abnormalities in systemic lupus erythematosus and sjögren's syndrome key: cord- -bhfghcby authors: zrzavy, tobias; kollaritsch, herwig; rommer, paulus s.; boxberger, nina; loebermann, micha; wimmer, isabella; winkelmann, alexander; zettl, uwe k. title: vaccination in multiple sclerosis: friend or foe? date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: bhfghcby multiple sclerosis (ms) is a debilitating disease of the central nervous systems (cns). disease-modifying treatments (including immunosuppressive treatments) have shown positive effects on the disease course, but are associated with systemic consequences on the immune system and may increase the risk of infections and alter vaccine efficiency. therefore, vaccination of ms patients is of major interest. over the last years, vaccine hesitancy has steadily grown especially in western countries, partly due to fear of sequelae arising from vaccination, especially neurological disorders. the interaction of vaccination and ms has been discussed for decades. in this review, we highlight the immunology of vaccination, provide a review of literature and discuss the clinical consideration of ms, vaccination and immunosuppression. in conclusion, there is consensus that ms cannot be caused by vaccines, neither by inactivated nor by live vaccines. however, particular attention should be paid to two aspects: first, in immunocompromised patients, live vaccines may lead to a stronger immune reaction with signs of the disease against which the patients have been vaccinated, albeit in weakened form. second, protection provided by vaccination should be controlled in patients who have been vaccinated while receiving immunomodulatory or immunosuppressive treatment. in conclusion, there is evidence that systemic infections can worsen ms, thus vaccination will lower the risk of relapses by reducing the risk of infections. therefore, vaccination should be in general recommended to ms patients. over the last years, especially in western countries, vaccine hesitancy has steadily grown and poses an increasing health concern ( ). the recent upsurge of measles in europe is an impressive example. anti-vaccinationists argue that possible side effects weigh out the benefits ( ) . especially sequelaes such as autism, multiple sclerosis (ms) and various neurological syndromes have been emphasized by the anti-vaccination lobby ( , ) . this alarming development is even partly supported by healthcare providers including some ms neurologists, who are afraid of iatrogenic deterioration of preexisting ms. indeed, studies linking vaccination and disease onset have been published. although these studies were often underpowered and lacked an adequate design in order to provide evidence of the suspected link, they caught public awareness leading to a drop of public vaccination coverage rates ( , ) . epidemiological studies and pharmacovigilance data have repeatedly demonstrated safety for the vast majority of vaccines. lately, a review concluded that there is no significant evidence for a causal relationship between the onset or deterioration of ms and vaccination against measles, mumps and rubella (mmr), influenza, hepatitis a, hepatitis b, human papilloma virus (hpv), diphtheria, tetanus, acellular pertussis, or meningococcal disease ( ) . some studies have even indicated a decreased risk for ms and reduced disease activity in preexisting ms ( ) . the aim of this review is to summarize data on vaccination and disease activity of both ms and acute disseminated encephalomyelitis (adem). moreover, vaccination-induced effects on the immune system are presented and potential interactions between ms and immunizations are discussed. vaccine-induced protection is a complex issue and depends on a cascade of mechanisms and mediators (figure ) . eventually, protection is accomplished either by antibodies or t celldependent factors or by a combination of both including neutralizing or antitoxic antibodies, cd + t cells, cd + t cells and corresponding cytokines (e.g., interleukin (il)- , , , , , , , , , and ) ( ) . generally, vaccines have to be capable of activating antigen-presenting cells (apcs) of the innate immune system, which subsequently present the vaccine epitope(s) to t cells-the so-called 'immunogenic potential' ( ) . in this context, dendritic cells play a pivotal role due to their enhanced capability to stimulate naïve t cells ( ) . the nature of vaccine-induced immunity depends on several parameters, of which the biological properties of the vaccine's epitope are of high importance ( ) . live vaccines are attenuated variants of pathogens that still can activate apcs, especially immature dendritic cells, patrolling through the body. this immunogenic potential is often lost by subcellular-or subunitbased vaccines ( ) , which is why these inactivated vaccine antigens are usually combined with so-called adjuvants to increase and modulate the vaccine's immunogenicity via a longer lasting and more effective activation of immune cells. one of the most widely used adjuvants are aluminum salts, which were originally thought to create a long-lasting depot of the antigen in order to provide its slow release, but have instead been shown to act on dendritic cells via prrs (pattern recognition receptors) leading to the secretion of pro-inflammatory cytokines ( ) . similarly, novel adjuvants like squalens or monophosphoryl lipid a (mpla-a detoxified lipopolysaccharide) aim to enhance the innate immune response, but never reach the immunogenic potential of live attenuated vaccines ( ) . adjuvants have been added to vaccines for more than years and over the last decades, considerable progress has been made in understanding their mode of action and to improve safety ( ) . besides the above mentioned aluminum salts, squalene and mpla, oil emulsions, saponin, toll-like receptor (tlr) agonists, enterotoxins, polysaccharides, and glycolipid adjuvants ( ) are used, all of which stimulate the immune system as well. aluminum adjuvants have now been used for decades and lots of experience has been gained on its use, effectiveness, and safety and they still remain the most frequently used adjuvants. their effects on the immune system comprise stimulation of macrophages and dendritic cells via prrs, inflammasome activation, il- β release and activation of th lymphocytes ( , ) . however, besides increased immunogenicity, aluminum adjuvants also increase reactogenicity and based on data from animal models and reports on narcolepsy, silicosis, guillain-barré-syndrome (gbs) and macrophagic myofasciitis, they are also discussed to induce autoimmunity ( ) . the second most commonly and long used adjuvants are oil emulsions. they have a strong reactogenic potential and can cause severe inflammatory local reactions such as ulceration and granulomas. the most well-known oil emulsion is complete freund's adjuvant. however, due to its potent reactogenicity, it is not suitable for human use. a possible association between oil emulsions and autoimmunity disorders has been hypothesized from animal models. oil emulsions are potent inducers of il- β and il- ( , ) . il- plays a major role in autoimmunity and ms and may trigger the migration of peripheral lymphocytes into the cns across the bbb ( , ) . frequently, a combination of adjuvants is used to increase immunogenicity of vaccines. as is an adjuvant emulsion containing squalene, dl-αtocopherol, and polysorbate . it is e.g., used for the pandemic swine flu vaccine pandemrix r ( ) or the fda-licensed h n monovalent influenza vaccine. in animal studies, autoimmunity was observed in connection with as ( ) and in humans, cases of narcolepsy have been reported ( ) . oil emulsions are often combined with tlr agonists such as mpla. generally, tlr agonist adjuvants activate the inflammatory transcription factor nfκb as is a combination of mpla and aluminum salts and is used as adjuvant in vaccines against hepatitis b (fendrix r ) and hpv, as well as in the new recombinant vaccine against herpes zoster. most polysaccharide adjuvants activate nfκb to induce immune processes (e.g., dextran, zymosan) ( ) . however, deltainulin for instance, a polysaccharide adjuvant used for advax r , acts via nfκb-independent mechanisms to enhance humoral and cellular immune responses. although the mechanisms are not yet fully understood, advax r has so far not shown inflammatory side effects and has proven safety in hepatitis b vaccination and influenza ( ) . after activation of the immune cascade and stimulation of dendritic cells, the latter increase their expression of mhc molecules and chemokine receptors such as ccr leading to their migration toward the draining lymph nodes in order to provide co-stimulatory signals for the differentiation of naïve t cells into immune effector cells ( ) . the activation of the immune cascade has various effects on t and b cells. in short, antigen-recognition by b cells leads to their activation and migration toward the t-b cell border of the lymph node, where they can subsequently receive additional stimuli by activated t helper (th) cells. these signals include cd interaction, secretion of cytokines by th or th cells, and finally the transformation of b cells into plasma cells predominantly secreting low affinity antibodies ( ) . later, the germinal center response contributes via affinity maturation (somatic hypermutation and affinity-based selection) and isotype switch to a sustained production of high affinity antibodies by predominantly plasma cells but also memory b cells. basically, in the lymph nodes, numerous b cells with various affinity compete for the antigens presented by follicular dendritic cells. these antigens are processed and further presented via mhc ii to follicular th cells, which provide costimulatory signals (e.g., cd , icos, and il- ) leading to survival and further proliferation of b cells with highest affinity for the antigen ( ) . in conclusion, vaccination-induced immune responses, including employed cell types and mediators, vary depending on the type of vaccine administration, kind of vaccine and choice of adjuvant. while antibodies will directly prevent and reduce infections, cd + and cd + t cells rather support the organism eventually reducing, controlling and clearing the pathogens. antibodies bind to their antigen, neutralize pathogens, activate macrophages and neutrophils as well as the complement system, while cd + and cd + t cells secrete cytokines, perforins, and granzymes ( ) . the choice of adjuvant seems to be critical, since some may cause problems in autoimmune diseases. thus, monitoring side effects regarding autoimmunity is essential. in the early days of vaccine development, louis pasteur used nerve tissue of infected animals to obtain a rabies virus vaccine ( ) . although saving countless lives it was recognized that active sensitization with neuronal tissue could occasionally lead to neuroparalytic autoimmune complications ( ) with self-limiting autoimmune encephalomyelitis that fulfilled the pathological criteria of ms ( , ) . advances in processing techniques and increasing insights in immunology led to modern vaccines devoid of neuronal tissue. ms is a chronic disease thought to be caused by immune-mediated mechanisms. thus, immune responses caused by vaccinations will affect the immune system. however, their effects on immunology per se, but especially those in ms patients, are scarcely understood. the same means by which infections can induce autoimmunity also apply for vaccination-induced immune activation. possible structural similarities between microbial epitopes and epitopes of the cns could lead to cross-reaction of antibodies via molecular mimicry as shown for streptococcal antibodies in heart tissue ( ) . additionally, epitope spreading is a mechanism leading to a broadening of the immune response from the dominant epitope to cryptic (intramolecular) or neighboring molecules (intermolecular) resulting in an increased antibody repertoire and cellular response ( ) . moreover, bystander activation, a process in which activated apcs stimulate autoreactive t cells, can occur ( ) . bacterial and viral infections can trigger relapses and mri activity in ms; vaccination has been proven to protect from or weaken infections, thus providing an "indirect" protection against ms disease activity ( ) . several reports on neurological disorders developing after immunization have been published including several cases on encephalomyelitic disorders (impaired consciousness, ataxia and optic neuritis) as well as demyelinating lesions in a patient with transverse myelitis after active immunization against influenza ( ) ( ) ( ) ( ) . immunization against rubella was associated with diffuse myelitis and recurrent relapses with optic neuritis, paraparesis and impaired motor function ( , ) . transverse myelitis ( ) as well as optic neuritis ( , ) were reported in patients vaccinated against measles, mumps and rubella. further cases with symptoms suggestive for disseminated encephalitis were reported after vaccination against diphtheriatetanus-poliomyelitis (dtp) ( ) and after immunization against smallpox, rabies or typhus ( ) . exacerbations of ms and demyelinating lesions were reported in ms patients and patients without a history of neurological conditions after immunization against hepatitis b ( ) . similarly, tourbah reported on patients with demyelinating lesions and clinical symptoms after vaccination against hepatitis b ( ) . in contrast to these case series, a case-control study (evidence class ii) ( ) including more than patients with ms or optic neuritis and controls without any underlying neuroimmunological disorder did not reveal an elevated risk for the development of ms or optic neuritis after immunization against hepatitis b, tetanus, influenza, measles/mumps/rubella, measles, or rubella ( ) . while hernan came to same results for immunization against influenza or tetanus in a case-control study (evidence class ii), active immunization against hepatitis b was reported to pose a higher risk for ms ( ) . the latter finding could, however, not be confirmed by confavreux in a large case-crossover study. additionally, no increased risk was seen for vaccination against tetanus and influenza as well ( ) . similarly, other class ii case-control studies did not report on an increased risk for ms after hepatitis b vaccination ( ) ( ) ( ) . an even decreased risk for ms was reported after tetanus immunization ( ) . in a large class i study, a patient register including , females vaccinated with the quadrivalent hpv vaccine was analyzed. thereof, , patients with ms and , patients with other demyelinating disorders were studied and no increased risk for cns manifestations was seen in this large cohort ( ). miller et al. performed a prospective class ii, randomized, double-blind, placebo-controlled study, which included ms patients, who received either standard influenza vaccination or placebo. for a months follow-up period, the occurrence of neurological symptoms or influenza was monitored and no differences were seen for relapse rates ( ) . a study by langer-gould reported on an increased risk for cns demyelinating diseases within the first days after vaccination. it was concluded that there is no increased risk for ms, but it seems that the transition from subclinical to overt autoimmunity in patients with existing disease is shortened ( ) . two major questions arise on the topic of "ms and vaccination": (i) can vaccines cause ms and (ii) can vaccines provoke or trigger relapses in patients with ms? (i) overall, the anecdotal reports associating ms onset and vaccination had limited reliability, lacked validity and could not be replicated in larger studies. therefore, there is consensus that there is yet no evidence that ms can be caused by vaccines neither by inactivated nor by live vaccines ( ). (ii) it is more difficult to assess the potency of vaccines to trigger relapses in ms patients. with respect to live vaccines it seems to be plausible that they may be able to provoke a deterioration of the disease, since they fulfill the criteria of an active infection with a replicative (although attenuated) organism. there is class iv evidence that at least the yellow fever (yf) d vaccine strain, which is derived from a natural occurring yf-virus and hasn't completely lost its neurotoxicity even after numerous passages, is able to provoke relapses in ms patients. however, it has to be kept in mind that the patient cohort had received immunomodulatory treatment and the sample size of this self-controlled case series study was rather small ( ) . the underlying potential immunologic mechanisms, which are responsible for this elevated relapse rate, are not understood yet and larger studies are necessary to confirm this association. hypotheses may be generated based on observations after infections with helminths, mycobacteria and epstein-barr virus, or by the immunologic properties of this particular vaccine strain ( ) . immunological analyzes showed that after immunization against yf, ms patients had a significantly increased mbp-and mog-specific response shown by increased numbers of cells secreting interferon, il- α, il- β and tumor necrosis factor compared to unvaccinated ms patients or ms patients vaccinated against influenza ( ) . still, there is no evidence for other live vaccines such as mmr to deteriorate ms ( , ) . for inactivated vaccines, there is already more evidence available that an association between ms relapses and different kinds of vaccines does not exist ( ) . even for vaccines, which were publicly accused to be associated with ms disease or relapse rate, like hpv or hepatitis b vaccines, there is no evidence to support any association between vaccination and clinical course of ms, as well as for vaccines containing inactivated neurotropic viruses like tbe ( , ) . it still remains unclear if inactivated vaccines may accelerate an upcoming relapse in patients with active ms by non-specific stimulation besides effects of vaccines on induction and the disease course of ms, potential immunological effects of adjuvants have to be considered as well. most experience on the possible induction of autoimmunity following administration of adjuvant-containing vaccines has been gained from animal models. however, results from experimental studies cannot be transferred to humans without reservation. first, the dose ratios tested in animal models are not the same as in humans and second, human immunology differs from animals. indeed, oil emulsions, aluminum salts and squalene have shown severe side effects in animal models, while they are considered to be safe in humans ( ) . an analysis performed by the european medicines agency (ema) ( ) investigated autoimmune disorders following vaccination against pandemic influenza a/h n between october and december ( ) . thirty percent of the million doses of the distributed vaccines contained aluminum salts and squalene-based adjuvants. overall, the study did not suggest a significant difference in the risk for autoimmune disorders for adjuvant and non-adjuvant vaccinations. adem was reported for people (adjuvant vaccines: , non-adjuvant vaccines: ), ms for people (adjuvant vaccines: , nonadjuvant vaccines: ), ms relapses for patients (adjuvant vaccines: , non-adjuvant vaccines: ), and one case of relapsing remitting ms was reported for adjuvant-containing vaccination ( ) . statistical analysis revealed only a nonsignificantly increased risk for gbs ( ) . also, a favorable benefitrisk profile of the vaccines was demonstrated ( , ) . in conclusion, following the reports from literature, all of the ema/fda-approved vaccines (with exception for yellow fever) and adjuvants do not show a significantly increased risk for ms and adem. constant improvement of basic immunological knowledge and technology will further improve the safety of adjuvants. table gives an overview of the recommendations of standard vaccinations in the general population and in ms patients. while there is a lot of literature on vaccination and risk for ms or ms relapses available, reports on vaccination and adem are scarce. yet, adem has been discussed to be a sequelae of vaccinations ( ) as well as to be preceded by infections. several cases of adem have been reported to be timely related to vaccinations against rabies ( ), hpv ( , ) , hepatitis a and b, diphtheria, tetanus and poliovirus ( ) , measles, rubella and booster immunization for japanese encephalitis ( ) . adem has been reported following vaccination against influenza, including eight cases after vaccination against h n . also, four adem cases after vaccination against yf can be found in literature ( , ) . besides case reports, there have been some observational studies, albeit all having their limitations. in out of reported cases of adem, patients had infections or vaccinations prior to disease onset ( ) . also, pellegrino et al. concluded a possible relation between post-vaccination adem in children and adults. four hundred four cases of adem were analyzed based on the data of the vaccine adverse event reporting system (vaers) database and the eudravigilance post-authorization module (evpm) ( ) . about % of the cases occurred between and days after vaccination, most commonly against influenza and hpv. a case-control study on vaccination against hepatitis b, influenza, polio, diphtheria, pertussis, tetanus, measles, mumps, rubella, japanese encephalitis, meningitis, hepatitis a, varicella and rabies did not reveal an increased risk for the onset of adem in the time spans of - days and - days after vaccination, but between and days ( ) . based on these reports, the risk for adem after vaccination cannot be completely ruled out. considerations on ms exacerbation and vaccination apply only for ms patients receiving no immunomodulatory/ immunosuppressive treatment. if any kind of immunosuppression is used for ms therapy, this choice of treatment will dominate the decision whether to vaccinate or not ( ) . in recent years, consensus statements on vaccinations during immunosuppressive treatments were published by various national and international societies and expert panels ( ) ( ) ( ) ( ) ( ) . there is consensus that inactivated vaccines will do no harm ( ) even in immunosuppressed patients. however, data on the efficacy of vaccinations in combination with the various available ms medications are missing. thus, for patients either receiving more than one immunomodulatory treatment or having underlying immunomodulating condition, the outcome is difficult to predict ( ). therefore, the success of vaccination should be verified by antibody testing if a valid test is available. except for a few treatments, which only lead to mild immunosuppression, live vaccines are contraindicated under immunosuppressive treatment. in some situations, risks and benefits of a live vaccine have to be weighed against each other, e.g., in varicella zoster virus (vzv)-negative ms patients under fingolimod treatment, varicella vaccination may be considered, since severe complications from natural varicella infection may outweigh the risk from this live vaccine. however, recommendations vary between different institutions even within the same country ( , , ) . a recent case report on a lethal vzv infection in an immunocompromised patient after vzv live vaccination drives the discussion on this issue ( ) . there is consensus about the timing of vaccination in patients, who will undergo immunosuppressive treatment: vaccinations should be given well in advance to the start of treatment (at least weeks for inactivated and ≥ weeks for live vaccines) and should be distinguished between primo-vaccinations and boosters. importantly, the refractory period after immunosuppression has to be considered as well, which may be up to year depending on the type of medication (e.g., rituximab or alemtuzumab) ( ) . vaccines will have various effects on the immune system, which greatly depend on the cell types typically engaged by the respective vaccines. the impact of immunosuppression on the various cell types (and possible mitigation of effects) should be taken into consideration. protective efficacy is mostly mediated by antibodies for the following vaccines: cholera, diphtheria toxoid, hepatitis a and b, haemophilus influenzae type b, influenza, japanese encephalitis, meningococcal ps and conjugates, papillomavirus, pneumococcal ps and conjugates, polio (sabin and salk), rabies, rotavirus, rubella, tetanus toxoid, typhoid ps, and yf. effects are solely born by t cells for tuberculosis (bcg), or by a combination of antibodies and t cells for measles and intranasal influenza vaccination. besides antibody-mediated protection, effects of t cells are discussed for pertussis ( ) . for patients receiving immunosuppressive treatment, vaccination control should be performed. for diphtheria, tbe (with caution), hepatitis a, b, haemophilus influenzae type b, measles, mumps, pneumococcus, polio, rubella, tetanus, rabies and varicella, standards are available and recommended to be tested. in general, to increase the validity of vaccination control, titers should be assessed in paired samples (before and after immunization) via the same method and at highquality standards ( ) . in general, patients should have received their recommended standard vaccines according to their region-specific vaccine guidelines. before certain immunosuppressive treatments are initiated, it is mandatory to exclude former infections and if necessary, vaccination should be considered according to the regulatory agencies. table provides an overview on necessary vaccinations according to fda/ema guidelines (extended vaccination reflects the authors' suggestion). for many immunotherapies, a prior exclusion of an ongoing vzv infection is required and vaccination should be offered to those, who haven't gained any immunity yet. additionally, vzv-seropositive patients undergoing immunotherapy should be offered vaccination as well to prevent zoster reactivation and late effects. recently, a non-live subunit vaccine has been authorized for vzv-seropositive patients. it possesses a better risk-benefit profile compared to the live vaccine and has already been approved by many countries ( ) . additionally, it should be considered to offer patients with upcoming fingolimod or alemtuzumab treatment the option of vaccination against hpv, as post-market surveillance showed increased reports of warts and cervical dysplasia due to these two ms therapies [ema; ( ) ]. furthermore, pneumococcal vaccine might be considered in patients receiving b cell-depleting therapies, as severe respiratory infections during phase iii studies were seen ( , ) . vaccine hesitancy is a major problem nowadays. the usefulness of active immunization is undisputed and has saved numerous lives. however, fear of possible, but also often unconfirmed, side effects has fostered this anti-vaccine sentiment. this has led to a recent outbreak of measles ( ) and curiously some viruses and disorders, which have been assumed to be eradicated, seem to become a hot topic for western health systems again. indeed, side effects upon vaccination may occur in rare cases, however, the benefits for individual people as well as the whole population will generally outweigh adverse effects. vaccine hesitancy results in a twofold problem: ( ) the missing protection for the unvaccinated people themselves but also ( ) a risk for people, who are not able to get vaccinated. the missing herd immunity poses a major problem for a group of patients with fragile health. for ms patients receiving immunosuppressive treatment, an acute infection can have dangerous sequelae. thus, if possible, ms patients should be vaccinated beforehand. the possible benefits outweighdependent on the individual case-the possible risks. an additional perspective raises the possibility of vaccination against ms. indeed, early approaches exploring vaccination with synthetic peptides in experimental animal models were successful, but translation into clinical treatment was so far unsatisfying ( ) ( ) ( ) . interestingly, it was recently shown that an anti-typhus vaccination (typhim vaccine) might have the potential to ameliorate the disease course of ms by targeting prohibitins on th cells. tested in an experimental ms model it led to decreased levels of il and increased numbers of foxp + regulatory t cells ( ) . further investigations are needed before studies should investigate treatment options for ms patients. still, it is a good example, how immunology of vaccination might overlap with and modulate the immunology of ms. theoretically, an increased immune response against different types of vaccines, such as live attenuated 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from patients with multiple sclerosis active human herpesvirus infection in patients with multiple sclerosis cerebrospinal fluid molecular demonstration of chlamydia pneumoniae dna is associated to clinical and brain magnetic resonance imaging activity in a subset of patients with relapsing-remitting multiple sclerosis multiple sclerosis, sporadic creutzfeldt-jakob disease and bovine spongiform encephalopathy: are they autoimmune diseases evoked by acinetobacter microbes showing molecular mimicry to brain antigens? the case for rhinoviruses in the pathogenesis of multiple sclerosis epstein-barr virus infection and multiple sclerosis: a review all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © zrzavy, kollaritsch, rommer, boxberger, loebermann, wimmer, winkelmann and zettl. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -x grfhov authors: sung, pei-shan; hsieh, shie-liang title: clec and clec a: pathogenic host factors in acute viral infections date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: x grfhov the protective roles of endosomal toll-like receptors (tlrs) and cytosolic nucleic acid sensors are well elucidated, but the pathogenic host factors during viral infections remain unclear. spleen tyrosine kinase (syk)-coupled c-type lectins (clecs) clec and clec a are highly expressed on platelets and myeloid cells, respectively. clec has been shown to recognize snake venom aggretin and the endogenous ligand podoplanin and acts as a critical regulator in the development and immunothrombosis. although clec has been reported to interact with type i immunodeficiency virus (hiv- ), its role in viral infections is still unclear. clec a binds to fucose and mannose moieties of dengue virus membrane glycans, as well as to n-acetylglucosamine (glcnac)/n-acetylmuramic acid (murnac) disaccharides that form the backbone of l. monocytogenes peptidoglycans. recently, we demonstrated that both clec and clec a are critical in microbe-induced “neutrophil extracellular trap” (net) formation and proinflammatory cytokine production. moreover, activation of clec by dengue virus (dv) and h n influenza virus (iav) induces the release of extracellular vesicles (evs), which further enhance netosis and proinflammatory cytokine production via clec a and toll-like receptor (tlr ). these findings not only illustrate the immunomodulatory effects of evs during platelet-leukocyte interactions, but also demonstrate the critical roles of clec and clec a in acute viral infections. the global outburst of severe acute respiratory syndrome coronavirus (sars-cov) infection in inspired our exploration of how a viral infection can activate a massive over-production of cytokines by the host immune system-a phenomenon known as a "cytokine storm"; this results in a severe inflammatory reaction and increases systemic vascular permeability within - days following viral exposure. the common features of sars-cov and other acute viral infections are the early onset of inflammatory reactions with elevated local or systemic vascular permeability, before the adaptive immune system is fully activated. these observations suggest that innate immune responses contribute significantly to the pathogenesis of acute viral infections. the identification of endosomal tlrs (tlr , tlr , tlr , tlr ) and cytosolic sensors led to the discovery that their activation by viral nucleic acids induces the production of interferons and proinflammatory cytokines. these intracellular nucleic acid receptors/sensors have been defined as "protective host factors" as they are critical for host defense against viral infections. however, the identity and contribution of "pathogenic host factors" to virus-induced severe inflammatory reactions and lethality, and how different viruses cause distinct clinical symptoms, has remained unclear. in contrast to the relatively conserved nature of nucleic acids (dna and rna), the proteins and glycans of viral envelopes are various have distinct structures; it is enveloped viruses, such as flavivirus, alphavirus, togavirus, coronavirus, orthomyxovirus, paramyxovirus, rhabdovirus, bunyavirus, and filovirus that are primarily responsible for severe inflammatory reactions following infection. therefore, we were interested to determine whether viruses activate membrane receptors on myeloid cells induce cytokine storms, and how these receptors contribute to complex cell-cell interactions during acute viral infections. in this review article, we illustrate the critical roles of c-type lectin receptors in innate immunity and discuss the potential of targeting these cell surface receptors in the treatment of acute viral infections. the common feature of acute viral infections is the rapid onset of inflammatory reactions after exposure to the virus. clinical symptoms range from inapparent and mild fever to severe inflammation and increased endothelial permeability in major organs. severe acute infections in human typically occur in response to enveloped viruses, such as dengue virus (flaviviridae), chikungunya virus (togaviridae), sars-cov (coronaviridae), influenza virus (orthomyxoviridae), measles virus (paramyxoviridae), rabies virus (rhabdoviridae), hanta virus (bunyaviridae), and ebola virus and marburg virus (filoviridae). this implies that membrane components may contribute to the severe inflammatory responses. acute viral infections begin with host exposure to virus and clinical symptoms become apparent gradually as the virus starts to replicate and innate immune responses are activated. abundant proinflammatory cytokines production leads to the characteristic symptoms in acute viral infections, including fever, aches, and pain. when host innate immunity fails to contain virus replication and remove infected cells, high fever and massive cytokine production occur in the host, leading to severe local and systemic changes in vascular permeability, functional failure of affected organs and shock syndromes. it is well-known most of pro-inflammatory cytokines are released from macrophages and severe acute infections are usually associated with the activation of macrophages by enveloped viruses such as dengue virus ( , ), h n viruses ( ) ( ) ( ) , and ebola viruses ( ) . in addition, activation of neutrophils leads to the formation of neutrophil extracellular traps (nets), which may further aggravate virus-induced inflammatory reactions. furthermore, acute viral infections frequently cause thrombocytopenia, and platelet-leukocyte interactions not only regulate inflammatory reactions, but also contribute to the pathogenesis of vascular injury, thrombosis, autoimmune reactions ( ) ( ) ( ) . however, the molecular mechanisms underlying platelet-mediated immunomodulation are still unclear. the human immune system has evolved to provide protection against a diversity of microorganisms, including viral infections. ones of the most important factors involved in suppressing the development of a viral infection is the production of interferons via the activation of intracellular nucleic acid sensors. the endosomal tlrs (such as tlr , , , and ) recognize diverse endogenous danger signals as well as nucleic acid components released from uncoated virions. activation of tlrs results in the production of type i interferons and proinflammatory cytokines, chemokines (including tnf-α, il- , il- , ip , and others) thereby initiating inflammatory responses. in addition, the intracellular rna sensors, such as mda- (melanoma differentiation-associated protein ) and rig-i (retinoic acidinducible gene i) as well as the dna sensors sting (stimulator of interferon genes) and aim (absent in melanoma ) that recognize conserved nucleic structures mediate the production of interferons and proinflammatory cytokines when activated. tlrs are central to the control of innate immune responses through the recognition of pathogen-associated molecular patterns (pamps) and endogenous damage-associated molecular patterns (damps) ( ) . therefore, tlrs are not only critical for host defense against pathogens but also contribute to the pathogenesis of autoimmunity ( , ) . several tlr agonists and antagonists are regarded as promising therapeutic agents for the treatment of sepsis, asthma, vaccine adjuvants, and autoimmunity ( ) . the cell surface tlrs (tlr , tlr , and tlr ) are activated by diverse pamps and damps, whereas the endosomal tlrs (tlr , tlr , tlr , and tlr ) recognize pathogen-derived nucleic acids and, in the context of therapeutic/experimental approaches, synthetic nucleic acids or nucleic acid mimetics. even though tlrs can bind ligand directly, recent studies have shown that high affinity and stable interactions with ligand also require tlr-associated proteins (i.e., co-receptors such as cd , lbp) ( ) . activation of all tlrs (except tlr ) initiates signaling via myd -dependent and trif-dependent pathways. it has been shown that activated myd triggers two signaling pathways: ( ) the traf -tbk -ikkε-irf /irf pathway to induce type i interferon (ifn-α and ifn-β) expression; and ( ) the traf -tak -ikk-nfκb pathway to induce proinflammatory cytokine expression. in contrast, activated trif primarily triggers the traf -tbk -ikkε-irf /irf pathway to upregulate type i interferon production. in addition to endosomal tlrs, cells are equipped with cytosolic sensors that detect invading viruses via recognition of viral dna and rna. viral rna is recognized by members of tolllike receptors (tlr , tlr / ) and rig-i-like receptors (rig-i, mda , and lgp (laboratory of genetics and physiology ). in addition, viral dna stimulate ifn production via tlr and intracellular dna sensors such as sting (stimulator of ifn genes), mpys (methionine-proline-tyrosineserine), mita (mediator of irf activation), or eris (er ifn stimulator) ( , ) . signaling pathways downstream rna and dna sensors induce the production of restriction factors (such as type i and type iii interferons) that prevent viral replication and give rise cell-intrinsic antiviral immunity ( - ). the superfamily of c-type lectin-like domain (ctld)containing carbohydrate-binding proteins comprises ∼ members that can be classified into groups in humans ( ) . while most ctld family receptors do not contain cytoplasmic domains capable of transducing signals, several are syk-coupled receptors have been reported to play critical roles in host immunity against fungal and non-microbial infections ( ) . there is ample evidence to demonstrate the critical roles of c-type lectins in the pathogenesis of dv infection ( , ( ) ( ) ( ) ( ) ( ) ( ) . macrophages, dendritic cells, and liver sinusoid cells are the major targets of dv in humans, where the c-type lectin receptors l-sign/cd (expressed in liver sinusoid cells), dc-sign/cd and mannose receptor/cd (expressed in macrophages/dendritic cells) have been shown to be crucial for dv entry into these cell types. dengue is a mosquitoborne virus and it is interesting to note that dv generated in mosquito cells infects human cells via dc-sign, while dv from human dendritic cells infects human cells via l-sign ( , ( ) ( ) ( ) ( ) ( ) ( ) . these observations led to the hypothesis that distinct glycans associated with dv derived from human or insect cell origins may contribute the differential usage of dc-sign and l-sign in target cell infection. despite the involvement of these c-type lectin receptors in the pathogenesis of viral infections, they do not contain intracellular motifs to enable the initiation of signaling cascades ( ) , nor do they transduce signals via associated adaptor proteins as observed in t cell receptors, bcell receptors, or syk-coupled receptors. as well as membraneassociated lectins, the soluble c-type lectin mannose binding lectin (mbl) not only binds dv generated from both human and mosquito cells, but also activates the complement system to neutralize dv infection ( ) . all the syk-coupled c-type lectin receptors (clrs) are type ii transmembrane proteins which belong to group ii or group v of the ctld superfamily. group ii includes four syk-coupled clrs (clec b/dcar , clec c/bdca- , clec e/mincle, and clec a/dectin- ), whilst group v (also known as the nk receptor-like lectin family) includes clec , clec a/mdl- , clec a/dectin- , and clec a/cd ( ) . among these sykcoupled c-type lectins, activation of clec a by flaviviruses and influenza virus induces the production of inflammatory cytokines and netosis, while these viruses also activate clec in platelets to elease extracellular vesicles (evs), which further enhance inflammatory cytokine release and netosis via clec a and tlr in macrophages and neutrophils. clec a, also known as myeloid dap -associating lectin- (mdl- ), is abundantly expressed in myeloid lineages, including neutrophil, monocyte, macrophage, osteoclast, microglia, and dendritic cells. while clec a does not contain a cytoplasmic motif, activation of clec a recruits the dap adapter protein, thereby triggering downstream signaling via syk. structural analysis shows that human clec a is an n-linked homodimeric glycoprotein expressed on cell surface ( ) . the ligand for clec a was unknown prior to the identification of its interaction with dengue virus ( ) . we showed that when clec a is activated by binding to the dengue virion this leads to dap phosphorylation and induction of proinflammatory cytokines via syk-mediated pathways. clec a is responsible for dvinduced hemorrhagic fever (dhf) and dengue shock syndrome (dss), which represent the most severe responses to dv infection and are characterized by plasma leakage due to increased vascular permeability. blockade of the dv-clec a interaction suppresses the secretion of proinflammatory cytokines, but without affecting interferon-α release. furthermore, blockade of dv-clec a interaction by anti-clec a monoclonal antibodies (mabs) efficiently alleviating plasma leakage and systemic hemorrhaging, thus protect mice from dv-induced shock and lethality in mice lack responsiveness to ifn-α and β (signal transducer and activator of transcription (stat )deficient mice). thus, blockade of clec a-mediated signaling in dv infected cells provides a promising strategy for attenuating systemic vascular leakage and increasing the survival rate of patients suffering from dhf/dss; this approach might also be applicable to other virus-induced inflammatory diseases ( ). however, even though blockade of clec a protects mice from lethal doses of dv, the protective effect is ∼ % at best. this suggests the presence of yet-discovered pathway in dv-induced lethality, which is the focus of ongoing research. we have shown that dv infection induces abundant il- β and il- production by inflammatory macrophages and also mediates pyroptosis in these cells as a consequence of upregulating pro-il- β, pro-il- and nlrp and enabling caspase- activation; blockade of clec a inhibits dv-induced nlrp inflammasome activation and cell death ( ) . we found that osteoclasts are highly susceptible to dv infection, which induces the production of cytokines and infectious virions at levels similar to macrophages and nuclear translocation of the transcription factor nfatc , via clec a. the transient inflammatory response that occurs in bone tissue during dv infection is associated with elevated osteolytic activity and release c-telopeptide of type i collagen (ctx- ) from bone. moreover, clec a −/− stat −/− mice are resistant to dvinduced osteolysis, and anti-clec a mab inhibits dv-induced osteolytic activity and ctx- releases in clec a ++ stat −/− mice. these observations indicate that dv osteoclast and increase osteolytic activity via clec a. the identification of osteoclasts as a novel target for dv provides a mechanism for some of the clinical symptoms experienced by dengue patients; i.e., as a consequence of transient upregulation of osteolysis ( ) . moreover, we demonstrated that japanese encephalitis virus (jev) binds clec a directly to trigger the phosphorylation of dap and the release of proinflammatory cytokines and chemokines in macrophages. even though blockade of clec a cannot inhibit jev infection to neurons and astrocytes, jev-induced cell death and proinflammatory cytokines in microglia-neurons-astrocyte mixed culture was dramatically inhibited by anti-clec a mab. this effect is achieved by blockade of jev-clec a interactions in microglia, thereby attenuating proinflammatory cytokines and toxic substances released from microglia. furthermore, peripheral administration of anti-clec a mab not only reduces jev-induced neuronal inflammation and lethality, but also restore blood-brain barrier (bbb) integrity after jev infection. this observation suggests that anti-clec a mab can penetrate bbb and attenuates jevinduced neuronal inflammation in brain, thereby restore bbb integrity and reduces neuronal toxicity and lethality. in contrast to conventional immunosuppressants, anti-clec a mabmediated immunomodulation does not inhibit host immunity to jev, as all surviving mice produce high titer of jev neutralization ab (both igm and igg) and develop protective cellular immunity against jev infection. these observations indicate that clec a play a critical role in jev-induced pathological changes, and blockade of jev-clec a interactions by anti-clec a mabs is a promising strategy to brain damage and neuronal sequalae after jev infection ( ) . in addition to flaviviruses, we further find that clec a interacts with the hemagglutinin protein of influenza viruses (h n , h n , h n ), and blockade of influenza virus-clec a interactions by anti-clec a antibodies reduces proinflammatory cytokine production without attenuating influenza virus replication in human macrophages. furthermore, reduced levels of tnf-α and ip- were found in clec a −/− bmdms (bone marrow-derived macrophages) than those in wild type cells. upon lethal challenges with a recombinant h n influenza virus, lower levels of proinflammatory cytokines and less pulmonary cell infiltration with higher survival rates are noted in clec a −/− mice, despite comparable viral loads are noted in both wt and clec a −/− mice. these results indicate that influenza virus activates clec a to enhance inflammatory reactions and cause severe tissue damage, and combination of current anti-viral drugs with anti-clec a mab will provide better effects to reduce tissue damage and lethality ( ). clec is highly expressed by platelets and megakaryocytes and low level expression of this syk-coupled clr has also been reported in mouse dendritic cells ( ) and neutrophils ( ) . human clec is a amino acid type ii transmembrane protein containing a putative n-linked glycosylation site in the extracellular domain. in contrast to clec a, clec contains a single yxxl motif (hemitam) in its intracellular domain, and dimerization of clec upon ligand engagement, activates platelets via syk and slp- ( , , ) . the o-linked glycoprotein podoplanin has been identified as the endogenous ligand of clec . podoplanin is a mucin-type transmembrane protein expressed in type i alveolar cells, kidney podocytes, and lymphatic endothelial cells ( ) . activation of clec by podoplanin during embryonic development is required for blood/lymphatic vessel separation ( , ) , but although clec is involved in thrombus stabilization, its absence does not significantly increase bleeding tendency ( ) . clec also interacts with aggretin (rhodocytin) ( ), a snake venom protein isolated from calloselasma rhodostoma ( ) that induces platelet activation and aggregation via its binding to clec ( ) . in addition to protein ligands, clec also binds to fucoidans ( ) , which are sulfated polysaccharides mainly comprised of fucose, but also containing other monosaccharides and uronic acid ( ) . clec have been shown to capture human immunodeficiency virus (hiv) via dc-sign and clec- , thereby facilitate viral dissemination in infected patients ( ) . moreover, clec is responsible for immunothrombosis in the context of bacterial infections ( , ) . it has been reported that the absence of clec increases clinical severity in a cecal ligation and puncture (clp) model of sepsis following injection of bacterial lipopolysaccharides ( ) , and deletion of clec in this model exacerbates cytokine storm and inhibits inflammatory macrophage recruitment to the infected peritoneum, resulting in increased bacterial load and organ injury ( ) . deletion of clec also enhances the severity of brain inflammation in the mouse experimental autoimmune encephalomyelitis (eae) model, where there is evidence that the podoplanin/clec axis promotes resolution of inflammatory reactions in autoimmunity ( , ) . recently, clec was shown to be a novel pattern recognition receptor for dv, where dv infection activates platelets to express cd p, cd and to release extracellular vesicles (evs), including microvesicles (mvs) and exosomes (exos) ( ) . we have shown that dv binds to clec on platelets, promoting the release of evs, including exos (dv-exos) and mvs (dv-mvs). while exos and mvs from resting platelets do not have any activity, dv-exos and dv-mvs are potent "endogenous danger signals" which trigger the activation of clec a and tlr , respectively, to promote netosis and production of proinflammatory cytokines in neutrophils and macrophages. while blockade of clec a offers ∼ % protection rate, simultaneous blockade of clec a and tlr further increase mice survival rate up to %. these observations indicate that clec a/tlr is not critical dv-induced pathogenesis, but also plays important roles in platelet-leukocyte interactions via recognizing platelets-derived exos and mvs. thus, targeting clec a/tlr have the potential to underpin novel strategies for treating acute viral infections. it has become clear that pathogens carry multiple pamps and activate immune cells via multiple receptors. for example, dv initiates inflammatory responses through activation of both clec a and tlr associated pathways, while listeria monocytogenes and staphylococcus aureus activate nalp (nacht, lrr and pyd domains-containing protein), nlr family nlrc (card domain-containing protein ) and aim (absent in melanoma ) inflammasomes and proinflammatory cytokine release via clec a and tlr ( ) . clec has been shown to form ligand-dependent multimers with other platelet receptors to activate inflammatory signaling pathways ( ) . viral glycans contain multiple terminal sugars, including mannose, fucose, sialic acids with or without sulfation; therefore, it is not surprising that multiple lectin receptors on host cells colocalize during engagement with these pamps. it has been demonstrated that dv interacts with clec a, dc-sign (dendritic dell-specific intercellular adhesion molecule- -grabbing non-integrin), dc-signr ( ), and mannose receptor (mr) ( ) . although dv binds with much lower affinity to clec a than to dc-sign or dc-signr, only clec a has been clearly shown to mediate downstream signaling pathways after engagement with dv. dv-induced activation of clec a is dependent on dc-sign and mr ( ) and imaging analysis has revealed that engagement of dv with myeloid cells triggers colocalization of clec a and mr/dc-sign to form a hetero-multivalent complex ( ) . the lectin heterocomplex would facilitate the formation of multivalence interactions between viral glycans and ctype lectins with distinct glycan-binding affinity to enable signaling via clec a. even though the interaction between dv and clec is weak ( ) , dv also binds platelets via dc-sign ( ) . thus, dv may also trigger the formation of dv-clec -dc-sign complex to enable signaling via clec (figure ) . figure | heterocomplexes of c-type lectins in myeloid cells and platelets. dengue virus (dv) and influenza virus (h n ) are captured by the high affinity receptors dc-sign and mannose receptor (mr). the formation of heterocomplexes enables syk-mediated signaling via low affinity clec a to activate the nalp inflammasome and induce the formation of carma /bcl /malt , thereby upregulating proinflammatory cytokine production (right). dv and h n are captured by the high affinity receptor dc-sign and receptor multimerization leads to activation of the low affinity receptor clec to stimulate production of extracellular vesicles (evs). aggretin, a high affinity ligand for clec , induces ev release and platelet aggregation independently of dc-sign (left). in addition to the formation of heterocomplexes between c-type lectins, we have shown that engagement of l monocytogenes with macrophages and neutrophils induces the formation of clec a/tlr heterocomplexes. while tlr binds lipoteichoic acid from the l. monocytogenes cell wall, clec a binds to glcnac-murnac disaccharides within the backbone of bacterial wall teichoic acids ( ) . coactivation of clec a and tlr by l. monocytogenes stimulates the production of inflammasomes and induces net formation, proinflammatory cytokine expression and γδ cd + th cells differentiation ( ) . in contrast to viral infection, clec a-deficient mice are highly susceptible to infections with bacteria including l. monocytogenes and s. aureus ( ) . whilst the host immune system is beneficial in promoting the clearance of many pathogens it can also have detrimental effects in some contexts (figure ) . one of the most cited examples is the high susceptibility to bacterial infection after acute viral infections, for example with influenza virus ( ) . potential explanations for this are the association of net formation with lung injury ( ) , and the central role of inflammasome activation in viral pathology ( ) ; blockade of clec a/tlr attenuates net formation and inflammasome activation and is thus beneficial to the host during acute viral infection ( ) . moreover, excessive nets cause damages of epithelium and vascular endothelium during bacterial infections, promotes vascular occlusion and tumor cell metastasis, and enhances autoantibody production ( ) . because clec a is also responsible for cona-induced shock syndrome ( ) and synovial inflammation in collagen-induced arthritis ( ), thus bloackade of clec a/tlr may reduce bacteria-induced systemic permeability change, inhibit tumor metastasis, and attenuate tissue damages during aseptic inflammation. following our identification of clec a as the pathogenic host factor in flaviviruses, we further found that clec and tlr are pathogenic receptors in dv, jev, and h n infections. these viruses are captured by high affinity receptors (dc-sign and mr) to induce inflammatory cytokine release via activation of clec a and clec . we have also demonstrated the complex interactions between platelets and leukocytes during viral infection. these studies have demonstrated the molecular mechanisms underlying virus-induced cytokine storms and lethality and revealed a new direction for the development of treatments for acute viral infections. moreover, excessive nets contribute to the pathogenesis of autoimmune diseases ( ) , where the risk of developing autoimmune diseases is increased in patients post dv infection ( ) . thus, blockade of clec a and tlr using a bi-specific anti-clec a/tlr mab has the potential to reduce the risk of autoimmunity post-dv infection, and have therapeutic effects in autoimmune diseases by limiting excessive net formation and persistent inflammasome activation. there has been a tendency to investigate host responses to pathogen from the perspective of single pamp-receptor interactions with a focus on endosomal tlrs and cytosolic sensors that recognize highly conserved dna and rna structures. however, the complex structures of viruses contain a diversity of pamps that stimulate multiple receptors on host immune cells and modulate innate immune responses to individual viruses. furthermore, viruses frequently infect multiple cell linages, including both immune and nonimmune cells, and induce cell-cell interactions during acute viral infections. our previous studies demonstrated virusmediated activation of platelets to enhance net formation, proinflammatory cytokine release, and inflammasome activation via membrane receptors that include clec a. this supported the idea that host cells not only recognize conserved ribonucleic acid structures to stimulate production of interferons, but also recognize viral membrane components and extracellular vesicles to further promote multiple innate immune pathways. the ability of dv-evs to enhance inflammatory reactions and netosis suggest that activated evs can be regarded as novel "endogenous danger signals" or "damage-associated molecular patterns" which activate innate immunity via clec a and tlr . in this regards, clec a/tlr heterocomplex may play important roles in the pathogenesis of aseptic inflammations. this argument is further supported by the observation that clec a + cells are responsible for collageninduced autoimmune arthritis ( ) and cona-induced lethal shock syndrome ( ) . it would be interesting to test whether ev-clec a/tlr interactions contribute to the pathogenesis of aseptic inflammation and autoimmune diseases in the future. in addition to mediating acute infections, some viruses interact with innate immune receptors to establish chronic infections. in this context innate immune receptors act as "pathogenic host factors" that transduce inhibitory signals after binding virus. human hepatitis b virus (hbv) frequently causes chronic and persistent infections following initial inflammatory responses. hbv is an enveloped dna virus that infects hepatocytes and causes persistent infection due to the presence of covalently closed circular (ccc)dna, which becomes inserted into the host genome in hepatocytes. the hbv envelope contains large, middle, and small hbv surface antigens (hbsags); the large hbsag is present on virions, known as dane particles, that contain the viral genome and are able to infect and replicate after entering hepatocytes, but this is not the case for the middle and small hbsags. interestingly, the middle and small hbsags outnumber the large hbsag and it has been speculated that these un-infectious hbsags may suppress host immunity and inhibit the production of anti-hbsag ab. identification of "inhibitory" innate immunity receptor(s) for hbv may provide an answer the un-resolved questions surrounding chronic persistent hbv infection and further our understanding of the roles of viral membrane components in the complex pathogenesis of viral infections. clec a is critical for dengue-virus-induced lethal disease distinct regulation of dengue virus-induced inflammasome activation in human macrophage subsets clec a-mediated enhancement of the inflammatory response in myeloid cells contributes to influenza virus pathogenicity in vivo induction of proinflammatory cytokines in human macrophages by influenza a (h n ) viruses: a mechanism for the unusual severity of human disease? ebola virus: the role of macrophages and dendritic cells in the pathogenesis of ebola hemorrhagic fever platelet-neutrophilinteractions: linking hemostasis and inflammation thrombosis as an intravascular effector of innate immunity the role of platelets in the recruitment of leukocytes during vascular disease toll-like receptors as potential therapeutic targets for multiple diseases the critical role of toll-like receptors-from microbial recognition to autoimmunity: a comprehensive review toll-like receptor co-receptors as master regulators of the immune response induction of type i ifns by intracellular dna-sensing pathways sting regulates intracellular dna-mediated, type i interferon-dependent innate immunity protect this house: cytosolic sensing of viruses sensing and responding to cytosolic viruses invasions: an orchestra of kaleidoscopic ubiquitinations cytosolic sensing of viruses chapter : c-type lectins c-type lectins in immunity and homeostasis dc-sign (cd ) mediates dengue virus infection of human dendritic cells dendritic cell-specific intercellular adhesion molecule -grabbing non-integrin (dc-sign)-mediated enhancement of dengue virus infection is independent of dc-sign internalization signals lectin switching during dengue virus infection dc-sign (cd ) promoter− a/g polymorphism is associated with dengue hemorrhagic fever and correlated to dc-sign expression and immune augmentation transmission-blocking antibodies against mosquito c-type lectins for dengue prevention the mannose receptor mediates dengue virus infection of macrophages the c-type lectin receptors clec- and dectin- , but not dc-sign, signal via a novel yxxl-dependent signaling cascade complement-mediated neutralization of dengue virus requires mannose-binding lectin crystallization and x-ray diffraction analysis of human clec a (mdl- ), a dengue virus receptor clec a is critical for dengue virus-induced inflammasome activation in human macrophages clec a is critical for dengue virus-induced osteoclast activation and bone homeostasis clec a regulates japanese encephalitis virus-induced neuroinflammation and lethality the expression of mouse clec- on leucocyte subsets varies according to their anatomical location and inflammatory state clec- is a phagocytic activation receptor expressed on murine peripheral blood neutrophils the platelet receptor clec- is active as a dimer clec- activates syk through dimerization podoplanin-positive periarteriolar stromal cells promote megakaryocyte growth and proplatelet formation in mice by clec- essential in vivo roles of the c-type lectin receptor clec- : embryonic/neonatal lethality of clec- -deficient mice by blood/lymphatic misconnections and impaired thrombus formation of clec- -deficient platelets clec- and syk in the megakaryocytic/platelet lineage are essential for development novel platelet activation receptor clec- : from discovery to prospects a novel syk-dependent mechanism of platelet activation by the c-type lectin receptor clec- aggretin, a novel platelet-aggregation inducer from snake (calloselasma rhodostoma) venom, activates phospholipase c by acting as a glycoprotein ia/iia agonist fucoidan is a novel platelet agonist for the c-type lectin-like receptor (clec- ) fucoidan structure and activity in relation to anti-cancer mechanisms dc-sign and clec- mediate human immunodeficiency virus type capture by platelets thrombomodulation via clec- targeting inflammation drives thrombosis after salmonella infection via clec- on platelets the podoplanin-clec- axis inhibits inflammation in sepsis podoplanin is a negative regulator of th inflammation pdgf upregulates clec- to induce t regulatory cells extracellular vesicles from clec -activated platelets enhance dengue virus-induced lethality via clec a/tlr robust, sensitive and facile method for detection of f-, cn-and ac-anions platelet receptors activated via mulitmerization: glycoprotein vi, gpib-ix-v, and clec- dengue virus infection is through a cooperative interaction between a mannose receptor and clec a on macrophage as a multivalent hetero-complex structural flexibility of the macrophage dengue virus receptor clec a: implications for ligand binding and signaling dengue virus binding and replication by platelets influenza virusinduced glucocorticoids compromise innate host defense against a secondary bacterial infection high level of neutrophil extracellular traps correlates with poor prognosis of severe influenza a infection delayed oseltamivir plus sirolimus treatment attenuates h n virus-induced severe lung injury correlated with repressed nlrp inflammasome activation and inflammatory cell infiltration neutrophil extracellular traps in immunity and disease activation of mdl- (clec a) on immature myeloid cells triggers lethal shock in mice myeloid dap -associating lectin (mdl)- regulates synovial inflammation and bone erosion associated with autoimmune arthritis neutrophil extracellular traps (nets) in autoimmune diseases: a comprehensive review increased risk of autoimmune diseases in dengue patients: a population-based cohort study the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © sung and hsieh. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -z r w k authors: stella, alessandro; lamkanfi, mohamed; portincasa, piero title: familial mediterranean fever and covid- : friends or foes? date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: z r w k familial mediterranean fever (fmf) and covid- show a remarkable overlap of clinical symptoms and similar laboratory findings. both are characterized by fever, abdominal/chest pain, elevation of c-reactive protein, and leukocytosis. in addition, colchicine and il- inhibitors treatments that are effective in controlling inflammation in fmf patients have recently been proposed for off-label use in covid- patients. thus, fmf may resemble a milder recapitulation of the cytokine storm that is a hallmark of covid- patients progressing to severe disease. we analyzed the sequence of the mefv-encoded pyrin protein – whose mutations cause fmf- in mammals, bats and pangolin. intriguingly, although pyrin is extremely conserved in species that are considered either a reservoir or intermediate hosts for sars-cov- , some of the most common fmf-causing variants in humans are present as wildtype residues in these species. we propose that in humans, pyrin may have evolved to fight highly pathogenic infections. the world health organization reported a novel coronavirus on december , as the cause of a cluster of pneumonia cases in the city of wuhan in the hubei province of china. since then, the severe acute respiratory syndrome coronavirus (sars-cov- ) has infected nearly million individuals worldwide causing more than , deaths during the past months of the covid- pandemic. among european countries, italy has been severely hit by covid- ( ) . since the initial outbreak, a huge body of clinical and scientific information has been accumulated on covid- , a multifaceted disease hitting not only lungs, but also other organs, with different defined stages ( , ) . in most cases, sars-cov- enters the human body through inhaled droplets and aerosols. although contact with contaminated surfaces has been hypothesized as a second possible infection route, the importance of this alternative mode of infection has not been assessed systematically ( , ) . upon infection, sars-cov- enters its target cells via: (a) binding of its spike protein (s) to angiotensin converting enzyme (ace ); (b) activation through proteolysis of the viral s protein catalyzed by the cellular transmembrane protease serine (tmprss ); and (c) fusion of sars-cov- virus with the host cell membrane. a wide variation in allele frequencies at ace expressions single nucleotide polymorphisms (esnps) loci can partly explain the differences in covid- prevalence across different countries ( ) . also, differential expression of ace occurs in several human cancers and chronic diseases, possibly influencing covid- susceptibility and severity ( ) . the involvement of ace and tmprss in viral cell entry has been exploited to plan experimental therapies based on using protease inhibitors such as camostat mesylate or nafamostat to block sars-cov- entry into cells ( , ) . once inside the host cells, the sars-cov- positive single stranded rna (ssrna) genome begins replication and cytoplasmic accumulation. the ssrna or its double stranded intermediate (dsrna) are recognized by the innate immune nucleic acid sensing systems whose activation exerts a first antiviral response through the production of type interferons and the secretion of pro-inflammatory cytokines. the immunomodulation at this early stage of infection might determine the growth of the viral load, with most infected individuals still being asymptomatic or paucisymptomatic, while almost % of sars-cov- positive patients develop fever, coughing, occasionally ageusia and anosmia and even gastrointestinal symptoms ( ), with or without hepatic involvement ( ) . patients progressing to the second phase show a strong immunological and hyperinflammatory response that is defined as a "cytokine storm" and which may lead to respiratory worsening and bilateral pneumonitis. in this second stage, covid- patients manifest symptoms mimicking those present in patients with auto-inflammatory diseases such as fever, arthralgia, leucopenia and myocarditis ( ) ( ) ( ) . we were intrigued by the remarkable overlap between these clinical manifestations and some of the typical manifestations of familial mediterranean fever (fmf), a largely recessively inherited monogenic inflammasomopathy (autoinflammatory disorder involving the inflammasome) caused by mutations in the mefv gene that is particularly prevalent in the mediterranean basin ( ) . while the previously mentioned clinical signs are not specific to fmf and shared with other hereditary recurrent fevers and inflammatory diseases, yet they represent an indication of similarities among fmf pathogenesis and the hyperinflammatory response observed in covid- patients. worthy of note, taste alteration has been reported amongst the prodromal manifestations preceding fever attacks in fmf ( , ) . we should stress that the cytokine storm observed in the inflammatory stage of covid , has been reported in other autoinflammatory diseases (aids) such as the macrophage activation syndrome (mas) and adult onset still's disease ( ) . however, authors have often reported genetic heterogeneity and overlap between these aids and hereditary recurrent fevers ( ) ( ) ( ) . colchicine, a natural alkaloid from colchicum autumnale, has a long history as a drug to treat pain and swelling since ancient egypt. it is nowadays used to treat fmf, gout, behçet syndrome and recurrent non-infective pericarditis. unsurprisingly, it is currently being investigated in several covid- therapeutic trials ( ) ( ) ( ) . notably, in colchicine-resistant or colchicineintolerant fmf patients, alternative treatments include biologics that neutralize the pro-inflammatory cytokine interleukin (il)- β directly (canakinumab) or inhibit il- β-mediated activation of the il- receptor (anakinra and rilonacept) ( ) ( ) ( ) . similarly to colchicine, several ongoing trials are evaluating the use of il- pathway inhibitors to treat covid- patients ( ) ( ) ( ) ( ) . although first results from covid- patients treated with these repurposed drugs have been conflicting ( ) ( ) ( ) , the role of pyrin (the inflammasome sensor protein that is encoded by mefv) in modulating severity and outcome of covid- is still unknown. the pyrin domain architecture shows intriguing features which may add insights into its possible role in covid- disease. pyrin has a n-terminal pyrin domain (pyd) that is frequently found in other innate immune pathogen sensors that mount inflammasome responses such as nlrp , nlrp , and aim . the n-terminal pyd domain engages in homotypic interactions with its pyd counterpart in the adaptor protein apoptosisassociated speck-like protein with a caspase recruitment domain (asc) to assemble asc specks, which recruit the inflammatory protease procaspase- inducing its self-cleavage and activation ( ) . caspase- in turn matures the proinflammatory cytokines il- β and il- and cleaves gasdermin d to trigger a lytic cell death mode termed pyroptosis that promotes secretion of aforementioned cytokines along with danger-associated molecular patterns (damps) such as il- alpha, hmgb and atp ( ) . pyroptosis is a double-edged sword with both antiviral and proviral activities during viral infections ( ) . in fact, cell death can lead to halting viral replication and infection, frequently at the price of increased inflammation. conversely, dead cells release a large number of viral particles contributing to viral dissemination. the mefv encoded pyrin sensor contains in its central region three domains: a bzip domain (aa - ), a b-box domain (aa - ) and a coiled-coil domain (cc, aa - ). the role of these three domains has not been thoroughly investigated and few fmf-causing variants localize to pyrin's central region ( ) . this region may have an autoinhibitory role precluding the pyd domain from interacting with asc and activating pyroptosis ( ) . the c-terminal b . (also known as pry/spry) domain is extremely important in fmf pathogenesis since most of the disease-penetrant mefv variants cluster in this region ( ) . although this uneven distribution of fmf-associated mefv mutations suggests that the b . domain is crucial in regulating pyrin inflammasome activity, the precise molecular mechanisms by which the b . domain regulates fmf pathogenesis have not been elucidated ( ) . likely, fmf-causing mutations in the b . domain may derail intramolecular interactions that keep pyrin in an autoinhibitory state. interestingly, the degree of amino acid conservation along the pyrin protein sequence is rather variable and may offer some insights in the dangerous liaisons between fmf and covid- . we aligned the mefv-encoded pyrin amino acid sequences from different species including the only pangolin and all bat species sequences available in genbank (figure ) . bats and pangolins have been considered the reservoir and intermediate host, respectively, of sars-cov- before transmission to humans. the alignment of the pyrin sequences presented unique evolutionary features (figure ). in fact, some of the most prevalent fmf-associated mutations in human pyrin were present as wild type in all bat species analyzed and in pangolin (v a, r h). this resembles previous findings that some fmf-associated mutations are retrieved as wild type in primates, suggesting evolutionary pressure on pyrin ( ) . in contrast, other fmf-associated amino acids residues that are largely prevalent in middle eastern populations (m i, m i, m v) were not observed in bats and pangolin. worthy of note m i, m v, m i, and r h were all associated with derailed pyrin-induced il- β secretion in a recently developed blood-based functional test for fmf alleles ( ) . it is tempting to speculate that fmf patients carrying v a and r h variants-which represents the wild type residues in all bats and pangolin sequences-might modulate better their cytokine response to sars-cov- infection. further, in a mouse knock-in model the v a variant causes a more severe fmf phenotype compared to m v, m i, and elevated production of multiple cytokines ( ) . given that the m i and m v/i alleles in patients provoke a hyperinflammatory response not different from v a and r h (at least in fmf), one may hypothesize that they could warrant a comparable attenuation of viral infection. an elaboration on this hypothesis would stem from the increased fmf severity associated to m i and m v/i mutations. this raised level of inflammation can either move the balance toward excess inflammation in covid- or be causing an even improved immunomodulation in covid- compared to v a, r h. moreover, historic pandemics and different pathogens may have selected for different mefv variants in their respective host species and populations. thus, a pathogen different from coronaviruses (i.e., yersinia pestis) might have selected the m i, m v/i mutations in humans but not in bats. indeed, confirming this hypothesis, and shortly after the submission of this work, genetic and experimental evidences have been reported linking these mefv variants with resistance to yersinia pestis ( , ) . the e q and r q mefv variants, which according to a current consensus are considered neutral polymorphisms, showed a response to colchicine challenge alike normal controls ( ) . these two variants presented rather divergent evolutionary features. while the glutamic acid at position was strictly conserved in all species analyzed, the arginine at position was conserved in pangolin and in of bat species, and changed to a glutamine in mouse and rats (figure ) . therefore, e q and r q which appear to be neutral in functional assays seem to be under apparently different evolutionary pressures. the frequency of mefv mutations and polymorphisms shows a great variability in countries where fmf has a high frequency. in fact, the m v and m i, are prevalent among turks, non-ashkhenazi jews, and arabs, while the m i is frequent in the armenian population ( ) ( ) ( ) ( ) . the v a and r h are generally associated with a milder phenotype, and generally reported in clusters of fmf patients of ashkhenazi jewish origin, and in the western mediterranean area ( ) . the e q variant, whose pathogenicity is still debated, also presents a wide variation in frequency across different populations ( ) . this peculiar distribution of the mefv variome led to the hypothesis that the mefv gene has been subjected to a balancing selection and nucleotide variation, particularly in the b . region, is adaptive ( ) . thus, the severity of covid- disease in fmf patients, once infected, might be influenced, at least partially, depending on specific mefv genotypes which shows country-specific differences. the mefv gene displays other intriguing evolutionary features. in fact, while the n-terminal pyd domain is conserved across all species, the b . domain appears of more recent origin. of note, when considering the entire pyrin amino acid sequence, human pyrin is more closely related to its bats and pangolin homologs than to other mammal species (figure ) . in contrast, the human nlrp protein presented a high level of homology in all analyzed species. it clustered with other mammalian nlrp proteins in the phylogenetic tree and was more distantly related to bat sequences (figure ) . in addition, differently from mefv, the most common human nlrp mutations-r w, d n, t m, and l p-which represent more than % of the total mutation burden for this gene ( , ) , were never present as wild type amino acid residues in all bat species and pangolin ( figure ) . therefore, considering the pyrin inflammasome a passive bystander in sars-cov- infection could lead to overlooking an important innate immune pathway that coordinates the response to pathogens. in fact, inflammasome-driven pyroptosis is one of the results of the cytokine storm which concurs to the high pathogenicity of both sars-cov, and sars-cov- ( , ). recent findings demonstrated that the sars-cov orf a protein can provoke a cytokine storm by activation of the nlrp inflammasome through traf -dependent ubiquitination of asc ( ) . a recent analysis of sars-cov- strains showed that non-synonymous substitutions in the sars-cov- orf a protein may alter virulence and infectivity ( ) . additional viral proteins are able to activate the nlrp inflammasome. the nlrp -mediated response to influenza a virus (iav) has been extensively studied and plays a crucial role in protecting the host while helping in clearing the infection. however, if the inflammatotory response is particularly prolonged and excessive it can increase disease burden ( ) . the nlrp inflammasome plays a critical role in guarding against iav infection and reducing lung damage consequent to infection ( , ) . other viruses are capable of activating the nlrp inflammasome. the viroporin b, released upon human rhinovirus infection (hrv), causes proteolytic activation of procaspase- and il- ß secretion in a nlrp dependent manner ( ) . several other viruses can provoke a sustained response from the nlrp inflammasome as extensively reviewed ( ) . similarly to the mefv-encoded pyrin, the n-terminal pyd domain of nlrp can recruit asc via homotypic interaction with its pyd counterpart on asc to assemble asc filaments. the assembly of asc filaments in macromolecular structures known as asc specks allow the recruitment of procaspase- to induce its self-cleavage and activation. caspase- in turn cleaves the proinflammatory and the answer is. . .. before the covid- pandemic, a recent report ( ) demonstrated that bats, when infected with different zoonotic figure | alignment of nlrp orthologs from amino acid to amino acid (human gene). the four residues whose mutations are responsible of more than % of nlrp -associated autoinflammatory diseases are boxed. frontiers in immunology | www.frontiersin.org september | volume | article viruses, can sustain high viral loads while presenting a dampened nlrp -mediated inflammatory response associated with a decrease in both asc speck formation and il- β secretion. hence, a dysregulation of the nlrp inflammasome can also contribute to the utterly complex individual immune response to viral infections ( ) . these findings suggest that the modulation of innate immunity rather than an enhanced antiviral defense might shape the different outcome of covid- disease. thus, we hypothesize that competition between pyrin and the nlrp inflammasomes for asc recruitment may tilt the balance between a cytokine storm and a finely adjusted protective inflammation (figure ) . fmf, in which pyrin activity and consequent asc oligomerization are increased because of mefv pathogenic variants, may therefore represent a unique opportunity as a disease model to investigate the regulation of the inflammatory response to novel emerging viruses. we presented here the unique evolutionary features of the mefv-encoded pyrin suggesting its putative contribution in shaping the individual risk to develop severe complications consequent to infectious diseases. several factors could have contributed to the rapid spreading of covid- pandemic, including access to adequate health care, aging demographic, metabolic dysfunctions, socio-cultural differences. it would be wise not to discard individual genetics, including the mefv gene, from the mix. future investigation on how carriers of different mefv genotypes have responded to coronavirus infections could help in refining existing and novel therapeutics in development for present and future challenges. in conclusion, the answer to the title question might not be blowing in the wind but could hopefully be found in a deeper knowledge of inflammasome regulation in well-known inflammatory diseases. the datasets presented in this study can be found in online repositories. the names of the repository/repositories and accession number(s) can be found in the article/ supplementary material. as designed the study and drafted the manuscript. ml and pp drafted the manuscript. all authors contributed to the article and approved the submitted version. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material covid- , internists and resilience: the north-south italy outbreak tocilizumab: from the rheumatology practice to the fight against covid- , a virus infection with multiple faces covid− : focus on the lungs but do not forget the gastrointestinal tract epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study clinical features of patients infected with novel coronavirus in wuhan comparative genetic analysis of the novel coronavirus ( -ncov/sars-cov- ) receptor ace in different populations systematic profiling of ace expression in diverse physiological and pathological conditions for covid- /sars-cov- 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responses to influenza a virus via the regulation of caspase- the nlrp inflammasome mediates in vivo innate immunity to influenza a virus through recognition of viral rna rhinovirus-induced calcium flux triggers nlrp and nlrc activation in bronchial cells response of host inflammasomes to viral infection dampened nlrp -mediated inflammation in bats and implications for a special viral reservoir host severe covid- : nlrp inflammasome dysregulated key: cord- -ecmayzr authors: savarin, carine; dutta, ranjan; bergmann, cornelia c. title: distinct gene profiles of bone marrow-derived macrophages and microglia during neurotropic coronavirus-induced demyelination date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: ecmayzr multiple sclerosis (ms) is a chronic inflammatory disease of the central nervous system (cns) characterized by demyelination and axonal loss. demyelinating lesions are associated with infiltrating t lymphocytes, bone marrow-derived macrophages (bmdm), and activated resident microglia. tissue damage is thought to be mediated by t cell produced cytokines and chemokines, which activate microglia and/or bmdm to both strip myelin and produce toxic factors, ultimately damaging axons and promoting disability. however, the relative contributions of bmdm and microglia to demyelinating pathology are unclear, as their identification in ms tissue is difficult due to similar morphology and indistinguishable surface markers when activated. the cd t cell-induced autoimmune murine model of ms, experimental autoimmune encephalitis (eae), in which bmdm are essential for demyelination, has revealed pathogenic and repair-promoting phenotypes associated with bmdm and microglia, respectively. using a murine model of demyelination induced by a gliatropic coronavirus, in which bmdm are redundant for demyelination, we herein characterize gene expression profiles of bmdm versus microglia associated with demyelination. while gene expression in cns infiltrating bmdm was upregulated early following infection and subsequently sustained, microglia expressed a more dynamic gene profile with extensive mrna upregulation coinciding with peak demyelination after viral control. this delayed microglia response comprised a highly pro-inflammatory and phagocytic profile. furthermore, while bmdm exhibited a mixed phenotype of m and m markers, microglia repressed the vast majority of m -markers. overall, these data support a pro-inflammatory and pathogenic role of microglia temporally remote from viral control, whereas bmdm retained their gene expression profile independent of the changing environment. as demyelination is caused by multifactorial insults, our results highlight the plasticity of microglia in responding to distinct inflammatory settings, which may be relevant for ms pathogenesis. introduction multiple sclerosis (ms) is a chronic inflammatory disease of the central nervous system (cns), characterized by demyelination and axonal damage. active demyelinating lesions are characterized by cd t cells, cd t cells expressing both th and th cytokines, bone marrow-derived macrophages (bmdm) and activated cns resident microglia ( , ) . myeloid cells activated by t cell effector functions are thought to participate in tissue damage by removing or "stripping" myelin ( ) , and secreting toxic factors, such as reactive oxygen species, nitric oxide and the pro-inflammatory cytokines, tumor necrosis factor (tnf), and il- β ( , ) . activated microglia also secrete chemokines, which recruit innate and adaptive immune cells into the parenchyma, further amplifying the destructive inflammatory response ( ) . however, both bmdm and microglia effector functions are highly heterogeneous depending on the environment and may not only contribute to disease progression but also to resolution ( , ) . for example, by removing apoptotic cells and debris, their phagocytic activity favors tissue repair and is essential for disease resolution ( ) . in addition, both cell populations secrete anti-inflammatory cytokines, such as il- and tgf-β, as well as trophic factors, which provide an environment that promotes tissue repair and neuronal protection ( ) . the heterogeneity of the inflammatory response associated with ms lesions at the cellular and functional levels, thus makes it difficult to establish detrimental versus disease resolving functions of bmdm and microglia in ms pathogenesis. in addition to the inherent limitations associated with sampling cns tissues for longitudinal studies, the individual role of bmdm versus microglia as pathological mediators remains ambiguous due to morphological similarities and lack of reagents uniquely identifying each population. however, increasing evidence from animal models supports the concept that microglia and bmdm comprise two effector populations with distinct origins (derived from progenitors in the embryonic yolk sac and circulating monocytes respectively) and functions during ms and other neuroinflammatory disorders ( ) . a variety of murine models, including autoimmune-and viral-induced demyelination, have been developed to study pathogenic features of ms ( ) . the most common is the experimental autoimmune encephalomyelitis (eae), an autoreactive cd t cell-induced autoimmune demyelination characterized by infiltration of myelin-specific th and th cells, bmdm and microglial activation ( , ) . pathogenesis during eae is associated with temporally distinct microglial activation and bmdm cns infiltration. early microglia activation is insufficient to trigger clinical disease, whereas delayed cns recruitment of bmdm directly correlates with disease progression. importantly, depletion of bmdm but not microglia inhibits eae ( , ) . similarly, mice deficient in ccl (ccl −/− ), a chemokine essential for inflammatory monocyte recruitment into the cns ( ) , are resistant to eae ( ) . in support of detrimental bmdm functions, a combined histological and gene profiling study showed that demyelination is mediated by bmdm associated with nodes of ranvier, whereas debris clearance is achieved by microglia ( ) . altogether, studies in the eae model demonstrate that bmdm recruitment into the cns is essential for the process of myelin loss and clinical manifestation. inflammatory demyelination is also induced following infection with two natural viral mouse pathogens, theiler's murine encephalomyelitis virus (tmev) and members of the neurotropic mouse hepatitis viruses (mhv). tmev infection induces an autoimmune disease in which bmdm are essential for both viral persistence and demyelination ( , ) . however, the function of bmdm as a main reservoir of active viral replication during chronic tmev infection, limits efforts to assess their role in demyelination independent of virus load ( ) . in contrast, infection with the non-lethal glia tropic mhv strain designated jhmv predominantly targets oligodendrocytes (olg) and to a lesser extent microglia and astrocytes. viral replication peaks at day post infection (p.i.), but infectious virus is reduced below detection by day p.i. acute infection initiates rapid cns recruitment of predominantly bmdm, but also neutrophils and nk cells, followed by infiltration of both cd and cd t cells, as observed in active ms lesions. the t cell response, which is essential to reduce viral replication, is highly th polarized with no evidence of il- or gm-csf production ( ) ( ) ( ) . importantly, t cell-mediated virus control coincides with initiation of demyelination, which peaks between days - p.i. after infectious virus is cleared ( , ) . although olg tropism is a requirement for demyelination, immunodeficient mice demonstrated that infection of olg in the absence of adaptive immunity is insufficient to cause demyelination. however, transfer of either virus-specific cd or cd t cells into virus infected immunodeficient mice leads to demyelination ( , ) . furthermore, ifn-γ dependent control of infectious virus within olg and no evidence for olg apoptosis, suggested that direct t cell-mediated cytolysis of olg does not play a major role in myelin loss ( ) . this implicates t cell activated bmdm and microglia as the most probable mediators of myelin destruction. moreover, both myeloid populations are abundant in lesions and occasionally associated with damaged axons ( ) . however, in contrast to eae, genetic or chemical depletion of monocytes during jhmv infection does not alter disease severity, virus replication or myelin loss ( , ) , suggesting that bmdm are dispensable for jhmv-induced demyelination. this study takes advantage of the distinct tissue environments established during eae and jhmv infection to characterize temporal alterations in gene expression profiles of bmdm versus microglia in a th biased demyelination model. to date, we are not aware of any reports evaluating the signature profile of microglia associated with pathogenic functions during demyelination. the results reveal that cns infiltrating bmdm rapidly establish a characteristic profile including m and m markers, which prevails throughout infection as the population declines. by contrast, gene expression in microglia is only prominently altered remote from viral control concomitant with demyelination; distinct from bmdm, the gene expression pattern is skewed to a highly pro-inflammatory and phagocytic profile. the results overall highlight the plasticity of microglia responses in distinct inflammatory settings, which may be relevant for ms pathogenesis at distinct stages of disease. wild-type (wt) c bl/ mice were purchased from the national cancer institute (frederick, md, usa). homozygous ccl deficient (ccl −/− ) mice were originally obtained from b. j. rollins (dana-farber cancer institute, boston, ma, usa). cx cr gfp/gfp (b . p-cx cr tm litt /j) and ccr rfp/rfp (b . (cg)-ccr tm . ifc /j) mice were purchased from the jackson laboratory (bar harbor, me, usa) and crossed to generate cx cr gfp/+ ccr rfp/+ mice. transgenic mice were bred and maintained at the biological research institute under sterile conditions. all procedures were preformed in compliance with the cleveland clinic institutional animal care and use committee approved protocols. the glia tropic jhmv neutralizing monoclonal antibody (mab)derived . v- variant was used for all infections ( ) . mice of both sexes between and weeks of age were infected in the left hemisphere with , pfu of jhmv diluted in endotoxin-free dulbecco's phosphate-buffered saline (pbs) in a final volume of µl. mice were monitored daily for clinical disease severity according to the following scale: , healthy; , hunched back and ruffled fur; , partial hind limb paralysis or inability to maintain the upright position; , complete hind limb paralysis; , moribund or dead. for analytical flow cytometry, anesthetized mice were perfused with ice-cold pbs, and resected brains and spinal cords homogenized using a ten-broeck tissue grinder as described ( ) . tissue homogenates were adjusted to % percoll (pharmacia, uppsala, sweden) and underlaid with ml % percoll prior to centrifugation at g for min at °c. cns mononuclear cell were recovered from the / % interface, washed and resuspended in facs buffer (pbs + % bovine serum albumin). cells were blocked with anti-mouse cd /cd (clone . g ) mab for min on ice prior to staining. staining was performed for min on ice using fluorescein isothiocyanate, phycoerythrin, peridin chlorophyll protein complex (percp), or allophycocyanin (apc) conjugated mab (all from bd biosciences except where indicated) specific for cd (clone ly- ), cd b (clone m / ), f / (serotec, raleigh, nc, usa) and major histocompatibility complex (mhc) class ii (clone g ). cells were then washed twice in facs buffer prior to analysis using a bd accuri flow cytometer (bd biosciences) and flowjo software (tree star inc., ashland, or, usa). for cell purification, spinal cords from pbs-perfused cx cr gfp/+ ccr rfp/+ mice were finely minced with a razor blade. minced tissues were enzymatically digested in rpmi medium containing % fetal calf serum, . % collagenase d ( mg/ml) roche, basel, switzerland and % dnase i ( mg/ml) (sigma aldrich, st. louis, mo, usa) for min at °c. collagenase was then inactivated by addition of % . m edta for min at °c prior centrifugation at g for min at °c. spinal cord-derived cells from seven mice were pooled and isolated using percoll gradients as described above and then stained with cd b-percp and cd -apc for min on ice. spinal cord-derived bmdm (cd hi cd b + ccr rfp+ ) and microglia (cd low cd b + cx cr gfp+ ) were purified using a facsaria cell sorter (bd biosciences) and resuspended in trizol. yields from -pooled mice ranged between . - × cells for bmdm and . - . × cells for microglia depending on the time p.i. microglia from naïve mice were used to assess baseline expression, whereas circulating monocytes were used as controls for cns infiltrated bmdm after infection. monocytes were isolated from blood treated with gey's solution to lyse red blood cells prior to staining and cell sorting. gene expression profiling using ncounter analysis rna was prepared by extraction with trizol reagent (invitrogen, carlsbad, ca, usa) and direct-zol rna mini prep (zymo research, irvine, ca, usa) according to the manufacturer's instructions. gene expression profiles were analyzed using the ncounter mouse myeloid innate immune panel comprising targets representing all major myeloid cell types and generated according to the manufacturer's protocol (nanostring technologies, seattle, wa, usa). the nanostring ncounter system directly captures and counts individual mrna transcripts using a multiplexed measurement system thereby omitting cdna based amplification ( ) . analysis was performed using nsolver analysis software v . and ingenuity pathway analysis (qiagen, hilden, germany). venn diagrams from individual gene lists and protein-protein interaction networks were constructed using genespring (agilent, inc.) and string software (http://www. string-db.org). to confirm validity of nanostring ncounter analysis, a small set of selected genes were analyzed by real-time pcr ( figure s in supplementary material). following rna extraction as described above, first-strand cdna was synthesized using reverse transcriptase (invitrogen) with oligo-dt and random primers (promega, madison, wi, usa) as described ( ) . gene expression analysis was performed using a fast real-time pcr system (applied biosystems, foster city, ca, usa), sybr green master mix (applied biosystems) and the following primers: gapdh, ′-catggccttccgtgttccta- ′ (forward) and ′-atgcctgcttcaccaccttct- ′ (reverse); il , ′-tg aggctggcattcatgtctt- ′ (forward) and ′-tccagtt ggcctctgttttagg- ′ (reverse); il rn ′-agatagacatg gtgcctattgacctt- ′ (forward) and ′-catctccagac ttggcacaaga- ′ (reverse) and arg ′-tgggtggatgct cacactga- ′ (forward) and ′-caggttgcccatgcaga tt- ′ (reverse). transcripts levels were normalized to the housekeeping gene gapdh and converted to a linearized value using the following formula: (c t gapdh-c t gene) × , , where ct represents the threshold cycle value. following pbs perfusion, spinal cords were fixed in % neutral buffered formalin, embedded in paraffin and sections stained with luxol fast blue as described to visualize demyelination ( ) . for analysis of iba + cells spinal cords from ice-cold pbs-perfused mice were quickly embedded in oct and kept at − °c until µm sections were prepared. sections were fixed with paraformaldehyde for min, treated with blocking solution for min and then stained with rabbit anti-iba mab (wako, osaka, japan) overnight at °c. goat anti-rabbit secondary ab (invitrogen) was added for h at room temperature and sections mounted with vectashield mounting medium (vector laboratories, burlingame, ca, usa). sections were analyzed using a leica tcs confocal microscope. to better characterize reactivity of microglia and infiltrating bmdm following jhmv infection, we initially monitored cns infiltration of bmdm, as well as upregulation of mhc class ii as an activation marker on both cns bmdm and microglia by flow cytometry. bmdm with a typical cd hi cd b + f / + phenotype comprised the majority of inflammatory leukocytes as early as day p.i. and then progressively decreased as virus replication is controlled by t cells. at the onset of demyelination at day p.i., the bmdm population stabilized at ~ % of the infiltrating leukocytes ( figure a ). bmdm initially infiltrated as mhc class ii lo expressing cells, but the vast majority upregulated mhc class ii by day p.i. mhc class ii expression on microglia was sparse at days and p.i., but rapidly increased by day p.i. and then gradually declined by day p.i. (figure a) . these kinetics supported that microglia and bmdm activation peaks delayed relative to peak bmdm accumulation and coincides with peak t cell ifn-γ production ( , ) . enhanced activation of microglia at day p.i., compared to earlier times p.i., was also supported by progression of morphological changes, evidenced by enlarged cell bodies and retracted and thickened processes ( figure b) . the decline of bmdm, but an ongoing activation phenotype of microglia at the time of evident demyelination implicated microglia as mediators of tissue damage during jhmv encephalomyelitis. biochemical depletion of peripheral monocytes indeed supported that bmdm are not essential to tissue destruction in jhmv-infected mice ( ) . data from our own laboratory further demonstrated that the chemokine ccl is essential for bmdm accumulation within the cns ( ) . the absence of ccl resulted in an ~ % reduction of bmdm at all time points, including day p.i. ( ) and ( figure c ) when demyelination is prominently evident in wt mice. nevertheless, microglia activation, as monitored by mhc class ii expression, was independent of ccl ( figure d ). most importantly, the absence of ccl -dependent bmdm within the cns did not alter demyelination ( figure e ). similar myelin loss at day p.i. comparing wt and ccl −/− infected mice supported the concept that microglia mediate demyelination during jhmv infection. we next evaluated effector functions of bmdm versus microglia associated with jhmv-induced demyelination by comparing gene expression profiles using ncounter analysis of mrna isolated from purified bmdm and microglia of infected cx cr gfp/+ ccr rfp/+ mice. characteristic expression of cx cr gfp and ccr rfp on cd high cd b + bmdm (population # ) and cd low cd b + microglia (population # ) is shown in figure throughout days - p.i. microglia were characterized by high expression of cx cr and undetectable ccr expression (figures b,c) similar to other inflammatory models ( , ) . in contrast, cns infiltrating bmdm expressed ccr and low levels of cx cr compared to microglia ( figure b ). co-expression of ccr and cx cr was maintained on bmdm at all time points p.i. and no cx cr − cells were detectable ( figure c ). as both microglia and infiltrating bmdm retained their phenotype throughout infection, cd low cd b + cx cr gfphi ccr − and cd hi cd b + cx cr gfplow ccr + populations were isolated by facs from spinal cords at days , , , and p.i. for subsequent mrna expression analysis. age-matched naïve animals were used to isolate microglia and blood circulating monocytes as precursors of cns-infiltrating bmdm. gene expression profiles for all purified populations were obtained using ncounter analysis and the innate myeloid immune panel. the respective naïve populations were used to assess signature gene expression profiles under homeostatic conditions (figure ) . figure a shows the top highly expressed genes within each population relative to three ncounter platform housekeeping genes, namely g pdx, polr b, and tbp, selected for three high, medium, and low expression, respectively, in this part of analysis platform. figure b lists the top enriched genes specific for microglia compared to monocytes, or monocytes versus microglia, respectively. among the top genes highly expressed in microglia, were also specific and included genes of the complement cascade (c qa, c qb, c qc, and c ar ) and trem , encoding a cell surface receptor involved in phagocytic functions and known to be expressed by microglia ( ) . other genes, such as adamts , f r or hpgds, found within the top enriched genes expressed by microglia ( figure b ) were also previously described as microglia specific ( , ) . cx cr mrna encoding the fractalkine receptor and used as a marker for microglia ( ) , was also among the top highly expressed genes ( figure a) , but not unique, consistent with the cx cr lo phenotype on circulating monocytes. similarly, ccr expression characteristic of monocytes was confirmed by ccr mrna as the second in place of the top expressed genes specific for circulating monocytes (figure b) . other specific signature genes of monocytes are related to motility and migration/tissue invasion, e.g., s a , s a , s a , fn , sema , mmp , and to a lesser extent mhc class ii antigen presentation, e.g., h -ab , cd , and fas. microglia and circulating monocytes also shared highly expressed genes, including genes characteristic of the myeloid lineage such as csf r, a gene coding for a cell surface receptor essential for hematopoietic figure a ). interestingly however, three genes among the common and highly expressed genes were regulated differently. ctss mrna, encoding for cathepsin s, a lysosomal cysteine proteinase participating in the mhc class ii molecule antigen presentation pathway as well as nociception ( , ) , was specifically upregulated within bmdm, with highest levels observed at days and p.i. (figure a) , when demyelination increases. by contrast, microglia transiently downregulated ctss mrna at day p.i. opposite regulation was also observed for dusp mrna (figure a ). dusp mrna encodes the dual specificity protein phosphatase , an enzyme involved in the cellular stress response and a negative regulator of cell proliferation ( ) . while dusp mrna levels were vastly upregulated early following bmdm accumulation, but declined by day p.i. and thereafter, levels were rapidly downregulated within microglia ( figure b) . finally, tyrobp mrna encoding for the trem adaptor dap and known to regulate microglia phagocytic functions ( ) , was downregulated in bmdm throughout infection, but specifically upregulated within microglia at days and p.i. (figure b ). this expression pattern on microglia correlated with the onset of myelin loss and supported trem signaling specifically by microglia in response to tissue damage. to determine whether apparently differential functions of bmdm and microglia associated with jhmv-induced demyelination are reflected in distinct gene profiles, we monitored overall up-and downregulation of gene expression relative to basal levels in each population. analysis times were chosen to correlate with innate responses (d p.i.), peak t cell effector function (d p.i.), resolution of infection and initiation of demyelination (d p.i.), and finally viral clearance and overt demyelination (d p.i.). at day p.i. a higher number of genes were differentially regulated within infiltrating bmdm compared to microglia ( versus ; figure a ). moreover, almost % of the genes showing altered expression in early infiltrated bmdm were increased compared to basal levels, while only % were increased in microglia; the remaining differentially expressed mrnas were decreased ( figure b) . the overall number of differentially expressed genes slightly declined in bmdm by day p.i., when t cells exert maximal effector function ( ) , and remained fairly constant throughout day p.i. (figure a) . furthermore, the relative decline in the proportion of upregulated mrnas coincided with an increased proportion of downregulated genes, reaching a roughly equal distribution at days - p.i., when virus is largely controlled (figure b ). in contrast, microglia altered their gene expression pattern extensively at day p.i. ( genes, figure a ) with % of differentially regulated genes showing increases ( figure b ). by day p.i., overall altered gene expression remained stable relative to day p.i., with equal proportions showing increases and decreases. however, as myelin loss progresses by day p.i., differentially regulated genes increased again in numbers, with the proportion of upregulated mrnas reaching % (figure a ). altogether these data show unique regulation of gene profiles in bmdm compared to microglia throughout the course of infection. while most changes were evident in bmdm following initial cns accumulation, microglia revealed most pronounced changes at the time of myelin loss. we further analyzed differential gene expression across time points focusing on upregulated genes using venn diagrams to reveal the relative proportion of genes that were commonly increased at all time points (figure c ). of the upregulated genes in bmdm at day p.i., were unique to day . on the other hand, genes (representing % of all upregulated genes) remained highly expressed at all other time points (figure c ). of note, not a single gene transcript was specifically upregulated at day p.i., and only three overlapped with sustained upregulation at days and p.i. similarly, only gene transcripts were specifically elevated at day p.i., were unique to both days and , and only one was unique to day p.i. these results indicate that the gene expression profile characterizing bmdm is established early following infection, with sparse unique alterations as bmdm decline during infection. in stark contrast, only of gene transcripts upregulated in microglia at day p.i. were unique to day , and no gene transcript was commonly upregulated across all time points analyzed. furthermore, distinct from bmdm, gene transcripts were figure | gene expression characterizing microglia and circulating monocytes under homeostatic conditions. spinal cord-derived microglia (cd low cd b + cx cr gfp+ ) and circulating blood monocytes (cd b + ccr rfp+ ) were purified from naïve cx cr gfp/+ ccr rfp/+ mice by facs and rna subjected to ncounter analysis using the myeloid cell probe panel. panel (a) depicts the top highly expressed genes and (b) the enriched genes uniquely characterizing each population. in (a) * highlights genes that are both highly expressed and enriched in each population, while # highlights genes highly expressed and common to both microglia and circulating monocytes. uniquely upregulated by day p.i., with none common to day p.i., and only six overlapping with those upregulated at day p.i. although no gene transcripts were upregulated uniquely at day p.i., overlapped with those upregulated at day p.i. a further genes, comprising % of all upregulated genes at day p.i., were specifically expressed at elevated levels at day p.i. coinciding with overt myelin loss ( figure c) . these profiles reveal a dynamic range of responses and extensive plasticity of gene expression profiles in microglia throughout jhmv infection ( figure c ). we next used a protein-protein network connection constructed based on differential gene expression to specifically examine upregulation of gene transcripts within bmdm and microglia correlating with demyelination at day p.i. for comparison, we also analyzed the network connection at day p.i., when expression profiles were most prominently altered in bmdm, but more modestly in microglia. this comparative analysis was chosen to provide clues about specific functions and involvement of microglia relative to bmdm in tissue destruction (figures and ) . our initial focus was on temporally altered networks in bmdm (figure ) . at day p.i., early infiltrated bmdm expressed a wide array of chemokines regulating cns infiltration of both innate (cxcl , ccl , ccl , ccl , ccl , and ccl ) and adaptive (cxcl , cxcl ) immune cells (figure a) . a large cluster of molecules regulating the innate immune response, essential to limit early viral replication ( , ) , was also expressed by bmdm (figure a) . these include pathogen recognition receptors such as tlrs (tlr - and tlr ) and molecules linked to the tnf pathway (tnf, traf , etc.). finally, bmdm expressed molecules involved in antigen presentation, including tap and tap , as well as t cell activating co-stimulatory molecules (cd and cd ) or il- , which induce th differentiation (figure a) . these results indicate that early infiltrating bmdm orchestrate the acute innate immune response crucial for limiting cns viral spread, as well as initiating the adaptive immune response by recruiting and activating t cells. at day p.i., correlating with peak demyelination, a more restrained number of mrna transcripts were upregulated in bmdm (figure b) . the cluster of chemokines mobilizing immune cells was sustained ( figure b) . in contrast, the molecular network extended from tnf was more limited at d p.i. compared to d p.i. (figures a,b) . molecules regulating primarily the cd t cell response were expressed at day p.i. and comprised gene transcripts involved in antigen presentation including mhc class ii (h -ab ) and co-stimulatory molecules such as cd and cd (figure b) . interestingly, among the more restricted number of gene transcripts upregulated at day p.i. in bmdm, several were transcripts encoding m molecules, which included arg , il rn and tgm (figure b) . in contrast to the vast number of genes upregulated early following infection in bmdm, a significantly lower number of upregulated genes characterized microglia at day p.i. (figure a) . transcripts for chemokines regulating migration of both innate and adaptive immune cells, such as ccl , ccl , ccl , ccl , cxcl , and cxcl , were expressed by microglia at day p.i. (figure a) . transcripts for tnf and other inflammatory cytokines generally associated with the innate responses, e.g., il a, il b, and il were also upregulated early in microglia ( figure a) . another extended network of tnf comprised psmb and psmb , subunits of the immunoproteasome, essential for antigen presentation by mhc class i molecules. by day p.i., the number of upregulated transcripts extensively increased in microglia (figure b) . the most clustered network comprised proteins like ccl , cxcl , cxcl , cxcl , cxcl , and cxcr , all chemokines and chemokine receptors regulating migration and arrest of adaptive immune cells within the cns during inflammation ( ) . this chemokine cluster was linked to tnf and inflammatory cytokines previously detected at d p.i., e.g., il a, il b, and il . another extended network of tnf comprised psmb and psm , also present at d p.i., ctnnb encoding b-catenin, a cellular adhesion molecule, and cdkn a, a cyclin inhibitor. other upregulated gene transcripts associated with class i antigen presentation, e.g., tap and tap , were also linked through a network associated with complement component genes (c , c ar , c b, c qa, c qb, c qc), which are highly expressed within microglia under homeostatic conditions (figure ) . similarly, tyrobp and trem , which formed phagocytic synapses ( ), are both highly expressed in microglia during myelin loss. finally, a wide variety of upregulated gene transcripts are associated with mhc class ii antigen presentation and modulation of t cell function. this includes h -ab , encoding for the mhc class ii molecules and h -dm, encoding for a second accessory protein, which facilitates peptide loading. similarly, genes associated with the invariant chain of mhc class ii were increasingly expressed within microglia, such as cd and ctss (cathepsin s, which cleaves invariant chain thereby promoting loading on mhc class ii). in addition, genes encoding for modulators of the cd t cell response, such as itgax (cd c), cd and cd were also expressed by microglia ( figure b) . overall, the upregulated networks are related to complement activation, enhanced class i and class antigen processing and presentation (potentially related to ifn-γ responses) as well as migration and phagocytic activity. however, there does not appear to be a bias toward phagocytic receptors over other components activated by pro-inflammatory mediators. pathogenic versus protective functions of myeloid cells following activation have also been correlated to expression of key molecules defined as m versus m markers. while the strict classification of myeloid cells into the m or m category has been tempered based on a more dynamic and mixed phenotype during inflammatory responses ( , ) , the m and m markers remain helpful to gage overall effector functions. among the analyzed gene transcripts in the nanostring myeloid panel related to m /m polarization ( m and m genes), between and % were upregulated across the course of jhmv infection with no difference comparing m versus m genes ( figure a) . among the total upregulated m markers, about % were commonly increased within both infiltrating bmdm and microglia; representative genes were il b and il ra (figures a,b) . however, while high levels of il b mrna were observed in both bmdm and microglia at d p.i., expression was only sustained in microglia at day p.i. and thereafter (figure b ). by contrast, il ra was increased in both bmdm and to a lesser extent in microglia at d p.i., but was decreased in both populations at later time points p.i. (figure b ). in addition, between and % of m markers were specifically expressed by infiltrating bmdm during the course of jhmv infection (figure a) , including cd , atf , ifng, ptgs , ccr , cxcl , and cxcl transcripts ( figure b and data not shown). however, only m related gene transcripts were specifically upregulated in microglia at the time of demyelination, including ccl , fas, cxcl , tnfsf , and psmb . (figures a,b and data not shown) . importantly, the most prominent difference between microglia and bmdm was noted in m marker regulation. among the - % m gene transcripts upregulated following jhmv infection, only a small proportion ( - %) was expressed by microglia ( figure a) . fn was the only m marker specifically expressed by microglia at the time of demyelination ( figure a) . although il rn transcript expression was elevated in both bmdm and microglia, the increase was at best modest in microglia ( figure c) . the majority ( - %) of m markers upregulated during jhmv infection were rather expressed by infiltrating bmdm, including arg , erg , il- , and ccl (figures a,c and data not shown). increased transcript levels were most pronounces at days and p.i., but dropped off thereafter. altogether, these data showed that while infiltrating bmdm express a mixed phenotype of m and m markers during jhmv infection, microglia expressed primarily pro-inflammatory genes while not expressing m markers. microglia and infiltrating macrophages are major components of ms active lesions ( ) . their effector functions are highly heterogeneous as evidenced by both pathogenic and protective functions during the course of ms ( ) . they can promote tissue damage by releasing toxic and pro-inflammatory molecules, mediate demyelinated axons through phagocytosis as well as propagate inflammation by recruiting and activating adaptive immune cells. on the other hand, both populations also display protective functions by clearing myelin debris, which facilitates remyelination, as well as releasing trophic and anti-inflammatory factors, which promote tissue repair. while it remains a challenge to distinguish infiltrating macrophages from microglia in ms lesions due to morphological and phenotypic similarities, they are disparate effector cells based on animal ms models ( , ) . questions relating to the pathogenicity of infiltrating macrophages and/or microglia in ms remain unanswered. can both populations display protective functions? do they display dynamic functions throughout the evolution of ms lesions? deciphering the respective roles of macrophages versus microglia in facilitating tissue damage and/or repair is essential to our understanding of ms pathogenesis and development of effective therapeutic strategies. in the murine eae model of ms, infiltrating bmdm are essential in mediating demyelination ( ) . gene expression profiles demonstrated that bmdm are indeed highly phagocytic and inflammatory at disease onset, while microglia display a repressed phenotype ( ) . by contrast, during jhmv-induced demyelination, recruited bmdm are dispensable for the demyelinating process ( ) . distinct from eae, where microglia activation precedes cns infiltration of bmdm ( ) , jhmv infection elicits early bmdm infiltration, prior to microglia activation. these distinct kinetics of bmdm recruitment relative to microglia activation thus appear to correlate with the apparently opposing roles of microglia as demyelinating populations. these data further suggest that early responses set the stage or imprint subsequent effector functions of bmdm and microglia. using a similar approach with nanostring analysis as in eae, the present study used gene expression profiling to characterize both bmdm and microglia myeloid functions at various times post jhmv infection. analysis of overall gene expression patterns revealed that the most extensive changes in bmdm were evident early after infection, while microglia showed a more dynamic profile throughout the course of viral encephalomyelitis. importantly, the most drastic gene upregulation in microglia was observed coincident with demyelination, at which time peak viral load and t cell effector function have substantially subsided ( ) . our data contrast with eae ( ) , where bmdm upregulated far more genes compared to microglia at disease onset, supporting opposing functions of bmdm and microglia in mediating demyelination in these two models. further, while bmdm exhibited a mixed expression profile of both pro-and anti-inflammatory markers, microglia expressed a highly pro-inflammatory profile and repressed most of the m markers across the entire time course of jhmv encephalomyelitis. analysis of protein-interacting networks within genes upregulated in microglia at the time of myelin loss revealed several key functions linked to promoting tissue damage. genes associated with complement activation were notably increased, although they were already highly expressed by microglia under homeostatic conditions. complement activation as a pathogenic component in ms has been reported following detection of deposits of the activated products of the complement component c in ms lesions ( ) . the classical complement pathway has also been shown to mediate olg death thus promoting demyelination ( ) . microglia phagocytic activity may also initiate tissue damage by directly removing myelin from axons, especially at the node of ranvier ( ) . genes associated with trem /dap signaling were also highly expressed by microglia at time of demyelination. trem modulates phagocytic capacity of myeloid cells via dap signaling ( ) and is expressed on myelin-loaded myeloid cells in ms lesions ( ) , supporting a role in ms pathogenesis. similar to jhmv infection, trem is predominantly expressed by microglia during eae and cuprizone-induced demyelination ( ) ( ) ( ) . however, trem -modulated phagocytic functions are essential for removal of myelin debris and remyelination implicating repair-promoting functions of microglia in the specific tissue environments defining these two demyelination models ( , ) . preferential trem expression within microglia compared to bmdm following jhmv infection support a more pathogenic role of trem- in jhmv-induced demyelination, potentially by promoting myelin stripping after recognition of glycolipids and phospholipids exposed on damaged myelin. in this context, it is critical to note that jhmv infection is associated with extensive transient production of ifn-γ and its inducible genes, i.e., inos, and cxcr ligands, which may drive a more phagocytic pathway in microglia in efforts to remove damaged proteins and lipids ( ) . further investigation is required to define inflammatory conditions under which trem- modulated or other phagocytic pathways promote tissue damage or repair and whether these are transient and reversible. microglia may also induce demyelination by secreting toxic factors including inflammatory cytokines that are highly expressed by microglia at time of demyelination, including tnf. tnf can induce olg death ( , ) and is expressed in active ms lesions, as well as elevated tnf in serum and cerebral spinal fluid correlates with enhanced ms pathology ( , ) . finally, microglia functions during jhmv infection were also associated with promoting adaptive immune response. an extensive network of chemokines and chemokines receptors relating to the recruitment and arrest of t and b cells within the inflamed cns were highly expressed by microglia. similarly, several genes associated with antigen processing and presentation by mhc class i and ii molecules were upregulated within microglia, suggesting that microglia promote t cell reactivation upon cns entry. however, microglia explanted during eae, tmev as well as jhmv infections failed to support myelin-specific cd t cell responses ex vivo, despite detection of internalized myelin ( ) ( ) ( ) . a potential deficit in antigen processing was supported by the ability of exogenous peptide to overcome the inability of microglia to prime myelinspecific cd t cells ( ) . nevertheless, this notion is opposed by our microglia profiling showing upregulation of genes involved in protein degradation and class ii peptide loading, e.g. cd (invariant chain), h -dma (peptide loading), ctss (cathepsin s), which cleaves invariant chain. the apparent inability of microglia to elicit cd t cell effector function ex vivo thus remains intriguing. our present study reveals new insights into the plasticity of microglia in adapting to inflammation and expressing pathogenic functions associated with demyelination, characteristics which have previously been ascribed to bmdm ( ) . moreover, altered bmdm expression profiles coincided with their early infiltration into the cns and remained largely similar throughout infection. while altered microglia gene expression coincided with the time of early, yet robust demyelination, it remains to be determined whether these changes are sustained at later time points during jhmv persistence, when clinical disease improves and remyelination occurs. it will be of specific interest to assess whether the microglia pro-inflammatory phenotype evolves to an anti-inflammatory, repair-promoting phenotype, as evidenced by the plasticity of myeloid cells in cns autoimmunity ( ) . furthermore, our study emphasizes that the distinct tissue environments during eae and jhmv infection drive opposite effector functions of microglia versus infiltrating macrophages. the interplay between t cells, infiltrating macrophages and microglia, as well as astrocytes drives ms pathogenesis, yet mechanisms ultimately leading to loss of repair remain unclear. taking advantage of demyelinating models characterized by distinct inflammatory factors such as both th and th cells in eae ( ) , strong th polarized responses during jhmv infection, distinct kinetics of bmdm recruitment versus glia activation promises to reveal essential new insights into the interplay of microglia and bmdm functions in debris clearance versus active myelin stripping and ongoing axonal damage. longitudinal studies will aid in developing efficient future therapies to combat ms pathogenesis. all procedures were performed in compliance with protocols approved by the cleveland clinic institutional animal care and use committee. author contributions cs designed and performed experiments, collected and interpreted the data, and wrote the manuscript. rd analyzed data and edited manuscript. cb designed the research, provided materials, interpreted the data, and wrote the manuscript. all authors approved the final manuscript. the authors would like to thank natasha towne for her exceptional technical assistance, as well as jennifer powers for performing facs purification. this work was supported by the national institutes of health grant ns and national ms society research grant nmss rg . rd is supported by the national institutes of health grant ns . the supplementary material for this article can be found online at https://www.frontiersin.org/articles/ . /fimmu. . / full#supplementary-material. figure s | validation of nanostring ncounter gene expression analysis by real-time pcr. to validate nanostring ncounter data, expression of il- , il rn, and arg was analyzed by q-pcr in naïve circulating monocytes and microglia, as well as in bone 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plasticity in the evolution of central nervous system autoimmunity the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -ds x yym authors: kim, young-seok; son, ahyun; kim, jihoon; kwon, soon bin; kim, myung hee; kim, paul; kim, jieun; byun, young ho; sung, jemin; lee, jinhee; yu, ji eun; park, chan; kim, yeon-sook; cho, nam-hyuk; chang, jun; seong, baik l. title: chaperna-mediated assembly of ferritin-based middle east respiratory syndrome-coronavirus nanoparticles date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: ds x yym the folding of monomeric antigens and their subsequent assembly into higher ordered structures are crucial for robust and effective production of nanoparticle (np) vaccines in a timely and reproducible manner. despite significant advances in in silico design and structure-based assembly, most engineered nps are refractory to soluble expression and fail to assemble as designed, presenting major challenges in the manufacturing process. the failure is due to a lack of understanding of the kinetic pathways and enabling technical platforms to ensure successful folding of the monomer antigens into regular assemblages. capitalizing on a novel function of rna as a molecular chaperone (chaperna: chaperone + rna), we provide a robust protein-folding vehicle that may be implemented to np assembly in bacterial hosts. the receptor-binding domain (rbd) of middle east respiratory syndrome-coronavirus (mers-cov) was fused with the rna-interaction domain (rid) and bacterioferritin, and expressed in escherichia coli in a soluble form. site-specific proteolytic removal of the rid prompted the assemblage of monomers into nps, which was confirmed by electron microscopy and dynamic light scattering. the mutations that affected the rna binding to rbd significantly increased the soluble aggregation into amorphous structures, reducing the overall yield of nps of a defined size. this underscored the rna-antigen interactions during np assembly. the sera after mouse immunization effectively interfered with the binding of mers-cov rbd to the cellular receptor hdpp . the results suggest that rna-binding controls the overall kinetic network of the antigen folding pathway in favor of enhanced assemblage of nps into highly regular and immunologically relevant conformations. the concentration of the ion fe( +), salt, and fusion linker also contributed to the assembly in vitro, and the stability of the nps. the kinetic “pace-keeping” role of chaperna in the super molecular assembly of antigen monomers holds promise for the development and delivery of nps and virus-like particles as recombinant vaccines and for serological detection of viral infections. introduction various types of viral vaccines have been developed over the last century with a wide spectrum of efficacy and safety ( , ) . the manufacturing of most conventional vaccines-live attenuated, inactivated, or subunit vaccines-invariably require the culturing of infectious viruses in cell substrates ( ) . despite dedicated efforts, conventional cell culture often fails to produce sufficient amounts of virus for evaluating the immunogenicity, protective efficacy, and safety of viral vaccines. moreover, some emerging viruses cause high-mortality rates, without options for treatment or prophylaxis, necessitating their manipulation, and manufacture under stringent bio-safety environment ( ) . not surprisingly, alternative technologies that circumvent these limitations are a high priority in the areas of vaccine development and production. nanoparticles (nps), virus-like particles (vlps), and assembly of multimeric peptides each provide attractive platforms for vaccine design ( ) . virus-like particles and nps structurally resemble infectious virions, but are non-infectious due to the lack of viral genomes. recombinant surface antigens from natural virions are assembled into highly ordered conformations as empty particles devoid of genetic material. antigenic epitopes are presented on the multivalent and highly repetitive outer structure of the nps, which leads to the crosslinking of b-cell receptors and the induction of long-lasting immune responses ( ) ( ) ( ) . by mimicking the morphology of the natural infectious virions, the regularly assembled particles are highly immunogenic, and are amenable to diagnostic and prophylactic exploitation. among the simplest targets are the vlps of non-enveloped viruses, such as hepatitis e virus or human papilloma virus, and are composed purely of viral capsid proteins ( ) ( ) ( ) . in contrast to non-enveloped viruses, where virion assembly is exclusive to capsid proteins, enveloped viruses (e.g., coronavirus or flavivirus), require an additional membrane component for assembly into mature virions. consequently, in enveloped vlps, the assembly of matrix proteins provides a molecular scaffold, and viral antigens are embedded into lipid membranes. different types of glycoproteins may be embedded in the lipid membrane as target antigens for generating immunological responses ( ) . however, this process requires multiple proteins (surface antigens and matrix proteins), and the enveloped vlps are not structurally uniform and are difficult to characterize. a promising alternative is to present the target antigens on the surfaces of self-assembled nps, which, in lieu of lipid membranes, serve as the macromolecular scaffold for the presentation of the antigens of interest. in developing np vaccines, consideration should be given regarding the selection of a robust and faithful system for np assembly that enables the cost-effective development and delivery of vaccines in a timely manner. structure-based approaches in silico and their underlying principles are relatively advanced for np assembly ( ) ( ) ( ) . most of the approaches consider the thermodynamic stability of the final assembled nps, without due recognition for the kinetic complexities controlling regular assemblage over random interactions that lead to misfolded aggregations. therefore, it is not surprising that most engineered nps are refractory to soluble expression, which presents practical challenges in production, both at a laboratory-scale and in commercial manufacturing processes. this problem becomes augmented when expressed in bacterial hosts because of a lack of folding assistance in the bacterial cytoplasm for viral antigens. therefore, due to advantages in assisted folding, posttranslational modifications, and the possibility of generating multiple-component nps and vlps, eukaryotic hosts such as yeast, insects, and mammalian cells have been favored over bacterial hosts ( ) ( ) ( ) . however, these systems are significantly more expensive than bacterial systems, are more time-consuming, and the down-stream processes are usually more complex. moreover, the purification of vlps from insect cell systems poses a challenge due to similar physicochemical properties between the vlps and the baculoviruses ( , ) . bacterial systems, if available, would provide a cost-effective means to develop and deliver vaccines, as well as sero-diagnostic antigen kits used to diagnose-specific infection diseases. middle east respiratory syndrome (mers) was first reported in saudi arabia in and has caused multiple cases of infection with high mortality in europe and asia ( , ) . mers is caused by mers-coronavirus (mers-cov), which can be transmitted from camels to humans, and from humans to other humans ( , ) . worldwide transmission is increasing in direct household and community-wide transmission, as well as in nosocomial settings, as exemplified in a outbreak in korea ( , ) . neither effective vaccines nor therapeutic interventions are currently available. because of this, assembly of mers-cov antigens into immunologically relevant conformation as nps would be of interest and may be helpful in developing vaccines, sero-diagnostic tools, and therapeutic monoclonal antibodies. in the current study, we present a novel bacterial np of mers-cov antigen using ferritin as a molecular scaffold for self-assembly. ferritin, which is present in most living organisms, has identical subunits that spontaneously self-assemble and form np complexes with internal and external diameters of and nm, respectively ( , ) . previous studies show that ferritins of helicobacter pylori from a human isolate can be used as scaffold for hiv and influenza np vaccines, using eukaryotic host cells such as human embryonic kidney cells (hek f or hek s) ( , ) . likewise, bacterioferritin (fr), which self-assembles into nanocages with octahedral symmetry, has also been evaluated as a potential drug delivery system ( ) . however, viral antigens of human pathogens are prone to misfolding into aggregates, which necessitates chemical refolding of the insoluble aggregates in order to regain solubility and to allow regular assembly of the antigen ( , ) . in addition, displaying antigens on the surface of multi-molecularly assembled scaffolds in bacterial hosts remains a daunting challenge. we hypothesized that nps displaying the receptor-binding domain (rbd) of the spike protein from mers-cov could be produced in a bacterial system by harnessing the function of a molecular chaperone. conventionally, protein folding and the prevention of non-functional aggregation have been ascribed to molecular chaperones ( ) ( ) ( ) . recently, it has been shown that rna molecules are able to provide novel functions as molecular chaperones ( ) ( ) ( ) . based on novel findings, the concept of chaperna (chaperone + rna) function was established ( ) . in this report, chaperna function was harnessed for the folding and assembly of hybrid ferritin monomers into nps using a bacterial expression system. we also demonstrated that the biophysical properties, including solubility, yield, and stability of mers-cov nps, could be improved by properly controlling the rna-binding affinity, and the concentrations of fe + and salts. the chapernabased np assembly may prove to be a versatile tool for developing and delivering recombinant vaccines and for serological detection of emerging/re-emerging viruses. the expression vector pge-hrid( ) was constructed from the parental vector pge-lysrs ( ) ( ) . the pge-lysrs( ) vector was enzymatically cut with ndei and kpni. the pcr product of hrid, which carries the tev protease cleavage site and a -histidine tag at the c-terminus, was cut using the same restriction enzymes and the digested fragment inserted into the vector to generate pge-hrid( ). fr (genebank accession no. nc_ . ) dna was synthesized by, and purchased from, cosmo genetech (korea). the dna was cleaved with sali and hindiii, and inserted into pge-hrid( ) to generate hrid( )-fr. the receptor binding domain (rbd), n-terminal residues - , of the mers-cov s protein (genbank accession no. afs . ), was generated by gene synthesis, cut with kpni and sali, and inserted into hrid-fr to generate pge-hrid( )-rbd-fr. linker ssg or asg was inserted into the c-terminus of the rbd using overlapping pcr, cleaved with kpni and sai, and ligated into hrid-fr, generating pge-hrid( )-rbd-[ssg]-fr or pge-hrid( )-rbd-[asg]-fr, respectively. the schematic diagrams of each expression vector are illustrated in figure b . the genes of mutant hrid( m) (k a and k a) and hrid( m) (k a, k a, r a, k a, k a, k a, k a, k a, and k a) were generated by gene synthesis, cleaved with ndei and kpni, and inserted into pge-hrid( )-rbd-fr, generating pge-hrid( m)-rbd-fr and pge-hrid( m)-rbd-fr, respectively. the mutation sites and amino acid sequences of the mutants are shown in table s in supplementary material. the resulting expression vectors were transformed into the escherichia coli strain shuffle ® t . the cells were grown in ml of lb medium with ampicillin ( µg/ml) at °c overnight. each type of transformant was inoculated into ml of lb medium with ampicillin, grown at °c until an optical density (od ) of . - . was reached. protein expression was induced with mm iptg for h. each sample was harvested by centrifugation, lysed by sonication in lysis buffer ( mm tris-hcl, ph . ; % glycerol; mm -mercaptoethanol; and . % tween- ). the soluble fraction of each lysate was purified on a ni-affinity histrap™ hp column by atka prime (ge healthcare) and concentrated with centriprep™ (merck millipore ltd.). the purified proteins were treated with tev protease to remove the fusion partner hrid. the assembled nps were purified by gel filtration on / superose™ increase columns (ge healthcare). to examine the size and structure of the purified nps, microscopic evaluations using tem and cryo-em were performed. for tem analysis, a drop of the nps was placed onto a formvar/carboncoated tem grid (spl). the grid was negatively stained with % uranyl acetate, dried, and examined using a jem- electron microscope (jeol) at an accelerating voltage of kv. the particle sizes were calculated using camera-megaview iii (soft imaging system-germany) for measuring the nps in random image fields. for cryo-em, the nps were placed onto plasma-treated formvar/ carbon copper grid (ems) and negatively stained with % uranyl acetate. the grid was accelerated at kv with an fei cryotecnai f cryo-em microscope made available through the korean institute of science and technology. the nps were examined and photographed in high resolution. nanoparticle samples ( ml) were placed into a dispo-h cell, and analyzed using a zeta-potential & particle size analyzer (els- ( , , and °c ) and the cell lysates were separated into total (t), soluble (s), and insoluble (p) fractions by centrifugation (left panel). the solubility of each protein expressed at °c was measured by a gel densitometer and the data were summarized and shown in the right panel (n = ). statistical significance (**p < . , ***p < . ) was indicated for the samples compared with the control using a two-tailed student's t-test. (d) illustration of mers-cov rbd-fr nps using the chaperna-based hrid fusion partner. the hrid facilitated folding of the aggregation-prone rbd-fr through interaction with rna. the monomer of rbd-fr formed a properly folded trimeric structure by cleaving hrid with tev protease. eight trimers assembled and formed into mers-cov-like nps. red triangles indicate the rbd trimer on the fr nps. of the nps was measured twice at °c in water as a solvent with the sample accumulation time at s. effect of salt and fe + concentrations on np assembly and stability cultured cells ( ml) were lysed with lysis buffer in the presence of various concentrations of nacl ( , , , , , , , , and mm) to evaluate the intracellular proteins. all samples were performed in triplicate. the cell lysates were separated into soluble and insoluble fractions by centrifugation, and the protein stabilities analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page). thus, the proteins from cell lysates ( ml culture) were purified using hispur™ ni-nta resin (thermo fisher scientific) in buffer a depending on nacl concentration ( - mm). to evaluate the effects on fe + on np formation, cells were cultured in lb media with various concentrations of fe + ( , , , and , µm). np formation was examined by size exclusion chromatography (sec), sds-page, tem, and dls at the various concentrations of nacl or fe + . the cells were harvested, sonicated with lysis buffer, and separated into soluble and pellet fractions by centrifugation. target proteins in the soluble fraction were purified using hispur™ ni-nta resin (thermo fisher scientific), following the manufacturer's instruction. t (total lysate), s (soluble fraction), p (pellet fraction), w (wash fraction), and e (the elution fraction were analyzed by sds-page. co-purification of the nucleic acids and proteins in the wash and elute were analyzed on a native agarose gel. the nucleic acids were visualized with ethidium bromide (etbr), and the proteins with coomassie staining. cultured cells ( ml) were harvested using the same method described above. the cells were lysed with µl of protein extraction reagent b-per™ ii (thermo scientific) and separated into soluble and pellet fractions by centrifuged , rpm for m. a µl aliquot of each soluble fraction was further treated with µg/ml of rnase a (intron biotechnology) and incubated at °c for min. the nuclease treated samples were clarified by centrifugation at , rpm for min and the soluble supernatants and the pelleted precipitates were analyzed on an sds-page gel followed by western blot analysis. to confirm the proper folding of rbd-fr and its variant (rbd-[ssg]-fr), the binding of the purified proteins with the mers-cov receptor hddp was performed by elisa. fr only and phosphate-buffered saline (pbs) were used as negative controls. nunc -well microtiter immunoplates (thermo fisher scientific) were coated with ng/well of hdpp proteins (abcam) and incubated at °c overnight. the plates were washed and blocked with µl/well of blocking buffer ( % bsa) for h at room temperature. rbd (ssg linker, wt, m, or m)-fr ( ng/ well) were added for h at °c. an anti-penta his antibody ( µl/well; qiagen) was serially diluted ( / to / , ) in tbst [ mm tris-cl (ph . ), . % tween- ], added to the wells, and incubated for h at °c. a secondary goat anti-mouse igg antibody conjugated with hrp in a -µl volume ( : , , sigma-aldrich) was added and incubated for h at °c. the plates were washed three times with tbst at the end of each step. after washing, µl/well of substrate tmb solution (bd biosciences) were added to the well and the plates were incubated at °c for min in the dark. µl of stop solution ( n h so ) was added to the well to stop the colorimetric reaction, and the absorbance at nm was measured using an elisa reader, fluostar optima (bmg labtech). . the coating antigens were removed, and the wells were blocked with pbst ( % skim milk in pbs and tween- ) for h at °c. after h, the blocking solution was removed. twofold serially diluted sera from four patients (cnnh- , , , and ) were added to each well and incubated at °c for h. the antigencoated wells were incubated with peroxidase-conjugated goat anti-human igg antibody (kpl, seracare life sciences, milford, ma, usa) at °c for h. the primary antibody was removed and , ′, , ′-tetramethylbenzidine (tmb; sigma-aldrich) was added to each well as colorimetric substrate. immediately after treatment of the reactions with stopping solution (sigma-aldrich), the od was read at nm. six-week-old female balb/c mice were immunized with µg/ mouse of the rbd-fr, rbd-[ssg]-fr, or rbd protein generated as described above, or with commercially available mers-cov rbd protein (mers-rbd- p; eenzyme) as antigen in bsl- facility in ylarc. antigens were diluted in pbs. for the first group, equal volume of mf adjuvant (addavax, cat. no vacadx- ) ( ) was mixed by pipetting. for the other group, equal volume of antigens and alum adjuvant (thermo fisher scientific) were mixed by pipetting following the manufacturers' protocol. pbs plus adjuvant and fr were used as negative controls. the immunized mice were boosted twice with intramuscular injections on days and . mice were anesthetized on days and for ocular bleeding from the orbital sinus ( figure s in supplementary material). immune sera were processed by centrifugation of the collected blood at , × g for min. the spleen and the balf (bronchoalveolar lavage) were obtained at days after the last immunization from sacrificed mice. balf was taken by washing the airways with ml of pbs. t-cell population from immunized mice were analyzed by flow cytometric analysis ( , ) . the spleens were taken at days after the last immunization from the sacrificed mice. to obtain single-cell suspensions, the tissues were homogenized and passed through µm cell strainers (spl). after centrifugation, erythrocytes were removed by red blood cell lysing buffer (sigma). the cells were washed and resuspended in iscove's modified dulbecco's media containing % fbs. for intracellular cytokine staining, the splenocytes were stimulated with µg/ml rbd protein or phorbol myristate acetate/ionomycin in the presence ng/ml recombinant human il- (biolegend) and brefeldin a ( : , ; ebioscience) at °c for h. after stimulation, the cells were blocked with rat anti-mouse cd /cd (bd biosciences) and surface stained with anti-cd (fitc, clone - . ; biolegend) and anti-cd (pe/cy , clone gk . ; biolegend) at °c for min. the stained cells were fixed in facs lysing solution (bd biosciences) at room temperature for min, and permeabilized with facs buffer ( . % fbs, . % nan in pbs) containing . % saponin (sigma) at room temperature for min. then, the cells were stained with anti-ifn-γ (pe, clone xmg . ; biolegend) and anti-tnf-α (apc, clone mp -xt ; biolegend) at room temperature for min. all data were collected by bd lsr fortessa (bd biosciences) and analyzed with flowjo software (tree star inc., ashland, or, usa). competition elisa was performed to determine whether mers-cov antigen [rbd-[ssg]-fr, rbd-fr, rbd, and fr (negative control)]-immunized mouse serum inhibited binding of rbd protein to hdpp receptor ( , ) . ng/well hdpp protein (abcam) was coated on nunc -well microtiter immunoplates (thermo fisher scientific) and incubated overnight at °c. plates were washed and blocked with µl/well of blocking buffer [ % skim milk in pbs and tween- (pbst)] for h at °c. at the same time, mouse sera immunized with rbd, rbd-[ssg]-fr, rbd-fr, and fr were serially diluted ( / to / ) with ng/well rbd protein (mers-rbd- p; eenzyme) in tbst [ mm tris-cl (ph . ), . % tween- ], added to new wells, and incubated for h at °c. µl solution was added to each well at °c and incubated for h. after that, µl of anti- xhis tag antibody conjugated with horseradish peroxidase ( : , , thermo fisher scientific) was added to each well and incubated for h at °c. plates were washed three times with tbst, and µl/well of substrate tmb solution (bd biosciences) was incubated at °c for min in the dark. µl of stop solution ( n h so ) was added to the well to stop the color reaction and measure the absorbance at nm using an elisa reader fluostar optima (bmg labtech). the hrid facilitated the solubility of mers-cov rbd-fr the spike glycoprotein (s) of mers-cov was used for the generation of mers-cov-like nps. s protein forms trimers, resulting in large spikes on the virus envelope ( ) . it is challenging to express the full-sized s protein (~ kda) in e. coli. thus, the s domain of s protein (~ kda), which includes the receptor-binding ability, was used. our initial attempt to express the s domain, either as s or as an s -fr fusion protein, failed; the expression level and solubility of the protein was below the lower limit of detection by sds-page and western blotting ( figure s in supplementary material). we therefore used the rbd ( - a.a.) of the s protein, which has a pivotal function as illustrated in figure b ( , ) . when expressed alone in e. coli, the rbd is not able to form the trimeric assembly (unpublished observation), due to the lack of the hr domain within the s domain ( ) . to overcome this problem, fr was used as scaffold for the assembly. fr is a spherical np whose subunits form trimers that subsequently result in octahedral structures composed of identical subunits ( ) . we therefore performed computational modeling to evaluate the potential of fr as scaffold for trimer formation of the rbd. possible trimer formation was analyzed by computational modeling using modeler ( , ) and cluspro ( , ) . various linkers, including ssg, asg, and d , were introduced between the rbd and fr with a goal to minimize steric hindrance between the two domains so as to enhance trimer and np formation. in silico analysis showed energy-stable trimeric models of rbd-fr, rbd-[ssg]-fr, and rbd-[asg]-fr, whereas rbd-d -fr failed to form a trimeric structure ( figure a) . the rbd-[ssg]-fr was predicted to be the most stable and well-structured compared with rbd-fr and rbd-[asg]-fr. initial testing of the rbd-fr constructs without hrid fusion showed that none of the constructs were solubly expressed, even under low-temperature culture conditions ( figure c ) ( figure b) . we previously confirmed that by using chaperna, the globular domain of influenza hemagglutinin (ha) is efficiently assembled into a trimeric complex with an immunologically relevant conformation (yang et al., in press) . as shown in figure c , the hrid fusion significantly increased the solubility of both rbd-fr ( . %) and rbd-[ssg]-fr ( . %), indicating that the chaperna platform effectively increased both the solubility and the folding of its fused target proteins. because of the poor expression level and low solubility of the rbd-[asg]-fr construct (figure c) , further experiments were performed using only the rbd-fr and rbd-[ssg]-fr constructs. after purification of the soluble proteins ( figure s in supplementary material), we determined the potential effects of using hrid as a fusion partner for the self-assembly of the nps. as shown in figure s in supplementary material, hrid-rbd-fr failed to form nps. because of this, we performed tev protease cleavage of the hrid. removal of the hrid domain facilitated the self-assembly of the rbd-fr monomers, and also eliminated the immune response against the non-self hrid domain in balb/c mice ( figure s in supplementary material). after hrid cleavage, rbd-fr and rbd-[ssg]-fr were purified using sec (figure a) . as expected, rbd-[ssg]-fr assembled into properly formed nps ( , kda) more efficiently than did rbd-fr nps, which were mainly detected in the void-volume fractions, suggesting they were irregularly assembled soluble aggregates. the size of the rbd-[ssg]-fr nps was further confirmed by tem. tem images of the rbd-[ssg]-fr np structures showed hollow, spherical particles that were more compact than the rbd-fr nps. the average diameter of the rbd-[ssg]-fr nps was - nm (figure b) . in contrast, dls analysis of the rbd-fr np structure without the ssg linker appeared to be smaller with an average intensity diameter of . nm, and this compared with rbd-[ssg]-fr that had an average intensity distribution diameter of . nm (figure c) . consistent with the sec analysis, rbd-fr without a fusion partner was mostly produced in a soluble aggregated form. therefore, we identified that the protein folding did not occur properly without hrid, and the formation of nps was confirmed by both sec and sds-page analyses. as shown in figure s in supplementary material, the purified nps retained their stability over an extended period of time at various temperatures ( , , and − °c). thus, these results indicate that the ssg linker allowed the rbd-fr to generate properly assembled nps. it should also be noted that the efficiency of protein folding and nps formation may be further enhance through appropriate linker selection. it has been reported that ionic strength plays an important role in the stability and self-assembly of ferritins ( , ) . we examined the effect of salt concentration on the formation and stability of the rbd-[ssg]-fr nps at various concentrations ( - mm). consistent with the previous studies, the stability of the protein was highly affected by the concentration of nacl in the lysis buffer by sds-page ( figure a ) (n = ). the solubility of the protein significantly decreased as the concentration of nacl increased from to mm, with the solubility being about . -fold lower at mm compared with mm. unlike previous studies, the solubility of the protein was gradually recovered at higher nacl concentrations (> mm); the solubility at mm was . -fold higher than at mm. furthermore, the yield of soluble of protein per liter of culture increased in a salt concentrationdependent manner (figure b) . to further investigate the effect of salt concentration, the physicochemical and morphological properties of the rbd-[ssg]-fr protein were examined by sec, tem, and dls. in mm nacl, most of the protein was aggregated during the purification process, and the purified protein failed to form spherical structures, but instead, existed predominantly as kda monomers (figures c,d; figure s in supplementary material). in contrast, the protein that was lysed in mm nacl and purified in mm nacl, developed well-structured nps according to tem and dls analyses (figures c,d; figure s in supplementary material). however, based on sec analysis, at high-salt concentrations (> mm), the protein failed to form stable structures with the proteins being eluted predominantly in the void volume, suggesting they were soluble aggregates under the high-salt concentrations ( figure c) . transmission electron microscopy images under the various salt concentrations clearly supported the conclusion, showing that the tendency for aggregation was dependent on the salt concentration ( figure d) . taken together, the results underscored the importance of salt concentration on the solubility of monomers and the quality of multimeric assembly of hybrid nps. ferritin has an intrinsic ability to interact with fe + to form ferritin-iron cores ( ) . thus, it was worth investigating the effect of fe + on the assembly and stability of rbd-[ssg]-fr nps. cells were grown in lb medium with various concentrations of fe + . as shown in figure a , the yield of purified protein was significantly increased from cultures with µm fe + , reflecting a . -fold increase compared with similar cultures µm fe + . the cell growth and purification yield at , µm fe + were slightly decreased, presumably due to the toxicity of ferric acid. np formation under the various concentrations of fe + was analyzed by sec ( figure b) . consistent with the previous results, the proteins were eluted mainly in the fractions expected for the size of assembled nps ( , kda). of note, the ratio between nps and soluble aggregates in the sec analysis showed that np formation was facilitated at high concentrations of fe + (figure b) . the formation of rbd-[ssg]-fr nps at an fe + concentration of , µm was confirmed by tem ( figure c ) and dls ( figure d) . the tem analysis clearly showed that the morphology of the proteins was more compact, and probably highly stable, when assembled at high fe + concentrations ( µm) than at lower concentrations ( µm) ( figure c ). as shown in figure d , the average diameter of nps examined by dls was . nm at high fe + concentration ( - , µm) and . - . nm at lower concentration ( - µm). these results suggest that both fe + and salts concentrations influenced the efficiency and quality of the regular assembly of hybrid ferritin monomers into nps. our previous studies show that an rna-protein interaction is crucial for transducing the chaperone function of rna into the folding of client proteins ( ) . consistent with that, our present study showed that rna facilitated the folding of its interacting proteins. the solubility of hrid(wt)-rbd-fr was . -fold higher than rbd-fr without hrid fusion (figure ) , strongly supporting the previous studies. in addition, the solubility of rbd alone was completely insoluble (figure b ; figure s in supplementary material). it has been shown that the positively charged residues of lysine moieties in hrid contribute to trna binding ( ) . in the current study, the trna binding induced the intrinsically disordered protein (idp) status of hrid to form alpha-helical structures ( figure a) . thus, two rna-binding (table s in supplementary material). the total e. coli lysate (t) was fractionated into the soluble fraction (s) and the pellet fraction (p) by centrifugation. as expected, both rbd and rbd-fr without fusion to hrid domain, were refractory to being produced as soluble proteins ( figure b) . interestingly, the solubility of the rna-binding mutants did not decrease, but actually increased to . % for the m mutant and . % for the m mutant compared with wild-type protein at . % (figure b) . considering that hrid is relatively unstructured in the absence of trna binding, the results are consistent with previous reports that the fusion with idps promotes the solubility of target proteins ( ) ( ) ( ) . following purification of wild-type hrid-rbd-fr (hrid(wt)-rbd-fr), electrophoretic mobility shift assays showed that greater amounts of nucleic acids were co-purified with hrid(wt)-rbd-fr protein than with the mutant hrid-rbd-frs ( and m) under non-denaturing conditions ( figure c ). the relative ratio of nucleic acid based on etbr staining and proteins based on coomassie staining in the eluted fraction confirmed the reduced affinity of mutants to nucleic acids. to test if rna had a role in maintaining the stability of the target proteins, the lysates were treated with rnase a to eliminate rna, and the solubility of each protein was analyzed by sds-page and western blotting. the soluble fractions of the lysates (s) were incubated at °c in the presence and absence of rnase a and the samples were further separated into soluble fraction (ss′) and insoluble fraction (sp′) by centrifugation. as shown in the left panel of figure , rnase a treatment completely abolished the effect of rna on protein solubility as compared with the control (rnase a−) or with samples prior to rnase treatment. parallel experiments with the and m mutants showed much less rna co-purified with the proteins, confirming the reduced affinity to nucleic acids and the complete depletion of rna by rnase a treatment (figure , left panel) . remarkably, the solubility of hrid(wt)-rbd-fr was greatly reduced by depletion of rna as reflected in the ratio of [ss′]/ [sp′] [ . and . for rnase (+) and rnase (−), respectively] by both coomassie staining and western blot analyses (figure , right panel). however, the solubility of the mutants ( and m), was not significantly affected by rnase a treatment, probably due to their lower affinity to rna (figure , right panel) . taken together, the results demonstrate that hrid(wt)-rbd-fr maintained a strong affinity for rna, and that affinity was pivotal for maintaining the solubility of the protein. to further define the rna dependence of solubility of the ferritin hybrids (figure ) , we investigated if the rna binding had a role in the formation of nps. rbd-fr and the various hrid-rbd-fr (wt, , and m) proteins were purified by nickel-affinity chromatography ( figure s in supplementary material) and their physicochemical properties analyzed by sec (figure a) , tem (figure b) , and dls ( figure c) . the soluble yields of rbd-fr (hrid fusion) was approximately . mg/l of culture, representing greater than , -fold higher levels than its hrid (−) counterpart (~ μg/l culture), again confirming the role of hrid as a robust enhancer for solubility and assembly. it was striking to note that the two mutant proteins, despite high solubility (figure b) , were detected at disproportionately higher amounts in the void fractions of sec, indicating that they failed to form nps of a defined size, and existed predominantly as soluble aggregates ( figure a) . however, hrid(wt)-rbd-fr predominantly formed nps of a defined size (~ , kda). it is also interesting to note that there was a slight shift of the rna-binding mutants ( and m) in the elution pattern, suggesting a larger size of nps compared with wild-type nps. overall, the ratio between soluble aggregates in the void volume and the nps of defined size clearly showed that rna binding was crucial for assembly of the monomers into nps. as a control, rbd-fr (without hrid fusion) existed predominantly as soluble aggregates ( figure s in supplementary material). consistent with these results, em analysis confirmed well-structured nps by hrid(wt)-rbd-fr, compared to largely aggregated structures by the mutant proteins ( figure b) . even if multi-molecular structure was formed, the structure becomes unstable, mostly as soluble aggregates. consistently, the intensity distribution diameter of the wild-type protein, as estimated by dls analysis, was nm compared with larger sizes of hrid( m) at . , . nm and hrid( m) at , . nm (figure c ; figure s in supplementary material). it is conceivable that soluble aggregates may shield the exposed -histidine tag, resulting in a decreased binding affinity to nickel resins and elution in earlier fractions compared with wt protein ( figure s in supplementary material). taken together, the data demonstrate that rna binding prevented aggregation into irregular conformations and guided the self-assembly of the hybrid ferritin monomers into nps of a stable structure. the immunological properties of ferritin nps were analyzed by elisa. the hddp (human dpp ) receptor has been previously identified as the receptor for mers-cov human infection ( ) . therefore, using hdpp as a coating antigen, elisa-binding assays between rbd nps and the receptor were performed (figure ) . fr without rbd fusion failed to bind, and was similar to the pbs negative control. strikingly, the binding ability increased in the same order as the rna-binding ability (hrid(wt) > m > m), with highest absorbance observed in the wt with the ssg linker (hrid(wt)-rbd-[ssg]-fr). the results show that the conformation of rbd in the wt nps better resembled the protective antigen of mers-cov rbd from cells, compared with the rna-binding mutants and m. again, judicious choice of linker between the ferritin carrier and the antigen was important for receptor binding and was reflected in its importance for np assembly into a stable conformation (figure ) . finally, the elisa results for np against human patients was investigated using the sera from four mers-cov-infected patients (figure ) . six different proteins, including five recombinant nps (hrid(wt)-rbd-fr, hrid( m)-rbd-fr, hrid( m)-rbd-fr, hrid(wt)-rbd-[ssg]-fr, and fr), and mers-cov rbd protein were compared by elisa using them as capture antigens. strong elisa signals were detected for the four recombinant nps and mers-cov rbd from cells (positive control). the wt form consistently showed a higher response than the rnabinding mutants (hrid(wt) > m > m), with hrid(wt)-rbd-[ssg]-fr being the best binder among constructs tested. these results address to the utility of the e. coli assembled mers-cov rbd-fr nps as useful tools for sero-diagnosis of mers-cov infection. taken together, the results confirmed the immunologically relevant conformation of the mers-cov rbd displayed on the hybrid ferritin particles, and the crucial role of rna in controlling the kinetic pathway for the assembly of viral antigen monomers into stable nps. to evaluate the immunogenicity of ferritin-based nps, balb/c mice (n = ) were immunized with rbd, rbd-fr, and rbd-[ssg]-fr nps antigens. the trnas were found to be removed from the hrid protein during the purification process. before immunization, potential rna contamination in the purified proteins was determined by gel electrophoresis. as shown in figure s in supplementary material, rna was below detection level, if any, after several purification steps, compared with the proteins purified in the first step. previously, mf -adjuvated and alum-adjuvated mers-cov antigen have been reported to increase the antibody and t-cell responses in mice ( , ) . thus, the first group and second group were immunized twice with . µg of antigen containing the equal volume of alum figure s in supplementary material and size exclusion chromatography was used to explore hdpp receptor-binding affinity to the protein. all data are shown as mean ± sd from triplicate samples. fr alone and phosphate-buffered saline were used as negative controls. figure s in supplementary material were used as coating antigens. fr alone and infected cell lysates were used as negative and positive controls, respectively. virus-infected sera from four patients were serially diluted from : (twofold dilution). all data are presented as mean ± sd of duplicate samples. higher than rbd, respectively. the antibody responses by rbd-fr and rbd-[ssg]-fr nps were much stronger than the rbd in all antibody subtypes tested (igg , igg a, and igg b) (figures b-d) . as a test of mucosal immune responses, the rbd-specific iga antibody levels from balf were also analyzed by elisa (figure e) . mf adjuvanted rbd-[ssg]-fr nps presented significantly higher od values than rbd and fr (negative control). these results suggested that rbd-[ssg]-fr nps induces local mucosal immune response stronger than rbd. in addition, it was confirmed that antibody responses of igg, igg (th ), igg a, and igg b (th ) against mf -adjuvated antigens were higher than those from alum-adjuvated antigens. in contrast, pbs and fr control groups failed to, or only weakly induce an antibody response against rbd protein. these results suggest that fr-based nps significantly enhance various antibody responses than monomeric antigens. the cellular immune responses were investigated in mice immunized with protein (rbd, rbd-fr, rbd-[ssg]-fr) and fr (negative control). splenocytes of mice (n = ) were harvested week after the last immunization, stimulated with | nanoparticles-immunized mouse serum inhibited interaction between middle east respiratory syndrome-coronavirus receptor-binding domain (rbd) and hdpp receptor. competition enzyme-linked immunosorbent assay showed that anti-rbd mouse sera ( : , from mice immunized with rbd-[ssg]-fr, rbd-fr, and rbd) blocked binding between rbd ( µg/ml) and hdpp receptor ( µg/ml). fr-immunized mouse serum ( : ) was used as a negative control. all sera were serially diluted from : (twofold dilution). all data are presented as mean ± sd (n = ) and p-values were obtained using student's two-tailed tests (***p < . ). figure | immune responses in receptor-binding domain (rbd) nanoparticles (nps) immunized mice (n = ). endpoint titer of igg (a), igg (b), igg a (c), and igg b (d) antibody binding to middle east respiratory syndrome-coronavirus rbd were detected using mice serum after two immunizations. rbd-specific antibodies were detected after immunizations of rbd nps, rbd, fr with adjuvant (alum and mf ) using enzyme-linked immunosorbent assay. (e). rbd-specific iga antibodies were detected using balf (diluted : ) after immunization of protein with mf . od, optical density. each endpoint titer was shown by individual. all error bars were shown as mean ± sd (n = ) and all p-values were obtained using student's two-tailed tests (**p < . , ***p < . ). rbd protein, and analyzed for cytokines by flow cytometry. in the rbd-immunized group, ifn-γ and tnf-α-producing cd + t-cell responses were detected at low levels. however, ifn-γ and tnf-α-producing cd + t cells were significantly increased in rbd-fr and rbd-[ssg]-fr-immunized groups compared with rbd and fr-immunized group ( figure s in supplementary material) . these results demonstrated that the rbd nps vaccination induced antigen-specific cd + t cells that produced ifn-γ and tnf-α upon antigen stimulation. anti-nps serum effectively blocked rbd protein binding to the hdpp receptor middle east respiratory syndrome-coronavirus infection is mediated by the interaction of rbd and the host receptor hdpp ( , ) . as a correlate of protection, a competition elisa was performed to investigate whether antibodies generated from nps immunization were able to interfere with the binding to hdpp . thus, after incubation of rbd protein with mouse serum ( : ), the binding of serum-mixed samples to hdpp protein was measured. as shown in figure , rbd-[ssg]-fr, rbd-fr, and rbd-immunized sera strongly abolished the binding of rbd to hdpp receptor ( . , . , and . %, respectively). interestingly, the relative efficiency of interference correlates with that of np assemblage (figure ). in contrast, the fr-immunized mouse serum (negative control) failed to inhibit the interaction. taking together, these results demonstrate that immunization of nps greatly stimulates mers-covspecific antibody response that effectively interferes with the cellular receptor binding, suggesting its possibility as a vaccine. however, protection efficacy should ultimately be tested in a live virus challenge model. having key immunologic features, like a highly repetitive nanostructure, provides a designing principle for nps in inducing potent and long-lasting antibody responses. for vlps of non-enveloped viruses, assembly is made purely by capsid proteins. for enveloped viruses, however, additional membrane components and matrix proteins are required to display the target antigens on the surface of assembled vlps. a promising alternative is to present target antigen on the surfaces of selfassembled nps, which, in lieu of lipid membranes and matrix proteins, serve as a macromolecular scaffold for the presentation of antigens of interest ( ) . ferritins, as a substitute for matrix proteins and membranes, have been used as scaffold for the regular assembly of target antigens. however, ferritin-based nps have been produced only in host cells of mammalian or insect origin ( , ) . previously, we showed that influenza ha could be assembled in a soluble, trimeric, and immunologically relevant conformation by exploiting chaperna activity ( ) . the present study is the first report of using rnas as molecular chaperone for supra-molecular structures. here, we present a novel bacterial system for np assembly of hybrid ferritin displayed surface antigens from mers-cov. the nps reacted strongly with sera derived from mers-cov-infected patients (figure ) confirming their utility in sero-diagnosis of infection. moreover, the antisera, generated from immunization of mice, were able to interfere with the binding to the cellular receptor hdpp (figure ), in part of essential protective immune responses. the efficiency of receptor-binding inhibition (figure ) , as well as the ability for inducing the mucosal responses (figure e) , correlated with the regular assembly of nps as examined by dls or em (figure ) , confirming that presentation of antigenic epitopes on a multivalent and highly repetitive structure is indeed important for the quality of immune responses. overall, the quality of nps and consequent immune responses were governed by the rna-mediated assembly of antigens. we hypothesized that chaperna function could be harnessed for presenting target antigens as highly repetitive nanostructures ( figure d) . the hrid is the n-terminal domain of hlysrs and was previously identified as a nucleic acid-binding domain ( figure ) ( ) . in this report, the hrid was exploited as a transducer for chaperna function (tcf) by serving as a docking-tag for cellular rna for the folding/assembly of the hybrid fr containing client antigen proteins [rbd of mers-cov (figure d) ]. the advantage of using hrid as a tcf could be many fold. first, hrid is small ( . kda), monomeric, and was flexible enough to allow the access of site-specific protease for the removal of hrid ( figure s in supplementary material). of note, hrid belongs to idps, which switches into stabile α-helixes upon binding with trnas. second, the bound rna, due to its highly negative charge, may resist uncontrolled intermolecular interactions among monomers into amorphous aggregation. finally, even the naked hrid (in the absence of rna binding), due to its intrinsically flexible nature, may not pose physical hindrance to multiple interactions among monomers, enabling assembly into stable super-structures, upon removal of the hrid. thus, the potential "pace-making" function harnessed with the rna molecule, allows a regular assembly of monomers as highly repetitive nanostructures. consequently, in the current study, hybrid fr was produced in soluble forms, could be purified by one-step affinity chromatography, and most remarkably, assembled into nps of defined sizes upon removal of the hrid ( figure s in supplementary material). consistent with the principles of design, the loss of rna binding by hrid significantly hampered the regular assembly of the ferritin monomers and increased the amount of non-functional misfolded proteins as soluble aggregates (figure ) . thus, the overall yield, as well as the quality of nps, were dependent on the chaperna function transduced by the hrid, which in turn was mediated by interaction with cellular rnas (likely to be trnas). the driving and controlling factors for de novo assembly of biomolecules are poorly understood. historically, host factors like groel/s were initially discovered as molecular chaperones for supporting viral growth in e. coli and supporting the assembly of viral capsid proteins ( , ) . moreover, groel/s also cooperates with rbcx in plant cells for the assembly of multicomponent rubisco, which is the most abundant protein in the biosphere responsible for photosynthesis ( ) . therefore, it is intriguing that rna could provide such a robust folding/ assembly of a supra-molecular structure. we recently confirmed that the present strategy could be successfully applied to the assembly of bacterially synthesized monomers of norovirus into vlps composed of monomers (unpublished observation, seong, b.l.). whether rna can substitute for, or collaborate with pre-existing protein-based molecular chaperones remains an exciting avenue for future investigations. it should be noted that the defined versatile functions are being expanded for rna molecules. as an engineered system for harnessing chaperna function, the present report may prove to be the tip of an iceberg for pivotal function of rna molecules as chaperones for the folding and supra-molecular assembly of proteins in living organisms ( , ) . various factors were identified as important for efficient assembly of mers-cov nps. as an extrinsic factor, the binding affinity of hrid to cellular rnas was crucial for the assembly and the quality of the assembled nps (figure ) . as intrinsic factors, the concentration of salts and fe + also influenced the assembly and stability of nps (figures and ) . the ionic strength played an important role in the stability and self-assembly of ferritins, and aggregation increased with increasing concentrations of nacl ( ) . the assembly of the hybrid mers-cov nps revealed an interesting change in salt dependence, with - mm nacl buffer as optimal condition as confirmed by em and dls analyses (figure ) . the change in salt dependence was probably due to the presence of electrostatic interactions among rbd domains ( , ) . the dependence on fe + was not surprising considering that ferritin has an intrinsic ability to interact with fe + to form ferritin-iron cores ( ) . based on our experience, to enhance the quality of nps, it is advisable to control fe + concentrations, both during the culturing of the bacterial cells and during the purification of the soluble monomer proteins (figure ) . first, the yield of the purified protein was increased in the presence of µm fe + (figure b) , up to . -fold greater compared with the control conditions lacking fe + . second, the ratio between nps and soluble aggregates in sec showed that nps formation was facilitated at high concentrations of fe + , and resulted in a more compact morphology under em (figures b,c) . thus, both the overall yield and the quality of nps were governed by their intrinsic ability to interact with fe + . finally, our data show that the presence and the nature of the linker between the ferritin and the rbd antigen was also important to the assembly of nps. it is possible that a linker with flexibility and sufficient length would accommodate the steric requirements for assembly of multimeric nps. however, it is difficult to precisely predict the effect of the linker, and therefore it is advisable to screen multiple constructs during the early stages of testing the assembly of nps displaying antigens of interest. in conclusion, the chaperna-based antigen assembly platform holds promise for the development and delivery of np-based vaccines to enhance rbd-specific antibody responses, and the serological detection of emerging viruses. various types of designing principles have advanced the structure-based approaches to np assembly ( , ) . however, most of the in silico methods consider the thermodynamic stability of the final assembled nps, but not necessarily the kinetic pathways leading to their successful folding into regular assemblages. consequently, most nps are refractory to soluble expression and fail to assemble as designed, resulting in significant, and practical challenges in the manufacturing process. the chaperna-mediated folding and the "pace-keeping" assembly of monomers into higher ordered structures will enable faithful production of np and vlp-based vaccines against emerging and re-emerging viral infections. this study was carried out in accordance with the recommenda- figure | elucidation of rna-mediated nanoparticle (np) formation of receptor-binding domain (rbd)-fr. (a) size exclusion chromatography analysis of rbd-fr nps purified from the tev protease-cleaved hrid(wt, , or m)-rbd-fr. the fractions ( - ml) estimated as nps were further analyzed by transmission electron microscopy (b) and dynamic light scattering (c) exploiting virus-like particles as innovative vaccines against emerging viral infections traditional and new influenza vaccines vaccine manufacturing: challenges and solutions management of accidental exposure to ebola virus in the biosafety level laboratory vaccine delivery using nanoparticles vaccine delivery: a matter of size, geometry, kinetics and molecular patterns virus-like particles as a highly efficient vaccine platform: diversity of targets and production systems and advances in clinical development the influence of antigen organization on b cell responsiveness structure of the hepatitis e virus-like particle suggests mechanisms for virus assembly and receptor binding self-assembly of human papillomavirus type capsids by 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domain-based subunit vaccines against middle east respiratory syndrome coronavirus protein/peptide-templated biomimetic synthesis of inorganic nanoparticles for biomedical applications self-assembling influenza nanoparticle vaccines elicit broadly neutralizing h n antibodies harnessing an rna-mediated chaperone for the assembly of influenza hemagglutinin in an immunologically relevant conformation host participation in bacteriophage lambda head assembly properties of a mutant of escherichia coli defective in bacteriophage lambda head formation (groe). i. initial characterization coupled chaperone action in folding and assembly of hexadecameric rubisco modeling the influence of salt on the hydrophobic effect and protein fold stability rational design of an epstein-barr virus vaccine targeting the receptor-binding site key: cord- -uu wlpmp authors: alberca, ricardo wesley; pereira, nátalli zanete; oliveira, luanda mara da silva; gozzi-silva, sarah cristina; sato, maria notomi title: pregnancy, viral infection, and covid- date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: uu wlpmp pregnancy comprises a unique immunological condition, to allow fetal development and to protect the host from pathogenic infections. viral infections during pregnancy can disrupt immunological tolerance and may generate deleterious effects on the fetus. despite these possible links between pregnancy and infection-induced morbidity, it is unclear how pregnancy interferes with maternal response to some viral pathogens. in this context, the novel coronavirus (sars-cov- ) can induce the coronavirus diseases- (covid- ) in pregnant women. the potential risk of vertical transmission is unclear, babies born from covid- -positive mothers seems to have no serious clinical symptoms, the possible mechanisms are discussed, which highlights that checking the children's outcome and more research is warranted. in this review, we investigate the reports concerning viral infections and covid- during pregnancy, to establish a correlation and possible implications of covid- during pregnancy and neonatal's health. pregnancy comprises a unique immunological condition, to allow fetal development and to protect the host from pathogenic infections. viral infections during pregnancy can disrupt immunological tolerance and may generate deleterious effects on the fetus. despite these possible links between pregnancy and infection-induced morbidity, it is unclear how pregnancy interferes with maternal response to some viral pathogens. in this context, the novel coronavirus (sars-cov- ) can induce the coronavirus diseases- (covid- ) in pregnant women. the potential risk of vertical transmission is unclear, babies born from covid- -positive mothers seems to have no serious clinical symptoms, the possible mechanisms are discussed, which highlights that checking the children's outcome and more research is warranted. in this review, we investigate the reports concerning viral infections and covid- during pregnancy, to establish a correlation and possible implications of covid- during pregnancy and neonatal's health. keywords: covid- , sars-cov- , pregnancy, neonatal, immunology pregnancy pregnancy comprises a unique immunological condition, to protect the fetus from maternal rejection, allowing adequate fetal development and protection against microorganisms ( , ) . the maternal immune system is challenged by paternal alloantigens expressed both by the fetus and the placenta. however, through a complex range of cells and molecules, the mother does not develop a classic response to this allograft ( ) . during pregnancy, fetal microquimerism occurs, where fetal cells, such as nucleated erythrocytes, trophoblastic cells, and leukocytes ( ), cross the placental barrier and expose the mother to fetal alloantigens. these cells can remain in the bloodstream and maternal tissues many years after delivery ( , ) . in comparison to the post-partum period, pregnancy increases monocytes, granulocytes, pdcs, mdcs in the blood, peaking during trimesters. simultaneously, during pregnancy occurs a reduction in cd , cd , and cd t cells in comparison with post-partum. b cells are decreased during the third trimesters. nk cells cd dim are reduced in the second and third trimester of pregnancy in comparison with the first trimester and post-partum period. during the second and the third trimesters, nk and cd t cells present a reduction in the production of ifn-γ, tnf, il- cells, compared with post-partum ( ) but the variability and contradictory reports are noted ( ) . maternal monocytes do not show differences in absolute numbers, however, they show some phenotypic changes including an increase in the expression of adhesion molecules (cd a, b; cd ), and the high-affinity igg receptor, fcγr-i (cd ) ( ) . the absolute number of nk cells in maternal blood increases in the first trimester of pregnancy ( ) . like lymphocytes, b cells are decreased during pregnancy and remain lower until month after delivery. in vitro, b cells of pregnant women were less responsive, with suppression of lymphopoiesis and exclusion of autoreactive b cells ( ). despite this, vaccine response during pregnancy remains effective ( , ) . from the th week of gestation, maternal peripheral blood monocytes also undergo phenotypic and functional changes. there is an increase in the ability to produce cytokines il- β and il- and a reduction in the potential for tnf-α secretion ( ) . the placenta is a transient chimeric organ that develops from the uterine wall and can express different receptors and dynamically delivered microvesicles through pregnancy ( ) . this organ mediates hormonal, nutritional, and oxygen support to the fetus while modulating maternal's immune response ( ) . the placental maternal face is formed from decidual cells, with the presence of wide range of immune cells, including uterine natural killer (unk), dendritic cells (dcs), and regulatory t cells (tregs). the fetal face consists of the placental villus, which contains fetal blood vessels surrounded by fibroblasts and placental villous macrophages of fetal origin, hofbauer cells ( , ) . treg cells are crucial for proper gestational development and are numerically elevated during pregnancy, in peripheral, deciduous and umbilical cord blood ( ) . paternal hla-c is a crucial molecule that can elicit allogeneic immune responses by maternal cell and aid in the development of maternal-fetal tolerance ( ) , also t reg may regulate cd + and cd + t lymphocyte activation through the expression of il- and tgfβ ( ) . another striking feature of the maternal-fetal interface is the accumulation of nk cells, which comprise up to % of deciduous leukocytes in early pregnancy ( ) . these cells are important for the regulation of cytokines production, especially il- , and act in the production of angiogenic factors, chemokines, controlling the invasion of trophoblasts and availability of adequate maternal blood at the implantation site ( , , ) . during pregnancy, hormonal variations can modulate immune responses, generating a reduction in the number of dcs and monocytes, and a decrease in the activation of macrophages, t, and b cells ( ) . to better establish the tolerogenic milieu, estrogen induces efficiently foxp t regs cells ( ) ( ) ( ) . changes in hormonal levels and immune system function generated by pregnancy may increase women's vulnerability to infections. pregnant women show higher mortality rates and complications associated with viral infections compared to the general population ( , ) . for example, varicella disease in children is mild, but primary infections during pregnancy can progress to varicella pneumonia and death ( ) . in , during the h n flu pandemic, an increased ratio of female to male cases was verified, in which pregnant women developed more complications, as severe acute respiratory syndrome, and higher mortality compared to the general population ( , ) . similarly, in the pandemic spanish flu, among , reported cases of influenza in pregnant women, % died as a result of the infection ( ) . in , with the h n pandemic, % of influenza deaths in women of reproductive age in minnesota occurred in pregnant women ( ) . although influenza viruses are restricted to maternal lungs, inflammatory cytokines can lead to fetal complications mainly preterm birth and fetus miscarriage ( , ) . in the ebola epidemic in , % of infected women (out of a total of ) were pregnant ( ) . some evidence suggests that during pregnancy there is a greater risk of developing serious illnesses, spontaneous abortion, hemorrhage, and death when infected with the ebola virus ( ) . additionally, infection by the lassa virus in pregnant women shows high levels of placental replication, and the risk of maternal-fetal mortality increases with the duration of pregnancy ( , ) . viruses can gain access to the decidua and placenta by ascending from the lower reproductive tract or via hematogenous transmission, viral tropism for the decidua and placenta is then dependent on viral entry receptor expression in these tissues as well as on the maternal immune response to the virus ( ) . a range of viral infections in pregnancy are associated with specific placental findings, including lymphoplasmacytic villitis with associated enlargement of villi and intravillous hemosiderin deposition in the setting of maternal cytomegalovirus infection ( ) , as well as rare reports of intervillositis in the setting of zika virus ( ) and dengue virus ( ), among others. although there is little knowledge about placental findings associated with the common coronaviruses, ng et al. reported placental pathology in seven women with sars infection in hong kong ( ) . in three placentas delivered in the acute stage of sars, demonstrated increased perivillous or subchorionic fibrin, while in two women who had recovered from third-trimester infection by the time of delivery, there were large zones of avascular villi, with one of the two additionally demonstrating a large villous infarct; both contained increased nucleated red blood cells in the fetal circulation. none of the seven placentas examined had any acute or chronic inflammatory processes ( ) . the covid- pandemic is still in its early stages, with preliminary case series of infection in pregnant women available. a study of three placentas delivered from pregnant women with sars-cov- infection, infected in their third trimester with emergency cesarean section, describe various degrees of fibrin deposition. the fibrin deposition occurred inside and around the villi with local syncytial nodule increases in all three placentas, multiple villous infarcts in one placenta, and a chorangioma in another case. all samples from three placentas were negative for the nucleic acid of sars-cov- ( ) . another study with placentas from patients with sars-cov- were examined and the most significant finding is an increase in the rate of features of maternal vascular malperfusion (mvm), most prominently decidual arteriopathy including atherosis, fibrinoid necrosis, and mural hypertrophy of membrane arterioles ( ). maternal hypertensive disorders, including gestational hypertension and preeclampsia, are the major risk factors for mvm ( ) , although only of the patients was hypertensive in this study. notwithstanding, sars-cov- is a virus that is expected to induce inflammation, it is relevant that neither acute inflammatory pathology (aip) nor chronic inflammatory pathology (cip) were increased in covid- patients relative to the controls. however, none of the covid- patients in this study were severely ill or undergoing a cytokine storm and it may be possible that cip could be induced in those cases of severe systemic inflammation ( ). there few knowledges about miscarriage in women with covid- , one case was a pregnant woman with symptomatic coronavirus disease who experienced a second-trimester miscarriage. a stillborn infant was delivered vaginally and swabs from the axillae, mouth, meconium, and fetal blood obtained within minutes of birth tested negative for sars-cov- and bacterial infection. the fetal autopsy showed no malformations, and fetal lung, liver, and thymus biopsies were negative for sars-cov- . furthermore, amniotic fluid and vaginal swabs sampled during labor tested negative for sars-cov- and bacterial infection. placental histology demonstrated mixed inflammatory infiltrates composed of neutrophils and monocytes in the subchorial space and unspecific increased intervillous fibrin deposition ( ) . during the worldwide sars-cov- (severe acute respiratory syndrome coronavirus- ) epidemic in , a notable increase in mortality and morbidity was documented in pregnant patients ( ) . agreeing with previous observations that the risk of viral pneumonia is significantly higher among pregnant women compared to the rest of the population ( ) . in , infection with the middle east respiratory syndrome (mers-cov) coronavirus in saudi arabia after the isolation of a male patient who died of severe pneumonia ( , ) . data on the effects of mers-cov on pregnancy are limited, whereas there is a description of stillbirth at months of gestation ( ) . between and , the ministry of health of saudi arabia reported the occurrence of , cases of mers-cov infection, five of which were pregnant ( ) . despite the few descriptions, the immunological changes in pregnancy may alter the susceptibility to mers-cov and the severity of the clinical disease ( ) . in a mice model of herpes virus infection, even in the absence of herpes virus placental passage, there was a marked increase in the levels of pro-inflammatory cytokines, including ifn-γ and tnf-α, as well as changes in fetal development ( ) . this scenario may result from the placenta's pro-inflammatory response generated by the infection, or it may be due to other physiological changes in the mother or placenta related to the infectious process ( ) . placental cells, predominantly trophoblasts, express tlr (toll-like receptors) and this expression varies according to the gestational age and the differentiation stage of these cells. viral infections can disturb the fine immune regulation at the maternal-fetal interface and lead to fetal damage, even without the virus reaching it directly ( ) . for example, tlr- expressed by trophoblasts in the first trimester of pregnancy ( ) , mediates rapid antiviral response ( ) , and induces the production of cytokines, type i interferon (ifn) and type iii ifn ( ) . tlr is also expressed in trophoblasts, which induces the synthesis of anti-viral cytokines and plays a role in preventing intrauterine transmission of hbv ( ) . however, these inflammatory responses can be associated with complications in pregnancy, such as pre-eclampsia and/or intrauterine growth deficit ( ) . in general, cytokines and ifns are important mediators in a healthy pregnancy, due to their role in the regulation of cell function, proliferation, and gene expression. however, when dysregulated, they have the potential to interrupt fetal and placental development pathways ( ). the world health organization (who) estimates that about . million children died within the first month of life in . every day ∼ , newborns die, amounting to % of all child mortality under the age of years ( ) . the majority of all neonatal's deaths are due to preterm birth, intrapartum-related complications (birth asphyxia or lack of breathing at birth), infections and birth defects. regarding the highest incidence of infection observed in early-life, it is generally attributed to an immature immune system during the transitional post-natal period ( ) . innate immune cells are composed of specialized cells, such as granulocytes (e.g., neutrophil), monocytes, macrophages, dcs and innate lymphocytes. around weeks gestation, neutrophils are present in human fetal liver parenchyma ( ) , when compared to the adult response, neonatal neutrophils have qualitative and quantitative impairments in the response under stress conditions, including reduced chemotaxis, respiratory burst, and extracellular traps formation ( ) . the cytokine profile produced by antigen-presenting cells (apcs) monocyte/macrophage and dcs in newborn differs from those produced by adults. typically, apcs from neonates produce less pro-inflammatory cytokines like il- β, tnf-α, il- p , and type i ifn upon stimulation on tlrs ( ) . otherwise, it produces great amounts of th -promoting cytokines (il- and il- ) when compared with adult cells ( ) . following, the importance of anti-inflammatory response in early life is highlighted through the great amount of il- produced by newborn monocyte/conventional dc (cdc) compared to adults ( ) . the pattern of innate cytokine response can be attributed to two mechanisms: (i) high mononuclear cell levels of intracellular cyclic adenosine monophosphate (camp), a secondary messenger that suppresses th but enhances th and anti-inflammatory cytokine production ( ) and (ii) altered dna binding capacity of transcription factors, such as irf to the promoter regions of cytokine genes secondary to age-specific chromatin ( ) . curiously, neonates' dcs activation with clr agonist dectin or macrophage-inducible c-type lectin (mincle), simultaneously with tlr / potently drives caspase- and nf-kb activation and th -supporting cytokine production (including il- p ), overcoming the age-specific epigenetic barrier in early life for irf function and leading to a th- phenotype ( , ) . on weeks of gestation, mature fetal αβ t lymphocytes can be detected. during the second and third trimesters of gestation, the repertoire of fetal t cell receptors diversifies ( ) . generally, neonates have a limited th profile response to some vaccines and pathogens, agreeing with a lower capacity of cd t cells to produce ifn-γ and of apcs to produce th -skewing cytokines ( ) . although there are some situations where the responsiveness of the th profile is efficient, for example, neonates and infants develop adult-like th responses to bcg or pertussis vaccines, and a fetus can develop th responses in congenital cmv infection ( ) ( ) ( ) . recent studies suggested that the early life immune system could present advantages for the elicitation of broadly neutralizing antibodies (bnabs), a response highly desired for an hiv vaccine. in fact, hiv-infected children develop bnabs responses earlier and more frequently than infected adults ( ) . congenital and perinatally acquired viral infections do occur and may lead to major disabilities in infancy and childhood, the main causes can be attributed to pathogens like toxoplasma gondii, rubella virus, cytomegalovirus (cmv), herpes viruses, syphilis, and zika virus ( ) . while congenital rubella virus syndrome is no longer seen in countries with compulsory immunization against this virus, an outbreak of zika virus (zikv) recently occurred in brazil resulting in the zikv syndrome, with brain lesions comparable to, but more severe than congenital cmv infection ( ) . neonates display an immature immune response, the first exposition to an environmental stimulus can shape the lung's immune response ( ) . furthermore, there is a predominant type immune response in the lungs ( ), these characteristics make infants susceptible to respiratory viral infections, a common cause of infant's death ( ) . rsv is an important cause of lower respiratory tract illness in infants globally and is responsible for one-third of deaths due to lower respiratory tract infections in children < year of age ( ) . pregnant women are considered at high risk for severe influenza disease, for this reason, influenza vaccination has been recommended for pregnant women and introduced into immunization programs ( ) . influenza vaccination is safe and protective on preterm birth (ptb) and low birth weight (lbw) ( ) . one of the benefits of maternal immunization has also been shown to extend to neonates through the transfer of maternal antibodies, providing passive immunization against the influenza virus ( ). on the severe pandemic h n influenza illness, some studies suggested an association between severe h n disease, preterm birth, and fetal death; however, these limited data do not permit firm conclusions ( ) . sars-cov- infected ∼ pregnant women during the pandemic ( ), causing a high lethality and miscarriage rate ( ) , but no neonatal infection has been reported ( ) . in , cynthia maxwell postulated possible intensive care and procedures to properly manage maternal and neonatal sars-cov- infections ( ) . vertical transmission of mers has not been documented. in a case report by alserehi et al., a mother was diagnosed with mers, treated and a cesarean section was performed to deliver a healthy preterm baby with weeks of gestation ( ) . hon et al. described children with mers, that presented persistent fever and cough, after treatment no fatal case was reported. all children in this report obtained the infection via adult-to-children transmission, and no children-tochildren transmission was reported ( ) . iqbal et al. reported a case of spontaneous vaginal delivery in covid- -positive pregnant, with no signs of neonatal infection up to -days post-partum ( ) . nevertheless, it is important to highlight contact precautions were made in this report to prevent post-partum transmission. in late , a respiratory infectious disease began to be investigated in wuhan, china ( ) . at first, contagion occurred through contact with some infected animals but, soon there were the first reports of human-to-human transmission ( ), the virus was identified as belonging to the coronaviridae family and was designated sars-cov- (severe acute respiratory syndrome coronavirus- ) ( ). like other members from this viral family, mers and sars-cov- , the new coronavirus causes a respiratory disease, named covid- (coronavirus disease− ) ( ) . although very similar, sars-cov- and sars-cov- impacted the world differently. sars-cov- emerged in and killed almost people in countries ( ) and, even without a vaccine, it was taken preventive actions as patient isolation. the new coronavirus has killed more than , people in just months and has spread to continent ( ). sars-cov- shares genetic similarities between sars-cov- and mers, and %, respectively ( ) . sars-cov- is an enveloped single-stranded rna virus and has a genome of ∼ , nucleotides that encode structural and accessory proteins-the largest known viral rna genome ( ) . sars-cov- and sars-cov- enter the host's cells via the ace receptor (angiotensin-converting enzyme ) ( ). in the lung, the most affected organ among those infected, the main target is the type alveolar cell ( ) . the ace receptor is also expressed in cells from kidneys, esophagus, heart ( ) . moreover, a small percentage of monocytes and macrophages express the ace receptor ( , ) . thus, there may be another alternative receptor or infectious pathway, such as antibody-dependent enhancement (ade). however, unlike other coronaviruses, limited to respiratory disorders, sars-cov- caused multiple organ failure. furthermore, this receptor is more expressed in the elderly, which associated with immunosenescence and other comorbidities common among the elderly may justify the high lethality rate in this age group ( ) . the viral load peaks occur during the first week of infection and then gradually decrease over the next few days. in addition, the viral load is correlated with the patient's age. igg and igm antibodies start to increase days after disease and most patients are seroconverted in the first days ( ) . moreover, in vitro assays, has shown that the serum from sars-cov- -infected patients were able to neutralize the virus ( ) . thereby, the humoral response can be another antiviral strategy via plasma transfer ( ) . in sars-cov- and mers, as a viral escape mechanism, the virus can suppress ifn type i response, either by cytosolic sensors of ubiquitination, inhibiting nuclear factors translocation or decreasing stat phosphorylation ( ) . neutrophils, c-reactive protein and several cytokines (as il- , tnf, il- ) are increased in covid- , and this elevation is correlated with disease severity and death ( ) . in serious illness, the same protein levels were detected and inflammatory cytokines increase is correlated with t cd + and t cd + lymphocytes decrease and lower ifnγ production. b-lymphocytes do not appear to be affected by the disease, regardless of severity ( , , ) . these characteristics observed in patients indicate that a covid- can be mediated by an intense inflammatory process that follows the disease severity. as with sars-cov- and mers, this increase in cytokine levels-known as a cytokine storm-can be involved with the pathogenesis of the disease ( ). to defend itself against an aggressive agent (such as infection, trauma, acute inflammation, among others) the body produces an exaggerated response to localize and then eliminate the damage. this response is known as the systemic inflammatory response syndrome (sirs) or, if the source infection sepsis ( ) , this process leads to the release of acute-phase proteins and endocrine, hematological and immunological changes, among them, the cytokine storm can lead to tissue damage and even death ( ) . cytokine storm is produced, mainly, by highly activated macrophages and can cause lung damage and start viral sepsis ( ) . this inflammation leads to other complications, such as acute respiratory distress syndrome (ards) and respiratory and cardiac failure ( , ) . studies in mice infected with sars-cov- , also demonstrate the cytokine storm dampening adaptive immunity ( ) . other factors may also influence the susceptibility for covid- infected persons, and some gene polymorphisms, well-documented for other viral infections ( ) . at the moment no vaccine or specific treatments are available for disease control of the sars-cov- . in pregnancy, pneumonia infections may trigger an increased mortality risk to the mother and fetus ( ) , which can also lead to complications as preterm birth and small for gestational age ( ) . placental syncytiotrophoblast cells express the ace receptor and this receptor is highly expressed in the first months of pregnancy. associated with placental immaturity, the early ace expression can make the first trimester the most likely period for sars-cov- -infection ( ) . a serine protease, tmprss , is also required for viral entry ( , ) and there is still no consensus about placenta expression. some studies report low, but present, mrna expression in human placentas ( ) , others describe that expression is not detectable ( ) . the association of tmprss and ace expression, in the first months of pregnancy, would make this phase more susceptible to sars-cov- -infection. blood tests in pregnant women revealed regular covid- markers, such as lymphopenia, neutrophilia, and elevated c-reactive protein level in pregnant women ( , ) . some reports also verified an increase in alt, ast, and d-dimer ( ) ( ) ( ) ). an important report verified that mothers developed anemia and dyspnea, which could potentially be a risk factor during c-section labor ( ) . chen and collaborators, verified alteration in calcium and albumin levels in the blood of pregnant women with sars-cov- infection ( ) , which could potentially increase the severity in covid- ( ) . furthermore, in a recent report involving maternal death in consequence to covid- , cases reported a low number of platelets, which is associated with an increase in mortality by covid- ( , ) . it is still under investigation the effects of sars-cov- infection in the maternal-fetal context ( table ) . some reports describe that symptomatic infected-mothers did not transmit the virus during pregnancy. in a case report of seven cases, showed that three babies were tested to sars-cov- and only was positive h post-partum ( ) . on the other hand, another report shows increase in inflammatory cytokines and virus-specific igm levels in newborns, from infected-mothers, h after birth ( ) , and in another report, newborns presented virus-specific igm and igg, but no sars-cov- -infection ( table ) ( ) . this lead to the possibility of the activation of the maternal immune system by sars-cov- may have some implication of the offspring's health and immune system development. although the number of pregnant women with covid- studies is limited, there is no conclusive report of vertical transmission ( table ) ( , ) . a recent case report, was described two cases of rashes and one with facial ulcerations ( ) . another important factor, besides the immune activation, the maternal usage of antiviral drugs can also permanently affect the offspring's immune response ( ) , as there is no current standard protocol of treatment regarding the usage of antibiotics or antivirals ( table ) ( ) . only a fraction of patients infected with sars-cov- develops severe respiratory disorders, it is unknown whether the pregnant could be more susceptible to pulmonary diseases. covid- can progress to a severe lung inflammation that can progress to life-threatening illness at the severe stage ( ) . this inflammatory process is associated with high plasma levels of cytokines, as cytokines storm, including il- , il- , il- , g-csf, ip- , mcp- , mip- a, and tnfα ( ) . this might play an important role in pregnancy as il- has been implicated to be upregulated in pre-eclampsia ( ) and miscarriage ( ) and il- /il- r signaling pathway in fetal miscarriage ( ) , due to the upregulation in the ratio of th /treg cells ( ) . another relevant aspect is the possible implication of polymorphisms in covid- diseases, as is well-documented for other viral infections ( ) . also, cytokines polymorphisms, such as tnf-α g/a (rs ) polymorphism is associated with recurrent miscarriage ( ) . in fact, tnf-α and tnf-α receptor play an important role in the development of the fetus, being present in the ovary, endometrium, placenta, and fetus, and in the amniotic fluid in different concentration ( ) . this increase in tnf-α during pregnancy may implicate in different health outcomes depending on the gestational period ( ) , leading to tissue necrosis in the placenta and hypoxia ( ) . interestingly, an acute increase of this cytokine during pregnancy in animals may cause abortion ( ) . moreover, alteration in the health status of the mother during pregnancy can have long-term effects on the offspring's health ( ) . inflammatory processes during pregnancy can also impact women's health, as the increase in tnf-a during pregnancy can also lead to impaired insulin sensitivity ( ) and gestational diabetes mellitus ( ) . in animal models, inflammation during pregnancy has been shown to alterations in the behavior ( , ) fetal brain development ( ) ( ) ( ) , metabolic disturbance ( , ) , and shape offspring's immune response to antigens and infections ( , ) . the physiological response, as stress and the control of temperature, during the infection may present a long-term effect in pregnant women with covid- . the increase in stressrelated hormones can also affect the offspring's immune system ( ) and fever during pregnancy increase the chances of neural disorders in the children ( ) . moreover, an increase in anti-inflammatory cytokine il- in covid- mothers is probably a regulatory mechanism crucial to regulate the inflammation ( ) and pregnancy maintenance ( ). even though no vertical transmission for covid- has been reported until now, several reports of early-life infections have been described with very low death rates ( , ) . reports with recommendations to the treatment of pregnant women with covid- ( ) and for neonates with covid- have been published ( , ) . another possible route for sars-cov- is oral transmission by fecal samples ( ) , and via breastfeeding from a sars-cov- infected mother. regarding breastfeeding, a small study found no evidence of covid- in breast milk, of six patients ( ) . however, the primary concern is whether an infected mother can transmit the virus through respiratory droplets during breastfeeding. other viruses in the past have also caused concern in pregnant women. the zika virus has been linked to several cases of microcephaly in newborns during an epidemic in in brazil ( ) . the infection had a high point in the first trimester of pregnancy, where there were more favorable conditions for its entry and replication in placental cells. in the case of sars-cov- , it has not yet possible due to the time of infection occurs in the world, to observe the consequences of infection in the firsttrimester pregnancy. taking into account the early pregnancy, the placental tissue immaturity together with the up-regulation of ace expression in placental cells, perhaps the more susceptible period for sars-cov- infection is around the first trimester of pregnancy. it is important to highlight that after the influenza pandemic there have been reports of reduced cytokine response to bacterial infections. this leads to the hypothesis that covid- can lead to impairments of the immune response to other pathogens and vaccines in the future. future investigations are needed to identify the possible implications of sars-cov- /covid- in pregnancy, the possible infection of the placenta in the first trimester of pregnancy and implications of the cytokine storm to the neonatal's health. ra and ms: write, conception, and review. np, lo, and sg-s: write and review. all authors contributed to the article and approved the submitted version. ra holds a post-doctorate fellowship from fapesp ( / - ) and lo also holds a post-doctorate fellowship from fapesp ( / - ). sg-s holds a master degree fellowship from fapesp ( / - ). the immune system in 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proposal of management care emergency plan for inter-hospital transfer of newborns with sars-cov- infection chinese expert consensus on the perinatal and neonatal management for the prevention and control of the novel coronavirus infection detectable sars-cov- viral rna in feces of three children during recovery period of covid- pneumonia zika virus in the americas: early epidemiological and genetic findings the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © alberca, pereira, oliveira, gozzi-silva and sato. this is an openaccess article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - y je authors: correa, isabel; ilieva, kristina m.; crescioli, silvia; lombardi, sara; figini, mariangela; cheung, anthony; spicer, james f.; tutt, andrew n. j.; nestle, frank o.; karagiannis, panagiotis; lacy, katie e.; karagiannis, sophia n. title: evaluation of antigen-conjugated fluorescent beads to identify antigen-specific b cells date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: y je selection of single antigen-specific b cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. to establish this, we selected folate receptor alpha (frα) as a model antigen and a mouse b cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (mov igg ) specific for frα, as test antibody-expressing cells. beads were conjugated to frα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-frα. bead-bound cells were single cell-sorted and processed for single cell rna retrotranscription and pcr to isolate antibody heavy and light chain variable regions. variable regions were then cloned and expressed as human igg /k antibodies. like the original clone, engineered antibodies from single cells recognized native frα. to evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (pbmcs) were spiked with test antibody-expressing cells. antigen-specific cells could comprise up to % of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was : or higher. in pbmc pools, beads conjugated to recombinant antigens frα and her bound antigen-specific anti-frα mov and anti-her trastuzumab antibody-expressing cells, respectively. from melanoma patient-derived b cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. this approach may be further developed to facilitate analysis of b cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires. the selection and study of single antigen-specific b cells and the identification of their expressed antibodies can help shed light on the nature and functions of the human humoral immune response. in our study, we present an antigen-conjugated fluorescent bead methodology designed to identify and isolate single antibody-expressing cells and to derive and clone matched heavy and light chain antibody variable regions into full length antibodies. we believe that our single cell-to-functional antibody strategy falls within the scope of this research topic, as it opens the way for opportunities to analyze b cells and their antibody profiles at the single cell level and may be potentially applied to help unravel diverse humoral immune repertoires in different health and disease states. the study of humoral immunity may be key to understand human health, aging, and disease, as well as to predict or monitor host immune responses to pathogen challenge and disease progression ( ) . moreover, dissecting b cell responses could help inform medical interventions such as preventative vaccines or therapeutic treatments ( , ) . currently, therapeutic monoclonal antibodies are part of the standard care of treatment for various conditions including diabetes, autoimmune and allergic diseases, and various types of cancer. a big proportion of these antibodies are fully mouse or mouse-human chimeras ( ) . therefore, identification of fully human, heavy (h), and light (l) variable regionmatched antibodies may be of interest for clinical applications, as administration of fully human antibodies is considered less likely to induce antidrug antibody responses compared with chimeric or humanized antibodies ( ) . immune monitoring of human b cells recognizing specific antigens, alongside identification, cloning, and production of their cognate monoclonal antibodies, may, therefore, be of substantial value to help evaluate reactivity and immunological response to these antigens. analyses of the antibody repertoires of b cells have been reported in the human setting and in animal models, with the most common applications in the study of autoimmune diseases ( ) , viral infections, and b cell malignancies ( ) ( ) ( ) . some studies have employed high-throughput sequencing of heavy chain variable regions or of paired h and l variable region sequences ( , ) . ultra-high-throughput sequencing techniques have also been developed, capable of analyzing repertoires of thousands of b cells from small human blood samples, with some allowing the mapping of paired h and l variable regions, in a few hours ( ) ( ) ( ) . b cells may also be a source of antigen-specific antibodies for clinical diagnostics or for therapeutic applications. existing methods to identify antigen-specific b cells and generate monoclonal antibodies include ex vivo b cell culture approaches, which could promote the survival and expansion of certain b cell subsets, screening of the culture supernatants to identify b cell reactivity and fluorescent-activated cell sorting ( ) ( ) ( ) ( ) ( ) ( ) . an essential element in the process of selecting antigen-specific b cells is detection of antibodies with a certain degree of specificity. this could be achieved by screening cell culture supernatants through elispot assays or elisa-based methods using immobilized recombinant antigens or cells ( , ) . screening cell culture supernatants by elisa, although highly sensitive, represents only a surrogate parameter and antigen reactivity should ultimately be confirmed only after sequencing and expression of the selected clone. for all these applications, the gold standard of identifying antigen-specific antibodies remains the expression of the recombinant antibody and further evaluation of its antigen recognition properties. workflows to facilitate selection of single human b cells without ex vivo growth, stimulation, and clone expansion, and which do not require sampling of cell culture supernatants could offer additional tools for the study of human b cell immunity. novel approaches to address these requirements involve the use of modified fluorescent tetramers for direct b cell screening by fluorescent-activated cell sorting ( , ) . in this study, we describe the design of a bead-based methodology to identify single antibody-expressing b cells, and to clone and produce antigen-specific antibodies. the workflow features bead-based identification and isolation of specific b cells using direct fluorescent-activated cell sorting, sequencing, and cloning of matched heavy and light chain variable regions in a single full sequence antibody expression vector system, and expression and testing the antigenic reactivity of the antibody clone. the workflow is designed to avoid ex vivo b cell expansion and secondary clone selection and to facilitate antibody generation and downstream evaluation. human immune cells were isolated from venous blood of healthy volunteers and patients with malignant melanoma. specimens were collected with informed written consent in accordance with the declaration of helsinki. the study was conducted at king's college london, king's college london, guy's and st thomas' nhs foundation trust ( /h / approved by london bridge nres committee; /lo/ approved by london-central nres committee). human peripheral blood mononuclear cells (pbmc) were isolated from ml blood using ficoll ® paque plus density centrifugation (ge healthcare). cell culture was performed using aseptic technique at °c in a humidified atmosphere in % co , unless otherwise specified. the human ovarian carcinoma cell line igrov naturally over-expressing folate receptor alpha (frα) was grown in rpmi glutamax™ medium (thermo scientific) supplemented with % fetal calf serum (fcs). the human breast cancer cell line mda-mb- was grown in dmem glutamax™ medium (thermo scientific) supplemented with % fcs. the permanently transfected murine myeloma cell lines sp / -mov specific for frα and sp / -sf , recognizing a colon carcinoma antigen ( ) , were cultured in dulbecco's modified eagle's medium plus % fcs as previously described ( ) . the human embryonic kidney cell lines, expi f cells, were cultured in serum-free expi expression medium (thermo scientific) on a stuart orbital shaker at rpm at % co . expi f cells were transfected with pvitro -hygro-mcs antibody constructs using the expifectamine transfection kit (thermo scientific) as per manufacturer's instructions. the anti-human epidermal growth factor receptor (her ) and the melanomaassociated antigen-specific chondroitin sulfate proteoglycan (cspg ) antibody constructs were previously described ( , ) . different avidin-or streptavidin-coated fluorescent beads of different sizes were used (table s in to biotinylate proteins of melanoma cell lines, confluent cell monolayers were washed with pbs and borate buffer ph . then, sulfo-nhs-lc-biotin at mg/ml in pbs was added and incubated for min on ice. the reaction was quenched with mm tris, mm nacl ph . , and the cells were subsequently extensively washed with pbs. cell lysis buffer (cell signaling technology) containing . % triton and supplemented with mm pmsf was added to the monolayer and the cells were harvested by scrapping. lysates were briefly sonicated and incubated on an orbital rocker for min at c. the lysates were cleared by centrifugation at × g for min, followed by a second centrifugation at , × g for min. supernatants were either used immediately or stored at − °c. recombinant folate receptor α (frα) (r&d systems, cat no -fr) and human epidermal growth factor receptor (erbb /her ) fc chimera (r&d systems, cat no -er) were reconstituted in pbs at . mg/ml as recommended by the manufacturer. biotins with three different arms were used: (i) sulfo-nhs-lc-biotin with a spacer arm of . Å; (ii) sulfo-nhs-lc-lc-biotin with a spacer arm of . Å, and (iii) nhs-peg -biotin with a spacer arm of Å (all from pierce-thermo fisher scientific). the three forms of biotin were reconstituted following the manufacturer's instructions and mixed with the recombinant proteins in pbs at a : (biotin:recombinant protein) molar ratio and incubated for min at room temperature (rt). free biotin was removed by dialysis in pbs. for binding of biotin-conjugated frα to lumavidin . µm microspheres, µl of microsphere solution was washed with ml of % bsa-pbs- . % sodium azide, spun at , × g for min, and pellets were resuspended in µl of % bsa-pbs- . % sodium azide. then, . µg of biotin-conjugated recombinant protein (in µl of pbs- % bsa) was added to the beads and mixed, by rotation, for up to h at rt. beads were then washed three times with ml of % bsa-pbs- . % sodium azide and resuspended in µl of % bsa-pbs- . % sodium azide. for binding of biotin-conjugated frα to all sphero fluorescent particles, µl of beads were washed with ml of % bsa-pbs- . % sodium azide, spun at , × g for min at rt, and the pellet was resuspended in the original volume of beads with % bsa-pbs- . % sodium azide. then, µg of biotin-conjugated recombinant protein was added and mixed, by rotation, for - h at rt. after binding, beads were washed three times with ml % bsa-pbs- . % sodium azide and resuspended in µl of % bsa-pbs- . % sodium azide. for binding of biotin-conjugated cell extracts to lumavidin . µm microspheres, µl of microsphere solution were washed with ml of % bsa-pbs- . % sodium azide, spun at , × g for min, and pellets were resuspended in µl of % bsa-pbs- . % sodium azide. then, µl of biotinconjugated cell extracts were added to the beads and mixed, by rotation, for up to h at rt. after binding, beads were washed three times with ml of % bsa-pbs- . % sodium azide and resuspended in µl of % bsa-pbs- . % sodium azide. for each test, µl beads were used. to check that frα/her proteins were attached on the bead surface, conjugated beads were stained with specific antibodies for frα/her . conjugated beads ( µl) were incubated in µl of facs buffer ( % fcs-pbs) for min at rt, then, µg of the monoclonal antibody mov (anti-frα), anti-her [previously described in ref. ( ) ] or isotype control antibodies were added. in all described experiments, the hapten-specific in-house produced monoclonal antibody anti-nip igg was used as an isotype control. the beads were incubated for min at °c, washed with ml of facs buffer, and incubated with anti-human immunoglobulin (ig) antibody conjugated to fitc (vector laboratories) for another min at °c. after a final wash with facs buffer, stained beads were analyzed on a facscanto™ flow cytometer (bd biosciences). beads coated with melanoma cell line extracts were tested by staining with a monoclonal igg antibody recognizing the melanoma-associated antigen cspg . sp / cells expressing mov igg or the control antibody sf were prestained with anti-mouse mhc class i-pe antibody to be used with streptavidin sa-blue . µm or lumavidin . µm beads (table s in ig variable region cloning pcr tubes containing sorted cells in lysis buffer were thawed and reverse transcription was performed in a thermal cycler using the superscript vilo cdna synthesis kit (thermo fis her scientific) following the manufacturer's recommendations. variable regions of ig heavy and light chains were amplified by two rounds of pcr amplification in µl mix, using µl of phusion flash polymerase mix per reaction (finnzymes), each primer at . µm, µl of the reverse transcriptase reaction as template for the first table ) , and the nested pcr protocol were previously described ( ) . pcr products were separated on agarose gels, purified using the qiaquick gel extraction kit (qiagen), and cloned using the zeroblunt pcr cloning kit (life technologies). six colonies were grown for each pcr band, plasmid dna purified using qiaprep spin miniprep kit (qiagen) and sequenced by source bioscience using the m forward primer. sequences were analyzed using imgt/v-quest ( ). heavy and light chain variable regions were cloned in pvitrohygro- (invivogen) using polymerase incomplete primer extension (pipe) cloning as previously described ( ) . in brief, pvitro-hygro- , containing pre-cloned human antibody heavy and light constant region cassettes (gamma /kappa) was used as a template in two separate pipe pcr reactions to amplify two linear plasmid fragments with partially single-stranded ″ ends. similarly, the ig heavy and light chain variable regions were pipe pcr-amplified, using the pcr-blunt constructs, generated previously, as pcr templates. next, the pcr products were diluted four times with ddh o and mixed in a ratio of we designed a process to allow the identification of antigenspecific antibody-expressing b cells, which can be performed without prior ex vivo growth or secondary screening of b cells. to establish this system, we selected folate receptor alpha (frα) as a model antigen. as the test antibody-expressing cells, we selected a b cell line expressing both the soluble and the membranebound [b cell receptor (bcr)] form of a human/mouse chimeric antibody (mov igg clone) specific for frα. the workflow (figure ) entails coupling of fluorescent polystyrene beads with the nominal antigen, folate receptor alpha (frα), followed by binding of frα-coated beads to anti-frα antibody-expressing b cells. single bead-conjugated cells were identified and isolated by facs sorting directly onto microplates containing lysis buffer. rna released from single cells was converted to cdna using reverse transcription followed by a semi-nested rt-pcr with ig specific primers. matched variable h and l chains were sequenced and cloned into a single expression vector containing the h and l constant igg region sequences as previously reported ( , ) . the full antibody was then expressed in a human expression host. the antibody was purified and antibody specificity for the antigen was tested on target antigen-expressing cells. we first established the workflow using apc fluorochrome lumavidin microspheres of . µm diameter (lumavidin . µm; table s in supplementary material). prior to interrogating the ability of these beads to bind antibody-expressing cells, we used flow cytometric evaluations to confirm that fluorescent beads could be successfully coupled to antigens. we tested the antigens frα and her conjugated to fluorescent beads. the anti-frα antibody mov igg bound to frα-conjugated beads, and the her -specific antibody trastuzumab bound to her- conjugated beads. on the other hand, the hapten-specific monoclonal antibody nip igg showed only background binding to frα-conjugated or to her- -conjugated beads, and no binding was detected on beads incubated with secondary antibody alone (figure a) . we also interrogated different coupling agents with varying biotin lengths (lc-biotin, . Å; lc-lc-biotin, . Å; peg -biotin, Å) to assess whether the distance between bead and antigen from the bead surface could affect antigen recognition by anti-frα-expressing b cells. we found no significant differences in mov igg antibody binding to bead-conjugated frα with respect to any of these coupling agents ( figure b) . we then established a model system using microspherecoupled frα and anti-frα antibody-expressing b cells. we confirmed that a small proportion of frα+ lumavidin . µm microspheres recognized single anti-frα antibody-expressing b cells (sp / -mov ) ( figure a) . when single bead-bound cells were isolated into lysis buffer-containing microplate wells by flow cytometric sorting, single cell pcr yielded matched h and l chain variable region dna products were detected to be of the expected size ( figure b) . antibody variable region sequences were cloned into a pvitro vector containing human igg /k constant region cassettes and the antibody was expressed in expi f™ cells. subsequently, the cloned igg antibody was used to assess binding to igrov cells, which express native cell surface human frα. flow cytometric evaluations confirmed that the anti-frα antibody cloned from a sorted single antibody-expressing b cell was able to specifically recognize antigen-expressing igrov cells. the cloned anti-frα antibody did not recognize mda-mb- breast carcinoma cells that do not express frα (figure c) . these findings confirmed that it is possible to extract matched variable region sequences from single antibody-expressing cells selected by recognition of specific antigen-coated microspheres. selection of single b cells allowed the identification of the expected matched variable regions and permitted the cloning and production of the corresponding monoclonal antibody from a single cell. this antibody was able to recognize natively expressed cell surface frα. influence of microsphere diameter and biotin length on specific recognition of antigen-expressing b cells since we observed that frα+ microspheres were able to bind only a small proportion of single anti-frα antibody-expressing b cells figure | workflow for identification of antigen-specific monoclonal antibodies derived from single cell cloning. biotinylated antigen (folate receptor alpha, frα) was conjugated to fluorescent beads conjugated to streptavidin or avidin. antigen-coated beads could bind cells expressing an antigen-specific immunoglobulin (ig) and bead-bound cells were purified using cell sorting. rna from sorted cells was reverse transcribed and ig variable regions were amplified by nested pcr, sequenced, and cloned using the dual expression plasmid pvitro containing human heavy and light chain constant region cassettes (gamma /kappa). antibodies were expressed in a human cell expression system, purified, and tested for antigen binding. (sp / -mov ), we investigated whether microsphere diameter or the frα-biotin on the beads could influence specific recognition of antibody-expressing cells. we confirmed that sp / -mov igg cells expressing mov igg on the cell surface could be recognized by human recom binant frα ( figure a ). as observed with recombinant antibody binding to frα-coated microspheres (figure ) , the distance between bead and antigen from the bead surface did not affect antigen recognition by anti-frα antibody-expressing sp / -mov cells ( figure b) . however, the proportion of the sp / -mov cells recognized by fluorescent beads coated with frα differed with bead size or type: microspheres of smaller bead diameters (sa-red . µm and sa-blue . µm) appeared to bind a higher proportion of b cells ( . and . %, respectively) compared to the lumavidin . µm beads ( . %) (figures c,d) . the length of the biotin arm had minor effects on the binding of the smaller sized (a-red . µm and sa-blue . µm) microspheres to sp / -mov cells. the peg -biotin arm showed a slightly higher proportion of microsphere-attached sp / -mov cells ( - % of cells) compared with beads attached to frα via lc-lc-biotin ( - %) or lc-biotin ( - %) arms ( figure e) . binding of frα-coated fluorescent beads appeared to be specific for antibody-expressing b cells, since frα-coated beads bound - % of sp / -mov igg cells, while frα-coated beads bound - % of sp / non-specific antibody-expressing cells used as controls ( figure e) . these findings suggest that bead size or type could influence the proportion of possible antibody-expressing b cells recognized by antigen-conjugated fluorescent beads. since alongside specific binding of beads to antibody-expressing b cells, we also detected non-specific binding of frα-coated beads to control sp / cells (figure e) , we further investigated the background binding of fluorescent beads to freshly isolated human pbmcs. a proportion (~ %) of human pbmcs (top panel) and also of human b cells (cd + cells, lower panel) could bind to frα-coated beads in most likely a non-specific manner ( figure a) . different blocking agents did not appear to reduce the levels of background binding (table s in supplementary material). furthermore, a-red . µm, sa-blue . µm, and lumavidin . µm frα-coupled (peg -biotin) beads were incubated with pbmcs and binding was directly compared to recognition of sp / -mov igg cells (used as positive controls). frα-coupled a-red . µm microspheres showed . % specific and . % non-specific b cell recognition, while sa-blue . µm microspheres showed . % specific and . % non-specific recognition of b cells. on the other hand, the larger sized lumavidin . µm microspheres showed . % specific and . % non-specific binding ( figure b) . this suggested that the smaller diameter beads were more likely to select antibody-expressing cells. in order to analyze whether, and at what frequency, anti-frα antibody-expressing b cells in pbmc samples may be detected by frα-coupled beads, we spiked human pbmc with sp / -mov cells at different frequencies and we detected double-positive (frα+/sp / +) events by flow cytometry (figure a) . frα-coupled a-red . µm fluorescent beads were incubated with pbmc ( µl beads/ pbmc, table s in supplementary material) in the presence of serially diluted ( : , : , : , ) sp / -mov cells pre-labeled with an anti-cd antibody. the actual dilution was calculated post acquisition ( figure b) . actual positive events were defined as anti-frα antibody-expressing b cells among all bead-bound cells and used to evaluate potential specific selection of real antigen-specific cells. the actual true events were defined as antigen-specific b cells bound to beads among all possible antibody-specific sp / + events and used to estimate the selection of antigen-binding cells compared to all specific cells in a sample. we observed that frα+ beads were able to identify % actual positive events (b cells) when the specific b cell frequency was ~ : . the proportion of actual positive events (anti-frα antibody-expressing b cells) was lower when the specific b cell frequencies in the pbmc pool were reduced (figures b,c) . the actual true events (b cells bound to beads among all possible antibody-expressing sp / + events) ranged between . and % (figures b,c) . beads of different types and diameter sizes varied in ability to detect actual positive events, and the proportion of actual positive events (among all bead-bound cells) decreased when the frequencies of frα+/sp / + b cells were lower among pbmcs. between and % of antibodyexpressing b cells recognized by beads were antigen-specific when b cells were found in high frequencies (≥ : ) in human blood. sa-blue . µm lc-lc biotin beads detected the highest proportion ( %) when b cells were found at a frequency of : ( figure d ; table s in supplementary material). to confirm selection of antibody-expressing cells by specific antibody, pbmc spiking showed that frα-coated beads were more likely than her -coated beads to be recognized by frα-specific sp / -mov cells (figure a ; figure s in supplementary material). anti-sf antibody-expressing sp / cells were also less likely to be detected by frα-coated beads in pbmc samples (figure a) . similarly, her -coated beads were more likely to bind anti-her (trastuzumab)expressing expi f cells compared with anti-cspg control antibody-expressing expi f cells. her -coated beads were also more likely to bind anti-her (trastuzumab)-expressing expi f cells compared with frα-coated beads, which showed low binding to anti-her (trastuzumab)-expressing expi f cells (figure b) . human ig expression on the surface of expi f cells is shown in figure s in supplementary material. together, these data suggest that antibody-expressing cells in pbmc preparations are able to recognize beads conjugated to their specific antigens. to interrogate this approach in the human setting, we evaluated whether fluorescent beads conjugated to melanoma cell surface antigens were able to identify antigen-reactive b cells. we prepared fluorescent beads coated with antigens extracted from human melanoma sk-mel- cells, which natively express the tumor-associated antigen chondroitin sulfate proteoglycan (cspg ). melanoma antigen-coated beads were recognized by an anti-cspg antibody but not by mov antibody specific for the antigen frα not expressed by sk-mel- cells ( figure s in supplementary material), suggesting that antigen-reactive antibodies can specifically recognize antigen bound on these beads. we then screened for antigen-reactive antibodies from human b cells. pbmcs from patients with melanoma incubated with melanoma antigen-coated beads were screened to identify mature cd /cd + b cells recognizing antigen-coated beads. single bead-bound b cells were isolated by single cell sorting ig heavy and light chain sequences were amplified, sequenced, and antibodies were cloned (figures a,b ). an antibody derived from a single bead-bound b cell could recognize melanoma antigen-coated fluorescent beads compared with secondary only or a non-specific antibody control. the b cell-derived clone showed binding comparable to that of a monoclonal antibody specific for the melanoma-associated antigen cspg (figure c) . taken together, these findings suggest that antigen-reactive b cells in human blood could be recognized by antigen-conjugated fluorescent beads and could be sorted by flow cytometry for the expression of monoclonal antibodies. in this report, we present the development of a fluorescent bead method for the selection of single antigen-reactive b cell clones and the production of the cloned antigen-specific antibodies for downstream testing. the workflow comprises: (a) conjugation of fluorescent microspheres with a recombinant antigen of interest; (b) identification of fluorescently labeled single antibodyexpressing b cells by flow cytometry using antigen-conjugated beads; (c) flow cytometric sorting of bead-bound single cells directly into lysis buffer; (d) single cell retrotranscription and sequencing of matched heavy and light chain antibody variable regions; (e) cloning and expression using a vector containing human constant region cassettes; (f) confirmation of antigen reactivity of the produced full-length antibody. we designed this process to allow the identification of antigen-specific antibodyexpressing b cells without the requirement of prior ex vivo growth or secondary screening of b cells, and we aimed to conduct the workflow from clone identification to antibody production and characterization in a timeframe of approximately days. to design this protocol, we employed a model system featuring human recombinant folate receptor alpha (frα) as the target antigen, a b cell line expressing both the soluble and the membranebound forms of mov igg , a human/mouse chimeric antibody specific for frα. we showed that fluorescent microspheres can be coupled with the recombinant antigen via biotin-streptavidin/ avidin bridging, and that immobilized antigens could be readily detected by antigen-specific monoclonal antibodies. recognition of the epitope on frα by the test antibody mov is thought to be dependent on the native folding of the target antigen. here, we demonstrated that it is possible to use antigen-coupled fluorescent beads of different sizes to identify and isolate single b cells expressing cell surface-expressed antibodies that recognize this conformational epitope. antigen-antibody recognition could be improved by evaluating beads with different characteristics and diameters, while varying the lengths of the avidin coupling agents did not significantly influence antibody-expressing cell recognition by the beads. the latter observation also suggested that engagement of the antigen to the bead surface via different biotins did not mask epitope recognition by specific antibody either in solution or when the antibody was expressed on the surface of a b cell. a key feature of this streptavidin-biotin bead-based approach is that, in principle, it may enable the coupling of virtually any known native or recombinant antigen to fluorescent beads and facilitate the detection of b cells reactive to this antigen. our strategy may offer different features compared with those of other detection methods. for instance, antibodies used for detection of bcr on b cells may also interact not only with bcrs but also with fc receptors on human b cells ( ) . tetramer technologies may be applicable to the selection of antigen-specific b cells, but fluorophores have been known to be released from antigen complexes and to bind non-specifically to immune cells, yielding false positive events ( ) . our protocol is also specifically designed to avoid the requirement for b cell culture and ex vivo expansion or for antibody selection from culture supernatants prior to sub-cloning, limiting dilution and isolation of antigen-specific b cell clones. as well as being laborious and lengthy, these steps may also be limited by specific expansion of distinct b cell populations not always representative of the original b cell repertoire, and which could suffer from potential loss of the antigen-specific clone during the cell ex vivo culture processes ( ) . following flow sorting of single cells directly into lysis buffer, we confirmed that it is possible to extract matched h and l chain variable region sequences from single antibody-expressing cells selected by specific antigen-coated beads. employing the frα-mov sp / b cell model, we confirmed that b cell selection by frα+ fluorescent beads allowed the identification of the matched variable regions and permitted the cloning and production of the corresponding monoclonal antibody from a single cell. this was greatly expedited with the use of a single vector expi f™ cell line-based expression system, which permitted antibody production within a few days ( , ) . while the vector used in this study contained the constant regions of human igg , in principle, this platform could be used for engineering of b cell-derived antibodies with constant regions of any isotype or species desired, potentially facilitating a wide range of downstream applications for this technology. importantly, we showed that cloned and expressed full-length antibody with variable regions extracted from a single b cell could recognize native cell surface-expressed human frα. the specificity of an identified antibody could ultimately be confirmed only by the expression of the antibody and subsequent testing against the natively expressed antigen. previous published studies report the number of detected b cells and the antigenic reactivity of antibodies secreted in supernatants of ex vivo b cell cultures without cloning, production, or testing of the derived antibody clones ( , ) . a similar b cell identification process to the one reported in our study described the frequency of antigen-specific b cells detected using a modified bead-based method ( ) . with this tool, it was possible to monitor the frequencies of anti-hla, anti-tetanus toxin-, and anti-ebna -committed b cells in different individuals. however, antibodies from selected b cells are often not cloned and expressed subsequently in order to analyze their ability to recognize natively expressed cognate antigens. our data confirming antibody sequencing, cloning, expression, and antigen recognition provide an early proof of principle that functional ig sequences could be recovered with this methodology. we ascertained that binding of frα-coated fluorescent beads can specifically single out antibody-expressing b cells. we found that frα-coated beads bound up to % of possible sp / -mov igg cells. however, alongside enhanced recognition of possible antigen-reactive b cells, frα-coated beads also bound to - % of non-specific sp / b cells. furthermore, a proportion (~ %) of human circulating b cells could bind to frα-coated beads in most likely a non-specific manner. together, these suggest that background non-specific binding of beads to cells may be a limitation of this methodology. we employed two approaches to evaluate whether antigen-coupled beads could detect antigen-reactive antibody-expressing cells in pbmc samples. our first approach was to "spike" human pbmc with sp / -mov b cells at different frequencies. we demonstrated that frα + fluorescent beads could identify antibody-expressing cells among pbmc populations when these antigen-reactive cells were present at higher frequencies in human blood. we also found that different bead types had different specific recognition and background recognition profiles. the proportion of actual positive b cells among all bead-bound cells decreased with lower frequencies of frα+/sp / + b cells among pbmcs. when b cells were found in high frequencies (≥ : ) in human blood, the proportion of actual positive events, the specific b cells recognized by the beads, ranged from to % of the total bound cells, with the sa-blue . µm lc-lc biotin beads able to detect the highest proportion ( %). although fluorescent activated cell sorting could enable separating a single cell from a heterogeneous population, the detection of cell populations with a low frequency remains challenging and presents a technical limitation with different protocols ( , ) . consistent with previous studies, here, we observed great variability in the acquired numbers of cells when analyzing samples with very low antigen-specific b cell frequencies. this implies that only active and high frequency humoral responses may be readily studied in the present context, while the detection of low frequency b cells represents an inherent limitation of this methodology and requires further optimization. as a next step, we asked whether using this protocol, fluorescent beads coated with melanoma cell line antigens may be able to identify antigen-reactive b cells in individuals suffering of malignant melanoma. using melanoma cell line surface antigen-coated fluorescent beads, we expressed a monoclonal antibody that bound to melanoma cell line protein-coated beads. this suggested that antigen-reactive b cells in human blood may be singled out using fluorescent beads. the selection of single antigen-specific b cells and the identification of their expressed antibodies is critical to gaining a deeper understanding of the nature and functions of active human humoral immune responses. identification of b cells, as well as cloning and production of their heavy and light chain matched antigen-specific monoclonal antibodies thus remain highly desirable in immunology research and antibody discovery. consequently, discovery of antigen-specific b cell clones forms the focus of numerous high-and low-throughput approaches ( ) ( ) ( ) . our bead-based protocol may provide an alternative, readily applicable means for exploring human b cells by facilitating the study of single b cell-derived antibodies and their functional profiles. since the frequency of antigen-specific b cells in the human circulation remains ≤ % of the total b cell populations ( ) , increasing the probability for more specific selection of such low-frequency b cells remains a major challenge with this and many other available technologies. future efforts may help improve specific selection by incorporating additional selection markers such as beads of multiple fluorophores ( ), or b cell activation markers to single out bcr-activated cells more likely to be matured antibody-expressing clones ( , ). an alternative strategy may entail an additional imaging tool to verify selection of single antigen-reactive b cells subsequent to cell sorting ( ) . furthermore, adjusting the cloning process to identify antibodies of different subclasses or specificities or from specific b cell subsets could improve the chance of detecting clones and clonal families in certain diseases, in which immunological conditions may promote specific antibody profiles ( ) ( ) ( ) ( ) . individually or combined, such approaches may help increase the chances of clonal selection. in summary, we describe the establishment of a methodology to identify single antibody-expressing cells and to produce and test their sequenced recombinant antibodies in a workflow that may be readily applicable in any basic and translational immunology laboratory setting. this single cell-to-functional antibody strategy may open the way for new opportunities to analyze b cells and their antibody profiles at the single cell level and may be potentially applied to help unravel diverse humoral immune repertoires in blood and tissues and in different health and disease conditions. human immune cells were isolated from the venous blood of human volunteers. specimens were collected with informed written consent in accordance with the declaration of helsinki. author contributions sk, pk, kl, and fn conceived the study, and ic, ki, sc, pk, kl, fn, at, and sk designed the methodology. ic, ki, pk, sc, sl, mf, and ac acquired data, generated materials, or helped with the data analysis and interpretation. sk, pk, ki, ic, and sc wrote the manuscript. ic, ki, sc, sl, mf, ac, js, at, fn, pk, kl, and sk discussed and interpreted the data and edited the manuscript. sk supervised the study, led and coordinated the project. we thank all volunteers who participated in this study. we acknowledge the biomedical research centre immune monitoring core facility team at guy's and st. thomas' nhs foundation trust and the nikon imaging centre at kings college london for assistance. assessment of b cell repertoire in humans age-related changes in human peripheral blood igh repertoire following vaccination vaccination-induced changes in human b-cell repertoire and pneumococcal igm and iga antibody at different ages monoclonal antibodies: a review monoclonal antibody therapeutics: history and future detection of expressed gene in 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manuscript. key: cord- - a tt f authors: barber-axthelm, isaac m.; kent, stephen j.; juno, jennifer a. title: understanding the role of mucosal-associated invariant t-cells in non-human primate models of hiv infection date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: a tt f chronic hiv infection causes systemic immune activation and dysregulation, resulting in the impairment of most t-cell subsets including mait cells. multiple human cohort studies demonstrate mait cells are selectively depleted in the peripheral blood and lymphoid tissues during hiv infection, with incomplete restoration during suppressive antiretroviral therapy. because mait cells play an important role in mucosal defense against a wide array of pathogens, fully reconstituting the mait cell compartment in art-treated populations could improve immunity against co-infections. non-human primates (nhps) are a valuable, well-described animal model for hiv infection in humans. nhps also maintain mait cell frequencies more comparable to humans, compared to other common animal models, and provide a unique opportunity to study mait cells in the circulation and mucosal tissues in a longitudinal manner. only recently, however, have nhp mait cells been thoroughly characterized using macaque-specific mr tetramer reagents. here we review the similarities and differences between mait cells in humans and nhps as well as the impact of siv/shiv infection on mait cells and the potential implications for future research. ]. while their role in cancer development and progression has not been fully elucidated, mait cells and specific populations of major histocompatibility complex class i-related (mr )-restricted t-cells have been shown to infiltrate the tumor microenvironment and are able to lyse cancer cells in vitro and in vivo, indicating a potential role in cancer immunotherapy ( ); reviewed in lukasik et al. ( ) . in contrast to conventional t-cells which recognize antigens through mhc-mediated antigen presentation, mait cells recognize antigens by mr -mediated presentation of vitamin b metabolites predominately through a vα . semi-invariant t-cell receptor (tcr) ( , ) . mait cells are also capable of being activated via a cytokine-mediated, antigen independent pathway, indicating they have properties of both innate and adaptive immune cells ( ) . these features of mait cells highlight their importance in rapid immune responses to a wide array of pathogens. while the ability of mait cells to mount an immune response against bacterial pathogens is well established, their role in viral infection is not as clear. viral replication cycles do not involve vitamin b synthesis pathways, which are necessary for tcr-dependent mait cell activation. multiple studies have shown increased mait cell activation in patients with active viral infections, including dengue ( , ) , influenza ( ) , chronic hepatitis b ( ) , hepatitis c ( ) , and severe acute respiratory syndrome coronavirus (sars-cov- ) ( ) . similar findings have also been made in mice following influenza infection ( ), as well as in vitro with hepatitis c ( ), influenza ( ) , and zika virus ( ) . virus-induced mait cell activation is mediated through tcr-independent pathways, as shown for influenza ( , ) , dengue ( ) , hepatitis c ( ), or zika virus ( ) exposure in vitro. this stands in contrast to mait cell stimulation with paraformaldehyde fixed e. coli, which is blocked by treatment with an anti-mr blocking antibody. beyond mait cell activation, data on mait cell contributions to viral immunity is limited. supernatant from activated mait cells suppresses hepatitis c virus replication in a dose-dependent manner ( ) . mr −/− mice, which are deficient in mait cells, have increased morbidity and mortality following influenza challenge; compared to wild type controls ( ). increased morbidity and mortality is ameliorated with adoptive transfer of murine pulmonary mait cells prior to viral challenge. adoptive transfer of pulmonary mait cells also reduced morbidity and mortality in rag −/− il- rγ −/− mice following influenza challenge ( ). taken together, these studies indicate that mait cells are capable of suppressing viral replication and providing clinical improvements during viral infections against certain viruses. additional studies are needed to better characterize the impact mait cells have, both beneficial and deleterious, on viral infections, as well as the breadth of viral infections that mait cells are able to mount an immune response against. a better understanding of mait cell function will require continued evaluation of human mait cell dynamics during various disease states, along with in vivo models that best represent the relevant pathologic processes. the mr gene, the primary receptor for mait activation through antigen presentation, is highly conserved among mammalian species, but is absent in non-mammalian species ( ) . additionally, there have been separate losses of functional mr among mammals, including in lagamorpha (rabbits), and in carnivora (dogs, cats, and ferrets) ( ) . mice carry a functional mr gene but have a relatively low abundance of mait cells in the peripheral blood (median: . %) necessitating the generation of transgenic mice expressing an invariant mvα -jα tcrα to increase mait cell frequencies ( , ) , or the boosting of tissue mait cell frequencies by administration of antigen and tlr agonists ( ) . in contrast, non-human primates (nhps) express a functional mr gene and maintain mait cells at frequencies more comparable to humans, providing a superior in vivo model to study mait cell immunological dynamics. herein, we discuss the current state of mait cell characterization in nhps [which has focused on rhesus macaques (rm), pigtail macaques (ptm), and mauritian cynomolgus macaques (mcm)] and the changes in mait cell populations that occur during simian immunodeficiency virus (siv) and simian-human immunodeficiency virus (shiv) infection, which are the critical animal models for hiv infection. human mait cells were originally identified as vα . + cd + cells among the bulk t-cell population [reviewed in garner et al. ( ) ]. recently, the development of antigen loaded mr tetramers has allowed for a more refined identification of mait cells by flow cytometry ( , ) . similar approaches have been utilized to phenotype macaque mait cells, via identification of vα . + and/or mr - -op-ru + t-cells ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . one important consideration for determining tetramer reactivity in macaque mait cells is the utilization of species specific mr tetramers. two studies have identified incomplete cross reactivity of human mr tetramers with macaque mait cells ( , ) . identification of these cells is improved with the use of macaque specific mr tetramers. furthermore, the inclusion or exclusion of vα . expression in the definition of a mait cell should be carefully considered. there is growing evidence of a unique vα . − mr tetramer + t-cell population in humans ( , ) , which has also been identified in the peripheral blood of ptms, rms, and mcms ( , , ) . additional work is needed to characterize these cells and to compare their phenotypic and functional properties to their human counterparts. human mait cells are predominately cd αα + or cd − cd − , with a minor population of cd -expressing cells ( ) . in contrast, nhp mait cells are almost uniformly cd α + , with studies noting an absence of cd − cd − mait cells in nhp ( , , ) . one additional study identified peripheral blood mait cells as predominately cd α + ( . %) or cd − cd − ( . %) in rms, with minor populations of cd + cd + ( . %) and cd + ( . %) mait cells ( ) . mait cells were identified based on reactivity to nhp-specific mr tetramers without concurrent expression of vα . , which may partially explain the presence of cd − cd − , cd + cd +, cd + mait cells that were not observed in other studies. it is presently unknown if nhp cd α + mait cells express a homodimeric (cd αα + ) or heterodimeric (cd αβ + ) receptor. the cause for this absence of cd − cd − mait cells in the majority of nhp studies is unknown, and additional studies are needed to characterize this variation from human peripheral mait cells. human cd + and cd − cd − mait cells have been shown to have distinct phenotypic and functional profiles ( ) . cd + mait cells express higher levels of cytotoxic and coactivating markers compared to cd − cd − mait cells, and produce higher levels of ifnγ and tnfα following stimulation. cd − cd − mait cell can be derived in vitro from cd + mait cells following tcr-dependent activation. potential causes for the relative paucity of cd + mait cells in captive nhps include species-specific variation in mait cell development or differentiation between humans and nhps, or environmental factors related to husbandry practices which drives the altered frequencies in nhp peripheral cd + and cd − cd − mait cells. a lack of cd − cd − mait cells in nhps may also impact the immune response to certain disease states, and should be considered when utilizing nhps as a model for humans. while nhps predominately lack cd − cd − mait cells, we caution against pre-gating on cd + t cells prior to identifying mr tetramer + cells in nhps, as this could hinder observing shifts in mait cell cd and cd co-receptor usage during disease states or experimental interventions. cd , a c-type lectin like-receptor, is almost ubiquitously expressed by human mait cells in the peripheral blood and has classically been used as a co-staining marker to gate on vα . + or mr tetramer + cells ( , , ) this marker is associated with innate-like, tcr-independent responses to il- + il- stimulation ( , ) . three studies have utilized cd expression to identify mait cells in nhps ( , , ) . however, two studies have noted poor cross-reactivity of antihuman cd antibodies against rm and ptm cd antigen ( , ) . additionally, cd downregulation has been identified in mait cells from chronic hiv infected individuals ( , , ) . as a result, we strongly caution against the use of cd to define the total mait cell population in nhps; rather, mr tetramer in combination with tcr vα . can distinguish major mr restricted t-cell populations, which can then be phenotyped for additional surface markers. plzf (zbtb ) is most consistently expressed on mait cells in nhps ( , - , , ) , as well as in humans ( , ) , and is a marker to consider when identifying the bulk mait cell population. additional markers that are expressed on a relatively high frequency of mait cells and may be considered for mait cell identification are ccr and il- rα, which have been shown to be expressed on mait cells from multiple nhp species ( , , , ) . mait cells constitute approximately . % (range: . - . %) of the total t-cell population in human peripheral blood ( table ) ( ) . by comparison, mait cell frequencies in the peripheral blood range from . to . % in rms ( , ) , . - . % in ptm ( ) , and . - % of cd + cd + t-cells in mcms ( ) . in general, nhps maintain peripheral mait cell frequencies at - % of the bulk cd t-cell population. while mait cell frequencies are lower than what is observed in humans, it is substantially higher than baseline mait cell frequencies in mice ( ) , and is sufficient to evaluate changes in peripheral mait cell frequencies and phenotype with different experimental interventions. similar to mait cell frequencies in the blood, nhps mait cell frequencies in tissues tend to be lower compared to the frequencies in humans, but higher compared to murine mait cell frequencies. both humans and nhps maintain relatively % of the bulk t-cell population, or % (range: - %) of the bulk cd + cd + t-cell population, in the lungs of rms ( , ) . additionally, mait cell frequencies are highly variable in rm bronchoalveolar lavage fluid (bal), comprising . - . % of the bulk cd + t-cell population ( , , , ) , or approximately - % of the bulk cd + cd + t-cell population ( ) . similar findings have also been made in mcms, with mait cell frequencies ranging from approximately - % of the bulk cd + cd + t-cell population in the bal ( ) . mait cells are consistently maintained at low frequencies in secondary lymphoid organs (lymph nodes and spleen) compared to the peripheral blood, in both humans and nhps. this is attributed to the relative lack of ccr and cd l expression, both required for lymphoid tissue homing, on peripheral mait cells [reviewed in kurioka et al. ( ) ]. additionally, it has been postulated that mait cells in the lymph represent a distinct population that recirculate from the tissues, separate from circulating mait cells in the blood ( ) . while similar patterns of tissue recirculation in nhps still need to be investigated, differential distribution of mait cell phenotypes between lymphoid and extralymphoid tissues has been described in rms. mait cells within secondary lymphoid tissues are predominately ccr + cd + (central memory), while mait cells in extralymphoid tissues are predominately ccr − cd + (transitional memory) or ccr − cd − (effector memory) ( ). species specific variations in t-cell functional activity are important to consider in the context of different animal models. stimulation of peripheral blood mononuclear cells (pbmcs) from ptm with the riboflavin metabolite-based antigen -op-ru results in a dose-dependent upregulation of cd and selective proliferation of mr tetramer + vα . + mait cells ( table ) ( ) , demonstrating that human and macaque mait cells are similarly activated by antigen (figure ) . surprisingly, -op-ru stimulated ptm mait cells produced relatively low amounts of tnfα and ifnγ (although ifnγ secretion could be augmented with il- and il- supplementation to the cultures). in contrast to the ptm data, stimulation of human peripheral mait cells with -a-ru resulted in significant production of tnfα and ifnγ ( ) . in contrast to humans and nhps, murine mait cells predominately produce il- following stimulation with -op-ru ( ) , highlighting the potential for species specific differences in mait cells functional activity, and the value of the nhps in being able to better model the human peripheral mait cell cytokine profile. the use of fixed e. coli to stimulate human mait cells is now a standard assay, and activates mait cells through both mr dependent and -independent mechanisms ( , ) . similarly, lps or e. coli stimulation resulted in significant ifnγ and tnfα production in rm and mcm mait cells ( , ) , as well as cd a upregulation in mcm mait cells ( ) . in contrast, mcm mait cell stimulation with mycobacterium smegmatis resulted in no significant increase in ifnγ and tnfα production, or cd a upregulation. following mitogenic stimulation with pma/ionomycin, both ptm and rm mait cells readily produce ifnγ, tnfα, and il- ( , , ) . variations in the amount of il- production have been reported, with ptm mait cells producing relatively blood mait cells during acute shiv infection ( ) . similarly, there was no change in cd expression on the effector memory (cd − cd + ) mait cell population of mcms following siv challenge ( ) . however, a transient but significant increase in cd , cd , t-bet, and rorγt was observed in the central memory (cd + cd + ) mait cell population of mcms at - weeks post-siv challenge ( ) . siv/shiv infection is associated with a transient increase in peripheral blood α β + mait cells in ptms at - weeks post infection ( ) . this increase correlated with peak viral load, as well as increased mait cell frequencies with rectal mucosa. one hypothesis is that early viral replication and increased antigen availability in the lymph node may drive α β expression on mait cells. peripheral blood mait cells upregulated α β when cocultured with mesenteric lymph node cells and -op-ru, demonstrating that circulating mait cells can upregulate α β when stimulated with antigen in the context of mesenteric lymph node cells ( ) . these findings are similar to what has been observed with conventional t-cells in humans and mice, where α β induction is mediated by retinoic acidproducing dendritic cells from gut-associated lymphoid tissues and mesenteric lymph nodes ( ) ( ) ( ) ( ) ( ) . similarly, α β expression is significantly increased on peripheral mait cells from chronic shiv-infected rms, compared to naïve controls ( ) . in humans, β integrin expression is increased in hiv + individuals, suggesting this relationship may be conserved between humans and nhp models [reviewed in saeidi et al. ( ) ]. this may also partially explain the mechanism for the transient increase in mait cell frequencies observed in the rectal mucosa of hiv + individuals during initial infection ( ) . alternatively, increased α β + mait cells in the peripheral blood following siv/shiv infection may be due to expansion and subsequent trafficking of mait cells into the blood from mucosal tissues sites. these changes correlate with peak viremia, suggesting that early viral replication may drive α β induction and increase mucosal tissue trafficking during acute infection. this is potentially driven by direct viral replication, or immune dysregulation with microbial translocation in the gastrointestinal tract. the lack of mait cell depletion during acute infection is consistent with what has been observed in humans during initial hiv infection and emphasizes the importance of the nhp-siv/shiv model to study mait cell dynamics during peracute infection. chronic hiv- infection in humans is associated with persistent mait cell depletion in the peripheral circulation [( , ) , reviewed in saeidi et al. and juno et al. ( , ) ]. in contrast, chronic siv/shiv infection has inconsistent effects on peripheral mait cell frequencies in nhps. one study reported a reduced frequency and absolute number of peripheral mait cells in rms during chronic siv infection ( ) . a reduction in ccr + mait cells was also noted during chronic siv infection, which was hypothesized to reflect impaired tissue trafficking capabilities of the remaining mait cells in the periphery. in contrast, a second study noted increased peripheral mait cell frequencies in rms during chronic shiv infection, which correlated with plasma viral load ( ) . despite the overall increase in frequency, mait cell frequencies in the peripheral blood were inversely correlated with plasma viral load, and directly correlated with peripheral cd + t-cell frequencies. human mait cells are not preferentially infected by hiv ( ) , suggesting that the observed correlation may reflect reduced mait cell frequencies due to reduced cd t-cell frequencies, which is secondary to virus-mediated cd t-cell depletion. other potential causes for the observed correlation includes increased mait cell consumption in response to higher amounts of virus mediated gastrointestinal inflammation and bacterial translocation. a third study identified no significant difference in peripheral mait cell frequencies during chronic shiv infection in ptms ( ) . increased ki- expression in the peripheral mait cell populations was identified in studies, despite the variability in mait cell frequency ( , ) . similar variability has also been observed in mait cell frequencies in tissues during chronic siv/shiv infection in nhps. two separate studies have reported increased and decreased mait cell frequencies in the bal fluid of chronic-shiv infected ptms and chronic-siv infected rms, respectively ( , ) . additionally, one study identified decreased mait cell frequencies in the inguinal lymph nodes, with concurrent maintenance of mait cells in the mesenteric lymph nodes of chronic shiv-infected ptms ( ) . in contrast, a separate study identified a reduction in mait cell frequencies in the mesenteric lymph nodes of chronic siv-infected rms ( ) . however, mait cell frequencies are consistently maintained in the liver, spleen, and gastrointestinal tract of chronic siv/shiv-infected macaques ( , ) . chronic hiv infection is associated with altered expression of a variety of cytokine and chemokine receptors on peripheral mait cells [reviewed in saeidi et al. ( ) ]. similar alterations have also been observed in chronic siv/shiv infected nhps. chronic shiv-infection is associated with reduced tbet and eomes expression of peripheral mait cells in ptms, while ccr and cxcr expression levels are unchanged ( ) . in contrast to humans where ccr and hla-dr expression is decreased and immune effector functions of peripheral mait cells are significantly impaired during chronic hiv infection. this is marked by reduced cytokine production and cd upregulation following e. coli stimulation, which is only partially restored with combination antiretroviral therapy [( ), reviewed in saeidi et al. ( ) ]. similar findings were also reported in one study evaluating chronic shiv-infected macaques, with reduced tnfα and il- production capacity following mitogenic stimulation ( ) . however, two studies looking at chronic siv and shiv infected mcms and ptms, respectively, report no alterations in cytokine stimulation capacity ( , ) . additionally, chronic shiv-infection in ptms is associated with increased cd expression on peripheral mait cells ( ) . siv/shiv infection does not consistently alter mait cells' ability to produce granzyme, with studies noting no significant changes in the frequency of granzyme b + mait cells in the peripheral blood during siv or shiv infection ( , ) . these findings are in contrast to what is observed in hiv + individuals, where peripheral granzyme b + mait cells are increased in frequency compared to uninfected control samples at resting state ( ) and have reduced perforin and granzyme expression following e. coli stimulation ( , ) . in one of the nhp studies, perforin + and perforin + granzyme b + mait cell frequencies were elevated following shiv challenge ( ) . the cause for the variability in mait cell frequencies, receptor expression, and effector functions observed in these studies is unknown. the authors hypothesize these may reflect species differences between rms and ptms, and/or differences in viral pathogenesis between different siv/shiv strains. additional sources of variation may be due to inter-study differences in gating strategies to identify mait cells, including the usage of cd expression and cd + pre-gating. there is also evidence of significant variability in mait cell frequencies within the peripheral circulation and different tissue compartments, both between different species and individual animals. this emphasizes the need for longitudinal studies, in order to evaluate changes in mait cell trends following different experimental manipulations, as opposed to cross sectional studies. it will also be important to consider standards for identifying nhp mait cells in future studies, including the usage of the vα . tcr and species specific mr tetramer reactivity, due to evidence of limited cross-reactivity with human mr tetramers. one important consideration for hiv + individuals is the risk of co-infection with secondary opportunistic bacterial and fungal pathogens, for which hiv-mediated dysregulation of mait cells may be a predisposing factor. while data on the impact of mait cell responses on fungal infections is currently lacking, mait cells have been shown to become activated in response to candida spp. [reviewed in wong et al. ( ) ] and aspergillus spp. ( ) . mycobacterium tuberculosis (mtb) is one of the most common opportunistic bacterial infection among hiv + individuals. individuals with active or latent mtb infections tend to have lower peripheral mait cell frequencies, compared to uninfected individuals ( ) . additionally, mait cells from hiv/mtb co-infected individuals tended to express lower levels of ccr compared to hiv infected individuals and healthy controls, though statistically significant differences were only seen between the hiv/mtb co-infected individuals and the healthy controls. this suggests that hiv-mediated mait cell dysregulation may predispose individuals to opportunistic infection with mtb. in rms, vaccination with bacillus calmette-guérin, a vaccine historically used to prevent tuberculosis, resulted in significantly increased expression of ki- and granzyme b in mait cells in the peripheral blood, the vaccine sites, and the draining lymph nodes, compared to pre-vaccination frequencies ( ) . while there was evidence of significant mait cell activation following vaccination, no significant changes in mait cell frequencies in the blood or tissue sites were observed. increased peripheral mait cell ki- expression, without an increased in peripheral blood mait cell frequencies, was also observed in rms following intrabronchial mtb infection ( ) . no significant difference in mait cell frequencies in the peripheral blood or tissue has been reported with siv/mtb co-infection, similar to what has been observed in humans ( , , ) . however, a weak negative correlation between mait cell frequency and bacterial burden was observed in siv/mtb co-infected mcms, which was not maintained in nhps mono-infected with mtb. there was also a significant reduction in tnfα producing capacity of mait cells from the peripheral blood and mtb granulomas of siv/mtb co-infected mcms, compared to mtb mono-infected controls ( ) . the results reflect the potential impact of mait cells on the susceptibility of mtb in hiv + individuals, in an nhp animal model. there is growing evidence that mait cells are capable of being activated in response to viral pathogens, despite the lack of vitamin b metabolites that are necessary for tcrdependent mait cell activation. additionally, there is evidence of altered mait cell frequencies, phenotypes, and functional activity during both acute and chronic hiv infection. however, the underlying pathophysiology for why these changes occur is not well understood. having a better understanding of this interaction may provide key insights into developing vaccines and/or therapies for hiv infection and related co-morbidities. the nhp animal model provides a valuable platform to study these changes, and understand the underlying mechanisms for why they occur. while siv/shiv infection in nhps does not reliably recapitulate all aspects of the mait cell changes that occur during hiv infection, notably the lack of mait cell depletion, it does model many aspects of hiv infection well. this includes an initial increase in mait cell frequency with upregulation of α β during acute viral replication, as well as evidence of impaired mait cell function in siv/mtb co-infected nhps. future studies should focus on characterizing the perturbations in the mait cell populations within different tissue compartments during siv/shiv infection; with a special focus on variables that may alter these perturbations, such as macaque species, age, and gender, as well as strain, dose, and route of administration of the virus. by better understanding how these factors impact mait cell dynamics during siv/shiv infection, we will ideally be able to refine the nhp model to better recapitulate the mait cell changes that occur during hiv infection in humans. additionally, the robust response that nhp mait cells generate to both antigen and cytokines, including cytokine independent cell activation, which is comparable to humans, may be advantageous for studying mait cell dynamics with other pathogens. ib-a wrote the initial draft of the manuscript. ib-a, sk, and jj revised the manuscript. all authors contributed to the article and approved the submitted version. we acknowledge funding through the australian national health and medical research council (nhmrc app ). the biology and functional importance of mait cells mait cells come to the rescue in cancer immunotherapy? mr presents microbial vitamin b metabolites to mait cells t-cell activation by transitory neo-antigens derived from distinct microbial pathways mr -independent activation of human mucosal-associated invariant t cells by mycobacteria mait cells are activated during human viral infections mait cells are activated in acute dengue virus infection and after in vitro zika virus infection mucosal-associated invariant t (mait) cells are more activated in chronic hepatitis b, but not depleted in blood: reversal by antiviral therapy mait cells contribute to protection against 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depleted and exhibit altered chemokine receptor expression and elevated granulocyte macrophage-colony stimulating factor production during end-stage renal disease mait cells reside in the female genital mucosa and are biased towards il- and il- production in response to bacterial stimulation key: cord- -jl zhg authors: khalaf, khalil; papp, natalia; chou, jadzia tin-tsen; hana, doris; mackiewicz, andrzej; kaczmarek, mariusz title: sars-cov- : pathogenesis, and advancements in diagnostics and treatment date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: jl zhg the emergence and rapid spread of sars-cov- in december has brought the world to a standstill. while less pathogenic than the – sars-cov, this novel betacoronavirus presents a global threat due to its high transmission rate, ability to invade multiple tissues, and ability to trigger immunological hyperactivation. the identification of the animal reservoir and intermediate host were important steps toward slowing the spread of disease, and its genetic similarity to sars-cov has helped to determine pathogenesis and direct treatment strategies. the exponential increase in cases has necessitated fast and reliable testing procedures. although rt-pcr remains the gold standard, it is a time-consuming procedure, paving the way for newer techniques such as serologic tests and enzyme immunoassays. various clinical trials using broad antiviral agents in addition to novel medications have produced controversial results; however, the advancement of immunotherapy, particularly monoclonal antibodies and immune modulators is showing great promise in clinical trials. non-orthodox medications such as anti-malarials have been tested in multiple institutions but definitive conclusions are yet to be made. adjuvant therapies have also proven to be effective in decreasing mortality in the disease course. while no formal guidelines have been established, the multitude of ongoing clinical trials as a result of unprecedented access to research data brings us closer to halting the sars-cov- pandemic. coronaviruses are widely known virulent pathogens affecting mammalian and avian species. previously, six globally distributed species of the virus have been identified to cause illness in humans. they are: human coronavirus oc (hcov-oc ), human coronavirus hku (hcov-hku ), human coronavirus e (hcov- e), human coronavirus nl (hcov-nl ), severe acute respiratory syndrome coronavirus (sars-cov), and middle east respiratory syndrome coronavirus (mers-cov) ( table ) . sars-cov first emerged in [ ] [ ] in china, presenting as an atypical pneumonia with febrile state, headaches, and a marked cough that may rapidly deteriorate into respiratory failure. the sars-cov outbreak was the first to be declared a pandemic, and ultimately infected , persons in countries, with a mortality rate of % ( ). epidemiological analysis posited an infectious zoonotic agent, and further serological studies identified the intermediate host to be the palm civet (paguma larvata). spillover from animals to humans was hypothesized to be via the horseshoe bat (genus rhinolophus). an adaptation of the sars-cov virus was later proven to be due to interspecies dwelling ( ) . a combination of ribavirin and pulse prednisolone showed positive results early in the outbreak and was adopted as the standard protocol. eventually, ribavirin was demonstrated to have increased cytotoxicity and lacked efficient antiviral action in vitro, and while pulse prednisolone showed efficacy in managing critically ill patients, its high dose administration was associated with disseminated fungal infections ( ) . later, the combination of interferon-alpha (inf-α ) and corticosteroids was found to yield a better prognosis, but did not prove beneficial for patients in the late stage of the disease ( ) . protease inhibitors produced an outcome similar to that of inf-α ( ) . a novel coronavirus later named mers-cov emerged in saudi arabia in . similar to sars, infected patients presented with a variety of clinical courses, from mild upper respiratory symptoms to fulminant pneumonia and multi-organ system failure. phylogenic and sequencing studies have proposed a bat origin, and surface protein modification was found to be derived from the intermediate hosts, dromedary camels. from to , more than , confirmed cases were reported, mainly in middle eastern countries, with a mortality rate of % ( ) . as with sars-cov, no definitive protocol was implemented, and most patients were treated with supportive therapy to preserve organ integrity ( ) . in a cohort study conducted by omrani et al., a treatment protocol involving ifn-α and ribavirin was initiated, and while survival improved significantly at days after treatment, this was not seen at day , necessitating further treatment options ( ) . sporadic outbreaks occur to this day, with patients undergoing largely supportive therapy. the sars-cov- outbreak began in december in wuhan, china where clusters of atypical pneumonia yielded evidence of a novel strain of coronavirus. as of august , , , , cases had been reported worldwide, with a fatality rate of %. although this novel virus is less severe than the first sars-cov outbreak, human-to-human transmission remains very high and the number of cases continues to rise exponentially in major urban areas, highlighting the urgent need to develop new containment, diagnostic, and treatment protocols. as time is of the essence, the pathogenic mechanism cannot be studied with the rigor and comprehension otherwise expected, and the opportunities to compare therapeutic protocols are few. origin of the virus during the initial outbreak, liu et al. obtained samples of blood, bronchoalveolar lavage fluid, and anal and oral swabs from patients in wuhan, china suffering from a pneumonialike illness resembling the - sars-cov. a pan-cov pcr was performed on samples from five patients, and were positive for a pathogen from the coronaviridae family. this was compared with a full-length sequence of viral rna from a bat coronavirus (bat-covratg ), and demonstrated . % similarity. thus, it is probable that the bat is the main reservoir of the novel coronavirus. identification of the intermediate host is an essential step in controlling the spread of disease, and became a priority for research teams. unfortunately, this was complicated by the many species of wild animals sold at the huanan seafood market, where the first cases were reported to have had contact. in , a sars-cov-like pathogen known to be widely distributed in the malayan pangolin samples was discovered. the receptorbinding domain (rbd) present on the spike protein (s) is a crucial determinant in host range, as its interaction with the host receptor is responsible for the infection. rbd sequences from bat-covratg , pangolin-sars-like cov and the novel sarslike pathogen were aligned. ninety three percentage similarity was demonstrated between the novel sars-like pathogen and the pangolin sars-like cov, and % similarity was demonstrated between the novel sars-like pathogen and the bat-covratg . thus, on the basis of the rbd, the pangolin-sars-like cov is determined to be more likely than the bat-covratg to infect humans, making this the possible intermediate host ( ) . xiao et al. conducted another study in which the pangolin-sars-like cov was isolated and amino acid sequence was compared to sars-cov- . this yielded , . , . , and . % similarity with the s, m, e, and n proteins, respectively, of the novel sars-cov, strengthening the previous assumption that the pangolin was the intermediate host ( ) . pathogenic classification is used to determine whether the pathogen is new or recurring in order to best implement safety and treatment protocols. while serological reactivity to viral proteins had been the mainstay of viral classification in the past, the process today now depends on replicated protein sequences. the international committee on taxonomy of viruses (ictv) maintains a study group for each viral family ( ) . after analysis, the novel virus was assigned to the order nidovirales on the basis of the following domains: polyprotein protease ( clpro), catalytic domain of rna polymerase (rdrp), nidovirus-associated rdrp (niran), zinc binding domain (zbd), and helicase (hel ) ( ) . subsequent next generation sequencing and phylogenic analysis placed the novel pathogen within the subgenus sarbecovirus of the genus betacoronavirus ( ) ( table ) . like other coronaviruses, the viral envelope is composed of a lipid bilayer derived from host cellular material, and the structural proteins are spike proteins composed of a trimeric glycoprotein projecting from the envelope like a crown. cryo-electron microscopy was used to compare the structural differences between the spike (s) proteins of sars-cov and sars-cov- . unlike the previous sars-cov which possessed a single arginine targeted by a trypsin protease, sars-cov- possesses an s /s protease cleavage site which is recognized by furin proteases. neuman et al. demonstrated the high level of protein organization and interaction within the envelope: the s protein was shown to be aligned with the npc, a crucial component of viral organization in protein-protein interactions that ensures high specificity and effectiveness for host invasion ( ) . sars-cov- is an enveloped virus with a , bp positivesense non-segmented rna genome. the non-structural proteins (nsps) are described in table . the remainder of the genome is responsible for accessory and structural proteins such as s, m, e and n. host cell recognition is the first and most essential step in viral pathogenesis. studies on the - sars-cov outbreak uncovered key interactions between the spike (s) protein, the rbd and angiotensin-converting enzyme (ace- ). due to the previously mentioned similarities between sars-cov- and its predecessor, a viral infectivity study was performed using hela cells with and without the ace- receptor, which showed that only cells possessing the ace- receptor were infected ( ) . the spike trimer is a class i fusion protein; upon infection the spike is cleaved by host proteases at the s /s site for division of the two domains. the s domain possesses the rbd which recognizes and binds the ace- receptor in a prefusion state. structural rearrangement of the s protein subsequently exposes a furin cleavage site on the s domain which enables viral entry by means of fusion after the s domain is shed ( ) . viral replication was hypothesized to occur via a process called autophagy: an evolutionary cellular process in which cytoplasmic proteins create isolation membranes surrounding materials destined for degradation ( ) . evidence of coronavirus autophagy was first demonstrated by prentice et al., who showed that in coronavirus mouse hepatitis viruses, atg -induced autophagy was required for the formation of double membrane vesicles (dmv) and for replication ( ) . another study confirmed the use of atg dependent autophagy of betacoronaviruses through nsp ( ). chen et al. previously showed that both sars-cov and mers-cov employ the use of papain-like proteases (plpro) to induce the formation of autophagosomes, but their fusion to lysosomes is inhibited which promotes the replication process ( ) . however, other studies have demonstrated that key autophagy proteins atg or atg were not required for coronavirus mouse hepatitis or sars-cov viral replication ( ) . reggiori et al. also yielded comparable results showing that the replication and release processes are not dependent on autophagy, but that the dmv's were coated with (lc )-i (non-lipidated microtubule associated protein i light chain ), thus showing the importance of this protein for viral replication ( ) . a recent study by benveunto et al. has shown that sars-cov- induced mutations within the nsp protein, which in turn induced autophagosome formation by inhibiting phagosomelysosome fusion ( , ) . sars-cov- may take advantage of this autophagy mechanism, as the acidic ph required for endosome maturation also functions to release the virion at the appropriate site. once all necessary proteins are translated and replication has taken place, virion assembly occurs in the golgi apparatus before release from the cell ( ) . symptoms attributed to sars-cov- shereen et al. determined a mean incubation period of days in the first patients testing positive for sars-cov- ( ). the time from infection to death varied from to days, with a mean of days ( ) . in january of , huang et al. examined all patients exhibiting pneumonia-like symptoms in order to determine the clinical features of sars-cov- ; importantly, this study did not discriminate between previously healthy patients and patients suffering from chronic illnesses. data was collected from the positive cases, and analysis yielded the following: fever was the first and most common primary symptom ( %), followed by dry cough ( %), lymphopenia ( %), dyspnea ( %), and fatigue/myalgia ( %). less common symptoms included sputum production ( %), headache ( %), hemoptysis ( %), and diarrhea ( %) ( ) . a recent systematic review of studies spanning nine countries disputes the above-mentioned data. in this analysis by grant et al., the most common symptoms were as follows: fever ( %), dry cough ( %), fatigue ( %), anosmia ( %), and difficulty breathing ( %) ( ) . another study by clemency et al. assessed the likelihood that sars-cov- infection presents with symptoms. the positive likelihood ratio of having symptoms with disease from highest to lowest was as follows: anosmia and ageusia, followed by loss of appetite, fever, muscle pain, fatigue, dry cough, productive cough, diarrhea, difficulty breathing, and sore throat ( ) . the stability of sars-cov- on various surfaces was compared to that of its predecessor sars-cov, and it was found that both viruses exhibit similar stability. the differences in epidemiological spread are therefore most likely to be dependent upon other factors such as high viral load and viral shedding in asymptomatic persons. the study is highly suggestive of an aerosolized pathogen as it remains viable and infectious in droplet form for up to h ( ). while resembling the previous sars-cov outbreak, this data confirms that sars-cov- is far more transmissible. kinetic studies employed to compare the two strains showed that sars-cov- binds to the ace- receptor with a -to -fold higher affinity than sars-cov, suggesting that this could be another main factor explaining the ease of transmission ( ) . to further support this hypothesis, computational structural predictions were established to differentiate between the rate of association between the ace- receptor and the spike protein of both sars-cov and sars-cov- . by incorporating the computer simulation into a mathematical model, it was shown that the novel pathogen has slower binding capacity than sars-cov, consistent with the varying life cycles of the two strains. in other words, the longer incubation period of sars-cov- was associated with the slower interaction between the spike protein and the ace- receptor. this allows for maintenance of an increased viral load in the body, thus contributing to increased human-to-human transmission ( ) . the renin-angiotensin-aldosterone system (raas) is responsible for inducing a coordinated cascade regulating both cardiovascular and renal functions. angiotensin-converting enzyme (ace) is responsible for converting angiotensin i to angiotensin ii, with the latter functioning as a pro-sympathetic molecule throughout the body to regulate blood pressure. wakahara et al. measured ace and ace- expression under various conditions. in addition to the ace- role in local regulation of raas through the conversion of angiotensin ii into vasodilators and anti-trophic peptide, it acts and is expressed in synergy with ace concentrations and is believed to possess counter-regulatory effects to ace ( ) . as the expression of the ace- receptor is vital for viral entry, it is essential to establish where this gene is expressed throughout the body to identify possible routes of infection and/or the extent of organ damage during infection. zou et al. employed the dataset systems of single-cell rna sequences from different body tissues to identify organs expressing the ace- gene in high concentrations. the symptoms reported to be associated with covid- (dyspnea, cardiac injury, kidney failure, diarrhea) were compared to ace- expression on target cells. it is on this basis that the organs at high risk of damage during viremia were recognized to be the lungs, heart, kidney, and upper respiratory tract ( ) . a minority of patients is seen to present with gastrointestinal involvement, and a further study by wong et al. on patients in wuhan demonstrated this complaint in % of their cohort. the virus was detected in stool by means of rt-pcr. staining of the n protein showed presence of the virus in the cytoplasm of duodenal, rectal and gastric epithelium thus indicating another possible mode of transmission and another sign of possible infection ( ) . zhao et al. also compared the symptoms and disease characteristics of covid- patients with that of other pneumonias, indicating that liver damage was more prominent in covid- patients. however, it was not clear whether liver enzyme elevation was due to drug toxicity or viral shedding ( ) . the ace- -receptor mapping experiment, while important, provides little information on the broad expressions of the receptor, as ace- concentration may differ due to various clinico-pathologies such as hypertension, diabetes, heart failure, smoking and kidney injury ( , ) . remuzzi and remuzzi in italy demonstrated that two-thirds of patients who died from sars-cov- were elderly, diabetic or suffered from cardiovascular disease ( ) . their first line drug of choice was angiotensin receptor blockers (arbs). ferrario et al. demonstrated in that selective blockade of angiotensin ii synthesis or activity has an up-regulatory effect of ace- receptors in the kidneys and heart ( ) . this ace- upregulation in the aforementioned comorbidities could therefore be responsible for the severity of infection in these patients. guo et al. demonstrated that in sars-cov- -positive patients, . % had an increase in troponin-t and crp levels that correlated with myocardial injury. sars-cov- patients who presented with an increase in cardiac markers exhibited a . % mortality rate compared to those with normal cardiac markers ( . % mortality rate). sars-cov- genomic material has also been isolated from cardiac biopsies. while the pathophysiologic development of myocardial injury is not yet fully understood, it is believed to be caused by either cytokine storm or direct myocardial damage through viral integration ( ) . to further understand the organ damage and hemostatic changes, han et al. assessed the change in coagulation proteins in both covid- patients and healthy patients. they found that antithrombin levels were lower in covid- patients, whereas fibrinogen, fibrinogen/fibrin degradation products and d-dimer were high ( ) . a recent cohort study by wichmann et al. reported a possible connection between covid- and incidence of thromboembolism. autopsies of covid- patients revealed the incidence of deep vein thrombosis (dvt) in % of cases, and a third of patients suffered a fatal pulmonary embolism. further studies should be conducted to further elucidate the formation of thromboembolism in the course of covid- ( ) . cd , also known as emmprin (extracellular matrix metalloproteinase inducer) is a highly glycosylated transmembrane glycoprotein shown to be involved in facilitating sars-cov endocytosis. this presents an alternative route of viral invasion ( ) . like the ace-receptor, cd is also present in various tissues such as lymphoid ( ) , testes, liver, cerebral, and renal tissues, as well as cardiac and skeletal muscle ( ) . in addition to enabling viral entry, cd has been linked to other pathologic conditions such as cancer ( , ) , heart disease ( ) and alzheimer's disease ( ) . furthermore, the speed of invasion may be increased as cd has been shown to be upregulated upon t-cell activation ( ) . wang et al. has since shown that the spike protein possesses high affinity for cd , thus mediating viral invasion and facilitating viral replication. it acts as a negative regulator of the immune system which may contribute to the significant cd + decline during covid- ( ) . under hypoxic conditions, this receptor is involved in the regulation of cell proliferation and apoptosis. the cd receptor, a marker of early-stage disease, can be useful for the assessment of pathological progression because it is often expressed on the surface of activated regulatory t cells ( ) . lung epithelium is the largest area of the body in constant contact with the external environment, and is responsible for processing inhaled air containing large volumes of bacteria and viruses. the immune response to sars-cov was widely studied, and sars-cov- is believed to induce the same responses. innate immunity is the primary countermeasure against infection, and is maintained by a variety of cell types found in airway epithelium, including dendritic cells, innate lymphoid cells and alveolar macrophages. the protective signaling cascade begins as the pattern recognition receptors (prrs) of innate cells recognize pathogen-associated molecular patterns (pamps) of viruses. after recognition, type i and iii interferon (ifn) and other proinflammatory cytokines undergo transcription. autocrine and paracrine signals in both healthy and infected cells subsequently ensure the translation of these cytokines ( ) . passive evasion refers to any mechanism of immune avoidance that does not directly interfere with the host immune system. many non-structural proteins (nsp) are translated in order to shield viral genomic material from the human immune system, as shown in table . nsp , , and are believed to function as modifiers of the intracellular membranes of organelles, where the virus may safely replicate. another passive mechanism is the conformational change of the viral mrna through either the addition of a '-cap (via formation of n- -methyltransferase in nsp ), or a '-o-methyl-transferase as with nsp , which marks the viral mrna to be of host origin. in another example, nsp induces endoribonuclease activity against viral mrna at certain stages of its development to avoid detection ( ) . nsp was shown to inhibit host mrna translation through the activation of exonucleases. these induce degradation of the host genome and prevent the ifn signal transduction that is necessary to activate viral clearance. nsp expresses plpro, which greatly resembles host cellular de-ubiquitinase action. this induces disruption of ubiquitin-mediated degradation and may also inhibit the innate immune response ( ). as with the previous outbreak, immune clearance of sars-cov- relies mostly on the activity of th cells via the extensive secretion of il- , il- , ifn-γ, and tnf-α/β. this cytokine microenvironment activates macrophages and causes cytotoxic t-cell proliferation, initiating pathogen clearance. following degradation and the presentation of viral antigens on apcs, the humoral response acts to limit future infections through antibody neutralization ( ) . in the lungs, alveolar macrophages are the first cells to make contact with microorganisms. their main function is to destroy any invaders without overstimulating the adaptive immune system, since foreign pathogens are ever-present. protective measures employed by alveolar macrophages include the surface expression of cd and tgf-β receptors, which function as negative regulators of dendritic cells and t cells ( , ) . this inactivation is exploited by sars-cov, leading to an increase in viral load. additionally, law et al. demonstrated that macrophages and dendritic cells are targeted by sars-cov, resulting in a very low expression of antiviral cytokines. this inhibits apoptosis and hinders the bridging of the innate and adaptive immune systems, leading to lymphopenia ( ) . in a cohort study by qin et al. of patients with confirmed sars-cov- infection, lymphopenia and an increase in neutrophil-to-lymphocyte ratio was routinely observed among infected patients and was especially evident in severe cases ( ) . it has been shown that these two parameters are indicative of systemic inflammation, and were associated with the worst prognosis ( , ) . because qin and colleagues did not observe any changes in either cd + or b cells, it was then hypothesized that sars-cov- plays a major role in indirectly damaging lymphocytes, with the release of proinflammatory cytokines and chemokines that results in the consumption of the lymphocytes necessary to prevent innate overactivation ( ). qin et al. also noted that the increase in neutrophil-to-lymphocyte ratio was accompanied by an increase in procalcitonin, indicating bacterial co-infection ( ). zhao et al. showed that mice depleted of alveolar macrophages and subsequently infected with a mouse-adapted sars-cov strain quickly developed activation of dendritic cells, which in turn activated cytotoxic t cells and initiated viral clearance ( ) . while most viral infections end with eradication and development of immunological memory, adaptive immunity does on occasion fail to develop adequately. callow et al. demonstrated in that patients previously infected with human coronavirus e showed a decline in antibody concentration and were capable of being re-infected after year ( ) . other studies involving mers-cov have come to the same conclusion, determining that the concentration of virusneutralizing antibody was dependent upon disease severity ( , ) . it is important to note that the failure of adaptive immunity could be due to either insufficient antibody response or decrease in t-cell durability ( , ) . appropriate adaptive immunity requires early cd + and cd + responses. in the case of sars-cov- , viral evasion of the innate immune system leads to an increase in cytokine production and late cd +/cd + response, which then leads to pathogenic inflammation in patients with high viral loads. due to rapid and unopposed sars-cov- replication, cd + t lymphocytes are quickly activated to differentiate into th cells and are responsible for releasing pro-inflammatory cytokines il- , gm-csf, and ifn-γ. gm-csf activates to further produce inflammatory monocytes (cd + and cd +) which release more il- . this disrupts the homeostasis of the immune system leading to cytokine storm ( ) . sars-cov- -related hyperinflammation involves very high levels of il- -β, il- , and tnf-α ( ). sars-cov- is thought to bind to the toll-like receptor (tlr), activating inflammasomes and resulting in the cleavage of pro-il- β to form il- β, a mediator of inflammation, fever and lung injury ( ) . the pathological immune response has a wide variety of clinical presentations, from mild symptoms to pulmonary failure and death. extensive lung damage is associated with neutrophil and macrophage infiltration while cd + and cd + count in peripheral blood are simultaneously lowered ( ) . croft et al. performed serological analyses in both healthy and sars-cov- -positive individuals. the cd + t cells in infected individuals demonstrated very high levels of cd , cd , and cd , indicating a hyperactive state compared to the healthy group. further analysis showed high levels of the t-cell activation marker ox on cd + cells, which has been proven to be crucial for cell expansion, survival and cytokine production in both t cells and innate cells ( ) . it is important to note that expression levels of ox also varied depending on the severity and progression of the disease, and may possibly be a marker of poorer prognosis. cd + t cells were also found in a hyperactive state, with higher expressions of cd , cd , and cd . in various deteriorating icu patients, there was an increase in expressions of pd- and tim- in both cd + and cd + t cells, indicating immune exhaustion ( , ) . aside from exhaustion antigens such as pd- , bellesi et al. ( ) revealed upregulated expression of the cd antigen on both cd + and cd + t cells. when cd apoptosis-related antigen is observed to be highly expressed, this is accompanied by a lower cd + and cd + count, which may partially explain the loss of lymphocytes in sars-cov- -positive patients. the loss of naive cells appears to be particularly important in this context. complement activation can be initiated via the alternative pathway, classical pathway or lectin pathway. innate immunity recognizes pathogen-associated molecular patterns (pamps) in host cells and initiates a destructive response. the mannosebinding lectin (mbl) pathway has been shown to be the first line of defense against sars-cov ( ). it is composed of pattern recognition molecules associated with serine protease from the mbl-associated serine proteases (masps), which circulate as zymogens and become active after recognition, binding to carbohydrate-based pamps ( ) . to further understand the triggers of the excessive immune response to sars-cov- , gao et al. showed that as with sars-cov, masp- contact with the n protein led to cleavage of c and c into c a/c b and c a/c b, respectively, and the mbl pathway proceeded as normal. none of the other pathways triggered complement activation ( ) . due to the high infectivity and severity of the virus, the world health organization (who) has been prompted to develop new tools to ensure the fast and accurate diagnosis of the viral infection. many governments have given an unprecedented level of freedom to nominated laboratories to establish reliable diagnostic tests. the increased demand for rapid testing has fueled research in sequencing, characterizing, and understanding sars-cov- , in order to definitively diagnose it in human samples. as of august , the recommended sampling sites are as follows: the upper respiratory tract (nasopharynx, oropharynx, anterior nares), lower respiratory tract (sputum, bronchoalveolar lavage, pulmonary tissue biopsy) as well as urine, feces or whole blood. according to centers for disease control (cdc) guidelines, material from the upper and lower respiratory tracts are preferred, and should be sampled if possible. material should be collected by an experienced specialist to ensure high standards, and should be tested as soon as possible; exceptions are serum samples which can be stored for up to - weeks, to be referenced in future follow-up ( ) . the overwhelming number of infected cases compounded with the shortage of healthcare personnel has led to the prioritization of testing of affected individuals. due to limited access and resources, the cdc has been compelled to define specific criteria for testing all -ncov patients under investigation (pui) . in order to be tested, the pui needs to manifest with: -"fever and symptoms of lower respiratory illness (cough, difficulty breathing) after days of travel to wuhan city or contact with a -ncov pui within the last days, " or -"fever or symptoms of lower respiratory illness (cough, difficulty breathing) after contact with a patient with a confirmed case of sars-cov- infection within days" ( ) . the introduction of restrictions not only guarantees good quality patient care and prioritizes acute and severe cases, but maintains the integrity of an already strained healthcare system. the high volume of cases demands fast and efficacious testing regimes for a variety of settings, including the hospital. depending on the type of technology and the personnel available, a few specialized diagnostic protocols have proven to be useful in the field. sars-cov- is an rna virus which sheds detectable genetic material in almost all excretions of an infected individual. this material can be detected by a simple nucleic acid test which is capable of identification and characterization of nucleotide sequences. in blood, sputum and other samples, the amount of genetic material is very sparse thus necessitating an additional amplification step in order to reach a particular detection threshold. this method is known as the nucleic acid amplification test (naat). another technique currently available is the polymerase chain reaction (pcr). real-time polymerase chain reaction (rrt-pcr) is the gold-standard molecular technique for detection of sars-cov- viral rna in all recommended samples. it is a primary diagnostic test that targets the following sequences that code for structural viral proteins: spike (s), membrane (m), envelope (e), nucleocapsid (n), and rnadependent rna polymerase (rdrp). the available literature suggests that the spike protein (s) may be the primary pivot in intracellular interactions with host cells, thus demonstrating high immunogenic character ( ) . high infectivity of sars-cov- has compelled the cdc to publish rrt-pcr primers and probes together with all relevant literature for public access ( ) . such advances in research were possible thanks to past experience with the previous betacoronavius epidemic. primer design was based on the nucleotide sequences that matched sars-cov and mers-cov with to % accuracy ( ) . the wide availability of protocols has accelerated progress in research and diagnostic measures. nevertheless, it should be noted that the high mutation rate and large genetic variability of the virus may negatively affect the performance of the assay, and may lead to an increasing number of false-negative results ( ) . additionally, the difficulty of the assay, complexity of the logistic analysis, and protocol duration ( min to a few hours) ( ) confer some limitations to this diagnostic tool ( ) . the full rna extraction protocol should be implemented in a biosafety cabinet at bsl- security level by trained and skilled personnel. it is recommended that none of the samples be heat-treated before rna extraction, which means that samples pose a high risk of infection to laboratory technicians ( ) . false-positive results may also be obtained in cases where the amount of viral material in a collected sample is too low for detection ( ) . based on a published summary report by the fda, the analytical sensitivity of rt-pcr is % with a limit of detection of . cp/ul. specificity showed no crossreactivity with the most common pathogens: bacterial (legionella pneumophilia, mycobacterium tuberculosis and m. pneumoniae, and streptococcus pneumoniae and s. pyogenes) and viral (adenovirus, parainfluenza, rhinovirus, rsv, etc.). the only detected cross-reactivity corresponded to the severe acute respiratory syndrome coronavirus (sars-cov), but this is not surprising since the n target gene in sars-cov- demonstrates more than % genetic similarity to other betacoronaviruses ( ) . the diagnostic protocol formulated by the cdc clearly states the process of confirmation of sars-cov- infection, though the criteria depend on the area where the pui is being diagnosed. in areas where the concentration of sars-cov- virus is high, a single positive naat result is required to diagnose the patient. in areas with low viral circulation more than one of the following is required: -positive naat for at least two different targets on the sars-cov- genome (one specific for the virus) - positive naat for betacoronavirus (sars-cov, mers-cov, sars-cov ) with sars-cov- genome sequencing. it is indicated that the sequenced fragment must be longer or different from the fragment used in the naat assay ( ) . in cases where all tested controls are positive, with all sars-cov- markers below growth thresholds, the naat result is negative. however, cases with high clinical suspicion may still produce a negative rt-pcr result. this may be due to a number of factors, such as poor quality and handling of specimens, specimen collection too early or late in the disease course, contamination, and/or technical errors ( ) . in other words, the lack of a positive result does not exclude covid- disease. in such instances identification of the infected individual is based on clinical observation, patient history and epidemiological information. given the number of tests done to date, in the case of discordant opinions, re-sampling and use of different markers is advised ( ) . the limitations of naat diagnostic techniques for sars-cov- have generated increasing demand for quicker and simpler tests based on serology. rt-pcr, while very reliable and precise, is only capable of identifying the presence of viral load, and cannot inform on the state or progress of the infection in a given patient. in contrast, serological testing and enzyme-based testing measure immunological responses to the virus, allowing for differentiation between exposed asymptomatic, acutely or mildly sick, and recovered cases. additionally, it is able to quantify the number of cases within a short period of time, and this is crucial for modeling the population-scale of infection, determining the level of prophylaxis, and has implications for the development of a vaccine against sars-cov- . there are many immunoassay techniques currently approved or pending. their versatility and creativity augment the number of ways with which one can detect the pathogen in a studied sample. they can be divided into serologic tests and enzyme-based immunoassays. serologic tests exploit the natural responses of the human immune system, while enzyme-based immunoassays detect the antigen with specifically manufactured monoclonal antibodies. all tests in this category detect either the antigen shed by the virus or the antibody that is produced by b-cells in response to the pathogen. the rapid diagnostic test (rdt) is a serologic type of diagnostic tool that allows for detection of the target after - min ( ) . it is a qualitative lateral flow type assay that resembles the standard rapid chromatographic test used for the detection of beta-hcg hormone in the urine of pregnant women. as a field-based test, it requires a drop of blood or other body fluid for identification of igg and/or igm antibodies, or the viral antigen itself ( ) . antigen based-rdts target the structural viral proteins (s, n, etc.) mainly found in secretions of the upper respiratory tract, and react to the antibodies that are produced - days after the initial infection. sufficient time is required for the concentration of antibodies to reach the threshold of detection ( ) . it is also advised to measure igg and igm titer baselines before or during the first few days after exposure ( ) . the non-governmental organization find recognizes cemarked rapid tests ready for use in the field ( , ) . another report by john hopkins center for health and security lists a few rdts currently approved for diagnostic use worldwide, but the antibody-rdt lateral flow assay by cellex inc. is the only rdt approved in the united states. this test was developed in the usa and china, with a sensitivity and specificity equal to . and . %, respectively ( ) . the statistical significance was determined from studies in two chinese hospitals on sars-cov- -positive patients and sars-cov- -negative patients ( ) . the test is currently available for purchase with a disclaimer from the fda that the test alone cannot be used for definitive diagnosis. in regards to the april who statement concerning immunoassays, the antibody-/antigen-detecting rdts are only to be used for research purposes ( ) . according to recent data, these diagnostic tests do not produce results with appropriate reliability. bruning et al. ( ) claims that immunodiagnostic testing for influenza infections in cases with a viral load comparable to sars-cov- showed test sensitivity that varied between and % ( ) . also, due to their abrupt development, the majority of available rapid tests appear not to have been properly validated by relevant institutions and may pose a serious risk to diagnostic efficacy. the simplicity of the rdt means it is neither capable of quantifying the number of antibodies and therefore the phase of infection, nor is it able to ascertain whether these antibodies are part of long-term immunity to the virus. as of august , it is unknown whether exposure imparts immunity after recovery. a day or more delay in diagnosis is a serious diagnostic limitation and may not be of increased benefit to acute-state patients. another type of serologic testing for sars-cov- is a neutralization assay. it is a valuable method that detects antibodies capable of clearing the infection. the procedure consists of the infection of a special type of cell (e.g., veroe ) with sars-cov- , followed by incubation for - days at variable concentrations. during this time cells are titrated with human serum antibodies in order to detect the quantity of antibodies necessary to stop viral replication ( ) . a study performed by zhou et al. demonstrated that : - : dilutions of igg-positive sample were capable of neutralizing tcid ( % tissueculture-infective dose) in sars-cov- cultures ( ) . other than this result, it remains a relatively novel area of research, and information regarding its usefulness in the diagnosis of covid- is sparse ( ) . table lists a number of exemplary testing kits currently available for diagnostic use. enzyme-linked immunosorbent assay (elisa) is currently one of the most popular commercially used enzyme based immunoassays. as indicated by the name, it is an assay using an enzymatic reaction to indicate a positive diagnostic result. plates covered with viral antigens, prepared by the manufacturer, are incubated with the patient's serum. in cases where the specific igg and igm antibodies are present, the antibody-antigen binding complex will be visualized through an enzymatic reaction (colorimetric change, fluorescence, etc.) the whole procedure may take up to h and requires a large sample of blood, plasma, or serum. it is both a qualitative and quantitative type of assay, thus has great potential for future diagnoses of sars-cov- infections. in a study of sars-cov- -specific antibody responses in covid- patients, nisreen et al. used elisa to demonstrate that severe viral infection resulted in higher antibody levels. additionally, they claim that igg seroconversion can be confirmed by applying the same technique in the second week of symptom onset ( ) . another team proved that the accuracy of elisa is dependent on the timing of the infection. on days - after symptom onset, elisa showed < % positive rate; the rate rapidly increased beyond that time frame for both igm and igg antibodies. liu et al. evaluated immunoassays for detection of antibodies against sars-cov- , and demonstrated that viral protein s (spike)-based igm elisa had statistically higher sensitivity than n (nucleocapsid)based elisa (p < . ) in detecting igm antibodies. it is suspected that the level of immunogenicity of the s protein is the reason for the relatively higher specificity compared to the n protein ( ) . based on this data, the antigen-based elisa will be a valuable diagnostic method for the fight against covid- . currently, the ngo find lists different immunoassay kits already commercialized, with additional test kits in development ( ) . with supplemental research, these have the potential to become a powerful rapid diagnostic tool for the hospital setting. however, due to their complexity they, unlike rdts, may not be used in the field. to reiterate, there is an insufficient amount of evidencebased research regarding elisa's specificity and sensitivity for use in diagnostic confirmation, to be comparable with rrt-pcr assays. a lesser known enzyme-based immunoassay that is nevertheless noteworthy is the chemiluminescent immunoassay (clia). very similar to elisa, it uses enzyme-labeled antibodies which activate an enzymatic reaction upon contact with their target. the photon of light emitted-luminescencecan then be quantified and directly corresponded to the volume of reagents. the benefit of this technique is its high sensitivity and the possibility of enhancing the reaction to allow for a larger threshold in samples with higher substrate concentration. clia can be used to detect versatile targets including igm, igg, and iga, and there seems to have been a recent increase in trust for this technique among clinicians. a systematic review by bastos et al. compared lfia, elisa, and clia tests, and the results suggest that clia exhibits the highest sensitivity and specificity for igm and igg in patients with covid- ( ) . however, bastos and colleagues also demonstrated that due to excessive discrepancies in test rests, none of the three techniques tested were reliable enough to be recommended for large-scale diagnostic purposes. after the - sars outbreak, chandwani and shuter conducted an in vitro study using an engineered prototype of sars-cov to test lopinavir/ritonavir, protease inhibitors indicated for dual-therapy prophylaxis and treatment of hiv- ( ). lopinavir has higher potency but is less bioavailable, thus co-administration with ritonavir, which additionally inhibits cytochrome p a, leads to prolongation of its action ( ) . they showed that this dual therapy inhibited viral cytopathic activity. furthermore, chu et al. conducted a non-randomized trial in , placing two groups of patients on different regimens: the first group received ribavirin, a nucleoside inhibitor, in dual therapy with a reducing dose of corticosteroids; and the second group received lopinavir/ritonavir/ribavirin. it was shown that treatment group two had a decrease in viral load, fewer nosocomial infections and an increase in circulating lymphocytes, indicating a favorable outcome ( ) . after the emergence of sars-cov- , a randomized trial was conducted involving severe cases. ninety nine of these patients received lopinavir/ritonavir while received standard supportive care. detectable viral load in both groups was the same, and time to improvement after lopinavir/ritonavir was only decreased by day, as compared to the group receiving standard care. the authors concluded that the treatment benefits of lopinavir/ritonavir were not established for severe illness ( ) . similarly, an open-label randomized control trial by cao et al. (chictr ), involving severe sars-cov- cases, compared lopinavir/ritonavir treatment with standard care alone, and they showed that the antivirals yielded no clinical benefits. further trials are thus recommended to establish any possible benefits for patients suffering from less severe illness ( ) . favipiravir, another rdrp inhibitor, is a broad antiviral with a mechanism of action that is hypothesized to either incorporate within viral rna leading to chain termination, or bind to a conserved region of the rdrp and prevent nucleotide addition ( ) . interferon-alpha (ifn-α) is an antiviral that binds to interferon receptors and activates signal modulators (jak / ). the phosphorylated interferon receptor binds to the signal modulators, resulting in immune modulation and antiviral protein transcription. in an open-label control study conducted by cai et al., the antiviral activity of favipiravir + ifn-α was compared to that of lopinavir/ritonavir + ifn-α in patients with confirmed sars-cov- infection. they demonstrated that patients receiving favipiravir + ifn-α exhibited faster viral clearance and radiological improvement, compared to the other study group. although yielding positive results, this was not a double-blind placebo-controlled randomized study, and more trials must be implemented before definitive conclusions can be drawn ( ) . umifenovir is a broad spectrum antiviral possessing dual properties: direct antiviral and virucidal action, as well as demonstrating virustatic effect through impedance of various stages of the viral life cycle ( ) clinical trials-mechanism of action unknown. uprifosbuvir, setrobuvir, balaprevir, '-c-methylcytidine, valopectibine bms- , mk , r , r , idx- , yak, psi- and psi- apparent in the umifenovir group, and no side effects were observed in either group ( ) . an open-label randomized control trial (chictr ) was conducted by chen et al. in which adult sars-cov- patients were administered either favipiravir or umifenovir. they showed that favipiravir significantly improved symptoms associated with cough and pyrexia. however, no clinical benefit could be observed when comparing viral clearance between the two therapies ( ) . after isolating the genomic sequence of sars-cov- , in particular that pertaining to rna dependent rna-polymerase (rdrp), elfiky showed that the rdrp of sars-cov- exhibits . % similarity with that of sars-cov. an rdrp model was engineered from the ncbi nucleotide protein database using the sars-cov rdrp genome as a template to test several antiviral drugs. to ensure high reliability of the model, a nucleotide comparison between the rdrp model and the sars-cov- rdrp was established, and was shown to yield . % homology. the study sought to establish which of compounds (table ) could bind to rdrp active sites and elicit inhibitory activity. drug docking site analyses were interpreted using protein-ligand-interaction-profiler (plip). five fda-approved drugs showed very high affinity for rdrp, and could prove to be beneficial against sars-cov- . additionally, three drugs from the clinical trial by elficky showed high affinity for rdrp, and these include idx- , setrobuvir and yak. drug side effects and toxicity are yet to be disclosed ( ) . additionally, one antiviral drug that sparked interest was remdesivir. in a small cohort study, a day course of remdesivir was administered to patients with severe infection, and clinical improvement was observed in %. fewer clinical benefits were observed in patients who received invasive ventilator support, and % of the patients, especially those who received invasive ventilation, died after the treatment course. multiple factors impede the accurate measurement of remdesivir efficacy and these include preexisting conditions and duration of intubation. as a result, any clinical benefits need to be further investigated in future placebo-controlled trials ( ) . in a double blind placebo-controlled randomized trial (ntc ) biegel et al. administered remdesivir to hospitalized sars-cov- patients presenting with lower respiratory tract involvement to assess its efficacy and safety. remdesivir was shown to be superior to placebo in decreasing recovery time ( ) . however, a multicenter, randomized, placebo-controlled trial conducted by wang et al. to assess the efficacy of remdesivir in patients with severe sars-cov- (ntc ) showed that remdesivir was of no clinical benefit compared to placebo ( ) . lastly, a multicenter analysis involving a double-blind placebo-controlled trial (ntc ) was implemented to assess the efficacy and safety of remdesivir in the treatment of hospitalized sars-cov- patients. preliminary results from this trial showed that patients receiving remdesivir had a faster time to recovery and a lower mortality rate when compared to the placebo group, and it is on the basis of this that the fda issued authorization of remdesivir for emergency use on may , . interferons (ifns) are important cytokines with critical antiviral activity. infected innate immune cells produce ifn which enable the jak-stat pathway, leading to recruitment of more nk cells and macrophages. as with sars-cov and mers-cov, nsp of sars-cov- exhibits anti-ifn activity by targeting proteins of the jak-stat signaling pathway ( ) . ifn inhibition has been correlated to disease severity ( ) . furthermore, ifn treatment has already proved effective against ss-rna viruses, and is widely used in the treatment of hcv and hbv. zhang et al. combined ifn-α and ifn-γ in a study conducted in vivo and in vitro. this combination demonstrated synergy, and smaller dosages were required than in ifn monotherapy. this suggests that reduction in unwanted side effects may be possible with the use of dual therapy ( ) . it was shown that sars-cov- evades detection from cytosolic rig-i and mda , preventing the activation of ifn-i and the subsequent stimulation of innate cells. it is at this point that the importance of toll-like receptors (tlrs) should be emphasized. negishi et al. demonstrated that viruses that evade cytosolic safeguards can be inhibited by tlr- action. tlr- functions to activate ifn-ii, which in turn elicits an antiviral response ( ) . the use of tlr- agonists in mice by shahabi nezhad et al. showed promising results; they were able to increase levels of ifn-α/β/γ, which compensates for the inhibitory actions of sars-cov on signaling pathways ( ) but further research is needed to determine if this may result in toxic overstimulation of the immune system ( ) . in an open-label randomized phase ii trial by hung et al., a triple combination of ifn-β- b, lopinavir/ritonavir and ribavirin was administered to covid- patients with mild to moderate disease. this combination proved to be safe and superior to lopinavir/ritonavir + ribavirin, with patients showing alleviation of symptoms, shortened duration of viral shedding, and shortened hospital stay. these promising results mean that future trials utilizing ifn-β- b are warranted ( ) . anti-c a-antibody, eculizumab, and bevacizumab the discovery of the sars-cov- specificity to masp- of the mbl pathway opened the potential for prophylactic treatment against cytokine-mediated lung damage. it was previously shown in the literature that acute lung injury due to viral infection could be prevented by the use of anti-c a-antibody treatment ( ) , and on the basis of this, gao et al. used a recombinant c a-antibody in an open-label trial involving severely ill patients the outcome of the first two recipients of the monoclonal antibody was described. both patients showed improved oxygen saturation, increased lymphocyte count, decrease in inflammatory proteins, improvement in liver function, and alleviation of pneumonia. although the use of anti-c a antibody shows great promise, the trial is still ongoing and the final efficacy is yet to be disclosed ( ) . another trial (ntc ) is currently assessing the potential of eculizumab to reduce mortality in patients. in a similar mechanism of anti-inflammatory action, eculizumab inhibits c cleavage thus preventing the release of c a. for the treatment of ards, a clinical trial (nct ) is comparing the therapeutic potential and side effects of the monoclonal antibody bevacizumab for critical covid- patients. by targeting vascular endothelial growth factor (vegf), an angiogenic factor, bevacizumab may prevent the disruption of the vascular barrier that causes edema and lung injury ( ) . a preliminary study conducted by wang et al. ( ) identified potential antibodies with the capacity to neutralize sars-cov- . the s protein ectodomains of both sars-cov and sars-cov- were expressed in hek- t cells using plasmid transduction. similarly, hek- t cells were transfused with plasmids containing sars-cov / in s protein subdomains tagged with either the mice or human fc portion of igg. h l mice antibodies were produced through gradual immunization with the sars-cov s protein ectodomain. the spleen and lymph nodes were then harvested to produce hybridomas. of the samples, only one of the hybridomas ( d ) exhibited cross-reactivity sars-cov and sars-cov- s proteins. the chimeric antibody was reformatted and expressed as a fully human igg. it was shown that this novel igg tightly binds the conserved rbd of the sars-cov- s protein in infected cells and neutralized the virus in veroe cells. this represents the very first human monoclonal antibody that is able to fully neutralize sars-cov- ( ) . this cross-neutralizing antibody, due to a conserved epitope region on the spike protein, could be the key to preventing future betacoronavirus outbreaks. d has recently completed phase i clinical trials (nct ) to establish dosing in hospitalized patients with sars-cov- under longterm follow up. now undergoing phase ii trials (nct ), the monoclonal antibody will be tested on ambulatory patients, with results estimated to be available in september. as stated earlier, the ace- receptor is crucial for viral attachment and entry. an in vitro experiment conducted by monteil et al. exposed veroe cells to varying concentrations of plaque forming units (pfu) from sars-cov- in the presence and absence of human recombinant soluble ace- (hrsace- ). the infection was inhibited h after introduction of the virus. the experiment was repeated using human capillary organoids and human kidney organoids, and the same inhibitory actions of hrsace- were observed. it is important to note the dosedependent nature of this inhibitory action. hrs-ace- has already undergone phase i and ii clinical trials for the treatment of ards ( ) , and monteil et al.' findings suggest that hrs-ace- could be a potential therapeutic agent against sars-cov- and phase ii trials (nct ) are currently underway in various european countries ( ) . in an open label trial by the recovery collaborative group, , hospitalized patients received either low dose dexamethasone or standard care alone by random assignment. it was observed that the group of patients on mechanical ventilation who received dexamethasone exhibited lower mortality rates compared to those receiving standard care alone ( ) . while other studies further support the role of glucocorticoids in the reduction of mortality ( ) some reported conflicting results and showed no clinical benefit or even harm to the patient ( ) . a systematic review of studies concluded that while critically ill patients are more likely to benefit from glucocorticoid therapy, their use was associated with increased mortality as it resulted in longer hospital stays and increased tendency toward serious nosocomial infections ( ) . clinical trials are currently ongoing to assess the risk vs. benefit for the use of glucocorticoids in the treatment of covid- . cytokine storm remains the main cause of acute lung injury and organ damage in the course of sars-cov- infection. it is chiefly caused by gm-csf and il- . il- receptors exist in two forms: the soluble form (sil- ) and the membrane bound (mil- ). to initiate pro-inflammatory action, il- binds to sil- and the complex binds to gp to complete signal transduction. due to this, the therapeutic use of the il- antagonist tocilizumab, which binds to both sil- and mil- , was suggested. xu et al. qualified critical patients in a trial with tocilizumab. clinical symptoms, radiological findings and laboratory values all improved after treatment, and patients were successfully discharged ( ) . in an open label study by morena et al., however, critically ill sars-cov- patients were treated with tocilizumab. all patients presented with decreased oxygen saturation and an increase in plasma il- . while positive results were observed with tocilizumab rapidly decreasing inflammatory markers, no clinical benefit was reported as patients quickly developed life-threatening bacterial and fungal infections ( ) . jordan et al. conducted another study administering critical patients with a single dose of tocilizumab. this resulted in significant decrease in inflammatory proteins and reduction in fever. twenty two patients receiving mechanical ventilation were able to be extubated and vasopressors discontinued. two patients died, and the authors report that these patients were already in severe septic shock due to sars-cov- -related pneumonia, and were unresponsive to vasopressors. four patients did not respond to the medication and had poorer outcomes. while the results are promising and in line with the findings by xu et al., limitations of this work include the lower-thanrecommended dose of tocilizumab, chosen by the authors due to drug shortage, and the absence of a control group ( ) . a placebo-controlled randomized clinical trial is still necessary before recommendations can be made. historically, il- has been shown to act as a pro-inflammatory cytokine with actions on several immune cells ( , ( ) . convalescent plasma has been employed and has shown promise in the treatment of sars, mers and influenza ( ) ( ) ( ) . in a study by duan et al., severely ill patients were transfused with ml of convalescent plasma harvested from donors who have recovered from sars-cov- and had antibody titers above : . within to days, symptoms had disappeared in all patients, and radiological investigation showed improvement after seven days. viral load was undetectable by rt-pcr in patients and no adverse side effects were detected ( ) . shen et al. treated five covid- patients with convalescent plasma. all five patients were critically ill and intubated, presenting with ards and pneumonia and experiencing high viral load despite treatment with antivirals. the outcome was largely positive: ards was resolved in four patients within days, and three patients were able to be weaned off mechanical ventilation within weeks. three patients were discharged while two remained stable in recovery ( ) . joyner et al. recently made an assessment of the safety of convalescent plasma in , covid- patients. the incidence of serious adverse events including transfusion reactions, cardiac events and thrombotic events was low. mortality rates were shown to be higher in critical patients receiving mechanical ventilation or those in septic shock. the conclusion drawn from the study provided evidence that the administration of convalescent plasma in a hospital setting was safe and that early administration is more likely to reduce fatality rates ( ) . it has been previously shown that sars-cov induces viroporin production in the host cell membrane to facilitate virion release. viroporin a has been associated with nlrp (nod-like r family, pyrin domain ) inflammasome activation ( ) , which induces the production of pro-inflammatory cytokines such as il- -β and il- via the gasdermin d (gsmd) pathway ( , ) . like sars-cov, sars-cov- induces the production of large amounts of il- -β ( ) . on the basis of this, a possible treatment could be il- , a member of the il- family. when placed with activated peripheral mononuclear cells, il- demonstrates suppressive and anti-inflammatory effects through the inhibition of il- , il- , and tnf production ( ) . another member of the il- family is il- . both in vitro and in vivo studies showed that il- acts as a negative regulator of inflammation, aiding in the protective actions exhibited by tgfβ on dendritic cells and thus attenuating the t cell response ( ) . both il- and il- could potentially be valuable in the treatment of covid- ( ) . baricitinib is a janus kinase (jak) / inhibitor used to treat rheumatoid arthritis. jak / inhibition prevents activation of pro-inflammatory cytokines such as gm-csf, il- , il- , il- , and il- . adaptor-related protein complex (ap )-associated protein kinase i (aak ) induces receptor-mediated endocytosis. baricitinib has been shown to have very high affinity for aak , thus could feasibly inhibit both cytokine storm and viral entry to the cell ( ) . in a small cohort study, titanji et al. administered baricitinib and hydroxychloroquine to patients with moderate to severe covid- . twelve of the patients recovered, and vitals and inflammatory markers were seen to improve after baricitinib was initiated. two patients however developed serious bacterial or fungal infections due to prolonged icu stay ( ) . a phase iii double blind placebo-controlled randomized trial involving baricitinib (nct ) is currently ongoing to assess the efficacy and safety of the drug as a potential immune inhibitor preventing cytokine storm and viral entry. sars-cov- is the most structurally and genetically similar to sars-cov, thus findings from monoclonal studies on sars-cov have been utilized to target the shared aspects between the strains. the monoclonal antibodies shown in table are engineered to specifically bind to different domains on s or s . though none have progressed to clinical trials, they mechanism of action r binds s and prevents interaction with ace- ( ) synergistic action; bind s and prevent interaction with ace- ; prevent immune escape ( , ) f g f g m bind linear epitope of s ; bind conformational epitope of s ; inhibit interaction of s with ace- ( ) binds hdr domain of s and prevents interaction of receptor in vitro ( ) binds s and prevents interaction with ace- ( ) monoclonal antibodies studied for the treatment of sars-cov- function igg that binds to a conserved region in the rbd of the s protein and fully neutralizes sars-cov and sars-cov- ( ) anakinra il- -inhibitor used to reduce effects of the cytokine storm in sars-cov- ( ) show promise in vitro and in vivo against sars-cov and sars-cov- ( ) . the immunomodulatory potential of mesenchymal stem cells (msc) was first identified by luk thromboembolism as a result of endothelial injury in the course of infection is a serious and fatal complication in critically ill patients ( ) ( ) ( ) . tang et al. enrolled covid- patients with severe disease for a study in which patients who received low molecular weight heparin for a week or more. these patients were associated with better prognosis than the patients who were not administered anticoagulant therapy ( ) . helms et al. followed icu patients in a multicenter prospective cohort study. thromboembolic events were observed in . % of patients despite prophylactic and therapeutic use of anticoagulants. the authors noted that pulmonary embolism was diagnosed a few days after icu admission and was more common in ards patients. they conclude that although other papers have reported the effectiveness of heparin, a higher anticoagulant target should be implemented with other anticoagulants such as anti-xa ( ) . the pathogenesis of thromboembolism in the course of covid- is still unclear. one of the proposed pathways implicates ards: the profound hypoxemia and vasoconstriction may lead to vascular occlusion ( ) . another proposed mechanism is that unlike in a healthy lung, a diseased lung is unable to maintain the balance between fibrinolysis and coagulation, thus resulting in decreased action of tissue plasminogen activators (tpa) ( ) . more randomized clinical trials are currently ongoing to assess the efficacy of various anticoagulants. in a pilot study by fowler et al., critically ill septic patients were given either a vitamin c infusion or a placebo. a dramatic decline in inflammatory markers was observed in the vitamin c group, and their sequential organ failure assessment (sofa) score was decreased compared to the placebo group. there were also no adverse events observed with the vitamin c group ( ) . in , fowler et al. published on the use of vitamin c vs. placebo in septic patients with ards, and they concluded that the vitamin c infusion did not improve either inflammatory markers or organ dysfunction score ( ) . several clinical trials are ongoing verifying the benefits of vitamin c in the treatment of covid- . widely used as an antimalarial drug, hydroxychloroquine has been shown to possess broad antiviral action, including effectiveness against hiv- and influenza type a and b. its antiviral activity has been tested on sars-cov- . in preventing the glycosylation of the ace- receptor, hydroxychloroquine effectively prevents viral entry ( ) . furthermore, it has been shown to alkalinize the organelle, which serves to prevent the formation of mature endosomes required to shield the virus from immune cells, and for replication ( ) . apart from its broad antiviral activity, hydroxychloroquine has proven to be adequately anti-inflammatory, interfering with nlrp activation and impairing the production of pro-inflammatory cytokines, specifically il- -β ( ) . in an open-label nonrandomized clinical trial by gautret et al., sars-cov- -positive patients were divided into three groups: group receiving hydroxychloroquine, group receiving hydroxychloroquine + azithromycin (antibiotics were given at the discretion of the physician to prevent opportunistic infections), and group not receiving hydroxychloroquine. the primary outcome in group and was viral clearance within to days, but greater results were achieved when hydroxychloroquine was combined with azithromycin ( ) . azithromycin, a bacteriostatic agent, has shown antiviral activity against zika virus, ebola virus ( ) and rsv. its antiviral mechanism of action, with regards to rsv, is hypothesized to be in decreasing the expression of fusion proteins in airway epithelium ( ) . in a study conducted by million et al., patients suffering from early sars-cov- infection were treated with hydroxychloroquine and azithromycin; this combination proved to efficaciously reduce the viral load and was deemed safe with minimal risk of complications ( ) . chen et al. also showed that hydrochloroquine was safe and efficacious in the treatment of mild disease ( ) . the efficacy of hydrochloroquine with or without azithromycin has since been disputed. in a recent open-label randomized control trial, the efficacy and safety of hydroxychloroquine + standard care vs. standard care alone was assessed by wei et al. in patients with mild to moderate covid- . the group receiving hydroxychloroquine was shown to suffer more adverse effects, and there was no observed clinical benefit compared to patients given standard care alone ( ) . molina et al. also concluded that while hydroxychloroquine proved to be beneficial in past studies, the combination of hydroxychloroquine and azithromycin for severe sars-cov- infections resulted in inadequate viral clearance, and the clinical benefits previously seen in patients with mild to moderate illness were not observed in hospitalized patients with severe disease ( ) . this broad spectrum antiparasitic agent has been shown to possess antiviral activity. in vitro studies indicate that ivermectin prevents non-structural proteins of both dengue virus ( ) and hiv- ( ) from interacting with the importin α/β on the host cell, which prevents viral integration. it has been shown that the sars-cov nucleocapsid (n) protein integrates with the nucleus and nucleolus, and prevents cytokinesis of the host cell via importin-α/β . the exact role of the n protein in the cell cycle is not known, but it is postulated that this structural protein enters the nucleolus to promote viral replication and encourage suitable conditions for viral packaging ( ) . an in vitro experiment by caly et al. showed that a single dose of ivermectin administered to inoculated vero cells effectively controlled viral replication within to days, which may prove beneficial for newly infected patients. it was hypothesized that like in other viruses, ivermectin's antiviral action against sars-cov- is derived from the inhibition of importin-α/β . ( ) . alam et al. treated mild to moderately ill patients with a combination of ivermectin and doxycycline. symptom improvement was observed after h following treatment, and no side effects were noted. this study however makes no conclusion on the efficacy and safety of this therapy as it is not a place-controlled randomized clinical trial ( ) . similarly, caly et al.' results, while promising, cannot be applied until safety margins have been established in further clinical trials. this glycopeptide antibiotic used in the treatment of grampositive bacterial infections has been shown to possess antiviral activity against ebola, mers, sars and hiv- . it is believed that teicoplanin interferes with endosome formation through alkalization. cleavage of the s protein by cathepsin in the late endosome is inhibited, which in turn prevents the release of viral rna ( ) . baron et al. showed that the cathepsin l sequence is conserved in sars-cov, suggesting that teicoplanin could be a key treatment in patients who are diagnosed early with sars-cov- ( ) . there have been tremendous advancements in the field of immunotherapy since the development of chimeric antigenreceptor t cells (car-t). these cells differentiate from our basic t-cells by overcoming t-cell control safeguards and therefore express fewer exhaustion markers pd- , tim , and lag . furthermore, they are capable of differentiating into terminal effector t-cells responsible for pathogen and tumor destruction. car-t cell treatment has already shown great advances in oncology, inducing long-term remission in patients suffering from acute lymphoblastic leukemia ( ) . it is currently being investigated for the treatment of viral infections such as hiv- , hbv, and hcv. development of car-t cells specific to hiv- infection has now entered clinical trials ( ) . crispr-cas (clustered regularly interspaced short palindromic repeats), a mechanism evolved to protect against bacteriophages has shown great promise as a genetic editing tool. in vitro studies have used lentiviral vectors consisting of cas (crispr-associated proteins) and sgrna specific ccr (single guided rna responsible for the detection of the genome of interest) against cd + t cells susceptible to hiv- infection. the viral vector was able to disrupt the ccr gene in the cd + cells thus inhibiting hiv- entry ( ) . in a novel study, the use of the crispr-cas system against human lung epithelium infected with sars-cov- yielded positive results: crispr-cas was able to be transfected in the lung epithelium to degrade the virus ( ) . this method was shown to possess protective actions against the known pathogenic coronaviruses. gene editing and car-t cell therapy open a new frontier in future treatment modalities. the rapid spread of sars-cov- poses a threat of global proportions. time is of the essence, and the discovery of accurate diagnostic methods and treatment protocols are imperative in preventing further spread of this pathogen. similarities between sars-cov- and its predecessor have formed the framework upon which diagnostic and treatment approaches to the novel virus are based. rt-pcr primers, based on sars-cov and mers-cov, have proven to be highly sensitive and specific, though not without their flaws. time consuming and prone to producing false negative results, this has led to the employment of more efficient testing methods such as serologic tests and enzyme-based assays, capable of quantifying infected patients on a large scale. to date, there are still no therapies specifically targeting sars-cov- . while many fda-approved antivirals on the market have had success in patients presenting with differing degrees of illness severity, the development of specific antivirals remains an area of active research. on the other hand, immunotherapy has been shown to be effective, particularly with the discovery of hrs-ace- and other promising immune modulators, the development of the d monoclonal antibody capable of neutralizing sars-cov- , as well as msc therapy. the non-traditional use of anti-malarial agents had previously showed great promise but have now proven to lack adequate antiviral action and have been associated with severe complications. until guidelines are updated following the multitude of ongoing clinical trials, standard care remains the main treatment modality. rigorous research with regards to this pandemic not only adds to the scientific literature, but is critical for public health policy surrounding future outbreaks. only with collaborative research efforts and dissemination of knowledge may we interrupt exponential transmission of disease and maintain human losses at the minimum. kk and jc: conceptualization. kk, np, and dh: investigation and resources. kk, np, jc, and dh: writing-original draft preparation. kk, jc, and mk: writing-review and editing. jc and mk: visualization. mk and am: supervision and project administration. all authors contributed to the article and 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dose-escalation trial car t cells beyond cancer: hope for immunomodulatory therapy of infectious diseases ccr gene disruption via lentiviral vectors expressing cas and single guided rna renders cells resistant to hiv- infection development of crispr as a prophylactic strategy to combat novel coronavirus and influenza key: cord- -oldy gta authors: barriocanal, marina; carnero, elena; segura, victor; fortes, puri title: long non-coding rna bst /bispr is induced by ifn and regulates the expression of the antiviral factor tetherin date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: oldy gta many long non-coding rnas (lncrnas) are expressed in cells but only a few have been well characterized. in these cases, lncrnas have been shown to be key regulators of several cellular processes. therefore, there is a great need to understand the function of more lncrnas and their regulation in response to stimuli. interferon (ifn) is a key molecule in the cellular antiviral response. ifn binding to its receptor activates transcription of several ifn-stimulated genes (isgs) that function as potent antivirals. in addition, several isgs are positive or negative regulators of the ifn pathway. this is essential to ensure a strong antiviral response and a later return of the cell to homeostasis. as the isgs described to date are coding genes, we sought to determine whether ifn also regulates the expression of long non-coding isgs. to this aim, we used rna sequencing to analyze the transcriptome of control and huh cells treated with ifnα . the results show that ifn-treatment regulates the expression of several unknown non-coding transcripts. we have validated two lncrnas upregulated after treatment with different doses of type i ifnα in different cells or with type iii ifnλ. these lncrnas were also induced by influenza and vesicular stomatitis virus mutants unable to block the ifn response, but not by several wild-type lytic viruses tested. these lncrna genes were named lncisg and lncbst as they are located close to isgs isg and bst , respectively. interestingly, inhibition experiments showed that lncbst is a positive regulator of bst . therefore lncbst has been renamed bispr, from bst ifn-stimulated positive regulator. our results may have therapeutic implications as lncbst /bispr, but also lncisg and their coding neighbors, are increased in cells infected with hepatitis c virus and in the liver of infected patients. these results allow us to hypothesize that several lncrnas could be activated by ifn to control the potency of the antiviral ifn response. the interferon (ifn)-mediated innate immune response provides a potent defense against pathogens ( ) . upon invasion, pathogenassociated molecular patterns (pamps) are detected by specific receptors in the cells. these can be located on the surface of the cell, as in the case of toll-like receptors (tlrs), or intracellularly, as in the case of the retinoic acid-inducible gene i (rig-i). pamp recognition triggers a series of signaling cascades that lead to the production and secretion of type i ifn. type i ifn (ifnα, ifnβ, and others) binds to ifn receptors present on the surface of all cell types and activates janus-activated kinase/signal transducer and activator of transcription (jak/stat) signaling. this gives rise to the nuclear translocation of the stat /stat /irf (ifn regulatory factor ) complex that binds ifn-stimulated response elements (isre) in the promoters of ifn-stimulated genes (isgs) and activates their transcription. a similar response is induced by type iii ifn (ifnλ) upon binding to its receptor ( , ) . in contrast, type ii ifn or ifnγ, produced by cells of the immune system, binds to the widely expressed ifnγ receptor ( , ) leading to nuclear translocation of stat homodimers, which bind to gamma-activated sequences (gas) in the promoter of immunoregulatory genes. ifn-stimulated genes are antiviral factors, positive regulators of the ifn pathway (stat and and irf ) or negative regulators that help ifn-induced cells to return to cellular homeostasis (socs and ups ) ( ) ( ) ( ) ( ) ( ) ( ) . among antiviral genes, there are factors that function to increase cell sensitivity to pamps (oas and pkr) or true antiviral effectors that block viral entry (mx, ifitm, and trim), virus replication, translation and stability (ifit, oas, pkr, and isg ), or viral release (viperin and tetherin/bst ) ( ) . while most ifn-induced factors known to date are proteins, ifn also activates the expression of several micrornas that contribute to the antiviral state or to the control of ifn response ( ) . few studies have been performed to address whether ifn could also regulate expression of long non-coding rnas (lncr-nas) ( ) ( ) ( ) . in recent years, viral infection has been reported to be able to induce the expression of cellular lncrnas. this has been shown for infection with enterovirus, influenza, hiv, hepatitis b and c viruses, and the sars coronavirus ( , ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) (carnero et al., in preparation) . the lncrna signature found after infection www.frontiersin.org should be a mixture of transcripts induced by the virus and transcripts that respond to the cellular antiviral pathways activated by the infection. in fact, activation of tlrs by pamps induces the expression of several lncrnas. tlr signaling leads to the activation of lncrna-cox , which regulates the expression of genes related to the immune system ( ) . activation of tlr results in increased neat , which increases the expression of genes such as il ( ) . tlr controls il b-erna and il b-rbt lncr-nas whose downregulation diminishes il b and accumulation of lps-induced rnas ( ) . likewise, the lps-induced inflammatory response is controlled by lnc-il r ( ) . innate activation also induces linc /thril, which controls tnfα and other genes involved in the immune response ( ) . in turn, tnfα induces lethe, a pseudogene that responds to nfκb and reduces inflammation by inhibiting nfκb dna binding activity ( ) . lncrna responses are also critical for the functionality of dendritic cells, cd + and cd + t-cells ( ) ( ) ( ) ( ) . thus, nest lncrna controls ifnγ locus in cd + t-cells leading to decreased salmonella enterica pathogenesis ( , ) . these studies illustrate the interest in identifying novel lncr-nas and elucidating their function and regulation. lncrnas are thought to be at least as numerous as protein-coding genes, but only a few are well characterized ( ) ( ) ( ) ( ) . lncrnas are transcripts similar to mrnas but with poor coding potential. they are more cell type-specific, less expressed, and less well conserved than mrnas ( , ) . interestingly, lncrnas are cell regulators that can function in cis, co-transcriptionally, or in trans. some control the expression of coding genes located in the same genomic region. therefore, the genomic location of lncrnas can provide hints as to their functionality. they can be sense or antisense (when overlapping with one or more exons of another transcript in the same or in the opposite strand, respectively); intronic (when derived from an intron of another transcript); divergent or bidirectional (when they share a promoter with another transcript in the opposite strand and therefore are co-regulated); or intergenic (when they are independent, located in between two other genes). several mechanisms are involved in the regulation of neighboring or antisense genes by lncrnas. these include transcriptional activation or interference, recruitment of chromatin modifiers and remodelers, regulation of imprinting, editing, splicing or translation, and stability ( ) ( ) ( ) ( ) ( ) . to address the issue of whether ifn could also regulate expression of lncrnas, which may play key roles in the antiviral response, we analyzed the transcriptome of cells treated or not with ifnα by rna sequencing (rnaseq). in this analysis, we identified two lncrnas upregulated in response to ifn in different cell lines. interestingly, these lncrnas are expressed from positions in the genome divergent from the well-characterized isgs isg and bst . therefore, we have called them lncisg and lncbst . these lncrnas and their coding counterparts are also induced in cells infected with mutants of influenza or vesicular stomatitis viruses (vsv) that fail to block the ifn response. surprisingly, they are also induced in culture cells infected with hepatitis c virus (hcv) and in the liver of patients with hcv infections. finally, according to hugo regulation, we have renamed lncbst bispr, from bst ifn-stimulated positive regulator, as we show that inhibition of lncbst expression by rnai leads to decreased levels of bst mrna, providing a new layer of regulation of the ifn response. the huh cell line, derived from a human hepatocarcinoma, was provided by dr. chisari's lab (scripps research institute, la jolla, ca, usa). a cells, from human non-small cell lung carcinoma, were kindly provided by estanislao nistal (cima, university of navarra, spain). human liver samples with or without hcv infection were obtained from the biobank of the university of navarra under approval from the ethics and scientific committees. liver tissue sections were snap frozen and stored at − °c. the clinical data from hcv-infected subjects are shown in table s in supplementary material. cells were grown in dulbecco's modified eagle medium (dmem) supplemented with % fetal bovine serum (fbs) and % penicillin-streptavidin and maintained at °c in a % co atmosphere. twenty-four hours before treatment with ifn, huh and a cells were seeded in six-well plates. then, , , , , , or units/ml of ifnα (sicor biotech, lithuania) were used in a final volume of ml. huh cells were also treated with ng/ml of il b/ifn-λ (r&d systems) in a final volume of ml. for treatment with ruxolitinib (selleckchem), cells were seeded out h before and treated with . µm ruxolitinib in a final volume of ml. one hour after treatment media were discarded and replaced by media containing units/ml ifnα. cells were harvested for rna extraction at the indicated times post-treatment. sirnas targeting lncbst /bispr were designed using iscore designer and rna scales ( , ) and purchased from dharmacon. the lncbst /bispr sirnas targeted the sequence gacuagugugagcaacaaa. for cell transfection with sir-nas, lipofectamine reagent (invitrogen) was used according to manufacturer's instructions. cells were seeded h before transfection. for each well of a six-well plate, pmoles sirna were used. the sirna was mixed with µl optimem. furthermore, µl lipofectamine were mixed with µl optimem media and incubated for min. then, lipofectamine and sirna solutions were mixed and incubated for - min at room temperature. after incubation, half of the volume of the cell media was discarded and , , or µl of the lipofectamine mixture were added to each well of , , or -well plates, respectively. six hours post-transfection the media from the cells was discarded and substituted with dmem media enriched with % fbs and antibiotics. hepatitis c virus jfh- was obtained from an initial viral stock from the genotype a jfh- plasmid (pjfh- ) previously described by wakita et al. ( ) . the virus was amplified as described ( ) . influenza virus strain a/pr / wt (pr ), a mutant lacking ns (∆ns ), vsv-gfp, and the mutant m r frontiers in immunology | molecular innate immunity were kindly provided by estanislao nistal (cima, university of navarra, spain) ( ) ( ) ( ) , semliki forest virus (sfv) was a gift from cristian smerdou (cima, university of navarra, spain), and adenovirus serotype (ad ) was amplified as previously described ( ) . vsv-egfp titration was performed in quadruplicates on a cells. the supernatant from infected cells was collected and : serial dilutions were performed. cells were seeded h before infection in -well plates and infected with µl of each dilution. twenty-four hours after infection, gfp expression was visualized by microscopy and used to determine the titer. cells were infected with hcv at a multiplicity of infection (moi) of . , with vsv at a moi of and with a moi of of influenza a, ∆ns , ad , and sfv. in the case of the lytic viruses, we used a moi of or as this causes cytopathic effects at h (for vsv, influenza and sfv) or h (for ad) in huh or a cells. after infection, the virus was removed and fresh medium was added to the cells. cells were harvested for rna extraction at the indicated times post-infection. two million huh cells were incubated in µl of cytoplasmic buffer ( mm tris hcl ph . , mm edta, and % np ) for min at °c. then, cells were centrifuged for min at g and the supernatant was used to isolate cytoplasmic rna. the pellet was washed with cytoplasmic buffer and centrifuged as before. the supernatant was discarded and the pellet was used to isolate the nuclear rna. rna from nuclear and cytoplasmic fractions was isolated with maxwell research system (promega). human tissue was homogenized using the ultra-turrax dispersing machine (t basic ika-werke) ( ) . total rna from the tissue was extracted in ml trizol (sigma-aldrich) and recommendations of the supplier were followed ( ) . dnase (fermentas) treatment was performed to eliminate dna from the samples before rt-pcr reactions. rna was extracted from cells with the maxwell research system from promega following the manufacturer's recommendations. rna concentration was measured using nanodrop spectrophotometer. the quality of the rna was analyzed by bioanalyzer (agilent technologies). reverse transcription (rt) was performed as described ( ) . the reaction was performed in the c touch thermal cycler from bio-rad. the samples were incubated at °c for min, then at °c for s and then immediately cooled to °c. qpcr was performed in the cfx real-time system from bio-rad as described ( ) . the results were analyzed with bio-rad cfxmanager software. gapdh levels were evaluated in all the cases as a reference. only the samples with similar gapdh amplification were analyzed further. the primers used are listed in table s in supplementary material and were designed with the primer program . rna of excellent quality, as determined by bioanalyzer (agilent technologies) was treated with the ribo-zero rrna removal kit http://frodo.wi.mit.edu (epicenter) to deplete from ribosomal rna. library preparation with truseq rna sample preparation kit (illumina) and sequencing was performed at the embl genomics core facility (genecore) in an illumina hiseq . sequences were paired-end, bases long, and strand specific. rnaseq data are available at the ncbi gene expression omnibus (geo) data repository . rna sequencing data analysis was performed using the following workflow: ( ) the quality of the samples was verified using fastqc software; ( ) the preprocessing of reads was performed by elimination of contaminant adapter substrings with scythe and by quality-based trimming using sickle; ( ) the alignment of reads to the human genome (hg ) was performed using the tophat mapper ( ); ( ) transcript assembly and quantification using fpkm of genes and transcripts was carried out with cufflinks ( ); ( ) the annotation of the gene locus obtained was performed using cuffmerge with gencode v as reference; and ( ) differential expression analysis was performed using cuffdiff ( ) . genes were selected as differentially expressed using a p-value threshold of . . further analysis and graphical representations were performed using an r/bioconductor ( ) . reads from all the differentially expressed sequences were visualized in the integrative genomics viewer (igv) ( , ) and the sequences were compared to the ensembl and encode databases and searched for in the genome browser from ucsc for more information ( , ) . candidates were divided into coding, non-coding (according to ucsc classification), or non-assigned, when the transcription of the sequence had not been annotated in the databases. functional enrichment analysis of gene ontology (go) categories was carried out using a standard hypergeometric test ( ) . biological knowledge extraction was complemented through the use of ingenuity pathway analysis (ingenuity systems) , with a database that includes manually curated and fully traceable data derived from literature sources. open reading frame finder (ncbi) was used to evaluate the length of all probable open reading frames (orfs) in lncisg and lncbst /bispr. coding potential was assayed with the coding potential assessment tool (cpat) ( , ) and by searching the lncipedia database ( ) for the presence of our candidates in the pride archive ( ) or in lists of transcripts associated with ribosomes ( , ) . phylogenetic codon substitution frequencies (phylocsf) were also used to predict the coding potential of lncisg and lncbst /bispr ( ). statistical analysis of the rnaseq data has been already described. remaining analysis was performed using graph-path. statistical significance of infected versus non-infected samples was calculated using a two-tailed non-parametric mann-whitney t -test or with a two-tailed students t -test when the samples followed a normal distribution according to the shapiro-wilk test. welch's correction was applied for samples with heterogeneous variance. for correlation studies, a two-tailed non-parametric spearman analysis was used. p values lower than . were deemed as significant. to identify lncrnas that respond to ifn, we treated huh cells with units/ml of ifnα for days. these conditions serve to induce the expression of well-known isgs such as gbp , irf , bst , oas, or isg ( ) . in addition, this treatment induces an antiviral effect, as hcv-infected huh cells treated with units/ml of ifnα , show decreased levels of viral proteins and viral genomes compared to untreated infected cells (data not shown). finally, the rna isolated from huh cells treated with units/ml of ifnα for days was used to hybridize an agilent array. analysis of the array showed that well-characterized isgs such as mx , stat , irf , isg , bst , and several members of the gbp, oas, and ifi families were upregulated with a very high statistical significance (b > ) ( ) . ingenuity analysis of the data showed that ifn signaling was the pathway with the highest enrichment followed by other antiviral responses. the microarrays were used to identify lncrnas regulated by ifnα ( ) . however, an array will only evaluate the expression levels of the transcripts that hybridize to probes spotted in the array. in the case of the lncrnas, the array used only addresses the expression of regions described as long intergenic noncoding rnas (lincrnas). however, it has been estimated that there could be as many lncrna genes as coding genes, and some authors consider that the number of lncrnas could be as high as ( , ) . therefore, to achieve a more complete identification of lncrnas that respond to ifn, we analyzed the transcriptome by rnaseq. rna isolated from control cells or huh cells treated with ifn as described, was sequenced after ribodepletion. around million reads were obtained per sample. analysis was performed using a bioinformatic workflow that includes tophat and cufflinks as described in the methods section. the analysis showed that, among the genes upregulated in response to ifn, there were several isgs such as mx , isg , bst , or members of the ifi and oas families ( figure a and table s in supplementary material). ingenuity analysis showed that ifn signaling is a top canonical pathway (p = . × − ), the top upstream regulator is ifnα (p = . × − ), and cell signaling and infectious and inflammatory diseases are among the main functions. the expression of~ coding genes was altered by ifn (table s in supplementary material). the rnaseq analysis also showed that the expression levels of many regions that do not correspond to coding genes were also significantly modified in response to ifn ( figure b) . out of the putative non-coding genes whose expression was significantly altered, half were upregulated (table s in supplementary material). all candidates where visualized using igv ( figure s in supplementary material) ( , ) . we also paid special attention to altered sequences located close to well-known isgs and to genomic regions that were highly expressed and deregulated in response to ifn. eight candidates that fulfill at least one of these two criteria were chosen for further validation ( table and figure s in supplementary material). to validate the eight candidates chosen, we treated huh cells with different doses of ifnα . rna was isolated from the cells at , , , or h post-treatment and the expression levels of the candidates were evaluated by quantitative rt-pcr (qrt-pcr) (table ; figure ). all the candidates were induced after ifn-treatment from to more than -fold. however, many of the candidates were detected at very low cycles in the pcr amplification. a closer examination of their sequences indicated that they contained repetitive sequences or sequences similar to mitochondrial or ribosomal rnas that could have led to an erroneous alignment of the rnaseq reads to the human genome. we believe that, even when the oligonucleotides used for amplification were specific, a partial homology to other sequences could allow cross-amplification and thus increased possibilities of misleading results. these candidates were not studied further. we focused on two lncrnas with no repetitive sequences whose expression was highly upregulated in response to ifn (table ; figure ). interestingly, database analysis showed that they are expressed from positions in the genome located close to isg and bst , both of which are well-characterized isgs. this may have functional relevance as some lncrnas have been described to regulate the expression of neighboring genes. therefore, we originally named these lncrnas after their neighbor, lncisg and lncbst . later, lncbst was renamed bispr to follow hugo regulations. when we evaluated the expression of these lncrnas and their neighboring transcripts, we observed that both were strongly upregulated at early times in response to ifn (figure a) . furthermore, they responded to ifnα doses as low as units/ml. these are similar levels to those found in the sera of some hcv patients ( ) . the induction was also observed at late times post-ifn-treatment. to evaluate further the robustness of the effect www.frontiersin.org of ifn on these lncrnas, we tested whether they also respond to ifnλ, a type iii ifn. huh cells treated with ifnλ for , , , , or h also showed increased levels of lncisg , lncbst /bispr, and their neighbors ( figure b) . in this case, all the transcripts showed a higher upregulation at later times post-ifnλ treatment. viruses activate the ifn response by several mechanisms. therefore, they have evolved to block ifn production and the activation of the ifn pathway. the molecular mechanisms involved in this ifn blockade have been characterized for many viruses. thus, for instance, ns protein from influenza virus and matrix protein from vsv are key factors in controlling ifn in infected cells ( ) ( ) ( ) ) . we sought to check whether lncisg and lncbst /bispr were induced by the physiological ifn induced by an influenza virus that lacks ns . therefore, we evaluated the expression of these lncrnas in cells infected with an influenza wild-type virus or a ns mutant. we also included cells infected with other rna viruses such as sfv and hcv or dna viruses such as adenovirus. all these viruses have developed mechanisms to block the cellular antiviral response and, with the exception of hcv, lead to a lytic infection. different times post-infection were evaluated. the last point was collected when the cytopathic effect was apparent. this occurred at h post-infection in the case of influenza and sfv or h post-infection, in the case of adenovirus. hcv-infected cells were collected at and h postinfection. the results showed that at later times post-infection with the influenza virus lacking ns , there was increased expression of lncisg , lncbst /bispr, and their neighboring coding transcripts (figure a) . this increase was not observed in cells infected with wild-type influenza virus, or with other wildtype lytic viruses, suggesting that the induction may be mediated by ifn. most lncrnas are tissue-specific. to determine whether lncisg and lncbst /bispr respond to infection only in huh cells or whether this effect is specific for influenza viruses, we infected alveolar epithelial a cells with vsv-gfp wild-type virus or with a m r matrix mutant that fails to control ifn. we chose a , because lung cells serve as the primary site for productive infection of vsv and many respiratory viruses ( ) . infection with the wild-type virus did not increase the expression of lncbst /bispr or bst (data not shown). however, a cells infected with the vsv mutant m r for , , or h did show increased levels of lncisg , lncbst /bispr, isg , bst , and other isgs such as gbp ( figure b) . surprisingly, infection with hcv also increased the expression of lncisg , lncbst /bispr, and other isgs, including isg , bst , and irf ( figure a and data not shown). to determine whether these genes were also upregulated in infected patients, we used liver samples from hcv-negative and hcv-positive donors. after quantification of the rna levels, we observed a significant increase in lncisg , lncbst /bispr, isg , and bst in hcvinfected patients compared to controls (figure a ). with the number of patients evaluated, a significant correlation was not found between expression levels and infection with a particular genotype of hcv, presence of hcv-induced hepatocellular carcinoma (hcc), liver cirrhosis, or with a particular cirrhosis stage. therefore, there were no significantly different levels of these transcripts in hcv-infected livers without hcc compared with the peritumoral tissue of hcv-infected livers with hcc. although most of the samples belong to patients that are still alive, no significant correlation was observed between the levels of the evaluated transcripts and survival post-diagnosis. finally, we performed correlation studies to analyze whether in the patients, the expression level of lncisg or lncbst /bispr correlates significantly with the expression level of their neighboring coding genes. the results show a highly significant positive correlation between lncisg and isg or lncbst /bispr and bst (figure b ). the experiments performed so far suggest that a general correlation could exist between the expression of lncisg and isg or lncbst /bispr and bst . each lncrna and its neighboring coding gene have similar induction patterns in response to ifn or to viral infection (compare their levels in figures and ) . furthermore, the levels of each coding/non-coding pair correlate significantly in patient samples (figure b) . to analyze this in more detail, we performed correlation studies of the coding/noncoding pairs in all the samples evaluated in figures and . the results show that the correlation of each pair was highly significant ( figure s in supplementary material). this suggests that they could be co-regulated, and therefore, they could share similar functions. however, expression of lncisg and lncbst /bispr also correlated significantly with the expression of other isgs such as oas, gbp , or irf (data not shown). to obtain more information on the relationship between the coding/non-coding pairs, we searched several databases. lncisg and lncbst /bispr genes are in head-to-head orientation with their coding neighbors ( figure s in supplementary material) and they could share the same promoter. this is based on the following facts: (i) the distance between the two genes is < bp, a cut-off for bidirectional promoters ( , ) ; (ii) there is a single dnase hypersensitivity region located between the genes, and (iii) polymerase ii (pol ii) chipseq analysis of k cells shows a single peak covering the h k ac region between both genes. interestingly, the peaks observed for pol ii chipseq are increased at min or h post-treatment with ifnα or ifnγ. finally, the promoter regions contain conserved isre sites and binding sequences for irf , irf , and irf . to discriminate whether lncisg and lncbst /bispr are induced directly by the jak/stat signaling pathway or by a secondary wave of the ifn response, we evaluated the expression of these lncrnas and their coding neighboring genes in huh or a cells incubated or not with the jak/stat inhibitor ruxolitinib. expression of gbp , a bona fide isg, was also evaluated as a positive control (figure ) . the results show that the levels of gbp , bst , and lncbst /bispr are significantly reduced liver samples from hcv-negative and hcv-positive donors were used to quantify the levels of lncisg , lncbst /bispr, isg , bst , and gapdh mrnas. statistical significance was calculated using a two-tailed non-parametric mann-whitney t -test for lncbst /bispr, isg , and lncisg and with a two-tailed students t -test with welch's correction for bst , which follows a normal distribution according to the shapiro-wilk test. (b) expression levels observed for lncisg , lncbst /bispr in patient samples were compared to the expression levels of their coding neighbors isg and bst , respectively. a correlation analysis was performed and statistical significance was calculated using a two-tailed non-parametric spearman analysis. www.frontiersin.org in the presence of ruxolitinib, indicating that their expression is stat-dependent. levels of bst and lncbst /bispr were also reduced in cells treated with sirnas targeting stat or by inhibition of irf , a transcription factor that acts downstream of ifn (data not shown). this indicates that bst and lncbst /bispr respond to stats but also to other transcription factors induced by ifn. these results agree with the possibility that bst and lncbst /bispr share a bidirectional promoter. in contrast, the effect of the jak/stat pathway on isg and lncisg expression was less robust. treatment with ruxolitinib decreased the expression of isg and lncisg , but in the latter, this effect was only observed in a cells. no effect on isg or lncisg expression was observed with a milder inhibition of stat or inhibition of irf using rna interference (data not shown). thus, although isg is induced very rapidly after ifntreatment, we do not observe a strong regulation of isg or lncisg by the stat pathway under the conditions used. in fact, it has been reported that a major regulator of isg is irf , a transcription factor activated in response to pamps, but also a downstream effector of the ifn response ( ) . we evaluated the coding capacity of lncisg and lncbst /bispr bioinformatically. orf finder (ncbi) was used to determine all possible orfs in these lncrnas ( figure s in supplementary material). the analysis shows that all putative orfs are shorter than amino acids. only two orfs could be translated according to their poor susceptibility to nonsense mediated decay. however, these orfs have non-consensus kozak sequences at the initiation codon and therefore a poor coding capacity. then, we evaluated the coding potential of lncisg and lncbst /bispr with the cpat ( , ) ( figure a) . cpat uses a model built with orf size and coverage together with codon (ficket score) and hexamer (hexamer score) usage bias. according to this program, lncisg and lncbst /bispr are non-coding as they have a coding probability of . and . , respectively, much lower than . , used as a threshold with the highest sensitivity and specificity to differentiate between coding and non-coding transcripts in humans figure | lncisg , lncbst /bispr have poor coding potential and accumulate preferentially in the nucleus. (a) bioinformatic analysis of the coding potential of lncisg and lncbst /bispr. results obtained from cpat and lncipedia. two transcripts have been evaluated for lncbst /bispr. lncisg and lncbst /bispr have a coding probability and a coding label of "non-coding rnas" according to these analyses. "lists" indicated the number of times that these transcripts have been found in the pride archive or in lists containing ribosome-associated rnas published by lee or bazzini. see the text for other details. (b) subcellular localization of lncisg and lncbst /bispr. huh cells were mock-treated or treated with units/ml of ifnα and divided into nuclear and cytoplasmic fractions. rna was isolated from each fraction and used to evaluate the expression levels of lncisg and lncbst /bispr by qrt-pcr. malat , gapdh, and isg mrna was also quantified and used as a reference to calculate the relative levels of each transcript and as a control to evaluate the subcellular fractionation. the ratio of cytoplasmic to nuclear levels is shown. the experiment was performed three times and each value shows the average of three replicas from a representative experiment. error bars indicate standard deviations. ( ) . lncisg and lncbst /bispr were also described as noncoding in lncipedia ( ) . this lncrna database shows that these lncrnas are not found in the pride archive, a database for proteomic data, or in lists of transcripts associated with ribosomes in ribosome profiling experiments ( ) ( ) ( ) . further, lncisg and lncbst /bispr were also described as non-coding by the analysis of phylocsf, which uses multiple alignments to calculate the frontiers in immunology | molecular innate immunity phylogenetic conservation score and determines whether a multispecies nucleotide sequence alignment is likely to represent a protein-coding region ( ) . finally, we evaluated the subcellular localization of lncisg and lncbst /bispr in huh cells mock-treated or treated with units/ml of ifnα. rna was isolated from nuclear or cytoplasmic fractions and quantified by qrt-pcr. we found that the coding gapdh or isg mrnas accumulate preferentially in the cytoplasm while the nuclear rnas malat or u are preferentially nuclear (figure b data not shown) . similarly, lncisg and lncbst /bispr, compared to mrnas, accumulate preferentially in the nucleus. this result, together with the bioinformatic analyses, strongly suggests that lncisg and lncbst /bispr are non-coding rnas. to address the role of lncbst /bispr, we used rna interference. huh cells treated or not with ifn, were transfected with sir-nas targeting lncbst /bispr and rna expression was evaluated by qrt-pcr. the results show that expression of lncbst /bispr was decreased compared to cells transfected with control sirnas ( figure a) . surprisingly, inhibition of lncbst /bispr also led to decreased levels of bst mrna. expression of lncisg , isg , gbp or expression of genes located in the genome close to bst or lncbst /bispr, such as gtpbp or mvb a, was not affected (figure a and data not shown). to determine whether this was a general phenomenon, we transfected the sirnas targeting lncbst /bispr into a cells infected or not with the vsv m r mutant or treated with ifn. similarly to what has been observed in huh cells, the sirna that targets lncbst /bispr leads to decreased levels of lncbst /bispr and bst mrna while the levels of isg mrna are not significantly affected (figure b) . similar results were observed with a different sirna targeting lncbst /bispr. rna sequencing analysis of human cells treated with ifnα and controls has allowed the identification of lncrnas induced in response to ifn (figure ) . analysis of the rnaseq data shows that several of the upregulated genes are well-known coding isgs such as isg or oas ( figure a , table s in supplementary material). ingenuity analysis confirms the enrichment of genes involved in the ifn response among the regulated factors. we have used rnas from similar ifn-treated and control cells to hybridize expression arrays ( ) . comparison of the datasets obtained in the analysis of the array and the rnaseq shows that only coding genes were identified in both studies, including oas, isg , mx , and some members of the ifi family. generally, overlap between microarray and rnaseq analysis is not high ( ) . furthermore, the overlapping decreases with sequencing depth and when low fold-changes or low abundance genes are analyzed. ( ) . this is because sequencing of low transcript abundances is characterized by high variance, which impedes their identification in rnaseq analysis. we believe that this may explain the poor correlation found between the array and the rnaseq datasets. in fact, we have determined by qrt-pcr that some ifnrelated low abundance transcripts are detected only in the array analysis. these are early responders to ifn, which increased only marginally days after ifn-treatment, when the analysis was performed. therefore, we believe that some lncrnas induced early post-ifn-treatment may have not been identified in our analysis. interestingly, in the process of writing this manuscript, a paper www.frontiersin.org was accepted describing the identification of ifn-induced lncr-nas by rnaseq in samples treated with ifn for short times ( ) . we believe that this study will be complementary to our work. together, the datasets should contain lncrnas regulated at early and later times post-ifn-treatment. similarly, the lack of correlation between the microarray and rnaseq datasets also indicates that they can complement each other. we have identified two lncrnas whose expression is highly upregulated in response to different doses of ifnα (table ; figure a ) or ifnλ (figure b) . our results show that induction of these lncrnas by ifnα seems faster than that observed for ifnλ. we cannot rule out the possibility that a fast response to ifnλ may also be observed when higher doses are used. encode analysis of polymerase ii binding to the promoters of these lncr-nas also shows that they may be induced by treatment with ifnα and ifnγ ( figure s in supplementary material). these lncrnas have been named lncisg and lncbst /bispr after their neighboring genes, which play a key role in the antiviral ifn response. our molecular and bioinformatic analyses strongly suggest that lncisg and lncbst /bispr are indeed lncrnas, as they accumulate preferentially in the nucleus of ifn-treated or untreated cells ( figure b ) and they have poor coding potential ( figure a and figure s in supplementary material). in general, the upregulation of lncisg and lncbst /bispr mimics that of their coding counterparts (figures - ) . in fact, analysis performed with all the expression data obtained in our studies, shows a highly significant correlation between lncisg and isg and between lncbst /bispr and bst . significant correlations also exist between these lncrnas and other ifn-induced genes such as oas, gbp , or irf ( figure s in supplementary material and data not shown). this may reflect the fact that all these genes are induced by ifn with a similar kinetics. in the case of the lncrnas and their coding counterparts, correlation of the expression may result from their transcriptional co-regulation. experimental and bioinformatic analyses indicate that bst and lncbst /bispr are bona fide isgs strongly induced by the jak/stat pathway in response to ifn (figure and figure s in supplementary material). furthermore, expression of bst , isg , and their neighboring non-coding genes is induced by downstream effectors of the ifn response. these studies allow us to suggest that lncisg /isg and lncbst /bispr/bst may share bidirectional promoters. other ifn-induced gene pairs may also be co-regulated by bidirectional promoters as these are enriched in stat binding ( ) . bidirectional promoters often couple genes involved in the same process, allowing for coordinated temporal and environmental responses ( , ( ) ( ) ( ) ( ) ( ) . non-coding rnas generated from bidirectional promoters may have functional roles that affect the bidirectional promoter, the neighboring protein-coding gene, or more distal genes ( ) . these effects could lead to activation or repression of the expression and could be mediated by either the transcription process itself or by the produced ncrna transcript ( ) . in this study, we show that post-transcriptional inhibition of lncbst /bispr leads to reduced levels of bst mrna (figure ) . therefore, lncbst /bispr should increase transcription or stability of its coding neighboring gene. our results demonstrate that this regulation seems specific for bst , as lncbst /bispr downregulation does not affect the expression of genes located nearby, which has been described for compact genomes ( ) . moreover, inhibition of lncbst /bispr does not affect expression levels of other ifn-related genes such as isg or gbp (figure and data not shown). we anticipate that inhibition of lncbst /bispr, and therefore of bst , could impact the antiviral effects of ifn. bst is also named tetherin, as it inhibits viral budding by using anchors that trap virions on the cell membrane ( ) ( ) ( ) . several enveloped viruses have been shown to be susceptible to the action of bst /tetherin and have evolved to develop evasion strategies ( ) . interestingly, hiv, influenza, hcv, and vsv are among the susceptible viruses and could be used to test the antiviral role of lncbst /bispr ( , ( ) ( ) ( ) ( ) ( ) ( ) ( ) . in fact, we show that lncbst /bispr and bst are induced after infection with hcv or influenza and vsv mutant viruses that activate the ifn response (figure ) . upregulation of lncbst /bispr, lncisg , and their coding neighbors was also observed in patients infected with hcv compared to controls (figure ) . similarly, a significant upregulation of lncbst /bispr and isg was also detected in human tlymphocytes infected with hiv compared to controls (data not shown). a non-significant increase in bst and lncisg was also observed in these samples. this leads to the possibility that interference with these factors could have therapeutic relevance. it is unclear why cells infected by these viruses, which employ several viral proteins to block the ifn pathway, show activation of these ifn response genes ( ) . in the case of hcv, it has been previously shown that patients with chronic hcv infections express isgs, including high levels of isg ( ) ( ) ( ) . in fact, hcv has evolved to use some isgs for viral replication ( , ) . this is the case for isg . isg is an ubiquitin-like protein that attaches to its targets in a process called isgylation ( , ) . protein isgylation may result in increased or decreased functionality depending on the target ( ) . isg preferentially conjugates newly synthesized proteins affecting more strongly viral proteins or cellular proteins translated into ifn-induced cells ( ) . viruses such as influenza, hiv, or vsv are susceptible to the action of isg ( , ) . in the case of hcv, a pro-hcv role for isg has been reported ( , ) . isg has been shown to negatively regulate rig-i and thus to inhibit the signaling process leading to ifn induction that affects hcv replication ( ) . furthermore, isg expression in the liver of chronically infected patients is considered a negative predictive biomarker of the ability of the patients to respond to ifn therapy ( ) ( ) ( ) (figure ) . in our study, we cannot address whether lncisg , bst , or lncbst /bispr are markers for the susceptibility of hcv patients to respond to ifn-treatment, as the hcv patients that we have studied are non-responders to ifn. we believe that, similar to lncbst /bispr, lncisg could affect the expression of isg or other genes. this lncrnamediated control has also been described for a lncrna located close to the isg viperin, which has been shown to regulate the levels of many ifn-inducible genes ( , ) . further experiments will be required to address the role of lncisg and to decipher the molecular mechanisms that allow the control exerted by lncbst /bispr on bst . we believe that these studies may be important to better understand the ifn response and its pro or antiviral functions on hcv and other viruses. frontiers in immunology | molecular innate immunity regulation of type i interferon responses dynamic expression profiling of type i and type iii interferon-stimulated hepatocytes reveals a stable 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biological relevance bidirectional gene organization: a common architectural feature of the human genome expression of the murine ranbp and htf -c genes is regulated from a shared bidirectional promoter during cell cycle progression divergent transcription is associated with promoters of transcriptional regulators functional consequences of bidirectional promoters induced ncr-nas allosterically modify rna-binding proteins in cis to inhibit transcription antisense expression increases gene expression variability and locus interdependency tetherin inhibits hiv- release by directly tethering virions to cells bst- /tetherin: structural biology, viral antagonism, and immunobiology of a potent host antiviral factor intrinsic cellular defenses against human immunodeficiency viruses bst- expression in human hepatocytes is inducible by all three types of interferons and restricts production of hepatitis c virus mechanism of hiv- virion entrapment by tetherin bst /tetherin inhibits hepatitis c virus production in human hepatoma cells vpu binds directly to tetherin and displaces it from nascent virions influenza virus partially counteracts restriction imposed by tetherin/bst- tetherin inhibits retrovirus release and is antagonized by hiv- vpu interferoninduced cell membrane proteins, ifitm and tetherin, inhibit vesicular stomatitis virus infection via distinct mechanisms regulation of hepatic innate immunity by hepatitis c virus interferon type i gene expression in chronic hepatitis c interferon signaling and treatment outcome in chronic hepatitis c intrahepatic gene expression during chronic hepatitis c virus infection in chimpanzees hepatitis c virus controls interferon production through pkr activation hepatitis c virus blocks interferon effector function by inducing protein kinase r phosphorylation interferon-induced isg pathway: an ongoing virus-host battle the antiviral activities of isg positive regulation of interferon regulatory factor activation by herc via isg modification the interferon stimulated gene functions as a proviral factor for the hepatitis c virus and as a regulator of the ifn response ifn-stimulated gene functions as a critical antiviral molecule against influenza, herpes, and sindbis viruses isg , a ubiquitin-like interferon-stimulated gene, promotes hepatitis c virus production in vitro: implications for chronic infection and response to treatment negative feedback regulation of rig-imediated antiviral signaling by interferon-induced isg conjugation the antiviral protein viperin inhibits hepatitis c virus replication via interaction with nonstructural protein a regulation of interferon-stimulated gene bst by a lncrna transcribed from a shared bidirectional promoter barriocanal contributed to the writing of the manuscript; victor segura was in charge of all the bioinformatic analyses; and puri fortes conceived the project and the required experiments, provided the budget, interpreted the data, and wrote the manuscript. we thank nerea razquin and celia prior for excellent technical assistance, paul miller for editorial work, estanis nistal, cristian smerdou, and rafael aldabe for influenza and vsv viruses, sfv, and hcv, respectively. we also thank pablo gastaminza for the huh cells sensitive to hcv infection used in all the experiments, esther larrea for ifnα and primers for isgs, elizabeth guruceaga for the initial studies on rnaseq data and ruben hernandez for helpful comments on the manuscript. we would like to thank patients for the generous donation of samples and virginia villar and the biobank of the university of navarra for their mediation. this work was supported by grants from ministerio de ciencia e innovacion bio / , and saf - , feder funding, funds from the "ute project cima" and by the project rnareg [csd - ], funded by the ministry of science and innovation under the program consolider ingenio . a manuscript from dr. valadkhan's laboratory has been accepted for publication that describes similar results ( ) . the supplementary material for this article can be found online at http://www.frontiersin.org/journal/ . /fimmu. . / abstract the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. the review editor saba valadkhan declares that, despite having collaborated on a publication in the last years with authors puri fortes, marina barriocanal, and elena carnero, the review process was handled objectively. key: cord- -v wnmg authors: flanagan, katie l.; best, emma; crawford, nigel w.; giles, michelle; koirala, archana; macartney, kristine; russell, fiona; teh, benjamin w.; wen, sophie ch title: progress and pitfalls in the quest for effective sars-cov- (covid- ) vaccines date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: v wnmg there are currently around sars-cov- candidate vaccines in preclinical and clinical trials throughout the world. the various candidates employ a range of vaccine strategies including some novel approaches. currently, the goal is to prove that they are safe and immunogenic in humans (phase / studies) with several now advancing into phase and trials to demonstrate efficacy and gather comprehensive data on safety. it is highly likely that many vaccines will be shown to stimulate antibody and t cell responses in healthy individuals and have an acceptable safety profile, but the key will be to confirm that they protect against covid- . there is much hope that sars-cov- vaccines will be rolled out to the entire world to contain the pandemic and avert its most damaging impacts. however, in all likelihood this will initially require a targeted approach toward key vulnerable groups. collaborative efforts are underway to ensure manufacturing can occur at the unprecedented scale and speed required to immunize billions of people. ensuring deployment also occurs equitably across the globe will be critical. careful evaluation and ongoing surveillance for safety will be required to address theoretical concerns regarding immune enhancement seen in previous contexts. herein, we review the current knowledge about the immune response to this novel virus as it pertains to the design of effective and safe sars-cov- vaccines and the range of novel and established approaches to vaccine development being taken. we provide details of some of the frontrunner vaccines and discuss potential issues including adverse effects, scale-up and delivery. there are currently around sars-cov- candidate vaccines in preclinical and clinical trials throughout the world. the various candidates employ a range of vaccine strategies including some novel approaches. currently, the goal is to prove that they are safe and immunogenic in humans (phase / studies) with several now advancing into phase and trials to demonstrate efficacy and gather comprehensive data on safety. it is highly likely that many vaccines will be shown to stimulate antibody and t cell responses in healthy individuals and have an acceptable safety profile, but the key will be to confirm that they protect against covid- . there is much hope that sars-cov- vaccines will be rolled out to the entire world to contain the pandemic and avert its most damaging impacts. however, in all likelihood this will initially require a targeted approach toward key vulnerable groups. collaborative efforts are underway to ensure manufacturing can occur at the unprecedented scale and speed required to immunize billions of people. ensuring deployment also occurs equitably across the globe will be critical. careful evaluation and ongoing surveillance for safety will be required to address theoretical concerns regarding immune enhancement seen in previous contexts. herein, we review the current knowledge about the immune response to this novel virus as it pertains to the design of effective and safe sars-cov- vaccines and the range of novel and established approaches to vaccine development being taken. we provide details of some of the frontrunner vaccines and discuss potential issues including adverse effects, scale-up and delivery. the recent emergence of severe acute respiratory syndrome coronavirus- (sars-cov- ), the cause of coronavirus disease , is wreaking havoc due to widespread dissemination throughout the world. on march , who formally declared that a global pandemic and by the end of august , almost million cases and over , deaths had been reported worldwide involving all continents, except antarctica ( ) . strategies to identify cases and limit spread by widespread testing and physical distancing have been challenging to implement, healthcare and public health systems have been overwhelmed, resulting in continued escalation in many countries and profound effects on lives and livelihoods. while the majority of people are either asymptomatic or experience a mild respiratory infection, ∼ % of cases are more severe and require hospital admission ( ) . reported mortality rates vary by geographic region, ranging from almost % in france to < % in many other countries ( ) . this wide discrepancy suggests selection bias due to differences in local testing strategies and capacity, consistent with differences in health system capacity, population demographics and other health determinants. certain groups such as the elderly and those with particular comorbidities are more likely to die of covid- ( ) . healthcare workers in particular have experienced significant morbidity and mortality from covid- ( ), causing clear psychological impacts and threatening delivery of healthcare services ( ) . there is no known effective treatment for this virus and currently no available vaccine, with the result being that sars-cov- continues to spread throughout the virus naïve population of the world. the urgent need for effective sars-cov- vaccines cannot be overstated. immunization can not only protect individuals but also, if provided to enough people in a timely way with (even) partially protective vaccines, induce sufficient herd immunity to curtail the spread of this virus and reduce morbidity and mortality across the globe. the race to develop safe and effective vaccines has seen sars-cov- candidate vaccines developed at a scale and pace never imagined before: currently almost potential vaccines are in various stages of development ( ) ( ) ( ) . a range of vaccine design approaches and platforms have been employed. however, since > % of candidate vaccines typically fail it is expected that the eventual number of successful vaccines may be only be a handful. they may also become available in different time frames and suitable for use in different populations. most vaccine candidates are currently in preclinical trials, but a number have entered phase or phase / studies, with plans to rapidly scale up to phase and . trials are being conducted at "pandemic speed" ( ) and using novel designs. this early success has already seen cooperation and collaboration as well as significant funding across the globe. for example, the coalition for epidemic preparedness innovations (cepi), a notfor-profit global coalition launched in to deal with the worldwide threat of epidemic outbreaks, is playing a pivotal role in supporting many of the frontrunner vaccines ( ) . herein, we review what is currently known about the immune response to sars-cov- and the various vaccine platforms being used to develop the sars-cov- vaccines. understanding the mechanism of action of the various candidate vaccines is the key focus of this review. we also discuss potential challenges at the immunological level, assessment of vaccine safety and scale-up and delivery. coronaviruses are enveloped positive-sense single stranded rna viruses belonging to the coronaviridae family. they infect birds and mammals causing a range of symptoms from respiratory to gastrointestinal disease ( ) . a number of relatively common seasonal coronaviruses are known to infect humans ( ) , causing mild respiratory illness ("the common cold"). two previous lethal human coronavirus diseases, namely severe acute respiratory syndrome (sars caused by sars-cov- ) ( ) and middle east respiratory syndrome (mers caused by mers-cov) ( ) arose in and , respectively. they have a high mortality rate of ∼ and %, respectively, but fortunately, neither reached pandemic levels. sars-cov- was able to be contained by public health measures to prevent human-to human transmission and then disappeared before vaccine development had progressed significantly. the genome of sars-cov- encodes for the structural proteins spike (s), envelope (e), membrane (m) and nucleocapsid (n) as well as a number of accessory and non-structural proteins (figure ) ( ) . the m and n proteins give the virion its shape, and the s proteins appear as spikes on the viral surface giving it a solar corona appearance. the s glycoprotein (spike) exists in trimeric form and is the structure by which binding to the host cells occurs. the virus receptor, angiotensin-converting enzyme (ace ), expressed on the figure | structure of sars-cov- and key antigenic components. illustration of sars-cov- which is a single stranded rna virus. the key antigenic components being targeted in vaccine design are shown on the right, consisting of the spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins. the main emphasis for human vaccines is based on the spike (s) protein, consisting of an s binding region and s fusion and cell entry region. the s domain contains the receptor binding domain (rbd) responsible for binding to the ace receptor on the surface of host cells. following fusion, the s protein sheds the s region and undergoes a dramatic structural change to its post-fusional state in order for the virus to enter the host cells. surface of multiple human cells is engaged via the receptor binding domain (rbd), which is part of the s subunit of the s glycoprotein ( ) (figure ) . the s subunit consists of two heptad repeat fusion regions, hr and hr , responsible for membrane fusion and cell entry. the rbd domain can either be down and buried or rotated up and primed for ace binding ( ) . the hidden down state is the predominant state of the s protein trimer in sars-cov- , and likely a mechanism the virus uses to hide the entry epitopes and evade the host immune response ( ) . two s trimers can concurrently bind to an ace dimer. following s protein binding in its prefusion state, the s subunit is cleaved and shed permitting the s dramatic conformational changes which constitute the post-fusion state required for viral entry ( , ) . innate immunity to covid- : protection or hyper-activation? sars-cov- stimulates an innate immune response via pattern associated molecular patterns (pamps) expressed by the virus, which in the case of sars-cov- consists of viral rna and its intermediates produced during replication ( ) . these conserved pamps stimulate multiple immune response pathways via pattern recognition receptors (prrs), including sensing by endosomal toll-like receptors (tlrs) and , cytosolic retinoic acid-inducible gene (rig- ) and melanoma differentiationassociated protein (mda ). local tissue damage in the lungs also releases damage-associated molecular patterns (damps) further contributing to local inflammation. the resultant inflammatory response provides immediate antiviral immunity via activation of antiviral type and interferon (ifn) pathways leading to an upregulation of inflammatory cytokines such as interleukin (il- ) and il- β, further recruiting neutrophils, other innate immune cells and stimulating anti-sars-cov- adaptive memory t cells and b cells ( ) (figure ) . there is still much to understand about the immune response to sars-cov- and the immunological differences in those with mild as compared to severe infection. emerging data suggest that the innate response to sars-cov- is aberrant ( , ) . for example, the early type i and iii interferon responses are relatively suppressed by sars-cov- , an immune evasion strategy employed by the virus, leading to early failure to control the virus. furthermore, uncontrolled local inflammation, or what has been described as "cytokine storm, " leading to tissue damage with inflammatory cell infiltration and the acute respiratory distress syndrome (ards) is thought to characterize late stage, severe manifestations of covid- ( ) ( ) ( ) . for example, patients with severe covid- had higher levels of il- , il- r, il- , and tumor necrosis factor alpha (tnf-α) than those with moderate disease, the former of which correlated with clinical severity and death ( , ) . those covid- patients requiring icu admission demonstrated greater plasma levels of il- , il- , il- , granulocyte colony-stimulating factor (gcsf), interferoninducible protein (ip- ), monocyte chemoattractant protein- (mcp- ), macrophage inflammatory protein- α (mip- α) and tnf-α than the non-icu covid- patients, all supporting enhanced innate immunity in the sicker patients ( ) . of note, the il- levels described in covid- patients are a log-fold lower than those described in classic cytokine storm ( ) . furthermore, il- is an immunosuppressive cytokine suggesting dysregulated immune homeostasis rather than pure inflammation. upregulated chemoattractant chemokines further recruit inflammatory cells including neutrophils, macrophages, natural killer (nk) cells and t cells resulting in further immunopathology ( ) (figure ). antibodies (abs) produced by activated b cells play a key role in anti-viral immunity via several mechanisms including viral neutralization, antibody-dependent cellular cytotoxicity (adcc), antibody-dependent cellular phagocytosis (adcp), and antibody-dependent complement activation (adca) (figure ) . it is thought that generation of high levels of neutralizing antibodies (nabs) against sars-cov- are required for a successful human vaccine ( ) . however, some patients recover without producing high levels of nabs and those with severe disease may experience an early rise in nabs ( ) . nevertheless, most vaccine efforts are focused on the induction of nabs to the s protein in order to block attachment of rbd to the ace receptor on host cells ( ) . as mentioned before, this domain is hidden in the s protein's prefusion state, presenting challenges to the success of rbd-based vaccines ( ) . for this figure | key components of the innate immune response to sars-cov- . antigen presenting cells (apcs), such as monocytes, macrophages, and dendritic cells (dcs), recognize pattern associated molecular patterns (pamps) expressed by sars-cov- via their pattern recognition receptors (prrs), such as toll-like receptor (tlr) and . this activates intracellular signaling pathways leading to the expression of type and interferons (ifns), which in turn activate innate immune cells to produce pro-inflammatory cytokines and chemokines. this leads to an influx and activation of neutrophils, further apcs and other innate immune cells, such as natural killer (nk) cells. reason, some groups have focused on eliciting nabs to the less immunogenic s subunit of the spike protein ( ) and sars-cov- vaccines based on other antigens, including the n protein, are also being developed. a recent report from the us describes outcomes among , covid- patients, many of whom had critical illness, transfused with the plasma from people who have recovered from covid- (convalescent plasma [cp]) ( ) . early cp infusion within days of illness had a lower -day mortality than those treated after or more days ( . % vs. . %). in this uncontrolled study, those who received higher levels of igg abs in the transfused plasma had a better mortality outcome. these data, alongside results from another uncontrolled study showing recovery in severely unwell covid- patients treated with cp, lends support to the notion that naturally acquired abs can be protective ( ) . however, other plasma factors such as cytokines, defensins and other non-specific abs may also play a protective role in these studies ( ) . sars-cov- -specific nabs recovered from infected humans also protected syrian hamsters and rhesus macaques in challenge studies ( , ) . ideally, a sars-cov- vaccine would induce long-lasting abs, but it is not yet known how long specific abs persist in sars-cov- infected individuals or how long they would persist after vaccination. indeed, several recent studies indicate rapid waning of sars-cov- nabs in some individuals following natural infection, although others maintained high levels to - days ( , ) , raising concerns about the persistence of nabs post-immunization; whether nabs plateau or continue to decline over time is yet to be determined. by contrast, nabs to sars and mers have been detected up to - years after infection in human survivors ( ) and a recent study reports nabs years after sars infection, suggesting that long-lasting coronavirus-specific nabs can be induced in some instances ( ) so hopefully ab mediated immunity to sars-cov- will be equally long-lasting. importantly, class-switched igg memory b cells to s and rbd have been demonstrated in covid- patients confirming the generation of b cell memory which can provide a rapid recall response on subsequent sars-cov- exposure ( ) . furthermore, we do not yet know what level of nabs are required for protection ( ) . the standardization of a range of assays to support vaccine studies, such as viral neutralization assays, to enable comparison of different vaccine candidates in different populations will be key to facilitating vaccine development, an issue which represents a current focus of the who ( ). t cell adaptive immunity: protection, immune suppression or disease enhancement? processing and presentation of sars-cov- epitopes by antigen presenting cells (apcs) via human leukocyte antigen (hla) class i leads to activation of naïve cd + t cells which differentiate into cytolytic effectors (cytotoxic t lymphocytes [ctl]) that kill virus-infected cells. activation of t helper (th ) cd + t cells via viral peptides presented on hla class ii alleles further enhances the cd + t cell response, while hla class figure | key components of the adaptive immune response to sars-cov- . the adaptive immune response is activated following viral uptake and antigen processing by a range of apcs. the apcs present viral antigen to b cells which then differentiate into antibody producing plasma cells. the neutralizing antibodies (nabs) then bind to key viral proteins, such as the spike protein, and neutralize their activity. other ab-mediated antiviral functions include antibody dependent cellular cytotoxicity (adcc), antibody dependent cellular phagocytosis (adcp), and antibody dependent complement activation (adca). cytotoxic cd + t cells kill virally infected cells via the production of granzymes and perforin and the expression of fas ligand (fasl), all of which mediate cellular apoptosis. a series of cd + t cell populations are involved in the adaptive cellular response to sars-cov- . follicular helper t cells (t fh ) and th cd + t cells both provide help for b cell antibody production. th and th cd + t cells are also thought to play a role in the inflammatory response and viral killing. cd + regulatory t cells have been implicated with an immunoregulatory role in sars-cov- infection via the production of anti-inflammatory cytokines and contact-mediated cellular suppression. whether cd + tregs and bregs play a role is not currently known. ii-restricted follicular helper (t fh ) and th cells enhance virusspecific antibody production (figure ) . effective viral clearance therefore requires a combination of cd + ctl and cd + t cell mediated enhancement of b cell and cd + t cell responses ( ) . pro-inflammatory th also play a role as evidenced by their high frequency in lung biopsy specimens of covid- patients ( ) (figure ) . severe viral disease, associated with an over exuberant t cell response, leads to local inflammation and toxicity, thus activation of immune modulatory factors or checkpoints is crucial in limiting the damage. indeed, regulatory t cells (tregs) play a key role in the control of inflammation and immunopathology in other coronavirus infections ( ) , but this has yet to be investigated for sars-cov- (figure ) . most activated t cells subsequently apoptose and die, but some persist and provide long-term virus-specific t cell immunity. natural infection with sars-cov- induced long-lasting memory t cells up to years after infection ( ) and memory t cells persisted to years after mers infection ( ) , supporting the potential for long-term persistence in sars-cov- infection. these should provide protective immunity on subsequent exposure to the pathogen, the ultimate goal of an effective vaccine. severe covid- patients exhibit immunological features associated with t cell anergy or exhaustion. for example, the lungs of severe covid- patients contain high levels of cd + t cells expressing classic markers and genes of exhaustion ( ) , while patients who have recovered from moderate or severe covid- seem to have robust memory t cell formation ( ) . these infiltrating cells also express high levels of markers of activation and cytotoxicity. other studies have similarly demonstrated that more activated and, in some cases, more exhausted t cells develop in symptomatic covid- patients compared to levels during the prodromal stage ( ) . those who experience mild-moderate disease maintain their lymphocyte counts and have more polyfunctional t cells; while studies in those with severe disease are contradicting, reporting both lower and higher cd + t cell cytotoxicity ( ) . severe disease is associated with profound lymphopaenia lending support to the theory that the disease also causes immunosuppression, although lymphocyte trafficking to the site of infection may also be responsible ( ) . a biphasic t cell response has therefore been proposed, characterized by an early cd + cytotoxic t cell response to control the virus followed by t cell exhaustion and depletion as a result of viral persistence over many weeks in some patients. this model could account for poorer outcomes in the elderly who have diminished t cell repertoires and better outcomes in children who have a diverse and abundant naïve t cell pool ( ) . nevertheless, the likely early protective role for cd + and cd + t cells in sars-cov- clearance suggests that their induction should be exploited in sars-cov- vaccine development strategies. no vaccine is licensed or available for sars or mers, however, considerable preclinical development studies have been performed. a range of vaccine development approaches were explored, including live attenuated vaccines and inactivated vaccines (such as inactivated whole virus), soluble protein vaccines, viral vectors, nanoparticles, and dna vaccines; most of these platforms are also being utilized for sars-cov- , as discussed below ( , ) . most of the sars and mers subunit candidate vaccines have targeted the s glycoprotein and been examined in studies conducted in animal models, although n, m, and e protein-based vaccines have also been tested ( ) . however, the fact that animal models, including mice and nonhuman primates, display only limited clinical manifestations of sars and mers, that it is not usually lethal, severely limit the translation of results into humans. to overcome this, animal adapted strains and transgenic animal models of severe disease have been developed ( , ) . in virus challenge studies in animals, high titers of nabs to both sars-cov- and mers virus correlated with protection ( , ) . relatively few studies have investigated the protective role of t cells, and while some correlate cd + and cd + t cell responses with protection, adoptive transfer and t cell depletion assays in mice suggest they are not necessary for protection in mice immunized with a dna-based sars vaccine ( ) . furthermore, vaccination of mice with sars and mers candidate vaccines has been shown to result in th lung pathology with eosinophilic infiltration following wild-type virus challenge ( ) ( ) ( ) ( ) . this immunopathological effect has been associated with whole virus vaccines with and without adjuvant and linked with responses to the n protein in particular; s protein-based vaccines may have a lesser effect. an n protein-based dna sars vaccine also elicited a delayed type hypersensitivity reaction in mice, further cautioning against using this protein for sars-cov- vaccines ( ). furthermore, s-based vaccines appear more immunogenic and protective in these studies and the duration of protection has been shown in challenge studies to last - months in mice immunized with a vesicular stomatitis virus (vsv)-based sars vaccine and animals immunized with dna and/or protein-based mers vaccines ( , ) . of note, in the former study, protection against viral challenge was complete in younger mice but limited in senescent mice, highlighting potential issues with successful vaccination in older individuals ( ) . the only sars vaccines to reach human phase clinical trials were based on inactivated virus, soluble s glycoprotein and dna approaches ( ) . mers vaccines tested in human phase clinical trials include a dna-based vaccine alone ( ) ; dna in combination with adenovirus or modified vaccinia ankara (mva) viral vectors as a prime-boost strategy ( , ) ; and a chimp adenovirus vaccine ( ) . efficacy against disease has not been tested in humans since it has not been deemed ethical to perform challenge studies with lethal viruses for which there is no effective treatment. the first sars-cov- genome was isolated from patients with pneumonia from wuhan, china in early and identified as a novel human coronavirus ( , ) . since that time, multiple viruses have been isolated and sequenced from patients throughout the world providing an understanding of the evolutionary capacity and diversity of this pathogen ( ) . structural genomics and molecular interaction analysis (interactomics) can be used to rapidly identify putative functional sites, facilitating rational vaccine design by identifying potential b and t cell epitope regions of key viral proteins ( ) . several groups have used computational programs and immunoinformatics to predict cd + and cd + t cell and b cell epitopes across multiple sars-cov- antigens from pathogen sequence data to design novel sars-cov- vaccines ( , ) . the genetic diversity and stability of these regions over time can then be characterized, and cross-reactivity predicted and examined to ensure a vaccine provides cross-strain immunity. the s gene of human coronaviruses is well-known to undergo genetic drift which could compromise sars-cov- vaccines based on this region ( , ) . reassuringly, comparisons between the sequences of known sars-cov- b and t cell epitopes from the n and s proteins have been shown that some of the epitopes map identically to proteins from sars-cov- ( ). importantly, these epitopes showed no mutations among available sars-cov- sequences suggesting that they reside in stable parts of the s protein. the t cell epitopes also covered a broad range of hla alleles and could therefore be future targets for vaccine design. one issue with this approach is that the epitopes will be specific for human hla alleles, making it difficult to test them for protective efficacy in animal models. one way to overcome this problem is to use humanized mouse models which have functional human immune systems ( ) , bearing in mind that mice can also be modified to express the ace receptor ( ) . also, there are often animal homologs of human cd and cd t cell epitopes so it may still be possible to screen for efficacy. however, it should be borne in mind that due to the urgent need to develop an effective sars-cov- vaccine, animal challenge data are not a mandatory pre-requisite to progression to clinical trials; nevertheless, ideally these should be conducted as part of the vaccine development strategy. the recent development of a laboratory-adapted sars-cov- strain that is pathogenic in mice, provides an ideal animal challenge model for testing the efficacy of sars-cov- candidate vaccines in mice ( ) . vaccine technology has been advancing rapidly and there is a plethora of approaches to constructing sars-cov- vaccines. these range from the original technique employed centuries ago in the smallpox vaccine development of using modified or killed whole organism, to protein and peptide-based vaccines, nucleic acid dna and rna vaccines and nanoparticle constructs. each approach has unique advantages and disadvantages including varying time to development and scalability, cost, stability, as well as anticipated safety and immunogenicity profiles; all approaches are represented among preclinical and clinical trials ( - ) (figure ). some vaccine candidates, particularly protein-based vaccines, require an adjuvant to boost their immunogenicity ( ) . to date, very few adjuvants have been licensed for human use since the original adoption of aluminum salts (alum) from the early s, which bias immunity toward a th response ( ) . several of the newer adjuvants consist of toll-like receptor agonists to stimulate innate immunity combined with alum, such as as which is based on the tlr ligand monophosphoryl lipid a (mpl). oil-in-water emulsion adjuvants including mf and as are stronger adjuvants than alum and have been adopted in several licensed influenza vaccines. the adjuvant as , present in the malaria vaccine rts,s, combines mpl and a saponin qs- ( ) . a wide variety of adjuvant approaches are being taken with the various sars-cov- vaccines in development, including those mentioned above, some of which are described in the relevant sections below ( ) . many vaccines are given via the intramuscular route, including the majority of the current covid- vaccines in development ( table ) . however, since sars-cov- causes infection via the respiratory tract, a vaccine targeting the mucosal immune system might be preferable ( ) . mucosal vaccines have the advantage of being needle-free and thus practical for mass-administration, but immune tolerance induction can be an issue. the formulation, including the use of mucosal adjuvants, is therefore important to ensure stimulation of adequate local and systemic immunity ( ) . several groups have developed mucosal sars-cov- vaccines. a deoptimized live attenuated whole virus vaccine is being developed for intranasal (i.n.) administration, and several adenovirus-based vaccines will also be administered via the i.n. route ( table ). an oral probiotic pill-based sars-cov- dna vaccine (bactrl) is also planned for clinical trials ( table ) . the sublingual route is also considered an attractive route for inducing mucosal immunity ( ) . an alternative approach is to combine parenteral vaccines with adjuvants such as retinoic acid and caf which are known to induce protective iga mucosal responses ( , ) . researchers are also investigating self-administered mechanisms for sars-cov- vaccines to overcome the need for a healthcare worker to administer the vaccine. for example, inovio tm have developed a novel hand-held cellectra r intradermal delivery device which uses a brief electrical pulse to reversibly open skin cell pores to allow the entry of their ino- dna plasmid vaccine ( ) ( table ). the university of pittsburgh school of medicine has developed an intradermal microneedle patch that can deliver sars-cov- protein antigens, either s or rbd (pittcovacc), through the skin which will enter human trials soon ( ) ( table ) . this approach delivers antigen and danger signals to the high-density dermal apcs stimulating a highly effective immune response, even in the absence of adjuvant. it requires only low doses of antigen thus reducing production costs. indeed, this method induced more potent s -specific nabs in mice than the traditional needle injections, with or without the inclusion of tlr ligand sequences ( ) . the development of live attenuated vaccines requires the organism to be modified via passage multiple times under unique conditions in the laboratory until it loses enough key virulent protein and peptide subunit vaccines are usually combined with an adjuvant in order to enhance immunogenicity. the main emphasis in sars-cov- vaccine development has been on using the whole spike protein in its trimeric form or components of it, such as the rbd region. multiple non-replicating viral vector vaccines have been developed, particularly focused on adenovirus; while there has been less emphasis on the replicating viral vector constructs. nucleic acid-based approaches include dna and mrna vaccines, often packaged into nanocarriers such as virus-like particles (vlps) and lipid nanoparticles (lnps). nanoparticle and vlp vaccines can also have antigen attached to their surface or combined in their core. the immune cell therapy approach uses genetically modified sars-cov- -specific cytotoxic t cells and dendritic cells expressing viral antigens to protect against sars-cov- infection. each of these vaccine approaches has benefits and disadvantages in terms of cost and ease of production, safety profile and immunogenicity, and it remains to be seen which of the many candidates in development protect against factors so as to not cause disease, but retains its ability to replicate in the vaccine recipient, and stimulate a robust immune response that protects against infection ( ) ( ) ( ) . these vaccines tend to be highly immunogenic, do not require an adjuvant and provide long-lasting immunity. however, they are usually contraindicated in those immunocompromised or pregnant. it is difficult to develop a live attenuated sars-cov- vaccine quickly because of the time and knowledge required to ensure that it is suitably attenuated and all virulence factors removed. reverse genetics can be employed for the rational design of the ideal attenuated sars-cov- vaccine virus, for example by determining key virulence factors, as has been done for other coronaviruses ( ). long-term maintenance of consistent live vaccine stocks is also problematic ( , ) . despite the challenges, three codon-pair deoptimized live attenuated vaccines are currently undergoing pre-clinical testing, but none have yet progressed to clinical trials ( ) ( table ) . these three candidates are being developed by codegenix/serum institute of india, indian immunologicals ltd/griffith university and mehment ali aydinlar university in turkey, respectively ( ). this approach relies on using a single virus strain which may not cross-protect against other strains, particularly as the virus continues to spread worldwide and mutates as selection pressure increases once immunity becomes more widespread. inactivated vaccines are produced by growing the virus and then killing or "deactivating" it so that it can no longer replicate. the whole virus can be used, although alternative approaches include splitting the virus with a detergent to disrupt it or purifying antigenic components to create a subunit vaccine ( ) . these vaccines are safer than live-attenuated vaccines but less immunogenic and often require an adjuvant. there are currently four inactivated vaccines in human clinical trials and a further nine in the preclinical stages of development (tables , ). an alum adjuvanted purified formaldehyde inactivated whole virus vaccine developed by sinovac biotech., called picovacc, has been shown to be immunogenic in mice, rats and non-human primates ( ) . it induced nabs to both vaccine and non-vaccine strains and provided partial or complete protection of macaques in challenge studies and has now entered human clinical trials in china ( pre-clinical data confirming induction of high levels of nabs in several animal models and protection against intra-tracheal sars-cov- challenge in rhesus macaques after doses of vaccine ( ) . the institute of medical biology/chinese academy of medical science has also developed an inactivated whole virus construct that is in a phase / trial; and bharat biotech international ltd also has one in a phase / trial ( table ) . rather than using the whole or a split fragment of the virus as the vaccine, antigenic proteins (or peptides) of the virus can be generated using recombinant technology in the laboratory instead ( ) . this is the most popular approach in the sars-cov- vaccine development field, with protein/peptide constructs in preclinical trials and that have entered clinical trials ( table ) . they are relatively simple vaccines to make and thus cheap to produce. a major advantage over whole virus vaccines is their relative safety since unnecessary reactogenic components such as lipopolysaccharides and toxins can be excluded. however, protein/peptide vaccines often require adjuvants and multiple doses in order to stimulate an adequate immune response. peptide vaccines usually consist of - amino acid b cell and t cell epitope regions of key antigens allowing for a precise and targeted immune response. peptidebased vaccines have been developed as candidates against several viral pathogens, but few have been licensed except for use in veterinary practice ( ) . this is in part due to their inherent limitations including a narrow breadth of immune response, potential for incorrect confirmation for epitope recognition, lack of b cell receptor cross-linking for b cell epitopes, hla restriction for t cell recognition, failure to induce cross-reactive immunity against different viral strains and failure to elicit longlasting immunity. the use of whole proteins in vaccines broadens their immunogenic potential, however the protein can also lose its native structure and thereby lose immunogenicity. in addition, as knowledge of the immune response to sars-cov- is still in its early stages, so too is the understanding of precisely what may be the most conformationally optimal, immunogenic and safe protein or peptide to utilize; this will be discovered as vaccine candidates using this approach are examined in clinical trials. the majority of sars-cov- candidate vaccines in development are based on the s protein or its rbd region (figure ) ( ) . indeed, five of the current protein/peptide vaccine candidates in clinical trials target the full s protein and the other two target rbd only ( table ) . some novel technologies have been employed to overcome the issue of the s glycoprotein losing its immunogenic conformational form. the university of queensland has developed a molecular clamp technique that uses proteins to stabilize the s protein in its coiled shape. this seeks to ensure that functional nabs are produced that will tightly bind to virus in its native form and thus prevent cell entry ( ) ( table ) . other groups have focused on using powerful adjuvants to ensure immunogenicity, for example novavax's recombinant s protein sars-cov- nanoparticle vaccine, nvx-cov , combined with an adjuvant called matrix-m tm . this is a saponin-based adjuvant that enhances apc recruitment and action and stimulates high levels of nabs and cell-mediated immune responses ( ) . the s protein in nvx-cov is present its native trimeric form and has been shown to stimulate high levels of nabs in mice and baboons in preclinical studies. primary phase clinical trial results have just been reported from a randomized, observer-blind, placebo-controlled trial of nvx-cov conducted in healthy adults ( ) ( table ) . safety and immunogenicity were assessed for doses of vaccine ( and µg) with (n = ) or without (n = ) the matrix-m tm adjuvant (a µg dose). a strong correlation was observed between nab titers and anti-spike igg to s protein with the adjuvanted, but not the unadjuvanted, vaccine. levels were comparable to those in convalescent sera (r = . ). both and µg adjuvanted doses elicited responses of similar magnitude and every participant seroconverted by both assays. t cell responses measured in participants showed that nvx-cov /matrix-m induces antigen-specific polyfunctional cd + t cell responses (ifn-γ, il- , and tnfα) in response to spike protein stimulation; there was a strong bias toward this th phenotype. the safety data are discussed in section comparative safety and tolerability results for current sars-cov- vaccine candidates in clinical trials and this vaccine is now progressing to phase trials. clover biopharmaceuticals's protein trimer tag© vaccine is combined with as adjuvant; the uq vaccine uses mf adjuvant; the vaxine pty ltd. s protein vaccine has a special advax tm adjuvant; and the medigen s based construct is combined with the adjuvant cpg ( table ) . generex biotechnology, in partnership with epivax, have developed a sars-cov- vaccine (epv-cov ) based on li-key technology with undisclosed sars-cov- peptides ( ). this uses synthetic peptides chemically linked to the -amino acid ii-key to ensure robust immune activation of th and th cd + t cells in particular, which in turn facilitate antibody production ( , ) . multiple viral vectors have been used for vaccine delivery due to their ability to infect cells and deliver the gene product encoding antigenic proteins for production inside the host cell following administration, usually via the intramuscular (i.m.) route ( ) . vector viruses are usually genetically modified to reduce their virulence and render them replication defective. adenoviruses and poxviruses are the most widely used non-replicating viral vectors, but many other viruses can also be adapted for vaccine delivery. replicating viral vectors, such as measles virus and vesicular stomatitis virus (vsv), can also be used for sars-cov- vaccine design; they mimic natural infection, rendering them self-adjuvanting and therefore more potent, and can be used in lower doses ( ) . while subunit vaccines are more focused on induction of humoral immunity, viral vector vaccines are able to induce robust cell mediated immunity (cmi) in addition to abs and have been shown in animal models to protect against challenge with pathogenic coronaviruses ( ) . a modified vaccinia ankara (mva) vector expressing a mers-cov recombinant protein induced t cell responses and nabs in mice, while a rabies virus-based sars-cov- vaccine protected mice against sars-cov- challenge ( ). human adenoviruses are the most common non-replicating viral vectors being used for sars-cov- vaccine development; constituting of the in clinical trials and of the in preclinical development ( table ) . the three in clinical trials include cansino's ad -ncov, janssen's ad covs ( ) and gamelaya's ad -based gam-covid-vac lyo vaccine ( table ) . all three encode the s protein. while adenovirus vectors are well-tolerated and highly immunogenic in most people, preexisting immunity to the viral vector can hamper the induction of immunity, particularly after repeated doses. animal adenoviruses can be used as vectors instead of human ones to overcome this problem, which is the approach used for the oxford university chimpanzee adenovirus-based candidate sars-cov- vaccine chadox ncov- (also called azd ) and the simian adenovirus-based reithera vaccine grad, both of which are in clinical trials ( table ) . anti-vector immunity can still develop after the first dose of vaccine even if a simian adenovirus vector is used. despite the concern about anti-vector immunity, of the adenovirus-based sars-cov- vaccines in clinical trials are planned to be given as a single dose, whereas almost all the other vaccines in clinical trials require at least doses ( table ) . phase and trials have now been completed and published for the cansino (ad -ncov) and oxford (chadox ncov- ) , the safety aspects of which will be discussed later in this article. the ad -ncov randomized, double-blind, placebo-controlled phase trial results involving volunteers ≥ years of age showed > % seroconversion by rbd-elisa, but only % seroconversion of nab responses in the higher dose group and % in the lower dose group after a single immunization ( ) . fifty two percent of volunteers had pre-existing anti-ad nabs, and those with higher anti-ad immunity had approximately half the rbd-specific elisa and sars-cov- nabs as compared to those with low pre-existing anti-vector immunity, supporting interference with vaccine immunogenicity. preliminary findings from a phase / , single-blind, randomized controlled chadox ncov- vaccine trial conducted in , healthy volunteers aged - years induced nabs in % of participants after a single dose of vaccine, rising to % after a booster dose ( ). neutralization titers were comparable to those observed in convalescent plasma from people who had recovered from covid- . both vaccine constructs elicited t cell responses which peaked at days post-immunization, as determined by interferongamma (ifn-γ) enzyme linked immunospot (elispot) assay ( , ). day responses appeared higher with chadox ncov- (median per million peripheral blood mononuclear cells [pbmc] ) as compared to the highest dose used of ad -ncov (mean per million pbmc) in the phase trial ( ); ad -ncov elicited a median of - sfu/million pbmc at day in the phase trial ( ). intracellular staining confirmed production of ifn-γ, tumor necrosis factor (tnf) and interleukin- (il- ) by both cd + and cd + t cells following ad -ncov immunization, with more cytokine detected in cd + t cells as compared to cd + t cells ( ) . institute pasteur replicating viral vector vaccine candidate tmv- , based on measles virus, is the only viral vector vaccine that has entered phase clinical trials to date ( table ) , but there are more at the pre-clinical stage of development. these include each based on influenza virus or vsv, more measles virusbased candidates, and each based on avian paramyxovirus, horsepox (called tnx- ), newcastle disease virus, and yellow fever virus ( table ) . the majority target the s protein or rbd, although one measles virus-based candidate also targets the n protein. two of the influenza-based candidates are designed for intranasal administration. a live attenuated yellow fever vector-based vaccine (yfs ) expressing the prefusion form of the s protein recently showed that a single dose protected most hamsters in a sars-cov- challenge model ( ) . nucleic acid-based vaccines (dna or mrna) offer a costefficient and scalable approach to sars-cov- vaccine development ( ) . the major nucleic acid-based approaches are described below. dna-based vaccines are stable and safe to handle. however, while there are multiple dna vaccine constructs targeting numerous viral infections in animal and human studies, to date none have been licensed for human use. naked dna can be injected and taken up by apc or apc can be directly transfected with plasmid dna encoding the target antigen. subsequent expression and presentation of the target antigen leads to the induction of antigen-specific cd + and cd + t cells and antibody production. however, dna vaccines tend to be poorly immunogenic, necessitating various strategies to improve immunogenicity such as the use of viral promotors, immunostimulatory sequences and adjuvants. nanocarriers such a virus like particles (vlps) can also be used to improve dna delivery, avoid its degradation and be immunostimulatory. there are safety concerns about potential integration into the host dna causing dysregulated gene expression although the risk of this is thought to be very low. a series of animal studies with sars-cov- nucleic acidbased vaccines have shown induction of nabs and cellular immunity, with partial protection against turkey cov challenge ( ) and vaccine-induced ab-mediated protection to the s protein in mice ( ) . twelve dna-based sars-cov- vaccines are currently in preclinical development and more have entered phase / clinical trials ( table ) . two dna-based candidates in clinical trials are administered i.m., whereas the other are administered via the intradermal (i.d.) route ( table ) . inovio pharmaceuticals is testing their cellectra r electroporation i.d. delivery device to administer their s protein-based dna plasmid vaccine ino- which induces nabs and t cells in mice and guinea pigs ( ) ( table ). an alternative approach currently in pre-clinical trials is being taken by symvivo with their dna b. longum vaccine bactrl which is administered as an oral pill ( ) . messenger rna (mrna) vaccines consist of an rna strand that codes for a target antigen ( , ) . the two main types are those packaged and delivered in non-replicating form or those packaged with other rna as an in vivo replicating vaccine. they offer a promising alternative to conventional vaccine approaches due to their high potency, capacity for rapid development and potential for low-cost manufacture, which could prove crucial for global accessibility for a covid pandemic vaccine. they also have a good safety profile, since they are not made with pathogen and do not integrate into host dna. several challenges encountered by mrna vaccines include the need for packaging, for example into particles or liposomes, as naked rna is otherwise rapidly broken down and the need for freezing or refrigeration for wide scale delivery. there are currently rna-based vaccines in pre-clinical trials and have entered clinical trials ( table ) . the clinical trial candidates include two lipid nanoparticle encapsulated rna sars-cov- vaccines that have already progressed to phase trials ( table ) . moderna/niaid have published preliminary results from their phase dose-ranging, safety and immunogenicity trial of doses of mrna- encoding the s protein conducted in healthy adults aged - years ( ) . the s protein in mrna- is stabilized in its prefusion state by proline substitutions in the s subunit; and the lipid nanoparticle coat consists of lipids. all participants developed rbd abs by elisa and sars-cov- -specific nabs by neutralization assay after doses in the upper range of those who have recovered from covid- (convalescent serum). t cell responses measured by intracellular staining for th (tnf, il- , ifn-γ) and th (il- , il- ) cytokines following stimulation with an s protein peptide pool showed a th cytokine bias and low-level cd t cell responses. an interim report from the phase / dose-ranging study of doses of biontech's mrna s protein vaccine candidate bnt administered to adults aged - years elicited rbdbinding igg and sars-cov- nabs in the order of . - . fold that of convalescent human plasma. t cell responses were not reported in this paper. this vaccine encodes trimerized rbd which is modified by adding a "foldon" trimerization domain to increase immunogenicity ( ) . both mrna- and bnt are now in phase clinical trials in adults ≥ years ( table ). the safety data for both will be reviewed in a later safety section. the remaining rna-based candidates in clinical trials include three in phase and one in phase / ( table ) . the imperial college london lnp-encapsulated rna vaccine consists of an s protein-based self-amplifying rna construct (lnp-ncovsarna); designed because sarna vaccines induce a more stable dna product and are more immunogenic than conventional nucleic acid vaccines ( ) ( table ) . nanoparticle-based vaccine approaches have received increasing interest in recent years due to their good safety profile and high immunogenic potential, with an ability to efficiently target dendritic cells (dcs) for processing and presentation, providing a clear advantage over less immunogenic dna and rna vaccines ( ) . materials used to construct nanoparticle vaccines include self-assembling viruses (virus like particles [vlps]), lipids (as liposomes), proteins, metals (e.g., gold) and polymers which also act as their own adjuvant ( ) . many nanoparticles are highly stable and less prone to degradation than other constructs such as "naked" protein, peptide, dna and rna vaccines ( , ) . selected antigens, which may be proteins or nucleic acid, can either be attached to the surface of the nanoparticles or combined in the particle core. they can be synthesized in a variety of shapes and sizes to induce robust innate as well as adaptive immunity. other modifications include altering the nanoparticle surface to target certain cells or enhance immunogenicity and packaging with tlr ligands and other immune modulators. one of the most popular approaches for viral vaccine development is engineering vlps consisting of self-assembled viral membrane in a monomeric complex which display the viral epitopes but lack multiple key viral components, ensuring they have no replication capacity ( ) . the widely used human papillomavirus (hpv) vaccines are an example of this approach. a nanoparticle polypeptide vaccine displaying sars-specific b cell epitope repeats from the c terminal heptad repeat region of the s protein in its native coiled formation was shown to elicit nabs in mice ( ) . a number of the protein and nucleic acid-based sars-cov- vaccine candidates use nanocarriers to package the vaccine product to improve stability and ensure efficient antigen processing ( ) . as mentioned above, novavax's full length s protein construct nvx-cov is a nanoparticle vaccine and the majority of mrna-based sars-cov- vaccines are packaged lnps, including mrna- and bnt . there are sars-cov- vlp vaccines in preclinical development and just one in a phase clinical trial ( table ) . the latter vaccine developed by mendicago inc. consists of plant-derived recombinant vlps made by transfecting viral genes into the cell nuclei of leaves permitting transient expression of viral proteins which form into vlps which are extracted and purified for clinical use ( ) ( table ) . sars-cov- vaccine approaches based on car-t cell concepts are also being tested in clinical trials. the shenzhen genoimmune medical institute in china is using a lentiviral vector system to create viral minigenes which express viral proteins (s, m, e and n proteins) and immune modulatory genes (p polyprotein protease) to modify dendritic cells (dcs). the lv-dc presenting sars-cov- specific antigens will activate cytotoxic t cells. novel dc (lv-smenp dc) and antigenspecific cytotoxic t cell vaccines are currently being tested by s.c. injection or i.v. infusion in a phase / multicenter therapeutic clinical trial in participants ranging from months to years of age ( ) ( table ) . the same company has also developed artificial dcs expressing sars-cov- mini-genes for various viral proteins which are being tested in a phase clinical trial as a s.c. injection in sars-cov- infected individuals in the same age range ( table ) . autologous dcs expressing sars-cov- antigens have also been developed by aivita biomed inc. in usa and are being tested as a s.c. administered vaccine in phase clinical trials in healthy adults > years of age ( ) ( table ) . whilst not suitable for large-scale delivery and use, immune cell therapy might be used in the context of pre-emptive therapy in high-risk patients such as the elderly and immunocompromised. there is increasing evidence that immunization with live vaccines can improve survival against non-vaccine targeted infections, a phenomenon termed "non-specific, " "off-target, " or "heterologous" effects of vaccines ( , ) . some of the best evidence comes from studies of immunization with the tuberculosis vaccine bacillus calmette-guérin (bcg); for example, bcg has been shown in three randomized trials to markedly reduce all-cause mortality in low birthweight neonates ( ) . bcg causes epigenetic modification of innate immune cells (monocytes and natural killer (nk) cells) thereby enhancing innate responses in a process called innate immune training ( , ) . experiments in both murine models and humans have further shown that bcg protects against certain viral pathogens ( ) . reports that people living in countries where childhood bcg is routinely given have lower covid- mortality have been posited as further evidence for protective effects of bcg ( ). however, these observations are fraught with confounders, including difficulty ascertaining covid- infection rates in these countries, the time the virus first entered the country and differences in national control strategies ( ) . furthermore, other studies contradict these findings, such as a geographic regression discontinuity analysis along the east and west german border showing that the covid- infection rates do not vary according to the bcg vaccination strategies employed during the cold war ( ) ; and a study from israel showing that sars-cov- infection rates in adults did not differ by bcg at birth status ( ) . even if childhood bcg immunization does not protect against covid- , a bcg dose as an adult may provide some short-term protection. indeed, it has been widely postulated that the nonspecific beneficial effects of bcg might protect against covid- , resulting in an explosion of interest in this vaccine as a protective strategy in recent months ( , ) . this has led to a number of clinical trials being established in north and south america, australasia, africa and europe, all testing the ability of bcg vaccine to protect against sars-cov- infection, mostly in high-risk exposed healthcare workers ( ) ( table ) . these trials collectively aim to recruit > , participants, with the largest trial of , healthcare workers currently recruiting across multiple sites in australia and europe. non-specific protective effects of live vaccines have also been postulated for oral polio vaccine (opv) ( ) and measles vaccine ( ) . as a result, a study of the protective effects of opv against covid- is due to commence in usa in june ( ) , and a clinical trial of measles-mumps-rubella (mmr) immunization and covid- protection is being conducted in egypt ( table ) . a consistent feature of the off-target effects of live vaccines has been their sex-differential nature; females often showing greater susceptibility to these immunomodulatory effects ( , , ( ) ( ) ( ) . indeed, sex differences in targeted vaccine immunogenicity and adverse events have also been widely described ( , ) . this raises the possibility that any off-target protective effect of live vaccines may differ between the sexes and that the sars-cov- vaccine candidates may show sex differences in immunogenicity and reactogenicity. it is possible, but uncertain, that strategies to use these already licensed vaccines may provide a modest level of protection against covid- , including those at highest risk, such as healthcare workers. it is important to recognize, however, that data from these studies will still need to accrue whilst we are waiting for sars-cov- targeted vaccines to come through the pipeline. given the unprecedented speed at which targeted vaccines are being developed, this approach may not be necessary or required; furthermore, these existing live vaccines are not currently recommended for this indication, so should only be utilized in this context in the setting of a clinical trial. even if an efficacious vaccine is developed there are a number of immunological challenges that need to be considered. furthermore, there is the enormous challenge of mass production and rapid and equitable delivery ( table ) . it is extremely unlikely that any of the sars-cov- vaccines will be % effective; while they may not prevent becoming infected, it is hoped that they will prevent progression to severe disease. indeed, the seasonal influenza vaccine is generally about % protective against infection but does decrease disease severity and hospitalization rates ( ) . a recent study in which macaques were vaccinated with the oxford university and astrazeneca adenovirus vaccine, chadox ncov- , found that the primates were protected from sars-cov- -induced pneumonia ( ) . however, the macaques still had high levels of virus replicating in their upper respiratory tract. it is hoped that even if the vaccines do not prevent infection in the upper airways, they may reduce viral load and disease severity and in turn, the amount of virus a vaccinated person transmits to others. even a modestly efficacious sars-cov- vaccine could mitigate the severity of this pandemic and be highly beneficial in a world struggling to contain this novel virus and its devastating social and economic effects. most vaccines are tested in healthy young adult males and non-pregnant women and, if safe, they are then tested in healthy children prior to licensure. this therefore raises the issue that any vaccines may initially have less empiric data available on use in certain key vulnerable populations such as the elderly, immunocompromised groups and pregnant women. it is plausible that vaccines may be considerably less immunogenic in older and frail elderly who experience the most severe outcomes from covid- , hence the importance of adjuvants in many of the vaccine candidates, both for dose sparing and enhancing immunogenicity ( ) . efficacy in this population is also likely to vary by the type of vaccine construct and adjuvant used. this has been seen for other diseases such as influenza, where the adjuvanted and high dose vaccines are more immunogenic than the standard inactivated vaccines in the elderly ( , ) . similarly, for herpes zoster, the efficacy of the live-attenuated vaccine in the elderly is approximately % compared with > % for a subunit adjuvanted vaccine (glycoprotein e and liposomebased as b adjuvant) ( ) . thus, ensuring studies can be conducted in these priority target groups, either pre-or earlypost deployment, will be critical and special formulations may be required to ensure adequate immunogenicity. throughout life, humans are repeatedly exposed to the endemic human seasonal coronaviruses and develop human cov (hcov) antibody repertoires that can potentially cross-react with sars-cov- -specific antigens ( ) . this early immune imprinting leads to preferential expansion of cross-reactive abs when a related antigen is encountered, in this case sars-cov- ( ). this long-recognized process is called original antigenic sin (oas) ( ) and is known to occur with several common viruses, such as influenza and respiratory syncytial virus ( , ) . oas can either lead to a less effective immune response or cause enhanced immunity and immunopathology ( , , ) . oas is one of the mechanisms responsible for the phenomenon of antibody dependent enhancement (ade) of immunity which is thought to occur in covid- , whereby non-neutralizing cross-reactive abs against other coronaviruses enhance host cell entry and viral infectivity and worsen disease severity ( , , ) . this was seen to occur in cats vaccinated against feline infectious peritonitis coronavirus ( ) ( ) ( ) and certain sars and mers vaccine platforms also appear to have shown worsened immunopathology in animal challenge studies compared with placebo ( ) . recently, it was shown in vitro using human cells that nabs to coronavirus s protein can also trigger ade by causing a conformational change of the s protein ( ) . antigen-experienced older individuals are more susceptible to the phenomenon of ade, while less antigen-experienced younger individuals mount more targeted antibody responses to viral neoantigens. this may in part account for the greater immunopathology of covid- in older individuals. indeed, it has been shown recently that covid- -naïve children and elderly have quite different cross-reactive sars-cov- -specific antibody signatures, which would play differing roles upon challenge with the wild-type virus ( ) . theoretically, a sars-cov- vaccine could skew the immune system toward production of less effective or even harmful cross-reactive hcov abs via these processes of oas and ade, rather than induce highly-targeted sars-cov- -specific abs as intended ( ) . indeed, anti-spike abs taken from critically unwell covid- patients with severe lung injury skewed macrophages in vitro into a pro-inflammatory phenotype, implicating the abs in driving the surge in lung-resident proinflammatory macrophages and subsequent pro-inflammatory cytokine release in the lungs ( ) . the authors attribute an aberrant igg glycosylation pattern in severe covid- patients with their more pro-inflammatory properties so hopefully the iggs induced by vaccination will not have this problem. indeed, none of the current sars-cov- vaccines in human clinical trials have reportedly caused ade to date, although the number of human subjects studied to date is still relatively small. since animal challenge data are not required to progress to clinical sars-cov- vaccine trials, the opportunity to screen for potential adverse events including ade at the pre-clinical stage of development may be missed. having said that, many of the sars-cov- vaccine candidates have undergone considerable pre-clinical testing, including challenge studies. there was no evidence of ade in pre-clinical sars-cov- challenge studies in rhesus macaques following immunization with dose of chadox ncov- ( ) or doses of the inactivated whole virus vaccine candidate bbibp-corv ( ), both of which were protective in these studies. it is postulated that basing the vaccine on the s rbd terminal subunit of the s glycoprotein should overcome this problem by inducing nabs only, although this has not been proven and, notably, only a few vaccine candidates have taken this approach. furthermore, the in vitro conformational changes causing ade described above occur in the rbd domain, so this approach may not be successful. aberrant manifestations of t cell responses were observed in the s for other viral vaccine candidates, such as inactivated vaccines against measles and respiratory syncytial virus (rsv), resulting in vaccine associated enhanced respiratory disease (vaerd) upon subsequent wild-type pathogen exposure ( , ) . a major driver of this was accentuation of th cytokines, with resultant allergic (eosinophilic) and airway hyperesponsiveness. this was compounded by another mechanism related to the conformation of the vaccine antigen, resulting in the generation of excessive non-neutralizing ab. having a high amount of binding, but not neutralizing, ab caused immune complex deposition and complement activation with local inflammation in the presence of a high viral load. it will therefore be important for vaccine candidates to mitigate the risk of vaerd by considering the t cell profile induced by vaccination, avoiding th biased cd + t cell immunity and biasing toward cd + cytotoxic t cell responses. the alum adjuvanted inactivated vaccine candidate picovacc could theoretically drive a th skewed immune response. while this was not observed in macaque studies when analyzing th and th cytokine profiles, it is not clear from the paper what samples were used to measure these cytokines, clarification of which will be crucial in vaccine safety assessment ( ) . many of the other vaccine constructs favor th immunity, as demonstrated in the published human trial results for several of the viral vector ( - ) and mrna vaccines ( ) and the novavax nanoparticle matrix m adjuvant vaccine ( ) . original antigenic sin also applies to t cell responses and therefore pre-existing cov-specific t cell memory may be enhanced by immunization with a sars-cov- vaccine leading to either suboptimal t cell responses or immunopathology ( , ) . vaccine safety assessment is integrated into all stages of the clinical development pipeline for candidate vaccines and continues once vaccines are deployed into the population. randomized controlled clinical trials examine safety, as well as immunogenicity and efficacy. decisions on the participant numbers to be included in phase studies will likely be powered on efficacy endpoints, but these studies also need to be of sufficient size and planned duration to ensure comparison of injection site, systemic and unanticipated or "unsolicited" adverse events between groups. comprehensive safety studies are particularly critical because some candidate vaccines use platform technologies that have not been examined extensively in human subjects to date, including some of the viral vectors, mrna and nanoparticle constructs, and because of the potential for enhanced disease and adverse events related to aberrant immune responses to be seen upon infection pre-and post-licensure. a list of adverse events of special interest (known as aesi) across all body systems, including immunological, cardiovascular, neurological, musculoskeletal and dermatological manifestations, have been agreed by the brighton collaboration in conjunction with cepi and the who with input from regulatory agencies and other experts, as have the associated case definitions and surveillance strategies ( , ) . listed conditions include anaphylaxis, vasculitides, myocarditis, generalized convulsions and meningoencephalitis, among many others. comprehensive data on safety are essential to ensure that the benefit:risk ratio of vaccination is favorable, to support decision-making by policy makers on wide-scale program implementation among healthy people, and to ensure that individuals are sufficiently confident to accept vaccination. some of these aesi will require post marketing (phase ) studies but are also undergoing evaluation in some of the preclinical and early phase evaluations, particularly the larger phase studies. interim phase / safety results have recently been published for two leading adenovirus-based vaccines, chadox ncov- ( ) and ad -ncov ( , ) ; two mrna based vaccine candidates, mrna- ( ) and bnt ( ) ; an aluminum adjuvanted inactivated whole virus vaccine ( ) ; and an adjuvanted recombinant protein nanoparticle vaccine, nvx-cov /matrix-m ( ) . all candidates exhibited acceptable safety profiles, and while local and systemic reactogenicity was common for the mrna, adenovirus and adjuvanted protein nanoparticle constructs, the inactivated whole virus vaccine was considerably less reactogenic ( table ) . reactions were generally mild to moderate and resolved within days. importantly, there were no serious adverse events reported for any of these vaccine candidates. pain at the injection site was a particularly common feature with the mrna vaccines (approaching % in some groups), and while it was the commonest reaction to the inactivated vaccine, it was still lower for this candidate than for the other vaccines (table ) . headache, myalgia and fatigue were the other most commonly reported symptoms for the mrna, adenovirus and adjuvanted protein nanoparticle candidate vaccines ( table ) . documented fever was relatively common for mrna and adenovirus candidates but ≤ % for the inactivated virus and adjuvanted protein nanoparticle candidates ( table ). the chadox ncov- trial underwent a protocol amendment during the trial allowing several sites to administer paracetamol prior to vaccination and hourly for h. this slightly reduced reports of pain, feeling feverish, chills, mylagia, headache and malaise but had no effect on confirmed fever. pre-existing adenovirus immunity, increased age and male sex were all associated with decreased fever following immunization with ad -ncov ( ) . in terms of blood parameters, immunization with chadox ncov- was associated with transient neutropaenia and bnt b with lymphopaenia. in the ad -ncov phase and trials reactogenicity was dose-dependent for pain, headache and fever; while it was dosedependent for almost all parameters for the mrna vaccines ( table ) . as a result of the high reactogenicity after a single dose of bnt b , it was decided not to give a second dose. interestingly, the ten participants given a booster dose of the chadox ncov- vaccine had less reactogenicity after the second dose. the opposite was found for the bnt- b mrna and nvx-cov /matrix-m vaccines for which reactogenicity was greater after the second dose ( , ) . these favorable safety results (alongside the good immunogenicity reported earlier in this article) have allowed the progression of the adenovirus and mrna candidate vaccines to phase clinical trials. whilst the sars-cov- vaccine pipeline is progressing at unprecedented speed, there is a concern that suppression of viral transmission in many countries will make evaluation of vaccine efficacy (ve) difficult, as phase studies need sufficient infection rates to compare disease incidence in vaccinated with control individuals. reassuringly, planned studies are currently expanding to higher burden countries where infrastructure to conduct complex adaptive clinical trials exists. nevertheless, the time needed to accrue sufficient data on efficacy will be a critical determinant impacting vaccine availability. in the setting of this uncertainty "human challenge models" for sars-cov- have been proposed. this methodology involves the intentional infection of research participants, providing safety data and pointing to immune correlates of protection. this approach has recently advanced vaccine development for infections like salmonella typhi (typhoid vaccine) and group a streptococcus ( , ) . the difference between these bacterial human challenge models and sars-cov- is that these pathogens have been researched for decades and have an effective antibiotic "rescue" treatment available. infecting a human with sars-cov- to test vaccine efficacy raises numerous ethical issues ( ) , including those around informed consent of the healthy volunteer, noting that while severe covid- is less common in young adults, deaths have still occurred in this age group. strict infection control and personal protective equipment (ppe) measures would also be required to limit "third-party" risk to staff co-ordinating any studies. while it is uncertain if this approach will be required, the who is progressing a framework, including key criteria, that will be required by ethical review boards in order to facilitate "human challenge" trials ( ). as noted by stanley plotkin, "extraordinary diseases require extraordinary solutions" ( ) . it normally takes decades for a vaccine to be developed and licensed for use. however, the race is on to develop a sars-cov- vaccine for worldwide distribution in an unparalleled timeframe to vaccinate the world's population and provide widespread herd immunity. this is already being facilitated by rapid progression through the normally slow bureaucratic regulatory and approval processes and unprecedented worldwide collaboration between governments, universities, pharmaceutical companies and philanthropic organizations; such efforts must continue. in addition, ensuring good public communication regarding the safety and effectiveness of the vaccine will be key to gaining public trust and acceptance of a vaccine that could be seen as rushed. additionally, having carefully designed post-marketing (phase ) surveillance is vital to detect rare or unexpected safety signals and aesi which may only be detected once the vaccine is rolled out on a mass scale. a key player in global access to a sars-cov- vaccine is the not-for-profit coalition for epidemic preparedness innovations (cepi) which is working with global health authorities and vaccine developers worldwide to support sars-cov- vaccine development ( , ) . cepi was founded in with the consensus that new and sustainable models of partnership are needed to respond to worldwide infectious diseases threats. founding members include the bill & melinda gates foundation (bmgf), wellcome trust uk, world economic forum, india department of biotechnology and the government of norway. cepi's goal is not just to advance development, but to also advance licensing, manufacturing, delivery and stockpiling of vaccines once an effective vaccine has been developed ( ). it has raised approximately $ . billion for sars-cov- vaccine development and initiated a series of partnerships in a bid to advance frontrunner candidate sars-cov- vaccines. cepi is investing across a range of vaccine technologies including novavax's nanoparticle vaccine (nvx-cov and matrix-m tm ), clover biopharma's s-trimer protein-based vaccine (scb- ), university of queensland's molecular clamp s protein vaccine, university of oxford's adenovirus vector vaccine (chadox ncov- ), inovio's dna plasmid vaccine (ino- ) and moderna's and curevac's mrna vaccines ( ) . many other government and philanthropic organizations are injecting large sums of money into the effort to develop effective sars-cov- vaccines. the european commission has registered an impressive $ billion in donations toward the development and deployment of vaccines, treatments and diagnostics against sars-cov- ( ). the us government agency biomedical advanced research and development authority (barda) has pledged almost $ million to accelerate the development of moderna's mrna- vaccine, with phase trials expected to begin soon ( ) . barda has also funded janssens's adenovirus (ad ) i.n. vaccine, astra zeneca's azd vaccine (formerly chadox ncov ), merck and iavi's rvsvg-cov and sanofi's recombinant sars-cov- protein vaccine and novavax's nanoparticle matrix m adjuvant vaccine ( ) , demonstrating the range of vaccine strategies being supported. importantly, these phase / studies are being conducted in countries with high disease burden (e.g., north and south america and south africa), so the vaccine efficacy (ve) and safety results can be obtained as rapidly as possible whilst meeting the vaccine regulators' requirements. the logistics of scaling up to produce the billions of doses required to immunize the world's population is an onerous task. it is not yet clear how long immunity will last and therefore whether booster doses or annual immunization will be required. furthermore, multiple initial doses may be needed and the vaccine may have to change as the virus evolves naturally. barda have pledged that they will scale up to million annual sars-cov- vaccine doses in the us each year under operation warp speed, while bmgf have recently awarded $ million to inovio's dna based vaccine ino- with the intention of providing over million doses by the end of . novavax has completed phase studies and is progressing rapidly to phase studies, with the aim of producing up to million doses by the end of and entering large-scale manufacturing of billions of doses in ( ) . curevac has a gmp-certified production facility that can produce million doses of their mrna vaccine in a single production run and million doses of azd (chadox ncov- ) will be available by july ( ) . even if sufficient sars-cov- vaccine doses can be manufactured, worldwide delivery presents another major logistic and financial hurdle. storage requirements will be enormous and the vaccine may need to be either frozen or refrigerated, presenting cold-chain issues. the decision about whether to prepare the vaccine in single-dose or multi-dose vials will impact manufacturing, storage, delivery and potential infection risks ( ) . infrastructure and manpower will be required to distribute and administer the vaccine. equity will be a major issue since richer countries may procure the vaccine for their citizens while poorer countries may not be able to afford it. cepi is negotiating global access upfront in order to ensure equitable access, hopefully averting "vaccine nationalism" ( ) . in early may, who launched the access to covid- tools (act) accelerator, which aims to handle the logistics of vaccine procurement and allocation ( ) . covax, co-led by cepi, gavi and who, is the vaccine pillar of act charged with accelerating vaccine development and manufacture and guaranteeing fair and equitable worldwide access. in an open letter, more than world leaders have united and called for a sars-cov- vaccine to be freely available to all people in all countries of the world in what they call "the people's vaccine, " which is surely the fairest way to tackle this unprecedented global pandemic ( ) . by contrast, pneumococcal conjugate vaccine which has been available for years, is still not included in the immunization programs of many countries due to lack of affordability, many children are left unprotected and about half a million deaths from pneumococcal disease each year. initially, as vaccine production commences, it will not be possible to deliver a sars-cov- vaccine to the entire world population and it is anticipated that key vulnerable populations will need to be targeted. these would likely include healthcare workers, the elderly and those with significant risk factors such as those with co-morbidities or the immunocompromised. it might even be used as a travel vaccine as borders re-open across the world, but is unlikely to be incorporated into infant schedules for some time given the very low risk of severe disease in that age group and time needed to conduct pediatric studies. while the world eagerly awaits effective sars-cov- vaccines as the solution to ending this pandemic, it should be borne in mind that there are many caveats even if robust sars-cov- specific nabs, cd + and cd + t cell responses can be induced by vaccination. the number of doses and dosing frequency will need to be determined, and, particularly if repeated or annual vaccination is required, the financial burden and logistics of delivery will have to be supported. a better understanding of what level of nabs correlate with protection and development of standardized viral neutralization along with other assays to compare vaccine candidates is eagerly awaited. potential disease enhancement and other theoretical safety concerns related to each type of vaccine need to be understood and carefully monitored for, while potential suboptimal immunity may need to be overcome. certain vulnerable populations may respond poorly to vaccination, or the vaccine may predominantly protect against severe disease, rather than infection. if t cells prove critical for protection, it will be necessary to ensure that the included t cell epitopes are recognized by enough hla types to ensure worldwide coverage. scale-up of production to billions of doses and delivery to all regions of the world is a massive logistic challenge. in the short-term the strategy will likely need to be targeted vaccination toward those most at risk (e.g., healthcare workers) with the strategy reviewed as vaccine production increases. on the positive side, given the multiple candidates being developed, there is considerable optimism that some of the vaccines currently in trials will prove effective and be available for use in with delivery scaled-up thereafter. kf proposed the project and coordinated the study group. kf, nc, and fr 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( ) available online at world leaders unite in call for a people's vaccine against covid- we would like to thank claudio rosa (www.claudiorosa.com) for medical illustration. key: cord- -y ji k authors: connell, anna r.; connell, jeff; leahy, t. ronan; hassan, jaythoon title: mumps outbreaks in vaccinated populations—is it time to re-assess the clinical efficacy of vaccines? date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: y ji k history illustrates the remarkable public health impact of mass vaccination, by dramatically improving life expectancy and reducing the burden of infectious diseases and co-morbidities worldwide. it has been perceived that if an individual adhered to the mmr vaccine schedule that immunity to mumps virus (muv) would be lifelong. recent mumps outbreaks in individuals who had received two doses of the measles mumps rubella (mmr) vaccine has challenged the efficacy of the mmr vaccine. however, clinical symptoms, complications, viral shedding and transmission associated with mumps infection has been shown to be reduced in vaccinated individuals, demonstrating a benefit of this vaccine. therefore, the question of what constitutes a good mumps vaccine and how its impact is assessed in this modern era remains to be addressed. epidemiology of the individuals most affected by the outbreaks (predominantly young adults) and variance in the circulating muv genotype have been well-described alluding to a collection of influences such as vaccine hesitancy, heterogeneous vaccine uptake, primary, and/or secondary vaccine failures. this review aims to discuss in detail the interplay of factors thought to be contributing to the current mumps outbreaks seen in highly vaccinated populations. in addition, how mumps diagnoses has progressed and impacted the understanding of mumps infection since a mumps vaccine was first developed, the limitations of current laboratory tests in confirming protection in vaccinated individuals and how vaccine effectiveness is quantified are also considered. by highlighting knowledge gaps within this area, this state-of-the-art review proposes a change of perspective regarding the impact of a vaccine in a highly vaccinated population from a clinical, diagnostic and public perspective, highlighting a need for a paradigm shift on what is considered vaccine immunity. muv is an enveloped, non-segmented, negative-sense, single stranded rna virus that varies between a spherical and pleiomorphic shape of ∼ nm ( - nm) ( , ) . muv is responsible for an acute viral infection, spread by respiratory droplets (via coughs, sneezes) and urine ( , ) . with an incubation period of - days, muv replicates in the nasopharynx and regional lymph nodes, with a secondary viremia occurring late in the incubation period ( , ) . muv can be detected from saliva up to days prior, and as late as days after clinical onset of parotitis ( ) . the muv genome of seven genes consists of , nucleotides, and encodes six structural proteins and at least two non-structural proteins; the nucleocapsid protein (np), v protein (v), phosphoprotein (p), matrix (m) protein, fusion (f) protein, small hydrophobic (sh) protein, hemagglutininneuraminidase (hn) protein, and large (l) protein. the role of the i protein is not known ( , , ) . the sh gene is the most variable region of the muv genome; a - % intravariation and - % inter-variation has been documented ( ) . this gene is used in molecular phylogeny for genotyping and to identify transmission patterns in populations ( ) . despite being serologically monotypic, muv genotypes (a to l) have been described to date (muv genotypes e and m are omitted, as the muv previously assigned to these groups were later re-assigned) ( , , ) . the geographic distributions of the muv genotypes varies worldwide but can co-circulate and thus drive temporal shifts in their distribution. genotype a was frequently isolated in europe until the 's. currently genotypes c, d, e, g, and h are prevalent in europe and the united states of america (usa) whereas genotypes b, f and i are more common in asian countries ( table ) ( , , , ) . since numerous mumps vaccines have been developed worldwide, varying in efficacy and safety profiles but primarily consisting of an attenuated live muv without an adjuvant ( , ( ) ( ) ( ) . currently in europe and for the majority of the g countries who have a mumps vaccine in their immunization schedule (table ) , the mumps vaccine is included as part of the trivalent measles, mumps rubella (mmr) vaccine, and is primarily administered in two doses ( , ) . the jeryl lynn (jl) vaccine, derived from the genotype a muv strain was first developed in the usa and has been used extensively in the united kingdom (uk), ireland and usa since it was licensed in ( ) . derived from a single clinical sample, and propagated in a chick embryo cell culture, two viral isolates (jl and jl ) are present, differing by ∼ nucleotides and amino acid changes ( ) ( ) ( ) . the rit mumps vaccine, developed from the dominant viral component (jl ) in the jl vaccine strain appears to have comparative safety and efficacy (seroconversion) profiles to the jl vaccine strain ( , ( ) ( ) ( ) . however, since no controlled clinical trials of efficacy have been published to compare the two doses of the two vaccines, the clinical significance of this observation is not known. despite the integration of the mmr vaccine into childhood immunization programs, cyclical outbreaks [defined as two or more cases linked by place and time ( ) ] of muv have been documented in several highly vaccinated populations such as ireland and the united kingdom ( , ( ) ( ) ( ) ( ) ( ) ( ) ( ) . between august -and january , , mumps cases were notified in ireland, primarily affecting individuals between the ages of - years. of the % of cases that stated vaccination status, % had received two doses of the mmr vaccine ( ) . an upsurge of mumps cases has also occurred in states of the united states over the last decades, primarily affecting people between and years in close contact/shared settings ( ) . in indiana, . % of mumps cases ( . % of university affiliated and % of community cases) had documented evidence of mmr vaccination ( ) . this results in a significant resource burden for public health departments to control. several reviews, both observational and systematic have demonstrated the clinical benefit of a mumps vaccine ( , ) , the pathogenesis and genomic diversity of the muv ( , , ) and the epidemiology surrounding the outbreak ( , , ) . it is not clear why these mumps outbreaks occur, although it has been alluded to be due to a number of interrelated factors, such as sub-optimal vaccine uptake ( , , ) , primary or secondary vaccine failure or failure of the mumps vaccine to protect individuals from infection (vaccine efficacy) ( ) (figure ). history depicts the remarkable public health impact of mass vaccination. previously inevitable childhood diseases with potentially debilitating or deadly outcomes have seen their rates plummet worldwide or become successfully eradicated. immunizations of vaccine preventable diseases are estimated to prevent ∼ - million deaths per annum and increase life expectancy by ∼ years ( ) . more recently there has been a shift in the public and media perception of vaccines to their safety, which has facilitated outbreaks such as mumps ( ) . organized opposition to vaccinations has a long history; public outcry and resistance following the introduction of the smallpox vaccine in the nineteenth century led to the introduction in england of the vaccination act of ( ) . with one in eight children in the usa under the age of currently thought to be unvaccinated due to parental choice, the who now considers vaccine hesitancy as one of the ten threats to global health in ( ) . vaccine hesitancy, defined as a "delay in acceptance or refusal of vaccines despite availability of vaccination services" involves a multitude of social, political, cultural and emotional factors in highly vaccinated, western populations ( , ) . one of the main issues is the parental concerns regarding the perceived risk of a vaccine to their child (such as timing/schedules of vaccines, associated pain of administration, and potential adverse effects) vs. the disease morbidity and mortality associated with the vaccine preventable disease ( , ) . the retracted paper published in the lancet in ( ) and "anti-vaccination" opinions on social media have also contributed to the persistent and insistent misinformation ( ) , despite vast follow-up epidemiological studies showing no relationship between the mmr vaccine and autism, or differing cognitive development/intelligence ( ) ( ) ( ) . however, the resultant reaction of the public led to the uptake of the first mmr vaccine falling sharply from , with uptake falling to below % in ( , ) . the age demographic that are experiencing the most cases of mumps in ireland during the current ongoing outbreak would have been scheduled to have received the first mmr vaccine between and . nevertheless, no deductions can be made, due to the lack of vaccination status information provided with reported cases ( ) . frontiers in immunology | www.frontiersin.org heterogeneity of immunization coverage in specific populations or geographic locations of susceptibility is also becoming an important epidemiological issue in maintaining proficient population immunity for mumps ( , , ) . the who recommends a > % mmr vaccine coverage for herd immunity. maintenance of such coverage is well-demonstrated in finland, where a country-wide -dose mmr vaccination program initiated in the 's eliminated measles, mumps and rubella within years ( , ) . recent publications from around the world indicate that the level of mmr vaccine uptake is far lower than what is recommended [reviewed in ramanathan et al. ( ) ] ( , ( ) ( ) ( ) ( ) . of the g nations that implement a mumps vaccine within their vaccination schedule, only countries have maintained vaccine coverage levels of > % (table ) . however, poor uptake/incomplete vaccination alone may not be the only issue relating to mumps outbreaks. in the netherlands, mumps outbreaks still occurred with an overall herd immunity threshold of - %, and where and % received the first and second mmr at months and years, respectively ( , ) . the clinical presentation of mumps is pathognomic (bi-lateral parotitis); therefore supporting laboratory diagnosis was rarely employed in the past. as the classical symptoms of mumps are not always typical, there may have been a significant number of individuals in the past who may have been infected but were not identified as such. when mumps vaccination was introduced in , the criteria the vaccine had to meet was the proof that it was clinically effective, i.e., that it reduced the risk of disease in vaccinated individuals in real-world conditions over a set period. such an example was seen the usa; the reported cases (i.e., diagnosis of clinical symptoms) of mumps declined from > cases per , population before (pre-vaccine era) to cases per , population in , a reduction of % ( , , , ) . to note, clinical efficacy was probably based upon the reduction of the "classical bilateral presentation" rather than the milder mumps presentation. therefore, one could argue that the original vaccine efficacy for clinical manifestations was over estimated. currently the laboratory diagnosis of mumps infection in ireland is based upon two approaches: detection of mumps rna by reverse transcriptase pcr (rt-pcr) in a buccal swab containing saliva, throat swab or urine specimen, and serological detection of immunoglobulin m (igm) using a capture assay ( , ) . both approaches for diagnosis are impacted significantly by the quality and timing of sample collection post-onset of symptoms and also if the subject is mumps naïve or had received mumps containing vaccine ( , , , ) . there are challenges in using standard serological laboratory diagnostic methods to reliably confirm mumps re-infection of individuals who had been previously naturally infected or vaccinated ( , ) . briefly, vaccinated individuals re-infected with muv may only generate a weak or undetectable igm response ( ) . although a rise in igg titer may also not occur in vaccinated individuals ( , ) , numerous studies have documented a rapid, variable increase in mumps-specific igg levels, with neutralization antibody concentrations present up to months post-infection ( , , ) . therefore, reverse transcriptase-polymerase chain reaction (rt-pcr) is recommended ( , ) , and was formally introduced in as the principle diagnostic tool in ireland to detect mumps in oral fluids ( ) . rt-pcr can identify current mumps infection more effectively in vaccinated individuals than serological techniques alone as it identifies the presence of the muv vs. the immunological response (igg, igm), and has been previously shown to % correlate with viral culture results ( , ) . the case numbers of more recent mumps outbreaks should always be assessed with this question in mind; are the number of mumps cases increasing, or/and are we better at diagnosing an acute infection? the latter seems to be the most probable, as many individuals who are being tested do not present with classical symptoms. in addition to enhanced surveillance of mumps cases, further optimizations of technologies are also occurring; the utilization of next-generation sequencing demonstrated that by editing one -fold degenerate nucleotide in the forward primer and three -fold degenerate nucleotides in the probe sequence optimized the fluorescence intensity and clinical sensitivity of the real-time rt-pcr when compared to the cdc-developed and who-recommended rt-pcr target [(np) gene] leading to ∼ % increase in clinical sensitivity (i.e., ct values that were ∼ . cycles lower) ( ) . much is not known about the immunological response to the mumps vaccine strain. however, a number of young adults who were vaccinated as children over the last two decades have demonstrated an increased risk of muv infection with time, which is assumed to be related to a decline of antibodies to sub-protective levels of immunity ( , , , , ( ) ( ) ( ) ( ) . primary vaccine failure is defined as the lack of a sufficient initial antibody response to a vaccine in a recipient resulting in a lack of protective immune responses ( , ) . although this type of vaccine failure may be because of improper storage/handling or administration of the vaccine, impacting its efficacy, it may also be due to the initial immunological response of an individual to the vaccine, which is usually quantified by the presence of antibodies that should be detectable in the weeks following vaccination. primary vaccine failure was attributed to primaryschool outbreaks of both mumps and measles in ireland, which subsequently resulted in reducing the age for the second dose of mmr vaccine from - years in to - years of age ( ) . with the cyclical outbreaks occurring, it has been proposed that primary vaccine failure could again be a factor. how is a response to a vaccine determined? in pre-licensure studies of the jl and urabe mumps vaccines, high seroconversion and low failure rates were observed in children after the first vaccine dose (> and . %, respectively), demonstrating that the vaccine induced an antibody response ( ) ( ) ( ) ( ) ( ) ( ) . a more recent study by ong et al. demonstrated that a ≥ fold increase in mumps antibodies -days post-vaccination was considered to be an adequate response of immunity ( ) . vaccine effectiveness (i.e., seroconversion post-vaccination) of vaccine doses has only been conducted on the jl strain; studies provided a median vaccine efficacy of %. these studies have shown that doses of mmr were more effective (but not statistically significant) than a single mmr dose to combat the incidence of mumps infection ( , , , , , ) . mumps-specific antibodies have been detected - years postvaccination and without substantial decline for years after mumps vaccination, with the immunogenicity and efficacy of the mmr vaccine showing comparable immunogenicity levels to post-vaccination levels at years ( , ) . however, most studies of this vaccine (involving either a mumps-specific vaccine or a combined vaccine) only followed-up to - days postvaccination ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . despite few follow-up studies estimating post-vaccination antibody titers specific to the vaccine mumps strain, the evidence of seroconversion post-vaccination in a number of studies indicate that primary vaccine failure does not seem to be a significant contributor to the outbreaks that have been recently observed ( , , , , , ( ) ( ) ( ) ( ) . it has been noted that a small percentage of the population do not seroconvert post-vaccination; < % who received the mmr vaccine were seronegative - years after the first dose of mmr (n = ) ( ) . poor immune responses to primary vaccination has been shown to be a good indicator of infection susceptibility ( ) . this is in agreement with the correlation of pre-outbreak jl virus neutralization titres and elisa results being significantly lower in individuals who became infected compared to non-infected individuals ( ) . further studies of these individuals may provide insights of which immunological process are integral to develop immunity. the current methods used to determine immunity against mumps cannot discriminate between primary and secondary vaccine failure; only the timing of these tests can assess whether an individual ever mounted an immune response postvaccination or whether the response is detectable years postvaccination. primary vaccine failure encompasses the failure to mount an immune response to a dose of a vaccine, secondary vaccine failure refers to a more gradual loss of immunity after a successful initial response that occurs over a number of years post-vaccination ( ) . several factors have been proposed to be implicated with secondary vaccine failure, such as waning immunity, a lack of cross-neutralization, and natural boosting. waning immunity is defined as a decline in immunological protection proportional to time since vaccination. potential waning immunity has been documented in the current mumps outbreaks seen in europe and the usa, mostly affecting young adults within highly vaccinated populations attending tertiary education who have received two doses of the mmr vaccine in early childhood ( , , , , , ( ) ( ) ( ) ( ) ( ) ( ) ( ) . a number of studies from the usa, where a jl vaccine has been used since have demonstrated waning immunity within the population. the risk of developing clinical mumps was shown to increase by - % for every year post-mmr vaccination ( ) , with the rates of mumps infection rising from . cases per , in those who received the second dose of the vaccine within years of the outbreak, to . cases per , in those who received it over years prior. using a mathematical model with analytical limitations, a recent metaanalysis of six studies estimated that vaccine-derived immune protection to muv wanes about years post-vaccination ( ) . kennedy et al. ( ) also demonstrated a decrease of ∼ % in mumps neutralizing antibody titers over years. in contrast, other studies appear to contradict, these findings, showing no link between mumps protection and time elapsed following administration of mumps vaccine ( , , , , ) . lebaron et al. ( ) and gothefors et al. ( ) demonstrated that - % of individuals still had detectable anti-mumps antibodies ∼ years after initial vaccination. cohen et al. ( ) also demonstrated minimal antibody level decline after two mmr doses - years after second vaccination. neutralizing antibodies against the jl- vaccine strain has also been detected in ∼ % for age groups - years, % for age group - years; and % for age group + years ( ) . implementation of a third dose of the mmr vaccine has been shown to be effective as a stop gap measure in limiting disease spread in outbreak settings situations ( ) . individuals vaccinated for the third time had a % lower risk of contracting mumps, with a decreased attack rate of . vs. . cases per , when compared to those who received a second dose. more than % of those who received a third dose of the mmr vaccine showed a -fold increase in mumps antibody titers ( , , , ) . an increase in mumps igg humoral immunity was also observed post-vaccine administration. however, this immunity boost has been shown to be a transient effect, with mumps antibody titers returning to pre-third dose of mumps-vaccination levels year after vaccination. therefore, as waning immunity is thought to be an important factor facilitating mumps outbreaks, the emphasis placed on the quantity/quality of mumps-specific antibodies may need to be re-assessed. it is yet undetermined if the total loss of detectable antibodies correlates to a loss of clinical protection, as the minimal level of neutralizing antibody required for protection against mumps has not yet been defined ( ). antigenic variation and thus reduced cross-neutralization between the vaccine and circulating strains of different muv genotypes have been cited as possible explanations for mumps outbreaks in highly vaccinated populations ( , , ( ) ( ) ( ) . recent outbreaks in europe and northern america (including ireland) have shown the circulating muv during the current outbreaks to be genotype g ( , , , ) . this muv genotype was first identified in , and has demonstrated intra-genotype diversity of up to % (table ) ( , ) . the jl vaccine strain (genotype a), differs phylogenetically to the circulating muv (genotype g) ( ) . in vitro studies of the genotypic distribution and temporal shift of muv suggest that cross neutralization between wild type and vaccine genotypes may be approximately half the concentration measured against the vaccine strain ( ) . pre-infection neutralization titers in mumps positive cases were also significantly lower against genotype g vs. mumps vaccine strain, potentially due to amino acid differences in b-cell epitopes and/or n-linked glycosylation sites on the hn and also within the f protein ( ) . santak et al. ( , ) also demonstrated that conformational changes within the f protein may lead to immunological escape. despite the decline/scarcity of cross-neutralizing antibodies, different mumps vaccines used worldwide have been shown to prevent significant clinical mumps infection during outbreaks ( , ) . dependent on the strain, a - -fold variation of patient sample titers has been shown to be protective in in vitro plaque reduction neutralizations ( , , ) . although the sera of one of these studies, was collected only weeks after mmr vaccination, a time point that may not signify the concept of waning immunity and antigenic differences, several other groups have shown that the most divergent strains of muv can be neutralized in vitro with only slight variations in titers, supporting the concept that muv is serotypically monotypic ( , , , ) . epitopes of the muv that are presented to cd + t-cells have been shown to be present in not only the circulating strains of virus but also in a number of vaccine strains ( ) . in addition, lewnard et al. ( ) also found no evidence that recent mumps outbreaks were due to the emergence of muv strains escaping vaccine-driven immunological pressure. therefore, the limited data does not suggest that antigenic drift of the muv leading to diminished neutralization capacity of the vaccine strain could fully explain the recent outbreaks ( ) . further studies into the cross-neutralizing capacity of the mumps vaccine strain administered - years previously to the current circulating strain of muv in countries where outbreaks are being observed will allow better deductions to be made. it is possible that differences in the neutralization capacity of vaccine-induced antibodies against different muv strains may be more significant when levels of neutralizing antibody are low and become "overwhelmed" when the mumps viral load challenge is high ( ) . several prominent mmr/mumps vaccine studies were undertaken at a time when there was still a high prevalence of circulating wild type virus, which enabled sub-clinical boosting to occur in an individual. such natural boosting is illustrated in belarus, where a subpopulation of vaccinated individuals only had a small amount of their overall mumps igg antibody levels specific to the vaccine-strain ( ) . neutralization antibodies against iowa-g/usa (the circulating wild type virus) were also present in pre-infection plasma of all mumps cases during a recent outbreak in the us ( ) . this indicates that the mumps vaccine alone is not solely responsible for the high levels of mumps antibodies ( ) , and that longterm antibody persistence or protective efficacy data of the vaccines used may not truly reflect the current circumstance of viral transmission/circulating within a highly vaccinated population ( ) . herd immunity increases the chance for natural mumps boosting for an individual is at a minimum, reducing the potential of the frequency of mumps outbreaks ( , , ) . with less opportunity for subclinical boosting (asymptomatic response to the circulating virus), the impact of other elements of waning immunity may play an increasingly critical role in the re-emergence of mumps outbreaks ( , ) . additionally, as the heterogeneous uptake of vaccines in this modern era is leading to susceptible individuals within the community, future work will need to encompass genotyping of circulating muv to examine how impactful subclinical boosting was on early measures of vaccine efficacy in current populations. why do we consider antibodies to be the best measurement of vaccine efficacy? the evolution of an individual's immune response differs between natural infection and vaccination, in particular the difference in the affinity and specificity of an immunological marker such as antibodies ( ) . true correlates of mumps immunity after vaccination have been poorly characterized; to date, there are no reliable correlates of protection from either symptomatic mumps infection (clinical immunity), or individuals previously exposed to muv ( ) . therefore, a serological surrogate/ substitute is used ( ). mumps vaccine efficacy is quantified by a single measure, igg which may not suffice to evaluate the magnitude of the actual humoral response. borgmann et al. ( ) proposed an increase in mumps-specific igg titer in sera as a diagnostic criteria of mumps reinfection ( ) . it has been suggested that vaccinated individuals have modified b-cell responses to muv that allow for the rapid generation of igg antibodies and a blunted or absent igm response ( , ) . in addition, emerging data in simian immunodeficiency virus studies suggests that not all antibody responses are equal, and qualitative features of antibodies may be key to defining protective immune profiles ( ) . despite its use, the correlation to mumps-specific igg concentrations and neutralization titers against the jl virus is poor, suggesting that igg concentrations do not adequately represent a sufficient surrogate correlate of protection ( ) . this is demonstrated in finland; only % of vaccinees had no detectable mumps antibodies after years ( , ) . data from the european sero-epidemiology network (esen ) project in reported that mmr immunization uptake in ireland in was % ( ), however it was also suggested that only - % of -to -year-olds in ireland had detectable antibodies to muv by either natural immunity or immunization ( ) . in , vaccine coverage of medical students in germany was reported to be . % ( ) . in children between the ages of - years, where . % had been vaccinated with the mmr vaccine at least once, only . % showed prevalence of antibodies ( ) . however, . % showed a prevalence of antibodies to measles and rubella in the absence of mumps-specific antibodies. therefore, previous measurement of anti-mumps-specific igg that represented immunity induced by the mumps vaccine appears to be overestimated ( , ) . antibody levels of other components of the mmr vaccine have seen similar trends. waning rubella antibody titers have been observed, despite the number of acute rubella and congenital rubella syndrome cases not increasing. it has also been shown that college students who received rubella vaccination during childhood and had low/no antibody response were able to mount a secondary response when challenged with rubella indicating that an individual's low antibody levels are not always indicative of susceptibility to infection ( ) . measles antibodies can also be detected for up to a decade post-vaccination, with > % of individuals still measles igg positive at - years of age ( , ) . however, as with mumps and rubella, waning measles antibody titers have been observed ( , ) . despite this, a recent longitudinal study of up to years demonstrates how effective the mmr vaccine has been in preventing diagnosed measles cases during the 's/ 's ( ) . similarly, three doses of the hepatitis b (hbv) vaccine in a cohort of alaskan natives showed > % seroconversion in children and young adult post-vaccination and provided long term and durable protection against chronic hbv infection. although no increase of hbv prevalence were observed % individuals had low to undetectable antibody levels after years. these observations suggest that an individual's antibody levels do not indicate susceptibility to infection, that either an antibody titer lower than recommended guidelines is still protective, or/and is an ineffective surrogate of protection. this is emphasized in a study by amanna et al.; ( ) responses to non-replicating protein antigens (tetanus and diphtheria) were shown to have approximate antibody half-lives of - years. in comparison, antibodies following wild type infection were shown to have half-lives of years or more which was thought until recently to confer a more prolonged lifelong protection ( , , ) . however, reinfections observed in individuals that were previously naturally infected have demonstrated that the quantitative measurement of antibodies do not indicate sterile immunity ( ) . it is also important to stress that seroconversion rates due to immunization/natural infection only reflects a change of antibody status from negative to positive, but not necessarily the intensity of antibody response. in addition, there is no consistency in the timing of sample collected post-vaccination to test vaccine efficacy, and between the serological tests utilized for detecting mumps antibodies. as a result, documented seroconversion rates of the mumps vaccines used vary widely (jl: - %, rit strain: - %, urabe am : - %, rubini: - %). this highlights that the assays used to detect immunity to muv may not always detect an adequate post-vaccination response. only a small number of serological commercial assays such as the detection of hepatitis b surface antibody (anti-hbs) ( ) and rubella igg ( ) have been designed using who reference material as a standard for quantification. however, even utilizing this reference standard demonstrates significant differences in the determined quantification of either anti-hbs or rubella igg depending on the assays used; although a value for anti-hbs of iu/ml is regarded as protective against significant hbv infection, the detection of this anti-hbs is significantly influenced by which anti-hbs assays is used ( ) ( ) ( ) ( ) ( ) . therefore, it is possible that the current assays/tests mechanisms utilized to measure mumps antibodies are too insensitive/inappropriate/crude to identify nuances in the immune response which could correlate with immunity against mumps. in addition, variation within neutralization epitopes i.e., the quality of the antibody present could be a more important correlate than quantity ( , ) . though labor-intensive, neutralizing antibodies are considered to be a better correlate of mumps immunity. antibodies against the haemagglutinin-neuraminidase protein (hn) and nucleoprotein (np) have been shown to neutralize muv, however, repeated attempts to define a titer that provides a protective threshold titer have been inconclusive ( , ) . in older studies, during field evaluations of the jl vaccine, neutralizing antibody titers of : - : in unvaccinated individuals was considered seropositive and protective from mumps infection ( , , ) . using a more contemporary wild-type isolate (iowa-g/usa ), a : neutralizing titer cut off was defined between case patients and exposed patients, despite the fact that no cut-off could fully discern between the two groups ( ) . however, that these results are dependent on the challenge virus strain used in the assay. rasheed et al. demonstrated a fold lower neutralization titer to the g-genotype when compared to the jl vaccine strain in - year olds ( ) . this has also been seen between mumps vaccine strains vs. circulating strains in india and china ( , ) . despite studies in more highly vaccinated populations demonstrating that hn-inhibiting titers after natural disease were : compared to : post-vaccination, neither appeared to prevent reinfection ( , ( ) ( ) ( ) ) . there is increasing evidence that the mumps-specific antibody response is broader than neutralization alone ( ) . avidity testing for virus-specific igg has been proposed ( , , ) . individuals who lack measurable mumps-specific antibody levels may be susceptible to infection but protected from significant illness as they may be protected by cell-mediated immune memory. prolonged t-cell responses are reported after other vaccinations; - years after a single dose of the rubella vaccine ra / , a t-cell proliferative response to neutralizing antibodyinducing peptides suggest t helper and b-cell interactions. this indicates that full vaccine effectiveness could be dependent on mounting both an antibody and cell-mediated immune response ( ) . although cell mediated immunity has not been as wellassessed in mumps infection, a lymphoproliferative response was induced in infants vaccinated at , , or months of age was induced ( ) with antigen-specific t-cells reported to appear within month of infection ( ) . lymphoproliferative responses to measles and mumps vaccine viruses were shown to persist in two thirds of the population at least years after immunization ( ), with t-and b-cell immunity persisting for years post-immunization ( ) . low levels of mumps-specific memory b-cells have also been documented suggesting that mumps infection or vaccination may not generate a robust b-cell memory ( , ) . two principal mechanisms for maintaining long-term humoral immunity have been proposed and reviewed by amanna et al. ( ) : associations between memory b-cell levels and antibody may reflect an epiphenomenon in which serum antibody levels and memory b-cells are equally stable but independently maintained. if memory b-cells and plasma cells are independently regulated, then multiple re-exposures to antigens may cause divergence between memory b-cell levels and antibody levels ( ) . antigens with the highest rates of boosting through vaccination or latent viral infection coincidentally showed the weakest association between memory b-cell titers and antibody titers ( ). although the role and efficacy of t-cell immunity to mumps infection is unclear, there is a possibility that certain muv strains may be capable of escaping vaccine induced t-cell responses, which may not be considered of significance until b-cell waning immunity comes into play ( ) . in individuals who did not respond to vaccination (i.e., had a ≤ -fold of mumps antibody titers days post-vaccination), several genes including those implicated in antigen presenting, processing, t-cell response and function showed significantly increased expression, with mhc class ii hla-drb and hla-dra, and cd induced when compared to responders day post-mmr vaccination. this may indicate that the stimulation of a rapid adaptive immune response limits antigenic presentation and hence prevent the differentiation of memory b-cells to antibody-producing plasma cells ( ) . differences in predicted b-cell and t-cell epitopes between jl vaccine strain and other vaccine strains may also be implicated in the outbreaks witnessed ( ) . although, it has also been shown that natural mumps infection or vaccination do not always induce both cellular and humoral immunity. de wit et al. ( , , ) has shown the presence of th -type cd + t-cells recognizing a muv epitope in a hlr-dr restricted manner. in addition, the response of ifn-γ and tnf producing cd + t-cells specific to muv epitopes are lower in vaccinated individuals when compared to individuals who were naturally infected ( , , ( ) ( ) ( ) . utilizing current knowledge and new technologies may help define a better surrogate correlate of protection and potentially determine a cut-off between the immunity of a vaccinated individual and a secondary mumps infection. this may potentially move the diagnostic preference from serological tests to more comprehensive functional assays. despite the large resurgence of mumps outbreaks, there is insurmountable evidence highlighting the benefit of the mumps vaccine ( table ) . routine childhood mmr vaccination has resulted in a dramatic decrease in the incidence of mumps cases, and has shifted the peak age-specific attack rates from a young children (manifesting between and years) to one that affects young adults, in particular those who have close interaction with other young adults ( - years) ( , ) . additionally, clinical manifestations and severity of disease in vaccinated vs. unvaccinated individuals differ ( , ) . although muv can be clinically asymptomatic in about - % of those who become infected, the vaccine against mumps confers protection in a dose response manner; unvaccinated individuals saw an attack rate of based on the reduction seen upon the introduction of a mumps vaccine, it has been proposed that mmr vaccination also prevents the transmission of the virus. there is limited knowledge regarding the shedding and transmission of muv, but it is thought that close contact and transmission of a certain viral load may induce clinical symptoms ( , , ) . modeling data suggests that infectious muv shedding decreases rapidly after the onset of symptoms, however - % are patients are thought to still be virally shedding days after the onset of symptoms ( ) . this could be the reason why the transmission of muv can be exacerbated by close social situations within a heterogeneously vaccinated population. outbreaks generally occur in situations of intense contact such as college dormitories, boarding schools, and youth summer camps ( ) , with up to a third reporting some contact with a mumps case ( ) . evidence of lower levels of viral replication also suggests a clinical benefit of the vaccine ( , ) . viral load and presence of the mumps vaccine genome in areas of viral replication was lower in vaccinated individuals vs. unvaccinated individuals ( ) . in addition, patients who contracted mumps but had two doses of mmr have been shown to shed less muv in their urine, with fewer experiencing bilateral parotitis or orchitis than unvaccinated individuals ( ), this suggests that immunity induced by mmr vaccination limits virus transmission and complications ( , ) . it should be noted also that individuals who received two doses of mmr, and had a positive correlation between viremia, salivary viral loads and systematic clinical mumps infection may have an increased risk of transmitting virus. these individuals also lacked mature functional responses, with low neutralizing antibody titers and avidity indexes ( ) . overall, evidence demonstrates a clinical advantage to receiving a mumps vaccine ( table ) . currently no global consensus exists for the measurement of mumps antibodies, mumps avidity or neutralizing titers that correlate to vaccine response and protection in healthy individuals. if a biomarker is discovered, it could be utilized as an international diagnostic reference standard to allow global harmonization and evaluation of the relative effectiveness of the different vaccination programs worldwide. such an attempt was conducted by andrews et al. ( ) , who reported on the european sero-epidemiology network project which was established to harmonize the seroepidemiology of five vaccine preventable infections including measles, mumps, and rubella in eight european countries. the study concluded that the development of an international standard for mumps would help in the standardization and comparability of mumps antibodies in the different enzyme immunoassays used in laboratories. however, to date, no international reference standard for mumps has been established. in response to infection, the human immune system launches a series of immunological responses with the goal of controlling or eliminating the pathogen. if the pathogen circumvents the frontline defense of the innate immune system, an adaptive immune response specific for the pathogen will become activated to respond, with the intention to generate humoral-and cell-mediated immunity. humoral immunity, represented by antibodies secreted by b-cells are not effective against pathogens that invade host cells. therefore, cell-mediated immunity instructed by the vaccination aims to stimulate the host immunological process and formation of cell-mediated immunological memory via the use of live-attenuated or of inactivated/subunit vaccine components to promote a cell-mediated immune response. extensive knowledge gaps significantly hinder improvements to the mumps vaccine and prospects for mumps eradication and maintaining proficient population immunity ( , , ) . few studies have collected data that examines different aspects of mumps immunity and are limited in their predictive value for future outbreaks ( ) . for example, the importance of t and b-cell responses in protective mumps immunity and how memory/plasma cell numbers are homeostatically maintained post-infection or vaccination is relatively unknown ( ) . it should be acknowledged that the mechanism of protection of infection may not be the same mechanism of recovery from infection, which may make the identification of a common correlate of protection and recovery difficult ( ) . therefore, if a correlate or surrogate correlate is unobtainable to define an individual's protection to mumps, should we re-consider and re-focus efforts on optimizing the vaccine using available historical clinical and trial data? it has been suggested that wild-type infection could confer a "better quality, " broader and prolonged immuno-activation than vaccine-induced immunity. this is reflected in mean neutralizing antibody titers detected post-mumps vaccination, which were over five times lower than those detected following wild type infection. similarly, hemagglutination-inhibiting titers after natural disease were : compared to : post-vaccination ( , , ) . the use of a live-attenuated virus vaccine is intended to mimic immunological reactions and responses between the host and wild type virus ( ) . the current liveattenuated mmr vaccine is intramuscularly injected, a route that significantly differs from the natural infection mode of transmission. however, emphasized by differing immunological kinetics between immunized and naturally infected individuals when subjected to wild type pathogens, injectable vaccines are considered not to be the best inducer of antigen-specific mucosal immune responses for mucosal pathogens, especially if the mode of administration is not the natural route (the respiratory tract) ( , ) . improvements on a broader range of antigen delivery systems will improve vaccination strategies and potentially prolong the effect of a vaccination by producing a localized immunological response in the relevant tissues ( , ) . mucosal vaccines such as intra-nasal vaccination have advantages over traditional injectable vaccines as they can induce an effective, more robust immune response without any physical discomfort and more closely replicate the natural route of infection for mumps ( , ) . b-cells induced by the mucosal response are also capable of secreting iga class of antibodies in the lumen, where the interaction and neutralization of specific antigens form iga-antigen complexes are easily able to be entrapped in the mucus and eliminated by cilial epithelial cells ( ) . activated mucosal lymphocytes can also reach other mucosal sites via the lymphatic system and have the capability to transfer immunity ( ) . such an example is the intranasal immunization of inactivated influenza. with a - % similar efficacy between the injectable and intranasal influenza in healthy individuals this intranasal vaccine can elicit the secretion of haemagglutinin and neuraminidase specific iga antibodies in the upper respiratory tract, and corresponding igg antibodies ( ) . live, cold adapted attenuated nasal influenza vaccine has been routinely used in russia for over years ( ) . other liquid live-attenuated intranasal vaccines are available; "nasovac r " in india, and "flumist r " in the us, uk and new zealand ( , , ) . inactivated vaccines consisting of heat/chemical or liveattenuating monovalent or multivalent pathogens in animals/cell lines were developed to protect against disease causing microorganisms ( ) . less emphasis was placed on understanding the mechanisms related to conferring immunological memory; the focus lay on the availability, mass production and administration of the vaccine to introduce herd immunity into populations ( ) . currently, the least expensive and time effective method to licensure is the comparison of serologic responses of the new vaccine to an existing licensed vaccine, which can lead to a bias on the development of novel vaccines ( ) . this methodology also does not account for the fact that each vaccine developed elicits its own immunological signature and may need to be considered on an individual basis ( ) . raymond et al. ( ) has suggested that embryonated chicken egg-based vaccines may induce antibodies that are more preferential to egg adapted strains better than wild type virus. amino acid substitutions/differences in key antigenic targets due to the passage of the growing virus within this environment may optimize the growth of the virus, but could lead to differences over time that could affect the immunogenicity or potency of the vaccine ( , , ) . the jl vaccine contains two isolates of the jl strain (jl and jl ) and whilst no immunological differences have been documented, jl grows to higher titers than jl in embryonic eggs and also demonstrates significant sequence variability ( , ) . zost et al. ( ) also demonstrated that an egg selected mutation within a glycosylation site in the - influenza vaccine strain led to the production of poorer neutralizing antibodies to the vaccine strain compared to wild type influenza virus. vaccine rit strain derived from one of the two distinct virus subtypes of the jl vaccine (jl ) showed comparable seroconversion rates despite inducing a significantly lower geometric mean antibody titer when compared to recipients of the jl vaccine, but does not have any longitudinal trials investigating its efficacy, even though there are populations who are currently receiving it ( , ) . the significant time gap between pathogen emergence and vaccine licensure, could potentially lead to antigenic drift. there is potential that modern biotechnologies could be utilized to design novel vaccine platforms ( , , ). clinically derived recombinant muv lacking the expression of the immunomodulatory v or sh protein are currently being investigated ( ) . in china, a vaccine consisting of the prevalent wildtype virus genotype (f) has recently been produced and is currently undergoing trials ( ) . in addition, despite being extremely pleomorphic, utilizing mhc epitopes as potential b-cell and t-cell vaccine candidates are also being investigated ( , , ) . vaccine design has involved the utilization and templating of epitopes that previously induced a b-or t-cell response during natural disease that are considered to be immunogenic enough to induce similar responses if administered in a vaccine. however, the appropriate b-cell and t-cell epitope/peptide candidates to induce a protective immunological response can be difficult to correctly identify and synthesize, as it may differ to the immunodominant epitope and host presentation of that antigen ( , ). prediction of mhc-peptide binding and cleavage has demonstrated mismatches in both vaccine tcell and b-cell epitopes in vaccinated individuals highlighting small number of distinguishing amino acid changes of the jl major strain ( ) . the importance of understanding tand b-cell responses and how antigen-specific memory cells numbers are homeostatically maintained post-infection is crucial to understand to ensure successful vaccine development ( , ) . since the 's, significant progress has also been made in developing flexible, amplifiable, scalable, inexpensive, and cold-chain free rna vaccines, such as synthetic mrna molecules encoding only the antigen of interest and selfamplifying rna (sa-rna) ( ) . such examples include an experimental mrna vaccine candidate (mrna- ) which encodes a stable form of the sars-cov- spike protein and has been accepted as a trial candidate for clinical trials in healthy male and female individuals ( , ) . in addition, sa-rna viruses as gene delivery and vaccine vectors have also demonstrated therapeutic efficacy in a number of preclinical studies. in the context of influenza, sa-rna vaccines have shown comparable results of protection at lower doses than mrna vaccines ( , , ) . exponential developments in the "omic" area has enabled further vaccine development and understanding of the immunological response and challenges surrounding this area ( ) . systems vaccinology, which includes immunoformatics, dna/rnaseq, microarrays, mass spectrometry proteomics, transcriptomics, and metabolomics have all shown huge potential in elucidating differences in vaccine strains, vaccine growth and individual response in depth and on an epigenetic level allowing the identification of new vaccine antigens with increased speed and sensitivity ( , , ( ) ( ) ( ) . adjuvants, a group of biological and chemical compounds could also be considered to enhance and improve the longevity of the immune response of a vaccine such as the mmr. adjuvants have been successful in significantly reducing overall antigen dose in vaccine formulations as well as alter and broaden the host response through epitope spreading and qualitatively shaping the effector function of antibodies through subclass selection ( , ) . the re-purposing of live-attenuated vaccines as tibv are also being investigated. trained immunity based vaccines (tibv) elicit heterologous protective effects by inducing a broader, lasting priming of innate immune cells, in addition to the intended specific immunological response and memory of conventional vaccines [reviewed in ( ) ]. mmr and bcg vaccines have been considered as potential tibv in the context of the current coronavirus disease (covid- ) pandemic ( ) , however further research is needed. the mumps component of a vaccine is an unpurified product whose potency is measured through a biological assay for the substance rather than through evaluation of integrity of physical form (quantitative pcr after cell culture) ( ) . a monovalent mumps vaccine lot is used to characterize the performance of the mumps potency assay with international reference standards. degradation products are neither identified nor quantified ( ) . currently, the minimum potency of the mumps vaccine used varies between brands used [summarized by su et al. ( ) ] ( ) . however, this potency measurement differs to other mmr vaccines strains previously used [reviewed in ( ) ]. in addition, the maximum required potency is not usually specified. atrasheuskaya et al. ( ) demonstrated that the four out of lots of vaccine associated with six cases of viral transmission postvaccination to previously vaccinated contacts were in fact twice as potent as the lots that were not associated with viral transmission post-vaccination ( , ) . this may impact the use and efficacy of specific vaccines. due to their neurovirulence and increased incidence of aseptic meningitis and mumps cases, the urabe am and rubini mumps vaccine strains were discontinued in many countries ( , , ) . comparing alternative culturing technologies and defining a viral potency range for vaccines could help reduce variability within the mmr vaccine ( ) . ensuring the use of a reference sample that had similar replication rate and composition as the virus to be tested will allow accurate determination of the quantity of virus present per lot of vaccine. investigating novel vaccine candidates shown to induce a similar quantity but qualitatively different antibodies will help segregate and reveal potential correlates of protection ( ) . incorporating more modern technologies such as microarray technology or antibody pattern/profiling (rather than single antibody measures) to investigate biomarkers of neutralizing antibody response and/or correlates of protective immunity, in addition to incorporating what has been accomplished in finland will allow further understanding of mumps immunity ( , , , , , ) . the efficacy of a vaccine is defined by disease prevention (sterile immunity, establishment of primary infection and shedding of mature virus particle), or complications associated with infection (orchitis, neurological issues etc.) ( ) . despite the well-documented success of the global immunization programs demonstrating how vaccines significantly attenuate disease and onward transmission of infection, they are rarely totally efficacious (demonstrated in pre-licensure clinical trials) or effective (determined by practical use) ( , , ) . therefore, does "immunity" refer to sterile immunity or solely to protection from symptomatic infection? what defines an effective vaccine, or what constitutes vaccine failure? does the medical profession and the "pro-vaccine" message contribute to the public skepticism regarding immunization? is it time to shift the medical and public perception paradigm from "protection of infection following vaccination" to "protection from serious clinical mumps manifestation"? the lack of definition leads to misinterpretation by health professionals and media of what is truly occurring. such an example is currently observed with influenza; individuals who have recently being vaccinated against influenza and subsequently become infected with influenza, assume that the vaccine has "failed" even though there is a reduction in symptoms. the current assertion that vaccines "protect against" or "eliminate" the risk of infection may contribute to the misperception about what level of protection a vaccine actually provides (vaccination efficacy) perpetuated by the witnessing of visible clinical disease and outbreaks despite vaccination ( , , ) . therefore, definition and consensus of what is termed a true "vaccine failure" is required to inform both the clinical and public perception of what the function of a vaccine is. deciding what the clinical endpoint of a vaccine is i.e., infection with mild clinical symptoms vs. natural infection/disease with its associated complications and assessing the impact of the vaccine in a heterogeneously vaccinated population will allow a better consensus of what is required. a paradigm shift in what is considered to be a good vaccine i.e. one that provides protection against serious clinical sequalae, in addition to identifying a reliable laboratory marker for this protection is required ( ) . by focusing on, and acknowledging that vaccines may not prevent infection but will attenuate the clinical complications/consequences that arise from infection in addition to reducing onward transmission will provide a more realistic view of the benefits of vaccination ( ) . immunity is therefore beneficial but does not necessarily mean protection. if we can decide whether the end point of a vaccine is either the prevention of infection or protection against serious sequalae of infection, its efficacy and impact can be determined and will have enormous implications on how vaccine failure can be studied, quantified and interpreted. this teasing out of the immunological response to muv will ultimately provide potential correlates with robust predictive power, suggest directions for further vaccine improvement, and enable the discovery of potential biomarkers to help create a more efficient diagnostic assay that can discern between different infectious diseases and vaccination vs. disease status. the identification and incorporation of a correlate into diagnostic protocols which can be widely accessible may potentially allow global harmonization of criteria defining immunological protection against mumps. the medical and scientific field needs to inform the public more accurately about what a good vaccine consists of, which may result in a more positive attitude toward vaccines. in the majority of individuals, a vaccine can prevent serious clinical sequalae and associated complications following wild type infections, but also significantly reduce onwards transmission in particular to the cohorts who are not vaccinated due to a contraindication to vaccination. this is the positive and realistic view of vaccination which should be presented rather than the current flawed message of "get the vaccine and be protected from infection." the public deserves, and will appreciate, a more accurate and informed message. ac, jc, and jh contributed to the conception and design of the review. ac wrote the first draft of the manuscript. jc, tl, and jh contributed to manuscript revision. all authors have read and approved the submitted version. this work was 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severe acute respiratory syndrome coronavirus (sars-cov- ) is the third coronavirus leading to a global health outbreak. despite the high mortality rates from sars-cov- and middle-east respiratory syndrome (mers)-cov infections, which both sparked the interest of the scientific community, the underlying physiopathology of the sars-cov- infection, remains partially unclear. sars-cov- shares similar features with sars-cov- , notably the use of the angiotensin conversion enzyme (ace ) as a receptor to enter the host cells. however, some features of the sars-cov- pandemic are unique. in this work, we focus on the association between obesity, metabolic syndrome, and type diabetes on the one hand, and the severity of covid- infection on the other, as it seems greater in these patients. we discuss how adipocyte dysfunction leads to a specific immune environment that predisposes obese patients to respiratory failure during covid- . we also hypothesize that an ace -cleaved protein, angiotensin - , has a beneficial action on immune deregulation and that its low expression during the sars-cov- infection could explain the severity of infection. this introduces angiotensin - as a potential candidate of interest in therapeutic research on cov infections. coronavirus (cov) is a single-stranded rna virus involved in human and animal diseases. the rare event of its transmission from avian and mammalian reservoirs (mostly bats) to the human host has led to widespread epidemics in recent years ( ) . indeed, over the last two decades, three cov outbreaks have forced human populations to change their perspectives regarding the control of emerging diseases and the importance of public health in general. the first outbreak caused by severe acute respiratory syndrome coronavirus (sars-cov- ) occurred between november and july , originating from china and then spreading worldwide ( ) . although the symptoms of sars-cov- infection were in most cases non-specific, including lethargy, myalgia, and headache, the high mortality rate of % in case series was mostly related to respiratory failure due to acute respiratory distress syndrome (ards) ( , ) . the physiopathology underlying the severity of sars-cov- infection remained unclear after the epidemic due to insufficient sampling. a second cov epidemic occurred in with middle east respiratory syndrome (mers)-cov, which has mostly led to small-size outbreaks in the years ever since ( ) . although it did not reach a pandemic status, mers-cov continues to infect humans, and the world health organization identified more than patients who have died of related complications since its discovery ( ) . indeed, mers-cov has a higher mortality rate in case series (case fatality rate of ∼ %), mostly from respiratory failure, which has led to the identification of unique strategies of cov infections to escape the immune response. due to the ending of the sars-cov- epidemic and the somewhat limited number of cases of mers-cov in the recent years, understanding the mechanisms of cov infections in humans has proven to be complex, and the conclusions drawn from in vitro experiments and animal models remain difficult to extrapolate. in november , cases of a pneumonia with atypical features were reported in wuhan, china; in january , sars-cov- was identified as the cause of this new cov-induced disease , which became a worldwide pandemic in the following months ( ) . although the mortality rates of this new covid- are still being debated, ranging between . and . %, it is still lower than those associated with sars-cov- and mers-cov infections. patients suffering from severe sars-cov- infection could be healthy or only have mild comorbidities such as hypertension or diabetes ( ) . most of all, severe cases due to respiratory failure occur - days after the first symptoms ( ) . studies on covid- have progressively stressed its similarities with previous cov infections, mostly sars-cov- , with the same unanswered questions regarding its physiopathology. one notable feature of this disease, already observed in previous cov infections, is the high prevalence of obese patients among the most severe cases. here we seek to explore what underlies the link between immune response and respiratory failure in cov infections on the one hand, and the current observation of obesity as a risk factor for severe outcome in covid- on the other. most of the time, the need for intensive care during covid- is secondary to the onset of ards ( ), as defined by the berlin criteria (bilateral shadowing on lung radiology, rapid deterioration of symptoms, and objective hypoxemia on blood samples). in the first published series, % of these ards cases were accompanied by septic shock or other organ dysfunction ( , ) . the nature of covid- -induced ards is still under discussion. interleukin (il) dosages are usually very high, and hypoxemia is severe in covid- -induced ards, which matches the hyperinflammatory profile described by calfee et al. ( ) . sars-cov- -induced ards was associated with vascular leakage and neutrophilic alveolitis ( ) , both of which are compatible with a hyper-inflammatory profile. in covid- , some experts observed ventilatory abnormalities suggestive of microcirculatory involvement such as hypoxic pulmonary vasoconstriction or distal thrombosis ( , ) . this points to the contribution of several factors in respiratory failure, with experts also citing the possible involvement of genuine viral pneumonia as well as capillary thrombosis by neutrophil extracellular traps (nets) ( ) . the reason for this respiratory outcome is most likely a complex interplay of multiple factors, which derive directly from cov virulence. the membrane protein angiotensin-converting enzyme (ace ) is used as an entry receptor by sars-cov- and sars-cov- ( , ) . it has been reported that sars-cov- has a greater affinity for ace than sars-cov- due to the specific amino-acid composition in the receptor-binding domain of the spike protein ( ) . ace is expressed at varying levels by most cells in the body but primarily in the small intestine, testis, kidney, heart, thyroid, and adipose tissue cells ( ) . the expression of ace in adipocytes seems to be promoted by high fat diets ( ) . in the lungs, it is expressed by % of epithelial cells, increasing with cell differentiation, and it is mainly located on the apical (or luminal) pole, serving as an accessible anchor point to airborne contaminants ( ) . ace is a key enzyme of the renin-angiotensin system, converting angiotensin (ang ) into ang - . ang binds to a receptor, the angiotensin type receptor (at r), a transmembrane g protein-coupled receptor, which is found in a large variety of cells, ranging from smooth muscle cells, endometrium, and myocardium to blood cells, renal interstitial, and glomeruli. the activation of at r has several effects: for example, vasoconstriction, vascular permeability, macrophage maturation, and pro-inflammatory cytokine release. during the resolution phase of the inflammation, ang promotes tumor growth factor beta production and fibroblast proliferation, leading to fibrosis and inadequate healing of the wounded tissue ( ) . an antagonistic pathway of the ang -derived effects results from the binding of ang - to the mitochondrial assembly (mas) receptor. mas receptor is a ubiquitous g-protein-coupled receptor, implicated, among others, in retina development ( ) , muscle wasting ( ) , and benign prostate hyperplasia ( ) . activation of the mas receptor by ang - induces vasodilatation by a nitric-oxide-dependent mechanism ( , ) and reduces oxidative stress induced by ang in vascular injuries ( ) . in macrophages, it promotes an anti-inflammatory profile ( ), for example, by lowering pro-inflammatory cytokine production, notably il- and tumor necrosis factor alpha (tnfα). ang - has also shown beneficial effects in inflammation resolution and fibrosis, notably in kidney and myocardial disease ( , ) . the binding of ace by sars-cov- prevents it from exerting its enzymatic activities, resulting in decreased anti-inflammatory ang - production and the accumulation of pro-inflammatory ang ( , ) . this results in high cytokine titers, neutrophil infiltration, and endothelial dysfunction in the lungs, potentially predisposing for ards. as early as , ace tampering was suggested to be an important mechanism in sars-cov- infection ( , ) . it was only later discovered that cov possesses very specific mechanisms to escape the host's immunity ( ) . these mechanisms, in addition to the pro-inflammatory response secondary to ace binding, might act as a trigger for a sustained and uncontrolled inflammatory response, leading to ards. in general, an efficient antiviral response is driven by t-helper lymphocytes (lth) with a specific polarization such as lth and lth . lth refers to a polarization in which lth primarily promotes cytotoxic lymphocytes (ctl) and natural killers (nk) for the control and destruction of infected cells as well as the release of specific cytokines, such as type interferon (inf- ) by innate immune cells. inf- is produced by infected cells and innate immune cells after recognizing the viral pathogen-associated molecular patterns (pamps), such as single-strained or uncapped rna, using cytoplasmic pattern-recognition receptors (prr). in particular, toll-like receptor (tlr ) induces toll/interleukin- receptor domain-containing adapter-inducing interferon-β (trif). hosts deficient in either tlr or trif are more susceptible to viral injuries and thus more at risk of developing ards during cov infections ( ) . inf- activates the janus kinase-signal transducers and activators of transcription (jak-stat) pathway, resulting in the modulation of hundreds of interferon-sensitive genes and notably in the synthesis of specific cytokines, preferably oriented toward viral control and clearance ( ) . most of these steps, involved in inf- signaling, are blocked by cov infections. this evolution trait is probably due to the presence of a constitutive inf- production in bats (principal reservoir of cov). cov infections are expert evaders of this antiviral response ( ) . their escape plan revolves around three main mechanisms: -first, hiding viral rna from cytoplasmic prr. after entering the cell, sars-cov- shields its rna by forming, inside the host's endoplasmic reticulum, a large network of doublemembrane vesicles isolated from the cytosol ( , ) . the modified capping of the viral rna ′ -o-methylation also prevents the binding to an important cytosolic prr ( ) . -next, direct tampering of the prr-related enzymes. for example, the papain-like protein in cov can modify the ubiquitinylation profile of tlr ( ) or other antiviralrelated prr ( ) . moreover, s protein triggers il- rassociated kinase and peroxisome proliferator-associated receptor gamma, subsequently downregulating interferon regulatory factor activity ( ) . in addition, the jamming of tlr phosphorylation reduces the prr activity, while blocking most of the inf- production pathways. -lastly, the non-structural protein in both mers and sars-cov- can selectively degrade host rna via endonucleolytic activity against which the viral rna is protected ( , ) . the many mechanisms used by cov probably leave the infected cells in a defensive cul-de-sac where they are incapable of developing an efficient antiviral response. on the one hand, viral pamps do not result in inf- production. on the other hand, non-viral pamps such as debris from cell lysis still stimulate the immune response. this could lead to inappropriate cytokine environments that lack inf- and are thus less effective against viruses, as seen in covid- ( ) . indeed, during covid- infection, most patients exhibit a specific cytokine profile, associating innate immunity chemokines (such as monocyte chemoattractant protein and interferon gamma-induced protein (ip- ), which are suggestive of macrophage activation and epithelial suffering), and pro-inflammatory macrophage-produced cytokines such as il- ( ). moreover, cov infections can directly induce the activation of nuclear factor kappa b (nfkb), notably by tampering with the tnf receptor-associated factor pathway (traf ) via its open reading frame a. activation by ubiquitination of traf also promotes the de novo development of the nod-like receptor pyrin domain containing protein (nlrp ) inflammasome and the production of il- β and il- ( ) . this cytokine production promotes macrophage activation and inf- , although it does not salvage a deficient polarization of the adaptive immunity toward lth and its subsequent efficient antiviral response. high plasma levels of il- and the absence of inf- have been noted in severe patients ( ) , illustrating a sustained innate response that fails to achieve viral clearance and triggers ards. however, this sustained inflammation without lth polarization might not be the only profile to bypass the antiviral cul-de-sac. some patients infected by mers-cov demonstrated a polarization of the immune profile toward a lth -mediated response. faure et al. compared two cases of mers with different outcomes ( ) ; the patient with a fatal outcome had an early increase in il- and il- titers (hallmarks of lth polarization), whereas the surviving patient had a spike in inf- but no indication of lth polarization. lth are effective actors in the clearance of extracellular microorganisms such as fungi and bacteria, but poorly effective against viral pathogens ( ) . in general, viral pamps do not usually polarize the immune response to lth . the association of severe outcome and inappropriate cytokine environment in cov infection suggests a link with immune polarization, as a result of the "cul-de-sac" of antiviral response induced by the cov escaping strategies. the resulting inefficient immune profile leads to a sustained viral exposure and persistent inflammatory state. in addition to the pro-inflammatory signals mediated by ace inhibition, this sustained and inappropriate immune activation might be strongly involved in the development of ards. obesity is a common condition, affecting up to % of adults in western countries. it is defined by a body mass index (bmi) > kg/m , irrespective of the location of the adipose tissue. however, all profiles of obesity are not equivalent in terms of their consequences. indeed, abdominal (or visceral) obesity (estimated by the waist circumference or waist-to-hip ratio), in which visceral fat predominates, is more associated with metabolic disorders such as type diabetes or hypertension, compared to "metabolically healthy" obesity, in which subcutaneous fat predominates. early observations in the sars-cov- epidemics suggested obesity to be a risk factor to covd- , or at least to severe forms of the disease ( ) . in our retrospective cohort, we observed more than % of patients with overweight or obesity (n = ) (figure ) . in a retrospective cohort, simonnet et al. showed an increasing risk of intensive care unit (icu) admission in covid- patients as bmi increased, independently of other metabolic disorders ( ) , which was subsequently confirmed by other teams ( , ) . thus, obesity appears to be a risk factor for presenting a severe form of covid- . it should be mentioned that once in icu, obesity is known to confer a survival advantage, termed the "obesity paradox" ( ) . patients with a bmi > kg/m seem to survive mechanical ventilation and severe septic states significantly better than patients with a normal or low bmi ( , ) , presumably due to their elevated muscle mass, which represents a metabolic reserve in the hypermetabolic state of icu patients ( , ) . it is not yet known whether once admitted to icu, obese covid- patients also present a better prognosis than patients of normal body weight. the scientific observations of the last two decades have placed obesity in a complex pathological framework centered around the deregulation of adipocyte, which is far from the naive idea of a simple diet-induced condition ( ). white adipose tissue (wat) is now recognized as an independent endocrine organ, whose main role is to regulate and store the energy provided by food. however, the hormones released by wat, specific to the adipocyte and known as adipokines, reach a large variety of organs and modulate an extensive range of functions, from appetite control to inflammatory response ( ) . leptin is the leading adipokine, whose anorexigen properties regulate satiety and food intake. leptin levels in blood are proportionate to the amount of wat and increase with bmi. interestingly, the leptin receptor (lepr) on immune cells mostly activates jak-stat and nfkb dependent pathways, except in neutrophils, macrophages, and antigen presenting cells, which all express a particular form of lepr. leptin promotes migration in the wat of resident macrophages and induces their polarization toward a pro-inflammatory profile or a classical activated macrophage (m ) profile, and unbalances the lth profiles, by reducing regulatory t-cells and promoting lth polarization ( ) . adiponectin is another adipokine, whose levels increase in proportion to subcutaneous fat but decrease with visceral fat accumulation. it favors whole-body insulin sensitivity, fatty-acid oxidation and diminishes the hepatic neo-glucogenesis pathways ( ) . adiponectin promotes primarily lth polarization, hence antiviral inflammation. other adipokines, such as lipocalin- , down-regulate inflammatory lth altogether by promoting regulatory lymphocytes. adipokines form a large family regularly counting new members over the last few years, all of which reveal complex and multiple implications in the regulation of energy storage and release, adipose tissue regulation and rather ubiquitous cellular metabolism ( ) . unlike subcutaneous fat, visceral fat accumulation, also described as "abdominal obesity, " is characterized by a dysfunctional profile of adipokines associated with a rise in pro-inflammatory signals. the triggers of this dysfunction is believed to be a metabolic stress in the presence of nutrient excess and a hypoxic stress caused by hypertrophic visceral adipocytes, due to an increase in cells' size and low neovascularization, via a mobilization of hypoxia inducible factor ( ) . unlike visceral fat, subcutaneous fat expansion is hyperplasic and is not correlated with low-grade inflammation ( ) . in severe abdominal obesity, the adipokine profile is unbalanced in favor of leptin production and low-grade inflammation at the expense of adiponectin, or lipocalin- . this deregulation of the adipokine profile links various disorders associated with metabolic diseases, such as insulin-resistance, to inflammatory manifestations, as described in rheumatoid arthritis ( ) . ang - takes an active role in regulating the effects of adipokines. its involvement was reviewed by lelis et al., with an exhaustive approach and emphasis on other adipokines that will not be described here, such as sirtuin and resistin ( ) . a strong interest in ang - has already arisen from these observations, particularly in the field of atherosclerosis and non-alcoholic fatty liver disease, in which ang - seems beneficial. in a concise article, mori et al. hypothesized that the disruption of the reninangiontensin system by the virus could impair the energetic functions of these pathways during sars-cov infections ( ) . we suggest that the tampering with such pathways could also lead to abnormalities in the inflammatory response observed in severe cov infections through their influence on immune regulation and cytokine production. adipocyte dysfunction in visceral fat is correlated to lowgrade persistent inflammation, known as meta-inflammation, which is suspected to be the starting point or an early factor in metabolic disorders associated with severe obesity ( ) . this meta-inflammation is mostly driven by the leptinactivated m macrophages in wat. wat-resident macrophages exhibit pro-inflammatory behavior, producing il- β, il- , and tnfα. the precursor of il- β is cleaved into bioactive il- β by the nlrp inflammasome, as a result of the nfkb pathway activation, which is induced by both pro-inflammatory and hypoxic signals originating from the adipocytes ( ). adiponectin can inhibit nfkb activation, but as mentioned above, depending on the obesity severity and profile, the effects of adiponectin can easily be overwhelmed by those of leptin ( ) . leptin also polarizes hematopoiesis directly in the bone marrow, promoting granulocyte, and erythroblast lines (the latter probably acts as a protective mechanism against hypoxia) at the expense of lymphocytes ( ) . when neutrophils are mature and circulating, leptin also promotes their survival on a dosedependent scale ( ) . higher levels of neutrophils have thus been observed in obese patients, possibly making the neutrophil recruitment during an inflammatory process more potent than in patients with a normal bmi ( ) . besides suffering from a pro-inflammatory environment, which favors macrophage activation and neutrophil production, obese patients exhibit abnormal responses to viral infection. as summarized by honce et al., during influenza infections, obese patients tend to have greater neutrophil activation and net development, contributing to capillary damage and thrombosis. such phenomena have been extensively found in covid- patients ( ) . their inflammatory response is also characterized by a lack of inf- production as well as a strong cytokine production, notably il- , ip- , and type inf, which are elevated in severe covid- . interestingly, patients with visceral fat accumulation also tend to have a lower tlr expression in adipocytes, muscle cells, and adipose tissue-resident macrophages, as well as a concomitant lower production of cytokines following exposure to viral pamps ( ) ( ) ( ) . this suggests that their baseline profile resembles that found in severe cov infections, in which the antiviral response is less efficient, but the overall inflammation is higher than in other viral infections. finally, both obesity and metabolic disorders are associated with vascular dysfunction. at the acute phase of lung infection, this could result in microcirculatory abnormalities, as suggested by intensive care physicians, and increased lung edema. patients with visceral fat accumulation, type diabetes ( ), and hypertension are not the only subjects at a higher risk of severe sars-cov- infection. when considering metabolic disorders separately, diabetes, non-alcoholic liver disease, and obstructive sleep disorders have been recently reported as risk factors for a severe outcome ( ) ( ) ( ) . this suggests that the metabolic dysfunction associated with these disorders more than obesity alone might be involved in the severity of the disease in these patients. when comparing the effects of ang - and the inflammatory environment of patients with adipocyte dysregulation and metabolic disorders, an interesting pattern emerges. all the immunological features arising from the adipocyte dysfunction-(i.e., m macrophage polarization with il- and tnfα production), and neutrophil promotion-may contribute to the development of ards and thus be countered by the activation of the ang - /mas receptor axis. ang - also favors figure | impact of severe acute respiratory syndrome coronavirus (sars-cov- ) on pathways promoting acute respiratory distress syndrome (ards). by inactivating the angiotensin conversion enzyme (ace ), sars-cov- leads to an accumulation of angiotensin and a lower dosage of angiotensin - , respectively resulting in the higher promotion and lower inhibition of pro-inflammatory signals. a strong capillary barrier and a beneficial oxidative profile, which are altered in patients with visceral fat activation and could help to prevent ards. this leads us to two hypotheses: either patients with metabolic disorders, primarily visceral fat accumulation, have a constitutional lower titer of ang - , as suggested by some observations ( ) , and a resulting higher inflammation; or the ang - levels in these patients are preserved and restrain the baseline inflammation. in the first case, the inappropriate inflammatory response, added to the diminished activation of tlr in obese patients, leads to unrestrained inflammation. however, if ang - is present in these patients and limits the meta-inflammation, acting as a guardrail, the antagonization of ace by sars-cov- and in addition to the lack of de novo ang - production could exacerbate the meta-inflammation and contribute to the severe septic states of obese patients with covid- , as illustrated in figure . in both cases, the supplementation of ang - in these patients might improve fitness upon sars-cov infection. ace deficiency has already been explored by some research teams to better understand the potential metabolic benefits of conversion enzyme inhibitors used in hypertension, among others. their studies highlighted the association between ace deficiency and higher titers of pro-inflammatory cytokines in obese mice, as well as in mice with glucose intolerance ( ) , which is closely correlated with metainflammation ( ) . other studies correlate ace deficiency with epicardial inflammation ( ) . this suggests that the ang - /mas axis allows a better control of inflammation in obese patients. tlr is a receptor to lps and leads to nfkb activation and (among others) hepatic inflammation. when administered orally to rats fed with a high-fat diet, ang - lowered hepatic inflammation, notably through a modulation of a metabolic pathway involving tlr ( ) . moreover, promoting the effects of the ang - /mas receptor axis using medication also improves the aforementioned cytokines and oxidative stress in obese mice, with a protective effect against diabetic cardiomyopathy ( ) . ang - is already in the spotlight of scientific research given its beneficial effects in preventing the development of metabolic disorders and obesity ( ) . we believe that our literature review highlights the beneficial effects of ang - on metainflammation in preexisting obesity and its potential involvement in inflammatory response and viral clearance, notably against sars-cov- . modulation of the renin-angiotensin system has been mentioned by others to explain the severity of covid- . a recent study found a lower mortality and intubation risk during covid- among elderly patients treated with nifedipine or amlodipine ( ) , although the study sample was small and most of the accessible data do not suggest a strong connection ( , ) . however, these drugs interfere with at r and not with the genuine production of ang - . in obese patients with covid- , this hypothesis should be considered. oral or parenteral ang - supplementation could be a therapeutic option to diminish the low-grade systemic inflammation due to adipocyte dysfunction and attenuate the severity of ace -mediated injuries consecutive to sars infection. parenteral ang - has already been used in human research on account of its property to enhance acetylcholine-mediated vasodilatation in endothelia, with safe outcomes ( ) . covid- is a viral disease with remarkable characteristics given its high severity in obese patients and its ability to tamper ace metabolism. we believe that more than being just an incentive to accelerate research on viral infection, covid- also presents an opportunity to respond to questions that were previously considered to be too intricate or complex, such as non-septic inflammation or the immune system communication underlying metabolic disorders. understanding the multiple and interrelated factors linking sars-cov- infection, angiotensin metabolism, global inflammation, and metabolic disorders such as type diabetes and obesity should provide us with a better insight into the way in which these conditions and 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insights into covid- renin-angiotensin-aldosterone system inhibitors in patients with covid- favorable vascular actions of angiotensin-( - ) in human obesity acknowledgments authors thank mrs. victoria grace, from english publications, for the careful editing of the publication. we kindly thank p. audoin, c. clape, a. metz, and i el amrani for their support regarding the reglementary procedures. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © méry, epaulard, borel, toussaint and le gouellec. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -b zev zb authors: sobocińska, justyna; roszczenko-jasińska, paula; ciesielska, anna; kwiatkowska, katarzyna title: protein palmitoylation and its role in bacterial and viral infections date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: b zev zb s-palmitoylation is a reversible, enzymatic posttranslational modification of proteins in which palmitoyl chain is attached to a cysteine residue via a thioester linkage. s-palmitoylation determines the functioning of proteins by affecting their association with membranes, compartmentalization in membrane domains, trafficking, and stability. in this review, we focus on s-palmitoylation of proteins, which are crucial for the interactions of pathogenic bacteria and viruses with the host. we discuss the role of palmitoylated proteins in the invasion of host cells by bacteria and viruses, and those involved in the host responses to the infection. we highlight recent data on protein s-palmitoylation in pathogens and their hosts obtained owing to the development of methods based on click chemistry and acyl-biotin exchange allowing proteomic analysis of protein lipidation. the role of the palmitoyl moiety present in bacterial lipopolysaccharide and lipoproteins, contributing to infectivity and affecting recognition of bacteria by innate immune receptors, is also discussed. geranylgeranyl cysteine thioether h-and n-ras ( ) rab proteins ( ) attachment of glycosylphosphatidylinositol anchor or phosphatidylethanolamine ( ) to the c-terminus of proteins is also a form of lipidation but is not shown here. a n-myristoylation is in most cases co-translational, but during apoptosis caspases can cleave some proteins, such as bid, exposing their n-terminal glycine residue, which is then modified by attachment of myristate ( ) . b hedgehog proteins are additionally modified by covalent attachment of cholesterol to their c-terminus ( ) . c o-acylation of wnt proteins is reversed by notum of the α/β hydrolase superfamily ( ) . exceeds the consumption of other saturated fatty acids and, in the usa it accounts for about % of the total intake of saturated fatty acids ( ) . a growing body of experimental and clinical evidence points to a link between a westernized diet, including a high intake of saturated fatty acids, and chronic inflammatory diseases ( ) ( ) ( ) . as dietary saturated and unsaturated fatty acids apparently modulate activity of immune cells, their influence on the immune responses triggered upon infection is also beginning to be investigated ( ) . these facts drive the interest in palmitic acid with an aim of elucidating the molecular mechanisms of its immunomodulatory properties. in this review, we focus on s-palmitoylation of proteins crucial for the interactions of pathogenic bacteria and viruses with the host. we emphasize novel data on the role of s-palmitoylated proteins in the invasion of host cells by pathogens and those involved in the host innate immune responses to the infection, which have been obtained thanks to the application of new technical approaches. recently, substantial progress in the understanding of protein palmitoylation was made possible by the development of methods allowing high-throughput analysis of cellular/tissue palmitoyl proteomes. we begin, however, by showing how unique protein s-palmitoylation is among other protein lipidations. s-palmitoylation is a posttranslational modification of proteins consisting in a potentially reversible covalent attachment of palmitoyl chain to a cysteine residue(s) of proteins through a thioester bond ( table ) . thus, s-palmitoylation resembles other reversible regulatory posttranslational protein modifications, including phosphorylation or acetylation, well-established factors affecting protein structure and functions. in particular, s-palmitoylation modifies cellular localization of proteins and their stability. the most dramatic changes of localization concern cytosolic proteins which upon s-palmitoylation acquire a hydrophobic anchor facilitating their docking into membranes (figure ) . however, several integral membrane proteins also undergo s-palmitoylation. it often occurs on cysteine residue(s) located in the proximity of the junction of the transmembrane and cytoplasmic domains of the protein. s-palmitoylated transmembrane proteins occupy various cellular compartments, such as endoplasmic reticulum, golgi apparatus, and the plasma membrane. in accordance, for some proteins, such as transmembrane adaptor proteins in leukocytes, s-palmitoylation was found secondary to the length and hydrophobicity of the transmembrane domain as a determinant of plasma membrane destination ( ) . s-palmitoylation also contributes to the compartmentalization of proteins to distinct domains of membranes-rafts and tetraspanin-rich microdomains. in fact, the interest in s-palmi toylation was boosted when it was found to be required for the targeting of some signaling proteins to rafts. rafts are nanodomains of the plasma membrane and some intracellular membranes, mainly of the trans-golgi apparatus, rich in sphingolipids, glycerophospholipids with saturated fatty acid chains, and cholesterol ( ) . the plasma membrane nanodomains are sites of signal transduction by distinct receptors of immune cells involved in both acquired immune reactions, such as t cell receptor (tcr), fcε receptor i, fcγ receptor ii, and in innate immune responses, such as toll-like receptor (tlr ) ( , ) . rafts are also sites of virion assembly and budding, as established, e.g., for influenza a virus and human immunodeficiency virus- (hiv- ) ( , ) . peripheral membrane proteins acylated with saturated fatty acids are likely to anchor preferentially between the ordered saturated lipids of rafts rather than between the disordered lipids of the surrounding membrane. it has been shown that, owing to their raft localization, s-palmitoylated kinases of the src family interact with raft-associating plasma membrane immunoreceptors and initiate signaling cascades fundamental to acquired immunity ( , , ) . it is worth noting that also the acyl chains attached to proteins can affect the membrane structure. studies on model membranes have revealed that palmitic and myristic acids facilitate formation of ordered lamellar membrane regions ( , ) . in accordance, s-palmitoylation of erythrocyte peripheral membrane protein called membrane-palmitoylated protein (mpp ) was found to be required for the proper lateral organization and fluidity of erythrocyte membrane. in the absence of mpp s-palmitoylation, raft assembly was disturbed and erythrocyte functioning compromised leading to hemolytic anemia in patients deficient in the enzyme catalyzing this reaction ( , ) . preferential raft association is a feature of some s-palmitoylated transmembrane proteins, e.g., adaptor proteins pag, lat, and ntal, which collaborate with the abovementioned immunoreceptors. in fact, palmitoylation is required for the raft association of most integral raft proteins ( , , ) . on the other hand, s-palmitoylation does not obligatorily confer raft localization on transmembrane proteins. certain s-palmitoylated proteins, such as transferrin receptor, glycoprotein g of vesicular stomatitis virus (vsv), and anthrax toxin receptor, tumor endothelial marker (tem ), are actually excluded from rafts. apparently, a combination of s-palmitoylation and the properties of the transmembrane domain of the protein contribute to its destination to the raft or non-raft environment ( , ) . it has also been proposed that the attachment of a fatty acyl chain at the juxtamembrane cysteine(s) of a protein can induce tilting of its transmembrane fragment, determining in which part of the membrane it will accommodate to avoid a hydrophobic mismatch potentially caused by the thickness of the bilayer ( ) . that not all s-palmitoylated proteins associate with rafts has been shown convincingly for macrophage-like raw cells, where only about half of those proteins were found in the triton x- -resistant membrane fraction enriched in rafts ( , ) . in accordance, proteomic data on the distribution of s-palmitoylated proteins in prostate cancer cells have revealed that several such proteins are recovered in the non-raft (triton x- -soluble) fraction and are likely localized to microdomains enriched in scaffold proteins called tetraspanins ( ) . the tetraspanins are small integral membrane proteins found in the plasma membrane and other cellular membranes, having four transmembrane helices and undergoing s-palmitoylation at several conserved cysteine residues. the tetraspanins interact with each other and with various transmembrane and cytosolic partners, often also s-palmitoylated, forming microdomains ("tetraspanin web") ( ) . it has been suggested that the amino acid composition of the s-palmitoylation site in some transmembrane proteins, such as the adaptor proteins involved in acquired immune responses, determines the association of those s-palmitoylated proteins with rafts or with the tetraspanin-enriched microdomains ( ) . an intriguing and still poorly addressed question concerns the relation between rafts and the tetraspanin-enriched microdomains, apparently of functional significance, e.g., during virus budding from host cells ( ) . this uncertainty stems partially from the fact that s-palmitoylation of tetraspanins governs their interactions with cholesterol and gangliosides leading at certain conditions to the recovery of tetraspanins in detergent-resistant membrane fractions enriched in rafts ( , ) . besides its involvement in targeting proteins to rafts or tetraspanin-enriched microdomain, s-palmitoylation has been found to govern accumulation of the transmembrane chaperone protein calnexin in the perinuclear domain of endoplasmic reticulum ( ) . s-palmitoylation also affects protein stability through its interplay with ubiquitination or phosphorylation, as found for the anthrax toxin receptor tem , antiviral interferon-induced transmembrane protein ifitm , calnexin, and zdhhc , one of palmitoyl acyltransferases described below ( ) ( ) ( ) ( ) . possibly the most intriguing is the reversible character of s-palmitoylation. enzymes catalyzing palmitoylation and depalmitoylation of proteins have been characterized ( , ) . palmitate is transferred onto the thiol group of cysteine from cytosolic palmitoyl-coa by palmitoyl acyltransferases, enzymes containing the zinc finger dhhc domain named after the highly conserved asp-his-his-cys peptide (figure ). this is a twostep reaction comprising transient autoacylation of zdhhc enzymes and transfer of the fatty acyl chain from this intermediate to a protein substrate ( ) . in mammals, the zdhhc enzyme family consists of proteins, and zdhhc proteins are also found in other eukaryotes but not in bacteria nor are they encoded by viral genomes. mammalian zdhhc enzymes, each having at least four transmembrane helices, are located in the plasma membrane, endoplasmic reticulum, and golgi apparatus ( ) . they display some specificity toward their protein substrates and also selectivity toward fatty acyl moieties other than palmitate, which contributes to the heterogeneity of lipids attached to proteins, such as viral glycoproteins described below ( ) . in the opposite process, the thioester bond is cleaved by acyl-protein thioesterases (apts) (apt and apt ) and palmitoyl protein thioesterases (ppts) (ppt and ppt ), which are localized in the cytosol and in lysosomes, respectively. apt and apt likely govern the dynamic functional changes of s-acylation of proteins ( ) while ppt and ppt depalmitoylate proteins during their degradation ( , ) . recently, serine hydrolases of the abhd family have also been identified as depalmitoylating enzymes, and their specific substrate proteins determined ( , ) . of note, the zdhhcs, apt /apt , and abhd proteins are s-palmitoylated themselves, and palmitoylation of zdhhcs and depalmitoylation of apt / can occur in a cascade manner ( , ) . the dynamic cycles of palmitoylation/depalmitoylation detected for several peripheral membrane proteins are often synchronized with intracellular trafficking of those proteins. they circulate between the plasma membrane and the golgi apparatus or endosomes, as exemplified by n-and h-ras, r -regulator of g protein and apts. in fact, it is proposed that palmitoylationdependent anchoring of apt in the plasma membrane allows it to depalmitoylate h-ras at this location, while subsequent autodepalmitoylation releases apt guiding it, alongside h-ras, for another round of palmitoylation at the golgi apparatus ( , ( ) ( ) ( ) . cycles of palmitoylation/depalmitoylation are crucial for signaling by distinct plasma membrane receptors and for their distribution ( ) ( ) ( ) . activation of tcr receptor or fas receptor in t cells was found to trigger quick and transient palmitoylation of lck kinase of the src family ( , ) , but the exact meaning of the dynamic protein s-palmitoylation for processes triggered during the host-pathogen interaction awaits elucidation. it is worth mentioning that although the zddhc enzymes catalyze bulk protein palmitoylation in eukaryotic cells ( ) , some proteins have a unique autopalmitoylation activity. these include bet , a component of a multisubunit transport protein particle complex involved in vesicular trafficking, tea domain transcription factors, and also bacterial evf protein ( ) ( ) ( ) ( ) . the palmitic acid residue is attached constitutively to a specific cysteine residue of those proteins, remains buried inside a hydrophobic pocked in their core thereby affecting the tertiary structure and, thus, interactions with other proteins ( , ) . an exhaustive discussion on the physiology of s-palmitoylated proteins in eukaryotic cells can be found in several recent reviews ( , , ) . it has been established that, in addition to palmitate, various other fatty acyl moieties, such as saturated stearate (c : ) or protein palmitoylation in host-pathogen interactions frontiers in immunology | www.frontiersin.org january | volume | article monounsaturated palmitoleate (c : ), and oleate (c : ) can be attached via the thioester linkage to proteins. the early reports on the heterogeneity of the fatty acyl moieties attached to cysteines obtained by analysis of selected immunoprecipitated proteins ( , , ( ) ( ) ( ) have recently been complemented by a comprehensive proteomic analysis of fatty-acylated proteins of macrophage-like raw cells ( ) . the latter study showed that an enrichment of culture medium of cells with monounsaturated fatty acids leads to their incorporation into a similar set of proteins as those normally modified with palmitate. among them, several proteins relevant to innate immune responses were found. all these data justify the use of a broader term s-acylation rather than s-palmitoylation ( table ). the physiological consequences of s-acylation of proteins with individual fatty acids are slowly being revealed. modification of fyn kinase with polyunsaturated fatty acid residue, such as arachidonate (c : ), disturbed its raft localization and, thereby, tcr signaling ( ) . a heterogeneity of s-acylation was also found in viral spike proteins, such as hemagglutinin (ha) of influenza a virus, and e and e of semliki forest virus, which are modified in host eukaryotic cells by attachment of both palmitate and stearate ( ) . in ha, stearate is attached at the transmembrane cysteine while palmitate is attached to two cysteine residues in a membrane-proximal region of the protein. the stearoyl chain seems to accommodate into a groove formed by amino acids of the transmembrane helix shaping the domain in a way that facilitates its fitting into rafts ( ) . s-stearoylation of human transferrin receptor at the juxtamembrane cysteine residues(s) is a key factor of the signaling cascade controlling mitochondrial morphology and functioning ( ) . of interest, the latter study also showed that dietary supplementation of stearic acid reversed the deleterious effects of a genetically determined mitochondria dysfunction in drosophila. taking into account that unsaturated fatty acids affect the profile of s-acylation of proteins in vitro ( , ) , it is of outmost interest whether a similar effect of unsaturated and saturated (palmitic) fatty acids could be achieved in vivo with respect to proteins of immune cells. beside s-acylation, less frequently palmitate can also be attached to the amine group of various amino acids (glycine, cysteine, and lysine) giving n-palmitoylation or to the hydroxyl group of serine or threonine in a process called o-palmitoylation ( table ) . as during s-palmitoylation, also other fatty acids can be utilized in these processes named then n-and o-acylation. thus, a type of protein n-acylation is n-myristoylation, a frequent modification contributing to membrane anchoring of peripheral proteins. the saturated myristate (c : ) is transferred to the protein from myristoyl-coa by n-myristoyl transferase (two isozymes in mammals). in a vast majority of cases, myristate is attached cotranslationally to the n-terminal glycine residue (after removal of the initiator methionine) via an amide linkage ( table ) . like most lipidations, this modification is irreversible. several viral proteins are n-myristoylated, such as gag of hiv- crucial for budding of newly formed virions from plasma membrane rafts of host cells, and proteins of parasitic protozoa plasmodium falciparum, trypanosoma brucei, and leishmania donovani (causing malaria, african sleeping sickness, and leishmaniosis, respectively). for this reason, n-myristoyl transferase is considered a potential drug target in the therapy of these diseases ( - , , ) . data on the n-and o-palmitoylation of proteins involved in the host-pathogen interactions are limited, but interesting conclusions can be drawn from the information concerning proteins taking part in other processes. n-palmitoylation of the n-terminal glycine of the α-subunit of a heterotrimeric g protein (gαs) has been described ( ) ( table ) besides the wellknown s-palmitoylation of this pivotal signaling protein. the n-palmitoylation of gαs is irreversible, and the enzyme responsible for this modification is unknown. it has been speculated that s-to n-palmitoyl migration can occur both in vivo and also in vitro during mass spectrometry analysis ( , ) . this suggests that caution is needed in interpreting results of this methodological approach, which is used with increasing frequency to study fatty acylation of proteins in immune cells (see next sections). probably the best-characterized is the n-palmitoylation of the n-terminal cysteine residue of hedgehog proteins (sonic, indian, and desert in mammals). it determines secretion of these proteins, which regulate embryonic patterning ( table ) . secreted wnt and ghrelin proteins are examples of o-acylation of serine residues with unusual fatty acid residues such as palmitoleate (c : ) and octaonoate (c : ) ( table ). the fatty acylation of hedgehog, wnt, and ghrelin is catalyzed by enzymes from the multipass membrane-bound o-acyl transferases family ( ) . besides these unusual fatty acid residues, attachment of palmitate to serine and threonine residues is found in secreted venom toxins of the spider plectreurys tristis, which selectively target neuronal ion channels ( ) . also histone h is o-palmitoylated at a serine residue in the nucleus by acyl-coa:lysophosphatidylcholine acyltransferase ( ) ( table ). the latter is of special interest in the context of innate immune responses since histone h o-palmitoylation regulates transcriptional activity, which is the final outcome of the pro-inflammatory signaling pathways triggered by receptors of the innate immune system. special attention should be devoted to ε-n-acylation consisting in the attachment of a fatty acid residue to the side chain of lysine by amide linkage ( table ) . ε-n-myristoylated are interleukin α (il- α) and tumor necrosis factor α (tnfα), the pro-inflammatory cytokines crucial in combating bacterial infections ( ) . the enzyme(s) catalyzing this reaction is unknown, but it has been established that sirtuins reverse this modification ( ) . the ε-n-acylation affects the release of tnfα by immune cells ( , ) . surprisingly, this rare modification is also found in toxins of so-called rtx (repeats-in-toxin) class released by some pathogenic gram-negative bacteria ( , ) . we describe these cases in more detail in the following sections. besides s-palmitoylation and n-myristoylation, s-prenylation is another common lipidation that endows proteins with a hydrophobic moiety and contributes to their association with membranes. this modification relies on the posttranslational and irreversible attachment of either farnesyl or geranylgeranyl chains to a cysteine residue in the c-terminal caax box (alternatively also cc and cxc motifs) via a thioether linkage. the process is catalyzed by protein prenyl transferases that use polyprenylpyrophosphate as the donor of the isoprenoid group ( table ) . in peripheral membrane proteins, the s-palmitoylation site is often located in proximity of n-myristoylation or s-prenylation sites or a polybasic motif, which all are likely to mediate initial weak binding of a protein to a membrane and thereby facilitate subsequent attachment of palmitate to the protein by the integral membrane zdhhc enzymes ( ) . in contrast to s-palmitoylation, data on the role of s-prenylation of proteins key to the host-pathogen interactions are scarce ( ) . however, since s-prenylation is typical for the ubiquitous small gtpases of ras superfamily, it is vital for proper functioning of b and t cells ( , ) . a glance at table indicates that palmitate can be covalently bound via oxyester, amide, and thioester linkages to respective amino acid residues creating an array of possible modifications. o-and n-palmitoylation of proteins seems to be stable, resembling in this regard the other common protein lipidations, n-myristoylation and s-prenylation. by contrast, there exist enzymes cleaving the thioester bond formed during s-palmitoylation. for a long time, our understanding of protein s-palmitoylation and its dynamics was poor in comparison with other reversible protein modifications due to technical difficulties. only recently have these difficulties been overcome with the introduction of methods allowing high-throughput identification of palmitoylated proteins, also those involved in the immune response to microbial pathogens, as discussed in the next sections. one of the basic problems hindering studies on protein s-palmitoylation lies in the fact that there is no identifiable consensus sequence for the palmitoylation site that could facilitate its prediction. from the technical point of view, the progress in a comprehensive survey of protein s-palmitoylation was also hampered by a lack of antibodies detecting this modification, with the sole exception of an antibody specific to palmitoylated psd- ( ) . a classical method used to demonstrate protein palmitoylation is based on metabolic labeling of living cells with [ h]-palmitic acid, subsequent immunoprecipitation of a selected protein and detection of the incorporated tritiated fatty acid by autoradiography ( ) . a major disadvantage of this method is its low sensitivity. only a minute fraction of the radioactive palmitate is bound to proteins, the majority being incorporated into lipids, which requires lengthy film exposure (counting in days). a methodological breakthrough in the identification of palmitoylated proteins came with the development of two nonradioactive methods based on so-called click chemistry ( ) ( ) ( ) and acyl-biotin exchange (abe) ( , ) . these techniques have paved the way for high-throughput mass spectrometry-based proteomic analysis of protein palmitoylation in various cells and tissues and facilitated identification of new palmitoylated proteins of both pathogens and host cells involved in the innate immune responses. in the click chemistry-based method, cells are metabolically labeled with a palmitic acid analog bearing an alkyne group at the ω carbon of the fatty acyl chain, such as -octadecynoic acid ( odya) or alk- (figure a) , and this step resembles the classic labeling of cells with [ h]-palmitic acid. however, in the click chemistry-based assay, the labeling and lysis of cells is followed by in vitro coupling of the function group of the palmitic acid analog to a reporter tag, which greatly enhances the sensitivity of detection of labeled proteins ( , ) . thus, after cell lysis, the labeled proteins are subjected to cu (i) -catalyzed cycloaddition known as "click" reaction with an azide-bearing detection tag. in this step, a triazol is formed between the alkyne group in the palmitic acid analog and the azide of the tag (figure a) . the azide-bearing tags can be either fluorescent, such as tetramethylrhodamine or dyes with infrared fluorescence, or carry a biotin moiety. depending on the tag used, subsequent sds-page separation of proteins allows global visualization of palmitoylated proteins by simple in-gel fluorescence or by blotting with a streptavidin-conjugated reporter ( , , ) . notably, proteins biotinylated via the click reaction can also be enriched on streptavidin-coated beads and then subjected to on-bead tryptic digestion (or in-gel digestion if eluted from the beads) followed by identification by mass spectrometry. such comprehensive click chemistry-based proteomic analysis has brought about identification of an array of palmitoylated proteins in dendritic cells ( , ) , macrophage-like raw cells ( ) , and t cells ( , , ) . some of the s-palmitoylated proteins newly identified in those studies, such as ifitm and tlr , are involved in the host-pathogen interactions regulating innate immune responses ( , ) , while many others are known to contribute to adaptive immunity ( , ) , as described below. recently, global profiling of toxoplasma gondii (the causative agent of toxoplasmosis) has been performed revealing that many components of the parasite's motility complex are palmitoylated ( ) . similar studies on cryptococcus neoformans (the fungus causing cryptococcal meningitis) have revealed a contribution of specific zdhhc palmitoyl acyltransferase, called pfa , to its virulence ( ) . moreover, application of analogs of various saturated and unsaturated fatty acids confirmed the heterogeneous nature of the fatty acylation of proteins in raw cells and suggested that dietary unsaturated fatty acids, after incorporation to proteins, can change their properties and thereby affect the functioning of immune cells ( ) . the major advantage of the click chemistry-based method is that it can reveal the time course of protein s-palmitoylation. by using click chemistry-based labeling in the pulse-chase mode, one can follow the dynamics of protein palmitoylation. with such an approach, it was found that the palmitate turnover on lck, an src-family tyrosine kinase, is accelerated by t cell activation ( ) . additional introduction of stable isotope labeling by amino acids in cells (silac) has provided quantitative proteomic data , and after cell lysis, the click reaction is conducted with azido-tagged biotin or fluorescent probes allowing enrichment and detection of labeled proteins in various ways. biotinylated proteins can be bound on a streptavidin resin and then released using, e.g., high concentrations of urea and sds ( ) . when a cleavable derivative of biotin, azido-azo-biotin, is used the labeled proteins are eluted from streptavidin beads with sodium dithionite, which cleaves the diazobenzene moiety in the linker arm of azido-azo-biotin, and analyzed by mass spectrometry or immunoblotting ( ) . (b) abe method. cells or tissues are lysed, free thiol groups of proteins are blocked by alkylation, and palmitoyl moieties are released with hydroxylamine. the newly exposed protein thiol groups are subjected to labeling with biotin-hpdp allowing selective binding, elution, and analysis of the originally s-palmitoylated proteins. the proteins can also be captured without biotinylation through a direct interaction of their thiol residues with a thiol-reactive resin (acyl-rac technique). on the dynamics of protein palmitoylation in the cell ( , ) . this approach revealed, rather unexpectedly, that in unstimulated t cell hybridoma, the palmitoylation of most protein species does not undergo turnover ( ) . another advantage of the click chemistry-based assay is its high specificity, because the alkyne group introduced in the analog of palmitic acid is not normally found in cells ( , ) . the click chemistry-based methods can also be used to follow the cellular localization of palmitoylated proteins by immunofluorescence when combined with the proximity ligation technique ( , ) . palmitoylation of individual proteins can also be studied after their immunoprecipitation ( , , , ). despite its unquestionable success, the click chemistry-based methods have limitations. they will detect only those proteins that undergo palmitoylation during the period of the metabolic labeling of cells. one should also bear in mind that the palmitic acid analog can be incorporated at s-, n-, and o-palmitoylation sites alike ( , ) . in addition, although odya (alk- ) is preferentially used to mimic palmitoylation of proteins, it can also be incorporated with low efficiency at n-myristoylation sites of proteins ( , ) . another group of proteins that will be labeled with the palmitic acid analog but are not s-palmitoylated are those bearing the glycosylphosphatidylinositol (gpi) anchor ( , ) . most of these limitations can be overcome using various fatty acid reporters, inhibitors, and by exploiting the sensitivity of the thioester bond to hydroxylamine treatment. given the large variety of chemical reporters preferentially mimicking distinct fatty acids, recent years have witnessed a plethora of chemistry-based proteomic studies not only on palmitoylated but also myristoylated proteins and proteins bearing the gpi anchor, including those of pathogens and immune cells ( , , , , ) the abe method reveals protein the abe method can be used as a complement to the click chemistry-based approach in cell studies but unlike the latter it is uniquely suitable for studying whole tissues. abe does not require metabolic labeling of proteins in living cells, thus some of the abovementioned limitations and difficulties do not apply. the abe method relies on in vitro exchange of thioester-linked palmitate to a derivative of biotin which allows subsequent affinity purification of the resulting biotin-labeled proteins on streptavidin-coated beads ( figure b) . the first step of the abe involves lysis of cells or tissues followed by irreversible blockage of free thiol groups in the solubilized proteins by alkylation, most often with n-ethylmaleimide. subsequently, the thioester bonds existing in s-palmitoylated proteins are broken with hydroxylamine, releasing palmitoyl moieties. the newly exposed thiol groups can now be tagged with sulfhydryl-reactive derivatives, such as biotin-hpdp, forming disulfide bonds with thiols. the biotinylated proteins are subsequently captured on streptavidin-coated beads and eluted with agents that reduce the disulfide bond between the protein and biotin-hpdp, such as β-mercapthoethanol, dtt, or tcep ( , , , ) . as an alternative to biotinylation, in the so-called acyl-rac technique, the newly exposed protein thiol groups in hydroxylamine-treated cell lysates are captured on a resin containing sulfhydryl-reactive groups ( ) . in both abe and acyl-rac, the eluted proteins can be separated by sds-page and visualized by gel staining or immunoblotting, or identified by mass spectrometry. furthermore, when the hydroxylaminereleased palmitoyl moieties are exchanged for a polyethylene glycol-maleimide derivative of a distinct molecular weight, a shift in-gel migration of tagged proteins is observed reflecting the number of fatty acyl residues originally s-bound to the protein ( , ) . the abe method has so far been used successfully for proteomic profiling of s-acylated proteins in immune cells, such as raw cells ( ), several types of blood cells, such as platelets, primary t cells, and immortalized b cells ( ) ( ) ( ) , pathogenic microorganisms such as t. brucei and t. gondii ( , ) , and tissues ( , ) . to quantify the aberrations in protein palmitoylation in a mouse model of huntington's disease, whole animal stable isotope labeling of mammals (silam) was applied followed by tissue isolation and abe procedure ( ) . in another approach, for quantitative analysis of the t-cell palmitoylome, abe was combined with labeling of proteins with various oxygen isotopes during their digestion with trypsin before mass spectrometry analysis ( ) . in addition, preselection of tryptic peptides obtained by abe on streptavidin-coated or sulfhydrylreactive resins greatly facilitates the identification of s-acylation sites by mass spectrometry ( , , ) . some aspects of the abe method deserve a comment. since the assay relies on the sensitivity of thioester bonds to hydroxylamine, abe detects all s-acylation without distinguishing between s-palmitoylation and the other cases. furthermore, there is a possibility of false-positive detection of proteins bearing a thioester linkage with compounds other than fatty acyl residues, such as ubiquitin in the e ubiquitin conjugase ubc ( ) . another source of false-positives is proteins in which free thiol groups were not completely alkylated before biotinylation. on the other hand, insufficient deacylation of bonafide fatty-acylated proteins with hydroxylamine results in their absence in the final sample ( ) . in summary, the click chemistry-based method relies on metabolic labeling of cells with a palmitic acid analog which incorporates into proteins and next tagging it with reporter molecules greatly enhancing the sensitivity of detection. it only reveals proteins undergoing s-palmitoylation during metabolic labeling of cells and allows revealing turnover of this modification. by contrast, the abe method is based on direct binding of sulfhydryl-reactive derivatives to thiol groups of cysteines unraveled by hydroxylamine treatment after lysis of cells or tissues. it allows the investigation of the whole but static palmitoylome. a comparative proteomic study of protein palmitoylation in p. falciparum found that the sets of proteins identified using these two approaches overlapped in . % ( ) , indicating that they provide complementary data on the cellular palmitoyl proteome. thanks to the application of the click chemistry-and abe-based methods numerous new palmitoylated proteins have been identified. in , a swisspalm database was launched, ( ) which provides an excellent, manually curated resource of information on palmitoylated proteins, palmitoylation sites, etc., available at http://swisspalm.epfl.ch/. all these efforts have greatly furthered our knowledge on molecular mechanisms regulating diverse aspects of cell functioning, including host-pathogen interactions and progress of infectious diseases, as highlighted below. bacteria lack protein palmitoyl acyltransferases of the zdhhc family and, therefore, are essentially devoid of s-palmitoylated proteins. yet, they have developed unique mechanisms utilizing fatty acids, such as palmitic acid, to modify their glycolipids and proteins. these modifications augment infectivity and help bacteria evade recognition by the host innate immune system. for example, the vast majority of gram-negative bacteria produce lipopolysaccharide (lps) as a part of their outer membrane. lps is composed of the variable polysaccharide o-antigen and more-conserved lipid a containing two glucosamine residues hexa-acylated with hydroxymyristic, myristic, and lauric acid. lipid a is recognized by cd protein and tlr receptor complexed with md protein on the plasma membrane of the host immune and some non-immune cells. activation of tlr triggers strong pro-inflammatory reactions aiming at eradication of the bacteria, but when exaggerated, eventually leading to sepsis ( ) . incorporation of an additional palmitoyl chain into lipid a markedly diminishes its ability to activate tlr and to induce the host pro-inflammatory responses, which is correlated with an increased survival of bacteria forming a biofilm ( , ) . this strategy is utilized among others by salmonella typhimurium, a causative agent of gastroenteritis, by bordetella bronchiseptica, a respiratory pathogen of human and other mammals, and by protein palmitoylation in host-pathogen interactions frontiers in immunology | www.frontiersin.org january | volume | article yersinia pestis causing plague ( , ) . the formation of the extra-acylated lps relies on the transfer of palmitate from phospholipids onto the hydroxymyristate chain at position of glucosamine of lipid a. the reaction is catalyzed by lipid a palmitoyltransferases (pagp in salmonella and its homologs in other bacteria) localized in the outer membrane of these pathogens ( , ) . in addition to causing steric hindrance preventing the binding to the tlr /md complex, the hepta-acylation of lps also protects bacteria from the lytic activity of cationic antimicrobial peptides, most likely by reducing the fluidity of the bacterial outer membrane ( , ) . apart from being incorporated into lps in diverse bacteria, palmitate has also been found to modify a virulence factor of gram-negative erwinia carotovora, the evf protein. the palmitoyl chain is linked via a thioester bond to the cys residue at the center of evf, plausibly by a self-palmitoylating activity of the protein. e. carotovora is a phytopatogen using insects such as drosophila as vectors for dissemination between plants. the palmitoylation of evf is required for infectivity of e. carotovora and its persistence in the insect gut, however, its mode of action of unknown. it has been speculated to be linked with an ability of evf to associate with lipid bilayers, but the lack of similarities between evf and any other bacterial protein of known function makes prediction on this subject difficult ( ) . a number of bacterial toxins of so-called rtx class released during infection of mammals by pathogenic gram-negative bacteria undergo ε-n-acylation of the side chain of internal lysines. these toxins include adenylate cyclase of bordetella pertussis, acylated with palmitic acid, and α-hemolysin of extraintestinal (uropathogenic) escherichia coli, acylated with myristic acid and also -and -carbon fatty acids. the acylation is catalyzed by an endogenous bacterial acyltransferase which, unlike its eukaryotic counterparts, transfers the acyl chain not from acyl-coa but from acyl-carrier protein. the acylated toxins secreted by the bacteria bind to the plasma membrane of the host cells, oligomerize and form pores causing cell lysis. in the case of the toxin of b. pertussis, essential is also the delivery of the adenylate cyclase moiety to the cell interior. acylation is required for virulence possibly being involved in oligomerization of the toxins ( , , ) . although lacking s-palmitoylated proteins (with the single known exception of evf), bacteria express a wide range of membrane-bound proteins modified by a complex lipidation at the n-terminus, with palmitate frequently being a component of the lipid moiety ( , ) . the bacterial lipoproteins are synthesized in a multistep process catalyzed by a unique set of lipoprotein processing enzymes, lgt, lspa, and lnt, absent in eukaryotic cells. the formation of these lipoproteins begins with the attachment of a diacylglycerol via a thioester bond to a cysteine residue located in the so-called lipobox motif of the signal sequence of the transmembrane lipoprotein precursor. the signal sequence is then cleaved next to the lipid-modified cysteine leaving it at the n-terminus of the mature protein ( ) . in gramnegative and less frequently also gram-positive bacteria, a third fatty acid residue is additionally attached via an amide linkage to the amino group of the cysteine in a reaction analogous to the n-acylation of hedgehog proteins (see table ). this di-and tri-lipidation ensures membrane anchoring of the lipoproteins. all such lipoproteins of gram-positive bacteria are exposed to the milieu while in gram-negative bacteria some face the periplasm. the lipoproteins of gram-positive bacteria, e.g., streptococcus pneumoniae (causing pneumonia), mycobacterium tuberculosis (tuberculosis), and gram-negative bacteria, such as neisseria meningitidis (meningitis), y. pestis (plague), the spirochaete borrelia burgdorferi (lyme disease) and treponema pallidum (syphilis) are crucial for their virulence. they control several aspects of the host-pathogen interactions, like adhesion and entry to host cells, protection against proteolysis and oxidative stress in the host cell, and regulation of expression of genes encoding cytokines both during initiation and progress of the disease ( ) ( ) ( ) . the surface exposure of the lipoproteins allows their involvement in the host cell invasion while on the other hand forming the so-called pattern signal recognized by the tlr receptor, which triggers the pro-inflammatory responses helping to combat the bacteria ( ) . of interest, tlr is s-palmitoylated, as discussed below. the involvement of lipoproteins in pathogenesis fuels studies on their properties. one such recent work employing click chemistry to profile the lipoproteins of e. coli identified lipoproteins with high/medium confidence, % of them predicted before by bioinformatics analysis ( ) . notably, in that study a -carbon alkynyl fatty acid analog alk- rather than alk- was preferentially incorporated into the lipoproteins, contradicting earlier studies using gas chromatography and tlc, which found that palmitate was predominantly used for bacterial protein modification ( ) . further studies are required to establish whether the fatty acid found in lipoproteins varies depending on culture conditions or is species specific. for example, odya labeling for click reaction confirmed incorporation of palmitate into pallilysin (tp ), a lipoprotein of t. pallidum. pallilysin is a metalloprotease that degrades human fibrinogen and laminin. it is suggested that its exposure on the bacteria surface enables degradation of host structural proteins to facilitate rapid dissemination of this highly invasive pathogen ( ) . bacteria occasionally high-jack the palmitoylation machinery of host cells to modify the environment so as to favor their internalization, survival, and replication inside the cells. bacillus anthracis (the causative agent of anthrax) is an example of such bacteria that modify s-palmitoylation of host proteins to their ends. the anthrax toxin produced by this pathogen binds to the tem and cmg (capillary morphogenesis protein- ) proteins which, under physiological conditions, are involved in cell-cell and cell-extracellular matrix interactions. they are s-palmitoylated at multiple (two to four) cysteines ( ) . the s-palmitoylation of tem was found to inhibit its association with plasma membrane rafts preventing its ubiquitination by the raft-associated e ubiquitin ligase cbl. the binding of anthrax toxin drives association of the receptor-toxin complexes with rafts possibly correlated with depalmitoylation of the receptor. this allows subsequent ubiquitination of the receptor, an uptake of the receptor/toxin complexes in a clathrin-dependent manner and eventual delivery of the toxin to endosomes. these events are facilitated by s-palmitoylation of partner(s) of the receptors, most likely including kinases of the src family ( , , ) . while b. anthracis utilizes palmitoylated host proteins to induce its internalization, a growing body of data suggests that protein palmitoylation in host-pathogen interactions frontiers in immunology | www.frontiersin.org january | volume | article also bacterial proteins can undergo s-palmitoylation inside the host cells. this type of modification concerns so-called effectors, bacterial proteins that are injected into the host cell cytoplasm either across the plasma membrane or the membrane of vesicles enclosing internalized pathogens, with the help of their secretion systems. these are secretion systems type iii and type iv, homologs of which have been described for pathogens and symbionts of mammals, insects, and plants ( , ) . the bacterial effectors can be s-palmitoylated to reach host cell membranes and thereby accumulate at a location most suitable for their activity. application of the click chemistry-based method utilizing an analog of palmitic acid (alk- ) for cell labeling has revealed s-palmitoylation of two effector proteins of salmonella enterica, such as ssph and ssei ( ) . s. enterica invades gut endothelial cells and is a leading cause of gastroenteritis and typhoid fever. ssph carries an e ubiquitin ligase domain while ssei shows sequence homology to bacterial proteins that have a deamidase activity, and inhibits migration of salmonella-infected cells. the latter activity requires s-palmitoylation of ssei. both proteins are stably s-palmitoylated, most likely by zdhh and zdhh of the host and bind to the plasma membrane in a palmitoylationdependent manner ( ) . also two effector proteins of the ipah family of shigella spp. were found to be s-palmitoylated in that study, suggesting that this modification can control the activity of effector proteins of other pathogens as well ( ) . indeed, gobx and lpda, effector proteins of legionella pneumophila, the causative agent of legionnaires' disease invading macrophages and lung endothelial cells, are s-palmitoylated as was found recently using click chemistry. lpda is a phospholipase hydrolyzing various phosphatidylinositols while gobx is an e ubiquitin ligase. gobx is targeted in a palmitoylation-dependent manner to the golgi apparatus, and lpda to the plasma membrane and a subset of intracellular vesicles ( , ) . thus, the diversified subcellular localization of bacterial effector proteins reflects that of eukaryotic proteins. it is worth noting that global profiling of acylated proteins with the application of click chemistry and an alkyne-functionalized analog of myristic acid, alk- , for cell labeling was effective in reveling the mechanism of action of shigella flexneri effector protein ipaj of type iii secretion system. this is a unique protease that cleaves off the n-terminal myristoylated glycine. this proteolytic demyristoylation activity of ipaj is specific toward golgi-associated arf/arl family of gtpases regulating cargo transport through the golgi apparatus, inhibition of which is apparently pivotal for virulence of the bacteria causing diarrhea in humans ( ) . in addition to the s-palmitoylation of the effectors of pathogenic bacteria of mammals mentioned earlier, double acylation, n-myristoylation and s-palmitoylation, has been reported of the so-called avirulence (avr) proteins (effectors of type iii secretion system) of pseudomonas syringae, a causative agent of diverse plant diseases. among them, avrrpm and arvb are n-myristoylated and s-palmitoylated by host acyltransferases at neighboring glycine and cysteine residues localized at the n-terminus of the proteins (similarly to eukaryotic kinases of the src family), while in avrpphb and two avrpphb-like effectors-orf and nopt, the double acylation motif is exposed after auto-cleavage of the proteins (similarly to some eukaryotic proteins cleaved by caspases). the acylation of the avr proteins ensures their anchoring in the host plasma membrane, which is required for their functioning. in disease-susceptible plants avr proteins contribute to successful infection; however, in plants expressing host resistance (r) genes they trigger plant defense signals, in both cases engaging plasma membrane-associated host proteins ( , ) . the importance of palmitoylation of bacterial effector proteins for their infectivity is only beginning to be uncovered, in no small part owing to the development of the click chemistry-based method for detection of this protein modification. however, the strategy of high-jacking the host palmitoylation machinery to modify own proteins seems to be much more commonly employed by viruses. viruses do not encode palmitoyl acyltransferases but exploit extensively the host palmitoylation machinery to modify their proteins essential for infection of host cells and own replication. in fact, s-palmitoylation of proteins was discovered in as a modification of envelope glycoproteins of sindbis virus and vsv. in those studies [ h]-palmitic acid was used for metabolic labeling of virus-infected cells and labeled proteins were identified by autoradiography ( , ) . subsequently, a number of other viral proteins have been found to be palmitoylated using this approach. the most-studied group of viral palmitoylated proteins is those found in enveloped viruses, i.e., viruses covered by a lipid bilayer obtained during their replication from a membrane of the host cell, such as the plasma membrane or endoplasmic reticulum. influenza virus, hiv- , hepatitis c virus (hcv), and herpes simplex virus (hsv) are the best known enveloped viruses. the envelope is rich in transmembrane, often s-palmitoylated, glycoproteins called spikes, which can bind to cognate receptors on the host cell plasma membrane triggering endocytosis of the virion, mediate subsequent fusion of the viral and cellular membranes allowing entry of the viral genome to the cytoplasm, and are also involved in the budding of newly formed virus particles from the cell. an example of such multifunctional palmitoylated transmembrane glycoproteins is ha present in the envelope of influenza virus together with another palmitoylated transmembrane protein, the matrix protein m , which forms a proton channel earning the protein the name viroporin. as mentioned earlier, ha of influenza a virus is s-stearoylated and s-palmitoylated, respectively, at one cysteine residue located in the transmembrane domain of ha and two cysteines found in the cytoplasmic (intraviral) tail in close proximity to the membrane ( ) . on the other hand, m is s-palmitoylated on the amphiphilic helix located in the cytoplasmic part of the protein. due to the s-palmitoylation and the presence of a cholesterol-binding motif the helix bends toward and associates with membranes ( , ) . during infection, ha binds to sialic acid residues of glycans localized on the surface of airway and alveolar epithelial cells. the bound virions are endocytosed and next the viral and endosome membranes fuse. the membrane fusion is driven by ha, which undergoes conformational changes induced by low ph of endosomes. acidification of endosomes activates also the m proton channel activity, protons entering viral core facilitate dissociation of the viral genome which then moves to the nucleus where rna replication occurs. the s-palmitoylation of ha is required for the fusion of the viral and endosome membranes at least in some subtypes of the virus while the ion channel activity of m is not dependent on its s-palmitoylation ( ) . newly synthesized viral proteins and rna are assembled into virions in the plasma membrane rafts which merge into lager platforms crucial for the virion assembly and budding off. the triple fatty acylation of ha is required for its targeting to plasma membrane rafts ( , ) . besides s-palmitoylation, also the amino acid sequence of the transmembrane domain of ha determines its association with rafts ( ) . on the other hand, among the amino acids of the cytoplasmic tail of ha no other than the two s-palmitoylated cysteines are required for viral assembly and replication, although it is still not clear whether raft targeting (in cooperation with the transmembrane fragment) is the only mechanism of their participation. it is proposed that they affect conformation of the ha tail controlling its interaction with structural matrix protein m lying beneath the viral envelope ( , ) . the budding off of the virion is facilitated by m which localizes at the edges of rafts as a result of a combination of its s-palmitoylation, cholesterol binding, and properties of the transmembrane fragment. m protein can create a "wedge" altering membrane curvature thereby facilitating membrane scission and release of the virion ( , ) . the influenza virus s-palmitoylated proteins are the archetype for many other viral proteins. thus, s-palmitoylated spike glycoproteins include s-protein of coronaviruses (e.g., severe acute respiratory syndrome virus), the fusion (f) protein of paramyxoviruses (e.g., measles virus), env of retroviruses [e.g., hiv- , feline immunodeficiency virus (fiv)], and filoviruses (e.g., ebola). other viral proteins modified with palmitate are viroporins, such as e protein of coronaviruses, and also peripheral membrane proteins or nucleocapsid proteins absent in influenza virus. it has been found that s-palmitoylation of f l, a peripheral protein of the envelope of vaccinia virus, controls the association of the protein with intracellular membranes, thereby the formation of the envelope ( ) . the core protein of the nucleocapsid of hcv resides on the surface of lipid droplets and binds in a palmitoylation-dependent manner to membranes of the droplet-associated endoplasmic reticulum. subsequently, it recruits viral proteins and newly synthesized rna for viral particle formation ( ) . besides the interest in the role of viral protein s-palmitoylation for infectivity and possible use of host zdhhc enzymes as targets of anti-influenza drugs ( ) , viral proteins often serve as a model to study the consequences of fatty acylation for protein functioning and localization in distinct membrane domains (see s-palmitoylation of proteins and its influence on protein localization, trafficking, and stability of this review). readers are referred to recent exhaustive reviews that consider these topics ( , , ) while we will focus here on the recent advances in the field of viral protein palmitoylation brought about mainly by proteomic studies. the click chemistry-based approach has led to the identification of s-palmitoylation in the cytoplasmic domain of the transmembrane spike protein env of fiv, considered to be the cat equivalent of hiv- . env comprises three transmembrane gp glycoproteins and three associated gp which bind to cd receptor and coreceptors on the surface of t lymphocytes allowing fusion of the viral envelope and the plasma membrane and entry of viral capsid. four cysteines in fiv env are s-palmitoylated vis-a-vis two found in the env of hiv- . the two most membrane-proximal cysteines, and , are required for the fiv membrane-fusion activity and incorporation of env into virions ( ) , in agreement with the importance of env s-palmitoylation for virion assembly of some hiv- strains ( ) ( ) ( ) . the assembly of hiv- virions takes place in plasma membrane rafts and is driven by n-myristoylated gag protein which anchors and oligomerizes preferentially in these plasma membrane domains due to the presence of the fatty acyl chain ( ) . the development of click chemistry-based methods allowed for the first time global profiling of acylated proteins in virusinfected cells. in addition to identifying acylated viral proteins this approach has also revealed how the viral infection modulates the acylation pattern of the host cell proteins. thus far, click chemistry has been used to study protein myristoylation and palmitoylation in cells infected with hiv- and with hsv. in the latter case, the standard metabolic labeling with alkynefunctionalized myristic and palmitic acid analogs followed by click chemistry and mass spectrometry was combined with silac to discern between the changes in the extent of protein acylation and those in their abundance following viral infection. this approach allowed an elaborate quantitative analysis of host protein acylation and has revealed an overall downregulation of the level of both host protein modifications in infected cells. while the decreased content of myristoylated proteins resulted mainly from suppression of host protein synthesis, the drop in several s-palmitoylated proteins ensued from the inhibition of their palmitoylation in infected cells. the affected proteins were localized mainly to the plasma membrane and the golgi apparatus and were involved in vesicle-mediated transport and ion transport. in addition, the study has expanded the list of hsv-encoded acylated (mostly palmitoylated) proteins that play different functions in the viral cycle, such as ge, gi, gk, us , and us ( ) . similar results pointing to global changes of host protein acylation were obtained upon analysis of protein myristoylation and palmitoylation in cells infected with hiv- . in that study, the cells were labeled with analogs of palmitic or myristic acid tagged with an azide moiety for click chemistry reaction; however, the following mass spectrometry analysis did not address the relation between changes of protein acylation vs. alteration of protein level. the study identified palmitoylated and myristoylated proteins significantly differing in abundance between hiv- infected and uninfected cells. several of the proteins affected by the infection were of host origin. the abundance of myristoylated proteins was in general increased while that of the palmitoylated ones-decreased in infected cells ( ) . in other words, the two studies have revealed that hsv and hiv- not only encode proteins that are acylated in the host cell but protein palmitoylation in host-pathogen interactions frontiers in immunology | www.frontiersin.org january | volume | article also alter the palmitoylation of host proteins, likely to adapt the cellular environment to favor their replication and budding. the majority of the acylated proteins affected by hiv- or hsv infection had not been described earlier in this context; therefore, further studies on these proteins could be crucial for better understanding of viral infection. thus, the click chemistry-based approach has been highly effective in revealing changes of the host protein palmitoylation and opening new possibilities for the identification of novel antiviral drug targets. the innate immune responses are the first line of active defense against microbial infections. the application of click chemistrybased and abe methods and their use for large-scale analysis of protein palmitoylation in murine dendritic cd . cells ( , ) , and murine macrophage-like raw cells ( , ) complemented by proteomic analysis of the raft fraction of those cells ( ) have contributed significantly to the understanding of the role of palmitoylation of host receptors and signaling proteins involved in innate immune responses. thus, the palmitoyl proteome analysis of murine dendritic cells unraveled s-palmitoylation of tlr , a receptor expressed in cells of myeloidal lineage, which heterodimerizes with tlr or tlr to bind bacterial tri-or diacylated lipoproteins, respectively, and also other microbial components, such as glycolipids (e.g., lipoarabinomannan) of mycobacterium and yeast zymosan ( ) . besides tlr , two other human tlrs out of ectopically expressed in hek cell, flagellin receptor tlr , and tlr , a unique tlr negatively regulating the pro-inflammatory activity of tlr , were also found to be palmitoylated. the s-palmitoylation site of human tlr was mapped to cys adjacent to its transmembrane domain. the modification was present in unstimulated cells and was linked with up-regulation of the cell surface localization of tlr . mutation of cys abolished the ability of the receptor to induce pro-inflammatory signaling in response to microbial ligands of tlr ( ) . further studies are needed to reveal whether s-palmitoylation of tlr controls its association with rafts as sites of tlr activation ( ) and/or affects endocytosis of the receptor, as found for the anthrax toxin receptor ( ) . one of the most extensively studied tlrs, tlr activated by bacterial lps, is not palmitoylated. yet, saturated fatty acids have been indicated to trigger pro-inflammatory signaling of tlr . thus, the tlr /md receptor complex is involved in the pro-inflammatory outcome of a diet rich in palmitic acid, as was found when analyzing markers of inflammation in the heart and adipose tissue of high fat diet-fed mice ( , ) . the molecular mechanisms underlying the pro-inflammatory properties of palmitic acid can involve its influence on the plasma membrane lipid order, hence raft organization, in a way that facilitates translocation of tlr (and tlr ) toward rafts ( , ) . palmitic acid also directly binds to the tlr -associated md protein ( , ) . an influence of palmitic acid on sphingomyelin/ceramide metabolism, which enhances the lps-induced responses, has also been considered ( ) . recent proteomic studies based on odya labeling of raw macrophage-like cells followed by click chemistry have revealed that stimulation of cells with lps induces profound changes of the abundance of palmitoylated proteins ( ) . the data are in agreement with earlier findings showing that lps induces accumulation of s-palmitoylated lyn kinase in the raft-enriched fraction of cells, allowing it to downregulate tlr signaling ( ) . one of the upregulated s-palmitoylated proteins was type ii phosphatidylinositol -kinase iiβ, which phosphorylates phosphatidylinositol to phosphatidylinositol -monophosphate. it was shown that palmitoylation determines the involvement of the kinase in lps-induced signaling ( ) . these data suggest that s-palmitoylated proteins, including enzymes catalyzing phosphatidylinositol synthesis and turnover, are important factors affecting the pro-inflammatory responses triggered by lps. notably lps induces production of tnfα, a pro-inflammatory cytokine that is s-palmitoylated itself. tnfα is synthesized as a transmembrane -kda precursor (tmtnfα) transported from the endoplasmic reticulum to the plasma membrane through the golgi apparatus and recycling endosomes ( ) . human tmtnfα is s-palmitoylated at cys located at the boundary between its transmembrane and cytosolic fragments, as was found independently by radiolabeling and by labeling with odya followed by click chemistry ( , ) . poggi et al. ( ) arrived at a complex model explaining how the s-palmitoylation of tnfα affects its activity ( figure a) . the modification was shown to favor the association of tmtnfα with rafts. upon cell activation, the extracellular domain of tmtnf is cleaved by adam metalloproteinase whereupon the soluble tnfα (stnfα) is released to the extracellular milieu and activates tnf receptor (tnfr) and tnfr . as adam localizes to both non-raft and raft regions of the plasma membrane, the s-palmitoylation of tmtnfα does not affect its cleavage and production of the soluble cytokine. however, s-palmitoylated tmtnfα interacts with tnfr in rafts thereby reducing the binding of stnfα and consequently reducing the sensitivity of the cell to this cytokine. in addition, the fragment of tmtnfα which remains after the release of stnfα in rafts if further processed by intramembrane sppl a and b proteases giving rise to icd (intracellular domain) of an own biological activity. by contrast, the non-raft fragment of the adam -cleaved tmtnfα is rapidly degraded ( ) . the transport and maturation of tnfα are also regulated by another posttranslational acylation, ε-n-myristoylation ( ) . as shown in figure b , myristic acid residues are attached to two lysines (lys and ) of human tmtnfα. this modification is reversed by sirtuin catalyzing the demyristoylation. depletion of sirtuin decreases the release of stnfα since the ε-n-acylated tnfα precursor is redirected to and accumulates in lysosomes ( , ) . it is worth noting that exogenous palmitic acid stimulates the ε-n-myristoylation of tmtnfα, thereby reducing the release of stnfα in favor of accumulation of tmtnfα in lysosomes ( , ) . this somehow surprising anti-inflammatory effect of palmitic acid can be explained by competitive binding between long-chain fatty acids (in this case, palmitic) and myristoylated substrates of sirtuin found in vitro- ( ) and adds a new dimension to the potential effects of palmitic acid. (a) non-palmitoylated tmtnfα is localized outside rafts while that s-palmitoylated on cys -in rafts of the plasma membrane. tmtnfα is cleaved by adam protease in both these plasma membrane environments giving rise to stnfα, which subsequently activates tnf receptor (tnfr) receptor leading to activation of nfκb and erk / . however, only the raft-residing tmtnfα is further processed by sppl b protease to yield icd, which activates the promoter of interleukin (il)- β and expression of il- . on the other hand, a pool of s-palmitoylated tmtnfα interacts in rafts with tnfr preventing its activation by stnfα. (b) tmtnfα is transported from the endoplasmic reticulum via golgi apparatus and recycling endosomes [ , ] to the plasma membrane [ ] . in the plasma membrane, tnfα is cleaved by adam giving rise to stnfα [ ] or is internalized [ ] and either returns from the endosomes to the plasma membrane [ , ] or is directed to lysosomes for degradation [ ] . ε-n-myristoylation of tmtnfα at lys and lys facilitates its degradation [ , ] at the expense of processing to stnfα [ ] . oligomerization of tmtnfα and tnfr is not shown. s-palmitoylation of host proteins is also vital in antiviral defense. viral nucleic acids, which are recognized by several tlrs and also cytoplasmic pattern-recognition receptors, induce robust production of type i interferons (ifns), mainly infα and ifnβ. the ifnα and ifnβ released from cells which first encounter viruses, e.g., dendritic cells, induce an antiviral reaction in an autocrine and paracrine manner upon binding to plasma membrane ifnα/β receptor (ifnar) consisting of subunits and . both human ifnar subunits are s-palmitoylated, as has been found by classical radiolabeling. the s-palmitoylation of ifnar on cys , localized near the cytoplasmic end of the transmembrane domain, is required for downstream activation of stat and stat and the following transcription of ifnα-activated genes ( ) . among the ifn-induced proteins, some have been shown to be palmitoylated, using click chemistry and abe. they include the immunity-related gtpase irgm , bst also known as tetherin, and ifitm and ( , ) . ifitms are potent restriction factors against a wide range of enveloped viruses, e.g., influenza, west nile, dengue, and zika viruses ( , ) . ifitms localize primarily to endolysosomal membranes where they inhibit viral replication by blocking their fusion with these membranes and also facilitate virus degradation ( ) . the exact mechanism of this antiviral activity is not clear, but it seems to rely on a perturbation of the organization of endolysosomal membranes. this can be linked with the intramembrane topology of ifitms and their s-palmitoylation. ifitm and likely possess two loops embedded in but not spanning the membrane with both the n-and c-termini facing the cytoplasm ( , ) . s-palmitoylation of conserved cysteine residues adjacent to these loops, cys , , and in murine ifitm , contributes to the membrane binding, similarly as found earlier for caveolins ( , ) . the s-palmitoylation also facilitates clustering of ifitm in the membranes, which is of potential significance for its antiviral activity ( ) . in support of the latter, the antiviral capacity was markedly reduced for non-palmitoylated mutant forms of ifitm ( , ) . however, s-palmitoylation did not affect the endolysosomal localization or stability of ifitm . subsequent studies have revealed that the localization and degradation of murine ifitm , both shaping its antiviral capacity, are orchestrated by numerous posttranslational modifications comprising polyubiquitination, tyrosine phosphorylation by the src-family kinase fyn, and methylation ( , ) . by contrast, s-palmitoylation alone of the closely related murine ifitm endowed it with an antiviral activity and enhanced stability by preventing proteasomal degradation ( ) , which indicates diverse effects of this modification on individual ifitm isoforms. the presented data are only beginning to fill the gap which existed in our understanding of the role of protein palmitoylation in innate immune responses. for a long time, it was lagging behind that on acquired immune responses, in which a plethora of s-palmitoylated proteins have long been known to be involved. they include receptors (cd and cd ), tyrosine kinases of the src family, transmembrane adaptor proteins (e.g., lat, ntal, and pag/cbp), and α subunits of heterotrimeric g proteins. their s-palmitoylation in most cases targets them to rafts and is a prerequisite for their involvement in the signaling pathways triggered by immunoreceptors [tcr, b cell receptor (bcr), and fcγ and fcε receptors] crucial for the acquired immune responses. an association of some components of these signaling pathways with tetraspanin-enriched domains has also been considered. these topics are discussed in several earlier reviews ( , , , ) . it is worth noting that large-scale proteomic analyses of fatty-acylated proteins of t cells ( , , , ) and b cells ( ) , identifying numerous new palmitoylated proteins, have been published recently. further studies will shed light on the possible engagement of those proteins in acquired immune responses and/or in the cross talk between the innate and the acquired immune system, in which phagocytic cells, such as macrophages and dendritic cells, are essential ( ) . protein s-palmitoylation affects their localization, trafficking, and stability. it has long been known as an important factor controlling signal transduction by the bcr and tcr receptors involved in acquired immune responses. it is now becoming evident that palmitic acid is also a key lipid affecting the diverse processes at the host-pathogen encounter. palmitate is a component of bacterial lps and lipoproteins; s-palmitoylation of viral, some bacterial, and numerous host proteins is recognized as a crucial factor affecting both the virulence of pathogens and the innate immune reactions of the host. our understanding of the latter has benefited greatly from the development of novel methods of detection of this protein modification. their application has led to the identification of numerous proteins involved in the host-pathogen interaction. the methods have also allowed highthroughput proteomic analysis of palmitoylation of proteins in infected cells, showing widespread changes of the host cell palmitoylome. future studies will tell whether complex feedback loops comprising palmitoyl acyltransferases and acylthioesterases, similar to those of kinases and phosphatases carrying out protein phosphorylation/dephosphorylation, are involved in controlling protein s-palmitoylation in infected cells. revealing how the s-palmitoylation of particular proteins is regulated during the host-pathogen interactions should allow its modulation to favor the host defense. all authors contributed to writing and critically revised the paper. the authors thank prof. andrzej sobota from the laboratory of molecular membrane biology of the nencki institute (warsaw, poland) and dr. jan fronk from the faculty of biology, university of warsaw for helpful comments and critical discussion. the work was supported by the national science centre, poland, grant number dec- / /a/nz / to kk. mechanisms of nutritional and hormonal regulation of lipogenesis atherothrombosis and coronary artery disease microbial induction of immunity, inflammation, and cancer nutritional modulation of metabolic inflammation the impact of western diet and nutrients on the microbiota and immune response at mucosal interfaces 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incorporation onto budding virus particles the cytoplasmic domain of human immunodeficiency virus type transmembrane protein gp harbors lipid raft association determinants bioorthogonal mimetics of palmitoyl-coa and myristoyl-coa and their subsequent isolation by click chemistry and characterization by mass spectrometry reveal novel acylated host-proteins modified by hiv- infection toll-like receptors and their crosstalk with other innate receptors in infection and immunity lipoteichoic acid and toll-like receptor internalization and targeting to the golgi are lipid raft-dependent dietary saturated fatty acids prime the nlrp inflammasome via tlr in dendritic cells-implications for diet-induced insulin resistance saturated palmitic acid induces myocardial inflammatory injuries through direct binding to tlr accessory protein md modification of pro-inflammatory signaling by dietary components: the plasma membrane as a target mechanisms for the activation of toll-like receptor / by saturated fatty acids and inhibition by docosahexaenoic acid palmitic acid is a toll-like receptor ligand that induces human dendritic cell secretion of il- β acid sphingomyelinase plays a key role in palmitic acid-amplified inflammatory signaling triggered by lipopolysaccharide at low concentrations in macrophages lps upregulates palmitoylated enzymes of the phosphatidylinositol cycle. an insight from proteomic studies cytokine secretion in macrophages and other cells: pathways and mediators transmembrane tnf (pro-tnf) is palmitoylated palmitoylation of tnf alpha is involved in the regulation of tnf receptor signalling palmitoylation of interferon-α (ifn-α) receptor subunit ifnar is required for the activation of stat and stat by ifn-α ifitm-family proteins: the cell's first line of antiviral defense the ifitms inhibit zika virus replication s-palmitoylation and ubiquitination differentially regulate interferon-induced transmembrane protein (ifitm )-mediated resistance to influenza virus phosphorylation of the antiviral protein interferon-inducible transmembrane protein (ifitm ) dually regulates its endocytosis and ubiquitination greasing their way: lipid modifications determine protein association with membrane rafts protein acylation and localization in t cell signaling (review) patterns, receptors, and signals: regulation of phagosome maturation key: cord- -pcsw vmx authors: liu, xiaosheng; cao, wei; li, taisheng title: high-dose intravenous immunoglobulins in the treatment of severe acute viral pneumonia: the known mechanisms and clinical effects date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: pcsw vmx the current outbreak of viral pneumonia, caused by novel coronavirus sars-cov- , is the focus of worldwide attention. the who declared the covid- outbreak a pandemic event on mar , , and the number of confirmed cases is still on the rise worldwide. while most infected individuals only experience mild symptoms or may even be asymptomatic, some patients rapidly progress to severe acute respiratory failure with substantial mortality, making it imperative to develop an efficient treatment for severe sars-cov- pneumonia alongside supportive care. so far, the optimal treatment strategy for severe covid- remains unknown. intravenous immunoglobulin (ivig) is a blood product pooled from healthy donors with high concentrations of immunoglobulin g (igg) and has been used in patients with autoimmune and inflammatory diseases for more than years. in this review, we aim to highlight the known mechanisms of immunomodulatory effects of high-dose ivig therapy, the immunopathological hypothesis of viral pneumonia, and the clinical evidence of ivig therapy in viral pneumonia. we then make cautious therapeutic inferences about high-dose ivig therapy in treating severe covid- . these inferences may provide relevant and useful insights in order to aid treatment for covid- . recently, the outbreak of a febrile respiratory disease caused by a novel beta-coronavirus, designated sars-cov- , has become the focus of worldwide attention. the event was declared a pandemic by the who on mar , , and the number of confirmed cases is still climbing worldwide. typical clinical features of covid- include fever, respiratory symptoms such as dry cough and shortness of breath, and fatigue ( ) . while the majority of infected individuals experience only mild symptoms or may even be asymptomatic, about to % of patients rapidly progress to severe conditions with acute respiratory distress syndrome (ards), which can result in shock, sepsis, and multiple organ dysfunction ( , ) . in addition to supportive treatment, several therapeutic approaches have been proposed, such as antiviral therapy, immunomodulators, blood products, and traditional chinese medicine (tcm) ( , ) . however, the optimal treatment strategy for severe covid- remains debated ( ) ( ) ( ) ( ) ( ) ( ) ( ) . to date, it remains an urgent requirement to identify the most effective treatment for severe and critically ill covid- patients, but a conclusion has not yet been drawn. the severity of respiratory viral infections depends on a fine balance between the virulence of the pathogen and the inflammatory response of the host's immune system ( ) . although an effective immune response is essential to eliminate viral pathogens, clinical observations and experimental data indicate that an excessive host immune responserather than direct viral damage-primarily account for the pathological injury and clinical deterioration of covid- ( ) . inflammatory cytokine storms and lymphopenia are the signature features of patients with severe covid- , indicating an intense systemic inflammatory response during sars-cov- infection ( ) . specific adjuvant immunomodulatory treatments have been considered for severe viral infections, but some of these were associated with a worse prognosis. therefore, it is vital to select appropriate immunomodulators in covid- patients and to carefully assess their benefits and risks ( , ) . based on the previous clinical experience in china, it was proposed that early initiation of high-dose intravenous immunoglobulins (ivig) and low-molecular-weight heparin might be effective in improving the prognosis of severe and critically ill covid- patients ( , ) . the national diagnosis and treatment protocol for covid- (trial version ) and recommendations from the peking union medical college hospital recommend appropriate application of ivig therapy in severe and critical covid- cases ( , ) . however, the specific molecular mechanisms and clinical effectiveness of ivig treatment remain unclear. here, we review the current knowledge of immunomodulatory mechanisms of high-dose ivig and discuss its potential role in treating severe viral infection. moreover, we summarize the clinical efficacy of ivig therapy in treating severe viral pneumonias such as that caused by sars, mers, influenza, and rsv disease, and its current application in covid- . a thorough understanding of the immunomodulatory mechanisms of ivig therapy is crucial for the timing and dosing of early intervention and may provide much-needed insights into the immunomodulatory treatment of severe and critically ill covid- patients. ivig is a blood preparation isolated and concentrated from healthy donors consisting of over % of igg and trace amounts of iga or igm. ivig has been used in clinical practice for many years. different doses of ivig serve diverse medical indications, and the clinical efficacy of ivig differs with dosage ( ) . at low and moderate doses, ivig can be administered as substitutional therapy for primary or acquired immunodeficiencies in order to improve plasma igg concentrations and provide passive immunity that allows for immediate protection against microbes in immunodeficient patients. in vitro and preclinical experiments confirmed that ivig contains multivalent pathogen-specific neutralizing igg antibodies against common opportunistic pathogens and toxins (e.g., enterococcus spp., haemophilus influenzae type b, streptococcus pyogenes, staphylococcus aureus, cytomegalovirus, and shiga toxins) ( ) ( ) ( ) ( ) ( ) ( ) . variance in pathogen-specific antibody titer and neutralizing activity is usually observed among various ivig preparations, and is most likely due to differences in the geographical regions where plasma samples were collected, reflecting differences in pathogen exposure ( - ). in addition to microbial binding, a wide range of endogenous antigen-specific igg are also present in ivig preparations. these endogenous antigens may include: inflammatory cytokines (e.g., il- α, ifn-α a, and gm-csf), chemokines (e.g., ccr ), apoptosis-related molecules (e.g., siglec- , siglec- , cd /fas, baff, and april), complement fragments (c a and c a), and anti-idiotypic antibodies (e.g., anti-amyloid β antibodies and anti-coagulation factor viii antibodies) ( - ). f(ab) ′ mediated neutralization of inflammatory cytokines, chemokines, and complement fragments, along with regulation of immune cell apoptosis might contribute to the conversion of proinflammatory to anti-inflammatory conditions. meanwhile, a possible explanation for the fact that only high-dose ivig can exert anti-inflammatory effects is that a sufficient amount of antigen-specific igg is required to achieve therapeutic activity. however, as previous research revealed that preparations with purified fc fragments of igg had an undamped effect compared with intact igg for some indications, f(ab) ′ -mediated mechanisms alone may not be sufficient for the extensive immunomodulatory effects of high-dose ivig therapy ( - ). therefore, fc-mediated mechanisms may play a decisive role in these processes. the fc fragment of igg is responsible for binding to fc receptors (fcrs) and complement (e.g., c q, c b, and c b). igg-specific fc receptors (fcγrs) belong to type i fcrs and are classified into activating fcγrs (fcγri, fcγriia, fcγriic, fcγriiia, and fcγriiib) and an inhibitory fcγr (fcγriib), depending on their intracellular motif and the function they mediate. type ii fcrs, including c-type lectins (dc-sign, dcir), type i lectins (cd ), and non-classical fcrs, such as fcrn and fcrl, also act as effector targets of ivig (figure ) . the fc-mediated mechanisms of high-dose ivig therapy include saturation of activating fcγrs and fcrn, upregulation of the inhibitory fcγriib, and scavenging of complement molecules. given the central role of activating fcγrs in mediating excessive antibody-dependent effector functions, a possible explanation of the potential anti-inflammatory function of high-dose ivig lies in that it significantly raises the concentration of igg above the normal plasma levels and contributes to a functional blockade of fcγrs, limiting access of immune complexes to these activating receptors. moreover, igg from ivig preparations has been proven to be active through inhibitory itami signaling in immune regulation ( ). the role of dimeric igg in ivig preparations is emphasized by an enhanced understanding of the optimal igg form to interact with activating fcγrs. in physiological conditions, monomeric igg is more likely to bind fcγri with high affinity rather than to other fcγrs with low affinity ( ). although monomeric igg at therapeutic levels are able to saturate lowaffinity fcγr in a dose-dependent manner, it has been suggested that the small amounts of dimeric igg bind with higher avidity to fcγrs, and therefore constitute the main active component of ivig mediating fcγr blockade, explaining the immunomodulatory function of high-dose ivig in itp, gbs, and cidp patients ( - ). endogenous pathogenic autoantibodies, mainly igg, play a predominant role in the immunopathogenesis of many autoimmune and infectious diseases. a hallmark characteristic of long serum half-life of endogenous pathogenic igg is mainly due to fcrn. fcrn is a non-classical fcr that can interact with igg in a ph-dependent manner, which is sufficient in rescuing igg from lysosomal degradation and recycling it to the cell surface ( , ). high-dose ivig therapy leads to an increased clearance rate of monoclonal antibodies and pathogenic igg in different mouse models, whereas this effect is not observed in fcrn deficient models, indicating that fcrn is involved in therapeutic effects of ivig therapy ( , ). influences of fcrn gene polymorphisms on different igg kinetics and therapeutic effects of ivig among patients have been observed ( ) ( ) ( ) . pharmacokinetic models predicted that the therapeutic effects of ivig relate to total serum igg concentration and fcrn-binding capacity ( ) . the substantial increase in igg concentration may saturate fcrn and reduce the half-life of pathogenic antibodies, contributing to the anti-inflammatory mechanism of high-dose ivig. a balance between activating and inhibitory fcγrs is critical for a well-regulated immune response, and a disbalance markedly influences immunopathology in autoimmune and infectious diseases. fcγriib is the only itim-containing fcγr and negatively regulates many aspects of immune and inflammatory responses. after efficient high-dose ivig therapy, an upregulation of fcγriib on immune cells is considered to contribute to the anti-inflammatory process ( , , ) . however, evidence of underlying molecular mechanisms remains sparse and this mechanism of therapeutic action is therefore controversial. in the last decade, a prevailing theory from murine studies argued that a minor portion of igg, with specific sialylation of the asn -linked glycan structure on the fc fragment, is essential for anti-inflammatory activities of ivig via direct interaction with myeloid regulatory cells expressing sign-r (mice) or dc-sign (human) ( , , ) . subsequent research revealed that administration of sialylated igg resulted in the production of il- from hdc-sign + macrophages or dendritic cells and ensuing expansion of il- -producing basophils, and these cytokines led to fcγriib expression on effector cells ( , ) . however, other studies have challenged this hypothesis. the evidence of a direct interaction between sialylated igg and dc-sign is not fully supported in the literature ( , ) . moreover, there is conflicting evidence suggesting that the sialylated fc fragment of igg is dispensable for the anti-inflammatory mechanisms of high-dose ivig ( ) ( ) ( ) ( ) ( ) . it should be noted that all evidence of this hypothesis derives from murine studies, which might not be readily translated to human conditions. these controversial results warrant further research to address the mechanisms and molecular basis of high-dose ivig upregulation of the inhibitory fcγriib. the complement system is activated through three pathways, namely the classical pathway, the lectin pathway, and the alternative pathway. besides the f(ab) ′ -mediated neutralization of complement c a and c a, the interaction between the fc fragment and complement c q, c b, and c b in a dosedependent manner, contributes to immunomodulatory effects of ivig on the classical complement pathway ( ) ( ) ( ) . the binding site of c b/c b is located on the residues - of the ch domain of the igg fc fragment, while the residues - of the ch domain are responsible for the binding of c q ( , ) . the binding domains may react with different n-glycan sialylation patterns on the igg structure and result in distinct anti-inflammatory effects through the complement pathway ( ) . as an extremely complex preparation, ivig contains a large number of bioactive moieties, and the entirety of effects from ivig is therefore not fully understood yet. the proposed antigen-specific f(ab) ′ -mediated mechanisms and unspecific fc-mediated mechanisms are not mutually exclusive, but is more likely to regulate the immune system in synergy, giving rise to the immunomodulatory effects of high-dose ivig in specific clinical settings. although an active immune response is essential for pathogen elimination in acute respiratory viral infections, excessive defensive reactions might wreak havoc on healthy cells and tissues. complications or mortality of respiratory viral infections are often associated with excessive production of proinflammatory cytokines and ensuing multiple organ dysfunction. although the immunopathogenesis of sars-cov- has not yet been fully described, the histopathological evidence strongly suggests a critical role of an excessive immune response in mediating extensive damage of the lung and other organs, similar to previous observations in sars, mers, influenza, and rsv disease, where hyper-inflammatory responses have been shown to be involved in the lung pathology. in this section, we review how a dysfunctional immune response may cause immunopathology in severe viral pneumonia, resulting in the current understanding of ivig therapy in modulating the hyperinflammatory conditions. the cytokine storm syndrome is a form of systemic inflammatory response common to severe acute viral pneumonias, and its presence has also been suggested in severe cases of covid- . there is a correlation between severity of the cytokine storm and prognosis of severe illnesses ( ) . at the initiation of infection, the host cells detect viruses through pattern recognition receptors (prrs), which in turn triggers an interferon (ifn) response and produces other pro-inflammatory mediators such as cytokines and chemokines, informing both innate and adaptive immune system to respond appropriately to infectious pathogens. a physiological cytokine and chemokine response induced by viruses is a sprawling network, which involves endothelial cells, mononuclear macrophages, dendritic cells, natural killer cells, and lymphocytes, contributing to pathogen clearance and immune protection. however, an uncontrolled positive feedback involving all the relevant players leads to pathogenic hyperinflammation and can cause extensive damage to tissues. endothelial cells can secrete inflammatory mediators, including cytokines, chemokines, histamine, matrix metalloproteinases, and adhesion molecules, which can lead to inflammatory cell infiltration and tissue damage, and plays an essential role in inflammation, hemostasis, and angiogenesis. sars-cov- infects host cells using the ace receptor, which is widely expressed on endothelial cells. endothelial cell infection and endotheliosis have been observed in some covid- patients ( ) . direct viral infection of the endothelium may result in extensive endothelial dysfunction and disseminated intravascular coagulation ( , ) . in addition, abnormal levels of complement factors and the deposition of terminal complement complex also suggested that the activation of complement pathways may contribute to endothelial damage and the associated pro-coagulant state in covid- ( , ) . although vasculitis and autoantibodies against endothelial cells have been reported and detected in sars patients, direct endothelial cell infection hasn't been demonstrated yet ( ) ( ) ( ) . these differences in endothelial infections may contribute to the different immunopathology between sars-cov- and sars-cov- . in influenza, the central role of the pulmonary endothelium for the regulation of innate immune cell recruitment into the lungs, along with the production of excessive pro-inflammatory cytokines and chemokines, has been demonstrated ( ) ( ) ( ) . the anti-inflammatory effect of high-dose ivig on endothelial cells is mainly inferred from in vitro observations. high-dose igg has been shown to specifically and completely inhibit tnf-αinduced secretion of pro-inflammatory cytokines (e.g., il- , g-csf, and il- β) and production of e-selectin in cultured human coronary artery endothelial cells ( , ) . these effects may predominantly be mediated by f(ab) ′ -mediated mechanisms ( , ) . the expression of endothelial adhesion molecules is also reduced by co-culturing endothelial cells with an ivig preparation ( ) . additionally, the inhibitory effects of the classical complement pathway by high-dose ivig may offer a potential protective effect on virus-induced endothelial damage. dendritic cells (dcs) are antigen-presenting cells of the innate immune system. plasmacytoid dendritic cells (pdcs) are unique sentinel cells that can recognize a virus through prrs, and enhance the secretion of ifn ( , ) . data from covid- patients and a ferret model reveal low levels of ifn with contrastingly high levels of il- and a strong chemokine response, suggesting a similar hyper-inflammatory response pattern to sars and mers ( ) . several sars-cov and mers-cov proteins have been shown to antagonize the ifn response in order to reach high viral titers after early infection ( ) ( ) ( ) ( ) ( ) . delayed and massive ifn signaling is suggested to further orchestrate extensive inflammatory monocyte-macrophage responses and induce t cell apoptosis, resulting in a dysfunctional immune response and cytokine storm during sars-cov and mers-cov infection ( ) ( ) ( ) . the immunomodulatory effects of ivig on dcs differ depending on dosage. ivig accelerates maturation at physiological doses, but inhibits the maturation, activation, and function at therapeutic doses ( ) . high-doses ivig treated immature dcs secrete increased levels of anti-inflammatory cytokines, and decreased levels of pro-inflammatory cytokines ( ) . c-type lectins expressed on dcs are considered as possible effector targets of ivig preparations, however, the exact mechanism of action of ivig on dcs remains controversial. as stated, interactions between fc-sialylated igg and dc-sign failed to reproduce in human cells, and the expression of fcγriib remains stable on dcs after ivig treatment ( ) . another c-type lectin, dric, has also been identified to bind to fc-sialylated igg and further mediates the induction of treg cells in a mouse model ( ) . additionally, an increased accumulation of lipids and decreased expression of mhc-ii, cd , cd /cd , and ifn-γr on dcs upon high-doses ivig treatment might suppress antigen presentation and allogeneic t-cell stimulatory capacity, contributing to a negative regulation of the immune response ( , , ) . whether infiltrating inflammatory monocyte-macrophages (imm) are beneficial or deleterious after a viral infection is mainly dependent on their ability to secrete inflammatory cytokines and chemokines. a dysregulated cytokine response can also promote excessive activation of imm, leading to immunopathological effects on healthy tissues. macrophage activation syndrome (mas) was reported in several covid- patients with severe respiratory failure, and the production of associated pro-inflammatory cytokines (il- and tnfα) might contribute to hyper-inflammatory conditions ( ) . it was also detected that sars-cov- infects macrophages and triggers secretion of il- , which might contribute to lymphocyte apoptosis, suggesting an excessive infiltration and intense pro-inflammatory activity of imm in covid- patients ( ) . increased pathogenic imm influx has been consistently observed in severe or lethal sars patients, along with mice and chinese rhesus macaque models ( , ) . the activation of macrophages by sars-cov occurs by tlr ligand recognition, subsequent activation of the nf-κb pathway and secretion of cytokines, contributing to the immunopathology of sars ( ) ( ) ( ) . high-dose ivig therapy is widely used for treatment of patients with autoimmune diseases complicated by mas ( ) ( ) ( ) ( ) ( ) . for inhibitory effects of ivigs, functionally activating fcγrs are required to acquire a cross-tolerant state of mouse macrophages ( ) . in vitro, ivig inhibits the production of pro-inflammatory cytokines by m macrophages and triggers macrophage polarization via fcγriiimediated mechanisms ( ) . ivig limits the differentiation of macrophages through inhibition of gm-csf-driven stat activation by fcγr-dependent mechanisms ( ) . an increased production of anti-inflammatory cytokines of lps-stimulated monocytes (especially il- ) is further enhanced by ivig through phosphorylation of erk /erk and p /mapk via fcγriia-mediated mechanisms ( ) . neutrophils are the most abundant type of granulocytes in peripheral blood and constitute an essential part of innate immunity. the release of cytokines, nitric oxide, reactive oxygen species (ros), and neutrophil extracellular traps (net) by neutrophils can help to neutralize pathogens. paradoxically, however, neutrophil reactivity can also enhance tissue damage in hyper-inflammatory conditions such as severe virus infection. excessive neutrophils and net production are considered a potential predictor of prognosis in influenza ( , ) . similarly, it has been considered that neutrophils in sars-cov- may induce nets and contribute to organ damage ( , ) . high levels of net biomarkers (e.g., cell-free dna, myeloperoxidase-dna, and citrullinated histone h ) are detected in severe covid- patients and may contribute to cytokine storm and respiratory failure ( ) . both fc and f(ab) ′ fragments account for the antiinflammatory activity on neutrophils. in patients with kawasaki disease, ivig therapy reduces the activation and nitric oxide production of neutrophils ( ) . in vivo studies revealed that ivig inhibits neutrophil recruitment and activation through the activation of shp- via fcγriii-mediated mechanisms ( ) . furthermore, ivig contains anti-siglec autoantibodies that can regulate neutrophil apoptosis in a dose-dependent cytotoxic manner via f(ab) ′ -mediated mechanisms ( , , ) . interestingly, high-dose sulfo-ivig preparations significantly reduce net formation in vitro as well as in rat models ( ) . natural killer (nk) cells are innate lymphocytes that play an essential role in the control of respiratory viral infections. within days after a respiratory viral infection, nk cells are activated, recruited to the lung, and act as effector cells in a classical fcγr-mediated function, namely antibody-dependent cytotoxicity (adcc). however, a dysfunction of nk cells may also be responsible for immunopathogenesis during infection. increased inhibitory receptor nkg a expression on nk cells and reduced production of cd a, ifn-γ, il- , and tnf-α, indicate a functional exhaustion of nk cells during sars-cov- infection, similar to that observed in sars ( ) . in patients with severe sars, the number of nk cells and levels of the functional nk-marker kir dl were significantly lower than in patients with mild sars, mycoplasma pneumonia, or healthy controls, manifesting a correlation between the severity of sars and the number and function of nk cells ( ) . during influenza virus and rsv infection, virus-induced apoptosis or modulation of nk cell cytotoxicity has also been proposed to serve as a viral evasion strategy, and eventually skew the inflammatory profile of the immune system ( , ) . after high-dose ivig therapy, a reduction in number and cytotoxic activity of nk cells is observed in patients with autoimmune diseases ( ) ( ) ( ) . the dose-dependent fcγriii blockade and circulating nk cells decline occurred following ivig treatment, suggesting that inhibition of nk cells by highdose ivig is associated with the saturation of activating fcγrs ( ) . the inhibitory effects of ivig therapy on nk cells may, however, not impair its functions for controlling viral infections and malignancies ( ) . cytokines secreted by different subclasses of cd + t cells trigger the immune response to pathogens and maintain immune homeostasis. however, an imbalance of th , th , th , and treg subclasses is associated with dysfunctional cytokine responses during severe viral infections. the profiles of serum cytokines revealed an increased concentration of th cells in covid- patients ( , ) . in severe sars patients, the activation of th -related cytokines and chemokines (e.g., il- , il- , il- ) was involved in hyperinflammatory conditions ( ) . furthermore, in mers patients, the significantly increased release of pro-inflammatory cytokines from th and th during the acute phase was considered to be at least partly responsible for the immunopathology ( ) . early abundant secretion of th -and th -related cytokines (e.g., il- and il- ) has furthermore been associated with complicated infections and mortality in severe influenza patients ( , ) . in addition to mediating expansion of tregs, which profoundly impacts immune cascades, high-dose ivig has also been shown to inhibit the activation and subsequent production of cytokines by th and th cells in several clinical studies and in vitro experiments ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . moreover, f(ab) ′ fragments, rather than fc fragments, have been shown to retain the function of intact antibodies in inhibiting th and th ( ) . it has been proposed that ivig may neutralize sphingosine- -phosphate (s p) receptors on the cd + t cell and downregulate the s p -mtor signaling axis, thereby inhibiting the differentiation and infiltration of th and th cells while favoring treg cells ( ) . interestingly, monomeric iga (miga) isolated from a ivig preparation has been revealed to interfere with the stat via its f(ab) ′ fragments and inhibits the differentiation and expansion of th cells ( ) . it is noted that the proposed mechanism of functional t cell-modulation by ivig also includes dc-mediated effects; ivig-primed dcs can steer non-treg cell precursors toward a treg differentiation, and c-type lectins expressed on dc cells may be critical in this process ( , ( ) ( ) ( ) . novel treg epitope peptides (tregitopes) on igg provide a further explanation of the regulatory effects of ivig on treg cells via dc-mediated internalization of ivig ( ) . tregitopes are natural t cell epitopes of igg, and the presentation of tregitopes on dcs in the context of mhc-ii can lead to the activation and expansion of treg cells, reinforcing the immunoregulatory effects on other conventional t cells ( , ) . cd + t cells contribute to pathogen clearance and immune protection. however, cytokines and cytotoxic granules produced by activated cd + t cells may exaggerate cytokine storms and are partially implicated in the immunopathology during respiratory viral infections ( , ) . both a substantial reduction of cd + t cell counts in peripheral blood, and hyperactivation with extensive expression of surface activation markers (hla-dr and cd ) and cytotoxic granules in cd + t cells are commonly observed in covid- patients ( , ) . an accompanying functional exhaustion of cd + t cells is furthermore detected in severe covid- patients ( ) . unlike influenza or rsv, the impaired function of cd + t cells during sars-cov- infection suggests that these cells may not be major contributors to the cytokine storm in severe covid- patients ( , , ) . however, overactivation and subsequent functional exhaustion of cd + t cells may correlate with disease progression ( ) . after effective high-dose ivig therapy, the expression of hla-dr and the proportion of extensive highly activated vβ elements of cd + t cells decrease in patients with autoimmune diseases ( , ) . in vitro and animal model studies report similar inhibitory effects ( , ) . as no fcγrs are expressed on t cells, the modulation of ivig on cd + t cells may largely depend on specific antibodies present in the ivig preparation, which directly bind to t cells, or through interactions between apc and tcr signaling ( ) . for example, it has been shown that the anti-b . - peptide antibodies in ivig may be able to inhibit hla class i-restricted cytotoxicity of cd + t cells via their f(ab) ′ fragment ( ) . apart from that, a saturation of activating fcγrs on apc results in reduced immune complex internalization and presentation, further contributing to the inhibitory effects of high-dose ivig on cd + t cells ( , ) . with assistance from helper t cells and stimulation from cytokines, b cells are activated and differentiated into plasma cells to produce antibodies and contribute to humoral immunity ( , ) . apart from the generation of antibodies, il- produced by activated b cells is also considered as a possible booster of the cytokine storm and immunopathology in autoimmune disease ( , ) . the rapid reduction of peripheral b cells is also a significant characteristic of severe coivd- patients, yet the exact mechanisms underlying this accelerated lymphocyte loss remain unclear ( , ) . although there is so far no reliable evidence correlating an overactivation of b cells with the development of cytokine storms during respiratory viral infections, the production of pro-inflammatory cytokines by b cells has been proposed to play a key role in the cytokine cascade in covid- ( ) ( ) ( ) . effects of ivig on b cells vary with the dosage: at low doses, ivig induces proliferation of b cells and the production of antibodies ( , ) . on the contrary, high-dose ivig inhibits the activation of b cells via both f(ab) ′ -mediated mechanisms (e.g., neutralizing april, baff, and fas) and fc-mediated mechanisms acting on inhibitory fcrs (e.g., fcγriib and cd ), as well as the tlr- signaling cascade. fcγriib is the only classical fcγr expressed on b cells, and its elevated expression levels after effective high-dose ivig therapy may be involved in inhibitory effects of ivig on b cells ( , , ) . however, ivig-induced upregulation of fcγriib in b cells is independent of classical fcγriib signaling intermediates, suggesting that it is likely a consequence rather than a cause of high-dose ivig therapy ( , ) . it has been proposed that sialylated igg binds to dc-sign in addition to cd , a c-type lectin which is also a known low-affinity ige receptor characteristically expressed on b cells, inducing the suppression of b cells ( ) . however, there has been disagreement as to whether there is a direct interaction between igg and dc-sign/cd ( , ) . a further proposed mechanism of action of high-dose ivig is the interaction between the sialylated fc fragment of igg and cd , an i-type lectin expressed on b cells, which results in the activation of the itim signaling cascade, subsequently promoting b cell apoptosis ( , , ) . except for fcrs, the tlr- signaling cascade may also be involved in inhibitory effects. ivig could mimic the effects of myd inhibitors and result in the suppression of the tlr-induced nf-κb signaling pathway and downstream production of cytokines ( , ) . a "paradoxical" phenomenon has been observed, where large amounts of immature plasma cells were mobilized after high-dose ivig therapy in patients with autoimmune diseases, suggesting de novo b cell activation. this is consistent with the immunomodulatory functions of ivig at low doses ( , , ) . although the potential roles of plasma blasts under different disease conditions remain unclear, this phenomenon highlights binary immunomodulatory effects of high-dose ivig to humoral immunity. to summarize, the cytokine storm observed during severe viral infection is associated with various immune dysfunctions. several therapeutic interventions targeting the host immune response have been attempted. although the proposed mechanisms of high-dose ivig on the modulation immune cells and cytokine cascades are mainly derived from research into autoimmune diseases, its use in reversing a cytokine storm, in addition to providing passive immunity during severe viral infection, is supported by recent evidence. the humoral immune response to invading pathogens by producing neutralizing antibodies is crucial in the host adaptive immune system. paradoxically, antibodies may provide an attractive means for a variety of viruses to enhance viral entry and replication in some cell types under certain conditions. the unique phenomenon that preexisting poor neutralizing antibodies facilitate viral access to fcγrs and lead to enhanced infection or immunopathology is also referred to as antibodydependent enhancement (ade). ade has been implicated widely in flavivirus infections, such as wnv and denv, and highdose ivig is commonly used in treating wnv encephalitis with satisfactory effect. however, little is known about ade in respiratory virus infection ( ) ( ) ( ) . ade is currently being considered as a potential contributor to the immunopathology of sars. people who succumbed to sars had significantly higher s glycoprotein-specific neutralizing antibodies in serum during the early stage of infection, indicating the possible presence of ade ( ) . similarly, recent results in sars-cov-infected chinese rhesus macaques consistently showed that anti-spike igg significantly amplified pro-inflammatory cytokine production in activated macrophages and enhanced pulmonary pathology. these effects could be reduced by the fcγrii blocking antibody ( ) . similarly, it has been shown that infection of macrophages is enhanced by anti-sars-cov spike immune serum but eliminated by the fcγrii antibody, suggesting an fcγrii-dependent ade of sars-cov ( ) . molecular signaling analyses investigating fcγriimediated infection by sars-cov revealed that an intact cytosolic domain of fcγr was required, and fcγriia was more prone to ade than fcγriib ( ) . although in vivo evidence of ade in mers is lacking, it has been shown that mers-cov infections depended on monoclonal antibody (mab) concentration, binding affinity of mab for the viral receptor dpp , and tissue expressions of dpp and fcγr, providing a molecular basis of ade in mers-cov ( ) . likewise, the presence of sub-neutralizing titers of antibodies is also a contributing factor to the unfavorable outcome in influenza infection. prophylactic treatment with monoclonal antibodies at a low dose following the h n virus challenge in mice has shown enhanced cellular infiltration and lung pathology compared to the control group ( ) . in contrast, a high dose prophylaxis showed protective effects ( ) . vaccinating pigs against h n resulted in the generation of cross-reactive anti-ph n ha antibodies, which exhibited poor neutralizing ability to the ha domain and enhanced ph n infection and lung pathology ( ) . besides influenza and potentially mers, the ade phenomenon has also been observed in rsv infection ( ) . it has been shown that rsv infections are significantly enhanced in nk cells previously incubated with sub-neutralizing titers of rsv-specific antibodies in vitro, promoting ifn-γ production of nk cells ( ) . it remains unclear whether such in vitro enhancement of rsv infection has a correlation with in vivo disease. nevertheless, the fc-mediated antibody effector functions are believed to participate in the antibody-dependent enhancement of rsv disease ( , ) . to date, the exact role of ade in sars-cov- infection remains unclear. regarding concerns that reappearance of cross-reactive antibodies from other serotypes of coronavirus may enhance the current infection, the risk of ade needs to be clarified when treating covid- patients ( , ). as described above, the occurrence of ade requires binding of the virion to antibodies at a sub-neutralizing concentration and subsequent uptake by fcγr-bearing cells ( ) . to inhibit potential phenomenon of ade in covid- , high-dose ivig therapy is therefore proposed ( ) . first, it is unlikely to have preexisting anti-sars-cov- antibodies in ivig preparations early in the novel epidemic. additionally, a high concentration of non-neutralizing antibodies, rather than a regular or diluted dose, would integrally saturate activating fcγrs and fcrn, which is beneficial to limit access of immune complexes and enhance clearance of inimical antibodies, to ultimately provide immunomodulatory effects in severe covid- patients ( , ) . based on its efficacy and supporting mechanisms in modulating the inflammatory response and improving serum igg levels, high-dose ivig therapy is considered for treatment of several severe viral infections. potential protective effects of ivig preparations were previously shown in influenza-and rsvinfected animal models ( ) ( ) ( ) ( ) ( ) ( ) . in this section, we review the previous clinical application of high-dose ivig therapy in treating viral pneumonia such as sars, mers, influenza and rsv disease, as well as its current application in covid- ( table ) . during the global outbreak of sars in caused by sars-cov, different therapeutic modalities (e.g., α-interferon, ribavirin, lpv/r, corticosteroids, convalescent plasma, and ivig) were empirically used ( ) . although strong evidence recommending the administration of ivig therapy is lacking and a previous systemic review concluded that the evidence for efficacy of improving prognosis with ivig therapy remained inconclusive, some studies commented that patients seemed to improve upon ivig treatment ( , ) . in a randomized controlled trial, ivig therapy efficiently improved the serum igg concentration of severe sars patients compared to the control group ( ) . however, possibly due to the inadequately controlled dosage of glucocorticoids and ivig therapy, which were given at the discretion of clinicians, there were no significant differences in the fatality rates and the rates of nosocomial infection of severe sars patients between the ivig group and the control group ( ) . apart from hypogammaglobulinemia, two further common features of severe sars are leukopenia and thrombocytopenia, and these features seemed to improve in sars patients upon ivig therapy ( , ) . the peripheral wbc and platelet count of severe sars patients significantly increased after receiving ivig therapy without steroids, suggesting a potential role of ivig in controlling leukopenia and thrombocytopenia in sars patients ( ) . furthermore, significant clinical improvement in pediatric sars patients was noted after ivig therapy ( ) . a declining wbc significantly recovered after ivig therapy, and the temperature of most patients normalized within days. compared to the historical controlled group, a significantly shorter time of chest radiographic absorption in these patients was observed and indicated the potential efficacy of ivig in clinical improvement and absorption of lung lesions. interestingly, an igm-enriched ivig preparation also showed significant beneficial effects in deteriorating sars patients who failed corticosteroid and ribavirin treatment ( ) . a significant improvement regarding the oxygen requirements and radiographic scores were observed after pentaglobin therapy, and most patients recovered. it has been proposed that abnormal cytokine levels (e.g., il- and tnf-α) are correlated with poor prognosis in sars patients, and that the inhibitory effects of pentaglobin on cytokine release might represent an essential mechanism of action in the treatment of sars ( , ) . mers is a highly fatal respiratory disease caused by mers-cov, with two major historic outbreaks in and . the rapid deterioration of health in a large number of patients made it unrealistic and unethical to perform randomized controlled treatment trials. therefore, the current availability of strong evidence is minimal, and only a few case series reported administration of ivig late in the course of mers. these studies discussed the possible efficacy of ivig in reversing severe thrombocytopenia through immunomodulatory mechanisms ( , ) . although influenza has been around for centuries, severe influenza remains a health challenge for humans. the complications or deaths are usually associated with the extensive induction of pro-inflammatory cytokines in severely affected influenza patients ( ) . immunomodulatory strategies or treatments, including amongst others corticosteroids, ppars agonists, s p receptor agonists, antioxidants, and ivig frontiers in immunology | www.frontiersin.org ards, acute respiratory distress syndrome; n.d., no data; spo , surplus pulse o ; crp, c-reaction protein; apache ii, acute physiology and chronic health evaluation score; urti, upper respiratory tract infection; lrti, lower respiratory tract infection. *all patients were treated with arbidol. † % patients were treated with ribavirin, % with oseltamivir, and % with arbidol. ‡ % patients were treated with lopinavir/ritonavir, % with interferon, and % with arbidol. § patients were treated with oseltamivir, and patients with steroids and lopinavir/ritonavir. ¶ patients received ivig at . - g/d for - d, average to g/d for d. these patients were treated with ribavirin. **all patients were treated with oseltamivir, and . % with zanamivir. † † all patients were treated with oseltamivir. frontiers in immunology | www.frontiersin.org therapy, have been widely considered as adjunctive treatments for cytokine storms in influenza ( ) . in a previous randomized controlled trial, patients infected with the severe pandemic influenza a (h n ) were randomized to receive . g/kg for one dose of flu-ivig (antiinfluenza hyperimmune intravenous immunoglobulin with high hai antibodies level) or ivig therapy ( ) . patients who received flu-ivig showed a greater rate of viral load reduction than the ivig group. however, there was no significant difference in the cytokine profiles (e.g., ifn-α , il- α, il- , il- , il- , il- ra, mcp- , mip- α, gm-csf, tnf-α) between the ivig and flu-ivig group on day . subgroup multivariate analysis of patients receiving treatment within days of symptom onset revealed that flu-ivig therapy, rather than ivig therapy, was the only factor that independently reduced mortality. in contrast, other rcts using ivig treatment at high doses have shown improved survival. in two similar studies, pediatric patients with the severe pandemic influenza a (h n ) were randomized to receive standard care or standard care plus g/kg/d high-dose ivig for days ( , ) . significant clinical improvement with regards to temperature normalization, reduction of cough, rhinorrhea, tachypnea, respiratory sounds (namely wheeze or rhonchi), and chest radiographic lesions were observed in the ivig group compared to the control group, revealing a possible clinical benefit of high-dose ivig therapy for the improvement of clinical symptoms in pediatric patients with severe influenza a h n ( , ) . rsv is a major respiratory pathogen that causes extensive respiratory symptoms, including both upper and severe lower respiratory tract infection (urti/lrti) in infants, the elderly, and immunocompromised individuals ( ) . available therapies are limited to rbv, ivig, and palivizumab, but the use of ivig for treatment of rsv infections remains controversial ( ) . a study showed that hypogammaglobulinemia was a significant risk factor for fatal outcomes of rsv infections in a hematology and transplant unit, and treatment with ivig at regular doses was unable to reverse the poor prognosis ( ) . by contrast, oral rbv in combination with high-dose ivig therapy (rather than lower doses), have been potentially attributed to the improvement of survival in rsv-infected allogeneic hematopoietic stem cell transplanted (hsct) patients ( ) . the dosage of ivig might be the critical factor for efficacy of therapy, since the combination of high-dose ivig and aerosolized rbv therapy has also been shown to be a safe and promising approach to prevent progression of rsv infections immunocompromised patients ( ) . several published studies suggest that a combined therapy of rbv and ivig improves the outcome of hsct and leukemia patients with rsv-urti, and additional recommendations are given for high-risk patients with rsv-lrti ( ) ( ) ( ) . however, it is noted that most studies on ivig efficacy are limited to pediatric or hsct patients, which may not explicitly be recommended in all situations ( ) . in summary, ivig therapy exhibits different levels of potential clinical benefits in sars, mers, influenza, and rsv infections, though currently there is no high-level evidence to support ivig use in these infections. of note, the presented results are partly limited to pediatric and immunocompromised patients, and the clinical benefits of ivig therapy may vary in different patient populations (e.g., infants, the elderly, and immunocompromised individuals). it is also noteworthy that most studies for these viral infections show possible benefits from ivig therapy at higher doses, which indicates that a high dosage might be key for clinical efficacy. considering the immunomodulatory effects of highdose ivig on the excessive immune cascade, high-dose ivig therapy, as stated above, may be considered a potential adjunctive treatment for severe covid- patients. the national diagnosis and treatment protocol for covid- (trial version ) and recommendations from the peking union medical college hospital have suggested the application of ivig therapy in severe and critically ill covid- patients ( , ) . several observational and interventional studies were conducted to evaluate the efficacy of ivig. a retrospective study (n = ) in china included severe and critical ill covid- patients that were administered ivig therapy after admission ( ) . ivig therapy was given at g daily, and of ( . %) patients died within days. for retrospective analysis, patients were split into two groups, based on whether they received ivig administration within or after h of admission to the intensive care unit (icu). the results showed that application of ivig within h of admission to icu reduced the use of mechanical ventilation, shortened duration of icu and hospital stay, and improved -day survival ( ). the -day mortality of the two groups (ivig within or after h) was . % (within h) and . % (after h), indicating that early initiation of high-dose ivig therapy may be beneficial in severe covid- patients. on the contrary, there were no significant effects of ivig therapy on the survival of severe covid- patients who developed ards in another study; here, the insufficient effects of ivig therapy may potentially be a result of timing and dosage of ivig administration ( ) . in another study, a combination therapy of a high dose of ivig ( g/d) and corticosteroid ( mg/d) succeeded in reversing the deteriorating condition of severe covid- patients who had previously failed low-dose ivig ( g/d) and corticosteroid treatment ( ) . however, as this study investigated a combined therapy, it is not possible to estimate the exact effect of high ivig doses in this group of patients. the administration of ivig therapy ( g/d for days) at the time of initiation of respiratory distress of severe covid- patients may improve the prognosis ( ) . it is noted that only one of three patients received corticosteroids, and these observations suggest that high-dose ivig therapy may be the main contributor to successful recovery from deteriorating conditions. however, more controlled trials of ivig therapy are needed to provide strong evidence for its beneficial effects in covid- . based on these clinical observations, the administration of high-dose ivig therapy at the appropriate point may be able to prevent disease progression and improve the prognosis of patients with severe covid- . currently ongoing randomized controlled trials of high-dose ivig therapy in severe covid- patients are evaluating the benefit of ivig compared to standard care (nct and nct ) and will be able to further provide more information about the clinical effects of ivig therapy in covid- . although a large number of clinical trials have demonstrated that ivig therapy is well-tolerated, various side effects have been reported ( ) . the majority of these effects are mild and transient, yet clinicians need to be vigilant about rare but serious adverse effects such as aseptic meningitis, renal impairment, thrombosis, and hemolytic anemia ( ) . it is noted that thrombosis is common in covid- , and whether highdose ivig therapy would further increase the risk of thrombosis remains unclear ( , ) . however, the combined treatment of low molecular weight heparin and high-dose ivig therapy at . - . g/kg/d for days in the early phase has been suggested to have proper efficacy in treating severe covid- patients ( ) . the ivig preparation is a widely used pooled human blood product that can provide passive immunity and modulate the immune functions. although, for coivd- the pathogenspecific effects of ivig are not relevant yet, since available preparations were collected from healthy donors without preexisting immunity early before the pandemic began. the anti-inflammatory and immunomodulatory effects on the various immune cells of high-dose ivig may account for its clinical benefits. based on these potential supportive f(ab) ′ and fc mediated mechanisms and the known clinical effects in treating severe virus pneumonia such as sars, mers, influenza, and rsv disease, the early application of high-dose ivig therapy may be considered in the management of severe covid- patients. 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allogeneic haematopoietic stem cell transplant recipients community acquired respiratory virus infections in cancer patients-guideline on diagnosis and management by the infectious diseases working party of the german society for haematology and medical oncology adverse effects of immunoglobulin therapy covid- and thrombotic or thromboembolic disease: implications for prevention, antithrombotic therapy, and follow-up the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © liu, cao and li. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -ocbygkyb authors: ye, zi-wei; yuan, shuofeng; poon, kwok-man; wen, lei; yang, dong; sun, zehua; li, cun; hu, meng; shuai, huiping; zhou, jie; zhang, mei-yun; zheng, bo-jian; chu, hin; yuen, kwok-yung title: antibody-dependent cell-mediated cytotoxicity epitopes on the hemagglutinin head region of pandemic h n influenza virus play detrimental roles in h n -infected mice date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: ocbygkyb engaging the antibody-dependent cell-mediated cytotoxicity (adcc) for killing of virus-infected cells and secretion of antiviral cytokines and chemokines was incorporated as one of the important features in the design of universal influenza vaccines. however, investigation of the adcc epitopes on the highly immunogenic influenza hemagglutinin (ha) head region has been rarely reported. in this study, we determined the adcc and antiviral activities of two putative adcc epitopes, designated e and e , on the ha head of a pandemic h n influenza virus in vitro and in a lethal mouse model. our data demonstrated that sera from the e -vaccinated mice could induce high adcc activities. importantly, the induction of adcc response modestly decreased viral load in the lungs of h n -infected mice. however, the elevated adcc significantly increased mouse alveolar damage and mortality than that of the pbs-vaccinated group (p < . ). the phenotype was potentially due to an exaggerated inflammatory cell infiltration triggered by adcc, as an upregulated release of cytotoxic granules (perforin) was observed in the lung tissue of e -vaccinated mice after h n influenza virus challenge. overall, our data suggested that adcc elicited by certain domains of ha head region might have a detrimental rather than protective effect during influenza virus infection. thus, future design of universal influenza vaccine shall strike a balance between the induction of protective immunity and potential side effects of adcc. introduction influenza viruses, as one of the major zoonotic pathogens, have continuously caused global health concerns due to their high propensity for unpredictable genetic mutation in major surface antigens, hemagglutinin (ha), and neuramindase. antivirals and vaccines are vital in combating the threat of influenza epidemics and pandemics. however, the increasing usage of licensed antivirals has resulted in the global emergence of amantadine-and/or oseltamivir- ( ) . on the other hand, seasonal influenza vaccines have to be updated annually due to the continuous antigenic drift and shift ( ) . otherwise, the mismatch between vaccinated formulations and that of natural selection would considerably limit the effectiveness of influenza vaccines. neutralizing antibodies have traditionally been thought to provide protection against influenza viruses. nevertheless, the effectiveness induced by such vaccines is limited by the emergence of mutant viruses that are resistant to antibodymediated neutralization ( ) . in this regard, the quest for universal influenza vaccines has fueled the interest in broadly reactive antibodies in addition to direct virus neutralizations. antibody-dependent cell-mediated cytotoxicity (adcc) uses effector arms of both innate and adaptive immune responses to eliminate target cells. natural killer (nk) cells, upon triggered by specific adcc antibodies, mediate the clearance of virusinfected cells ( ) . the nk cell cd receptor recognizes the fc portion of igg antibodies that in turn bind to antigens on virus-infected cells ( ) . this interaction induces degranulation of nk cells to release perforin/granzymes as well as secretion of antiviral cytokines such as interferon-γ (ifn-γ) and tumor necrosis factor-α (tnf-α) ( ) . since adcc could invoke protective immune response against infections from a broad array of viruses, the adcc antibody response was incorporated as one of the most important features of potential universal vaccine candidates by the world health organization. notably, multiple components of influenza viruses are known to induce adcc, including the viral surface proteins such as ha ( ) and m ectodomain ( , ) , as well as the internal proteins including np and m ( ) . the glycoprotein ha consists of two functional domains, the immunodominant globular head domain and the more conserved stalk domain ( ) . conventionally, neutralizing antibody response to influenza virus is dominated by antibodies that target the head region, which block the virus receptor-binding site. compared with the bulky globular ha head, the ha stem region is far less immunogenic, and antibodies directed to this domain occur at a relatively low frequency. however, a rare class of neutralizing antibodies that target a conserved site in the ha stem was reported recently, which might shed new light for the development of universal influenza vaccines ( ) . we have previously identified two putative adcc epitopes that mapped to the ha head of a pandemic h n influenza virus ( ) . both epitopes, designated e and e , revealed by epitope mapping of convalescent-phase plasma igg antibodies from six h n -infected human subjects, are dominant and highly conserved ( ) . in this study, we further dissected the function of the two adcc epitopes in vitro and in a lethal mouse model. surprisingly, our results demonstrated that the adcc response elicited by the e epitope triggered a detrimental rather than protective effect against influenza virus infection. while the antiviral efficacy provided by the stalk-specific adcc antibodies has been confirmed ( ) , our data raised concerns on the side effect of certain ha head epitopes in devising a universal influenza vaccine. in this regard, our study suggested that a delicate balance between protective immunity and over induction of adcc should be maintained, which should be an important consideration in evaluating vaccine safety. the la cell line, which was derived from mouse lung adenoma, was maintained in dmem/f- medium (gibco) supplemented with % heat-inactivated fetal bovine serum (fbs), u/ml penicillin, and μg/ml streptomycin (p/s). peripheral blood mononuclear cells (pbmcs) were prepared by ficoll-paque separation ( ) of heparinized whole blood obtained from healthy balb/c mice ( - weeks old). to prepare the adcc target cells, la cells were transfected with an ha expression plasmid that based on the cdna fragment of influenza virus strain a/hong kong/ / (h n )pdm . specifically, the full-length ha fragment was cloned into a mammalian expression vector peak plasmid containing a mouse igg fc gene (ch + ch ) ( ) . the pandemic h n influenza virus strain a/hong kong/ / (h n )pdm was used for in vitro virus infection; while its mouse-adapted version, a/hong kong/ md/ (h n )pdm was propagated in embryonated hens' eggs and utilized for in vivo experiment ( ) . the viruses were stored in − °c in aliquot and titrated by standard plaque assay. all experiments with live viruses were conducted using biosafety level facilities as described previously ( ) . balb/c female mice, - weeks old, were kept in biosafety level housing and given access to standard pellet feed and water ad libitum. all experimental protocols followed the standard operating procedures of the biosafety level animal facilities and were approved by the animal ethics committee in the university of hong kong ( ) . vaccinations were carried out to immunize the mice with e or e or ha epitopes by dna priming and protein boost. pbs was used as a negative control. the specified vaccination scheme was listed in table . to prepare the dna plasmids, either of the e or e fragment ( ) was cloned into the mammalian expression vector peak as described for the ha plasmid construction. priming of the mice by electroporation. to prepare protein for vaccination, recombinant ha, e , and e fusion proteins were expressed in freestyle ft™ transient expression system (invitrogen) and purified by protein a affinity (ge healthcare). subsequently, proteins were dialyzed and concentrated with vivaspin centrifugal concentrator (ge healthcare), followed by protein boosting through intramuscular route. each mouse received μg protein at each protein boosting. sera were obtained at day postimmununization before virus challenge. antibody titers raised against e , e , and ha in mouse sera were evaluated by elisa as previously described with some modifications ( ) . mouse sera collected from the pbs-treated group were taken as a background control. immunized mice ( mice/group) were inoculated with five % lethal dose (ld ) of mouse-adapted pandemic h n influenza virus by intranasal route, i.e., , pfu/mouse. animal survival and body weight were monitored daily for days after virus challenge. a body weight loss of % was set as the humane endpoint. three mice per group were randomly selected and euthanized on day and post-challenge, respectively. mouse lungs were collected from the euthanized mice. half of the lung tissues were harvested for virus titration by rt-qpcr methods ( ) , while the other half were immediately fixed in % of pbs buffered formaldehyde for histopathological analyses as described previously ( ) . slides were examined in a blinded manner and scored with a semiquantitative system according to the relative degree of inflammation and tissue damage ( ) ( ) ( ) ( ) . inflammation was scored as follows: , no inflammation; , perivascular cuff of inflammatory cells; , mild inflammation (extending throughout % of the lung); , moderate inflammation ( - % of the lung); , severe inflammation involving over one half of the lung. antibody-dependent cell-mediated cytotoxicity activity, reflected by the rate of cell death, was measured by a flow cytometry-based assay that described previously with some modifications ( ) . generally, the pkh- dye (sigma) was utilized to label the target cells, i.e., ha-transfected la- cells; while -aminoactinomycin d ( -aad; invitrogen) was used to stain the dead cells that mediated by adcc activity. experimentally, pkh- -labeled target cells and unlabeled effector cells (i.e., pbmcs) were prepared in rpmi medium (gibco) containing % fbs and % p/s with a cell density of and cells/ml, respectively. subsequently, μl of target cells were dispensed into a round-bottom -well plate, followed by addition of μl of mouse serum. mouse serum concentration in each group was normalized before addition according to their titers determined by elisa. one hour after incubation, μl of effector cells were added to incubate with the target cells. three hours later, another μl of -aad was added to each well before performing the flow cytometry. cell death was determined with a facsaria iii flow cytometer using the bd facs software (bd biosciences). percent cell death was calculated by software analysis of four identifiable cell populations: live effector cells (no dye), dead effector cells ( -aad positive), live target cells (pkh- positive), and dead target cells (pkh- and -aad double positive). assay controls used to define cell populations included target cells alone (background cell death) and target cells with μl triton x- added (maximum cell death). percent adcc was calculated as [(percent experimental lysis − percent spontaneous lysis)/(percent maximum lysis − percent spontaneous lysis)] × %, in which percent spontaneous lysis refers to the percent lysis of infected cells with effectors but in the absence of serum, while percent maximum lysis refers to the percent lysis of infected cells with effectors in the presence of % triton x- . experiments were performed in triplicate and repeated twice for confirmation. immunostaining was performed as previously described to visualize perforin expression in mouse lung tissues ( ) . rat anti-mouse perforin (abcam ab ) and goat anti-rat alexa were used as primary and secondary antibodies, respectively. images were acquired with a carl zeiss lsm system. table s in supplementary material. statistical comparison was performed by student's t-test using graphpad prism . differences were considered statistically significant when p < . . results adcc responses are enhanced by the sera of e -vaccinated mice in our previous study, we mapped two putative adcc epitopes, e and e , on the ha head region. by depleting e and/or e from clinical plasma igg antibodies, the adcc activity dropped significantly, which suggested that the two epitopes played potential roles in eliciting adcc ( ) . in this study, we sought to confirm the function of these putative epitopes in the induction of adcc activity using a mouse model. immunization of mice gave raise to igg titers against the e , e , or ha protein, as quantified by elisa (figure ) . a prime/boost immunization strategy was adopted, and mice that immunized with pbs or ha were included as a negative or a positive control, respectively ( table ). our results indicated that serum samples from mice vaccinated with e ( figure a ) or e ( figure b) both exhibited strong dilution-dependent antibody responses, reaching a titer of more than : . additionally, using ha as the coating antigen for elisa, we demonstrated that e and e sera could bind fulllength ha at a comparable efficiency ( figure s in supplementary material). taken together, our data suggested that the vaccination was successful and the resultant serum samples could be utilized for further investigations. next, adcc activities in serum samples from e -, e -, or ha-immunized mice were evaluated by flow cytometry-based adcc assays. to this end, the ha-transfected la cells were labeled with the cell-membrane marker pkh and utilized as target cells for adcc-specific antibody binding. subsequently, the vaccinated mouse serum was added to bridge the interaction between target cells and pbmc effector cells (figure ) . the presence of adcc-mediating antibody was determined by analyzing the cytotoxicity rate within the cell mixture that contained the target cells, serum, and effector cells, in which the dead target cell population was revealed by the cell death marker, aad (figure ) . as shown in figures a-g , sera from the e -vaccinated mice consistently triggered the highest aad + rate among all evaluated groups, suggesting that a higher percentage of cell lysis was induced in the e group in comparison to the other groups. the percentage of cytotoxicity was normalized using the formula reported by srivastava et al., with spontaneous and maximum cell cytotoxicity rate taken into consideration ( ) . quantitatively, sera from the e -vaccinated mice elicited a significantly increased (p < . ) adcc activity in comparison with the pbs-vaccinated group (figure h) . of note, though sera from the e -vaccinated mice triggered a subtle increase in adcc activity, the difference was not statistically significant ( figure h) . intriguingly, albeit ha contained the e epitope, sera from the ha-vaccinated group did not induce a significantly elevated cytotoxicity response in comparison to that of the pbs control group (figure h ). since e was capable of inducing adcc activities, we hypothesized that e -vaccinated mice could potentially be protected by the elicited adcc activity after influenza virus challenge. to this end, we inoculated the vaccinated mice with pandemic h n influenza virus in a lethal mouse model ( figure a) . as shown in figure b , mice in the ha-vaccinated group, as a positive control, demonstrated a substantial reduction of viral load on both day and day post-inoculation in comparison to the pbs-vaccinated group. importantly, we detected an approximately one log decrease of viral load in the mouse lungs of the e -vaccinated group in comparison to that of the pbs-vaccinated group on day post-inoculation, while no significant difference could be observed between the two groups on day post-virus challenge (figure b ). in addition, the viral load in the lungs of the e -vaccinated mice was notably lower on day when compared with that of day , suggesting that the adcc effect was triggered between day and postinoculation ( figure b) . figure | schematic diagram of the antibody-dependent cell-mediated cytotoxicity (adcc) assay. adcc activity was determined by a flow cytometrybased assay using two fluorescent dyes. pkh- , a membrane-labeling dye, was used to specifically identify the ha-transfected target cells. aad was excluded by viable cells but could penetrate the cell membrane of dead or dying cells and intercalate into double-stranded dna. briefly, μl of pkh- -labeled target cells ( cells/ml) was dispensed into a round-bottom -well plate, followed by addition of e /e /ha/pbs sera and effector/pbmc cells. following a -h incubation, -aad was added. cell death was determined on a facsaria iii flow cytometer using bd facs diva software (bd biosciences). percent cell death was analyzed by the flowjo software. in parallel, we measured the survival rate and body weight changes of the mice. as shown in figure c , all mice from the ha-vaccinated group survived the course of infection while all mice received pbs-treatment died, indicating that the virus challenge was successful. unexpectedly, our results demonstrated that the mice in the e -vaccinated group succumbed to influenza virus infection at a significantly earlier time (p < . ) postinoculation when compared with that of the pbs-vaccinated control group (figure c ). in line with the survival rate, mice from the e -vaccinated group suffered from an apparently accelerated weight lost starting on day post-inoculation in comparison to mice from the pbs-and e -vaccinated groups, although the difference did not reach statistical significance ( figure d) . next, we carried out histopathological examinations on the lung sections of the virus-infected mice. using uninfected mouse lung tissues as a control (figures i,j) , our observation showed that on both day and day , mice from the ha-vaccinated group (figures g,h) exhibited ameliorated alveolar morphology changes when compared with those from the e (figures c,d) , e (figures e,f) , and pbs (figures a,b) groups. importantly, the lung pathological scores of mice from the ha-vaccinated group on both day and day were significantly lower than those of the pbs-treated mice (figures k,l) ; while mice from the e -vaccinated group suffered from a significantly more dramatic interstitial inflammatory infiltration than that of the pbs-treated mice on day ( figure l ). this result indicated that the detrimental lung damage of e -vaccinated mice, possibly triggered by adcc, might account for the reduced viral load in lungs as well as the earlier drop in survival. to address whether the severe lung damage in the e -vaccinated group could be attributed to the adcc effect, we performed immunofluorescence staining to visualize the expression level of perforin among different mouse groups. perforin is released by activated nk cells and is known as a marker of adcc activation ( ) . as quantitated in figure m , the e -vaccinated mice (figures d-f) demonstrated the highest perforin expression level in the lung sections amongst the other three groups on day post infection (figures a-c,g-l) . however, the mrna expression level of perforin was not significantly different across all evaluated groups ( figure n) . binding of fc receptor (fcr) on effector cells triggers the secretion of cytotoxic granules as well as antiviral cytokines and chemokines, such as ifn-γ and tnf-α ( ). to investigate if elevated expression of proinflammatory cytokines might contribute to the lung damage, we measured the mrna expression level of representative cytokines including tnf-α (figure a) , il- β (figure b) , and ifn-γ (figure c ) in the mouse lungs samples. our results showed that the gene expression of all three proinflammatory cytokines were significantly enhanced in the e -vaccinated group when compared with those of the pbstreated group (figure ) , which were in line with the perforin protein expression pattern in figure . together, our data suggested that the e epitope from the ha head domain mediated unfavorable adcc that resulted in a more severe lung damage and exacerbated mouse mortality despite a successful control of the h n influenza viral load. antibody-dependent cell-mediated cytotoxicity, as a bridge of the innate and adaptive immunity, plays important roles in humoral and cellular immune response ( , ) . since adcc antibodies are known to recognize a wide range of viral proteins that lead to the lysis of virus-infected cells, a better understanding on the adcc mechanism during influenza virus infections will facilitate the development of universal influenza vaccines with broader protections ( , , ) . the conserved viral proteins or domains, such as m extracellular domain and ha stem domain, have been widely studied as potential targets of domain-based universal influenza vaccines. jegerlehner and colleagues have demonstrated that the protective role of m adcc-mediating antibodies was dependent on fcr ( , ) . dilillo et al. provided further support that the influenza-specific adcc antibody, though elicited by the ha stem, also required fcrs interaction for protection against lethal influenza infection ( ) . collectively, both studies highlighted the functional importance of fcr during adcc-mediated virus clearance. on the other side, unexpected cases have been reported that young adults without prior exposure to the h n virus produced robust adcc-mediating antibody response upon infection of this virus strain. some individuals even possessed cross-reactive adcc-mediating antibodies toward avian h n and h n strains in the absence of prior exposure ( ) . however, the mechanism of such antibody responses remains unclear to date. in this study, we investigated the adcc effect of the two putative ha head epitopes in vitro and in vivo. our data demonstrated that e -induced adcc activity against h n influenza virus infection in vitro (figure ) . unexpectedly, although e vaccination decreased the viral load in h n -infected mice (figure b) , it induced exacerbated lung damage ( figure ) and a higher level of nk activity (figure ) that accelerated mouse death ( figure c ). nk cells, which offer the first line of defense against virus infection, have been widely considered to be beneficial to the host during viral infections. however, a recent report by zhou et al. revealed that adoptive transfer of nk cells from influenza virus-infected lungs, but not uninfected lung, resulted in a more rapid weight loss and increased mortality of virus-infected mice ( ) . this finding was in line with our observation that e -induced adcc exhibited deleterious impact to promote mortality during influenza virus infection. most healthy donors have a persistently low level of crossreactive adcc-mediating antibodies, while these cross-reactive antibodies are found in individuals in the absence of detectable neutralization ( , ) . in our previous study, both e and e epitopes were identified as putative regions that could induce adcc activity. the depletion of such antibodies in human plasma significantly decreased the adcc effect. however, for certain samples, it appeared that more diluted plasma exhibited higher adcc activity than less diluted plasma, and the use of igg antibodies at a low concentration led to a higher adcc activity than the use of igg antibodies at a high concentration ( ) . to date, there is no conclusive study on the correlation between antibody concentration and adcc activity, neither was the optimal concentration of adcc antibodies that could protect against virus infection elucidated. in this context, we demonstrated here that an overwhelming production of adcc antibodies in the absence of neutralization might not play a protective role against influenza virus infection. indeed, multiple factors such as saturation of antibodies or interference from non-adcc antibodies may contribute to the induction of adcc ( , ) . in this case, the threshold level of protective adcc-mediating antibodies should be investigated in further studies. various adcc assays that mainly differ in the choice of effector cells and measurement of adcc activity have been reported ( , ) . for example, some studies used ha-transfected or virus-infected a cells as target cells, which were susceptible results are presented as bar charts with mean values ± sd. differences between groups were compared using the t test. *p < . and **p < . as compared to the pbs-immunized group. survival rate (c) and body weight (d) of the mice ( mice per group) vaccinated with pbs (red), e (blue), e (green), and ha (purple) were monitored for days. the body weight values are shown as means ± sd for the mice that were alive at each time point (***p < . ). to nk cell-mediated adcc after incubating with the sera from healthy donors or clinical blood samples ( , , ) . in our case, we isolated pbmcs from healthy mice as effector cells and measured the death rate of target cells in the presence of vaccinated mouse sera (figure ) . at the same time, utilization of flow cytometry for quantitation of cell cytotoxicity provided an efficient and precise way to assess the adcc responses (figure ) . importantly, the h n -infected la cells showed a low background of cell death in the absence of antibodies (figure c) , which suggested la as an ideal cell line for the mouse-specific adcc assay. collectively, the established in vitro adcc assay, together with the balc/c mouse model, might be applied for the evaluation of other influenza-specific adcc epitopes. experimental mouse models are an invaluable scientific resource that allow comprehensive investigation of key biological questions in vivo and provide an essential platform in the study of many human diseases. it has been widely acknowledged that the mouse and human antibody repertoire share a general similarity ( ) ( ) ( ) . however, differences in germline antibody repertoire exist between species, and the number of mature naïve b cells from mice is smaller than that from humans ( ) . both variations may contribute to the dissimilarities in the antibodies elicited by the e -containing fragments in humans compared to those in mice. due to the diversity of the b cell antigen receptor repertoire between the mouse and human model, the antibodies bind to the same fragments in distinct host models might potentially have slightly different epitopes. alternatively, there may be fundamental differences between murine and human in terms of the regulation in nk cell cytotoxic granule secretion ( ) . surprisingly, distinct expression patterns of perforin were detected between protein ( figure m ) and mrna (figure n ) levels. the discrepancy might be explained by a previous finding that resting murine nk cells are "pre-armed" with high amounts of perforin mrna, which can be rapidly translated into protein upon activation in vitro and in vivo ( ). this mechanism of murine nk cells facilitates a better control of perforin expression, allowing a rapid production of effector proteins without the need of de novo gene transcription ( ). upon activation, adcc effector cells produce various cytokines such as tnf-α, il- β, and ifn-γ. further, cytokines may represent one of the arming pathways that stimulate the translation of perforin ( ). notably, human nk cell is minimally cytotoxic at rest, expresses little perforin protein, and becomes potently cytotoxic only after cytokine activation ( , ) . our result showed that tnfα, il- β, and ifn-γ were significantly elevated in e -vaccinated group when compared with those of the pbs-treated group (figure ) . in this regard, the upregulation of these cytokines may activate the cytotoxic perforin to cause the detrimental damage in mouse lungs. in our previous study, e and e epitopes were located on the ha head ( figure s in supplementary material), both of which are conserved in h n strains from ( _h n ) ( ) . to date, however, the role of ha head during influenza virus infection and adcc activation has not been fully delineated. in a recent study, dilillo and colleagues reported that neutralizing antibodies targeting the ha stem but not the ha head were capable of conferring influenza-specific adcc ( ) . they proposed that only the anti-stem antibodies could bind in a correct conformation to ligate fcrs, which was based on the observation that a strain-specific anti-ha head antibody (py ) was unable to mediate fcγr binding and to protect mice. on the other hand, ha head-induced adcc activities were reported by a number of other groups ( ) ( ) ( ) , which was in agreement with our findings. the discrepancy between these observations might be due to the sequential and structural variations among subtypes/strains of influenza virus used. interestingly, although both e and e epitopes are located on the ha head, serum from the ha-vaccinated group did not trigger a significantly elevated cytotoxicity response (figure h) , implying that additional regulators exist to control the adcc activity. in summary, we provided in vitro and in vivo evidence to verify the effect of the ha head epitope e -mediated adcc. importantly, our data suggested that e -mediated adcc alone caused detrimental effect during influenza virus infection, which raised concerns on using this conserved non-neutralizing region of the ha head in future designs of a universal influenza vaccine. in fact, the "headless ha" has been recommended in several vaccine designs that aimed to make use of adcc antibodies ( ) ( ) ( ) ( ) . our data provided further evidence in support of this "headless ha" vaccine design strategy. emerging influenza antiviral resistance threats influenza vaccine -outmaneuvering antigenic shift and drift variable influenza vaccine effectiveness by subtype: a systematic review and 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challenge with divergent influenza viruses stalking influenza by vaccination with pre-fusion headless ha ministem key: cord- -t r xp authors: coughlan, lynda title: factors which contribute to the immunogenicity of non-replicating adenoviral vectored vaccines date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: t r xp adenoviral vectors are a safe and potently immunogenic vaccine delivery platform. non-replicating ad vectors possess several attributes which make them attractive vaccines for infectious disease, including their capacity for high titer growth, ease of manipulation, safety, and immunogenicity in clinical studies, as well as their compatibility with clinical manufacturing and thermo-stabilization procedures. in general, ad vectors are immunogenic vaccines, which elicit robust transgene antigen-specific cellular (namely cd (+) t cells) and/or humoral immune responses. a large number of adenoviruses isolated from humans and non-human primates, which have low seroprevalence in humans, have been vectorized and tested as vaccines in animal models and humans. however, a distinct hierarchy of immunological potency has been identified between diverse ad vectors, which unfortunately limits the potential use of many vectors which have otherwise desirable manufacturing characteristics. the precise mechanistic factors which underlie the profound disparities in immunogenicity are not clearly defined and are the subject of ongoing, detailed investigation. it has been suggested that a combination of factors contribute to the potent immunogenicity of particular ad vectors, including the magnitude and duration of vaccine antigen expression following immunization. furthermore, the excessive induction of type i interferons by some ad vectors has been suggested to impair transgene expression levels, dampening subsequent immune responses. therefore, the induction of balanced, but not excessive stimulation of innate signaling is optimal. entry factor binding or receptor usage of distinct ad vectors can also affect their in vivo tropism following administration by different routes. the abundance and accessibility of innate immune cells and/or antigen-presenting cells at the site of injection contributes to early innate immune responses to ad vaccination, affecting the outcome of the adaptive immune response. although a significant amount of information exists regarding the tropism determinants of the common human adenovirus type- vector, very little is known about the receptor usage and tropism of rare species or non-human ad vectors. increased understanding of how different facets of the host response to ad vectors contribute to their immunological potency will be essential for the development of optimized and customized ad vaccine platforms for specific diseases. adenoviruses (ad) represent a promising vector platform for the development of vaccines for infectious disease, largely due to their safety and ability to stimulate robust cellular and/or humoral immune responses in multiple species ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , as compared with other genetic vaccine platforms ( , ( ) ( ) ( ) ( ) . adenoviruses derived from humans and non-human primates (nhp) belong to the family adenoviridae and the genus mastadenoviridae, and are further subdivided into species a-g (i.e., for species a viruses, these are denoted hadv-a followed by the type number). accounting for the inclusion of many ad recombinants ( , ) , ∼ human ads (http://hadvwg.gmu. edu/) and > non-human ad serotypes have been identified to date. adenoviruses are non-enveloped viruses which contain a double-stranded dna genome. the virion exterior is composed of three major structural proteins, the fiber, the penton base and the hexon [ ( ) ( ) ( ) ( ) ; figure ]. recombinant ad (rad) vectors can easily be rendered replication-incompetent (non-replicating) through deletion of the essential viral gene e from their genome and can be vectorized for easy manipulation ( , ( ) ( ) ( ) . further improvements to these first-generation ad vectors have been developed in which the e region is also deleted, to accommodate a larger heterologous transgene capacity of ∼ . kbp. ad vectors display a number of desirable characteristics which make them particularly well-suited to prophylactic vaccine applications. their genome is stable and easy to manipulate, they can be amplified and produced to high titers using various complementing cell lines which adhere with good clinical practice (gcp) procedures ( ) , and they have an outstanding track-record as safe and immunogenic vaccines in numerous human clinical trials ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . historically, the most commonly used rad has been human mastadenovirus c, human adenovirus type- (hadv-c , referred to ad throughout this manuscript). however, despite its well-characterized biology and robust immunogenicity, high seroprevalence has limited its widespread use in humans and has prompted the development and investigation of novel ad species, either rare species human ads ( , , ) or those derived from nhps ( , , ) , many of which have very low seroprevalence ( ) ( ) ( ) ( ) ( ) . for the purpose of this review, the nomenclature of ad types will use reference to the vertebrate species from which the vector was derived (i.e., h for human or ch for chimpanzee) followed by the virus type number, as outlined in table . human ad vectors will include their assigned adenovirus species group (i.e., a-g). this nomenclature has been proposed to ictv by dr. don seto, george mason university and dr. james chodosh, harvard university (personal communication). the use of non-replicating viral vectors as a vaccine platform has several advantages over other vaccine formulations (i.e., recombinant protein, inactivated particles). viral vectored vaccines retain some characteristics of a live attenuated vaccine in terms of their ability to enter target cells, engage intracellular trafficking pathways to deliver their genome and facilitate antigen (ag) expression and subsequent ag-presentation in vivo, but ( ) and was created with ©biorender -biorender.com. proteins are not to scale. possess additional safety features. furthermore, in order to drive the expression of substantial quantities of transcripts which correspond to the encoded vaccine ag, non-replicating ad vectors make use of powerful exogenous promoters, such as the cytomegalovirus (cmv) promoter ( ). unlike recombinant protein or inactivated vaccines in which antigen quantity is limited to the input vaccine dose, the use of exogenous promoters facilitates more sustained transgene antigen expression in vivo. in general, ad vectors are well-established to stimulate cd + t cell responses directed toward transgene ag, with selected ad types confirmed to elicit robust cellular immunity in both animal models ( , ( ) ( ) ( ) ( ) ( ) and humans [( , , , , , ) ; see table ]. memory cd + t cell responses elicited following vaccination with ad vectors exhibit an extended contraction phase ( ) . importantly, the persistent ag expression following immunization with ad vaccines enables the induction of sustained immune responses ( , , ( ) ( ) ( ) , making them very attractive vaccine vectors for conferring long-lasting immunity. it is believed that the prolonged expression of vaccine ag facilitates the maintenance of effector cd + t cells while simultaneously permitting their differentiation into central memory populations ( ) . improved understanding into how ad vectors prime and maintain such long-lived responses will be crucial not only in designing improved ad vaccines, but also other vaccine adenovirus classification for human and non-human ad vectors referred to in this review. the nomenclature of ad vectors derived from non-human primates, including chimpanzees, is not standardized, resulting in the confusing use of multiple names assigned by individuals who vectorized these constructs. in this review text, we propose to follow current standards for human ad vectors, such as hadv-c , as outlined by ictv, for descriptions of ad vectors derived from chimpanzees or non-human primates. abbreviated "alternative" names are used in table platforms which are optimized for diverse disease targets. however, the precise factors which contribute to the robust immunogenicity associated with particular ad-vectored vaccines are currently unclear. it is widely appreciated in the ad vaccine field that ad vectors can act as a "self-adjuvant, " allowing the stimulation of multiple innate immune signaling pathways upon viral entry, which can augment the immunogenicity of the encoded ag (although conversely, stimulation of certain signaling pathways can also be detrimental to their immunogenicity, as discussed below). although we have some understanding of how individual pathways work in vitro in defined cell types (i.e., dendritic cells, macrophages), understanding how these pathways intersect, or cooperate in the development of protective immunity in vivo, is complex and our understanding is incomplete. additionally, it is apparent that there is a clear hierarchy of immunological potency when evaluating distinct ad species and serotypes as vaccine vectors in animal models. although a few selected vectors display robust immunogenicity in vivo which is comparable to that of ad , most are less immunogenic ( , , , , ) , and there are considerable differences in the phenotype and functionality of immune response elicited ( , , ) . in recent years, investigators have begun to identify several crucial factors which could contribute to these profound disparities in immunological potency. it is now believed that differences in (i) cellular receptor and/or co-receptor usage, viral entry, trafficking, endosomal escape, and in vivo tropism can contribute to the (ii) differential activation of innate immune signaling which influences subsequent immune responses ( ) . in addition, it is apparent that the (iii) magnitude and persistence of transgene expression can also shape the ensuing immune response ( ) and all of these factors are in turn affected by the (iv) vaccine dose ( ) and route of administration ( ) . increased understanding of, and implementation of efforts to overcome these striking differences in immunological potency or quality, will absolutely be required for the development of optimal ad vectors for clinical use. as a result of extensive study over the past few decades, we have a clear understanding of the in vitro tropism determinants of vectors derived from species c hadvs (i.e., hadv-c /ad ) ( ) . the classical entry pathway of rad -based vectors in non-immune cells is mediated by binding of the fiber knob domain (figure ) to the coxsackie and adenovirus receptor (car). following this "docking" interaction, viral internalization is facilitated through interactions between the arginine-glycineaspartate (rgd) motif within the viral penton base and cellular integrins (namely αvβ and αvβ ) on the surface of cells ( , ) . adenovirus capsid disassembly then proceeds in a systematic and stepwise process. initial binding to car is a motile interaction, whereas the subsequent interaction with immobile αv integrins results in the ripping or shedding of fibers from the virion, initiating partial disassembly of the virion at the plasma membrane ( , ) . it was previously believed that endosomal escape was ph-dependent ( ) . however, it has subsequently been demonstrated that exposure of protein vi from the capsid interior ( ) at the cell surface, as a result of mechanical strain induced by the antagonistic car:integrin interaction ( ) , facilitates access to the cytoplasm through the action of its ph-independent membrane lytic activity ( ) ( ) ( ) . following endosomal escape, the virion is transported to the nuclear pore complex via the microtubule network ( , ) . once the virion has docked at the nuclear pore complex, interactions with cellular proteins trigger further capsid disassembly and allow the viral dna to extrude into the nucleus for subsequent gene expression ( ) . however, unlike ad , adenoviral types derived from species b or d viruses such as hadv-b , hadv-b , hadv-d , or hadv-d can use alternative binding/entry factors or receptors to car, such as cd ( - ), desmoglein- (dsg- ) ( ), or sialic acid ( ) . the post-entry steps of these rare species ad viruses in diverse cell types are not as well-characterized as the car-mediated entry of ad . however, it is considered that the use of alternative entry pathways or different receptors can not only result in differences in endosomal escape, trafficking to the nucleus and subsequent transgene expression ( ) , but can also impact on the in vivo tropism of the vector following different routes of vaccine administration (i.e., intramuscular vs. intranasal). as a result, triggering of innate immune signaling pathways may also differ at each step of the entry process ( , ) . less efficient trafficking pathways could result in weak or limited induction of cytokines/chemokines ( ), or increased uptake in cell types which result in vector degradation with minimal transgene expression ( ) ( ) ( ) ( ) . consequently, such differences between diverse ad vectors can impact on the magnitude and phenotype of the ensuing adaptive immune response when they are used as a vaccine platform ( , , ) . macrophages. in addition to the classical in vitro entry pathways described above, ad vectors can infect mononuclear phagocytes efficiently both in vitro and in vivo, independently of their described surface receptors (i.e., car, dsg- ) ( ), which are absent on murine macrophages. in vivo interactions with tissue resident macrophages, such as kupffer cells in the liver or alveolar macrophages in the lung, can result in scavenging and degradation of significant amounts of input ad vector ( ) ( ) ( ) . not only can these interactions result in limited transgene expression which could affect the therapeutic efficacy, but the phagocytosis of ad particles can trigger inflammatory responses ( , ) , leading to undesirable off-target toxicity ( ) . this is a particularly important consideration for therapeutic applications which require systemic administration or are designed for use in immunocompromised individuals (i.e., oncolytic viral therapy for disseminated metastases) ( ) . as a result, efforts have been made to characterize the mechanisms of ad viral entry in macrophages and to better understand how these interactions contribute to the induction of inflammatory responses within defined anatomical compartments following different routes of administration (i.e., intramuscular, intranasal vs. intravenous). opsonization of ad viral particles by complement or antibodies (natural or anti-viral) can bridge entry into macrophages by engaging fc-receptors (fcrs) or complement receptors ( ) ( ) ( ) . additionally, a role for scavenging receptors in facilitating viral entry into murine macrophages, both in vitro and in vivo, has been outlined ( , , ) . scavenging receptors are a heterogenous and structurally diverse family of receptors capable of interacting with endogenous proteins and lipids, microbial ligands, and non-opsonized particles, including viruses ( ) . in addition to contributing to the clearance of particulate ag, scavenging receptors have been implicated in innate immune sensing, due to their ability to recognize pathogen-associated molecular patterns (pamps). murine sr-a ( ), sr-aii ( ) , and marco (sr-a ) ( ) have been described as receptors for rad vectors, and marco + marginal zone macrophages in the spleen have been shown to accumulate ad -based vectors following intravenous (i.v.) delivery in mice ( , ) . the fiber knob (i.e., sr-a ), or hexon protein has been implicated in mediating these interactions (i.e., sr-aii and sr-a ). interestingly, sr-a (marco) was shown to not only facilitate entry and efficient gene transduction with ad , but also with hadv-c , hadv-b , and hadv-d ( ). the mechanism of interaction between ad and sr-aii/sr-a was proposed to involve the negative charge conferred by specific hypervariable regions (hvrs) of the viral hexon, namely hvr . in support of this, preferential scavenging of negatively charged particles has previously been shown to contribute to the differential recognition of ad vectors by macrophages in vivo ( , , ) . dendritic cells. dendritic cells (dcs) are a specialized subset of professional ag presenting cells which are central to the development of protective immunity. dcs can process ag using several methods; (i) direct presentation, in which an infected dc presents peptide:major histocompatibility complex class (mhc) complexes directly to t cells, (ii) cross-presentation in which ag derived from other infected cells is phagocytosed by dcs, processed and then presented to t cells, and (iii) cross-dressing ( ) , in which peptide:mhc complexes are acquired from other professional or non-professional antigen presenting cells (apcs) and are transferred to the dc through a process of trogocytosis, in which fragments of the plasma membrane containing the mhc complex merges with the recipient cell. cross-dressing is also hypothesized to occur through the intercellular transfer of pre-formed peptide:mhc complexes by extracellular vesicles, such as exosomes [( - ); figure ]. a critical role for dcs in the robust induction of cd + t cell responses following immunization with rad vectors has been demonstrated ( , ) . it has been shown that cd + , but not cd + t cell responses elicited by ad vaccines, are dependent on cross-presentation by specific sub-populations of dcs, including cd α + dcs ( ) . in support of this, ag-specific cd + t cell responses in batf -deficient mice (batf −/− ), which preclude the development of the latter dc population (cd α + dcs), were shown to be ∼ % lower following immunization with several ad vectors ( ) . dcs infected with ads upregulate mhc, as well as co-stimulatory molecules including cd , cd , or cd , leading to the activation or maturation of dcs ( ), in a manner which is believed to be dependent on nuclear factor kappa-lightchain-enhancer of activated b cells (nf-κb) signaling ( ) . the maturation of murine dcs has been proposed to be mediated by the fiber knob domain of ad ( ) . in vivo experiments in mice have shown that uptake of rad -based vaccines in draining lymph nodes (dlns) following intramuscular (i.m.) or subcutaneous (s.c.) vaccination is highest in cd c + cd − b − dcs, although cd c + cd + b − dcs were the most potent for eliciting naïve ag-specific t cell proliferation ( ) . as previous studies have shown that targeting vaccine ag to defined populations of dcs can improve immunity and vaccine efficacy ( , ) , efforts are ongoing to better understand the precise interactions between diverse ad types and dc subpopulations in vivo, as well as how we can engineer ad vectors which are targeted to specific receptors on the surface of dcs ( , ) . receptors on the surface of dendritic cells (dcs) can permit entry of ads independently of the classical car receptor, which is largely absent on dcs ( ) . receptors proposed to be involved in ad viral entry into human or murine dcs include dendritic cell-specific intercellular adhesion molecule- -grabbing nonintegrin (dc-sign) ( ), cd ( , , ) , or cd /cd ( ) . a vector based on chimpanzee ad vector , chadv- , was shown to efficiently transduce cd -expressing murine dcs in vitro ( ) . recombinant ad vectors pseudotyped with the fiber knob from ad ( ) or porcine adenovirus type ( ), can increase entry into human dcs, via cd /cd and surface glycans, respectively. similarly, pseudotyping rad with the fibers from species b, hadv-b or species d, hadv-d displayed increased entry into murine dcs compared with unmodified rad ( ) . interestingly, in the latter study, increased entry into dcs was not associated with improved cellular immunity following subcutaneous immunization. in support of this, species b ad vectors, including ad , were long considered to hold great potential as potently immunogenic vaccine vectors due to their increased ability to target both myeloid and plasmacytoid human dcs via cd . however, these vectors were subsequently shown to be some of the least potent ad vaccines in vivo ( , ) . these observations and findings are important in highlighting that multiple parameters, such as post-entry intracellular trafficking kinetics or differential activation of innate immune signaling pathways, not just viral tropism, likely play key roles in the induction of robust immunogenicity. the ability of diverse rad vaccines to elicit robust cellular immune responses and confer protective immunity in animal models makes them an attractive vector platform for vaccine development ( table ). in addition to this, their safety in clinical applications, compatibility with clinical-grade manufacturing and scale-up ( , ) and their suitability for long-term storage ( ) , or thermo-stabilization ( ) ( ) ( ) and stockpiling for cold-chain free storage, has solidified their appeal as vaccines for major infectious diseases ( ) . it is this clear translational potential which has emphasized the importance of improving our understanding of the mechanisms which underlie differences in the immunological potency of diverse ad types. ad vectors are capable of triggering multiple innate immune sensors at several steps in the viral entry pathway ( , ) , in a process which does not require viral replication or gene expression ( ) . viral penton rgd:cellular integrin-mediated internalization and subsequent escape from the endosome is considered to be a crucial step in activating many innate immune responses to ad vaccines ( ) . it is considered that preferential stimulation (or avoidance) of defined innate immune signaling pathways could impact on the downstream immunogenicity of distinct ad vectors. with regard to assessing the differential stimulation of innate immune signaling pathways, this is complicated by the fact that many vectors have not been compared side-byside, and published data proposing roles for these pathways in the immunological potency of different ad vectors are often contradictory. however, a number of pathways which have been implicated in innate immune sensing of ad vectors and the caveats associated, are described below. recombinant ad vectors contain pamps which can be sensed by cell-surface or endosomal pattern recognition receptors (prrs) such as the toll-like receptors (tlrs). tlrs implicated in the sensing of ad vectors include tlr ( ), tlr ( ) , and endosomally located tlr ( , ) , which can trigger the down-stream activation and transcription of anti-viral genes including nf-κb, mitogen-activated protein kinases (mapk) and interferon-regulatory factors (irfs). the intracellular adaptor protein myd has been reported to play a major role in the induction of ag-specific cellular immune responses following tlr-mediated sensing of ad vaccines ( , ) . importantly, the ability to engage multiple myd -dependent signaling pathways simultaneously, is believed to contribute to the robust immunogenicity associated with ad vaccines. however, their immunological potency is also attributed to the fact that innate immune activation by recombinant ad vaccines can occur not only via tlr-dependent mechanisms, but also through numerous tlr-independent pathways ( , , , , ) . the viral dna itself can play a crucial role in triggering innate immune responses. in recent years it has been demonstrated that following rupture of the endosomal membrane, ad viral dna can also be sensed by the cytosolic dna sensor cgas ( , ) . the engagement of cgas triggers a signaling cascade involving the adaptor sting ( ) and activation of the kinase tbk , which initiate the induction of irf -responsive genes ( ) , such as type i interferons (ifns). it has been shown that the absence of cgas or sting results in reduced activation of early innate immunity (i.e., ifn-β, cytokines, chemokines) but does not impact adaptive anti-vector immune responses in mice. however, the latter studies were performed in the context of i.v. delivery and anti-vector, not transgene-specific immunity ( ) , and as such, the relative importance of dna sensor pathways in the immunogenicity of ads as vaccine vectors is less clear. it has recently has been suggested that ag expression is a more crucial predictor of ag-specific memory t cells ( ) , as abrogation of sting and type i ifn responses during ad vaccination in mice merely altered the early kinetics of cd + t cells, but did not impair the magnitude of t cell memory responses ( ) . in the latter study, it was shown that sting could act as a dominant innate prr sensor for many ad vectors. interestingly, abrogation of sting accelerated the kinetics of ag-specific t cell responses following vaccination with chadv- , a chimpanzee ad vector, but was dispensable for the early induction of cd + t cell responses for ad and rare species hadv-d based vaccines ( ) . this supports the idea that a complex interplay between multiple prr-mediated signaling pathways exists, and that different ad vectors are differentially impacted by these pathways. our understanding of this is confounded by differences in receptor usage, in vivo tropism, engagement of prrs in diverse hematopoietic and non-hematopoietic cells, and by differences in putative pamps on diverse ad particles. in addition to inducing the expression of anti-viral genes, infection with ad vectors also triggers pro-inflammatory responses through cytosolic dna-sensing mechanisms which are independent of tlr and irfs ( ) . in macrophages, recognition of ad viral dna has been shown to be mediated by the innate cytosolic molecular complex, or inflammasome, in a process involving nlrp and (asc), which is independent of viral gene expression or replication ( ) . the multi-protein inflammasome complex mediates caspase- activity, resulting in the processing of pro-interleukin- β into its active and secreted form. il- β subsequently induces signaling cascades of proinflammatory cytokines and chemokines through the il- ri both in vitro and in vivo in response to ad infection. however, alternative and contradictory mechanisms of immune activation in macrophages by ads have also been identified which are independent of the nlrp inflammasome and its components. di paolo et al. showed that direct interactions between the rgd motif within the penton base of the ad virion and the β subunit of integrins on the surface of macrophages were responsible for activating il- α. the authors also proposed that il- α, not il- β, was the predominant activator of innate immune responses to ad in vivo ( ) . one very important caveat which complicates our ability to systematically investigate how innate immune responses contribute to downstream adaptive immunity to ad vaccines, is that many studies are performed in vitro, using defined non-immune epithelial/endothelial cells or cultured immune cells including macrophages or dendritic cells ( , , ) . these cell type-specific findings often contradict subsequent in vivo studies using transgenic mice in which these "critical" mediators of innate immunity are knocked-out ( , ) . for example, despite numerous reports describing an important role for tlr in vitro, comparisons of wildtype and tlr −/− mice have demonstrated that the impact of tlr in innate immune sensing of ad particles in vivo is minimal ( ), at least for i.v. administration of ad . similarly, although cgas or sting were shown to be pivotal in early immune sensing of ad in vivo, studies using cgas −/− or sting −/− mice showed that these molecular effectors have little impact on subsequent adaptive immunity and antibody production ( ) . these discrepancies are obviously further complicated by the known capacity of ad vectors to engage multiple innate signaling pathways simultaneously, rendering individual pathways at least partially redundant in vivo ( ) . in addition to this, differences in the route of ad vector delivery (i.e., i.m, i.v., or intranasal) and access to different cell types, the multiplicity of infection (moi) or injected dose, the timing or method of analysis reported in published work and the use of non-human or rare species ad vectors with differential receptor usage, also limits our ability to fully dissect out the key contributing pathways ( , ) . collectively, these factors highlight the many challenges facing the field and explain why we currently lack consensus on precisely which innate signaling pathways could contribute to protective immunity following vaccination with diverse ad vectors. it has been proposed that minimal induction of type i ifns ( , , ) , in conjunction with sustained transgene expression ( ) , are hallmarks of potently immunogenic ad vaccine vectors in vivo. excessive stimulation of type i ifn pathways at early time-points following immunization has been shown to lead to decreased transgene expression and subsequently reduced ag-specific antibody (ab) responses, following immunization with a chimpanzee ad vector, chadv- ( ) . the authors demonstrated that these effects could be reversed by immunizing mice which have a defective type i ifn receptor ifnar −/− , resulting in an increased ab response, thereby confirming that type i ifn stimulation can have a detrimental impact on humoral immunity directed toward the ad-encoded transgene ag ( ) . in support of these findings, quinn et al. also showed that abrogation of type i ifn and sting could increase transgene expression from rad vaccines, and that the development of protective cellular immunity correlated with this increased transgene expression ( ) . following immunization with rare species and non-human ad vectors, the authors used a systems biology-based, gene expression analysis approach at several time-points to confirm the differential modulation of ifn responsive genes. they determined that type i and type ii ifns were upregulated at h post-immunization, which was followed by the induction of isgs by h. interestingly, the most protective rads identified in their study (i.e., ad and chimpanzee ad, chadv- ) exhibited the weakest transcriptional activation of these pathways. however, only the impact of innate gene activation on cd + t cell responses, but not transgene-specific abs, was investigated ( ) . nonetheless, collectively, these studies support the concept of robust, persistent ag expression combined with low innate gene stimulation in contributing to the potency of rad vaccines ( , ) . this is also supported by the knowledge that relative to rare species human and non-human ad vectors ( , ) , immunization with the potently immunogenic ad vector is well-established to result in robust, persistent ag expression ( , , , , ) , while triggering minimal type i ifn responses in vivo. future studies which aim to comprehensively characterize the contribution of early innate immune activation and correlate this with the downstream immunological potency and efficacy of lead ad vaccine platforms will be required. non-replicating ad -based vectors are well-established for their ability to confer robust transgene expression following immunization ( ) . furthermore, low level transgene expression can persist long-term ( , ) , with transcriptionally active ad vector genomes being maintained in muscle at the injection site, or within draining lymph nodes ( , ) , depending on the route of administration. as previously outlined, it has been proposed that it is this magnitude and persistence of transgene ag expression which is crucial for the induction of robust and protective t cell responses following ad vaccination ( ). as described above, quinn et al. demonstrated that strong activation of innate immune gene expression profiles in the draining lymph nodes (dlns) correlated with limiting ad-mediated transgene expression for many rare or non-human ad types ( ) . the ad vectors with the highest levels of transgene ad expression in dlns, ad , and chimpanzee ad vector chadv- , also had the most attenuated ifn induction. the latter vectors were both potently immunogenic following immunization in mice. in agreement with this, similar comparative immunogenicity studies in mice have shown that transgene expression levels within muscle and dlns are lower following immunization with a chimpanzee ad vector, chadox , when compared with ad , and that this translates to superior immunogenicity observed with ad ( ) . to directly address the question of how persistence of ag contributes to the induction of the robust immunogenicity of ad vaccines, finn et al. constructed an ad vaccine with a doxycycline-regulated transgene expression cassette ( ) . by switching off transgene expression at early vs. late time-points post-immunization, the authors confirmed the importance of presence of ag in expanding and maintaining memory t cell responses up to d , but showed that the maintenance of memory responses at later time-points (d ) is independent of transgene expression. as discussed above, it is apparent that a combination of multiple parameters influences the extent of transgene expression. these factors include the receptor usage and cellular tropism of each ad vector, the presence and accessibility of specific cell types at the site of injection, in addition to differences in the induction of early innate immunity by diverse ad vectors. collectively, these parameters shape subsequent adaptive immune responses. the successful transduction of cells at the site of vaccine administration, and subsequent engagement of defined and desirable prrs which result in robust transgene expression, depend on the cell type and the specific ad vector being studied. with this in mind, much of the evidence to date has focused on characterizing the induction of innate immune responses in vitro in apcs such as dcs or macrophages, which play important roles in initiating anti-viral immune responses. it is considered that inflammation induced at the injection site, can lead to an influx of apcs (monocytes or dcs) which carry ag to the dlns ( ) . immature dcs residing at the site of vaccination respond to innate immune signals (i.e., stimulation of tlr pathways) by undergoing maturation, upregulating co-stimulatory molecules and migrating to dlns where they present ag to naïve t cells ( ) . however, non-professional apcs expressing mhc i ( , ) , such as tissue-specific epithelial or endothelial cells, could also contribute to the immune sensing of ad vectors at the site of injection ( ) . therefore, it is clear that the route of vaccine administration ( , , ) , the abundance and accessibility of cell types at those locations, as well as the surface expression of suitable entry receptors, will profoundly affect the immunological potency and protective efficacy of a chosen ad vector. as a result of the long history of experimental use of ad vectors as oncolytic agents aimed at treating disseminated metastases, a significant amount of information exists in the literature regarding interactions between ad vectors, immune cells ( , , ) , parenchymal cells ( , , , ( ) ( ) ( ) ( ) ( ) , and stromal cells ( ) following i.v. delivery of ad vectors ( , , ) . however, i.v. vaccination would be impractical for widespread population use and so immunization by i.m. or i.n. administration is preferable, particularly for vaccination against respiratory pathogens. unfortunately, less is known about the precise interactions which occur at the site of injection or within dlns in vivo following i.m. or i.n. immunization with ads in animal models, and these questions are even more difficult to address in humans, without the use of invasive procedures (such as fine needle aspirates of lymph nodes) ( ) . the cell types present when vaccine is administered i.m. include myocytes, skeletal muscle cells, fibroblasts and endothelial cells, with apcs such as dcs or macrophages representing a minority when compared with the abundance of murine skeletal muscle cells ( ) . early studies by mercier et al. demonstrated that transduction of different cell types can modulate the outcome and phenotype of the humoral immune response following ad vaccination. the authors transduced dcs (professional apcs), myoblasts (progenitor cells which give rise to muscle cells) and endothelial cells ex vivo with ad expressing a model antigen β-galactosidase (β-gal) ( ) , and vaccinated mice i.m. with the ad-transduced cells. the authors found that all transduced cell types elicited humoral immune responses to the β-gal transgene to a similar extent (albeit with differences in their temporal kinetics), but that the igg isotype subclass profile differed. injection of transduced dcs or endothelial cells resulted in production of ag-specific abs which were exclusively igg a , whereas vaccination with ad-transduced myoblasts elicited a more balanced ab response with equivalent igg :igg a . interestingly, only mice immunized with ad-transduced dcs elicited robust cd + t cell responses, as did vaccination with control virus ad-β-gal, suggesting that ad interactions with different cell types in vivo could influence divergent arms of the adaptive immune response. bassett et al. also demonstrated that ag presentation by non-lymphoid cells, in cooperation with hematopoietic apcs, contributes to the kinetics of primary cd + t cell expansion, the maintenance of memory responses and to the functional phenotype of the cellular immune response following ad vaccination ( ) . through a series of investigations, the authors showed that although dlns act as the site of immunological priming in response to ad vaccination, primary expansion of the ag-specific cd + t cell response requires a source of sustained ag expression outside of dlns ( ) . they hypothesized that this cell type was of non-hematopoietic origin, due to their prior findings that a radioresistant population of cells was capable of priming cd + t cell responses in mice leukopenic mice ( ) . therefore, it is feasible that several modes of ag presentation take place following i.m. immunization with ad vectors, all of which contribute to different facets of the ensuing immune response. these interactions are summarized in figure , showing that ag presentation could take place not only within dlns, but also at the site of injection, with or without the involvement of professional apcs. it is important to note that the majority of detailed mechanistic studies into the immunogenicity of ad vectors have been performed using ad -based vaccines and as such, similar information regarding the in vivo cellular tropism of rare species or non-human ad vectors, is much more limited ( ) . it will therefore be crucial to outline the precise factors which confer a hierarchy of potency between ad vectors in the future, as many ad vectors are already under clinical investigation, or are advancing rapidly into clinical trials. this improved knowledge would allow us to engineer optimal platform vectors which combine multiple attributes associated with potent immunogenicity and long-lived protective efficacy. mucosal i.n. or aerosolized (a.e.) delivery of ad vectors is particularly attractive for the development of vaccines against respiratory pathogens ( , , ) . ad vectors are capable of eliciting both humoral and cellular immune responses following i.n. ( , ( ) ( ) ( ) and a.e. ( ) vaccination in animal models, both in the bronchoalveolar lavage (bal) and within the lung interstitium ( , ) . many wildtype adenoviruses are common respiratory pathogens (i.e., hadv-c , hadv-e ), highlighting their potential suitability for targeting vaccine ag to mucosal surfaces within the respiratory tract. however, several chimpanzee ad vectors, which are not associated with respiratory infections, have displayed superior immunogenicity to ad when non-professional apcs such as parenchymal cells at the site of injection (muscle cells shown as an example) can present antigenic peptide on mhc i to infiltrating cd + t lymphocytes, outside of secondary lymphoid organs. figure is updated from coughlan et al. ( ) and holst and thomsen ( ) and was created with ©biorender -biorender.com. administered by the i.n. route in mice, including a chadv- based vaccine against pulmonary tuberculosis (tb) ( ) and a chadv- vaccine against pseudomonas aeruginosa ( ) . a major limitation in the application of rad vaccines to mucosal respiratory surfaces may be the rapid scavenging and degradation of ad vaccine vector particles by tissue resident alveolar macrophages in the lung, as it has previously been estimated that ∼ % of ad vector genomes are lost within the first h following lung delivery in mice (intratracheal) ( ) . furthermore, alveolar macrophages were found to be the predominant cell type responsible for initial inflammatory cytokine induction ( ) . however, depending on the balance of innate immune stimulation and the retention of a certain level of transgene expression within the respiratory tract, these interactions could be beneficial for the induction of subsequent adaptive immune responses. alveolar macrophages may play a role in trafficking to dlns to facilitate ag priming, or the inflammatory cytokines they release could signal the recruitment of lymphoid cells which could further potentiate immune responses to ad-encoded transgene ag. the many studies which have confirmed the induction of protective immunity following i.n. vaccination with ad vectors support this possibility, but the precise mechanistic factors which underlie these effects and the specific cell types which contribute to vaccine efficacy are not extensively described. as differences in the phenotype and trafficking potential of cd + t cells has been observed when comparing i.m. vs. i.n. vaccination of mice with ad vectors, it will be important to identify the optimal ad vaccine platform which elicits the correct correlates of protection for a specific disease target, before deciding on the ideal route of vaccine administration ( ) . the ability to engage and activate multiple, diverse innate immune signaling pathways simultaneously (excluding type i ifn) can allow rad vectors to act as an effective selfadjuvant, relative to other vaccination platforms ( , , ) . these attributes suggest that ad virions themselves possess several pamps. in fact, it is considered that the native receptor determinants or entry factors of a particular ad vector may, at least in part, regulate innate immune activation. in support of this, binding of the fiber knob domain to car is sufficient to activate p mapk, p / mapk (erk / ), and nf-κb pathways ( ) , resulting in the transcription of proinflammatory cytokines both in vitro and in vivo ( ) . similarly, ad vectors which are capable of using cd as a receptor have been shown to display preferential activation of tlr to carbinding ad vectors in vitro ( ) . however, cd distribution is limited to the expression in the testes in mice, rats and guinea pigs, in contrast to its widespread expression in humans ( ) . therefore, this likely has little contribution to the immunological potency of cd -using ad vectors in murine models. the fiber knob domain of ad -based vectors has been shown to contribute to the maturation of murine dcs, as recombinant knob protein, full-length fiber protein and penton capsomers (penton plus fiber), but not hexon or penton alone, were capable of exerting this effect ( ) . in support of this, teigler et al. previously suggested that fiber-receptor interactions were important for triggering innate immune responses to rare species ad vectors ( ) . furthermore, studies using hadv-b vectors pseudotyped with the fiber knob from ad were shown to be more immunogenic in mice and nhp, suggesting that the fiber knob domain could contribute substantially to immunological potency ( ) . however, similar studies in which the entire fiber and penton rgd motif from rad were swapped into chimpanzee ad vector chadox , failed to increase the immunogenicity of chadox relative to ad , suggesting that factors beyond fiber/penton capsomer interactions confer immune responses to chadox ( ) . more recently it has been shown that a member of the vitamin-k dependent protein family, growth arrest specific protein (gas ), can bind differentially to the fiber of rad vectors ( ), interacting with a putative epitope which is outside of the fiber knob domain, such as the fiber shaft. interestingly, gas bound efficiently to the fiber of car-binding ad , without affecting its ability to enter cells, but significantly reduced the induction of type i ifns, resulting in prolonged transgene expression. conversely, gas failed to bind to the fibers of non-ad and non-car binding viruses, such as a species d ad vectors hadv-d , which has been shown to display reduced immunogenicity in mice when compared with rad ( ) . in support of this, hadv-d has been shown to elicit robust ifn-α and had increased stimulation of ifn-responsive genes when compared with ad ( ) . importantly, immunization of ifnr −/− mice with hadv-d resulted in robust cellular immune responses, comparable to ad ( ) . these data support the hypothesis by several other groups that ad vectors which trigger robust type i ifn responses exhibit reduced transgene expression and impaired vaccine immunogenicity ( , ) . therefore, one strategy to overcome stimulation of ifn-α could be to identify the precise amino acid binding epitopes for gas and genetically engineer this region into the fiber of non-gas binding ad vectors in an effort to increase their immunogenicity ( ) . in addition to interactions with car, interactions between the rgd motif within the penton base and cellular integrins can contribute to the induction of innate immune signaling pathways both in vitro ( ) and in vivo ( ) . following i.v. administration of ad , interactions between the β subunit of cellular integrins and the rgd motif on marco + marginal zone macrophages in the spleen were required for triggering il- α-dependent innate immune signaling in mice ( ) . however, it is unclear how these interactions affect immune responses following immunization by other routes of administration. finally, the ad virion itself and its major capsid protein, the hexon, has been described as a potent adjuvant, capable of activating cd + and cd + cellular immune responses to a model immunogen mixture in mice ( ) . although the precise mechanism underlying this effect has not been identified, subsequent studies have suggested that the hvr regions, particularly hvr , are involved in enhancing interactions with scavenging macrophages ( , ) . one approach to identifying lead vaccine candidates is to perform head-to-head comparisons of immunogenicity in animal models to identify the most immunogenic vectors, followed by detailed and systematic experimental studies to try to identify the underlying mechanisms which contribute to immunological potency (i.e., transgene expression magnitude and duration, innate immune stimulation). however, in the interim, efforts are ongoing to try to maximize immune recognition of the ad encoded ag, in an effort to compensate for the reduced immunogenicity associated with certain ad vectors which are otherwise an ideal platform (i.e., low seroprevalence, high titer growth, stability). furthermore, this type of modification could permit dose-sparing, which would have cost saving effects, as well as minimizing any vector-induced reactogenicity, without compromising on immunogenicity. such approaches include the use of molecular or genetic adjuvants, namely in the form of fusion proteins with the vaccine antigen of interest, which help to potentiate immunogenicity by enhancing ag presentation or dissemination [reviewed in detail by neukirch et al. ( ) ]. one such approach which has previously been described includes the generation of fusion-ag cassettes which enhance mhc presentation. this can be achieved by fusing vaccine ag to β-microglobulin for enhanced mhc i presentation ( ) , or to the invariant chain of mhc ii ( ) ( ) ( ) ( ) . these approaches were capable of enhancing antigen-specific cd + t cells responses in mice following immunization with an ad vector encoding the ii or β -microglobulin fusion antigen. in a separate approach, we recently have shown that encoding vaccine ag cassettes in which a model antigen, enhanced green fluorescent protein (egfp), is fused to a protein domain known to tether proteins to the surface of extracellular vesicles (evs), can dramatically improve the humoral immunogenicity of both ad and chimpanzee ad vector chadox in mice following i.m. and i.n vaccination. evs play important roles in regulating immune responses and are conveyors of cellular communication signals ( ) . as evs are frequently implicated in host-pathogen interactions and have been shown to transfer antigenic material to apcs ( , ) , we reasoned that directed targeting of a vaccine ag to their surface could potentiate immune responses. although cellular immunity was largely unaffected, ag-specific humoral immune responses were increased up to -fold when compared with unmodified egfp ( ) . the choice of molecular or genetic adjuvant will depend on the predicted correlates of protection for a specific disease target: in certain cases, a robust humoral immune response will be more important than a cellular immune response. further to this, some adjuvanting technologies will work for one ag but not for another, and structural constraints may limit the ability to modify complex, multimeric ags. this will require the optimization and selection of different components to be combined and assembled into customized ad vectors which are tailored to specific pathogens. challenges a number of promising animal studies solidified the appeal of ad as a platform vaccine vector for infectious diseases. in particular, a study by sullivan et al. demonstrated that a singleshot, low dose ( × vp) immunization with ad expressing ebola virus glycoprotein (gp) could provide % protection from challenge in nhps ( ) . similar studies using ad based vectors expressing siv gag demonstrated its superiority to plasmid dna or modified vaccinia ankara (mva) in attenuating viremia following virus challenge ( ) . on the basis of these promising early results, a multicenter phase ii clinical trial called the merck step study was initiated, in which a vaccine composed of a mixture of ad vectors expressing hiv- gag, pol, and nef genes was administered to participants at high risk for hiv- acquisition ( , ) . phase i safety and immunogenicity studies in healthy, hiv-seronegative adults showed that this vaccine could elicit antigen-specific ifn-γ elispot responses in both ad seronegative and seropositive individuals ( ) . however, the phase ii study was terminated prematurely due to futility and failure to meet pre-defined endpoints: an interim analysis determined that the vaccine would not be efficacious in preventing hiv- infection, or in reducing viral-load setpoint in seroconverters, despite eliciting t cell responses in most participants ( , ) . subsequent to this, a post-hoc analysis of the study suggested an association between vaccination with the ad vaccine and increased acquisition of hiv- ( ). on multivariate analyses, this increase was largely restricted to a defined sub-set of participants: uncircumcised men with high baseline antibody titers against ad . several hypotheses were proposed to explain the increase in hiv- acquisition, including the formation of immune complexes (ic) between anti-ad antibodies and dcs, which were shown to enhance hiv- infection of t cells in dc-t cell co-cultures in vitro ( ) . an alternative hypothesis suggested that ad immunization induced the expansion of ad-specific memory cd + t cells which upregulate ccr expression and/or homing markers for mucosal sites, thereby increasing the pool of hiv- susceptible cells at the site of infection ( ) . although the latter hypothesis has been challenged ( ) , the precise mechanisms underlying the increased acquisition of hiv- in the merck step trial remain inconclusive ( ) . however, it is important to note that the effects were shown to wane over time ( ) . nonetheless, this outcome led to a dampening in enthusiasm for the broad application of ad -based vaccines for major infectious diseases, prompting the investigation of novel rare species human or non-human ad viruses as alternative vaccine platforms. several rare species or non-human ad vaccines are now leading the way in human clinical trials, namely species c vector hadv-c and species d vector hadv- , as well as chimpanzee ad vectors chadv- , panadv- , chadv- , and chadox , which cluster phylogenetically with species c or e human ads (see table ). many of these vector candidates had previously been identified in animal studies as being potently immunogenic, and in some cases were comparable to the potency of ad [( , ); table ]. in particular, hadv-c and chadv- appear to possess attributes which make them an attractive platform vector ( %, % seroprevalence, respectively) ( , ), and as a result, have been developed for clinical testing as vaccines against major global pathogens, hepatitis c virus (hcv) ( ) and hiv- ( ) . both hadv-c and chadv- were shown to be safe and immunogenic in humans when used as a vaccine to elicit immunity against hcv, although hadv-c appeared to be superior in its ability to cellular immune responses with increased magnitude and breadth at lower doses (i.e., × vp) ( ) . with regard to chadv- , part of its appeal includes its ability to elicit long-lived cellular and humoral immune responses directed toward the encoded vaccine antigen. immunization of nhps with chadv- expressing hiv gag resulted in cellular immune responses which persisted for more than years ( ). a heterologous boost with panadv- at week facilitated an expansion of gag-specific ifn-γ secreting t cells, in addition to boosting antibodies to hiv- gag. as such, there is broad interest in using these rare vectors in heterologous prime:boost vaccination regimens in an effort to confer long-lived protective immunity against challenging pathogens. in support of this, a phase i clinical trial to test the hadv-c and chadv- vectors expressing hcv antigens demonstrated that heterologous boost immunizations resulted in long-lived, polyfunctional effector, and central memory t cell responses which were sustained for up to year in humans ( ). panadv- expressing respiratory syncytial virus (rsv) fusion (f), nucleocapsid (n), and matrix (m - ) antigens has also been tested in humans following i.n. and i.m. administration ( ) . neutralizing antibodies to rsv f were increased following i.m., but not i.n. prime immunization. similar trends were observed for antigen-specific t cell responses to the vaccine inserts, although increases were minimal following panadv- i.m. prime only. this vaccine was also evaluated in older adults ( - years) who are at increased risk of severe rsv disease, with similar results ( ) . immune responses elicited by panadv- were improved upon boosting with an mva vector which also encoded the rsv transgene antigen. chadv- , also identified as a clinically viable ad vector with low seroprevalence which displayed protective efficacy in animal studies ( table ) , has been shown to be safe and immunogenic in children and adults ( ) ( ) ( ) ( ) . when chadv- has been used in a prime:boost vaccination regimen with mva expressing malaria antigens, promising efficacy was observed in uk and kenyan adults ( , ) , but has recently been associated with disappointing efficacy in field trials in young children in burkina faso, a highly endemic malaria transmission region ( ) . chadox- is a species e chimpanzee ad vector developed by the jenner institute at university of oxford which has been tested clinically as a vaccine for influenza virus as a standalone vector or for use in prime:boost with mva ( , ) , and for several other infectious disease targets such as chikungunya fever (nct ), malaria (nct ), and tuberculosis (nct ). numerous additional trials are currently ongoing or actively recruiting participants, including a recently initiated study to test a novel vaccine for covid- (nct ). in addition to vectors derived from nhps, promising advances have been the development of hadv-d vectors. with a number of clinical trials registered on clinicaltrials.gov, this platform has advanced into clinical studies as a vaccine against ebola virus ( ) ( ) ( ) , rsv ( ), hiv- ( , , ) , and has also very recently been announced as a candidate vectored vaccine against covid- . the first-in-human testing of hadv-d expressing hiv- env demonstrated that the vaccine was safe and well-tolerated and elicited env-specific antibodies and antigen-specific elispot responses in all participants ( ) . although hiv- specific neutralizing antibodies were not detected, the study reported multi-functional readouts for the non-neutralizing antibodies elicited, including effector functions such as antibody-dependent cell-mediated phagocytosis (adcp), antibody-dependent cell-mediated virus inhibition (adcvi) ( ) . this vaccine platform was subsequently improved upon by encoding polyvalent "mosaic" hiv- antigens, env, gag and pol, representing computationally optimized sequences aimed at maximizing recognition of t cell epitopes ( ) . evaluation of the hadv-d platform in various prime:boost regimens is ongoing and preliminary data suggest that it is immunogenic, capable of eliciting env-specific antibodies which exhibit adcp and cellular immune responses out to week . importantly, these assays were found to be correlates of protection in a parallel shiv challenge model in rhesus monkeys (macaca mulatta) ( ). the hadv-d mosaic hiv- vaccine is currently in phase iib efficacy studies in sub-saharan africa (nct ). based on the literature, it appears that ad vectors derived from species c, d, or e are most likely to be immunogenic vectors. requirements for selecting specific vectors will vary depending on whether the required application is as a stand-alone vaccine or as part of a prime:boost regimen. for standalone vaccination regimens aimed at eliciting a rapid, protective response during an emerging pandemic, the magnitude of response to an identified correlate of protection following a single shot is crucial. secondary considerations for an evolving pandemic scenario would be rapid immunogenicity at a low dose, and the capacity for lyophilization or stabilization, to facilitate dose-sparing, vaccine cost-effectiveness and pandemic preparedness. in this regard, of the ad vectors evaluated to date, chadv- and hadv-c appear promising. for protection against more complex pathogens which require long-lived polyfunctional responses, or sustained humoral immunity with extensive breadth (i.e., universal influenza vaccine, hiv- or hcv), heterologous prime:boost vaccination regimens should be evaluated using diverse ad vectors, or ads in combination with mva or protein based immunogens at different intervals. it is difficult to predict which ad vectors should take precedence as the encoded antigen will need to be tailored to elicit the correct phenotype of immunity against a defined correlate of protection for each specific disease target. however, underpinning the evaluation of all ad-based vaccines in pre-clinical animal studies, should be the inclusion of species c ad vector controls to represent a benchmark of immunological potency. in addition, ad vaccine candidates should be compared at several doses to evaluate the maintenance of vaccine potency upon dose de-escalation. the field should also make efforts to improve our understanding of the basic biology of many of these novel ad vectors, as insights into the receptor usage, interactions of ad vectors with different cell types following immunization and subsequent stimulation of differential innate signaling pathways will all impact on their downstream immunogenicity and ability to confer protective efficacy. unfortunately, one major challenge in performing these types of head-to-head comparisons is the lack of widespread availability of many of these rare species or non-human ad vectors to academic investigators, as many of these are being developed by large pharmaceutical companies. it is clear that a hierarchy exists in the immunological potency observed between rare species human and non-human ad vectors in various animal species. as outlined above, ad vector immunogenicity is most likely dependent on a complex combination of factors, rather than any particular factor in isolation. an ideal ad vaccine platform will combine the following attributes; (i) low 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encoding rsv-f protein induce durable and mucosal immunity in macaques after two intramuscular administrations firstin-human evaluation of the safety and immunogenicity of a recombinant adenovirus serotype hiv- env vaccine (ipcavd ) characterization of humoral and cellular immune responses elicited by a recombinant adenovirus serotype hiv- env vaccine in healthy adults (ipcavd ) mosaic hiv- vaccines expand the breadth and depth of cellular immune responses in rhesus monkeys thanks to prof. peter palese, ismms, for helpful and constructive discussion of this review. figures were created with ©biorender -biorender.com. the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © coughlan. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - zx rm authors: barbieri, antonio; robinson, nirmal; palma, giuseppe; maurea, nicola; desiderio, vincenzo; botti, gerardo title: can beta- -adrenergic pathway be a new target to combat sars-cov- hyperinflammatory syndrome?—lessons learned from cancer date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: zx rm sars-cov- infection is a new threat to global public health in the (st) century ( ), which has now rapidly spread around the globe causing severe pneumonia often linked to acute respiratory distress syndrome (ards) and hyperinflammatory syndrome. sars-cov- is highly contagious through saliva droplets. the structural analysis suggests that the virus enters human cells through the ligation of the spike protein to angiotensin-converting enzyme (ace( )). the progression of covid- has been divided into three main stages: stage i—viral response, stage ii—pulmonary phase, and stage iii—hyperinflammation phase. once the patients enter stage iii, it will likely need ventilation and it becomes difficult to manage. thus, it will be of paramount importance to find therapies to prevent or slow down the progression of the disease toward stage iii. the key event leading to hyperinflammation seems to be the activation of th- immunity response and cytokine storm. b( )-adrenergic receptors (b( )ars) are expressed on airways and on all the immune cells such as macrophages, dendritic cells, b and t lymphocytes. blocking (b( )ar) has been proven, also in clinical settings, to reduce th- response and negatively modulate inflammatory cytokines including il- while increasing ifnγ. non-selective beta-blockers are currently used to treat several diseases and have been proven to reduce stress-induced inflammation and reduce anxiety. for these reasons, we speculate that targeting b( )ar in the early phase of covid- might be beneficial to prevent hyperinflammation. coronaviruses are a variable group of enveloped, positive-sense, single-stranded rna viruses ( ) . they cause several diseases involving respiratory, enteric, hepatic, and neurological systems with high severity among humans and animals ( , ) . in the last two decades, the entire world witnessed two novel types of coronavirus, severe acute respiratory syndrome cov (sars-cov) and middle east respiratory syndrome cov (mers-cov), causing severe human diseases ( , ) . in december an outbreak of pneumonia of unknown cause occurred in wuhan, hubei province, china that rapidly spread around the globe, reaching to pandemic dimensions, and it was later named as sars-cov- or covid- ( , ) . the main symptoms of covid- include fever, fatigue, and cough, which are similar to that of sars-cov and mers-cov infected cases. sars-cov- is closely related to other coronaviruses ( % identity) which likely originated from bats. sars-cov- enters the human cells through ace receptors after ligation of spike protein. these receptors are highly expressed in the lung but are also found in other organs such as the heart, kidney, endothelium, and intestine ( ) ( ) ( ) . notably, ace is highly expressed on the luminal surface of intestinal epithelial cells, functioning as a co-receptor for nutrient uptake, in particular for amino acid resorption from food ( ) . for this reason, the intestine might also be a putative entry site for sars-cov- and that the infection might have been initiated by eating food from wild animals. to date the virus has infected hundreds of thousands of people, and about half of the hospitalized patients show clinical signs of comorbidities such as hypertension ( . - %), diabetes mellitus ( . %), coronary heart diseases ( . %), and cerebrovascular disease ( . %). also, a high rate of mortality has been reached when one or more comorbidities coexist. covid- infection associated symptoms, including acute respiratory distress syndrome (ards) and septic shock seem to be associated with hyperinflammation and cytokine storm as in patients' serum the levels of several cytokines are elevated ( ) . in particular il- , il- b, il- , tnf, gm-csf, ip- (ifninduced protein ), il- ,mcp- , and il- ra are mainly involved both in mild and severe forms of disease and altered circulating leukocyte subset and cytokine secretion ( ) . as many of these cytokines are involved in the th type response, wu and yang suggested that targeting the th pathway may counteract the covid- symptoms ( ). this hypothesis relies on different pieces of evidence: il- , tnfa, and il- b promote th response and are associated with inflammatory symptoms including fever, and the two latter are also associated with vascular permeability and leakage; il- has a broad inflammatory effect and together with gm-csf is involved in inflammatory and autoimmune disease; covid- patients have a significantly increased number of ccr + th cells ( ) ; elevated th and il- related pathways are increased in sars-cov, mers-cov, and h n influenza virus patients ( ) ( ) ( ) ; in mers-cov patients, il- and low ifng are associated with worse prognosis ( ) . targeting beta- adrenergic pathway was shown to reduce inflammatory cytokine and th response in different settings such as cancer and autoimmune diseases. for this reason we believe that beta- adrenergic pathway should be more deeply investigated as a possible target to reduce inflammation-related symptoms of sars-cov . b-adrenergic receptors are g-protein coupled transmembrane proteins. there are three subtypes of b-adrenergic receptors (b -ars, b -ars, and b -ars) that mediate a wide range of physiological responses to catecholamines epinephrine and norepinephrine, and thus play an important role in regulating cardiovascular responses in health and disease. b-adrenoceptors regulate many aspects of airway function, including airway smooth muscle tone, mast cell mediator release, and plasma exudation. b -adrenergic receptors are located mainly in the heart and in the kidneys ( , ) . b -adrenergic receptors are located in the lungs, gastrointestinal tract, liver, uterus, vascular smooth muscle, and skeletal muscle ( , ) . b -adrenergic receptors are located in fat cells. more than % of all breceptors in the human lung are located in the alveoli where the b -subtype predominates ( %) ( ) . however, b and b subtypes also coexist and are distributed uniformly in the alveolar walls. b -adrenergic receptors are expressed by all the cells of the immune system, including t and b lymphocytes, dendritic cells (dcs) and macrophages ( ) . the specific role of adrenergic signaling in regulating immune responses and inflammation is still under debate. however, there are many pieces of evidence supporting a proinflammatory action and promotion of th response. manni et al. showed that triggering b -adrenergic signal stimulates murine dc to secrete il- and promote a th response ( ) . in dc, the b adrenergic signal also reduces il- and ifng production but promotes il- . b -ar signaling plays a pivotal role in macrophage activation and proinflammatory cytokine production. similarly, in other immune cells, the overall effect of b -adrenergic stimulation is an exacerbation of inflammation, promotion of b cell antibody production, and stimulation of dc and macrophages to secrete proinflammatory cytokines. very interestingly, chiarella et al. showed that b -ar on alveolar macrophages is responsible for il- secretion and promotion of inflammation and prothrombotic state in a murine model of particulate-matter-induced thrombosis. administration of b -ar agonist induces mitochondria-dependent reactive oxygen species (ros) generation which results in camp response element-binding protein (creb) that results in il- activation. huang et al. investigated the effect of lymphocyte-derived catecholamines on the differentiation and function of t helper (th) cells, suggesting that this shifted the th /th balance in the direction of greater th polarization ( , ) . panina-bordignon et al. also showed that beta -adrenergic signaling inhibits the production of il- , thereby promoting th differentiation and inhibiting th development associated with antitumor immunity. catecholamines are also known to impact the immune response via down-regulation of ifn-g production ( ) . functionally, ifn-g can exert direct antiviral effects on infected cells as well as neighboring cells ( , ) . it can also activate local immune cells, like tissue-resident dendritic cells, macrophages, and nk cells, to augment antiviral functions ( , ( ) ( ) ( ) . furthermore, ifn-g can also control the antiviral state by modulating the differentiation and maturation of t cells and b cells ( , ) . khalili et al. showed that propranolol, a nonselective beta-blocker, synergized with an hsp- -rich tumor lysate vaccine to increase ifn-g production in a murine model of fibrosarcoma ( ) . treated animals showed lower rates of tumor growth (p < . ) and increased levels of ctl activity (p < . ). beta-blockers are a class of medications that are prevalently used to manage abnormal heart rhythms, hypertension and to protect the heart from recurring myocardial infarction. there are two main categories of beta-blockers: non-selective and selective. the first group includes older molecules such as propranolol, nandol, timol, etc., which have different degrees of affinity for b -ar and b -ar; second-generation drugs that are more selective such as metoprolol and acebutolol, etc. are specific for b -ar. after the introduction of cardioselective beta-blockers, the non-selective are not used frequently to treat heart conditions as the selective ones have fewer side effects. however, they are still in use to treat several other conditions such as long qt syndrome, aortic dilation in marfan syndrome, liver cirrhosis to reduce portal hypertension and bleeding esophageal varices. propranolol, a non-selective beta-adrenergic blocker, has been extensively used for over years in the treatment of many cardiovascular problems such as ischemic heart diseases, arrhythmias, and heart malfunction. recently, several clinical trials are supporting its benefit in a number of conditions, including cancer, hemorrhage, sepsis, and hypermetabolic syndrome associated with severe burns, akathisia associated with alzheimer's disease or psychosis, aggression associated with brain injury or disease, and anxiety ( ) ( ) ( ) . two different reports on cancer patients show that propranolol treatment reduces inflammatory cytokines including il- and tnfa, inflammation-related transcription factors such as nfkb and stat and reduces the activation of treg lymphocytes ( , ) . they also showed the potential benefit of propranolol on cancer recurrence (cr) and overall survival (os) ( , ) . in these trials on breast cancer patients, propranolol treatment wellpreserved the anticancer immunological profiles of peripheral blood mononuclear cells, reduced emt and prometastatic and proinflammatory transcription factors in tumor samples. in addition, shaashua and colleagues ( ) have suggested a positive synergistic effect of anti-inflammatory and betablockers, when these drugs are co-administered. beta-adrenergic receptor antagonists are also known to have effects on platelet aggregation, and a meta-analysis published in showed that they decreased platelet aggregation by % ( % ci = - %, standardized mean difference = − . , % ci = − . to − . , p < . ) ( ) . in particular non-selective lipophilic beta-blockers (including propranolol) decreased platelet aggregation more than that of the selective nonlipophilic beta-blockers. in addition, nonselective beta-blockers have been proved effective on the acute prothrombotic response to psychosocial stress and elevated plasma levels of factor viii:c in patients with deep vein thrombosis ( , ) . we had previously reported that propranolol reduces the effects of hypothalamic-pituitary-adrenal (hpa) axis stimulation induced by chronic stress. in particular, in a melanoma mouse model, propranolol treatment a week prior to the induction of stress delayed tumor growth. furthermore, propranolol-treated mice showed lower levels of vegf and enos with respect to untreated mice proving that it reduces the production of proangiogenic factors involved in tumor growth ( ) . our reported findings also show that propranolol exerts antimetastatic effects on du prostate cell lines xenograft in mice. it decreased metastatic foci in the inguinal lymph nodes, significantly reducing mmp and mmp in tumor samples. moreover, a norepinephrine-dependent increase in epithelial to mesenchymal transition (emt) can be rescued by propranolol treatment ( ) . our preliminary data (figure ) in mouse lung colonization model of melanoma show a significative reduction of il- and il- a levels and an increase of ifn-g in mice treated with selective b -ar inhibitor ici , (ici). moreover, our data show an increase of il- as shown in icitreated mice with respect to the controls. the patients with sars-cov- at the late stages of the disease suffer from many abnormalities, which are the result of immune system imbalance and malfunction which can lead to proinflammatory reactions and immunopathological conditions, presented by lethal inflammation in the lungs and vascular leakage ( , ) . several pieces of evidence support the pivotal role of il- in driving these phenomena. indeed, clinical evidence from china and italy in different hospitals ( ) showed that the il-r blocker tocilizumab, normally used for the treatment of rheumatoid arthritis can effectively improve clinical conditions of patients. the b -ar blockade can reduce il- and other inflammatory cytokines in the patient's serum contributing to rebalancing the immune system. in , kaplan et al. suggested a possible role of propranolol in the treatment of rheumatoid arthritis ( ) . moreover, the stress-induced inflammation, which likely occurs in patients diagnosed with covid- , could worsen the clinical symptoms; non-selective beta-blockers could prevent this phenomenon and at the same time reduce anxiety in those patients. furthermore, the pericyte injury due to virus infection may result in capillary endothelial cell dysfunction, inducing microvascular dysfunction ( ) . indeed, tang et al. proved that anticoagulant treatment is associated with decreased mortality in severe covid- patients with coagulopathy ( ) . beta-blocker might counteract this by reducing prothrombotic response, vascular tone, and vegf secretion ( ) ( ) ( ) . notably, some authors demonstrated that propranolol inhibits choroidal neovascularization (cnv) in vivo and b -ar blockade reduces vascular endothelial growth factor (vegf) expression in mouse retinal pigment epithelium and choroidal endothelial cells in culture ( ) . since beta-blockers may cause bronchospasm, in some cases, their use is not recommended in asthma or chronic obstructive pulmonary disease (copd). however, a recent study on a large number of patients in denmark showed that in patients taking beta-blockers (including non-selective) the risk of hospitalization for copd is reduced ( ) . similarly, in asthma, a recent systematic revision of the literature suggests that the escalating figure | summary of possible benefits of b -ar targeting in patients with sars-cov- . explanation step by step of different phases of sars-cov- pathogenesis and possible tool offered by beta-blockers to prevent and counteract uncontrolled cytokine storm and thrombus formation in late phase iii that lead to the death of patient. figure | proinflammatory cytokines in mice untreated (control) or treated with beta- -inhibitor (ici , ). cytokine multiplex assay was performed through the quantification in serum of il- a, il- b, il- , il- , il- , il- , il- , il -a, ifn-g, tnf-a, g-csf, gm-csf, in a lung colonization model of mice bearing murine melanoma cell line b f . error bars depict means ± sd. one-way anova and bonferroni post-hoc analysis were used to examine the significance of differences among groups (graph pad prism . ). a probability value with p < . was considered to be statistically significant. barbieri dose of beta-blockers is well tolerated and could be beneficial for airway inflammation and hyperresponsiveness ( ) . the patients positive for the sars-cov- did not describe any worsening of the symptoms. however, more accurate data are necessary to establish the safety of adrenergic targeting in these patients. many supporting pieces of evidence show that the major symptoms of covid- infection are associated with hyperinflammation over-activation of th response. indeed, treatments known to reduce th response such as tocilizumab and ruxolitinib have been already used to treat covid- patients in phases ii and iii. b -ar signals have been described to have a central role in rheumatoid arthritis in promoting inflammation and th response. non-selective beta-blockers have been used in clinical settings to reduce inflammation and th response. in addition, the non-selective beta-blocker propranolol blocks at induced expression of il- , nfkb, tnf, ifn-g, vegf, and metalloprotease engagement. we believe that b -ar signals might be a valuable target for new strategies aiming to block or slow down the transition from the early phases (i and iia) of covid- to phase iii by reducing the activation of th response and inflammatory cytokine release and prevent venous thromboembolism ( figure ). for these reasons we would like to point out the importance to perform further preclinical and clinical studies to explore this opportunity. the authors acknowledge that the data presented in this study must be deposited and made publicly available in an acceptable repository prior to publication. frontiers cannot accept a manuscript that does not adhere to our open data policies. ab and vd have thought and wrote the manuscript. nr critically revised the manuscript. gp revised immunological aspects and made corrections. nm made important contribution on beta-blockers. gb critically revised the manuscript and supervised the study. all authors contributed to the article and approved the submitted version. this work was funded by an "ricerca corrente" grant from the italian ministry of health. coronaviruses-drug discovery and therapeutic options the emerging novel middle east respiratory syndrome coronavirus: the "knowns" and "unknowns severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease a novel coronavirus from patients with pneumonia in china multiple organ infection and the pathogenesis of sars organ distribution of 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anti-il r role in treatment of covid- -related ards propranolol and the treatment of rheumatoid arthritis the ace expression in human heart indicates new potential mechanism of heart injury among patients infected with sars-cov- anticoagulant treatment is associated with decreased mortality in severe coronavirus disease patients with coagulopathy b-adrenergic receptors (b-ar) regulate vegf and il- production by divergent pathways in high b-ar-expressing breast cancer cell lines norepinephrine and adenosine- '-triphosphate synergize in inducing il- production by human dermal microvascular endothelial cells norepinephrine upregulates vegf, il- , and il- expression in human melanoma tumor cell lines: implications for stress-related enhancement of tumor progression b -adrenergic receptor antagonism attenuates cnv through inhibition of vegf and il- expression therapy and risk of chronic obstructive pulmonary disease -a danish nationwide study of · million individuals beta-blockers: friend or foe in asthma? we thank dr. sandra mazzoli for paper formatting, figure and test suggestions and dr. vincenzo quagliariello for elisa test on cytokines. key: cord- -qaagup d authors: flicker, sabine; zettl, ines; tillib, sergei v. title: nanobodies—useful tools for allergy treatment? date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: qaagup d in the last decade single domain antibodies (nanobodies, v(h)h) qualified through their unique characteristics have emerged as accepted and even advantageous alternative to conventional antibodies and have shown great potential as diagnostic and therapeutic tools. currently nanobodies find their main medical application area in the fields of oncology and neurodegenerative diseases. according to late-breaking information, nanobodies specific for coronavirus spikes have been generated these days to test their suitability as useful therapeutics for future outbreaks. their superior properties such as chemical stability, high affinity to a broad spectrum of epitopes, low immunogenicity, ease of their generation, selection and production proved nanobodies also to be remarkable to investigate their efficacy for passive treatment of type i allergy, an exaggerated immune reaction to foreign antigens with increasing global prevalence. type i allergy, an ige antibody mediated hypersensitivity disease, represents a common health problem affecting almost % of the population worldwide ( ) . the recognition of allergens by specific ige antibodies that are generated after sensitization is a key event for the initiation of allergic inflammation ( ) . allergic patients suffer from a variety of allergic symptoms including rhinoconjunctivitis and asthma ( ) but also food allergy and skin inflammation ( ) . these clinical manifestations cause a major burden by reducing the quality of life of affected persons ( ) . while anti-inflammatory treatment based on pharmacotherapy reduces allergic symptoms and is the most commonly prescribed medication for treatment of allergic patients ( ) , only allergenspecific immunotherapy (ait) represents a causative treatment of type i allergy. in fact, ait induces a protective immunity in allergic patients based on the modification of cellular and humoral responses to the disease causing allergen ( ) . besides the inhibition of ige binding to their specific allergen, the immune deviation from a th to th response, and the decreases in numbers of effector cells in target organs, the generation and maintenance of allergen-specific regulatory t and b cells and the involvement of their suppressive cytokines are essential for the induction of allergen tolerance ( ) ( ) ( ) . beyond doubt the improvement of allergic symptoms is further caused by ait-induced igg antibodies found in serum and nasal secretions ( , ( ) ( ) ( ) ( ) . for many years ait was conducted with aqueous natural allergen extracts and patients experienced considerable side effects due to the unpredictable composition and poor quality of the injected extracts ( ) . recent developments like next-generation forms of ait based on molecular approaches may overcome the limitations of current forms of ait ( , ) . the last generation of improved vaccines, i.e. peptide carrier vaccines, induces an igg response that targets ige binding sites on allergens. induced igg antibodies effectively block ige binding and are termed blocking antibodies ( , ) . however, the efficacy of such blocking antibodies was long questioned because it revealed to be cumbersome to isolate reproducible defined, i.e. monoclonal allergen-specific antibodies comprising the capacity to inhibit allergen-induced allergic reactions. a recent proof of concept study re-stimulated the idea to generate monoclonal allergen-specific antibodies and to evaluate their feasibility for allergy treatment. the authors could show that a single subcutaneous injection of a mixture of two human monoclonal allergen-specific igg antibodies significantly reduced allergic symptoms in allergic patients ( , ) . moreover, validated in a pca mouse model, the mixture of these two monoclonal antibodies proved to be more potent in inhibiting mast cell degranulation than igg antibodies purified from patients' sera who underwent successful ait ( ) . furthermore, these human monoclonal igg antibodies recently completed the phase ii clinical trial in treatment of cat allergic patients (https://clinicaltrials.gov/ct /show/nct ). these results proved for the first time that allergy treatment with monoclonal allergen-specific antibodies is a well-tolerated, rapid, and effective approach to reduce allergic inflammation and rekindled the blocking antibody concept ( , , ) . nevertheless, the generation and identification of blocking conventional human or humanized antibodies is connected with high costs for production, validation and application ( , ) . therefore, cost-effective alternatives are currently sought. the nanobody technology represents such an alternative implying a significant improvement to the laborious methods to obtain monoclonal blocking conventional antibodies. due to their beneficial properties of small molecules and monoclonal antibodies, nanobodies in general are an attractive agent for development of novel therapeutic strategies ( , ) . the ease of their generation and production, the single domain organization, their beneficial biochemical properties and their feature to recognize small cavities on the surface of antigens and hence bind to epitopes inaccessible for conventional antibodies ( ) have raised the particular interest of allergologists recently. can the nanobody technology provide enhanced opportunity to generate a panel of antigen-binding molecules with various epitope specificities for certain allergens different to conventional antibodies? will these identified allergen-specific nanobodies be more efficient in blocking than conventional igg antibodies due to their pronounced cleft recognition? will it be possible with this technology to find single nanobodies that are able to abrogate ige-mediated allergic inflammation? these questions and our wish to answer these questions attracted our attention. within this review, we focus on the powerful nanobody technology to generate allergen-specific nanobodies and report on their evaluation for prospective application for passive allergy treatment. if allergologists are asked why the search for effective protective allergen-specific monoclonal antibodies is complex and laborious, they will describe this issue by the typical quest for a needle in a haystack. through intense and precise molecular and immunological exploration of available allergen-specific monoclonal antibodies in the past it was proven that epitope specificity and affinity are decisive for their inhibitory potential to block ige binding and thus ige-mediated reactions ( , ( ) ( ) ( ) . the commitment to find and isolate monoclonal antibodies with specificity and high affinity for certain allergens and even more for certain epitopes always started with several fundamental decisions. amongst them the choice for the perfect source to gain dna coding for antibodies and the applied technology to generate allergen-specific antibodies are two of the most critical ones. regarding the dna source both animals, mainly mice, and humans served as blood, spleen, tonsils and even bone marrow donors in the last decades to isolate b cells or plasma cells and thus dna coding for antibodies ( ) ( ) ( ) . for the proof of principle, murine igg antibodies overlapping with human ige binding sites are valuable tools to investigate the effects to inhibit ige epitope recognition on allergens and consequently to contribute to the design of hypoallergenic derivatives suitable for ait ( ) . however, the direct therapeutic use of these murine monoclones in humans is limited by the high incidence of harmful immune responses against these administered foreign proteins ( ) . to mitigate this limitation numerous murine monoclonal antibodies have been re-engineered by chimerization and humanization technologies. these expensive procedures are justified for fatal diseases like different forms of cancer but were barely applied for allergen-specific murine antibodies so far with a few exceptions ( , ) . this was one of the main reasons why allergologists in the recent past endeavour to focus on human donors including allergic patients, ait-treated patients and even healthy individuals depending on the research question ( , , ) . various methods were utilized to generate allergen-specific genuine, i.e. native antibodies with the preservation of the natural vh and vl pairing including hybridoma technology, epstein-barr-virus (ebv) transformation, single b cell sorting and cloning and humab mice (transgenic mice that produce fully human antibodies) ( , ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . in parallel, versatile approaches were developed to generate non-genuine antibodies by random combination of vh and vl chains, i.e., combinatorial fab/scfv libraries or (semi-) synthetic libraries ( , , ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . based on pcr amplification as strong tool to depict large antibody repertoires and phage display to screen these large repertoires, many recombinant allergen-specific antibody fragments (fabs or scfvs) were isolated ( , , ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . all mentioned technologies have definitely contributed to the isolation and evaluation of monoclonal allergen-specific igg, ige antibodies and fragments thereof and furthermore to assess their feasibility for allergy treatment. nevertheless, all mentioned technologies are also reported to have some limitations. while the hybridoma technology and ebv transformation are generally unsuitable for a comprehensive screening of large antibody repertoires because of their inefficient fusion and transformation events, the single b cell sorting was long hampered by inadequate staining technologies to clearly identify allergen-specific antibody producing cells ( , ) . the main drawback of combinatorial libraries is that they usually rely on random combination and thus most likely unnatural vh and vl antibody pairings. additionally, it turned out independent of the applied technology to be very difficult to isolate monoclonal igg and ige antibodies with a broad epitope spectrum for each allergen. it also revealed that besides several blocking antibodies also many non-blocking or even enhancing antibodies were isolated ( , ( ) ( ) ( ) . while all three types of monoclonal antibodies were unambiguously supportive to study the structural requirements for efficient effector cell activation and hence contribute to elucidate the underlying mechanisms of type i allergy, non-blocking and enhancing antibodies were fully useless for the prospective application as protective antibodies. these insights forced allergologists to look beyond the conventional antibody horizon. about years ago, a group of belgian scientists made an unexpected discovery, which was patented and later presented to the scientific community in the form of the well-known discovery publication in the journal nature in ( ) . they found that a significant amount of non-canonical types of antibodies is naturally present in blood of camelidae in addition to conventional antibodies. this exceptional type of antibody called heavy chain-only antibody (hcab) lacks light chains and consists of a homodimer of shortened (without ch domain) heavy chains. the antigen-recognition region in hcabs is formed by only one variable domain (v h h) that is directly linked via a hinge region to the fc-domain ( ) . later on, similar non-canonical hcabs were found in some cartilaginous fishes such as sharks and ratfish ( ) ( ) ( ) . the antigen-binding variable domain of these antibodies was named vnar as opposed to v h h in camelids. a recombinant protein version of the v h h-or vnar-domain is usually called "single domain antibody" or "nanobody". the very popular term "nanobody" is the commercial name given by the belgian biopharmaceutical company ablynx, a pioneer in hcab-based therapeutic applications that was acquired by sanofi in . the nanobody generation technology was proven to be a very efficient machinery to generate nanobodies with required properties and offered crucial advantages compared to traditional techniques utilized to produce murine or human conventional antibodies. after the typical initial immunization (of camelids) step, the full repertoire of cdna coding for functional nanobodies can be efficiently cloned from peripheral blood lymphocytes of immunized animals using pcr amplification and then a panel of nanobodies of required specificity can be easily selected using phage (or other type of) display-based methods ( , ( ) ( ) ( ) . in addition, there are different in vitro affinity maturation approaches to improve features of initially selected nanobodies ( , , ) . in some cases, especially if the antigen of interest is toxic, unstable, non-immunogenic or not available in sufficient quantity, other types of libraries (naive, semisynthetic or fully synthetic libraries) can be efficiently used instead of immune libraries for generation of nanobodies ( ) ( ) ( ) ( ) ( ) . synthetic libraries can be made using special predesigned scaffolds such as humanized scaffolds optimized for intracellular stability ( ) or optimized for bacterial expression ( ) . non-immune libraries are typically much larger than immune libraries and a ribosome display was suggested for the initial selection round of such large libraries to work with higher concentrations of nanobody variants than in case of phage display ( , ) . synthetic libraries combined with different selection procedures were successfully used to obtain conformationally selective nanobodies against g protein-coupled receptors ( ), sybodies against very challenging targets such as the heterodimeric bacterial abc exportertm / ( ) or the intracellular kdel receptor ( ) to name a few examples from many others. nanobodies comprise unique features that distinguish them from classical antibodies. nanobodies are the smallest known antibody fragments ( × . x nm, - kda) of natural origin that are able to specifically bind their cognate antigens. due to their often extended cdr loop they can form unusual paratopes, i.e. finger-like extensions and thus recognize special native antigenic epitopes (small cavities, concave surfaces, conformational epitopes, active sites of enzymes) that are hidden for conventional antibodies (figure ) . indeed, nanobodies have proven to be useful tools for modulating the activity of enzymes ( , , ) . it could be therefore speculated that allergen-specific nanobodies that modulate or inhibit the proteolytic activity of certain allergens (e.g., phl p , der p ) might reduce their penetration capacity through mucosal surfaces. furthermore, nanobodies are able to bind small peptides with high affinity ( ) ( ) ( ) ( ) ( ) . their high affinity, solubility and stability over a wide range of temperatures and ph, ease of producing in bacteria or other expression systems make them convenient molecules for different applications, as well as for all possible engineering modifications e.g., development of complex constructs and conjugates. nanobodybased tools are therefore increasingly used for research, molecular visualization, diagnostics and development of new treatment options for various pathologies, including cancer and other socially significant diseases ( , , ( ) ( ) ( ) ( ) ( ) . so far, only one allergen-specific nanobody is described in the literature. this nanobody is reported to be specific for the major peanut allergen, ara h and was isolated from a synthetic library of humanized nanobodies via phage display ( ) . the interaction between ara h and the ara h -specific nanobody resulted in a dissociation constant of nm representing medium affinity binding and was further investigated by the structural determination of formed co-crystals ( ) . the authors acknowledged that additional work is needed to improve the affinity of the isolated nanobody to make it an attractive tool for the development of biosensors for peanut allergen detection. this finding clarifies that the selection procedure is only one part of the successful discovery of potent ige-blocking nanobodies, thus the evaluation of selected nanobodies is critical as well. nevertheless, we are confident that soon more allergenspecific nanobodies will arise to be studied for their potential to abrogate ige-mediated allergic inflammation. similar to the evaluation of conventional antibodies with the focus to identify effective protective monoclones, generated nanobodies have to be assessed first for their allergen specificity, epitope recognition, cross-reactivity to homologous allergens present in related species, for their affinity to their corresponding allergens and most importantly for their ability to inhibit patients´ige binding to these allergens (figures a-c) . after the allergen specificity of isolated nanobodies is confirmed, the proof for cross-reactivity (figure a) is of great importance because ige antibodies from allergic patients often displayed cross-reactivity to allergens from other allergen sources ( , ) . high affinity and slow dissociation of formed nanobody/allergen complexes will be critical prerequisites for allergen-specific nanobodies to be chosen as suitable candidate ( figure b) . however, the pivotal characteristics for an allergen-specific nanobody to be attractive for further processing will be the determination of its potential to block patients' ige binding and hence ige-mediated effector cell activation ( figure c) . additionally, specific nanobodies have to be tested as well for their cross-protectivity to homologous allergens. all these properties are crucial requirements for allergen-specific nanobodies to be selected for further essential investigations concerning half-life, clearance and safety. nanobodies are considered as proteins of weak immunogenicity due to the shared similarities with variable vh domains of human immunoglobulins (igg subclass), and they can be further improved by a humanization approach ( ) ( figure d) . consequently, no immune response against applied nanobodies was raised in mice or humans that were injected with nanobody-containing constructs ( ) ( ) ( ) . safety of nanobody-based drugs is confirmed by several completed phase and phase clinical trials ( ) and recent approval by the us food and drug administration (fda) and the european medicines agency (ema) of the first therapeutic nanobody, caplacizumab, a bivalent nanobody designed for the treatment of thrombotic thrombocytopenic purpura and thrombosis ( ) . though advantageous for in vivo imaging, the small size of nanobodies could be seen as a disadvantage for passive treatment of allergy due to a quick renal clearance of nanobodies from blood (approx. min). many different strategies to extend the in vivo half-life of nanobody-based construct have been developed ( ) . they include increasing the hydrodynamic radius of a protein by attaching highly flexible and hydrophilic molecules such as polyethylene glycol (peg) and carbohydrates or by genetic fusion with polypeptide chains mimicking the biochemical properties of peg, fusion of v h h to the fc region of igg, fusion or non-covalent binding to albumin ( ) ( figure d ). nanobodies can also be used as modules to engineer larger molecules with several valencies and/or specificities, such as multivalent ( ) ( ) ( ) ( ) , bispecific ( , ) , and other ( , ) constructs that may acquire considerably higher specificity, binding efficiency and biological activity ( , , ) . nanobodies were also considered as possible ligands to design new highly specific immunosorbents ( ) ( ) ( ) . different types of nanobody-based tools/approaches can be envisaged to be potentially profitable for an allergy treatment: a) bispecific nanobodies for topical application to capture allergens before they penetrate epithelial mucosa in airways, b) very stable nanobodies to capture food allergen in gastrointestinal tract, c) anti-idiotypic nanobodies mimicking allergenic epitopes as a possible replacement for a complex natural allergen for a new kind of ait vaccine development, d) multivalent nanobody-based constructs for systemical administration to efficiently block allergen interaction with ige on mast or basophil cells, e) efficient immunosorbents to remove ige from the blood by immune apheresis. correspondingly, different administration approaches for nanobody-based constructs can be developed: aerosol or topical applications, oral route or subcutaneous administrations. temporary blocking of allergen-ige interaction (i.e. by topical or systemic administration of specific nanobodies) or a subtraction of ige from the periphery blood (i.e. apheresis) may give a short-term treatment effect. for a long-term treatment effect we could hypothesize the use of anti-idiotypic nanobodies to ige. such nanobodies may represent "internal images" of an allergen and mimick hypoallergenic b cell epitopes. to efficiently induce igg response that targets ige binding sites on allergens, these nanobodies should be fused to a viral coat protein as it was described for next-generation forms of ait ( ) . the generation of allergen-specific nanobodies unambiguously represents a reasonable progress in the field of allergy. with their well-documented qualities including their ability to recognize unusual "hidden" epitopes, high affinity binding, solubility, extreme stability and low immunogenicity, nanobodies attracted the interest of allergologists to study their suitability for passive allergy treatment. the chance to find allergen-specific nanobodies with this powerful technology that ideally comprise high affinity and bind to epitopes partly or fully overlap with ige binding sites on allergens is tempting. however, so far no allergen-specific nanobody fulfilling these criteria was reported indicating that it might be rather difficult to raise allergenspecific nanobodies of sufficient affinities. whether the current lack of such nanobodies is owed to some inherent structural or functional properties of nanobodies and/or the camelid immune system or the simple reason that the current research focus in the allergy field is on ait and its improvement has to be resolved. if allergen-specific nanobodies are identified that competitively block allergen binding to ige and thus abrogate ige-mediated allergic inflammation, we assume that they will represent appropriate tools for future allergy treatment. their economic properties, i.e. low production costs encouraged researchers to elaborate antibody engineering of these single-domain 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immunization with adenoviral vector expressing chimeric nanobody-fc molecules as therapy for genital infection caused by mycoplasma hominis multivalent anchoring and oriented display of single-domain antibodies on cellulose single-domain antibody-based ligands for immunoaffinity separation of recombinant human lactoferrin from the goat lactoferrin of transgenic goat milk a method for the parallel and sequential generation of single-domain antibodies for the proteomic analysis of human blood plasma the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © flicker, zettl and tillib. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -y lvcjqd authors: eichinger, katherine m.; kosanovich, jessica l.; gidwani, sonal v.; zomback, aaron; lipp, madeline a.; perkins, timothy n.; oury, tim d.; petrovsky, nikolai; marshall, christopher p.; yondola, mark a.; empey, kerry m. title: prefusion rsv f immunization elicits th -mediated lung pathology in mice when formulated with a th (but not a th /th -balanced) adjuvant despite complete viral protection date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: y lvcjqd respiratory syncytial virus (rsv) remains the most common cause of lower respiratory tract infections in children worldwide. development of a vaccine has been hindered by the risk of developing enhanced respiratory disease (erd) upon natural exposure to the virus. generation of higher quality neutralizing antibodies with stabilized pre-fusion f protein antigens has been proposed as a strategy to prevent erd. we sought to test whether there was evidence of erd in naïve balb/c mice immunized with an unadjuvanted, stabilized pre-fusion f protein, and challenged with rsv line . we further sought to determine the extent to which formulation with a th -biased (alum) or a more th /th -balanced (advax-sm) adjuvant influenced cellular responses and lung pathology. when exposed to rsv, mice immunized with pre-fusion f protein alone (pref) exhibited increased airway eosinophilia and mucus accumulation. this was further exacerbated by formulation of pref with alum (aluminum hydroxide). conversely, formulation of pref with a th /th -balanced adjuvant, advax-sm, not only suppressed rsv viral replication, but also inhibited airway eosinophilia and mucus accumulation. this was associated with lower numbers of lung innate lymphocyte cells (ilc s) and cd + t cells producing il- + or il- + and increased ifnγ+ cd + and cd + t cells, in addition to rsv f-specific cd + t cells. these data suggest that in the absence of preimmunity, stabilized pref antigens may still be associated with aberrant th responses that induce lung pathology in response to rsv infection, and can be prevented by formulation with more th /th -balanced adjuvants that enhance cd + and cd + ifnγ+ t cell responses. this may support the use of stabilized pref antigens with th /th -balanced adjuvants like, advax-sm, as safer alternatives to alum in rsv vaccine candidates. respiratory syncytial virus (rsv) is the most common cause of lower respiratory tract infections (lrti) in children worldwide with nearly every child infected by years of age ( ) ( ) ( ) . in children > years of age, rsv causes an estimated million acute lrti annually, with over million episodes requiring hospitalization ( ) . in , the global cost estimate for inpatient and outpatient rsv lrti management in young children (< years of age) was ∼ . billion euros (equivalent to ∼$ . billion usd) ( ) . in addition to young children, rsv is a common cause of severe respiratory disease in the elderly and those who are immunocompromised ( ) ( ) ( ) . given that severe rsv disease affects ages spanning infancy to geriatrics, it is clear that natural rsv infection does not induce long-lasting immunity and individuals are re-infected throughout their lives ( , ) . thus, rsv immunization has the potential to boost rsv immunity and alleviate the morbidity associated with repeated rsv infections across all age groups. however, despite the immense economic and healthcare burden posed by rsv infection, there is currently no licensed rsv vaccine. the discovery and stabilization of the prefusion conformation of the rsv f protein (pref) re-ignited hopes for an rsv vaccine due to its ability to elicit potent neutralizing antibodies ( ) . a number of studies have demonstrated the protective potential of high levels of rsv neutralizing antibody and as such, boosting serum neutralizing antibody levels has been an important objective of rsv vaccine research ( , ) . the incorporation of adjuvants into vaccine formulations can enhance the vaccine's effect as well as reduce antigen concentrations and the number of immunizations required for a protective effect ( ) . differential stimulation of various pattern recognition receptors can further shift the immune response toward vaccine antigens to promote th -or th -type immune responses. based on the known th -bias associated with early rsv infections, it is imperative to understand the extent to which preventative rsv vaccine adjuvants shift the th /th balance. in use since the 's, aluminum salts have long been recognized for their ability to enhance immunogenicity and boost antibody production ( ) . alum adjuvant was used in the formalin-inactivated rsv (fi-rsv) trials of the 's that produced enhanced respiratory disease (erd) requiring hospitalization upon natural rsv exposure in % of vaccinees ( ) . subsequent investigations into the cause of fi-rsv-induced erd have suggested that alum exacerbated the t helper type (th ) pathology associated with erd ( ) . other studies have disputed alum's role and have instead suggested that formalin inactivation of rsv resulted in poor neutralizing antibody development and immune complex deposition ( , ) . reports have also demonstrated that formalin-inactivation of rsv resulted in post-fusion f (postf) protein being the predominant protein presented on the surface of the virion ( ) . the implication of this data is that pref is more representative of live rsv and therefore, may be less likely than postf subunit vaccines to induce pathology. moreover, in a naïve cotton rat model, both pref and postf immunization elicited protective rsv immunity without inducing alveolitis when paired with the th -skewing toll-like receptor agonist (tlr ), glucopyranosyl lipid a (gla) ( ) . these results suggest that more th -biased adjuvants may provide a safe alternative to alum in models of pref immunization. importantly, th skewing adjuvants, including tlr agonists, have recently been fda-approved for use in other vaccine systems, like hepatitis b ( ) . advax-sm is an adjuvant comprised of delta inulin polysaccharide formulated with the tlr agonist, cpg oligodeoxynucleotides (cpg odn). the advax-sm adjuvant system has demonstrated greater th -skewing properties when formulated with live rsv immunization ( ) and ameliorated th -related airway eosinophilia in a model of immunization against severe acute respiratory syndrome (sars)-associated coronavirus ( ) . in the immunization studies presented here, we conducted a detailed evaluation of the protective capacity of rsv pref antigen alone or combined with the th /th -balanced adjuvant, advax-sm (pref/advax-sm), or the th -skewing adjuvant, alum (pref/alum/aluminum hydroxide) in naïve balb/c mice. these studies evaluated immunogenicity, efficacy, innate and adaptive immune responses, and safety at acute and convalescent time points following rsv challenge. despite pref/alum immunization generating higher neutralizing antibody titers, both pref/advax-sm and pref/alum had undetectable viral replication, while pref alone lacked the immunogenicity to fully protect from rsv infection in naïve animals. pref/advax-sm induced more balanced th /th immunity characterized by the generation of neutralizing antibody, a mean pref-specific igg a/igg ratio > , rsv f-specific and cytotoxic cd + t cells, and th cd + t cells. importantly, pref/advax-sm immunization protected from increased inflammation and mucus production, even when compared to pbs controls. in contrast, pref alone and pref/alum generated robust th immunity evidenced by pref-specific igg a/igg ratios < and increased il- + and il- + cd + t cells. interestingly, despite undetectable viral replication at dpi, pref/alum immunization induced large populations of type innate lymphoid cells (ilc ) in the lung producing the th -type cytokines, il- and il- . combined, the th immunity of pref/alum was consistent with enhanced inflammation featuring airway eosinophils and increased mucus production. overall, our observations have significant implications for the rsv vaccine field demonstrating that higher neutralizing antibody titers, while protective, are not implicitly tied to rsv pref vaccine safety and more th /th balanced adjuvants may generate protective responses while eliciting a desirable safety profile in naïve mice. animal studies were carried out in accordance with the university of pittsburgh's iacuc guidelines for the use and care of laboratory animals. seven to eight week old balb/cj female mice were purchased from the jackson laboratory (bar harbor, me). female mice were immunized via intramuscular (i.m.) injection with mcl of vehicle (naïve and pbs), stabilized rsv prefusion protein (pref; mcg; calder biosciences) alone, or formulated with advax-sm tm (pref/advax-sm; mg/mouse; vaxine pty ltd, bedford park, australia) or alum (pref/alum; mg/ml). specifically, alhydrogel adjuvant %, an aluminum hydroxide wet gel suspension from invivogen, was used in these studies. advax-sm is composed of microparticles of polyfructofuranosyl-d -glucose (delta inulin) combined with cpg . -odn ( ′ atcgactctcgagcgttctc- ′ ), which was synthesized by genedesign (osaka, japan). the immunized mice were boosted weeks later with their respective vaccine formulations. at weeks post-prime, mice were challenged intranasally (i.n) with rsv l ( × pfu/gm) and culled at or days post-infection (dpi) using % isoflurane and cervical dislocation. rsv l was propagated and viral titers quantified as previously described ( ) . all animal studies were approved by the university of pittsburgh institutional animal care and use committee; protocol # . bronchoalveolar lavage (bal) was collected through intratracheal instillation of hbss + edta. bal samples were centrifuged and the soluble fraction stored at − • c for cytokine analysis and the cellular fraction analyzed via flow cytometry. cytokine concentrations were determined using the bio-plex pro tm mouse cytokine -plex assay (biorad, ca), per manufacturer's protocol. the right lung was harvested and enzyme-digested into a single cell suspension for flow cytometry, as described previously ( , ) . where indicated, bal cells and lung homogenate were stimulated ex-vivo for intracellular cytokine detection. briefly, live cells from bal and lung homogenate were enumerated using a hemacytometer and trypan blue. for intracellular cytokine staining of lung homogenate or bal, million cells in duplicate (singular for bal) were plated in a cd -coated ( mcg/ml, biolegend) -well flat-bottomed tissue culture plate in mcl of % rpmi supplemented with cd ( mcg/ml) and incubated at • c overnight. after overnight stimulation with cd /cd , lung homogenate underwent a secondary stimulation with pma ( : , ), ionomycin ( : , ), and brefeldin a ( : , ) for h prior to t cell surface and intracellular flow staining. to obtain intracellular ilc cytokine staining, lung homogenate ( million cells) was plated in -well tissue culture plates and stimulated with pma ( ng/ml), ionomycin ( ng/ml), and brefeldin a ( : , ) in % rpmi at • c for h prior to surface and intracellular flow staining. bal cells were surface stained with combinations of the following (clone): molecular probes live/dead fixable blue, cd / ( . g ), siglec-f (e - ), f / (t - ), cd b (m / ), ly g ( a ), cd (gk . ), cd α ( - . ), cd (im ) (bd biosciences, ca), cd c (n ), cd ( d ), and tcr β (h - ) (biolegend, ca). lung homogenate was surface stained with combinations of the following antibodies: cd / ( . g ), lineage cocktail, cd ( -f ), st (dih ), il- rα (a r ), cd ( d ), tcr β (h - ) (biolegend), cd (gk . ), and cd α ( - . ) (bd bioscience). following surface staining, cells were fixed and permeabilized for intracellular staining with bd cytofix/cytoperm tm solution kit (bd biosciences) according to the manufacturer's protocol. intracellular cytokines were stained with a combination of the following: cd (c c ), il- (trfk ), ifnγ (xmg . ), granzyme b (qa a ) (biolegend), and il- (ebio a) (thermofisher scientific, ma). where indicated and prior to surface staining, bal samples were incubated with rsv a strain f-protein − mhc i pentamer (h- kd kyknavtel; proimmune, fl) to identify rsv f − -specific cd + t cells. samples were run on a bd lsrfortessa managed by the united flow core of the university of pittsburgh. data was analyzed using flowjo v software (flowjo, llc, or). cell populations were defined as follows: eosinophils (siglec f+/ f / +/ cd lo/-/ cd b+); neutrophils (siglec f-/ cd bhi/ ly g+/ cd c-/lo); monocytes (siglec f-/ f / +/ cd c+/ cd b+); ilc s (lin-/ cd +/ st +/ il- rα+); t cells (± cd -/ tcr β +/ cd + or cd +). a lps-treated negative control was used to set the gate for rsv a strain f-protein − mhc i pentamer+ cd + t cells. pre-challenge serum was collected via submandibular bleed - days prior to rsv challenge and separated using gel-z serum separator tubes (sarstedt, germany). serum was stored at − • c until heat inactivation ( • c for min) and neutralizing antibody titers were performed. serial dilutions of heat inactivated serum ( mcl in phenol-free mem supplemented with % fbs and pen/strep, invitrogen) were incubated for h in a • c co incubator in a well plate format with pfu/well line rsv-renilla luciferase virus (provided by martin moore) in mcl phenol free mem medium as above. after h, hep- cells were trypsinized and a total of . × cells were added per well in mcl of phenol free mem with fbs and antibiotics as above. cells were incubated for a total of - h at • c, % co and luciferase readout was then obtained using the renilla-glo luciferase kit (promega) according to the manufacturer's instructions. luciferase activity (luminescence) was measured using a novostar plate reader after a min incubation at • c. all plates were run in duplicate and averaged. co-star -well, high binding elisa plates were coated with rsv pref at a concentration of mcg/ml overnight at • c. each plate included standards of either mouse igg or igg a (invitrogen) at and mcg/ml in a -fold dilution series for intra-plate quantification of signal on uncoated wells. plates were then washed with pbs, and blocked for h at • c with % bsa in pbs. heat-inactivated serum samples were diluted : in % bsa in pbs for the first well, and then -fold serially diluted a total of times. serum was incubated on the plates for h at • c, followed by three washes with pbs . % tween- , and secondary antibody incubation with anti-igg or anti-igg a (isotype specific, bd pharmingen), respectively at a : , dilution for min at • c in % bsa. -step tmb (thermo scientific) was used to develop the plates and the reaction was quenched by the addition of n h so . plates were read at nm in a novostar plate reader. data analysis was performed in excel and data points were interpolated from the linear region of the standards on each individual plate. samples were run in duplicate and the data presented represents the average values from both runs. left lungs were gravity filled with % formalin at and days post rsv challenge, as previously described ( ) . the mcgowan institute for regenerative medicine (university of pittsburgh, pa) stained and processed the preserved lungs. periodic acid-schiff (pas) stained lungs were assessed by two pathologists blinded to treatment groups to quantify airway mucus production, according to previously published methods ( ) . briefly, a score of - was given to all airways (average ) with the following scale: = no pas+ cells; = - % pas+ cells; = - % pas+ cells; = - % pas+ cells; = - % pas+ cells. scores were averaged and the total percentage of pas+ airways were graphed along with a more detailed breakdown of the proportion of each severity score ( - ) (# of airways of individual severity scores/total airways scored). standard hematoxylin and eosin (h&e) staining was performed on lung sections and scored by two pathologists (oury and perkins) blinded to treatment groups, according to previously described methods ( ) . in short, each field (average fields) in the lung was observed with a light microscope (x magnification) and scoring was based on the percentage of lung tissue affected according to the following scale: = no inflammation, = up to %, = - %, = - %, and = - %. scores were averaged and reported as a proportion of the sum of scores divided by the total number of fields counted. statistics were performed with graphpad prism software (graphpad software, la jolla, ca). results in the figures are displayed as the mean ± sem. neutralizing antibody data was analyzed by nonlinear regression to obtain ic values, which were compared between immunization groups using anova with a tukey's post-test, with pbs serving as the control. statistical significance was determined between each immunization cohort using anova with tukey's multiple comparison test between all groups (note: naive animals were not included in analysis but are graphed for reference). comparisons of the inflammatory and % pas+ scores within immunization groups over time were made using t-tests corrected for multiple comparisons with the holm-sidak method (α = . ). p-values < . were considered significant. data is representative of separate experiments. the th -biased humoral immunity of pref/alum and the greater th -skewed humoral immunity of pref/advax-sm protect against rsv replication the objective of this study was to determine the capacity of rsv pre-fusion (pref) vaccines formulated with different adjuvants to generate neutralizing antibody, prevent virus replication, and protect from pulmonary pathology following rsv challenge. to that end, week old balb/cj mice were immunized twice with pbs (vehicle control), pref alone, or pref formulated with the th /th -balanced adjuvant, advax-sm (pref/advax-sm), or the t helper type (th )-skewing adjuvant, aluminum hydroxide (pref/alum). six weeks after their final immunization, sera were collected from mice in each group prior to viral challenge with rsv line ; a control group of pbs-immunized mice received vehicle only (naïve) ( figure a) . a comparison of pref-specific igg a and igg in prechallenge sera provided initial evidence supporting the roles of advax-sm as a more th -biased adjuvant and alum as a th polarizing adjuvant; production of igg a and igg subclasses are reflective of their respective th and th biased immune responses in mice ( ) . pref/advax-sm immunization produced higher titers of igg a than all immunization groups tested ( figure b ) and greater igg compared to pbs ( figure c ). the mean igg a/igg ratio of pref/advax-sm mice was > and significantly higher than pref or pref/alum animals, indicating a more th -skewed humoral response ( figure d ). in contrast, pref/alum immunization produced negligible titers of igg a ( figure b) and instead elicited increased titers of igg ( figure c) , with a resulting igg a/igg ratio < ( figure d ). similar to pref/alum, pref alone elicited an igg a/igg ratio < , indicating th -dominant antibody responses in both groups. to assess differences in protective antibody responses between immunization groups, neutralizing antibody titers were measured in pre-challenge sera and rsv lung titers were quantified at days post-infection (dpi). mice immunized with pref/alum generated greater neutralizing antibody titers than all other immunization groups ( figure e) . despite lower neutralizing antibody titers in mice immunized with pref/advax-sm, as compared to pref/alum, both immunization groups had undetectable rsv in their lungs at dpi ( figure f ). in contrast, pref alone generated measurable neutralizing antibody titers in % (n = / ) of the animals and only % (n = / ) had undetectable rsv lung titers ( figure f ). all immunized mice had lower viral lung titers when compared to pbs controls following rsv challenge, however, only mice figure | the th -biased humoral immunity of pref/alum and the th skewed-humoral immunity of pref/advax-sm protect from rsv challenge. at - weeks of age, female adult balb/cj mice were immunized with phosphate buffered saline vehicle control (naïve & pbs), prefusion rsv f protein alone (pref), or pref formulated with advax-sm (pref/advax-sm) or alum (pref/alum) as depicted in (a). six weeks post-boost, pre-challenge serum was collected and mice were subsequently intranasally challenged with vehicle (naïve) or × pfu/gram of rsv line . animals were culled for sample collection at or days post infection (dpi) (a). pre-challenge serum was analyzed for pref-specific igg a (b), igg (c), igg a to igg ratios (d) and neutralizing antibody titers (e). at dpi, left lungs were harvested and virus quantified using standard h&e plaque assays (f). data are represented as mean ± sem (n = - mice per group);*p < . , **p < . , and ****p < . . due to a lack of pre-existing pref-specific antibody, pbs was not included in the analysis in (d). immunized with pref/alum or pref/advax-sm achieved full viral protection at dpi. to evaluate potential immunopathology in mice that received th -skewing immunizations (pref alone and pref/alum) as compared to the greater th -biased regimen (pref/advax-sm), left lungs were harvested from immunized mice at dpi and stained with h&e to evaluate inflammation. representative images ( x) taken from animals in each immunization group demonstrated increased inflammation in all immunization groups relative to pbs controls, with pronounced perivascular inflammation seen in pref-and pref/alum-immunized animals (figures a-e) . inflammatory scores increased at dpi in all groups that received pref-formulated immunizations but only pref alone and pref/alum elicited significantly greater inflammation compared to pbs (figure f) . to determine the contribution of innate cellular responses to the enhanced figure | pref and pref/alum immunization elicited enhanced pulmonary inflammation characterized by robust airway eosinophil recruitment. naive mice were immunized and challenged with rsv as described in figure . at dpi, left lungs were formalin filled, paraffin embedded and sectioned for staining with h&e. each panel represents an individual mouse from the indicated group (scale bar µm) (a-e). two blinded, independent pathologists scored all slides as described in the methods. scores between the two investigators were averaged and data is represented as mean ± sem (n = mice) (f). at dpi, bal was collected and eosinophils (g), neutrophils (h), and monocytes (i) were identified via flow cytometry. data are represented as mean ± sem (n = - mice per group);*p < . , **p < . , ***p < . , and ****p < . . yellow arrows highlight perivascular inflammation and blue arrows highlight peribronchial inflammation. inflammation observed in pref-and pref/alum-immunized groups, bronchoalveolar lavage (bal) was collected at dpi and analyzed via flow cytometry (gating strategy for discriminating innate immune cells is shown in supplementary figure ) . immunization with pref alone and pref/alum elicited a dramatic recruitment of eosinophils to the airways following rsv challenge, whereas eosinophil populations remained at baseline in pref/advax-sm-immunized mice ( figure g ). neutrophils were increased in the bal of pref-and pref/advax-smimmunized mice compared to pbs controls ( figure h ) and total monocytes were greater in pref-immunized animals as compared to all other groups tested (figure i) . taken together these results show that increased airway eosinophils paralleled increases in inflammation scores in mice immunized with pref alone and pref/alum, whereas neutrophils and monocytes were more pronounced in the unadjuvanted pref group. human rsv disease is characterized by both inflammation and extensive mucus production. thus, to determine if the enhanced inflammation seen in pref-and pref/alum-immunized groups was also associated with enhanced mucus production, left lungs were harvested at dpi and periodic acid-schiff (pas) stained to analyze mucus metaplasia. representative images ( x) were taken from a sample within each immunization group to visualize the extent of mucus production (figures a-e) . a majority of airways from naïve ( figure a) , pbs (figure b) , and pref/advax-sm immunization groups ( figure d ) had little to no pas+ staining, while pref alone ( figure c ) and pref/alum ( figure e ) groups had extensive pas+ staining. consistent with the representative lung sections, pref-and pref/alumimmunization groups had higher percentages of pas+ airways compared to all other groups tested ( figure f) . to discriminate the degree of airway mucus production, the level of severity was reported for each group, whereby no pas+ staining in the airway yielded a score of " , " the frequency of airways with - % pas+ staining received a score of " , " - % pas+ yielded a score of " , " - % pas+ airways received a score of " , " and - % pas+ staining received a score of " ." four days after diluent (figure g ) or rsv challenge, pbs ( figure h ) and pref/advax-sm ( figure j ) immunization groups had the largest percentage of unaffected airways and a small proportion of airways with mild pas+ staining ( score). in contrast, pref-( figure i ) and pref/alum-immunized mice ( figure k ) had smaller percentages of unaffected airways and greater frequency of airways with higher severity scores ( - scores). pref/advax-sm immunization produced th -dominant immunity with increased rsv f-specific cd + t cells to delineate the relationship between distinct th cell subsets and associated pulmonary inflammation and mucus production, figure | increased mucus production parallels enhanced inflammation in mice immunized with pref alone or pref/alum. mice were immunized and challenged with rsv as described in figure . at dpi, lungs were formalin filled, paraffin embedded and sectioned for staining with pas. each panel represents an individual mouse from the indicated group (scale bar µm) (a-e). to quantify the extent of pas staining, lungs were scored as previously described in the methods. scores were averaged and the total percentage of pas+ airways were graphed (n = ) (f); **p < . and ***p < . . a more detailed breakdown of scores for each cohort is provided, calculated as a proportion i.e., number of airways of each severity score ( - ) divided by the total number of airways, according to the methods (g-k). th -and th -type cytokines were quantified from bal. ifnγ, the canonical th cytokine, was highest in pref/advax-sm mice at dpi (figure a) , whereas levels of the th -associated cytokines, il- ( figure b ) and il- ( figure c) , were similar between pref-advax-sm-immunized mice and control groups (naïve and pbs). conversely, pref-and pref/alum-immunized mice had increased concentrations of il- and il- with no appreciable increase in ifnγ levels in these groups. to identify cellular sources contributing to the th associated cytokine bias of pref/advax-sm and the th associated cytokine bias of pref and pref/alum immunization, ilc and cd + t cell populations were analyzed from lung homogenate at dpi via flow cytometry (gating strategy to discriminate t cell populations and ilc s are shown in supplementary figures , , respectively). consistent with their th cytokine profiles, pref/alum immunization elicited greater ilc populations ( figure d ) following rsv challenge compared to all other groups. though ilc s trended higher in pref alone immunization than control and pref/advax-sm groups, the difference was not significant. moreover, pref/alum-immunized mice had increased il + ( figure e ) and il- + ( figure f ) ilc populations as compared to pref/advax-sm-immunized mice and pbs controls; once again, increased trends in the pref alone group did not achieve significance. in conjunction with increased ilc s, pref/alumimmunized mice had marked increases in il- -producing cd + t cells in lung homogenate ( figure h ). pref-immunized mice had similar trends in increased il- + cd + t cells but were not significantly different compared to pbs controls. in contrast, pref/advax-sm-immunized mice had the largest population of ifnγ+ cd + t cells (figure g ) with distinctly increased ifnγ+:il- + cd + t cell ratios (figure i ) as compared to pref-and pref/alum-immunized mice. a similar increase in the ratio of ifnγ+:il- + cd + t cells was observed in the pbs group. lastly, due to the importance of cd + t cells in clearing rsv, general cd + t cell populations and rsv f − -specific cd + t cells were compared in mice from each immunization group. mice immunized with pref/advax-sm had increased total cd + t cells as compared to pref-immunized mice and pbs controls ( figure j) . moreover, only pref/advax-smimmunized mice had increased rsv f − -specific cd + t cells, which were greater than all other groups tested ( figure k) . collectively, these results identified ilc s and cd + t cells as figure | pref/advax-sm immunization produced th -dominant immunity with increased rsv f-specific cd + t cells. at dpi, first wash samples were harvested from the airways for quantification of ifnγ (a), il- (b), and il- (c) by luminex. right lungs were harvested and homogenized for quantification of ilc s (d) and ilc intracellular cytokine staining of il- (e) and il- (f) by flow cytometry. intracellular production of ifnγ (g) and il- (h) by cd + t cells from lung homogenate were quantified by flow cytometry and the ratio of ifnγ/il- -producing cd + t cells was calculated (i). total cd + t cells (j) and rsv f − -specific cd + t cells (k) were measured from bal. data are represented as mean ± sem (n = - mice per group);*p < . , **p < . , ***p < . , and ****p < . . frontiers in immunology | www.frontiersin.org cellular sources of th -type cytokines associated with pref-and pref/alum-immunized mice following rsv challenge. in stark contrast, pref/advax-sm immunization elicited an increase in ifnγ-producing cd + t cells and rsv f − -specific cd + t cells in response to rsv exposure that likely contributed to viral protection. to determine if immunization with pref, pref/alum, or pref/advax-sm expedites disease resolution, innate inflammatory cells and lung inflammation were examined later at days post-rsv challenge. representative images ( x) were taken from h&e stained lung sections from each immunization group (figures a-e) . each rsv-challenged group displayed enhanced inflammation that was predominately perivascular in nature with non-significant reductions in inflammation in the immunization groups compared to the pbs group ( figure f ). similar to the -day time point, pref/alum mice maintained greater airway eosinophil populations ( figure g ) as compared to pref/advax-sm-immunized mice and pbs controls. neutrophils were elevated in pbs controls and the pref/alum group compared to pref/advax-sm-immunized mice, although no significant difference was observed in the pbs group (figure h) . pref-immunized and pbs control groups had the greatest number of monocytes in the bal when compared to pref/advax-sm-immunized mice ( figure i) . overall, populations of innate inflammatory cells had largely resolved in the bal of pref/advax-sm-immunized mice by dpi. in pbs controls, increases in neutrophils and monocytes likely contributed to the dramatic increase in inflammation between and dpi, whereas inflammatory scores for the groups immunized with pref remained largely unchanged (figure j) . these results suggest that inflammation and recruitment of inflammatory cells dramatically increased in pbs controls, innate inflammatory cells largely resolved in the pref/advax-sm group, and pref/alum immunization failed to resolve airway eosinophilia by days post-rsv challenge. to determine if mucus production resolved or worsened in immunized mice over the course of infection, left lungs were collected and pas-stained at dpi. representative images ( x) were taken from samples in each group (figure a-e) . readily discernable mucus was observed in pbs ( figure b ) and pref/alum ( figure e ) lung sections, with a lower frequency of pas+ staining seen in mice immunized with pref-alone ( figure c ). quantitative analysis of the percentage of pas+ figure | inflammation worsens over time in unimmunized compared to immunized groups. at dpi, left lungs were formalin fixed and stained with h&e to assess inflammation. each panel represents an individual mouse from the indicated group (scale bar µm) (a-e). two blinded, independent pathologists scored all slides and scores were averaged. data is represented as mean ± sem (n = - mice) (f). bal was collected at dpi for quantification of eosinophils (g), neutrophils (h), and monocytes (i) via flow cytometry. data are represented as mean ± sem (n = - mice). inflammatory scores from and dpi were compared for each immunization groups over time (j). data are represented as mean ± sem (n = - mice); *p < . and **p < . . airways revealed that pbs controls and pref/alum-immunized groups had the highest proportion of pas+ airways (figure k) . pref/advax-sm immunized mice maintained a low percentage of pas+ airways through dpi. a more detailed analysis revealed that > % of airways were pas+ in pbs-( figure g ) and pref/alum-immunized mice ( figure j ) and these groups had the largest proportions of airways that received higher severity scores (scores of - ) relative to all other groups (figure f-j) . at dpi, the majority of airways in the prefimmunization group were unaffected and when pas+ was observed, it was generally mild (score of ; figure i ). mice immunized with pref/advax-sm (figure j ) had the largest percentage of pas-free airways, with a smaller proportion of airways receiving higher severity scoring (scores of [ ] [ ] [ ] . similar to what was seen with inflammation over time, pbs controls demonstrated a distinct enhancement of mucus production between and dpi (figure l) , while pref/alum immunization elicited a high degree of pas+ staining throughout the rsv time course. in contrast, by dpi, pref-immunized mice resolved some of the high proportion of pas+ airways observed at dpi, while mice immunized with pref/advax-sm maintained low levels of mucus production. together, these data suggest that the high degree of mucus produced by dpi in pbs controls is mitigated more effectively in mice immunized with pref/advax-sm as opposed to pref/ alum immunization. based on the changes in lung pathology observed between immunization groups at dpi, we asked whether there was an associated change in th phenotypes among immunization figure | mucus production persisted in the lungs of pref/alum-immunized mice at dpi and remained low in mice immunized with pref/advax-sm. at dpi, left lungs were formalin fixed and stained with pas to assess mucus production. each panel represents an individual mouse from the indicated group (scale bar µm) (a-e). a more detailed breakdown of scores for each cohort is provided, calculated as a proportion i.e., number of airways of each severity score ( - ) divided by the total number of airways, according to the methods (f-j). to quantify the extent of pas staining, lungs were scored as previously described in the methods. scores were averaged and the total percentage of pas+ airways were graphed. total percentage pas+ airways from dpi are shown in (k) (n = - ) and are further compared over time between and dpi in each group in (l); **p < . . (figure a) , while mice immunized with pref alone and pref/alum had larger populations of il- + cd + t cells ( figure b) . additionally, il- + cd + t cell populations in pref/alum immunized mice were significantly higher than pref/advax-sm animals ( figure c ). examination of cd + t cell populations revealed greater granzyme b+ ( figure d) and ifnγ+ cd + t cells + (figure e ) in the airways of pref/advax-sm immunized mice and pbs controls, with pbs mice having significantly more ifnγ+ cd + t cells than the other immunization groups tested. in addition to cellular analysis in the bal, t cells and ilc s were measured in lung homogenate to address potential differences in cell localization. in accordance with their role as early immune responders, ilc populations ( figure f ) had contracted by dpi and no differences were detected between immunization groups. moreover, populations of il- +-( figure g ) and il- +-producing ilc s ( figure h) were similar between all groups. finally, th phenotypes were examined at dpi in lung homogenate. pref/advax-sm immunized mice had the highest number of ifnγ+ cd + t cells (figure i ) compared to other groups, while il- + cd + t cells ( figure j) were greatest in pref-vaccinated mice. consistent with the data at dpi, the cd + ifnγ+:il- + ratio ( figure k ) was higher in pref/advax-sm immunized mice as compared to all other groups tested. this demonstrates the persistent th -dominant immune response elicited by pref/advax-sm immunization. here, we explored the ability of a rsv pref subunit proteinbased immunization to protect naïve balb/c mice from rsv infection and pulmonary pathology when formulated with either a th -or more th /th -balanced adjuvant. alum has been in use in human vaccines since the early th century and is well recognized for its th -skewing and antibody boosting properties. as expected, pref/alum immunization produced th -biased immunity with high titers of pref-specific igg and neutralizing antibody, which were associated with undetectable viral replication following rsv challenge. while pref alone lacked the immunogenicity to offer complete rsv protection, likely due to low neutralizing antibody production, prefimmunized mice generated th -type immune responses similar to mice immunized with pref/alum. on the other hand, advax-sm is a delta-inulin polysaccharide adjuvant formulated with cpg . odn, a tlr agonist. the combination of delta inulin and tlr agonism has been reported to generate a th /th balanced adjuvant response in models of sars-associated coronavirus, japanese encephalitis virus, and west nile virus ( , , ) . in models of rsv immunization, tlr agonists formulated with formalin-inactivated rsv have increased immunogenicity while ameliorating pulmonary pathology and reducing airway hyperresponsiveness normally exacerbated by fi-rsv immunization ( , ) . in one report, an intramuscular immunization of live rsv formulated with advax-sm protected from rsv infection with increased neutralizing antibody titers and greater rsv-specific igg a/igg , but had similar lung inflammation as unadjuvanted control mice, suggesting the th polarization of the live rsv vaccine may have overcome the greater th bias of the cpg oligonucleotide ( ) . these studies support the idea that tlr agonists may be combined with stabilized pref proteins to induce a more th -biased response with an acceptable safety profile and highlight the importance of evaluating adjuvant safety as well as efficacy when developing vaccine strategies. in our study, mice immunized with pref/advax-sm elicited a greater th -type response, while still generating high levels of pref-specific igg a and igg antibody. despite producing lower neutralizing antibody titers than pref/alum immunized, pref/advax-sm mice were resistant to rsv infection. moreover, the discreet th profiles produced by pref/alum and pref/advax-sm generated distinct differences in pulmonary pathology. the th profile of pref/alum-immunized mice was associated with enhanced pulmonary inflammation and mucus production at dpi and continued to have the greatest proportion of pas+ airways as late as dpi. the immunopathology associated with pref/alum immunization occurred in spite of high neutralizing antibody titers and undetectable viral replication. previous studies of fi-rsv-associated erd have demonstrated that poor avidity and affinity maturation resulted in non-protective antibody development and th -associated immunopathology ( ) . furthermore, it has been suggested that alum did not alter the erd profile induced by fi-rsv immunization ( ) . however, in our study, alum adjuvant exacerbated the pathology induced by immunization with pref alone despite protection from rsv infection. in contrast, the th -type responses generated by immunization with pref/advax-sm elicited less overall inflammation with low levels of mucus production. importantly, pref/advax-sm immunization protected mice from the worsening inflammation and mucus production that occurred between and dpi in pbs controls. taken together, these data indicate that rsv pref immunization formulated with th -skewing adjuvants, like advax-sm, provide protection against rsv infection in naïve balb/c mice as compared to alum by inhibiting viral replication without eliciting enhanced pulmonary pathology. though ige was not measured in these studies, alum, as opposed to cpg-odn, adjuvanted antigens have been associated with an increased production of ige ( ) . future studies will be needed to address the role of ige following immunization with rsv pref adjuvanted with alum. neutralizing antibodies have been shown to reduce the severity of rsv disease ( , ) . moreover, the risk of reinfection is inversely correlated to the level of serum neutralizing antibodies ( ) . as such, boosting serum neutralizing antibody titers has been an important objective of rsv vaccination, especially since the discovery and subsequent stabilization of the pre-fusion conformation of rsv f protein ( ) . rsvneutralizing activity in human sera is primarily derived from pref-specific antibodies ( , ) . thus, the use of stabilized pref as a vaccine antigen has sparked new hope that high neutralizing antibody titers can be produced to provide long-term protection. pre-clinical studies in young and aged mice have demonstrated the neutralizing antibody boosting-potential of rsv pref when formulated with both th -and th /th -skewing adjuvants ( ) . in agreement with our data, pref immunization alone has been shown to lack sufficient immunogenicity to increase neutralizing antibody titers above a protective threshold ( ) . our study expanded these findings by directly challenging pref-immunized mice and demonstrated incomplete protection and enhanced lung pathology following rsv infection in association with little to no measurable neutralizing antibody production (i.e., only % of pref-immunized mice produced any measurable neutralizing antibody). earlier work found that higher titers of neutralizing antibody were associated with rsv pref immunization formulated with th /th -balanced adjuvants ( ) . in contrast, our results show that the th -dominant immune response of pref/alum immunization produced higher neutralizing antibody titers as compared to the th -biased immune response elicited by pref/advax-sm immunization. importantly, however, both pref/alum and pref/advax-sm immunization fully protected mice from rsv infection with a complete absence of detectable viral replication in the lungs at dpi. additional studies will be required to determine if lower neutralizing antibody production, as seen in pref/advax-sm, affects the durability of protection against rsv challenge. despite evidence of the correlation between neutralizing antibody in the serum and protection from severe rsv disease ( , , , ) , other data suggests that high serum levels of neutralizing antibody provide insufficient protection from rsvdisease or reinfection in some individuals ( ) ( ) ( ) . therefore, rsv vaccines that rely on neutralizing antibody as the sole correlate of protection against rsv disease may not provide universal or long-lasting immunity. numerous studies have demonstrated the importance of t cells in clearing rsv in primary rsv infection ( ) ( ) ( ) . in mouse models of primary infection, cd + t cells are critical for viral control and both the number of cd + t cells and their production of ifnγ are crucial for effective clearance ( ) ( ) ( ) . despite conferring protection against rsv replication, immunopathology has been linked to ifnγ-producing cd +t cells during primary infection ( ) and in memory responses resulting from immunization ( ) . in contrast, our data shows that immunization with pref/advax-sm protects against rsv infection and elicits rsv f-specific and ifnγ-producing cd + t cells without inducing additional pulmonary pathology. alternatively, by dpi, a robust ifnγ+ cd +t cell response was induced in pbs-immunized control mice that was further associated with increased inflammation. these divergent outcomes in lung pathology associated with ifnγ+ cd +t cells may be explained by the presence of pre-existing neutralizing antibody in pref/advax-sm as compared to the lack of pre-existing neutralizing antibody in pbs controls. another model of rsv vaccination has demonstrated that pre-existing neutralizing antibody can protect mice from severe immunopathology caused by potent memory ifnγ-producing cd +t cells ( ) . therefore, rsv vaccination approaches that combine pref with th /th -balanced adjuvants may prove to be efficacious and safe through the combination of increased neutralizing and rsv-specific antibody production and memory ifnγ+ cd +t cell responses. like cd + t cells, research has demonstrated the importance of cd + t cells in the control of rsv infection and their role in inducing pathology ( ) ( ) ( ) . the 's vaccine trials of formalin-inactivated rsv formulated with alum (fi-rsv) demonstrated the sobering potential for vaccine-associated erd. upon natural rsv exposure, % of vaccinees developed erd, requiring hospitalization and two children died ( ) . investigations into the causes of erd using multiple animal models have revealed th cd + t cells to be closely linked to the increased inflammation, mucus, and airway hyperresponsiveness observed in erd ( ) ( ) ( ) ( ) ( ) . in our study, both pref alone and pref/alum immunization elicited th cd + t cell profiles associated with enhanced inflammation, airway eosinophils, and extensive mucus production. interestingly, despite their similar th phenotypes, pref-immunized mice resolved airway neutrophils, eosinophils, and mucus more rapidly than pref/alum immunized mice. this resolution occurred in pref-immunized mice despite their poor neutralizing antibody production and detectable rsv replication, which was likely the cause of increased inflammatory innate cell recruitment as compared to pref/alum immunization. whereas, mice immunized with pref/alum had high titers of neutralizing antibody and undetectable rsv in the lung, yet persistent airway mucus and inflammation. in contrast to data showing the ability of pre-existing antibody to temper the immunopathology induced by ifnγ+ cd +t cells ( ) , our data suggests that pre-existing neutralizing antibody alone may not modulate the immunopathology associated with th cd + t cells in a similar manner. in addition to increases in th cd + t cells, pref-but especially pref/alum-immunization was associated with large populations of ilc s producing il- and il- in the lung. ilc s are the dominant innate lymphoid cell in the lung and are well recognized for their ability to produce large amounts of type cytokines upon stimulation ( ) . in primary rsv, ilc expansion and activation result from damage to airway epithelial cells via rsv infection and subsequent release of il- ( ) and/or thymic stromal lymphopoietin (tslp) ( ). however, in pref/alum immunized animals, ilc s increased and became activated in spite of effective rsv neutralization and undetectable virus in the lung at dpi. our data suggests that an alternative mechanism may contribute to the induction of ilc responses in pref/alum immunized mice. a murine model of nippostrongylus brasiliensis demonstrated that crosstalk between ilc s and antigen-specific th cd + t cells occurs, promoting ilc proliferation and il- production ( ). this ilc -cd + t cell crosstalk was shown to be mutually beneficial to both cell types' maintenance, expansion, and cytokine production. future studies are necessary to understand how pref/alum immunization contributes to ilc expansion and activation. however, this is the first study to suggest a relationship between ilc activation and th -associated immunopathology following pref/alum immunization in naïve mice. in conclusion, our studies demonstrated that pref/advax-sm and pref/alum immunizations provided equivalent protection from rsv infection but elicited dramatically different outcomes regarding lung pathology. the th immunity induced by pref/alum immunization was associated with increased inflammation and abundant mucus production that persisted through dpi. in addition to il- -and il- -producing cd + t cells, pref/alum immunization was also associated with increased il- -and il- -producing ilc populations. this previously unrecognized relationship between pref/aluminduced th immunity and ilc activation in the absence of rsv replication may have important implications for rsv vaccine development. it suggests that despite eliciting high titers of neutralizing antibody, the extensive th immune bias generated by pref/alum may still lead to overt pathology in naïve individuals. it has been suggested that rsv exposure prior to immunization may mitigate the overwhelming th immunity and lung pathology observed here with pref/alum, however, considering the important safety concern, further studies are needed to confirm this hypothesis. in contrast to the th -skewed immunity of pref/alum, the combination of neutralizing antibody production, th immunity, and cytotoxic ifnγproducing cd +t cells induced by pref/advax-sm provided vigorous protection from rsv replication without inducing pathology. taken together, these data suggest that rsv pref protein subunit vaccinations formulated with th /th -balanced adjuvants may provide complete rsv protection with a desirable safety profile. future studies will be needed to elucidate safety and protection of preimmune mice when immunized with pref alone or when formulated with a th -vs th -biased adjuvant. the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. the animal study was reviewed and approved by the university of pittsburgh institutional animal care and use committee. kei, jk, and kem contributed to the overall study design, execution, and interpretation of data and writing of the paper. sg and az contribute through development of novel assays for generation of data. ml contributed to data interpretation, figure development, and writing. my contributed to data generation, study design, and analysis. tp and to contributed to the analysis and interpretation of data. cm and np contributed to study design. all authors contributed to the article and approved the submitted version. viral and host factors in human respiratory syncytial virus pathogenesis respiratory syncytial virus-associated hospitalizations among children less than months of age global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in : a systematic review and modelling study cost of respiratory syncytial virus-associated acute lower respiratory infection 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combined actions of distinct cd t cell subsets vaccine-enhanced respiratory syncytial virus disease in cotton rats following immunization with lot or a newly prepared reference vaccine respiratory synctial virus infection in balb/c mice previously immunized with formalin-inactivated virus induces enhanced pulmonary inflammatory response with a predominant th -like cytokine pattern group innate lymphoid cells in pulmonary immunity and tissue homeostasis for clinical pharmaceutical sciences, university of pittsburgh school of pharmacy. this work benefitted from special bd lsrfortessatm funded by nih s od - (pi: borghesi). np (vaxine pty ltd., bedford park, australia) generously provided advax-sm but did not otherwise provide financial assistance for this project. development of advax-sm adjuvant was supported by national institutes of health contracts hhsn c, hhsn c and ai . this publication's contents are solely the responsibility of the authors and do not necessarily represent the official views of the national institutes of health, national institute of allergy and infectious diseases. we would like to pay special thanks to dr. kevin legge for his thoughtful and constructive feedback during the writing of this manuscript. conflict of interest: np is affiliated with vaxine pty ltd., which hold commercial interests in advax adjuvants cm and my are affiliated with calder biosciences, which holds commercial interests in the stabilized pref protein. the remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © eichinger, kosanovich, gidwani, zomback, lipp, perkins, oury, petrovsky, marshall, yondola and empey. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - x j iqg authors: bösl, korbinian; ianevski, aleksandr; than, thoa t.; andersen, petter i.; kuivanen, suvi; teppor, mona; zusinaite, eva; dumpis, uga; vitkauskiene, astra; cox, rebecca j.; kallio-kokko, hannimari; bergqvist, anders; tenson, tanel; merits, andres; oksenych, valentyn; bjørås, magnar; anthonsen, marit w.; shum, david; kaarbø, mari; vapalahti, olli; windisch, marc p.; superti-furga, giulio; snijder, berend; kainov, denis; kandasamy, richard k. title: common nodes of virus–host interaction revealed through an integrated network analysis date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: x j iqg viruses are one of the major causes of acute and chronic infectious diseases and thus a major contributor to the global burden of disease. several studies have shown how viruses have evolved to hijack basic cellular pathways and evade innate immune response by modulating key host factors and signaling pathways. a collective view of these multiple studies could advance our understanding of virus-host interactions and provide new therapeutic perspectives for the treatment of viral diseases. here, we performed an integrative meta-analysis to elucidate the different host-virus interactomes. network and bioinformatics analyses showed how viruses with small genomes efficiently achieve the maximal effect by targeting multifunctional and highly connected host proteins with a high occurrence of disordered regions. we also identified the core cellular process subnetworks that are targeted by all the viruses. integration with functional rna interference (rnai) datasets showed that a large proportion of the targets are required for viral replication. furthermore, we performed an interactome-informed drug re-purposing screen and identified novel activities for broad-spectrum antiviral agents against hepatitis c virus and human metapneumovirus. altogether, these orthogonal datasets could serve as a platform for hypothesis generation and follow-up studies to broaden our understanding of the viral evasion landscape. viruses are one of the major causes of acute and chronic infectious diseases and thus a major contributor to the global burden of disease. several studies have shown how viruses have evolved to hijack basic cellular pathways and evade innate immune response by modulating key host factors and signaling pathways. a collective view of these multiple studies could advance our understanding of virus-host interactions and provide new therapeutic perspectives for the treatment of viral diseases. here, we performed an integrative meta-analysis to elucidate the different host-virus interactomes. network and bioinformatics analyses showed how viruses with small genomes efficiently achieve the maximal effect by targeting multifunctional and highly connected host proteins with a high occurrence of disordered regions. we also identified the core cellular process subnetworks that are targeted by all the viruses. integration with functional rna interference (rnai) datasets showed that a large proportion of the targets are required for viral replication. furthermore, we performed an interactome-informed drug re-purposing screen and identified novel activities for broad-spectrum antiviral agents against hepatitis c virus and human metapneumovirus. altogether, these orthogonal datasets could serve as a platform for hypothesis generation and follow-up studies to broaden our understanding of the viral evasion landscape. viruses continue to be a major contributor to the global burden of disease through acute and chronic infections that cause substantial economic impact in addition to increased mortality and morbidity ( ) . despite the tremendous improvement in the understanding of the antiviral immune response and the availability of therapeutics, existing and emerging viral diseases are an ever-growing problem, particularly in developing countries. development of antiviral resistance of hepatitis c virus (hcv), influenza a virus (iav), herpes simplex virus (hsv), human cytomegalovirus (hcmv), human immunodeficiency virus (hiv), and other viruses is a major concern ( - ). one of the main reasons for increasing resistances is the accumulation of mutations in the viral genome caused by multiple factors including the polymerase infidelity ( , ) . therefore, the world health organization (who) and the united nations have urged for better control of viral diseases. this has led to turning the focus on the host for therapeutic intervention. targeting the host factors has been proven to be useful for restricting viral infections ( , ) . the small molecule ccr inhibitor maraviroc and the anti-cd monoclonal antibody ibalizumab are examples of successful use of host-directed therapies for combating hiv in clinic ( ) ( ) ( ) . viruses have evolved to evade the host antiviral response at various stages starting from viral sensing to antiviral proinflammatory responses ( ) ( ) ( ) . multiple studies attempted to understand global principles of the viral evasion employed by various viruses, including dengue virus (denv), ebola virus (ebov), iav, and hiv ( ) ( ) ( ) ( ) ( ) ( ) . global systems-level approaches including functional rnai screens, interactome mapping technologies such as affinity-purification mass spectrometry (ap-ms), quantitative proteomics, and crispr/cas -based screens have provided unparalleled details and insights into the dynamics of host proteome in immune cells ( ) ( ) ( ) ( ) , host-virus interactome ( - , , ) , and also identified important host dependency factors of various viruses ( , , ) . meta-analyses of such high-dimensional datasets have been crucial for identifying novel host factors as drug targets such as ubr in iav infection ( ) . moreover, some of these factors represent drug targets for multiple viruses ( ) . we hypothesized that combining a meta-analysis of host-virus protein-protein interactions of multiple viruses and functional rnai screens would provide novel insights for developing broadspectrum antiviral strategies. for this, we assembled a host-virus protein-protein interactome of , host-virus interactions (hereafter referred to as "hvppi") covering viral proteins from different viruses and , host proteins. we performed extensive bioinformatics and network analysis and integrated this dataset with genome-wide or druggable-genome rnai screen data from published studies. this resulted in the assembly of critical nodes of viral evasion and identification of core cellular processes and druggable nodes that were verified by a drug re-purposing screen using broad-spectrum antivirals. host-virus protein-protein interactions were downloaded from published studies ( - , , - ) we performed gene-set enrichment analysis using david bioinformatics resources [version . , ( ) ]. for all enrichment analysis, a p-value cutoff of ≤ . was used as significant. protein disorder analysis was performed using iupred a software. we used the offline version with protein sequences downloaded from uniprot. statistical analysis of disordered region distribution was performed by kolmogorov-smirnov test in r statistical environment. annotation of human proteins was mapped from uniprot id to ensembl using ensdb.hsapiens.v . the index of subcellular localization of interaction partners of single viral proteins was calculated for all viral proteins with ≥ five host targets. localization of host targets was mapped using compartments ( ) , filtered for a minimum evidence score of in the knowledge channel, excluding non-experimental based localization predictions. evidence for all protein was subsequently divided by the absolute number of host-targets per viral protein. multiple sequence alignment was performed using clustal x [version . , ( )]. genome-wide rnai screen data for hcv ( ) and hpv ( ) , through genomernai database [( )-gr ], as well as druggable rnai screen data for hpv ( ) , vacv ( ) , and sv ( ) were integrated in the existing network as z-scores. drug-gene interaction data was downloaded from dgidb and drugbank. the identifiers were mapped to uniprot ids and then compared with hvppi. for the hmpv nl/ / -gfp screen, approximately × human retinal pigment epithelial (rpe) cells were seeded per well in -well plates. human non-malignant rpe cell line represents excellent model system for studying replication of many viruses including respiratory ( , , ) . the cells were grown for h in dmem-f medium supplemented with % fbs, . % nahco , and µg/ml streptomicine and iu/ml penicillin. the medium was replaced with virus growth medium (vgm) containing . % bovine serum albumin (bsa), mm l-glutamine, . % nahco , and µg/ml l- -tosylamido- -phenylethyl chloromethyl ketone-trypsin in dmem-f . hcv screen-associated cell culture conditions are described in kim et al. ( ) . the compounds were added to the cells in fold dilutions at seven different concentrations starting from µm. no compounds were added to the control wells. the cells were mock-or virus-infected at a multiplicity of infection (moi) of one. after h of infection, the medium was removed from the cells. to monitor cell viability, celltiter-glo reagent was added ( µl per well). this assay quantifies atp, an indicator of metabolically active living cells. the luminescence was measured with a plate reader. to determine compound efficacy, hmpv nl/ / -mediated gfp expression was measured. the half-maximal cytotoxic concentration (cc ) and the half-maximal effective concentration (ec ) for each compound were calculated after non-linear regression analysis with a variable slope using graphpad prism software version . a. the relative effectiveness of the drug was quantified as the selectivity index (si = cc ec ). cytotoxicity and antiviral activity of the compounds against gfp-expressing hcv in huh- . cells was determined as previously described ( ). to provide new and critical insights into viral evasion mechanisms we performed a comprehensive meta-analysis of the host-virus interaction landscape. we assembled the hostvirus protein-protein interaction data ("hvppi") from published studies ( figure a ) ( - , , - ) . this dataset comprised of protein-protein interactions from two different types of experimental methods-affinity purification mass spectrometry (ap-ms) and yeast two-hybrid screens (y h). altogether, this combined dataset includes viral proteins, , host proteins, and , protein-protein interactions ( figure b and figure s ). many interactome networks including yeast and human are scale-free networks, where a large portion of the nodes (e.g., a protein in the network) have few interactions and only a few nodes have large number of interactions. the latter are often referred to as "hubs" which are crucial in keeping the network intact ( ) . we performed network topology analysis to infer the properties of the host proteins targeted by the viral proteins in the context of the human protein interactome. we considered two important parameters-relative betweenness centrality (which reflects the amount of information that passes through this protein in the human interactome) and degree (number of binding partners in the human interactome) of the host proteins targeted by each virus. the targets of all the viruses showed higher betweenness centrality and degree as compared to an average protein in the human interactome (figures c,d) . this shows that viruses, by targeting "hubs" and proteins that serves as key communication nodes, have evolved the best way to disrupt the scale-free human interactome. this topological property thereby shows how viruses having small genomes achieve the maximal effect in rewiring the human interactome to benefit viral survival and replication. our analysis is in agreement with several previous studies, which have highlighted this property ( , , , , ) . we propose that this could be a general principle for all viruses. proteins typically fold into stable three-dimensional structures that mediate specific functions. in addition, there are substructures in proteins termed "intrinsically disordered regions (idrs)" which lack stable structures under normal physiological conditions. idrs are required for multiple cellular functions even though they lack these defined structures ( ) . many studies have highlighted the presence of such idrs in viral proteins ( ) ( ) ( ) , such as e from human papilloma virus, that are crucial for hijacking the cellular machinery. we analyzed the host proteins from the hvppi for presence of idrs using the prediction software iupred ( ) . it is estimated that the human proteome more than , short linear binding motifs in idrs ( , ) . proteins with idrs are often signaling hubs and might form dynamic complexes with other proteins through specific motif based interactions ( ) . we found a statistically significant enrichment (p-value < . × − ) of idrs in the host proteins targeted by viruses (figure a and figure s ). we then identified the subnetwork in the hvppi which contained the top host targets with high disorderness score ( figure b) . the top five proteins with large idrs include cd antigen (cd ), serine/arginine repetitive matrix protein (srrm ), myristoylated alaninerich c-kinase substrate (marcks), bag family molecular chaperone regulator (bag ), and mitochondrial antiviralsignaling protein (mavs; figure c ). cd is a marker of exhausted cd + t cells ( ) and replication of hcv in t cells was shown to decrease cell proliferation by inhibiting cd expression and signaling ( ) . srrm is a serine/arginine-rich protein involved in rna splicing ( ) . srrm is differentially phosphorylated in hiv- infected cells and absence of srrm lead to increased hiv- gene expression, since it regulates the splicing of hiv- ( ) . in the hvppi, srrm is targeted by multiple viral proteins including the tat protein from hiv- . tat protein has an important role in the stimulation of the transcription of the long terminal repeat (ltr) ( ) . in addition, ns protein from influenza b virus has also been reported to interact with srrm ( ) . proteins of the marcks family are involved in a range of cellular processes including cell adhesion and migration ( ) . marcks is a negative regulator of lipopolysaccharide (lps)-induced toll-like receptor (tlr ) signaling in mouse macrophages ( ) . mavs is an adaptor protein in the rig-i signaling pathway involved in the sensing of rna. ablasser et al. ( ) reported that double-stranded dna serves as a template for rna polymerase ii and is transcribed into a ′ triphosphate containing double-stranded rna, which activates the rig-i signaling pathway. in the hvppi, mavs is targeted by several proteins from dsdna viruses such as ebv and hpv. altogether, our analysis shows that the idr-high part of the human proteome is an essential part of the viral evasion strategy and some of the selected targets highlighted here could show novel insights into the viral evasion mechanisms. however, the very flexible protein structure of disordered proteins also makes them also difficult to target with drugs. to assess the signaling pathways and cellular processes within the hvppi, we identified highly connected subnetworks within hvppi network. we constructed a host-host interaction network based on the host targets in the hvppi and identified a number of highly connected subnetworks/clusters (figure ) . we then performed a gene-set enrichment analysis of significantly enriched biological processes. we found one or more enriched processes for each of this subnetwork including core cellular processes such as proteasome, spliceosome, protein translation, protein/rna transport, and cell cycle. next we listed the viruses that target one or more of these processes, and found that almost all the core pathways and processes are targeted by all the viruses that are part of the hvppi (figure s ). this analysis highlights the core components of the cellular process subnetworks which are targeted as part of the viral evasion strategies and thus could be broad-spectrum antiviral hot-spots from a therapeutic point of view. given that the viral proteins were interacting with a large number of host proteins, we analyzed the sub-cellular location of the host proteins. we performed gene-set enrichment analysis of sub-cellular localization information provided by uniprot database. we binned the localization into compartments and estimated the percent of host proteins in a given compartment as compared to the total number of host proteins targeted by a given figure | clusters of hvppi involved in core cellular processes. network view of the "clusters" or highly-connected sub-networks and their associated cellular processes. each cluster is marked in a unique color. virus. we found that the viral targets were distributed across multiple subcellular compartments with cytoplasm being the most common ( figure s a ). the hvppi includes two different strains of iav-pr (h n ) and udorn (h n ). the subcellular localization analysis showed that both strains were enriched for nuclear proteins. non-structural protein (ns ) from both the strains had the highest number of nuclear targets but their targets were very different ( figure a) . ns of udorn was enriched for a large number of histones as compared to ns of pr that had large number of heterogeneous nuclear ribonucleoproteins (hnrnps), such as hnrnpu−a known restriction factor for many viruses. this corroborates with the observation that ns protein has short linear histone mimicry motifs that can suppress the host antiviral response ( ) . in our analysis, we found that it is ns of udorn that has a histone mimicry motif "arsk" (figure s b) . similarly, hpv and hpv e proteins interact more often with host proteins located in the endoplasmic reticulum (er). we found both common and specific subsets of er proteins targeted by the e protein ( figure b ). hpv e protein er targets were enriched for phospholipid biosynthesis as well as gpi anchor related proteins, such as phosphatidylinositol glycan anchor biosynthesis class s/t/u (pigs, pigt, and pigu), glycosylphosphatidylinositol anchor attachment (gpaa ) and phosphatidylserine synthase (ptdss ). hpv e protein er targets were enriched for er-associated ubiquitindependent protein catabolism involving host proteins such as er degradation enhancing alpha-mannosidase-like protein (edem ) and er lipid raft associated (erlin ). er targets common to hpv and hpv e protein were enriched for unfolded protein response, n-linked glycosylation and protein folding involving host proteins such as srp receptor alpha/beta subunit (srpra/srprb) and catalytic subunits of the oligosaccharyltransferase complex (stt a and stt b). two independent crispr/cas screening studies identified multiple er associated components including stt a and stt b as host factors for denv, zika virus (zikv) and japanese encephalitis virus (jev) ( , ) . the non-canonical function of stt a and stt b is required for denv replication and that ns protein of denv interacts with these proteins ( ) . our orthogonal approach can lead to the identification of critical host factors, and similar functions of er components, such as stt a and stt b, are used by hpv and hpv as well. thus, targeting the non-canonical function of stt a and stt b could be a broad antiviral strategy. overall, the enrichment analysis clearly shows that there is commonality and specificity in the subcellular targets of the viral proteins and that detailed interrogation of these targets can give vital clues into the viral evasion mechanisms. rnai screens have been a powerful high-throughput method to identify various cellular functions, including for identification of host restriction factors of viruses ( ) . in order to explore the functional relevance of the host targets in the hvppi, we integrated it with five published rnai screens that performed genome-wide or druggable-genome-wide rnai screens for identifying host factors of hcv ( ), hpv ( ), hpv ( ), sv ( ) , and vacv ( ) . we found that host targets from the hvppi were spread across the spectrum of genes with proviral as well as antiviral phenotype (figure s ) , thus showing that targeting of the host protein by the virus could lead into any direction that favors the virus. we then investigated the top proviral genes that are also targeted by the viral proteins as seen in the hvppi. we identified host proteins ( figure a ) that were significantly enriched for coatomer protein complex (copi), protein translation/transport and proteasome ( figure b) . this further substantiates the findings from the earlier section on the core cellular processes targeted by the viruses. network analysis of these top hits showed high level on connectivity and crosstalk-for example between the translation and proteasome machinery (figure c ). vesicle carriers are involved in the transport of membranes and proteins. copi system is one of the three vesicular carrier systems that is involved in the early secretory pathway ( ) ( ) ( ) . moreover, it has been pointed out that there is a strong similarity between vesicular transport and viral transport [viral entry to budding process, ( ) ]; thus making copi system important for the viral life cycle. in addition to the findings of the present study, sirna-based silencing of copi lead to a decrease in entry and subsequent gene expression of iav, vsv, lcmv, and hpiv and disruption of the copi complex inhibited the production of infectious progeny virus ( , ) . copi coatomer inactivation results in a direct decrease of vsv attachment and uptake, but not for membrane fusion or rnp release; however the direct mechanism remains unclear ( ) . altogether, these top hits including the copi system could serve as targets for developing therapeutic antiviral intervention strategies for a broad group of viruses. our analyses pointed out that viral evasion mechanism observed in one virus could also be relevant for other viruses. to test this, we obtained known drug-gene interactions from dgidb ( ). we selected investigational/experimental/approved antivirals compounds ( ) which had dgidb annotated targets that are part of the hvppi. we added agents as controls (table s and figure s ) . we tested broad-spectrumantivirals against gfp-expressing human metapneumovirus (hmpv) nl/ / strain ( ) . seven different concentrations of the compounds were added to hmpv or mock-infected cells. hmpv-induced gfp expression and cell viability was measured and after h to determine compound efficiency and toxicity. after the initial screening, we identified five compounds, which lowered gfp-expression without detectable cytotoxicity (with si > ). we repeated the experiment with these compounds. the experiments revealed novel activity of azacytidine, lopinavir, nitazoxanide, itraconazole, and oritavancin against hmpv ( figure s a and table , table s ). similarly, we examined toxicity and antiviral activity of broad-spectrum-antivirals against gfp-expressing hcv in huh- . cells using previously described procedures ( ) . our test identified azithromycin, cidofovir, oritavancin, dibucaine, gefitinib, minocycline, and pirlindole mesylate as novel anti-hcv agents with si > ( figure s b and table , table s ). in summary, our metaanalysis approach of the hvppi could provide novel and faster approaches for the re-purposing of existing drugs as antiviral agents. using integrative analysis of orthogonal datasets our study provides a comprehensive view of viral evasion mechanisms. in particular, our analysis of the hvppi network revealed that all the viruses have evolved to target proteins that are central and have strong control over the human interactome. host proteins targeted by viruses contain a high proportion of intrinsically disordered regions. we identified the core cellular processes and associated proteins that are targeted by all viruses. detailed comparative analysis of the subcellular localization of the host proteins showed commonality and specificity both between viral proteins from different strains of the same virus; and between viruses. integrating hvppi with functional rnai screens showed that % of the hvppi are host factors of one or more virus. hvppi data-based drug re-purposing screen identified novel activities for various broad-spectrum antivirals against hmpv and hcv. this unique dataset can be used for further detailed interrogation of the mechanisms behind viral evasion. this could serve as a starting point for identifying novel host targets and generating hypothesis in the context of viral evasion and development of pan-viral therapeutic intervention strategies. the methods described here also provide unique ways of dissecting the orthogonal datasets. various analyses from this study have highlighted the existence on pan-viral evasion points that could be utilized for the development of host-directed antiviral therapies. it is also intriguing to see that there is commonality and specificity at the level of sub cellular localization of the viral targets. our analyses have underlined some salient features in the context of iav, hpv, denv, and hcv. further detailed analysis in this context along with protein sequence features, such as short linear motifs [slims; ( ) ] would provide novel insights as well as deeper understanding of how small sequence features are involved in the hijacking of the host machinery. integration of such data with known drug-gene interactions provides a clear estimate of the druggable proportion in the hvppi. our meta-analysis approach of the hvppi could provide novel avenues of re-purposing existing drugs for antiviral targeting strategies. our meta-analysis approach of the hvppi could provide novel avenues of re-purposing existing drugs for antiviral targeting strategies. to prove the concept, we tested bsas against hmpv, hcv, sindbis virus (sinv), cytomegalovirus (cmv), and hepatitis b virus (hbv). importantly, bsas have dgidb annotated targets that are part of the hvppi, whereas were used as controls. these safein-man drugs have already been used as investigational agents or experimental drugs in different virus infections (table s ) . we demonstrated novel antiviral effects of azacytidine, itraconazole, lopinavir, nitazoxanide, and oritavancin against hmpv, as well as cidofovir, dibucaine, azithromycin, gefitinib, minocycline, oritavancin, and pirlindole against hcv. azithromycin, is an fda-approved antibiotic of the macrolide family. it is also an investigational agent against rsv and experimental agent against ebov, hrv-a, zikv, and rsv. cidofovir is an fda-approved anti-cmv drug. it is also investigational agent against adv, bkv, hpv, hsv- , hsv- , and experimental drug against b v. dibucaine is an fdaapproved amide local anesthetic. in addition, it is experimental anti-hev-a, hev-b, hev-d, and ebov agent. gefitinib is an fda approved anticancer drug. it has also antiviral activity against bkv, cmv, and vacv. minocycline is a broad-spectrum antibiotic and experimental anti-denv, hiv- , and wnv agent. oritavancin is a semisynthetic glycopeptide antibiotic used for the treatment of gram-positive bacterial skin infections. it also inhibits ebov, mers-cov, and sars-cov infections. pirlindole is an antidepressant, which is also experimental anti-hev-a, hev-b, and hev-d agent. azacitidine is a chemical analog of cytidine, which is used in the treatment of myelodysplastic syndrome. it is also an experimental anti-adv, the measurements were repeated three times (p < . ). fluav, rvfv, hiv- , and hiv- agent. itraconazole is an antifungal medication. it is also used as experimental anti-hev-b, hrv-b, hrv-a, par-a , and safv agent. nitazoxanide is a broad-spectrum antiparasitic drug, which is also investigational agent against fluav and hcv and experimental anti-chikv, rsv, hbv, hiv- , vacv, rv, jev, mers-cov, nov, ruv, and zikv agent. lopinavir is an fda-approved antiretroviral of the protease inhibitor class. it is also investigational anti-mers-cov and experimental anti-zikv agent (table s ). in addition to inhibition of viral proteases (table s ) , lopinavir was reported to induce host rnasel production in infected and non-infected cells ( ) . rnasel is endoribonuclease that is a part of interferon (ifn) antiviral response, which is the most critical node of virus-host interactions. although, the antiviral mechanisms of action of other compounds are still unknown, these agents could inhibit steps of viral infections, which precede reporter protein expression from viral rna. in summary, our results indicate that existing bsas could be re-purposed to other viral infections. to further expand a spectrum of their activities, these bsas could be tested against other viruses. re-purposing these and other safe-inman antiviral therapeutics could save resources and time needed for development of novel drugs to quickly address unmet medical needs, because safety profiles of these agents in humans are available. effective treatment with broad-spectrum-antivirals may shortly become available, pending the results of further pre-clinical studies and clinical trials. this, in turn, means that some broad-spectrum-antivirals could be used for rapid management of new or emerging drug-resistant strains, as well as for first-line treatment or for prophylaxis of acute virus infections or for viral co-infections. the most effective and tolerable compounds could expand the available therapeutics for the treatment of viral diseases, improving preparedness and the protection of the general population from viral epidemics and pandemics. all datasets used for this study are accessible as stated in the materials and methods section . . we thank christian sinzger and group for the egfp-expressing tb e cmv strain. we thank vironovative and erasmus mc for the gfp-expressing hmpv nl/ / strain. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material redefining chronic viral infection antiviral drug resistance as an adaptive process emerging virus diseases: can we ever expect the unexpected? emerg microbes infect herpes simplex virus resistance to acyclovir and penciclovir after two decades of antiviral therapy mechanisms of viral mutation complexities of viral mutation rates host-directed therapies 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jalalvand, farshid; thegerström, john; riesbeck, kristian title: the interplay between immune response and bacterial infection in copd: focus upon non-typeable haemophilus influenzae date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: yfh d io chronic obstructive pulmonary disease (copd) is a debilitating respiratory disease and one of the leading causes of morbidity and mortality worldwide. it is characterized by persistent respiratory symptoms and airflow limitation due to abnormalities in the lower airway following consistent exposure to noxious particles or gases. acute exacerbations of copd (aecopd) are characterized by increased cough, purulent sputum production, and dyspnea. the aecopd is mostly associated with infection caused by common cold viruses or bacteria, or co-infections. chronic and persistent infection by non-typeable haemophilus influenzae (nthi), a gram-negative coccobacillus, contributes to almost half of the infective exacerbations caused by bacteria. this is supported by reports that nthi is commonly isolated in the sputum from copd patients during exacerbations. persistent colonization of nthi in the lower airway requires a plethora of phenotypic adaptation and virulent mechanisms that are developed over time to cope with changing environmental pressures in the airway such as host immuno-inflammatory response. chronic inhalation of noxious irritants in copd causes a changed balance in the lung microbiome, abnormal inflammatory response, and an impaired airway immune system. these conditions significantly provide an opportunistic platform for nthi colonization and infection resulting in a “vicious circle.” episodes of large inflammation as the consequences of multiple interactions between airway immune cells and nthi, accumulatively contribute to copd exacerbations and may result in worsening of the clinical status. in this review, we discuss in detail the interplay and crosstalk between airway immune residents and nthi, and their effect in aecopd for better understanding of nthi pathogenesis in copd patients. the lungs are vital organs involved in gas exchange between the vascular system and the external environment, thus they are greatly exposed to the environment-derived microorganisms, including fungi, viruses, and bacteria. the bronchial tree and parenchymal tissues of the lungs, that until recently were considered as sterile, are colonized by phylogenetically-diverse microbes. the genera of firmicutes, bacteroidetes, and proteobacteria are the most common phyla identified and represent % of the total bacterial microbiome in the healthy airway ( , ) . the majority of the lung microbiota belongs to the normal flora that play an important role in the pulmonary epithelial integrity, colonization resistance, and homeostasis of the immune system in the respiratory tract ( ) . a small fraction of them are, however, potentially pathogenic microorganisms that are involved in a variety of lung diseases, as exemplified by the genus haemophilus. non-typeable haemophilus influenzae (nthi) is a gram-negative coccobacillus that are commonly residing in the human airways. uniquely and yet unexplained, nthi is a commensal when colonizing the nasopharynx or throat, but pathogenic in the lower airways triggering a robust inflammatory response [for reviews see ( , ) ]. nthi is considered a potential opportunistic pathogen as it frequently infects the lower respiratory tract of lungs with structural damage as a consequence of non-infectious lung diseases or mechanical injuries. moreover, nthi occasionally causes bronchitis and pneumonia ( ) . in addition, lower airway colonization by nthi has been associated with disease progression of several more or less non-infectious lung diseases such as bronchiectasis ( ), cystic fibrosis ( ) , interstitial lung diseases ( , ) , but mostly in chronic obstructive pulmonary disease (copd) ( , ) . copd is a severe inflammatory lung disease characterized by airflow limitation with a range of pathological changes. both genetics and environmental factors trigger the onset of copd, however, microbes including nthi play an important role in the acute exacerbations. this review describes the disease progression of copd in the context of host immuneinteractions linked to nthi, and the overall impact in disease exacerbation. copd is the third leading cause of morbidity and mortality worldwide expected to affect more than million people by ( , ) . according to the global initiative for chronic obstructive lung disease (gold), copd is a pulmonary disease that is manageable, but significant exacerbations and co-morbidities may, however, contribute to the overall severity in individual patients ( ) . copd is characterized by chronic airflow limitation of the peripheral airways with a range of pathological changes in the lung that are not fully reversible, and usually become progressively worse over time. the progression of copd is associated with an abnormal inflammatory response of the lung to noxious particles or gases. from a pathological point of view, copd comprises a group of pulmonary abnormalities related to the inflammatory reaction of the airways, alveoli, and pulmonary vessels ( ) ( ) ( ) ( ) . these include (i) pulmonary emphysema, (ii) chronic bronchitis, and (iii) disease in the small airways. the pulmonary abnormalities progressively affect all parts of the lung, resulting in increased resistance of the conducting airways and thus chronic airflow obstruction that eventually will lead to a declined lung function. emphysema is a permanent loss of elastic lung recoil caused by elastolytic destruction and enlargement of the alveolar wall distal to the terminal bronchioles. this consequently results in the loss of alveolar attachments to the small airways and thus limitation of airflow and gaseous exchanges. chronic bronchitis is characterized by consecutive and chronic cough with expectorations that last for more than months within years. it is associated with inflammation of the bronchial walls with increased inflammatory infiltrates, hyperplasia of goblet cells, hypertrophy of tracheobronchial submucosa, increased mucous secretion and, finally, dilatation of the airway ducts (airways of about - mm in internal diameter). the majority of the ciliated epithelium lining the airways are also either compromised or dysfunctionnal, and may be replaced by nonciliated squamous epithelial cells. small airway diseases, on the other hand, involve hyperplasia and metaplasia of mucosal glands and goblet cells, hypersecretion of intraluminal mucus, macrophage bronchiolitis, and accumulation of lymphocytes in the small bronchioles (airways of ∼ mm or less in diameter and terminal bronchioles). in addition, distortion, fibrosis, stenosis, tortuosities, hyperplasia, and hypertrophia of the small airway smooth muscles also contribute to the loss of elasticity in the lung parenchyma. although copd mainly affects the lungs, it also produces significant extrapulmonary consequences as a results of an escalated inflammatory response orchestrated by airway cells and immune mediators ( , ) . the comorbidities are commonly seen in copd patients despite the actual mechanism responsible for the systemic inflammation remains to be elucidated. the development of copd is multifactorial, with cigarette or tobacco smoking being the primary cause of copd ( , ) . other risk factors that may promote the onset and progression of copd includes prolonged occupational exposure to particles/gases in mining and textile industries, air pollution resulting from biomass combustion, and bronchial hyperresponsiveness ( , , ) . the variability of copd incidences among smokers is also explained by a genetic predisposition, such as α -antitrypsin deficiency and cutis laxa [mutation of the elastin gene (eln)] ( , ) . the α -antitrypsin deficiency is caused by deleterious homozygous mutations in serpina which contributes to - % of copd cases. the deficiency results in increased neutrophil elastase activity that ultimately leads to the degradation and collapse of the alveoli. importantly, meta-analyses of genome-wide association studies (gwas) and other genotyping studies have revealed that multiple single nucleotide polymorphism (snps) in at least genes from different pulmonary genomic loci are associated with copd susceptibility ( ) ( ) ( ) ( ) . airway epithelium exposed to cigarette or tobacco smoke has compromised tight junctions and delayed epithelial wound repair ( ) ( ) ( ) ( ) . moreover, cigarette smoke alters basal cell differentiation and subepithelial extracellular matrix (ecm) composition, and thus causes airway remodeling (i.e., goblet cell hyperplasia and small airway squamous metaplasia) ( ) ( ) ( ) . this results in mucus hypersecretion, impaired mucocilliary clearance, and airway obstruction. tobacco or cigarette smoke also enhances proliferation and ecm deposition by activating the extracellular signal related kinase (erk) and the p signaling pathway ( ). the alteration of major ecm components are widespread in all lung compartments in copd patients with a total increase of type i and iii collagens, fibronectin, and laminin in parallel with reduced concentrations of proteoglycans, perlecan decorin, versican, biglycan, tenascin and elastin ( , ). cigarette induced-overexpression of matrix metalloproteases (mmps- , , , , , and ) and elastase has also been reported, and may contribute to the airway tissue destruction and fibrosis ( - ). in addition, harmful volatile chemicals derived from cigarette smoke (i.e., acetaldehyde, acrolein, and crotonaldehyde) are prone to form carcinogen adducts with dna and various proteins (i.e., apoliprotein e and surfactant protein a). they also dysregulate airway epithelial ion transport, disrupt the phagocytic activity of airway phagocytes, and diminish the airway surface liquid volume ( - ). numerous proteomics and transcriptomic analyses have unveiled the crucial impact of cigarette or tobacco smoke and copd disease progression on airway gene expression ( , ). the differential gene expression studies were done using copd experimental models or clinical samples [i.e., bronchial epithelial cells, sputum, plasma, blood, and bronchoalveolar lavage (bal) fluid]. collectively, most of the altered genes are involved in oxidative stress, xenobiotic metabolism, antioxidant responses, dna repair, ecm remodeling, inflammatory responses, and immune defenses, which the latter two are our major interest of discussion in this review. the omics data aid in the increased knowledge of molecular mechanisms in copd. they may reflect the dynamic response and attempts by the airway epithelial cells to repair the cytotoxic injury primarily triggered by inhaled irritants. deleterious and irreversible alterations occurring and interfering with the airway epithelial homeostasis and immune defense may promote copd development and progression. notably, gene alterations in phagosomal-and leukocyte transendothelial migration pathways (ltm) are significantly correlated with the level of t cells and airway obstruction in smokers ( ). the ltm, however, were found to be further dysregulated in copd patients. hence, in addition to clinical/physiology variables, a number of gene products with significant differential gene expression may be targeted as specific proteomic signatures or biomarkers for early copd detection, patient monitoring, disease subgrouping, and finally treatment selection ( , ). tobacco or cigarette smoke regulates airway gene expression via two main mechanisms, by altering the status of (i) chromatin remodeling, and (ii) dna methylation of the target genes (figure ) ( - ). chromatin remodeling is a result of a disrupted balance in histone acetylation/deacetylation ( ). excessive activation of more than transcription factors including nf-κb, and lipoprotein peroxidation products (peroxinitrite, acrolein, and hne from tobacco smoking) contributes to such anomaly. nf-κb is a key inflammatory and redox-sensitive transcription factor that plays a direct role in cigarette smoke-induced airway inflammation. nf-κb has been described as a "smokesensor" due to its sensitive activation by tobacco residues ( ). stimulation of multiple signaling cascades [p mitogenactivated protein (mapk) kinases, mitogen and stress-activated kinase (msk ), protein kinase c zeta (pkcζ), and iκb kinase (ikk) complex (ikkα, ikkβ, and nemo)] by tobacco residues promotes the activation and nuclear translocation of transcription factor nf-κb rela/p ( , - ). this is followed by a complex formation of nf-κb/cbp-p [coactivator, crebbinding protein (cbp) or cbp/p ] at target dna sequences. it should be noted that cbp/p also has intrinsic histone acetyltransferase (hat) activity. subsequent acetylation and phosphorylation of the subunit p in the nf-κb/cbp-p complex by the activated msk /pkcζ-signaling pathways (and other different kinases), and cbp/p , respectively, are required for the full activation of nf-κb ( , , ). this enhances the dna binding affinity of the complex. histones h and h in the chromatin complex of target sequences are then being acetylated (histone h at lys ; h at lys and lys ) and phosphorylated (histone h at ser ) by the subunit cbp of the nf-κb/cbp-p complex, and the activated msk and pkcζ, respectively. the hyperacetylated core histones, however, fail to be neutralized or deacetylated by a dysfunctional histone deacetylase (hdac ). peroxinitrite nitrates the tyrosine residues of the hdac and causes inhibition of activation and reduced expression of the protein. of note, peroxinitrite is a by-product generated from the immune cell-derived nitrite oxide (no) and reactive oxygen species (ros) of cigarette smoke ( , ). cigarette or tobacco smoke disturbs the dna methylation status of target genes through several mechanisms. firstly, dna damage caused by cigarette smoke stimulates the dna methyltransferase (dnmt) to actively induce cpgs methylation at the damaged site ( ). the hypermethylation is prone to introduce error of methylation in some target genes, resulting in reduced gene expression. secondly, activation of nicotine signaling pathway by tobacco smoke causes camkii/iv and erk/mapk pathway activation that subsequently induces the activity of cbp to suppress the expression of dnmt . this may result in reduced dna methylation and thus altered level of gene repression by dnmt ( - ). finally, enhanced activities of transcription factors such as hypoxia inducible factor due to the high level of carbon monoxide and hypoxia have also been reported to influence airway gene expression ( ) . consequently, the combinatorial effect from both aberrant acetylation of histone and dna methylation promotes the transformation of chromatin from a condensed structure to an activated open conformation. this facilitates irregular accessibility of dna for transcription machineries, hence figure | cigarette and tobacco smoke has several effects on gene regulation. nicotine and other compounds in the smoke alter gene expression by two pathways, firstly, chromatin remodeling (left) and secondly, dna methylation (right). chromatin remodeling involves activation of kinases signaling pathways, activation and nuclear translocation of transcription factor nf-κb (rela/p ), and complex formation with cbp/p on specific dna sites. cbp/p is intrinsically a histone acetyltransferase (hat). subunit p is further phosphorylated at ser and ser , respectively, by msk and pkcζ, whereas cbp acetylates p at lys . the phosphorylation and acetylation enhance the interaction within the nf-κb/cbp/p complex while stabilizing the dna binding of nf-κb. the complex of nf-κb/cbp/p then modifies the histones through cbp-mediated acetylations of histone h (at lys ) and h at lys and lys , and phosphorylation of h at ser by msk and pkcζ. this results in the structure change of chromatin, from a condensed structure (repressed) to an activated open conformation. the transcription of target genes is therefore increased. in the second mechanism, several side effects resulting from cigarette smoking such as dna damage and nicotine signaling could trigger the hypermethylation or decreased methylation of target dna. this may lead to dna methylation anomalies and thus altered dna expression. resulting hypoxia due to high concentrations of carbon monoxide also contributes to altered gene expression. the aberrant gene expression by cigarette smoke mostly occurs in pro-inflammatory genes with resulting increased production of inflammatory mediators, and amplified inflammation in the copd lung upon exposure. irregular gene expression by various cell types in the airway. the mechanisms reported are responsible for increased expression of nf-κb-dependent proinflammatory gene products [i.e., il- β, il- , il- , ccl- cyclooxygenase (cox)- , and mip- /cxcl ] in both pulmonary structural cells (bronchial, small airway, and alveolar epithelial cells) and immune cells (alveolar macrophages), increased vegf and inos in nasal fibroblasts and lymphocytes (jurkat t cells), respectively, and decreased activity of antioxidant transcription factor nrf and α -antitrypsin in bronchial epithelial cells ( , , , , - , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . these may contribute to the anatomical anomalies in the airway and excessive inflammatory responses among smokers during the course of copd. copd is associated with chronic inflammation in the peripheral airways orchestrated by both innate and adaptive immune responses that are interconnected via dendritic cells ( ) . increasing numbers of inflammatory cells (neutrophils, macrophages, t and b lymphocytes, mast cell, eosinophils, and dendritic cells) and inflammatory mediators are accumulated in the airway lumen/wall in the lung parenchyma ( , ) . these immune cells and inflammatory mediators can hence be detected in the sputum and bal fluid of copd patients. the level of accumulation is positively correlated with disease severity. an increasing number of studies using animal models and clinical tissues have reported the nature of excessive airway inflammatory responses in copd. despite this, the heterogeneity in symptoms progression among copd patients remain unexplained. the overall mechanism of copd inflammatory immune response is depicted in figure . the first line of defense in the lung-the innate immunity and inflammasome lung structural cells (epithelial and endothelial cells, fibroblasts, and airway smooth muscle cells) are activated by inhaled irritants through the stimulation of several pattern recognitions receptors (prrs), with toll-like receptor (tlr)- being reported as the key player in most of the inflammatory responses ( ) ( ) ( ) ( ) . this causes an increased expression and release of an array of pro-inflammatory mediators and chemokines through the oxidative pathway by the activated bronchial epithelial cells and immune cells (alveolar macrophages). the inflammatory mediators [(interleukin (il)- β, il- , il- , il- , c-x-c motif chemokine ligand (cxcl) , granulocyte-macrophage colonystimulating factor (gm-csf), granulocyte-colony stimulating factor (g-csf), tumor necrosis factor (tnf)-α, fibroblast growth factor and (fgf / ), transforming growth factor (tgf)-β , c-c motif chemokine ligand (ccl) , ccl , and thymic stromal lymphopoietin (tslp)] act on recruited immune cells and resident cells to initiate a series of innate immune responses ( , ( ) ( ) ( ) ( ) ( ) . meanwhile, activated alveolar macrophages, which are usually patrolling the lung parenchyma, further release more pro-inflammatory mediators and chemokines [il- β, il- , il- , il- , tnf-α, ccl , cxcl , cxcl (ena- ), cxcl , cxcl , cxcl , ccl , leukotriene b (ltb )], ros, elastolytic enzyme [matrix metalloprotease protein (mmp)- ,− , and− ; and cathepsin-k,-l, and -s], gm-csf, and g-csf ( , , ) . the enhanced levels of ccl and cxcl result in recruitement of blood monocytes expressing ccr and cxcr (receptors for ccl and cxcl , respectively), to the lung and differentiate locally into macrophages. interestingly, there are higher expression levels of the ccr and cxcr found on blood monocytes in copd subjects ( ) . this may explain the rapid recruitment and excessive accumulation of monocyte-derived interstitial macrophages in the lung tissue of copd patients ( , ) . upregulation of neutrophil chemoattractors (ltb , cxcl , cxcl , il- , and tnf-α) induces a massive migration of circulating neutrophils into the lung parenchyma ( ) . the transmigration of blood neutrophils occurs through adherence of the granulocytes to e-selectin of endothelial cells that is found to be upregulated in copd ( ) . this results in airway neutrophilia in several copd patients ( , , ) . the recruited neutrophils (to the lung) are then activated to secrete granule proteins [myeloperoxidase (mpo) and neutrophil lipocalin] while releasing its own il- for further neutrophilic recruitment and amplification of the inflammation ( ) . in addition to the macrophage-derived proteases, neutrophils also secrete serine proteases [neutrophil elastase (ne), cathepsin g, proteinase- , mmp- , and mmp- ] that are associated with serious alveolar destruction in emphysema ( ) . the protease activity may be further enhanced in conditions with genetic deficiencies or suppressed expression of α -antitrypsin by tobacco smoke. in addition, ne, cathepsin g, and proteinase- are involved in the stimulation of mucus secretion from submucosal glands and goblet cells, resulting in airway mucus hypersecretion and airway obstruction in copd ( ) . the nlrp (nlrp : nucleotide-binding domain, leucinerich-containing family, pyrin domain-containing- or nod-like receptor protein ) inflammasome is a cytosolic multi-protein complex (consisting of the inflammation sensor protein nlrp , adapter protein asc, and the effector protein caspase- ) ( ). the nlrp inflammasomes are involved in the copd airway inflammation by regulating the production of pro-inflammatory cytokines il- α, il- β, and il- . these cytokines are important for neutrophil survival and activation of t helper (th) cells ( ) . interestingly, local airway nlrp inflammasome activation is positively correlated with acute exacerbations and lower airway microbial colonization in copd patients ( , ) . moreover, in an elastase-induced emphysema model, the nlrp inflammasome is activated in addition to hyperproduction of mucin muc ac by diesel extract particles, extracellular atp, and inflammatory protein s ( , ) . the adaptive immunity is initiated at a later stage, and is recognized by the increased number of t and b lymphocytes and pulmonary dendritic cells. dendritic cells are the major antigen-presenting cells (apc) in the airways, and link the innate and adaptive immunity. circulating dendritic cells (expressing receptors ccr and ccr ) are recruited to the airway via dendritic chemoattractants ccl and ccl released by activated airway epithelial cells in response to cigarette smoke ( , ) . dendritic cells act by endocytosis of inhaled irritants that subsequently are processed into antigen peptides during maturation and further migration to lymph nodes. uncommitted t lymphocytes are thereafter primed by the presented antigen. these important cells are activated by il- released from dendritic cells for subsequent commitment into antigen-specific t cell lineages, i.e., t helper (th ; cd + cd + ) cells, whereas immature dendritic cells in the airway promote th differentiation ( , ) . interestingly, in copd patients, pulmonary th and cytotoxic t cells (tc; cd + cd + ) express more cxcr receptors compared to healthy individuals ( , ) . this enhances their migration toward chemoattractants cxcl , cxcl , and cxcl that are actively released by alveolar macrophages in copd subjects. activated cd + t cell subset type (tc ) releases perforins, granzyme b, and tnf-α to induce alveolar cells apoptosis, contributing to the emphysema ( ) . in parallel, pulmonary th t cells are activated by alveolar macrophage-derived il- and il- to secrete il- a and il- causing neutrophilic inflammation ( , ) . inflammatory cytokines are also released by type figure | non-typeable h. influenzae-dependent immune responses in the lower airway of copd patients result in inflammation. airway epithelium exposed to cigarette or tobacco smoke display an increased permeability with compromised tight junctions, and airway remodeling (goblet cell hyperplasia and small airway squamous metaplasia). cigarette smoke causes the activation of airway epithelium and alveolar macrophages. the activated airway structural and resident immune cells release an array of chemotactic factors responsible for recruitment of inflammatory and immune cells to the lung. activated epithelium produces tgf-β and fgf that triggers the production of ecm molecules by fibroblasts. increased deposition of ecm causes progression of fibrosis and air flow limitation. the chemokines cxcl and il- , and ltb attract the circulating neutrophils through the receptors cxcr and blt , respectively. meanwhile, cxcl and ccl targeting the receptors cxcr and ccr on blood monocytes are also released. recruited blood monocytes differentiate into macrophages in the airway tissue. activated alveolar macrophage and epithelium cell also release inflammasome ( l- β and il- ) for neutrophils survival and activation of helper t cells th . the chemokine il- are released by macrophages to attract t helper cell subset th , and ilc . both th and ilc will release il- and il- that will act on the alveolar epithelium to release cxcl and il- for enhanced recruitment of neutrophils, resulting in neutrophilic inflammation. activated neutrophils are thereafter degranulated and release myeloperoxidase (mpo), lipocalin, neutrophil elastase (ne), cathepsin-g (cg), proteinase- (prot- ), and matrix metalloprotease (mmp) and . the granulated products are proteolytic and elastilolytic to aveolar, causing alveolar destruction and emphysema. in addition, ne, cg, and prot- are also targeting goblet cells and submucosal glands to induce hypersecretion of mucus. dendritic cells carrying the receptors ccr and ccr are recruited to airway tissue via chemottractants ccl and ccl . the dendritic cells uptake the antigen (smoke residues), and present the antigens to the naïve t cells at lymph nodes. uncommitted t lymphocytes are thereafter primed to the presented antigen and activated by il- derived from dendritic cells (professional antigen presenting cells; apc). mature/activated t cells expressing receptor cxcr are chemotactic toward cxcl , cxcl , and cxcl and are recruited to the lung tissue. cytotoxic cd + t cell subtype tc releases perforin and granzyme b resulting in epithelial apoptosis contributing to emphysema progression. for the humoral immune response, b cells are activated by th , enter the circulation via high-endothelial venule (hev)-like vessel and transported to lung tissue, and organized into lymphoid follicles at peripheral airway. b cell-derived plasma cells from lymphoid follicles release iga, and secreted into airway lumen as secretory iga (siga) via the polymeric immunoglobulin receptor. mucosal antibodies play an important role to eradicate pathogens and noxious antigens via immune exclusion. however, the airway defense by siga is diminished by nthi iga protease that degrade the antibodies. tlr and tlr of the airway phagocytes and epithelium following exposure to cigarette smoke are not responding to p and los of nthi. this results in defective phagocytosis and delayed bacterial clearance from the airway. the suppressed tlr induction in t cells has also lead to th predominant immune response, with low production of ifn-γ and reduced t cell-mediated immune killing of nthi. moreover, nthi downregulates foxp of tregs and thus impairs the anti-inflammatory/pro-inflammatory balance of tregs. the extensive immunosuppressive activity by tregs diminishes the response of effector t to (continued) figure | nthi stimulation. lastly, plasma cells from copd patients fail to produce nthi-specific antibodies and compromised immunoglobulin class switching. the impairment of the host immune response in copd toward nthi infection are labeled in blue. in total, nthi infection in copd lung adversely reduces the production of il- β, il- , il- , cxcl- , il- , tnf-α, antimicrobial peptide (amp), and ifn-γ. this may explain the inefficient eradication of airway pathogens in copd patients whereby persistent nthi infection concomitantly escalates the inflammation and thus exacerbation in copd. innate lymphoid cells (ilc ) ( ) . the ilcs are involved in the homeostasis of lung immunity and are regulated by epithelially produced il- and tslp ( , ) , and are further stimulated in response to cell damage. the accumulation of b lymphocytes in the peripheral airway and within lymphoid follicles is associated with airway autoimmunity in the progression of copd ( ). airway tissue damage in conjunction with impaired t-regulatory cells (tregs), both related by cigarette smoke, contributes to the formation of autoantibodies against airway components. autoantibodies against elastin, epithelial, endothelial, carbonylated, and citrullinated proteins are found in the circulation of copd patients ( ) ( ) ( ) ( ) ( ) ( ) . the generation of autoantibodies might activate plasma exudate-derived complement components resulting in a chronic inflammation, and consequently damage of the airways with emphysema progression ( ) ( ) ( ) ( ) . from a physiological point of view, a modulated inflammatory process is important for a protective and optimal immune response. however, the prolonged airway inflammation in copd as a results of impaired homeostasis leads to serious side effects since it amplifies the tissue damage and impairs the local immune defenses. the abrogated local immune system may make the airways of copd patients susceptible for opportunistic or recurrent infections by viruses and bacteria that in turn might exacerbate the disease. acute exacerbations of copd (aecopd) are episodes of acute symptom worsening that usually are associated with both respiratory (increased airway inflammation) and non-respiratory (system inflammation/co-morbidities) effects ( ) ( ) ( ) . the typical symptoms of an aecopd include increased production of purulent sputum, dyspnea, cough, wheezing, and symptoms of a cold that may last from days up to weeks ( , , ) . it commonly occurs in patients with advanced copd and results in additional therapy based on the level of exacerbations. exacerbations are classified in three levels according to gold. there is the mild disease that can be treated with short acting bronchodilators (sab); moderate disease with sab combined with antibiotics and/ or oral corticosteroids; and finally severe exacerbations with acute respiratory failure which requires emergency room visit and eventually hospitalization ( , ) . aecopd is a complex yet multifactorial consequence of copd. most of the exacerbations could be triggered by infectious (up to %) or non-infectious agents (∼ %) (aecopd with known etiology), whereas up to % of cases are of unknown etiology ( , ) . respiratory tract infections are the major causes for aecopd with known etiology and are mainly attributed to infections by viruses, bacteria, and atypical bacteria (not detected with conventional gram-staining) ( , , ) . non-infectious causes of aecopd include air pollution, environmental factors, meteorological effects, and comorbidities of the patients, all of which are partially contributing to copd exacerbations ( , , ) . respiratory viral infections are often the primary cause in the infection-dependent aecopd, and virus was identified as single or multiple infecting strains from up to % of copd patients with exacerbations recorded between years - ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the most common infecting viruses are, by far, human rhinovirus, influenza virus a, and respiratory syncytial virus, whereas parainfluenza virus, coronavirus, echovirus, human metapneumovirus, and adenovirus are considerably rare. bacterial infections contribute to an average of % of infective acute exacerbations with a prevalence being reported ranging from to % ( , , ( ) ( ) ( ) . the most commonly pathogenic bacterial species isolated from the lower airway of copd patients during aecopd are nthi, moraxella catarrhalis, streptococcus pneumoniae, staphylococcus aureus, pseudomonas aeruginosa, and klebsiella pneumoniae ( , , , , ( ) ( ) ( ) ( ) . it has been suggested that infection with new strains of the infecting species, rather than a new species, is highly associated with an increased risk of exacerbation ( , , ) . atypical bacteria that cause exacerbations are chlamydia spp., legionella pneumophilia, and mycoplasma spp. in contrast to viral infections that are diagnosed in - % of copd patients with stable disease and increase to . - % during copd exacerbations, bacterial colonization in the airways are more common with the same species during both stable disease ( - %) and exacerbations ( . - %) ( , , , , , ( ) ( ) ( ) ( ) . hence the precise or direct role of bacterial infection as the primary cause in triggering aecopd remains controversial although a significantly increased bacterial load is observed during exacerbation in several patients. this further suggests that bacteria might be more involved as secondary invaders after an initial viral infection. viral infections have been reported to cause several physiological changes in the lung that in turn facilitates secondary bacterial invasion. the mechanism of bacterial superinfection has been described for h. influenzae, s. pneumoniae, s. aureus, and many other airway pathogens ( ) ( ) ( ) . firstly, viral infections destroy the tight junctions of the airway epithelial barrier while inducing epithelium apoptosis. this results in the onset of airway epithelium lining repair whereby the sloughed off dead cells would become a rich nutrient source for growth of infecting bacteria. the damaged epithelium lining also enables bacterial adherence to the exposed basement membrane and ecm. secondly, the demolished ciliated clearance as a result of the virus-damaged airway epithelium lining further promotes bacterial colonization and subsequent epithelial transmigration into deeper tissues ( ) ( ) ( ) . lastly, viral infections are also detrimental to the airway immune defense by causing degradation of antimicrobial peptides (amp), and by triggering ifn-γ secretion by immune cells. this results in suppressed macrophage and neutrophil responses to infecting bacteria, and thus enables bacterial evasion of the airway immune defense ( ) ( ) ( ) ( ) . nevertheless, viral and bacterial coinfection have greater impact in the aecopd airway inflammatory responses than bacteria or virus infection alone ( , ) . this is in parallel with the co-isolation of both respiratory viruses and bacteria from to % of aecopd patients ( , , , ( ) ( ) ( ) ( ) ( ) . infective aecopd is also attributed to impaired functions of amp, macrophages, and neutrophils triggered by inhaled irritants such as tobacco smoke. expression of microbialinduced amp (human β-defensin ) is suppressed in airway epithelial cells when exposed to cigarette smoke ( , ) . both the alveolar and monocyte-derived macrophages in patients with copd are defective in phagocytosis of bacteria such as h. influenzae and s. pneumoniae ( , ) , and in efferocytosis of apoptotic neutrophils and epithelial cells. in addition, neutrophils from copd patients are aberrant in chemotactic response with defective accuracy ( ) . all these factors contribute to the failure to resolve inflammation in copd leading to facilitated chronic microbial colonization, also during exacerbations. the low number of cultivable bacteria found in healthy individuals previously led to the conclusion that healthy and normal lungs are virtually sterile. this hypothesis is currently being revised, since the introduction of s rdna based molecular diagnostics has shown that even healthy lungs have a distinct microbial community, different from that seen in the upper respiratory tract ( , ) . this has led to the concept of a core human lung microbiome which can be altered in copd stable disease and during exacerbations ( ) . the role of the lung microbiome in the pathogenesis of copd by influencing host immune response has also been suggested ( , ( ) ( ) ( ) ( ) ( ) ( ) . the stability of the lung microbiome has profound impact on maintaining local immune homeostasis ( ) . according to the "vicious circle" hypothesis, airway inflammation and impaired immune defenses caused by either viral infections or irritant inhalation have ecological influence on the airway microenvironment and growth conditions that would eventually lead to dysbiosis of the lung microbiota ( , ) . the changed lung microbiome would then cause a maladaptive immunological response resulting in further inflammation and damage of the lung immune defenses, and additional alteration of the lung microbiome. the chain of events thus generates a vicious circle that contributes to copd progression and exacerbation. several studies have documented that copd progression from stable state to an exacerbation could induce microbiota shift in the lower airway (bronchioles), sputum, and throat ( , ( ) ( ) ( ) ( ) ( ) ( ) ( ) . alteration in the microbiome complexity or richness is associated with the inflammatory process and changes in ecm protein expression in the lung, as observed in copd ( , ) . declined diversity in the lung microbiome has been reported to be related to disease severity, inflammation and decreased lung functions in copd. this includes the increased emphysematous destruction, bronchial tissue remodeling, lymphoid follicle formation, elevated autoantibodies, and il- a production, and finally increased neutrophil extracellular traps (net) formation in the airway of animal models or aecopd patients ( ) ( ) ( ) ( ) . it has recently been reported that lung microbiome diversity is also associated with genetic factors. mannose-binding lectin (mbl) deficiency has also been associated with disease severity and exacerbations in patients with cystic fibrosis and bronchiectasis ( ) . however, copd patients with a genetic deficiency in mbl are less susceptible to haemophilus spp. colonization, lowering the risk of exacerbations while their lung microbiota is more diverse than normal copd patients ( ) . in this review we will focus on nthi, one of the dominant genera that is relatively abundant in the total copd-dependent lung microbiome, due to its role of infection in copd immunological responses ( , , - , , - ) . the microbiology of h. influenzae has recently been reviewed in detail by our group and others ( , , ) . it is a gramnegative coccobacillus that commonly colonizes the human nasopharynx, and is typed as capsulated (type a-f) or nonencapsulated strains (nthi). h. influenzae may cause both invasive and mucosal disease ( ) . since the introduction of capsule polysaccharide conjugate vaccines against type b (hib), nthi dominate, followed by capsule type f (hif) ( , ) . mucosal infections, including acute otitis media, sinusitis, and exacerbations in copd, are nowadays mainly associated with nthi. there has also been a significant shift in the epidemiology of severe invasive disease, from hib infections in small children to nthi in adults ( , ) . the most common principal infection focus by h. influenzae is now community acquired pneumonia (cap), whereas the incidence of historically common diagnoses such as meningitis and epiglottitis have significantly decreased ( , ) . patients with underlying conditions, notably copd, seem to be at higher risk for invasive infections ( ) . there is consensus that h. influenzae is one of the key bacterial pathogens involved in pathogenesis of both stable copd disease and acute exacerbations ( ) . however, the relative abundance and significance of nthi in copd varies between different studies. several factors, such as sampling methodology, choice of microbiological analysis and, if the patient has a stable disease or an exacerbation, or has been subject to previous antibiotic therapies, tend to affect the outcome of the studies ( ) . common sampling methods from the lower respiratory tract include both bronchoscopy techniques such as protected specimen brush (psb) and collection of bal fluid as well as non-invasive methods like sputum sampling ( ) . all of these methods, particularly sputum, are to some extent subject to the risk of contamination from the normal microbial flora of the oro-and nasopharynx, which might reduce their specificity ( ) . however, several studies still show a distinct association between lower respiratory tract samples and clinical parameters in copd patients, making the information valuable ( ) . cultivable bacteria are seldom found in the lower airways of healthy individuals ( ) , whereas copd patients show bacterial growth in - % of cases even during stable disease ( table ) . on top of that, several studies have shown a significant increase in the proteobacteria phylum, which includes haemophilus spp., in individuals with both stable disease and aecopd ( table ) . nthi is consistently one of the predominating bacterial species isolated in those cultures; other important pathogens include s. pneumoniae, m. catarrhalis, and p. aeruginosa ( ) . during aecopd, the bacterial load is increased even further, and nthi continues to be the predominating species ( ) . furthermore, acquisition of a new nthi strain has, in one study, been linked to the onset of aecopd ( ) . moreover, the growth and dominance of h. influenzae following rhinovirus infection was observed in the sputum microbiome of patients with copd ( ) . the chronic inflammation that characterizes copd pathogenesis causes significant changes to the pulmonary tissue. the lower respiratory tract of patients suffering from this disease is marked by epithelial denuding, hypersecretion of mucus, disproportionate phagocyte presence and imbalances in antioxidant/oxidants ( ) . this altered milieu selects for specific bacterial species that are genetically equipped to competently address these environmental stressors ( , , ) . nthi is the most common pathogen isolated from the sputum of copd patients, and the primary cause of exacerbations ( ) , indicating a unique ability to colonize and persist in the chronically inflamed lower respiratory tract. in recent years, great efforts have been made in understanding how nthi colonizes the pulmonary tissue. in addition to the regular arsenal of virulence factors associated with nthi ( ), the bacterial pathogen undergoes specific adaptations to increase its fitness in the copd setting. specific genetic islands that include ureabcefgh, lic b, hgba, iga, hmw , and hmw have been reported to be enriched in nthi strains isolated from copd patients compared to commensal nthi ( ) . these genes are involved in raising the ph of the environment, lipooligosaccharide (los) synthesis, iron uptake, immune evasion, and attachment to host tissue. the validity of these findings is strengthened by previous work identifying upregulation of many of the same bacterial gene products during growth in copd sputum ( ) . moreover, peroxiredoxinthioredoxin, an antioxidant enzyme, was found to be one of the most enriched proteins in nthi during growth in copd sputum, suggesting that the bacteria upregulate oxidative stress-countermeasures when facing oxidative imbalances in the diseased lung ( ) . oxidative stress resistance has previously been shown to be vital for nthi survival in infection models ( ) . in a seminal investigation by pettigrew et al., wholegenome sequencing (wgs) was conducted to follow the in vivo adaptation of nthi to the copd environment over time ( ) . several interesting findings were reported in this work. firstly, the median duration of persistence by the pathogen was found to be days, but it could persist in patients for up to as many as , days. secondly, slipped-strand mispairing-mediated phase variation was identified as the primary genetic adaptation to the niche. poignantly, the genes affected by the regulation mechanism encoded for (among others) the hmw adhesins, los biosynthesis, and iron uptake, that is, the same processes identified in the previous studies as important for copd adaptation ( , ) . thirdly, and somewhat surprising, it was observed that a very limited number of genes were gained/lost during persistent colonization, meaning that selection for strains that thrive in the inflamed lower airways occurs at the very onset of colonization. finally, the authors reported that genetic changes occurred in of the investigated vaccine antigens during persistent infections, a fact that might be taken into consideration for potential vaccine development against nthi. another virulence factor that has been reported by murphy and co-workers to play a pivotal role for nthi survival in copd settings is iga-protease, a hydrolytic enzyme that cleaves secretory iga (siga) antibodies in the mucosal epithelium ( ) ( ) ( ) . four genes encode for the same number of different variants of the endopeptidase with various cleavage site specificities: two igaa (igaa and igaa ) and two igab (igab and igab ). the igaa is present in all nthi whereas igab is present in ∼ % of the strains ( ) . the igab gene has been reported to be more prevalent in copd exacerbation-causing strains, although the in vivo expression levels did not differ from asymptomatic colonization strains that also carried the gene ( ) . however, iga-protease b and b have been found to promote the intracellular survival of nthi in human epithelial cells, providing a secondary function (in addition to hydrolysis of iga antibodies) that could facilitate nthi growth in inflamed environments ( ) . while a majority of the persistent nthi strains that dwell in copd patients continuously express one or more variants of the enzyme, it has recently been found that a phase variation to an off-state can occur via slipped-strand mispairing over time ( ) . this suggests that during certain conditions, there is a fitness benefit in not expressing iga in the airways of copd patients, albeit the specifics of this process are currently unknown. another interesting aspect of nthi colonization of copd patients is with regard to biofilm formation ( ) . nthi strains that colonize the eustachian tube causing otitis media are known to build up biofilms in situ ( ) . however, strains isolated from copd patients tend to have significantly diminished ability to form biofilm compared to invasive strains or those isolated from otitis media patients ( ) , suggesting that this mechanism is not important for survival in the copd niche. as biofilms tend to protect the bacterial community from external assaults, these findings could indicate that the hypermucoid milieu in the copd airways is severely impaired in its ability to deliver an apt immune response for optimal clearance of residing microorganisms. in light of this impairment, biofilm formation might not be necessary for nthi to persist in this particular environment. infections with nthi have also been shown to reduce cellular levels of e-cadherin, a protein required for tight junction formation and epithelial cell integrity in human cells ( ) . considering that perturbations in the epithelial cell barrier caused by the loss of e-cadherin is a common symptom of copd, nthi-mediated exacerbations likely contribute to this step of copd pathogenesis. the subsequent denuding of the epithelium could facilitate microbial colonization of the basal lamina, a well-established virulence mechanism employed by nthi and other pathogens ( ) . it is currently unknown which bacterial virulence factor(s) that induce the reduction of e-cadherin levels in the host. in summary, investigations from recent years show that the environment of the lower respiratory tract of copd patients selects for nthi strains that can upregulate adhesins, modify los biosynthesis pathways, increase antioxidant stress responses and cellular invasion strategies, and, finally, trigger tolerance against acidic ph. these important colonization mechanisms thus provide researchers with viable targets for developing novel therapies. nthi is a commensal in the nasopharyngeal site but is often associated with strong inflammatory responses in the lower respiratory airways, especially in patients with copd, bronchiectasis, cystic fibrosis, pneumonia, or idiopathic pulmonary fibrosis ( , ) . colonization and subsequent infection of nthi in the lower airways of copd patients elicits episodes of immune responses orchestrated by both the innate and adaptive immunity. nthi infection is thus commonly associated with inflammation that is mainly mediated by transcription factor nf-κb-dependent production of proinflammatory mediators. the activation of nf-κb requires induction of cross-signaling networks and cascades via activation of prrs (pattern recognition receptors) of host innate immune cells ( ) . unresolved or prolonged (chronic) inflammation or failure to restore the homeostatic inflammatory status potentially contributes to exacerbations. this is clearly shown in murine copd simulation models with nthi-triggered inflammation ( - ). mice exposed to nthi lysates display inflamed airways loaded with increased levels of inflammatory mediators and phagocyte infiltrates. moreover, multiple exposures to bacterial lysates which may represent a chronic nthi infection caused extremely high infiltration of phagocytes and lymphocytes in the airways of this particular mouse model. in addition, the airway walls of the infected animals were also thickened due to increased collagen deposition (fibrosis) that reflects the typical copd features. the host immune response and specific interactions during nthi infection in copd is summarized in figure . the epithelium and alveolar macrophages are predominant cell types in the airway compartment. they comprise the first line of defense in the cellular immune response against potential inhaled pathogens and antigens. the sensing of bacteria, and particularly nthi in the lower airways is initiated via prrs expressed on innate immune cells and endothelium in addition to epithelial cells ( ) ( ) ( ) . tlrs are prrs that sense stimulation by nthiderived pathogen-associated molecular patterns (pamps), and play a primary role in initiating effector cellular responses and intracellular signaling for nf-κb activation ( ) . among the different tlrs, most of the studies on nthi infection have by far been focused on tlr and . lipoproteins including nthi p , and los are potent immunomodulators for activation of tlr and tlr , respectively, and has been described in several studies on airway epithelial cells and alveolar macrophages ( ) ( ) ( ) ( ) . interaction of nthi lipoprotein p with tlr on human epithelial cells [type ii alveolar a and human middle ear epithelial cells (hmee)] causes nf-κb-dependent activation via two distinct tlr-signaling pathways, that is, the nf-κb translocation-dependent, and -independent pathways ( ) . the nf-κb nuclear translocation-dependent pathway requires activation of nf-κb-inducing kinase ikk complex. in the second pathway, the mkk / -p mapk signaling cascade is recruited for direct nuclear phosphorylation, and thus activation of nf-κb. the branching of both pathways may occur at the tgf-β activated kinase (tak ) signaling junction. nthi stimulation via tlr and downstream activation of p mapk/nf-κb-dependent pathways result in expression of cox- and prostaglandin (e ) (pge ) that promote inflammatory responses ( ) . tlr stimulation by nthi los also contributes to the activation of nf-κb via two signaling pathways, the primary activating pathway of myd cascade and the alternative pathway of toll/il- r domain-containing adapter-inducing interferon-β (trif). both pathways activate nf-κb through phosphorylation and degradation of inhibitor iκbα ( , ) . nthi-tlr signaling mediates an effective innate immune response that leads to upregulation of tnf-α, il- β, il- , macrophage-inflammatory protein (mip)- α, mip- , and neutrophil infiltration in the airways of mice. the tlr response promotes efficient pulmonary clearance of bacteria in tlr expressing animals compared to cd /tlr knockout mice ( , ) . a recent study by jungnickel et al. revealed that, in parallel with the infection-induced pulmonary neutrophilic inflammation, nthi-dependent stimulation of both tlr and tlr in a transgenic mouse [(kras la ) with oncogenic kras allele in the lung epithelium] additionally promotes the proliferation of kras-induced early adenomatous lesion in the lung in an tlr-dependent manner ( ) . the association or role of nthi-induced airway inflammation in lung cancer progression, however, is not supported by another recent cohort study showing the lack of differences in nthi specific-antibodies between cancer-and non-cancer copd patients ( ) . lastly, dectin- and the epidermal growth factor receptor (efgr) pathway also have proinflammtory effects upon interaction with nthi ( , ) . activation of the dectindependent proinflammatory response requires nthi-induced phosphorylation of the dectin- hem-immunoreceptor tyrosinebased activation motif (hemitam) ( ) . direct activation of efgr in alveolar cells and hmee by nthi-derived egflike factor has been shown to contribute to nf-κb activation. the efgr-dependent nf-κb activation is mediated via an nf-κb nuclear translocation-independent pathway, which involves both mkk / -p and pi k/akt signaling pathways ( ) . surprisingly, the interaction of efgr and nthi also results in negative regulation and suppression of the induction of tlr via the src-mkk / -p α/β map kinase-dependent signaling cascade, and this in turn may facilitate nthi infection ( ) . the actual components of nthi that exhibit the egf-like factor activity have, however, yet to be defined. the efgrdependent negative regulation of tlr may thus suggest a novel mechanism targeted by nthi for immune evasion by attenuating the responses of host prr, despite the contradicted role of efgr in proinflammatory and innate immune responses of the airway epithelium ( ) . nthi infection also upregulates the nrlp -inflammasome during nthi-induced inflammation in the airway epithelium and alveolar macrophages, leading to increased secretion of il- β and il- , and thus neutrophilic influx to the lung ( ) . some of the endogenous inflammatory mediators that are produced in response to nthi infection, including tnf-α, il- α, and tgf-β , may act synergetically with nthi on the airway epithelial and immune cells. the synergetic interaction drives a positive feedback loop to amplify the nf-κb transcriptional activity on proinflammatory genes and further augments airway inflammation. the synergetic activation of nf-κb by nthi and tnf-α in hmee and normal human bronchial epithelial (nhbe) cells occurs via nf-κb nuclear translocation-dependent and independent pathways. the latter pathway involves mapk/extracellular signal regulated kinase kinase kinase (mekk )-dependent activation of mapk kinase / -p mapk pathway ( ) . however, the synergetic action of nthi with tgf-β is mediated by another mechanism which involves smad / -protein kinase a (pka)-p -dependent signaling cascade. the pathway components, pka and p , phosphorylates residue ser and acetylates lys of the nf-κb subunit p , respectively. this results in enhanced dna-binding activity of nf-κb ( ) . the synergetic action of nthi with both tnf-α and tgf-β enhances the production of tnf-α, il- β, and il- from airway epithelial cells and interstitial polymorphonuclear infiltrates. recently, it has been reported that co-infection of human rhinovirus and nthi on the airway epithelial cells (nhbe cells and the beas- b cell line) also results in synergetic induction of ccl and il- , albeit the exact mechanism remains to be elucidated ( ) . of note, activated macrophages also release increased concentrations of tnf-α and il- α ( ) , further enhancing the inflammatory synergetic effect of surrounding immune cells. finally, il- α acts synergetically with nthi to upregulate the expression of amp β-defensin (defb- ) via the p /mapk pathway ( ) . of note, il- α could also act individually to upregulate the expression of defb- via the src-dependent mek / -erk / signaling pathway ( ) . taken together, the synergetic action may aid in the expansion of the inflammatory response and in some cases worsen the clinical outcome. alveolar macrophages located in the air-parenchyma interface are the primary professional phagocytes in the lung ( , ) . these cells are responsible for infection eradication through its phagolysosomal machinery while releasing a plethora of inflammatory cytokines and chemokines for promoting a local inflammatory response and recruitment of neutrophils. neutrophils are the first responder cells recruited from circulation to the airway for efficient killing of pathogens through an array of microbicidal strategies ( , ) . during nthi lung infection, both alveolar macrophages and neutrophils are the main innate immune cells involved in the pulmonary bacterial clearance through phagocytosis. they are also an important source of cytokine secretion required for induction of other immune cells and enhanced bacterial killing. eradication of nthi by alveolar macrophages involves adhesion or contact, phagocytosis and phagolysosomal processing of bacteria, in addition to secretion of tnf-α. phagocytic clearance of nthi by alveolar macrophages is orchestrated through actin polymerization, plasma membrane lipid rafts, and phosphatidylinositol -kinase (pi k) signaling cascade upon induction of macrophage prrs by nthi ( ) . interestingly, in response to nthi infection, human alveolar macrophages, and blood neutrophils produce extensive amount of intracellular and extracellular ros as a component of the antimicrobial defense. this leads to the formation of macrophage and neutrophil extracellular traps (mets and nets, respectively), with co-expression of mmp- for enhanced bacterial killing ( , ) . nevertheless, the overexpression of mmps may adversely result in a protease imbalance and contribute to alveolar emphysematous destruction and bronchiectasis in copd ( ) . moreover, excessive endogenous ros production could also introduce airway oxidative stress that is detrimental by causing chronic inflammation and tissue damage in the lung, and thus contributing to the copd exacerbation ( , ) . the net formation is elicited mainly by nthi los in addition to other haemophilus pamps ( ) . several studies by king et al. have revealed that t cellmediated adaptive immune responses against nthi airway infection in patients with idiopathic bronchiectasis and copd has been predominated by a th /tc response ( ) ( ) ( ) . the activated t cells produce reduced level of the cd ligand and ifn-γ, and increased levels of tnf-α, il- , and il- , as well as altered igg subclass production by plasma cells. it is to be noted that the th /tc -mediated immune response is less effective in suppressing nthi infection. redirecting the th /tc -mediated immune response to th /tc dominant (which is more protective) by adding the th /tc mediators (cd ligand and ifn-γ) has helped to restore the t cellmediated immune killing of nthi ( ) . however, a separate study in a copd mouse model by lu et al. reported that nthi infection causes increased production of airway type interferon ( -ifn) ( ) . it was further reported that dna of nthi acts as a pamp in stimulating the sting/tbk /irf pathway, and thus the production of -ifn. the impact of the bacterial dnainduced -ifn in host immune/inflammatory response, which may potentially induce a th /tc response requires further investigations. copd patients also have abnormally higher number of treg cells, myeloid-derived suppressor cells (mdsc), and exhausted effector t cells (pd- + ) than healthy individuals ( , ) . cigarette smoke-induced anti-inflammatory activity of tregs in a copd model is further suppressed by nthi infection. the pathogen causes downregulation of foxp (biomarker of tregs), and thus impairs the anti-inflammatory/pro-inflammatory balance of tregs ( , ) . this may lead to the extensive immunosuppressive activity by tregs on the proliferation of nthi p -specific effector t cells, causing a diminished response of effector t cells to sputum il- and il- induction, and increased levels of il- and tgf-β ( , ) . recently, it has been reported that mucosal-associated invariant t cells (mait) from copd patients are more effective in response to nthi stimulation and thus produce increased levels of ifn-γ, -, to -fold more than the copd th (cd + ) and tc (cd + ) cells ( ) . however, the pulmonary mait cell immune responses are compromised in the presence of corticosteroids that are commonly used for the treatment of copd. this may potentially prone the t cell-mediated immunity to a th /tc response in copd patients treated with corticosteroids ( ) . interestingly, antigen-specific th cells from nthi-immunized non-copd mice model recognize both homologous and heterologous strains of nthi, and are able to confer protection upon adoptive transfer ( ) . however, it is unclear whether the th cell which is prone to the inflammatory response could be "trained" to counteract the nthi infection in copd patients, particularly during exacerbations. during the systemic humoral immune response in nthiinfected copd patients, greater concentrations of nthi-specific igg, iga, igm, and ige serum antibodies are produced compared to non-infected controls ( , ( ) ( ) ( ) . some of the nthispecific serum immunoglobulins are specific to p , p , and p ( , , ) . however, decreased mucosal antibodies associated with siga deficiency, or decreased total igg in the small airways have been reported in copd patients, and might be associated with disease severity ( , ) . importantly, nthi-specific mucosal siga has been found to be lower in the airways of nthi-infected copd patients than the non-colonized patients ( , ) . the epithelial polymeric immunoglobulin receptor (pigr) is essential for the generation of mucosal siga. it is, however, downregulated in copd patients with a positive correlation to disease severity and increased level of tgf-β ( ) . the combinatorial effects of downregulated plgr and elevated tgf-β contribute to an impaired mucosal iga immunity in copd patients. a mouse model lacking the pigr ( −/− ) is therefore devoid of siga and are susceptible to airway stimulation by an nthi lysate resulting in increased inflammation and airway neutrophilia. interestingly, introduction of exogenously added siga mitigated the airway inflammation ( ) . nthiinfected copd patients with greater airway inflammation have also decreased nthi-specific mucosal igg in the bal fluid compared to the non-colonized patients ( ) . interestingly, the phenomenon with decreased nthi-specific antibodies seems to be restricted to the airways, since the specific serum antibodies are not affected. therefore, the reduced mucosal igg is unlikely to be associated with hypogammaglobulinemia (igg deficiency), despite the latter was reported as a contributing factor in nthi infection ( ) . decreased airway iga might be attributed to the expression of iga proteases by nthi. the bacterial iga protease degrades the local airway iga during airway colonization to avoid immune exclusion by siga ( , ) . reduced mucosal antibodies might promote host immune evasion and resistance to complement-mediated killing of nthi, thus enable persistent colonization of nthi in the airways of copd patients, in addition to a plethora of various other virulence mechanisms ( , , ) . in a cohort study of stable copd patients, augmented airway inflammation and plasma fibrinogen, but not systemic inflammation, were found to be constantly correlated with the increased bacterial load ( ) . higher numbers of nthi has a greater impact than s. pneumoniae and m. catarrhalis in triggering inflammatory responses as measured by the augmented levels of inflammatory cytokines in sputum including il- , mpo, and l- β. the increased inflammatory response in affected patients is potentially attributed to the persistent colonization of nthi in the lower airway ( , ) . the compromised innate immune response in copd, particularly the decreased microbicidal activity, has been regarded as one of the culprits for persistent airway colonization by nthi, and is highly associated with copd exacerbations (figure ). whilst the role of macrophage extracellular traps (met) for killing of nthi remains unknown, it has been reported that blood neutrophils and net from copd patients are defective in the killing of planktonic or biofilm/net-entrapped nthi, respectively ( , , ) . a series of studies by berenson et al. revealed that alveolar macrophages derived from copd patients are basically dysfunctional in eradication of nthi ( , ( ) ( ) ( ) . intriguingly, tlr and tlr expressed on alveolar macrophages from copd patients are intrinsically unresponsive to the potent immunomodulatory lipoprotein p and los, respectively. this causes decreased los/p -induced expression of tlrs, reduced nf-κb nuclear activation and consequently diminished il- , tnf-α, and il- β responses by alveolar macrophages from copd patients. the compromised tlr expression and signaling potentially contribute to the defective complement-dependent and independent phagocytosis of nthi. the defective phagocytosis is greater for nthi than for m. catarrhalis, and correlates with disease severity. interestingly, the phagocytosis disability was not detected in monocyte-derived macrophages in copd. in contrast, however, taylor et al. reported that monocyte-derived macrophages from copd patients are also defective in phagocytosis of nthi and s. pneumoniae. the author also suggested that the defective monocytederived macrophages are not attributed to the alteration in cell surface tlr or tlr expression, macrophage receptor with collagenous structure (marco), cd , cd or the mannose receptor ( ) . the unresponsive tlr and tlr in copd alveolar macrophages to nthi lipoprotein and los might be explained by the recently reported phenomenon of tlr tolerance ( ) . repetitive stimulation of copd alveolar macrophages with the same tlr ligands, pam csk and lps desensitizes the tlr and tlr , respectively, and generates tlr tolerance. moreover, the repetitive tlr stimulation further reduced the production of tnf-α, ccl , and il- without affecting the constantly augmented level of il- and il- in alveolar macrophages. this may provide alternative explanations for diminished immune responses against the recurrent/repetitive infection by nthi. the intrinsically reduced expression of tlrs in copd patients may also contribute to the impaired pulmonary immune response thus facilitating nthi persistent colonization. expression of tlr or tlr are found to be lower on sputum neutrophils, alveolar macrophages, nasal epithelium, and t cells in copd patients despite high concentrations of il- and mmp- ( ) ( ) ( ) ( ) . the lack of the more protective th /tc immune response in copd patients against nthi infection might be attributed to upregulated antagonists (a , irak-m, and myd s) of the myd /irak/mapk signaling pathway in copd t cells ( ) . it should be noted that the myd /irak/mapk pathway is required for expression of tlr in th , whereas production of ifn-γ in th /tc is tlr -dependent via the tlr /trif/ikke/tbk signaling pathway. the antagonists prevent the nthi los-induced tlr expression in th and tc and thus a reduced secretion of ifn-γ. in addition, unusual high numbers of tregs in copd patients have also contributed to effector t cell dysfunction or a th /tc predominant immune response ( ) . however, freeman et al. reported that tc (cd + ) cells from copd patients have increased expression of tlr , tlr , tlr , tlr , and tlr / as well as tc cytokines (ifnγ and tnf-α) compared to healthy individuals that may imply the auto-aggressive response of lung tc cells in copd lung inflammation ( ) . however, the copd tc cells can only be stimulated by ligands for tlr / (pam csk ) yet tolerant to other agonists, indicating the dysfunctional tlrs or tlr tolerance on t cells despite their high level of receptor expression. inversely, peripheral blood neutrophils isolated from copd patients have increased expression of tlr , tlr , and nlrp ( , ) . nevertheless, the increased tlrs expression might not improve the microbicidal ability of copd peripheral neutrophils probably due to the inaccurate responses to cytokines ( ) . in addition, certain types of snps (snps) in tlr and tlr have also been associated with decreased lung function, enhanced inflammatory responses and increased immune cell infiltration in copd ( ) . interestingly, the diminished il- responsiveness of copd alveolar macrophage to nthi infection has a strong association with the carriage of tlr (t c) polymorphism instead of tlr (arg gln), tlr (thr ile; asp gly), and tlr (t c) ( ) . the carriage of tlr (t c) is also positively correlated with diminished lung function. of note, the activation of tlr -signaling cascade in pro-inflammatory cytokine response requires stimulation from microbial dna ( ) . the microbicidal malfunction in both innate and adaptive immune cells is also potentially linked to the deleterious effect of tobacco smoke, the major risk factor for copd. it has been reported that, exposure of tobacco or cigarette smoke can impair phagocytosis/engulfment of nthi by alveolar macrophages isolated from copd patients ( , ) . moreover, the chemical exposure also suppressed the tlr-induced tnfα, il- , and il- production in copd alveolar macrophages that have been pre-stimulated with tlr , , or ligands (pam csk , lps, or phase i flagellin, respectively), or whole nthi bacteria ( ) . this may potentially delay the macrophagedependent bacterial clearance. the suppressive effect of cigarette smoke in macrophage-dependent phagocytosis is due to the suppression of the pi k signaling cascade which is required for optimal phagocytic activity and movement ( ) . meanwhile, the cigarette smoke also inhibits the activation of the p -erk signaling pathway and p /nf-κb, thus dampens the nthi losinduced cytokine production of copd alveolar macrophages ( ) . the diminished alveolar macrophage responsiveness could also be related to anticholinergic agents used by copd patients that results in lower concentrations of nthi-induced tnfα ( ). nevertheless, the impaired phagocytosis of nthi by copd alveolar macrophages could be improved in the presence of nuclear erythroid related factor and microrna mir- ( , ) . interestingly, in addition to the constant exacerbated inflammatory effect observed in different murine model studies, gaschler et al. observed a rapid pulmonary clearance of nthi in mice upon exposure to cigarette smoke, and this was positively correlated with an increased neutrophilia in the animal bal fluid ( , ( ) ( ) ( ) . however, in other copd animal studies, cigarette smoke also impaired the il- production that has a potential anti-bacterial activity while delaying the airway clearance of nthi ( ) ( ) ( ) . interestingly, il- might play a protective role in copd exacerbation as supplementation of il- manages to restore the homeostasis of airway immune response and improve nthi clearance ( ) . the increased airway neutrophilia might be due to the enhanced production of pulmonary il- triggered by cigarette smoke ( , , , ) . this may imply the important microbicidal role of neutrophils (neutrophilia) in compensating the copd-or cigarette smoke-associated dysfunctional alveolar macrophages ( , ) . however, such compensation may not be adequate to provide optimal immune defense to eradicate persistent nthi lower airway colonization, since the cigarette smoke also has profound suppressive effect on the host adaptive immunity, thus constantly risking the copd patients to episodes of exacerbation and relapsed infection. in adaptive immunity, cigarette smoke impairs the antigen-specific b and t cells responses to nthi infection. it suppresses the secretion of ifnγ and il- by nthi-specific t cells. antibody production by b cells has also been attenuated, with lower levels of specific anti-p antibodies and compromised igg , igg a, and iga class switching ( , ) . a recent and some previous cohort studies revealed that the level of airway antimicrobial cathelicidin (hcap /ll- ) in copd patients increase gradually from the stable disease to exacerbation states ( , ) . moreover, higher levels of cathelicidin are positively associated with nthi airway colonization, sputum neutrophilia, and higher concentrations of il- , particularly in the nthi-infected copd patients. of note, cathelicidin and other amps play important roles in the innate immune defense against different pathogens and persist immunomodulatory properties ( ) ( ) ( ) . ironically, it is plausible that the increased level of cathelicidin could diminish or alter the balance in lung microbiota, and the immune/inflammatory response. this might contribute to the "vicious circle, " thus considerably increasing the risk for nthi infection during copd exacerbations ( , ) . moreover, the microbicidal property of cathelicidin could be compromised by the inflammatory conditions in the airway, such as low ph, or the effect of cigarettes that causes peptide citrulination and modification ( , ) . finally, expression of amps (human beta defensin and s a ) by copd airway epithelium in response to nthi infection, is also disturbed by cigarette smoke. the insulted airway cells have also a reduced expression of tlr and il- , and impaired nthi-induced nf-κb activation ( , ) . thus, a large body of evidence exists on the deleterious effects of tobacco smoke. antibiotic treatment of aecopd has been shown to significantly reduce the risk of treatment failure, especially for in-patients with severe exacerbations and patients requiring intensive care ( ) . the efficacity of antibiotic treatment for out-patients with exacerbations is less clear ( , ) . recommendations on which empirical treatment to use for aecopd varies between different countries, but common antimicrobial agents that are frequently used as definitive therapy against nthi include aminopenicillins (with or without a beta-lactamase inhibitor), tetracyclines, trimethoprimsulfamethoxazole, and fluoroquinolones. in addition, the clinical and laboratory standards institute (clsi) has developed clinical breakpoints for the macrolides azithromycin and clarithromycin ( ) , whereas the european committee on antimicrobial susceptibility testing (eucast) have not set any clinical breakpoints against this class of antibiotics due to lack of clinical data ( ) . one study shows that nthi frequently develops resistance to macrolides during prolonged treatment and that treatment failure may occur, making fluoroquinolones more reliable for eradication in copd-patients ( ) . as for aminopenicillins, resistance is also common, with up to - % of nthi isolates expressing beta-lactamases and an additional - % of the isolates having amino acid substitutions in penicillin-binding protein (pbp ), which reduces their susceptibility to these agents ( , ) . the fraction of isolates expressing beta-lactamases has been stable during the last years, whereas an increase has been seen in isolates displaying altered pbp ( , ) . this is worrisome, since some of these amino acid substitutions also confer resistance to third generation cephalosporins ( ) . moreover, there seems to be a correlation between isolates expressing altered pbp and increased invasiveness. studies have shown that strains that express a mutated pbp with certain key amino acid substitution have a significantly higher rate of invasion of bronchial epithelial cells compared to strains with a wild type pbp ( ) . however, when such mutated pbp was cloned into a susceptible wild type strain, invasion efficacy did not increase, suggesting that pbp is only indirectly linked to invasion ( ) . besides using antibiotics for acute management of copd exacerbations, some studies have considered the use of continuous prophylactic antibiotics in the management of patients with copd ( ) . there is some evidence that continuous administration of macrolide antibiotics would prevent future exacerbations in a selected population of the most severely ill patients, but a cochrane review revealed no support for a reduced all-cause mortality or less hospital readmissions ( ) . however, more recent studies have shown a significant decrease in both the frequency of exacerbations and hospitalizations when long-term azithromycin treatment was chosen ( ) . the fact that macrolide antibiotics display not only antimicrobial effects, but also have anti-inflammatory and immunomodulatory properties, has made them interesting to use as prophylactic therapy ( ) . it has been shown that azithromycin inhibits mucus hypersecretion in the respiratory tract by significantly inhibiting tnf-α induction of the muc ac mucin secretion from human nasal epithelial cells ( ) . more specifically, it has been shown that azithromycin can reduce the nthi-dependent induction of muc ac expression by suppressing the transcription factor activator protein- ( ) . apart from affecting mucus secretion, it also seems that low-dose azithromycin has the ability to improve phagocytosis of bacteria by airway macrophages ( ) . one study showed that azithromycin concentrations that were unable to kill nthi still increased the uptake rate of the bacteria into alveolar macrophages by enhancing their phagocytic function ( ) . however, the risk of development of antimicrobial resistance limits the use of low-dose azithromycin solely for its immunological properties. this has triggered an interest in finding new macrolide substances that lack antibiotic effect and solely interact with the airway immune system ( ) . the considerable clinical problems caused by nthi with regard to copd exacerbations and otitis media has prompted the scientific community to investigate whether a vaccine can be developed against the pathogen ( , , ) . the search has been intensified due to a steady increase in antibiotic resistance and a trend of more invasive infections caused by nthi over the last decade ( ) . whereas, a highly efficient glycoconjugate vaccine has previously been developed against hib, an identical strategy cannot be employed against nthi due to the lack of a polysaccharide capsule. vaccine developments efforts have thus been concentrated on identifying nthi surface structures that are immunogenic, have low antigenic variability, and are conserved across this genetically highly heterogeneous species. several promising vaccine candidates have been identified in the last years, as excellently reviewed elsewhere ( , ). two of these antigens, fused into one protein, protein e-pila, are together with protein d currently being tested by glaxosmithkline in a phase iib proof-of-concept clinical trial (randomized, observer-blind, placebo-controlled, and multicentric) for infection prophylaxis in copd patients ( - years old) ( ) . notably, the m. catarrhalis ubiquitous surface protein a (uspa ) is also included in the vaccine so that an immune response against both exacerbation-causing pathogens could be elicited by the same preparation. this clinical study (nct ) is the only one currently being conducted on nthi (and m. catarrhalis) according to clinicaltrials.gov, and as the investigations are on-going, the results are currently unknown. due to an increase in the difficulty to treat nthi infections, an efficient and protective nthi vaccine likely considerably raises the quality of life of copd patients. since nthi-mediated exacerbations contribute to the progression of the disease and a steady deterioration of the pulmonary capacity of those patients, prevention against nthi infections potentially slows down the debilitating effect of the disease. it is therefore critical to continue this line of research until such a vaccine has been obtained. it could also be worth targeting non-conventional structures with a vaccine, such as the secreted enzymes urease and iga protease that have proven important for nthi infections in copd patients in several studies ( ) . copd is a multifaceted airway disease. several factors influence the clinical outcome of copd. importantly, the crosstalk between intrinsic factors (the stability and integrity of the airway immune response and structure in addition to hereditary factors), and the extrinsic factors (lung microbiome, viral and bacterial infections, meteorological factors, and noxious inhalation) determines the fate of lower airway opportunistic infection by h. influenzae. intriguingly, nthi has been one of the most isolated pathogens at both stable and exacerbation states of copd. such persistent airway colonization of nthi costs virulence fitness to counteract with the bactericidal effect of the host immune response. adversely, the impaired defense mechanisms in copd are not only unable to protect the lung structure from inhaled physical assaults, but they also fail to suppress nthi infection. the disoriented immune response in copd instead allows the pathogen to cause more harm and inflammation in the airways. the currently used bronchodilator and inhaled corticosteroid therapies have limited efficacy in preventing disease progression in copd. moreover, the inhaled corticosteroid therapies might have side effects that may weaken the immune response. hence, more investigations are needed to garner a more adequate knowledge regarding the variabilities in immune networking of copd. this knowledge will be an important platform for a more efficient drug design. in addition, a vaccine targeting nthi is another important approach in controlling the infective exacerbations in copd as the antibiotic treatment is also getting dampened by the emergence of nthi antibiotic resistance. y-cs coordinated and drafted the major part of the manuscript, and prepared the figures; fj participated in the literature study of virulence and vaccine research of nthi; jt prepared the review section for nthi epidemiology in copd and antibiotic studies; y-cs and kr edited and critically 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extended-spectrum cephalosporins ampicillin-resistant non-beta-lactamaseproducing haemophilus influenzae in spain: recent emergence of clonal isolates with increased resistance to cefotaxime and cefixime an amino acid substitution in pbp- in haemophilus influenzae associate with the invasion to bronchial epithelial cells genotypically defined beta-lactamase-negative ampicillin-resistant isolates of non-typable haemophilus influenzae are associated with increased invasion of bronchial epithelial cells in vitro prophylactic antibiotic therapy for chronic obstructive pulmonary disease (copd) clinical and safety outcomes of long-term azithromycin therapy in severe copd beyond the first year of treatment azithromycin inhibits mucus hypersecretion from airway epithelial cells azithromycin inhibits nontypeable haemophilus influenzae-induced muc ac expression and secretion via inhibition of activator protein- in human airway epithelial cells low-dose azithromycin improves phagocytosis of bacteria by both alveolar and monocyte-derived macrophages in chronic obstructive pulmonary disease subjects relationship between azithromycin susceptibility and administration efficacy for nontypeable haemophilus influenzae respiratory infection nonantibiotic macrolides restore airway macrophage phagocytic function with potential anti-inflammatory effects in chronic lung diseases vaccines for nontypeable haemophilus influenzae: the future is now we thank the following funding agencies for their financial support during the preparation of the manuscript. they are the alfred Österlund, the anna and edwin berger, the swedish medical research council (grant number k - x- - - , www.vr.se), the physiographical society (forssman's foundation and, endowments for the natural sciences, medicine and technology), skåne county council's research and development foundation, and heart lung foundation (kr: grant number , www.hjart-lungfonden.se). the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © su, jalalvand, thegerström and riesbeck. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -qk xb a authors: hanada, shigeo; pirzadeh, mina; carver, kyle y.; deng, jane c. title: respiratory viral infection-induced microbiome alterations and secondary bacterial pneumonia date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: qk xb a influenza and other respiratory viral infections are the most common type of acute respiratory infection. viral infections predispose patients to secondary bacterial infections, which often have a more severe clinical course. the mechanisms underlying post-viral bacterial infections are complex, and include multifactorial processes mediated by interactions between viruses, bacteria, and the host immune system. studies over the past years have demonstrated that unique microbial communities reside on the mucosal surfaces of the gastrointestinal tract and the respiratory tract, which have both direct and indirect effects on host defense against viral infections. in addition, antiviral immune responses induced by acute respiratory infections such as influenza are associated with changes in microbial composition and function (“dysbiosis”) in the respiratory and gastrointestinal tract, which in turn may alter subsequent immune function against secondary bacterial infection or alter the dynamics of inter-microbial interactions, thereby enhancing the proliferation of potentially pathogenic bacterial species. in this review, we summarize the literature on the interactions between host microbial communities and host defense, and how influenza, and other acute respiratory viral infections disrupt these interactions, thereby contributing to the pathogenesis of secondary bacterial infections. influenza and bacterial pneumonia are the leading cause of morbidity and mortality from infectious diseases worldwide. influenza and other respiratory viral infections predispose patients to secondary bacterial super-infections, which are frequently associated with a more severe clinical course. it is estimated that the so-called "spanish flu" pandemic of h n influenza a virus from to resulted in more than million deaths, with many caused by bacterial superinfection leading to secondary pneumonia ( ) ( ) ( ) ( ) ( ) ( ) ( ) . even in the antibiotic era, over half of patients with severe infections in the h n and h n pandemics had bacterial complications ( ) ( ) ( ) . bacterial co-infection was also detected in ∼ % of cases in the h n pandemic, with high mortality rates despite administration of appropriate antibiotics ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . thus, it is evident that a better understanding of the pathogenesis of secondary bacterial pneumonia following viral infections is needed in order to make therapeutic strides for this devastating complication. the mechanisms of post-viral bacterial infection are complex, comprising multifactorial processes mediated by interactions between viruses, bacteria, and the host immune system. the pathogenesis of super-infection has been attributed to direct mucosal/epithelial damage by influenza virus, increased bacterial colonization of the upper and lower respiratory tracts (urt and lrt, respectively), and dysregulation of immune responses, which all lead to increased susceptibility to secondary bacterial infections. however, emerging evidence suggests that our microbial communities residing on our mucosal surfaces likely shape the rigor of our immune responses and shape the ecological relationships between host and pathogens. over the past years, intense interest has focused on examining how the microbial communities which inhabit our bodies-which some consider to be a separate "organ system" given the sheer physical bulk, number of genes, and metabolic activities-govern the balance between health and susceptibility to diseases, including infections. this raises the possibility that disruptions in the normal microbial communities by an acute viral infection might contribute to the development of post-viral bacterial pneumonia. the recent development of culture-independent methods of microbial identification has enabled the study of microbial communities on mucosal surfaces of the human body, referred to as "microbiota." the microbial communities of mammalian hosts are diverse, comprised of bacterial, viruses, archaea, parasites, and fungi. the human microbiome project (hmp) and other similar large-scale sequencing projects worldwide have characterized the distinct microbial communities that have adapted to the unique environmental niches within our bodies, such as the gut, skin, airways, genitourinary tract, and oral cavity. the gut microbiome, in particular, has been shown to play an integral role in shaping the immune system starting early in life, with continued influence on priming the nature and robustness of immune responses throughout one's lifetime. the respiratory tract also harbors distinct communities of microbes, with multiple discrete ecological niches (e.g., nasal cavity, oropharynx, upper airways) that vary in terms of temperature, ph, oxygen tension, mucus production, and other factors. the effects of viral infections on both the gut and respiratory microbiome have recently undergone examination. surprisingly, influenza infection has been found to result in significant changes in the gut microbiome, despite the lack of detectable virions in the gi tract. by comparison, the effects of viral infection on the respiratory microbiome appear to be relatively modest, but detectable. while the effects of these alterations on risk of secondary bacterial pneumonia have not been studied, potential mechanisms by which these changes might modulate susceptibility to secondary bacterial infections include alterations in the nature and magnitude of the immune response in the host (microbiome on host effects) and facilitating growth of pathogens in the absence of normal commensals (inter-microbial effects). in this article, we review the current understanding of how alterations in the microbiome following viral infection might alter host immune responses and increase susceptibility to secondary bacterial infections. although the term "microbiome" encompasses all microbial communities, there is currently a paucity of studies on how the mycobiome (fungal microbiome) and the virome (viral microbiome) affect host defense against respiratory infections and vice-versa; thus, this review will focus on the bacterial microbiome literature. of the niches in the body, the gut microbial community has been the most intensively studied, with over , publications to date. while the virome and mycobiome (fungi) are also being analyzed, the bulk of the literature has focused on the bacterial component of the microbiome, and thus most of our understanding of the relation of the gut microbiome to host immunity and pathogenesis of chronic diseases comes largely from studies of the bacterial community. during health, the human gut bacterial community is diverse, with each individual harboring over trillion bacteria, comprised of over different species. the gastrointestinal microbiota is dominated by firmicutes (e.g., lactobacillus, bacillus, and clostridium) and bacteroidetes (e.g., bacteroides), with lower abundances of proteobacteria (e.g., escherichia) and actinobacteria (e.g., bifidobacterium) ( , ) . wild-living mice exhibit more diverse microbiomes, with significant abundance of proteobacteria as well as firmicutes and bacteroidetes ( ) . the gut microbiome, in addition to its metabolic functions in the host, plays an integral role in the development, instruction, and priming of the immune system. germ-free (gf) mice (which lack microbiota) have markedly underdeveloped gut-associated lymphoid tissues, decreased number and smaller-sized peyer's patches and mesenteric lymph nodes, and defects in antibody production, compared to specific pathogen free (spf) mice. not surprisingly, germ-free animals exhibit increased susceptibility to multiple types of infections, including viruses, bacteria, and parasites ( ) ( ) ( ) ( ) ( ) ( ) . however, compared to free-living mice or laboratory animals exposed to gut flora from wild mice, spf animals have a more limited microbial community and are also more susceptible to inflammatory diseases, with a reduced immune repertoire including deficits in memory responses ( , , ) . although an extensive discussion of the healthy gut microbiome and its impact on host immunity is beyond the scope of this review, we will highlight a few important aspects of how the intestinal bacterial community microbiome maintains a healthy host immune environment. first, bacterial metabolites generated by gut commensals contribute to the maintenance of intact epithelial integrity, regulatory t-cell development, and a relatively anti-inflammatory immune state. in particular, short-chain fatty acids (scfas) such as acetate, propionate, and butyrate are fermentation products of dietary fiber and carbohydrates by large intestinal bacteria ( ) . in addition to being a major energy source for intestinal epithelial cells, scfas promote the development of naive cd + t cells into regulatory t cells ( , ) , induce "tolerogenic" dendritic cells in the intestinal mucosa ( ), and limit autoimmity ( , ) . at the same time, microbial metabolites are integral for promoting immune responses in the gut against pathogens, including inducing secretion of il- ( ) and defensins ( , ) . thus, the products of microbiome metabolism are integral to the appropriate regulation of mucosal barrier integrity and immune homeostasis. in addition, specific members of the bacterial community have been shown to foster the proper maturation and development of the immune system. while this is still an area undergoing intense investigation, one notable example is the discovery that segmented filamentous bacteria are critical promoters of intestinal mucosal iga production ( , ) and th cell induction ( , ) . dysbiosis, or an imbalance in the normal composition of the microbiome, is associated with a variety of chronic diseases, many of which are characterized by chronic inflammation or abnormal metabolism, including inflammatory bowel disease, cardiovascular disease, and diabetes. thus, fostering appropriate levels of diversity and composition of the gut microbial community is critical for promoting health and immune homeostasis. during health, the composition of the microbiome is governed by a number of selective pressures unique to each anatomic niche, including temperature, nutrient availability, ph, oxygen tension, and the local immune environment. shortterm perturbations in the gut microenvironment caused by illness, antibiotic usage, or dietary changes (e.g., starvation) can alter the gut microbiome and subsequently lead to transient alterations in immune responses. thus, investigating whether influenza and other respiratory viruses alter the gastrointestinal microbiome could have mechanistic implications for viralmediated suppression of antibacterial immune responses. although the composition of the gastrointestinal microbiome is largely influenced by dietary patterns, respiratory viral infections could also contribute, along with other stress inducers such as broad-spectrum antibiotics exposure and chronic inflammation. using animal models of pulmonary infections by influenza and respiratory syncytial virus (rsv), multiple groups have shown that the gut microbiome is clearly impacted by respiratory viral infections, despite the lack of detectable respiratory virus in the gut ( ) ( ) ( ) ( ) ( ) . in a murine model of influenza infection, the investigators found that although the total numbers of bacteria in the gut did not decrease, there was a reduction in the quantities of segmented filamentous bacteria (sfb) and lactobacillus/lactococcus, accompanied by increases in enterobacteriaceae. interestingly, although sfb have previously been shown to induce th cells ( , ) , flu-infected mice had increased il- a levels and numbers of th cells in the small intestine and colon, which appeared to contribute to intestinal injury ( ) . in this study, antibiotic treatment prior to influenza infection ameliorated the degree of intestinal injury, but not lung injury, suggesting that gut dysbiosis contributed to local but not systemic inflammation. other groups have similarly reported increased proteobacteria (the phylum of which enterobacteriaceae are members) ( , ) , decreased firmicutes (which include sfb, lactobacillus and lactococcus species), and increased bacteroidetes ( ) following infection by flu or rsvs but not after administration of live attenuated influenza vaccine (laiv), indicating that live viral infection is required for these changes ( ) . the increase in proteobacteria appears to be mediated by type i interferons (ifns) ( ) , which not only depleted anaerobic bacteria but also increased susceptibility to secondary salmonella colitis. however, caloric restriction also figure | shifts in the mouse gut microbiome in the setting of influenza infection. during an acute respiratory viral infection, changes in the bacterial composition of the gut microbiome can be observed despite the absence of detectable virus in the gastrointestinal compartment. this suggests that systemic immune signals, physiologic changes (e.g., weight loss), and other still unknown factors are disrupting the normal ecology of the gut, thereby leading to dysbiosis. however, the majority of these studies have been conducted in laboratory animals housed under spf conditions. it remains to be determined whether human patients and mammalian hosts with more diverse baseline gut microbiota (i.e., mice in the wild), exhibit similar qualitative or quantitative changes. results in increased relative abundance of proteobacteria and increased bacteroidetes to firmicutes ratio, raising the possibility that decreased oral intake during influenza may contribute to changes in the microbiome ( , , , ) . it has also been shown that influenza infection alters intestinal microbiota composition through type ii ifn produced by lung-derived t cells recruited to the intestine ( ) . thus, changes in the gut microbiome appear to result not from direct viral effects but from systemic inflammatory signals that travel from the lung and trigger local inflammatory responses in the gut (figure ). interactions between respiratory tract infections and the gut microbiome are bidirectional. while respiratory viral infections can change the gut microbiome, the gut microbiome also shapes the adaptive immune responses against respiratory pathogens. mice pretreated with an antibiotic cocktail showed increased morbidity and mortality during influenza infection ( , ) . the severity of infection was associated with reductions in dendritic cell migration rate and the number of local t cells. mice given a week oral course of broad-spectrum antibiotics before respiratory viral infection mounted an attenuated anti-pr antibody response, were incapable of inducing cd + t cell-mediated ifn-γ response to pr antigen, and had fewer influenza-specific cd + t cells ( , ) . these mice also had higher viral titers in their lungs ( ) . germ-free mice and antibiotic-treated mice also exhibit impaired antibody responses to seasonal influenza vaccination, which was restored by oral administration of flagellated e. coli, demonstrating a dependence on tlr -mediated sensing of the host microbiota ( ) . the gut microbiome is essential for priming innate immune responses against pulmonary infections as well. during viral infections, the degree of macrophage response to respiratory viruses depends on the presence of gut microbes. macrophages from animals treated with antibiotics exhibited defective responses to type i and ii ifns and impaired capacity to limit viral replication, suggesting that intestinal microbiota provide immune stimulation that establishes an "activation threshold" for innate antiviral immune responses ( ) . a comparison of c bl/ mice from the jackson laboratory (which lack sfb in the stool) and taconic biosciences (which are sfb positive) revealed that sfb-deficient animals have increased lung bacterial burdens and more severe pneumonia when challenged with methicillin-resistant staphylococcus aureus (mrsa) ( ) , which was associated with decreased il- -mediated responses in the lung. another study using broad-spectrum antibiotic treatment followed by intranasal administration of s. pneumoniae in mice demonstrated that microbiome depletion led to decreased survival, increased lung bacterial burden, and increased systemic dissemination of bacteria ( ) . antibiotic-pretreated animals displayed altered cytokine profiles in the lung compared to untreated controls following s. pneumoniae infection, including significantly decreased tnf-α levels at and h after infection. additionally, in the microbiota-depleted group, alveolar macrophages and blood neutrophils exhibited decreased phagocytic activity, and decreased inflammatory cytokine production following ex vivo stimulation by toll-like receptor (tlr) ligands such as lipoteichoic acid (lta) ( ) . these effects might be mediated in part by decreased nod sensing of meso-dap (diaminopimelic acid)-containing peptidoglycan found in gut microbiota, which previously was shown to be essential for priming innate immune responses to s. pneumoniae ( ) . thus, antibiotic-induced disruptions in the normal gut microbial community alter multiple aspects of normal host defense against acute respiratory pathogens (figure ). collectively, the studies above suggest that modulation of the gastrointestinal tract microbiome plays an important role in acute respiratory infections, but precisely how the microbiome should be manipulated to promote appropriate immune responses during acute respiratory infections is unclear. currently, clinical studies have shown that although probiotics do not influence the incidence of respiratory tract infection, they do reduce the severity of symptoms and duration of the illness ( , ) . pinpointing which members of the gut microbial community are essential for proper immune priming is challenging, but necessary for guiding further microbiome-based therapies. clostridium orbiscindens, a member of the human gut microbiome, has been found to produce desaminotyrosine (dat) from metabolism of flavonoids and amino acids. antibiotic-treated mice exhibited markedly decreased fecal and serum dat levels, which was associated with attenuated type i ifn responses to influenza infection and increased mortality ( ) . thus, identification of dat-producing microbiota might serve as a modality for priming type i ifn responses against viral infections. another group demonstrated that oral administration of lactobacillus plantarum enhanced the type i ifn response and lowered viral titers in the lungs in a murine model of influenza infection ( ) . other lactobacillus strains are known to enhance tnf-α and ifnγ production by nasal lymphocytes upon influenza infection ( ) . oral administration of a probiotic cocktail containing lactobacillus restored the immune response and enhanced the activation of signaling pathways associated with recognition of single-stranded rna virus ( ) . an alternative approach to administering probiotics is to alter the local metabolic environment to regulate immune responses. a recent report demonstrated that animals fed a high fiber diet had increased generation of scfas, leading to enhanced antiviral cd + t cell immune responses and attenuated neutrophil-mediated lung injury during influenza infection, resulting in improved survival ( ) . thus, one strategy for decreasing the incidence of post-viral bacterial infections is to limit the severity of the primary viral infection. however, activation of antiviral immune responses, including type i and type ii ifns, have been associated with increased susceptibility to secondary bacterial pneumonia ( , ) . thus, another strategy is to enhance immune responses against common bacterial causes of pneumonia. one group re-colonized antibiotic-treated or germ-free mice with groups of cultivatable commensal bacteria, and found that administration of lactobacillus reuteri, enterococcus faecalis, lactobacillus crispatus, and clostridium orbiscindens, which are strong stimulators of nod (i.e., cytosolic receptor for muramyl dipeptide, which is found in cell walls of certain bacteria), are able to protect against bacterial pneumonia by enhancing gm-csf production ( ) . whether viral-induced changes in the gut microbiome is associated with immune defects that promote secondary bacterial pneumonia, or whether the impaired antibacterial defenses observed in virally-infected hosts can be restored by augmenting certain components of the microbiome are important areas to be investigated. the microbiome of the respiratory tract has also been investigated in the context of viral infections. its role in the development of secondary bacterial pneumonia following influenza and other acute respiratory viral infections is unclear. the respiratory tract is the main site of continuous contact with frontiers in immunology | www.frontiersin.org figure | effects of antibiotic pre-treatment on immune responses to influenza, streptococcus pneumoniae, and lipoteichoic acid (lta). the effects of the gut microbiome on immune responses to respiratory pathogens have been investigated by administration of oral antibiotics to generate alterations in the gut flora, followed by acute infection, and analyzing host immune responses compared to non-antibiotic-pretreated animals. multiple aspects of innate and adaptive immune responses are altered in antibiotic treated animals, including decreased antibody production, decreased phagocytic activity, and decreased inflammatory cytokine production by innate immune cells (e.g., alveolar and peritoneal macrophages) following ex vivo stimulation with tlr ligands. exogenous microbes. as is the case with the gut, immunity at the mucosal interface of the respiratory tract is a constant balance of tolerance of commensal and non-invasive microbes and immune activation against pathogens. the urt and lrt have similar microbial community compositions, although microbe densities are much higher in the former in healthy hosts. several factors are known to influence airway microbiome composition including infection history, age, genetics, and structural lung disease. the urt is an interconnected system consisting of the anterior nares, nasal cavity, nasopharynx, sinuses, eustachian tube, middle ear cavity, oral cavity, oropharynx, and larynx, each of which serve as distinct niches with their own microbial communities. in healthy adults, bacteria present in the nasal cavity are typically those associated with skin, predominantly members of the actinobacteria (e.g., corynebacterium spp., propionibacterium spp.), followed by firmicutes (e.g., staphylococcus spp.), and proteobacteria ( ) ( ) ( ) . the oropharynx contains members of firmicutes, proteobacteria, and bacteroidetes, including streptococcus, neisseria, haemophilus, and lachnospira spp. ( , , ) . skin and oral cavity lineages are represented in the nasopharynxe.g., streptococcus, staphylococcus, corynebacterium, and prevotella ( , , ) . a limited number of pathogens including streptococcus pneumoniae, neisseria meningitides, and haemophilus influenzae are commensal bacteria of the urt. in healthy individuals, the microbial community richness (i.e., the total number of bacterial taxa) is lower in the lrt than that in the urt ( , ( ) ( ) ( ) . contrary to dogma that normal healthy lungs are a sterile environment, a distinct, and somewhat dynamic lung microbiome can be identified using sequencing technology, with microaspiration serving as the primary route of microbial immigration from the urt to the lrt ( , ) . the major phyla in healthy lungs are bacteroidetes and firmicutes, which mainly include prevotella, veillonella, and streptococcus ( ) ( ) ( ) . individuals with chronic airway diseases (e.g., cystic fibrosis, copd) have increased bacterial populations in the lungs ( ) and differences in the relative abundance of certain species ( ) . impaired airway clearance due to intrinsic or extrinsic factors leads to the proliferation of bacterial species that can exploit this growth opportunity ( ) . how respiratory viral infection affects the diversity of microbial communities and whether viral-induced dysbiosis influences immune functions is being examined. nonetheless, bacterial colonization of the urt is generally considered as the first step in the development of invasive bacterial infections ( , ) , including secondary bacterial infections following respiratory viral infection. bacterial abundance, species diversity, and factors that shape the immune response to subsequent infections are discussed in greater detail below. respiratory viruses enter the human body through the urt and are the most common type of acute infections of the respiratory tract. one possible mechanism by which influenza and other viral infections might predispose infected hosts to secondary bacterial pneumonia is by altering the microbial composition of the upper respiratory tract, fostering enhanced growth of pathogens, and facilitating the subsequent entry of large bacterial loads into the lrt ( ) . this section will examine recent literature on how acute respiratory viral infections have changed the urt microbiome. given the effects of viruses on enhancing bacterial adherence to the epithelium ( ) ( ) ( ) , it is perhaps not surprising that multiple studies of human subjects as well as in animal models have shown that viral infections are associated with increased colonization by potentially pathogenic bacteria (known as "pathobionts"). a comparative analysis using qpcr to detect specific bacteria in adult patients with or without influenza a infection showed that staphylococcus aureus, s. pneumoniae, and h. influenzae were present in , , and % of infected patients, respectively as compared to , , and % of uninfected patients ( ) . in experimental in vitro models, viral infections increase the colonization rates of various bacteria in the urt ( - ), including s. pneumoniae and h. influenzae ( ) ( ) ( ) . in children, influenza is associated with a -fold increase in nasopharyngeal titer of s. pneumoniae ( ) . animal models have similarly confirmed that viral infection, particularly influenza, increases bacterial colonization rates in the urt, enhancing the risk of secondary bacterial infections ( ) ( ) ( ) . higher pneumococcal colonization density has been linked to respiratory virus coinfection and invasive pneumococcal pneumonia, after adjusting for age and sex ( ) . another case-control study comparing nasopharyngeal bacteria with and without pneumonia also found an association between nasopharyngeal load of s. pneumoniaebut not of h. influenza and m. catarrhalis-and viral coinfection and pneumonia ( ) . in addition, viral infections potentially may enhance transmission of bacteria. in a study of mice colonized with s. pneumoniae and then infected with influenza a virus days after, s. pneumoniae transmission occurred only when all mice were infected with influenza and was blocked by an influenza-neutralizing antibody ( ) . however, while specific bacteria might gain a competitive advantage during viral infections, this does not universally translate to all bacterial taxa. a recent study of subjects with and without respiratory viral infections demonstrated lower overall bacterial reads from nasopharyngeal samples in virally-infected subjects compared with uninfected controls ( ) . the relationship between acute viral infections and bacterial colonization appears to be bidirectional. bacterial carriage or their ligands can increase or decrease viral infectivity rate, thereby positively or negatively influencing the subsequent host immune response to viral infection. viral replication in the respiratory tract can be enhanced by exposure to s. pneumoniae ( ) . patients harboring s. pneumoniae are more likely to experience subsequent acute respiratory illness episodes than those without colonization ( ) . in addition, bacteria present in the airways can modulate host responses against viral infection. the presence of a nasopharyngeal commensal protected mice against rsv-induced airway hyperresponsiveness. rsv-infected mice who underwent antibiotic-mediated depletion of streptococcus viridans in the nasopharynx exhibited increases in number of inflammatory lymphocytes and airway hyperresponsiveness, and decreases in regulatory t cell number and transforming growth factor-β production ( ) . others have shown that colonization of the urt with s. aureus drastically reduced influenza-induced acute lung injury and mortality in mice by recruiting a c-c chemokine receptor type + cluster of differentiation (cd) b + monocyte subset to the lungs and inducing an m macrophage phenotype ( ) . with the availability of next-generation s rrna sequencing, microbiome-based studies have attempted to discern global patterns of change in the bacterial community of each anatomic niche during viral infections, such as changes in diversity. diversity can be assessed using a variety of indices, such as total number of unique species of the microbiome (i.e., richness) or other measures that account for both richness and the evenness of relative abundance of the members of the community (e.g., shannon index). results from microbiome analyses have not demonstrated consistent changes in diversity when comparing virally infected subjects with healthy controls. this is not surprising given the variability of the subjects sampled, differences in type and severity of viral infections, type and timing of sample collection, and analysis methodology. in some studies, increased bacterial diversity appeared to be associated with influenza severity. a french study of children admitted to the hospital with influenza revealed increased diversity of the nasopharyngeal microflora with increased influenza severity ( ) . children with severe influenza showed decreased relative abundance of s. aureus and increased abundance of prevotella, streptobacillus, porphyromonas, granulicatella, veillonella, fusobacterium, and haemophilus. a recent chinese study in patients with h n avian influenza demonstrated significantly increased diversity in the oropharyngeal microbiome of h n -infected patients compared to healthy controls, particularly h n patients with secondary bacterial pneumonia ( ) . conversely, a french study of nasopharyngeal samples and a south korean study of oropharyngeal samples from patients with acute respiratory viral infections both displayed decreases in diversity indices during viral infections compared to healthy controls ( , ) . both studies included subjects ranging from infants to adults > years of age, limiting conclusions about age-related effects. longitudinal studies conducted in healthy volunteers who underwent experimental self-innoculation with rhinovirus also failed to demonstrate significant changes in diversity of the urt microbiome, while administration of laiv vaccine to healthy adults led to increases in diversity measures following viral challenge ( , ) . thus, unlike other diseases where decreased diversity is considered deleterious to the host, the effects of viral infections on diversity per se are variable and not presently considered a good indicator of risk for complications, including secondary bacterial pneumonias. microbiome sequencing studies also enable investigators to identify changes in abundance among multiple bacterial taxa simultaneously, beyond just what can be cultured individually. this allows investigators to determine what groups of bacteria are changing in unison during viral infection and which are existing in competition with one another. this information may have implications for the development of probiotic therapies (as discussed below). a recent metagenomics-based study in france reported enrichment of s. aureus, s. pneumoniae, h. influenzae, moraxella catarrhalis and klebsiella pneumoniae in nasopharyngeal samples of subjects with confirmed respiratory viral infections compared to healthy controls ( ) . an examination of the oropharyngeal microbiome of pneumonia patients with and without influenza a h n pandemic viral infection showed that firmicutes (which include staphylococcus and streptococcus spp.) and proteobacteria (mainly pseudomonas amygdali, p. fluorescens, pseudomonas sp. uk , acinetobacter baumanii and a. junii)-were significantly enriched in patients with influenza ( ) . another study of patients with pandemic h n influenza infection revealed that the predominant phyla of the upper respiratory tract (nasal and nasopharyngeal samples) in patients harboring pandemic h n were actinobacteria, firmicutes, and proteobacteria although normal controls were not included; however, the authors suggested that flu is associated with an expansion of proteobacteria ( ) which is generally less abundant in healthy hosts. these findings are supported by another group who found that moraxella and enterobacter spp. (which are classified as proteobacteria) were the most highly represented bacteria in nasopharyngeal samples obtained from patients with pandemic h n influenza ( ) . however, these studies demonstrated that there was considerable inter-subject variability, highlighting the need for longitudinal studies to decipher changes following viral infection. investigators have also sought to determine whether specific viruses are consistently linked to enrichment of certain bacterial taxa. in the nasopharyngeal compartment of aboriginal and non-aboriginal children in australia, positive associations were detected between hrv and s. pneumoniae, h. influenza, and moraxella catarrhalis carriage as well as between adenovirus and m. catarrhalis ( ) . another study examining the presence of respiratory viruses by pcr panel and prevalence of bacterial carriage in the nasopharynx of children found a strong positive association between s. aureus colonization and influenza virus ( ). moreover, s. pneumoniae colonization was positively associated with the presence of hrv and enteroviruses; h. influenzae was positively associated with hrv and rsv; and m. catarrhalis colonization was positively associated with coronaviruses and adenoviruses. a s rrna sequencing-based study conducted in infants with acute rsv or hrv respiratory infections reported that infants with rsv had significantly higher abundance of staphylococcus spp. compared to hrv-infected infants ( ) . an analysis of the urt bacterial content of healthy asymptomatic individuals and patients with influenza virus, parainfluenza, hrv, rsv, coronavirus, adenovirus, or metapneumovirus by culture-independent pyrosequencing revealed six distinct bacterial profiles-i.e., streptococcus + prevotella + veillonella, streptococcus + haemophilus + neisseria, streptococcus, moraxella, haemophilus, and klebsiella. these profiles, however, were not associated with virus type but were linked to the age of subjects ( ) . given that many human studies are cross-sectional in nature, it remains unclear whether post-viral bacterial pneumonias might be the result of viral infections enhancing bacterial colonization or acquisition, colonizing bacteria influencing host susceptibility to respiratory viral infections, or a combination of both. another complicating factor particularly in cross-sectional studies examining the microbiome during viral infections is that the groups are not well-controlled and the sample numbers are relatively small considering the number of variables that could affect the respiratory tract microbiome-such as age, gender, oral hygiene and nose-picking habits, healthcare-based employment status, smoking status, medication use, exposure to small children, etc. the underlying type of viral infection, sampling timepoint after onset of infection, severity of infection, and concomittant antimicrobial usage are other confounding factors. this may underlie the highly variable and sometimes discrepant observations from microbiome studies in patients with viral infections. there have been few clinical studies comparing baseline pre-and post-infection microbiomes in otherwise healthy individuals with acute viral infections due to the difficulty of sampling before infection. however, the relatively few studies available provide insights into the dynamicity and stability of bacteria colonization patterns over time, and whether and how perturbations brought on by acute viral infections alter these patterns. in healthy children, the major phyla among nasopharyngeal microbiotas are proteobacteria, firmicutes, bacteroidetes, actinobacteria, and fusobacteria, with moraxella, haemophilus, streptococcus, flavobacteria, dolosigranulum, corynebacterium, and neisseria as predominant genera. changes in nasopharyngeal microbiome diversity were observed across seasons, with a predominance of proteobacteria and fusobacteria in fall-winter and bacteroidetes and firmicutes in spring; these differences were independent of recent antibiotics and viral co-infection ( ) . however, another analysis of two nasopharyngeal washes collected . - . months apart from children and adolescents with asthma showed no significant differences in nasopharyngeal microbiome diversity across seasons, although mean relative abundances of haemophilus, moraxella, staphylococcus, and corynebacterium varied significantly between summer and fall samples and between age groups. moreover, in . % of patients, operational taxonomic units (otus) in patients varied significantly between time points ( ) . an investigation of the frequency and seasonal variation in bacterial and viral load in asymptomatic healthcare professionals during the winter and summer months showed that of the subjects tested during the winter, were colonized with at least one bacterial species and tested positive for at least one virus. the most frequently detected pathogens were methicillinresistant staphylococcus aureus (mrsa), m. catarrhalis, and coronavirus. in contrast, of the subjects tested during the summer, harbored at least one bacterium (mainly mrsa and k. pneumoniae) and four tested positive for one virus ( ) . several larger scale surveillance studies of mainly pediatric populations have examined the natural temporal patterns in bacterial colonization during viral infections. one clinical investigation assessed the presence and density of s. pneumoniae, h. influenzae, and m. catarrhalis in the nasopharynx of children during urt infection and in the healthy state, and reported that the proportion of children colonized with these bacteria was higher during infection than during asymptomatic surveillance visits. mean density of all bacterial species was significantly higher at each visit when a virus was detected. interestingly, the percentage of colonized children and bacterial density were also higher at asymptomatic visits in which virus was detected than at those in which virus was not detected ( ) . another study of families with small children using longitudinal nasal swab sampling demonstrated that rhinovirus infection was associated with increased acquisition of s. pneumoniae from the community as well as increased transmission of s. pneumoniae within the family ( ) . other groups have examined the effects of experimental innoculation of hrv into the urt (nares) (figure ) . these studies reported no significant changes in total read counts or of the main phyla (e.g., actinobacteria, firmicutes, and proteobacteria) over time in nasopharyngeal samples ( ) or throat swabs ( ) . in the oropharyngeal compartment, rhinovirus infection was associated with a strong trend toward transient increases in the relative abundances of h. parainfluenzae, neisseria subflava and a weak trend toward an increase in s. aureus ( ) . by days, abundance of these bacteria had returned to baseline. nasopharyngeal sampling showed completely opposite results, with decreased relative abundance of haemophilus and neisseria spp., but an increase in the normal nasal commensal, propionibacterium, in subjects following hrv infection ( ) . no differences in staphylococcus were observed. however, the number of subjects were small in both studies, limiting the power to detect changes over time. nasopharyngeal microbiota composition has been shown to be altered by influenza vaccination (figure ) . administration of live attenuated influenza vaccine (laiv), which is nasally instilled, to healthy children increased the nasal colonization density of s. pneumoniae in subjects who harbored this bacterium at the time of vaccination, and transiently increased rates of colonization by h. influenza ( ) . in healthy adult volunteers, it was demonstrated that intranasal laiv administration induced an increase in the diversity of the nasopharyngeal microbiome, figure | changes in the human upper respiratory tract microbiome following viral exposure. given that bacterial pneumonia frequently arises as a result of aspirated bacterial pathogens, a potential mechanism by which viral infections might increase the risk of secondary bacterial infections is through increased colonization of the upper respiratory tract by bacterial pathogens. in human subjects, live attenuated influenza vaccine (laiv) and human rhinovirus (hrv) have been shown to disrupt the local host bacterial community, with increased relative abundance of potential pathogens (or pathobionts), such as staphylococcal and neisseria species. the major changes in the upper respiratory tract microbiome are highlighted here. as well as relative abundances of staphylococcus and bacteroides ( ). these changes were not observed in subjects given saline nasal spray. in a mouse model, bacterial density in the nasopharynx after laiv administration was increased as much as , times compared to influenza-naive hosts, and the duration of carriage of s. pneumoniae or s. aureus was also increased to -fold ( ) . however, systemic vaccination can also alter the urt microbiome. a longitudinal study of healthy subjects found a significant association between the presence of lactobacillus helveticus, prevotella melaninogenica, streptococcus infantis, veillonella dispar, and bacteroides ovatus and influenzaspecific h and h iga antibody response ( ) . thus, it is remarkable that a relatively mild viral stimulus such as flu vaccine can lead to detectable changes in the urt microbiome. although the data are still preliminary, animal studies have suggested that antiviral immune activation contributes to changes in the urt microbiome and facilitate colonization by potential pathogens, such as s. aureus. in a mouse model of s. aureus nasal colonization, the absence of type i ifn receptor was associated with decreased persistence of bacteria ( ) . type iii ifn, which is also induced during influenza infections, led to changes in the nasal microbiome, including increased numbers of culturable bacteria. increased upper respiratory tract persistence of s. aureus as well as increased risk of s. aureus pneumonia was observed in flu-infected wildtype mice compared to mice lacking the type iii ifn receptor ( ) . currently, however, is it unclear to what extent viral-induced changes in the urt microbiome alter subsequent immune responses against secondary bacterial infections. compared to studies of the urt microbiome, studies of the lrt microbiome following viral infections are relatively scarce due to the difficulty of obtaining uncontaminated samples from the lung. samples of convenience, such as sputum, suffer from oral contamination, but bronchoscopic samples are invasive and expensive to obtain on a regular basis. moreover, it is unclear whether outside of patients with chronic lung disease (e.g., copd), the lung microbial burden is of sufficient magnitude to exert robust effects on immune responses and risk of secondary bacterial infection during viral infection. data from a mouse model of influenza infection seem to indicate that flu infection has only a modest effect on bacterial counts, diversity and composition of the lung microbiome ( ) . in subjects with chronic obstructive pulmonary disease (copd) after hrv infection but not in healthy individuals, there was an increase in bacterial burden and growth of bacteria present at baseline, particularly h. influenzae ( ) . the researchers observed that the growth of bacteria seemed to arise from the existing community. s. pneumoniae intranasally inoculated into mice pre-infected with influenza virus first colonized the nose, followed by the trachea and lungs several days later with purulent inflammation. however, this effect was not observed in uninfected animals. this suggests that pneumococcal infection may sequentially develop from the urt to the lrt in influenza virus-infected subjects ( ) . thus, it is possible that some individuals with influenza infection might develop changes in their lung microbiome as a result of changes in their urt microbial communities. respiratory viruses not only alter the bacterial community in the urt, but also promote bacterial colonization of the lrt by a variety of mechanisms that impair bacterial clearance. first, mucus production in the respiratory tract is increased to facilitate viral clearance during infections. however, excessive mucus production can lead to airway obstruction by impeding mucociliary clearance ( ) . second, viral infections can also reduce ciliary beat frequency and the number of ciliated cells, disrupt the coordinated movement of cilia, and impede the repair of respiratory epithelial cells, further leading to reduced mucociliary clearance ( , ) . third, respiratory viral infections impair innate immune responses against bacteria ( ) ( ) ( ) . innate immune cells including macrophages and neutrophils are recruited to the lung by cytokines and chemokines for phagocytosis and bactericidal activity. prior viral infections dysregulate both alveolar macrophages ( , ( ) ( ) ( ) ( ) ( ) ( ) and neutrophils ( , , ) , thereby inhibiting bactericidal activity. thus, with multiple aspects of pulmonary host defense impaired, it would not be entirely surprising if a subset of influenza infected patients developed secondary bacterial pneumonia as a result of being unable to clear aspirated pathobionts from the urt. in addition to enabling us to determine what is present during states of health, large-scale sequencing-based microbiome analyses have also revealed who is not present during disease. it has long been appreciated that mechanisms have evolved in bacteria that confer competitive advantages, permitting them to survive in an otherwise inhospitable host environment. however, interspecies competition also maintains homeostasis of the microbial community, either through their abilities to capture scare resources (e.g., iron), or targeted killing of other bacteria (e.g., bacteriocins), preventing one microbe from dominating the community. thus, it is possible that the immune response incited by acute viral infections, changes in the host epithelial surface caused by the virus, or the virus itself might lead to elimination of a host commensal that is responsible for keeping pathobionts in check. for example, s. epidermidis and propionibacterium acnes abundance in the nares has been shown to be negatively associated with s. aureus carriage ( ) . understanding these interactions may create new avenues for therapeutic interventions aimed at reducing colonization by pathogenic bacteria during influenza epidemics or pandemics. one group of commensals that has been examined for its role in inhibiting nasal carriage by s. aureus and s. pneumoniae is corynebacterium spp. an early study in japan reported on the effects of introducing a corynebacterium strain into the nares of healthy adult hospital workers who were persistent carriers of s. aureus, with successful eradication in % of subjects ( ). the mechanism appeared to be bacteriocin-independent. in comparison, s. epidermidis implantation did not have an effect. whether the s. epidermidis strain used expressed the serine protease esp, which inhibits biofilm formation by s. aureus and nasal colonization ( ) , is unknown. subsequent studies by another group reported that c. pseudodiphtheriticum inhibited s. aureus growth, whereas c. accolens and s. aureus appeared to support each other's growth ( ) . conversely, other investigators observed that corynebacterium spp. were enriched in children who were not nasally colonized with pneumococcus, and demonstrated that c. accolens inhibit s. pneumoniae growth in vitro by expressing a lipase that releases free fatty acids from skin surface triacylglycerols, which inhibit pneumococcal growth. thus, painstaking identification and mechanistic interrogation of interspecies competition between commensals might lead to novel insights as to how viral infections might confer competitive advantage to pathobionts, and how to exploit natural strategies employed by commensals to restore homeostasis to the host microbial niche. interestingly, a recent preclinical study using a murine model of rsv and s. pneumoniae superinfection employed nasal priming by a c. pseudodiphtheriticum strain to augment host defense against the viral infection, which enhanced clearance of secondary bacterial challenge and reduced lung injury measures ( ) . finally, direct effects of the infecting virus on bacteria that comprise the microbiome may facilitate the transition from pathobiont to pathogen. a metagenomic analysis showed that ph n -associated airway microbiotas were enriched in genes associated with cell motility, transcriptional regulation, metabolism, and response to chemotaxis compared to the same bacteria in non-infected patients ( ) . these data imply that influenza infection perturbs the respiratory microbiome, leading to the production of secondary metabolites including immune-modulating molecules. viruses have also been found to impair bacterial biofilm formation and disrupt existing biofilm ( ) ( ) ( ) ( ) . influenza has been shown to affect the s. pneumoniae transcriptome in terms of downregulating expression of genes associated with the colonizer state and upregulations of bacteriocins ( ) . thus, direct effects of viruses on bacterial transcriptional patterns might be a mechanism by which colonizing bacteria acquire invasive potential, thereby leading to bacterial superinfections. there are several areas that must be addressed by future respiratory microbiome research. first, it is necessary to standardize protocols used to analyze the respiratory microbiome, including sampling, processing, and bioinformatics methodologies. for example, sputum may be an appropriate material for investigations of respiratory diseases since it contains components of the lrt and can be obtained easily. however, more reliable information on the lrt requires invasive samples such as bal or protected specimen brush frontiers in immunology | www.frontiersin.org or bronchial/lung biopsies. second, most studies are limited to experiments conducted in animal models. even in human studies, most analyses have been performed in a small number of patients and have described bacterial communities in the urt. the role of microbial communities outside of the lungs including gut, sinus, and skin should be considered in the context of airway diseases. third, most studies on the microbiome have focused on the bacterial component, and have largely omitted fungi and viruses. the role of viruses-including the vast number of phages that infect bacteria-and fungi in respiratory diseases cannot be examined through s rrna gene analyses, and there are no studies describing the composition and role of the respiratory virome due to the difficulty of comprehensive analyses for viruses. fourth, it is not sufficient to study microbial communities based on species composition; a functional characterization through transcriptome and proteasome analyses is necessary to understand mechanistic role of microbiome on outcomes of infection. finally, mucosal microbiome manipulations by vaccines, antibiotics, and probiotics in the gastrointestinal and respiratory tract niches represent novel approaches for the prevention, treatment, and management of acute and chronic lung diseases. however, given that antibiotic therapy could affect commensal bacteria and hasten the emergence of drug-resistant bacteria, more research is needed on the long-term effects of this therapy. animal models should be developed to study the influence of the urt and lrt microbiomes on immune responses to respiratory viral infections; only then will it be possible to consider the clinical application of microbiome modulation strategies. respiratory viral infections can initiate a cascade of host immune responses that alter microbial growth conditions in the urt, lrt, and the gut (supplemental table ). activation of influenzainduced antiviral interferon pathways can lead to inadequate innate immune cell responses during host defense against secondary bacterial infections, resulting in the proliferation of potentially pathogenic bacterial species. concomitant changes in the gut microbiome caused by the initial viral infection may also alter immune cell priming against secondary bacterial challenge, although this has not been examined to date. although the picture is incomplete, recent microbiome literature provides additional insights into the pathogenesis of dysregulated immune responses following acute viral infections, that may promote the development of secondary bacterial pneumonias (figure ) . clarifying the differences and dynamics of respiratory microbiota in healthy subjects and chronic lung diseases during acute respiratory viral infections can elucidate pathogenesis of viralbacterial interactions and provide a basis for developing novel approaches for the prevention, treatment, or management of acute respiratory infection and exacerbation of chronic lung diseases. sh and jd co-wrote the manuscript. sh, jd, and mp designed the figures and table. kc and mp edited and provided critical revisions of the manuscript. all authors approve the final version and agree to be accountable for the content of the manuscript. jd is supported by a research grant from the national institutes of health (grant no. r hl ). the views expressed in this article are those of the authors and do not necessarily reflect the position or policy of the department of veterans affairs or the us government. we thank cat meyer for her assistance with the figures. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material interactions between influenza and bacterial respiratory pathogens: implications for pandemic preparedness bacterial pathogens and death during the influenza pandemic updating the accounts: global mortality of the - "spanish" 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germline/maturation hypothesis, elicitation of broadly neutralizing antibodies against hiv- and cord blood igm repertoires date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: xiyeceib we have previously observed that all known potent broadly neutralizing antibodies (bnabs) against hiv- are highly divergent from their putative germline predecessors in contrast to bnabs against viruses causing acute infections such as henipaviruses and sars cov, which are much less divergent from their germline counterparts. consequently, we have hypothesized that germline antibodies may not bind to the hiv- envelope glycoprotein (env) because they are so different compared to the highly somatically mutated hiv- -specific bnabs. we have further hypothesized that the immunogenicity of highly conserved epitopes on the hiv- envelope glycoproteins (envs) may be reduced or eliminated by their very weak or absent interactions with germline antibodies and immune responses leading to the elicitation of bnabs may not be initiated and/or sustained. even if such responses are initiated, the maturation pathways are so extraordinarily complex that prolonged periods of time may be required for elicitation of bnabs with defined unique sequences. we provided the initial evidence supporting this antibody germline/maturation hypothesis, which prompted a number of studies to design vaccine immunogens that could bind putative germline predecessors of known bnabs and to explore complex b cell lineages. however, guiding the immune system through the exceptionally complex antibody maturation pathways to elicit known bnabs remains a major challenge. here, we discuss studies exploring the antibody germline/maturation hypothesis as related to elicitation of bnabs against hiv- and present our recent data demonstrating the existence of germline-like precursors of vrc antibodies in a human cord blood igm library. elicitation of broadly neutralizing antibodies (bnabs) targeting the hiv- envelope glycoproteins (envs), the key to an effective hiv- vaccine, remains elusive. previous studies have demonstrated several properties of the hiv- envs that could limit their ability to elicit bnabs. these include protection of the conserved structures by variable loops ( ) ( ) ( ) , remarkable genetic diversity ( ), a glycan shield ( ) , steric occlusion ( ) , and conformational masking ( ) . until years ago, only a handful of bnabs, including b , g , f , and e , were known. although the structural and functional studies of those bnabs revealed some important neutralization epitopes ( ) , such bnabs have not been successfully elicited by any vaccination approach. in , we first noted that in hiv- specific bnabs the number of amino acid mutations from their closest corresponding germline sequences was significantly higher than that of bnabs against the sars cov coronavirus, and hendra and nipah viruses, which cause self-limiting acute infections ( ) . using a large nonimmune igm library, we identified several hiv- env specific antibodies and found that they had fewer somatic mutations than the hiv- bnabs, as well as limited neutralizing activity ( ) . these findings indicated that elicitation of hiv- bnabs would require far more extensive maturation processes than those needed to generate the bnabs against the sars cov and henipaviruses. so, we have suggested that the difficulty of eliciting these bnabs may be due, at least in part, to the complex and prolonged maturation pathways required for the development of bnabs against the hiv- , which can take long time ( ) . we thus speculated that this may represent another significant challenge in the development of effective hiv- vaccines. we quantified the number of mutations in human monoclonal antibodies (mabs) that we selected from phage libraries generated from an hiv- -infected patient with a known time of infection ( ) . we calculated the number of amino acid mutations per heavy chain v gene, and defined it as antibody somatic mutational diversification (asmd). we compared the extent and dynamics of the asmd between hiv- -specific mabs and a panel of sars covspecific mabs. our experiments based on the asmd predicted that elicitation of hiv- -specific bnabs would take at least years. an illustrative mathematical model using the asmd rate based on www.frontiersin.org an exponential time dependent function suggested that a much longer time would be needed for the required maturation, unless somatic diversification had already been initiated from an intermediate antibody. thus, all these initial studies corroborated our hypothesis that the infrequent occurrence or absence of bnabs in hiv- -infected patients could be due, at least in part, to the lack or limited availability of b cell receptors that rapidly mature into bnabs. therefore, we suggested that appropriate immunization protocols of long duration need to be developed using the knowledge gained from the exploration of antibody maturation pathways in humans ( ) . from the striking observation that all known potent hiv- bnabs are highly divergent from their putative germline predecessors in contrast to bnabs against henipaviruses and sars cov coronavirus, we hypothesized that, since the germline antibodies are so different compared to the highly somatically mutated hiv- bnabs, they may not bind to the env. this led us to the hypothesis that the immunogenicity of the highly conserved epitopes on the hiv- native envelope glycoproteins (envs) is reduced or eliminated by their very weak or absent interactions with germline antibodies, which could not initiate and/or sustain immune responses leading to elicitation of bnabs: even if immune responses are initiated and sustained, the maturation pathways are so complex that help and long times may be needed for their elicitation. to test our antibody germline/maturation hypothesis, we designed germline-like antibodies corresponding to the known bnabs b , g , f , x , m , and m (the latter three antibodies were discovered in our laboratory and possess hiv- cross-reactivity with moderate neutralizing activities) and evaluated them for binding to envs ( ) . we found that while germline-like x , m , and m bound to envs with relatively high affinity, the germline-like precursors of b , g , and f failed to bind envs in an elisa assay although their corresponding mature bnabs bound strongly. these results provided initial evidence that the env structures containing conserved epitopes might not initiate humoral responses due to limited or absent binding to the germline precursors of bnabs. these germline precursors may also be of limited availability as recently reported ( ) . following that initial study, we expanded our investigation to different variants of the two different antibodies (b and x ) including their closest germline counterparts and several germline-like intermediates ( ) . the experiments showed that b intermediate antibodies neutralized only some hiv- isolates with relatively weak potency. in contrast, intermediates of x neutralized a subset of the tested hiv- isolates with efficiencies comparable to those of the matured x . these results helped explain the relatively high immunogenicity of the coreceptor binding site on gp and the abundance of cd -induced (cd i) antibodies in hiv- -infected patients (x is a cd i antibody) as well as the maturation pathway of x . in the case of b , germlinelike intermediates along the maturation pathway were shown to not only bind some envs but also human self antigens, suggesting that antigens other than the envs could help guide the immune system through the b maturation pathway. therefore, we proposed a conceptually new vaccination approach, in which it is critical to identify primary immunogens that bind to the germline antibodies that are predecessors of bnabs. if needed, these immunogens should be combined with secondary immunogens that recognize intermediate and/or matured antibodies to guide the immune system through the prolonged, complex maturation pathways ( ) . in this respect, we envisioned that the knowledge of human antibodyomes would become indispensable to elucidate the origin, diversity, and maturation pathways of bnabs and discover germline-like intermediates of bnabs that could provide a basis for the design of novel hiv- vaccine immunogens ( , ) . in recent years, several groups have reported a number of new bnabs that were identified from multiple hiv- infected individuals using designed novel antigen baits and advanced technologies implemented in isolating human mabs and high-throughput sequencing ( ) . particularly, haynes, kwong, stamatatos, scheif et al. have dealt with a large amount of data delineating structural, genetic determinants, and maturation pathways of different bnabs. these studies not only confirmed our previous findings that the envs fail to engage germline versions of bnabs but also suggested possible holes in b cell repertoires and demonstrated the implications of our antibody germline/maturation hypothesis for finding germline-like precursors, intermediates as well as for designing immunogens that could potentially bind to such bnab intermediates. in this report, we discuss the recent advancements in hiv- vaccine research in the context of the antibody germline/maturation hypothesis, and highlight critical factors to be considered when exploring germline-like precursors and intermediates of bnabs. we also report for the first time using sequencing data analysis of a human cord blood igm library to identify putative germline precursors of the heavy and light chains of vrc -like antibodies. these naturally occurring cord blood-derived vrc -like heavy and light chains may be useful as putative templates for designing novel vaccine immunogens that can lead to the elicitation of vrc -like antibodies and for understanding the maturation pathways of this bnab. still there are major challenges to be overcome. new empirical and semiempirical approaches could be successful; recently, new paradigms were discussed that could better fit our increased knowledge of hiv immunopathology and which could possibly be more helpful in guiding future vaccine research than did past unsuccessful approaches ( ) . dna isolation, amplification, and sequencing of the human cord blood igm library were previously described in detail ( , ) . for quality control, we trimmed the sequence reads and retained only sequences with lengths of more than nucleotides, covering the entire antibody variable domains consisting of all three complementarity determining regions (cdrs) along with framework regions (frs). we used imgt/highv-quest for immunogenetic and statistical analyses ( ) . the output results from the imgt/highv-quest analysis were stored in a local postgresql database, and structured query language (sql) was used to retrieve the data for further analysis. statistical calculations were carried out using jmp ® statistical software (sas institute, cary, nc, usa). antibody sequences from ighv - and igk - lineages were retrieved from our local antibodyome database consisting of immunogenetic data derived from sequencing of the human cord blood igm library using sql statements. amino acid sequence identities between each of the selected lineage sequences from the sequence data and pertinent germline sequences were calculated based on the pairwise alignment using local blast as implemented in bioedit v . . ( ) . phylogenetic analysis was carried out using the archaeopteryx software ( ) . our earlier observation of the extensive maturation of hiv- bnabs in contrast to those against some viruses causing acute infections led to the antibody germline/maturation hypothesis ( - , , ) . according to this hypothesis, it is critical to identify immunogens that would bind to germline and/or intermediate antibodies of bnabs, as well as the exploration of antibodyomes could be useful for identifying such immunogens ( ) . figure describes the timeline involving some of the key developments in current hiv- vaccine research focused on antibody germline-like intermediates and maturation pathways of bnabs. major research efforts in this direction were spearheaded by deep sequencing and structural biology studies of vrc -like and other cd -binding site (cd b) antibodies from hiv- -infected individuals. these studies delineated possible maturation pathways of such antibodies with high levels of somatic mutations and convergence in antibody recognition ( , ) . both studies revealed that the putative germline precursors of these antibodies had weak or no apparent affinity for env, and acquisition of a large number of somatic mutations were needed for the breadth and potency of these antibodies. these studies also explored antibody diversity and found many intermediates of similar lineages of the heavy chain genes from the two ighv families vh - and vh - that paired with different light chain genes. thus, analysis of the vrc -related antibodyome from hiv- infected patients revealed b cell maturation pathways that may help guide the vaccine-induced elicitation of such antibodies. however, if we could find germline-like intermediates of such bnabs from a naïve antibody repertoire, then www.frontiersin.org potential vaccine immunogens developed based on those templates would stimulate an adequate b cell immune response in healthy humans. to this end, we identified vrc -like intermediate antibodies from a naïve antibody library of human cord blood, which is presented later in the text. we previously analyzed the igm repertoires of healthy individuals and identified several intermediates of b from the vh - gene family ( ) . sequence analysis of , unique sequences from the igm repertoires revealed a cdrh with a length ( amino acids) and sequence similar ( %) to that of the b cdrh , but the v gene associated with that cdrh was found to be hv -b ( ) . this finding indicates that long cdrh s may not be a limiting factor for the development of bnabs ( ) although long cdrh motifs with certain amino acid preferences and/or associations with particular heavy or light chain families favoring polyreactivity may not be undermined. stamatatos and coworkers have conducted experiments screening a large panel of recombinant envs for binding to the germline predecessors of b , nih - , and bnc to test how env immunogens interact with the predicted germline versions of known bnabs ( ) . they found that the mature bnabs reacted with diverse envs but the corresponding germline antibodies did not. they examined in detail the germline b and its chimeric forms -either the germline heavy chain paired with the mature light chain and vice versa -to test whether they could interact with any of the recombinant envs derived from clade a, b, and c viruses. among all the recombinant envs tested, at least one env (qh ) was found to bind a b chimera with a mature heavy chain. however, this chimera failed to mediate calcium mobilization, indicating no bcr activation via bcr-antigen engagement. in other studies, they found that the elimination of certain conserved glycosylation sites on envs led to the binding of germline versions of vrc and nih - and bcr activation ( ) but that the modified envs did not interact with pg and - d germlines ( ) . haynes and coworkers have succeeded in finding envs capable of engaging the germline versions of a cd bs bnab, ch , while studying the co-evolution of the antibody in an hiv- infected patient ( ) . they found that ch is less mutated than most other cd bs bnabs, and importantly that the unmutated common ancestor of the ch lineage avidly bound the transmitted/founder hiv- envs. this finding suggests that early founder envs could bind optimally to the germline and intermediate versions of ch , and therefore, are promising vaccine immunogens, representing an important step forward in hiv- vaccine development. similarly, the maturation pathway of the potent v v -directed hiv-neutralizing antibody, cap -vrc , has been described, in which a germline-like intermediate with a -amino acid residue long cdrh was shown to bind and neutralize the superinfecting virus weakly, but did not bind or neutralize heterologous viruses ( ) . these results suggest that the cap -vrc lineage could be initiated by using a rare superinfecting-virus-like v v env. in another successful effort in identifying an env that could engage the germline versions of bnabs, scheif and coworkers devised a computation-guided approach combined with in vitro screening to engineer a gp outer domain. the designed protein not only bound to multiple vrc -class bnabs and their germline precursors but also activated b cells expressing diverse intermediates of the bnabs ( ) . therefore, priming with the protein and subsequent boosting with more native immunogens could help induce early somatic mutations and the ultimate elicitation of vrc -class bnabs. interestingly, nussenzweig and coworkers' study showed that somatic mutations of the frs and insertions of some bnabs are required for their broad and potent hiv- neutralizing activity ( ) . based on structural information, they made different germline versions of vrc , nih - , a , and bnc , and found that mutations in frs were also essential for binding, breadth, and potency of most bnabs. this suggested that certain framework mutations could be critical and should be preserved for designing the intermediates of such bnabs. several other studies mining the hiv- infected donors' antibodyomes ( - ) revealed putative intermediates of bnabs. many of them with lower levels of somatic hyper mutations could bind to selective envs; for example, intermediates of pgt - were able to preferentially bind native envs relative to monomeric gp ( ) . we also identified f -like antibodies (m and m . ) with much fewer mutations than f and suggested their use as a model system for elicitation of such antibodies ( , ) . all these newly discovered bnabs raise the hopes for effective hiv- vaccine development as they reveal characteristic features of bnabs that could help us understand the immunological basis critical for their production and also serve as templates for rational vaccine design. therefore, the focus has been dramatically shifted to explore and overcome the immunological hurdles associated with the elicitation of bnabs, namely, extensive somatic mutations of bnabs. major challenges remain in identification of intermediates with a minimal number of mutations, and appropriate env immunogens that would bind such intermediates and activate bcrs, which can lead to the maturation of the intermediate antibodies to bnabs. recently, new paradigms that better fit our increased knowledge of hiv immunopathology and which may be more helpful in guiding future vaccine research than did past unsuccessful approaches were discussed ( ) . we previously characterized the human cord blood cell-derived igm antibodies using sequencing to study gene diversity and somatic mutations ( ) . naïve germline antibody repertoires, particularly from babies, may be quite unique for understanding the b cell maturation pathways, as they can also mount an immune response against hiv- as recently found ( ) . our earlier gene usage analysis of the cord blood igm repertoire showed the biased ighv gene usages ( ) as similar to adult igm repertoires ( ) . however, we already noted that the ighv - gene usage was significantly higher in the cord blood igm repertoire, i.e., an overall contribution of % as compared to % in adult igm repertoires. this suggested that the cord blood igm repertoire may be advantageous for the exploration of the ighv - * lineages when studying germline precursors and intermediates of vrc heavy chain. a total of , heavy chain and , light chain sequences of ighv - and igkv - lineages, respectively, were used to frontiers in immunology | hiv and aids select the top sequences as closest intermediates for vrc in each heavy and light chain categories by using local blast searching. we performed phylogenetic analysis of the selected sequences to identify genetic relationships among vrc -like intermediates of heavy (figure a) and light (figure b ) chains. we found two of the antibody heavy chains, hwav and jhedt, which were % identical to the ighv - * germline sequence. remarkably, their cdrh sequences had the same length ( amino acids) as that of the vrc heavy chain. for these heavy chain sequences, the cdrh lengths ranged from to amino acids with sequence variations at the junctions. one of the germline sequences, jhedt, had a point mutation at cys tyr (kabat numbering) of cdrh that exactly mimicked the residue tyr of cdrh in vrc . the residue tyr at cdrh of vrc is most likely contributed by the ighd - * germline with a point mutation cys tyr. the other heavy chain sequence i at, which was the closest to vrc heavy chain, also had the same mutation at cys tyr. one of the other germline sequences, hwav , had trp b (kabat numbering) of cdrh that exactly mimicked the residue trp b of cdrh in vrc . intriguingly, the trp b residue is a junctional amino acid of the cdrh in germline hwav , and it exactly replicates the trp b junctional residue of cdrh in vrc . this suggests a possible maturation mechanism involved in the vrc -like intermediates where junctional amino acids could determine the maturation pathway far preceding the somatic hypermutation required for affinity maturation ( ) . most of the closest ighv genes, out of shown in the figure a , have at least one mutation in the v region, and www.frontiersin.org two sequences, g w t and gd c, have two mutations at each of the cdrh . the pre-existing amino acid mutations found in the v region and cdrh sequence information may inform the design of heavy chain germline-like precursors and intermediates, and help naturally reconstruct the b cell clonal lineages in the maturation pathways of vrc . light chain recognition of envs by vrc and vrc -related antibodies has been studied in detail using structural and sequencing data ( ) . the vrc light chain commonly uses the igkv - lineage and has a characteristic five amino acid long cdrl and a distinctive two amino acid deletion in cdr l . therefore, we selected the igkv - lineage sequences with five amino acid length cdrl s, but no sequences were found with a two amino acid deletion in cdr l ( figure b ). all of them had either framework or cdr mutations or both. four of them had a point mutation at cdrl and seven of them had a point mutation at cdrl . the structural basis for germline gene usage of vrc -related antibodies targeting the cd bs has been previously described ( ) , which revealed a set of signature features for these antibodies that were verified by mutagenesis. these signature features explained the origin of the igvh - gene and antibody resistance for some env sequences. we found that characteristic residues including the trp b of heavy chains were conserved while light chains did not have any characteristic residues as reported previously ( ) . however, other pre-existing amino acid mutations in light chains could have implications for the vrc -related intermediates with a characteristic cdrl of five amino acid length. we analyzed the amino acid length distributions of cdrh and cdrl sequences that were of vrc origins, namely, ighv - and igkv - for heavy and light chains, respectively, as derived from the human cord blood igm library (figure ) . the cdrh lengths ranged from to amino acids, indicating high cdr length diversity (figure a ). vrc has a cdrh length of amino acids, which is shorter than those of most other anti-hiv- antibodies ( ) . the lcdr lengths ranged from to amino acids ( figure b) . the cdrl of vrc has a characteristic length of five amino acids with a mature genetic signature ( ) . analysis of the human cord blood igm repertoire showed only a fraction of such light chains with a shorter length of five amino acids ( figure b) respectively. these plots show that there are position specific variations in the cdrh and cdrh regions of ighv - genes. these could indicate possible ighv - specific pre-existing amino acid mutations in cdrh and cdrh , as observed in several naïve antibody heavy chain sequences, which could inform the design of germline precursors and intermediates of vrc -like antibodies. we previously observed that the v-d-j rearrangement patterns occurred at different frequencies with , v-d-j combinations in a human cord blood igm repertoire ( ) . figure a shows the v-d-j diversity associated with ighv - gene sequences using a bubble plot for comparison with different d and j genes. the vrc heavy chain uses ighd - and ighj genes to recombine with ighv - . however, other vrc -related antibodies exhibit a skewed usage of ighj genes although at least three different ighj genes (ighj , ighj , and ighj ) are involved ( ) . as the human cord blood igm library has a large functional v-d-j diversity, it can be used to identify potential vrc -like heavy chain germline precursors and intermediates. in jawed vertebrates the expressed heavy chains may use any of the six ighd reading frames (rfs); however, rf is thought to be the preferred one as it mostly encodes tyrosine and glycine. the remaining five rfs encode either hydrophobic or charged amino acids, but the use of inverted rf , rf , and rf are discouraged. preferential usage of ighd rfs has been long implicated in b cell development and antigen-specific antibody production ( ) ( ) ( ) , and selected based upon its amino acid content ( ) . genetic control of igdh rf preference over the regulation of repertoire development has been recognized ( ) . here, we have analyzed the productive ighd rf usages in a human cord blood igm library. frequency distribution of rfs is plotted using a pie chart as depicted in figure b . we noted that there were not any highly restricted usages of the ighd rfs although some preferential usages depending on the ighd genes were found. this clearly indicates that ighd rfs diversity could add more diverse amino acid contents leading to enormous cdrh diversity. it may also be possible that intermediates with different rf choices play a critical role in selecting certain maturation pathways efficiently. the antibody germline/maturation hypothesis led to a paradigm shift in the design of immunogens for bnab elicitation, as well as the realization of the importance of the complexity of the bnab maturation pathways, and exploration of human antibodyomes ( ) . in fact, human antibodyome exploration is also promising for other fields of science and medicine ( , ) . this antibodyome approach is now a major direction of research in the hiv- vaccine field ( , ) . an important goal is to precisely identify naturally occurring germline-like precursors and intermediates of bnabs that could help designing novel immunogens, which could activate the corresponding bcrs and drive the immune system to produce bnabs within a short period of time. we presented an approach using a human cord blood igm library to identify putative germline precursors and intermediates of vrc -like heavy and light chains, which could be useful in reconstructing the b cell clonal lineages in the maturation pathways of vrc -related bnabs. this method has the potential to help in the identification of naturally occurring germline-like precursors and intermediates of any known bnab and in the development immunogens based on hiv- envs ( ) and peptides ( ) , as well as non-hiv- molecules ( ) . however, major challenges remain and new paradigms that better fit our increased knowledge of hiv immunopathology could possibly be more helpful in guiding future vaccine research than did past unsuccessful approaches ( ) . we thank the laboratory of molecular technology of saic-frederick, inc., for providing roche sequencing service. we thank tina ju for critically reading the manuscript. this research was supported by the intramural research program of the nih, national cancer institute, center for cancer research, the intramural aids targeted antiviral program (iatap) of the nih and by federal funds from the nih, national cancer institute, under contract nos. no -co- and hhsn e. the www.frontiersin.org the antigenic structure of the hiv gp envelope glycoprotein crystal structure of a soluble cleaved hiv- envelope trimer cryo-em structure of 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neutralizing antibodies revealed by structures and deep sequencing sequence and structural convergence of broad and potent hiv antibodies that mimic cd binding immunologic basis for long hcdr s in broadly neutralizing antibodies against hiv- recombinant hiv envelope proteins fail to engage germline versions of anti-cd bs bnabs engineering hiv envelope protein to activate germline b cell receptors of broadly neutralizing anti-cd binding site antibodies diverse recombinant hiv- envs fail to activate b cells expressing the germline b cell receptors of the broadly neutralizing anti-hiv- antibodies pg and - d co-evolution of a broadly neutralizing hiv- antibody and founder virus developmental pathway for potent v v -directed hiv-neutralizing antibodies rational hiv immunogen design to target specific germline b cell receptors somatic mutations of the immunoglobulin framework are generally required for broad and potent hiv- neutralization multidonor analysis reveals structural elements, genetic determinants, and maturation pathway for hiv- neutralization by vrc -class antibodies mining the antibodyome for hiv- -neutralizing antibodies with next-generation sequencing and phylogenetic pairing of heavy/light chains de novo identification of vrc class hiv- -neutralizing antibodies by next-generation sequencing of b-cell transcripts the effects of somatic hypermutation on neutralization and binding in the pgt family of broadly neutralizing hiv antibodies cross-reactive hiv- -neutralizing human monoclonal antibodies identified from a patient with f -like antibodies structural basis for hiv- neutralization by f -like antibodies m and m . early development of broadly neutralizing antibodies in hiv- -infected infants precise determination of the diversity of a combinatorial antibody library gives insight into the human immunoglobulin repertoire junctional amino acids determine the maturation pathway of an antibody structural basis for germ-line gene usage of a potent class of antibodies targeting the cd -binding site of hiv- gp forced usage of positively charged amino acids in immunoglobulin cdr-h impairs b cell development and antibody production preferential use of dh reading frame alters b cell development and antigen-specific antibody production b cell development regulated by gene rearrangement: arrest of maturation by membrane-bound d mu protein and selection of dh element reading frames the restricted dh gene reading frame usage in the expressed human antibody repertoire is selected based upon its amino acid content regulation of repertoire development through genetic control of dh reading frame preference the promise and challenge of high-throughput sequencing of the antibody repertoire b-cell-lineage immunogen design in vaccine development with hiv- as a case study immunogen design for hiv- and influenza recognition of synthetic glycopeptides by hiv- broadly neutralizing antibodies and their unmutated ancestors content of this publication does not necessarily reflect the views or policies of the department of health and human services, nor does the mention of trade names, commercial products, or organizations imply endorsement by the u.s. government. the guest associate editor marc h. v. van regenmortel declares that, despite having collaborated with the author dimiter s. dimitrov, the review process was handled objectively and no conflict of interest exists. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord- -vwrxentv authors: shivarov, velizar; petrov, peter k.; pashov, anastas d. title: potential sars-cov- preimmune igm epitopes date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: vwrxentv while studying the human public igm igome as represented by a library of , linear mimotopes, three exact matches to peptides in the proteins of sars-cov- were found: two in the open reading frame ab and one in the spike protein. joining the efforts to fast track sars-cov- vaccine development, here we describe briefly these potential epitopes in comparison to mimotopes representing peptides of sars-cov, hcov e and oc . the covid pandemic has put to test the capacity of vaccinology to produce as fast as possible relevant vaccines. a number of recent reports predict possible sars-cov- epitopes for vaccine development but there are no reports on experimentally defined b cell epitopes ( ) ( ) ( ) ( ) ( ) . the closest to identification of actual epitopes is the finding of pentapeptide sequences from the viral proteome in other known epitopes form iedb ( ) . a library of , mimotopes corresponding to the human public igm repertoire as represented in a plasma pool from , healthy donors was recently designed ( ) . the mimotopes were selected from a commercial amino acid random peptide phage display library (ph.d. , new england biolabs). conceptually, this mimotope library reflects at a certain level of detail, the repertoire of igm specificities in the plasma focusing on the recurring ones. the latter can be just natural antibodies or they may represent the product of fast extrafollicularly expanding igm clones that may serve as precursors of highly specific, somatically mutated, class-switched b cells. the preimmune repertoire has to be quasi-complete to provide for rapid expansion of clones reactive with any newly encountered antigen. the same may not be true for our library although, due to the polyspecific binding, most of the available public repertoire may be partially represented in it ( ) . here we report that the igm mimotope library contains heptapeptides identical to peptides in the proteome of sars-cov- . one of them may serve as a potentially neutralizing epitope on the spike protein. the design and the properties of the mimotope database has been published elsewhere ( ) . the available sequences of the genomes of sars-cov (nc_ . ), sars-cov- (asm v ), hcov e (nc_ . ), and hcovoc (ay . ) were split into consecutive overlapping heptamers shifted by one residue and the resultant sequence sets were compared to the sequences in the database of natural mimotopes. only exact matches were considered. the homologous sequences in the non-redundant databases of the human proteome and viridae (taxid: ) were blast searched using the ncbi blastp suite (https://blast.ncbi.nlm.nih. gov/blast.cgi?page=proteins). as part of an ongoing analysis, the natural mimotope database was represented as a graph by connecting the sequences having at least exact matches (i.e., of maximal hamming distance ). the graph had one giant component containing approximately % of the sequences which was further considered as the graph of interest. for the present study, the degrees of the vertices representing the natural sars-cov- epitopes, all of which belonged to the giant component, were used as the number of adjacent mimotopes parameter. for a set of words of length l based on an alphabet of l symbols, the theoretical average number of neighbors n at hamming distance d was calculated using the following formula for the number of neighbors: l d for the present study, l = , l = , and d < . for the first layer of neighbors n = and for the second n = . under the hypothesis that the database is a random sample from the set of heptamer peptides, the probability of the occurrence of each neighbor is: and the expected mean number of distinct neighbors at d < was calculated as p.(n( , )+n( , )) ≈ . . the value of p was used subsequently also in a binomial test to calculate the probabilities of finding equal or higher number of adjacent mimotopes ( table ) . the structure of the spike of sars-cov- was recently published [ vsb.pdb ( ) ]. it was used to visualize the molecular context of the spike epitope found. the visualization of the structure and the calculation of the relative solvent exposed surface were done using ucsf chimera, developed by the resource for biocomputing, visualization, and informatics at the university of california, san francisco, with support from nih p -gm . to demonstrate linear b cell epitope prediction uncertainty, we have reanalyzed data from he et al. ( ) on patients' sera reactivity to sars-cov peptides comparing them to bepipred (http://tools.iedb.org/bcell/help/#bepipred ) scores of the same sequences. a simple comparison for exact matches to peptides from the sars-cov- proteome yielded heptapeptides-two in the open reading frame ab ( aqtgiav and tkgphef ) and one in the spike protein ( ttldskt ). the expect value (e) is a parameter that describes the number of hits one can "expect" to see by chance when searching a database of a particular size. essentially, the e value describes the random background noise (https://blast.ncbi.nlm.nih.gov/ blast.cgi?cmd=web&page_type=blastdocs&doc_type= faq#expect). the e value of search results with so short sequences is very high and the mere number of sequences is not statistically significant. yet, this does not refute the fact that heptapeptides which are operationally defined mimotopes of human preimmune antibodies, are part of the viral proteome and, thus, represent (parts of) possible epitopes. on the other hand, the mimotopes in the database sometimes form non-random clusters of homologous sequences much like the mimotopes selected by a single monoclonal antibody. each one among , randomly selected heptamers should have on the average . homologous sequences in that same database that differ from it by up to mismatches. as seen from table , all sars sequences but not those from trivial hcov were members of clusters significantly greater than random (binomial test, p < . , false discovery rate adjusted). this is an indication that the presence of these sequences is non-random and they represent clusters of mimotopes representing well-represented individual (poly)specificities. an important prerequisite for the functionality of these epitopes is their degree of exposure to the solvent. the recently published structure of the spike (s protein) of sars-cov- ( ) shows that ttldskt forms a loop exposed to the solvent (figure a) . the relative solvent exposed surface greatly exceeds the threshold of % for participating in contacts ( figure b) . this loop is adjacent to the loop representing the epitope of the neutralizing antibody lca on the sars-cov spike ( , ) . presumably, it is similarly exposed further in the open conformation of the spike domains. the adjacent n-glycosylation sites are n and n . dependent on the size of the carbohydrate sidechains, they may partially occlude the epitope. the closest sequences in the human proteome are tltldskt of the prostate-specific transglutaminase (tgm ) and ttldski of mucin- [also known as ovarian tumor marker ca , q wxi . , ( ) ]. both are on tumor associated antigens ( , ) . while tgm is an intracellular antigen, mucin- is highly accessible on cell surfaces and in a soluble form. the mucin sequence ttldski is t/s biased, represents part of the highly o-glycosylated n-terminal part of mucin- and is predicted to be o-glycosylated itself. normally, such mucin protein core epitopes are occluded by glycosylation and thus, cryptic with respect to immune tolerance. yet, monoclonals to similar epitopes turned out to bind specifically to tumor expressed mucins ( ) ( ) ( ) ( ) which are aberrantly/hypo glycosylated. the sequences ttldskt has several exact matches in viruses outside the family coronaviridae in hypothetical proteins ( )]. the two predicted natural epitopes are overlaid in red. there is antibody reactivity in patients' sera to these epitopes although one of them has bepipred score far below the threshold of . . of various phages. at least one of them infects l. plantarum which is a common species in the gut microbiome. it is not surprising that the public igm repertoire has clones potentially capable of binding to non-conserved regions of novel viruses. similarly, the igm igome contained sequences found also in sars-cov, although the epidemic was too restricted to be reflected in the antibody repertoires of the donors (table ) . furthermore, no signs of persistent antibody titers after exposure were observed. the representation of clones reactive with the trivial human coronaviruses e and oc was rather narrower than that of the unknown strains. some of the epitopes were conserved between sars-cov and sars-cov- (aqtgiav and tkgphef) but they were found in non-structural proteins and are hardly targets for neutralizing antibodies ( table ) . on the other hand, all potential epitopes found could play a role in targeting the viral proteins to specific b cells which produce the bulk of natural igm. the latter are known to be excellent antigen presenting cells able to prime cd + t cells, and initiate th immune responses ( ) ( ) ( ) in antigen specific manner much like activated specific b cells ( ). it has been shown that b cells secreted igm is a non-redundant and essential arm of the humoral responses to influenza in mice ( ) . this implies that natural antibody epitopes might be essential components of subunit vaccines even though they may not represent typical dominant epitopes. the role of overlapping t and b cell epitopes is not clear except when the b cell receptor has a high enough affinity for the epitope to protect it during processing ( ) , but it is interesting that one of the sars-cov natural epitopes ( ttstalg ) is also part of a cd t cell epitope in the context of hla-dr b * : ( ) . using the iedb preferred method the epitope ttldskt is predicted to overlap a potential class ii epitope in the context of hla-drb * : , while two other potential epitopes just up-and downstream overlap it partially (in the context of hla-dpa * : /dpb * : and hla-drb * : , hla-drb * : and hla-drb * : , respectively). in this respect, maybe a more useful epitope would be the continuous sequence niirgwifgttldsktqsllivnnatnv . the current thinking separates the repertoire of natural and induced antibodies ( ) . the preimmune igm mimotopes we describe could represent also epitopes of naïve b cell clones which may have undergone extrafollicular expansion poised to initiate also follicular immune responses. as to the capacity of these epitopes to induce fully mature antibody response, it is interesting to note that the two preimmune igm epitopes found for the spike of sars-cov ( ttstalg and vkgddvr ) are proven antibody targets in approximately one fourth of the sars patients ( ) . thus, our mimotope library has the capacity to identify potential true precursor epitopes and not only natural antibody epitopes. furthermore, a recent report indicates the importance of igm antibodies in the control of the diseases in mild cases of covid ( ) . thus, it is quite possible that the sars-cov- spike epitope ttldskt is bound by b cells that will contribute to the induced immune response. none of the in silico predicted epitopes ( - ) overlaps with ttldskt which is also specific to sars-cov- . the correlation between the actual reactivities in sars-cov patients' sera and the bepipred score ( figure c) confirms the low power of linear b cell epitope predicting algorithms, and underlies the necessity to base the proposals of new epitopes as much as possible on actual binding data. these considerations make the novel sars-cov- epitopes valid targets in the search for a vaccine for covid- . the whole paradigm followed here focuses exclusively on the relatively rare linear epitopes. a lot more information about conformational epitopes may be hidden in the natural mimotope database but the approaches for sorting out clusters of mimotopes defining a conformational epitope are still being developed. the proposed actual preimmune igm epitopes of sars-cov- can be instrumental both as parts of subunit vaccines or in the design of nanoparticlebased vaccines but also in the development of therapeutic monoclonal antibodies. the datasets analyzed and the scripts for this study can be found in the github repository (https://github.com/ansts/ sars-cov- ). vs and ap: conceptualizing, manuscript preparation, and data analysis. pp: data analysis. novel antibody epitopes dominate the antigenicity of spike glycoprotein in sars-cov- compared to sars-cov structure, function and antigenicity of the sars-cov- spike glycoprotein candidate targets for immune responses to -novel coronavirus (ncov): sequence homology-and bioinformatic-based predictions preliminary identification of potential vaccine targets for the covid- coronavirus (sars-cov- ) based on sars-cov immunological studies epitopes for a -ncov vaccine diagnostic profiling of the human public igm repertoire with scalable mimotope libraries identification of immunodominant sites on the spike protein of severe acute respiratory syndrome (sars) coronavirus: implication for developing sars diagnostics and vaccines cryo-em structure of the -ncov spike in the prefusion conformation unexpected receptor functional mimicry elucidates activation of coronavirus fusion muc (ca ): tumor biomarker to cancer therapy, a work in progress overexpression of transglutaminase and prostate cancer progression: a potential predictor of less favourable outcomes effect of modification of carbohydrate side chains on the reactivity of antibodies with core-protein epitopes of the muc gene product development and characterization of breast cancer reactive monoclonal antibodies directed to the core protein of the human milk mucin epitope mapping of anti-muc mucin protein core monoclonal antibodies epitopes of muc tandem repeats in cancer as revealed by antibody crystallography: toward glycopeptide signatureguided therapy an overview of b- cells as antigenpresenting cells b cells are the dominant antigen-presenting cells that activate naive cd + t cells upon immunization with a virus-derived nanoparticle antigen b- and b- cell-derived immunoglobulin m antibodies are nonredundant components of the protective response to influenza virus infection modulation of antigen processing by bound antibodies can boost or suppress class ii major histocompatibility complex presentation of different t cell determinants searching immunodominant epitopes prior to epidemic: hla class ii-restricted sars-cov spike protein epitopes in unexposed individuals inherent specificities in natural antibodies: a key to immune defense against pathogen invasion breadth of concomitant immune responses prior to patient recovery: a case report of non-severe covid- the authors wish to thank prof. angel galabov for critical reading of the manuscript. key: cord- - ewvwg o authors: rudd, christopher e. title: gsk- inhibition as a therapeutic approach against sars cov : dual benefit of inhibiting viral replication while potentiating the immune response date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: ewvwg o the sars-cov (covid- ) pandemic and uncertainties in developing a vaccine have created an urgent need for new therapeutic approaches. a key question is whether it is possible to make rational predictions of new therapies based on the presently available scientific and medical information. in this regard, i have noticed an omission in the present analysis in the literature related to the exploitation of glycogen synthase kinase (gsk- ) as a therapeutic approach. this is based on two key observations, that gsk- inhibitors can simultaneously block sars viral replication, while boosting cd + adaptive t-cell and innate natural killer (nk) responses. firstly, it is already clear that gsk- phosphorylation of sars cov n protein on key serine residues is needed for viral replication such that small molecule inhibitors (smis) of gsk- can inhibit viral replication. in comparing protein sequences, i show here that the key sites in the n protein of sars cov n for replication are conserved in sars cov . this strongly suggests that gsk- smis will also inhibit sars cov replication. secondly, we and others have previously documented that gsk- smis markedly enhance cd + cytolytic t-cell (ctl) and nk cell anti-viral effector functions leading to a reduction in both acute and chronic viral infections in mice. my hypothesis is that the repurposing of low-cost inhibitors of gsk- such as lithium will limit sars-cov infections by both reducing viral replication and potentiating the immune response against the virus. to date, there has been no mention of this dual connection between gsk- and sars cov in the literature. to my knowledge, no other drugs exist with the potential to simultaneously target both viral replication and immune response against sars cov . the sars-cov (covid- ) pandemic and uncertainties in developing a vaccine have created an urgent need for new therapeutic approaches. a key question is whether it is possible to make rational predictions of new therapies based on the presently available scientific and medical information. in this regard, i have noticed an omission in the present analysis in the literature related to the exploitation of glycogen synthase kinase (gsk- ) as a therapeutic approach. this is based on two key observations, that gsk- inhibitors can simultaneously block sars viral replication, while boosting cd + adaptive t-cell and innate natural killer (nk) responses. firstly, it is already clear that gsk- phosphorylation of sars cov n protein on key serine residues is needed for viral replication such that small molecule inhibitors (smis) of gsk- can inhibit viral replication. in comparing protein sequences, i show here that the key sites in the n protein of sars cov n for replication are conserved in sars cov . this strongly suggests that gsk- smis will also inhibit sars cov replication. secondly, we and others have previously documented that gsk- smis markedly enhance cd + cytolytic t-cell (ctl) and nk cell anti-viral effector functions leading to a reduction in both acute and chronic viral infections in mice. my hypothesis is that the repurposing of low-cost inhibitors of gsk- such as lithium will limit sars-cov infections by both reducing viral replication and potentiating the immune response against the virus. to date, there has been no mention of this dual connection between gsk- and sars cov in the literature. to my knowledge, no other drugs exist with the potential to simultaneously target both viral replication and immune response against sars cov . (tocilizumab), inhibitors of the co-receptor serine protease tmprss (such as camostat mesylate), soluble receptor ace as a decoy, amongst others ( ). a key objective will be to inhibit sars cov transcription while potently boosting the immune response against the viruses and limiting the cytokine release syndrome (crs) associated with severe disease. crs is mediated predominately by t-cells and inflammatory myeloid lineage cells, the latter pathogenically licensed primarily by cd + tcells. in this light, the potential for the use of inhibitors of the serine/threonine kinase glycogen synthase kinase (gsk- ) has passed somewhat unnoticed. the hypothesis underlying this article is that gsk- inhibitors will both inhibit sars-cov replication and potentiate cd + t-cell responses for enhanced viral clearance. it should therefore be considered as a new therapeutic approach. the sars-cov has four structural proteins, known as the s (spike), e (envelope), m (membrane), and n (nucleocapsid) proteins. the n protein holds the rna genome and is needed for transcription of the viral genome, while the s, e, and m proteins together create the viral envelope ( ). the sars cov n protein is phosphorylated by gsk- , an event that is needed for viral replication ( - ). the inhibition of gsk- with small molecule inhibitors (smis) prevents sars cov n phosphorylation, and in the process, limits sars cov viral replication ( , , ). as seen in figure a , in comparing sequences, it is clear that the sars cov n protein sequence has the same conserved key serine residues (serine and ) as found in the sars cov n protein. most residues surrounding these key phosphorylation sites are identical between sars cov and sars cov . it therefore seems likely that gsk- phosphorylation of the serine residues in sars cov will occur as in related sars cov . gsk- inactivators also inhibit the coronovirus protease (m pro ) (or c-like protease) ( ) that cleaves the sars-cov- encoded polyproteins (pp a and pp ab) needed for viral replication and transcription ( ) . from these two angles, it is a reasonable hypothesis that the inhibitors of gsk- that inhibit sars cov replication will inhibit sars cov replication. to date, this has not been interrogated, but could be rapidly tested in in vitro assays using vero or t cells. secondly, studies from my lab and others have shown that gsk- negatively regulates t-cell proliferation and function ( ) ( ) ( ) . gsk- is most active in resting t-cells, keeping cells in a quiescent state. this function is unlike of other kinases such as p lck which initiate the activation of t-cells ( ) . as a consequence, the inhibition of gsk- with the small molecule inhibitors (smis) such as sb markedly enhance adaptive cd + cytolytic cell (ctl) function ( , ( ) ( ) ( ) (figure b) . this potentiating effect is due in part to the upregulation in expression of the transcription factor t-bet (tbx ) ( ), a central regulator of th differentiation ( ) . t-bet, in turn, inhibits the transcription and expression of inhibitory receptors pd- and lag , while promoting the expression of cytolytic effector molecules in cd + t-cells, granzyme b, perforin and interferongamma ( , ) . further, and salient to this hypothesis, gsk- smis help resolve viral infections in mice, acute infection by the murine gamma-herpesvirus , and chronic infection with the lymphocytic choriomeningitis clone (lcmv) ( ) . the effects of gsk- were preferentially seen in cd ctls, and to a lesser extent, cd + t-cells, the latter contributing to the crs seen in the more severe clinical manifestations of covid- . gsk- inhibitors have also been found to induce the suppressive cytokine interleukin (il- ) in cd + t-cells which might dampen crs in severe disease ( ) . il- limits the immune response and prevents tissue damage in infection and autoimmune disease ( ) . lastly, gsk- inhibition drives the maturation and function of natural killer (nk) cells ( ) . natural killer (nk) cells are effector cells of the innate immune system and also important in the control of viral infections ( ) . the inhibition of the gsk- pathway, therefore, plays central roles in promoting both the adaptive and innate immune responses against viruses. the evidence presented here strongly suggests for the first time that gsk- inhibitors could constitute an effective therapy in restraining the progression of sars cov- infections. to my knowledge, no other drugs exist which might simultaneously target both sars cov- viral replication, and the immune response against the virus. it would be a novel and affordable therapeutic approach with the potential dual targeting both the virus and immune system in the treatment of covid- patients. in the immediate term, lithium chloride could be administered to patients on a compassionate basis given that citrate, orotate, and carbonate salt formations of the drug are in wide clinical use for the treatment of bipolar disorders. various side effects such as nausea have been reported, although these can be minimized by gradually increasing doses to the desired strength. renal side effects seen in some patients can also be ameliorated with proper drug monitoring ( ) . relative to the life-saving potential of gsk- inhibition in the treatment of covid- , the side effects are a minor consideration. with time, more specific gsk- reagents such as sb could be tested in clinical trials. both atp competitive and potentially more selective allosteric non-competitive inhibitors could be used. the inhibitor tdzd- has been tested in preclinical models of cancer ( ) , while another inhibitor tideglusib has been in phase ii clinical trials for alzheimer's disease and progressive supranuclear palsy where it is welltolerated ( , ) . others recently noted the potential of gsk- inhibitors given effects on other viral infections, but failed to note the homologous regulatory phosphorylation sites in sars cov and cov ( ) . it is these known and previously unknown target effects which i argue are key to the potential success of gsk- inhibition in the treatment of covid- . all risks should be understood prior to administration of any treatments and precautions taken under the close supervision of a physician. the original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s. the sars-cov- (covid- ) pandemic and uncertainties in developing a vaccine have created an urgent need for new therapeutic approaches. in this opinion or hypothesis article, i propose the exploitation of glycogen synthase kinase (gsk- ) as a therapeutic approach. this is based on two key observations, that gsk- inhibitors can simultaneously block sars viral replication, while boosting cd + adaptive t-cell and innate natural killer (nk) responses. my hypothesis is that the repurposing of low-cost inhibitors of gsk- such as lithium chloride will limit sars-cov- infections by both limiting viral replication and potentiating the immune response against the virus. the author confirms being the sole contributor of this work and has approved it for publication. sars-cov- vaccines: status report news feature: to counter the pandemic, clinicians bank on repurposed drugs analysis of therapeutic targets for sars-cov- and discovery of potential drugs by computational syndrome coronavirus nucleocapsid protein and viral replication identification of phosphorylation sites in the nucleocapsid protein (n protein) of sars-coronavirus inhibition of cell proliferation by sars-cov infection in vero e cells structure of m(pro) from sars-cov- and discovery of its inhibitors the crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor glycogen synthase kinase : a kinase for all pathways? glycogen synthase kinase inactivation drives t-bet-mediated downregulation of co-receptor pd- to enhance cd (+) cytolytic t cell responses negative regulation of t cell proliferation and interleukin production by the serine threonine kinase gsk- the cd receptor is complexed in detergent lysates to a protein-tyrosine kinase (pp ) from human t lymphocytes two strings in one bow: pd- negatively regulates via co-receptor cd on t cells small-molecule inhibition of pd- transcription is an effective alternative to antibody blockade in cancer therapy glycogen synthase kinase inactivation compensates for the lack of cd in the priming of cd (+) cytotoxic t-cells: implications for anti-pd- immunotherapy. front immunol t-bet in disease small molecule inhibition of gsk- specifically inhibits the transcription of inhibitory co-receptor lag- for enhanced anti-tumor immunity glycogen synthase kinase- controls il- expression in cd (+) effector t-cell subsets through epigenetic modification of the il- promoter the regulation of il- production by immune cells gsk inhibition drives maturation of nk cells and enhances their antitumor activity nk cells in host responses to viral infections what we need to know about the effect of lithium on the kidney rapid and selective death of leukemia stem and progenitor cells induced by the compound -benzyl, -methyl, , , -thiadiazolidine, , dione (tdzd- ) treatment of alzheimer's disease with the gsk- inhibitor tideglusib: a pilot study gsk- inhibitors: preclinical focus on cns international group for the study of lithium treated, lithium's antiviral effects: a potential drug for covid- disease? the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © rudd. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -a pahdr authors: maron-gutierrez, tatiana; rocco, patricia r. m. title: cell-free therapies: novel approaches for covid- date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: a pahdr nan first described in late , coronavirus disease (covid)- , caused by the severe acute respiratory syndrome coronavirus (sars-cov- ), rapidly escalated into a global pandemic with a high case fatality rate. covid- patients with acute respiratory failure exhibit some distinctive pathological characteristics, which to some extent resemble the acute respiratory distress syndrome (ards), sars, and middle east respiratory syndrome (mers); these include hypoxemia, diffuse alveolar damage with cellular exudates, extensive pulmonary inflammation, lung edema, and hyaline membrane formation ( ) . in addition to respiratory failure, these patients present with a dysfunctional systemic host response that affects multiple organs, including the central nervous, cardiovascular, renal, and gastrointestinal systems ( ) ( ) ( ) , as well as a wide range of coagulation disturbances, such as thrombocytopenia, sustained systemic clotting activation, massive thrombin and fibrin formation, and disseminated intravascular coagulation ( ) . few therapies are effective for covid- patients, and to date there are still no vaccines available. there are vaccine candidates in development and ongoing clinical trials, but they are expected to be available, at best, in early ( ) . thus, novel effective and safe therapies are urgently required to treat covid- patients ( ) . in this context, several clinical trials have begun pursuing new therapies as well as repurposing existing ones, largely through drug repositioning, including of antiviral, antimalarial, and anti-inflammatory agents. these therapies aim to target entry of the virus into host cells, multiplication of the viral genetic material, and/or the immune response and inflammatory process ( ) . although some therapies shows promising results, they raise some concerns, such as limited cohort sizes, non-randomized trials, lack of considerations for gender, comorbidities, concurrent treatments, and route of drug delivery, among others ( ) . early in the covid- pandemic, corticosteroids were not recommended because of the adverse effects previously observed in influenza, sars-cov, and mers-cov infections ( ) . nevertheless, a recent controlled, open-label trial of dexamethasone for up to days resulted in lower -day mortality in mechanically ventilated patients ( ) . mesenchymal stromal cell (msc)-based treatment has been proposed as a suitable therapeutic approach for covid- ( ) . as mscs have the potential to interact directly with immune cells, their transplantation may improve outcomes in covid- patients through modulation of the immune response, mitigation of the inflammatory cascade, and promotion of tissue repair and regeneration ( , ) . in addition, cd , the second entry receptor for sars-cov- , can be expressed by tissue-specific stem cells ( ) . together with the loss of airway epithelial cells by viral infection and replication, the additional loss of regenerating stem cells may be responsible for diminished cellular and lung regeneration ( ) . cell-based therapies have been quite extensively studied for potential applicability in covid- , especially given the short time since the onset of the pandemic ( , ) ; however, due to the risk of macro-and microthrombosis, cell-free therapies may be more appealing. cell-free therapies might decrease injury to different organs, such as lung, heart, kidney, liver, and brain, as well as reduce thrombus formation and endothelial inflammation (figure ). cell-free therapies, such as the msc secretome (obtained as conditioned medium) and extracellular vesicles (evs) from mscs, have been studied in ards ( ) and multiple organ dysfunction syndrome (mods) ( , ) for their antiinflammatory and anti-fibrogenic effects, as well as their epithelial and endothelial regenerative properties. however, figure | potential effects of pharmaceutical preparations of msc secretome on different organs involved in covid- . the msc secretome, in the form of conditioned medium containing extracellular vesicles (evs) and mitochondria, could be transformed into a stable product for the treatment of patients with covid- . secretome-based therapies might mitigate cardiac, kidney, liver, nervous system, and lung injury; decrease macro-and micro-thrombus formation and endothelial inflammation; and repair lung epithelial and endothelial cells. many researchers and international societies, including the international society for extracellular vesicles (isev) and the international society for cellular and gene therapies (isct), have expressed concern regarding the use of evs-whether derived from mscs or from other cell sources-in the treatment of covid- ( ) . clinical trials are encouraged; however, the use of evs for any purpose in covid- is not endorsed by isev and isct until proper regulation of manufacturing, quality control protocols, and clinical trial design are in place, in order to avoid the stem-cell industry trying to sell unregulated msc treatments ( ) . in this context, the implementation of computer-controlled bioreactors ( ) and the development of standard operating procedures (sops) for obtaining a good manufacturing practice (gmp)-grade msc secretome and its components are necessary for clinical applications ( ) . these must be reproducible, scalable, and well-controlled to limit heterogeneity and enhance predictability in the composition and function of secretome-derived products ( ) . further studies are still needed to better understand the best route of cell-free therapy delivery, dose, and timing of administration ( ) . moreover, important factors should be taken into consideration such as the culture medium, cell and tissue source, donor variability, and culture conditions (cell priming with hypoxia, biochemical or mechanical stimuli, three-dimensional spheroid culture, among others), as well as the timing and method of msc-secretome harvesting ( , ) . in short, cell-free therapies could be a more suitable treatment for covid- than mscs, but additional investigations are required ( ). the msc secretome is a complex mixture of soluble components (growth factors and cytokines), a vesicular portion that comprises evs, and cell organelles (e.g., mitochondria) ( ) ( ) ( ) . considering that sars-cov- infection is being associated with an increased inflammatory process ( ), we believe that msc secretome products might help reverse covid- related immune dysregulation, due to their anti-inflammatory, immunomodulatory, and regenerative effects ( ) . the msc secretome has properties similar to those of its parent mscs ( ) . moreover, the secretome is generally considered safer than parent cells, since it ( ) lacks the potential for endogenous tumor formation, as it cannot self-replicate; ( ) can be classified as non-immunogenic, due to the limited number of antigenic components; and ( ) may lead to less formation of emboli when injected intravenously ( , ) . as recently reported elsewhere, the msc secretome (in the form of conditioned medium) can be stored more easily than mscs ( ) , which is an important consideration given the lack of adequate facilities in developing countries. transforming msc-secretome components into a freeze-dried, stable powder product which can be reconstituted for intravenous injection or inhalation might be a suitable approach for the treatment of patients with covid- (figure ) ( ) . mitochondria are intracellular organelles that play a vital role in cellular homeostasis and enable stress adaptation ( ) . most cellular energy generation takes place in the mitochondria ( ), and excessive mitochondrial dysfunction leading to defects in energy flow leads to unsustainable maintenance of life and adaptation to stress ( ) . one of the main mechanisms associated with the pathophysiology of sepsis is mitochondrial dysfunction ( , ) . in , the first evidence that mscs restore alveolar bioenergetics through cx -dependent alveolar attachment and mitochondrial transfer was observed in experimental ards ( ) . in , phinney et al. observed mitochondrial transfer from mscs to macrophages in response to oxidative stress ( ) . recently, court et al. investigated the effect of mscmediated transfer of mitochondria on lymphoid cells. they observed mitochondria-labeled mscs mainly in cd +t cells, paving the way for exploration of organelle-based therapies in immune diseases ( ) . interestingly, mscs are not the only cell type able to transfer mitochondria. lipopolysaccharide (lps)stimulated monocytes release free and microvesicle-associated mitochondria as part of their secretome ( ) . these studies demonstrate the complexity of cell-to-cell communication by identifying mitochondria as a source for target cells to restore their bioenergetics, enable immunomodulatory effects, and suppress inflammation. clinical trials failed to show efficacy of immunomodulatory therapies in sepsis ( , ) . since bacterial sepsis shares some similarities with covid- , we may consider a new route for therapeutic intervention focused on mitochondrial cell transfer. another option is therapy with engineered evs containing mitochondria. the immunomodulatory and regenerative potential of mscs may be independent of direct cellular cross-talk ( , ) . mscs act through a paracrine mechanism based primarily on evs, which interact with neighboring target cells or can reach distant organs ( ) . distinctions between the subtypes of evs were previously based on subcellular origin, with exosomes being of endosomal origin and microvesicles derived from the cell membrane. however, given the historically contradictory definitions and inaccurate expectations of biogenesis associated with these terms, in , isev recommended the use of new terms for ev subtypes that refer to their physical characteristics, such as size (small and medium/large evs) or density; their biochemical composition (cd +/cd +-evs, annexin a stained evs, etc.); or their cell of origin (msc evs, podocyte evs, etc.) ( ) . ev biochemistry varies according to composition and cell source ( ) . evs can carry membrane and cytosolic proteins, transcription factors, dna, coding and non-coding rnas and various signal transduction molecules ( , , , ) , acting on both physiological and pathological events, e.g., modulating the inflammatory response ( , ) . evs also carry different cytokines and growth factors, such as interleukin (il)- and il- , transforming growth factor (tgf)-β, and hepatocyte growth factor (hgf) ( ) . in addition, evs contain matrix-remodeling enzymes, such as matrix metalloproteinases (mmps), heparanases, hyaluronidases, and tissue inhibitors of metalloproteinases (timps); ev-mediated proteolytic activities have also been described, which might modulate the remodeling process and contribute to tissue repair ( ) . in this context, our group demonstrated that mscs increased mmp- expression and decreased timp- expression in an experimental model of ards, suggesting an effect on the extracellular matrix ( ) . rather than suppressing immune responses, evs appear to act as true modulators, inducing regulatory responses and tolerance in order to restore homeostasis ( ) . administration of evs has proven safe and effective in preclinical studies of lung injury and sepsis models ( , , , ( ) ( ) ( ) . in preclinical studies, ev therapy ameliorated acute lung injury ( , ) and was equally or even more effective than mscs in mitigating lung inflammation and pathological damage ( , ) . evs have also been shown to attenuate e. coli and influenza infections ( , , ) , including a mixed swine (h n , h n ) and avian (h n , h n ) influenzainduced lung injury model ( ) . the beneficial effects of evs have further been observed in an ischemic stroke model ( , ) . since covid- may be associated with damage to other organs in addition to the lungs and has been associated with ischemic stroke, evs could be a particularly promising therapy in this context ( ) . however, only one prospective study has evaluated the effects of evs (specifically, exosomes from bone marrow-derived mscs) in covid- patients ( ) . even though the inflammatory response was reduced significantly, and no adverse events were observed, this study encountered limitations regarding ev characterization and biological properties, the actual dose of ev administered, and how the injection of evs was monitored. at the time of writing, there are three ongoing clinical trials of msc-derived evs for covid- treatment, to be administered intravenously (chictr ) or by inhalation (nct , chictr ); however, recruitment has not yet begun. it is important to consider both the source of mscs from which evs are derived, which can be obtained from different tissues and donors, and their preparation. depending on these factors, mscs and their evs can have different therapeutic properties. compared to bone marrow-derived mscs, for instance, adipose tissue-derived mscs express more tissue factor (an important initiator of coagulation in sepsis) and reduce hemocompatibility, which has been shown to vary according to donor and culture handling conditions ( , ) . therefore, evs obtained from adipose tissue-derived mscs may have greater thrombogenic activity than those from bone marrowderived mscs ( , ) , and thus should not be considered for use in covid- patients ( , ) . furthermore, the therapeutic effects of evs are known to vary according to their preparation method, even when obtained from the same msc source ( ) . moreover, differences in donor parameters, including age, have been associated with significant variations in cytokine content, thus resulting in different effects on injury mitigation ( , ) . in addition to their natural cargo, evs can be loaded with biochemical compounds or genetically engineered to target infected cells, thus providing additional perspective for covid- treatment beyond msc-derived evs, including ev-based drug delivery, inhibition of ev biogenesis and uptake, and evbased vaccines ( ) . the latter might be a particularly promising cell-free approach to covid- treatment. exosome vaccines may contain membrane-anchored ectodomains of sars-cov- components on their surface, facilitating cross-linking of the bcell receptor ( ) . exosome-based vaccines containing the spike (s) proteins of sars-cov- , one of the structural proteins that mediate viral entry into the host cells ( ) , could induce high levels of neutralizing antibodies ( ) . in addition, the cargo of ev-based vaccines can be modified to include proteins and mirnas to help modulate the immune response ( ) . however, further research is needed to assess the safety and clinical pharmacology of ev-based therapies in order to provide guidance for manufacturing, storage, dosing, and administration ( ) before these potential treatments can be made more accessible worldwide ( , ) . in the specific setting of covid- , administration of msc-evs may have several advantages over mscs: (i) there is no risk of emboli formation in the injured microcirculation; (ii) no risk of mutagenicity or oncogenicity is observed; (iii) nebulized delivery can be used (despite several controversies regarding this route of administration); and (iv) tolerance of longer storage periods allow for later therapeutic use, reducing the stringency of storage and transportation requirements. in short, cell-free therapies should be considered a promising alternative for covid- treatment. clinical trials of ev-based therapies for covid- should clearly describe the dose, route of administration, characteristics of the 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cells compatible with human blood? stem cells the effects of cell type and culture condition on the procoagulant activity of human mesenchymal stromal cell-derived extracellular vesicles effect of mscs and msc-derived extracellular vesicles on human blood coagulation differential effects of extracellular vesicles from aging and young mesenchymal stem cells in acute lung injury msc-derived exosomes: a novel tool to treat therapy-refractory graft-versushost disease detection of antibodies against sars-cov in serum from sars-infected donors with elisa and western blot exosomal vaccines containing the s protein of the sars coronavirus induce high levels of neutralizing antibodies the authors thank filippe vasconcellos, b.a., for his assistance in editing the manuscript. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © maron-gutierrez and rocco. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -v dfel l authors: adachi, shun; koma, takaaki; doi, naoya; nomaguchi, masako; adachi, akio title: commentary: origin and evolution of pathogenic coronaviruses date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: v dfel l nan one of major study concerns in virology is viral adaptive evolution due to its highly replicable and mutable nature in changeable environments. some viruses with this property are severely pathogenic for animals and also for humans. virologists thus must prepare the ground for clinical applications of their findings obtained by structural and functional analyses on viruses. among viruses, some coronaviruses (covs) are notorious for causing the severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers). markedly, the very now covid- outbreak is brought about by a new coronavirus designated sars-cov- ( - ). in this commentary, we focus on the titled review article and mainly introduce evolutionary aspects of the coronaviruses. the said article has successfully predicted today's covid- outbreak by pointing out that novel pathogenic variants will readily emerge from very diversified severe acute respiratory syndrome-related coronaviruses (sarsr-covs) of the bat origin through their close coexistence and high genetic recombination ability (figure ) . therefore, it is very appropriate and timely to introduce this excellent review article here in the general commentary article. as basal knowledge for coronaviruses, they form an envelope structure at the outer surface of virions and their morphology is spherical with - nm in diameter. their genome is positivesense (+) single-stranded (ss) rna and - kb in size. coronaviruses have extra accessory genes in addition to those for viral structural proteins. following entry into cells via the specific interaction of viral envelope glycoprotein spike (s) and cellular receptor, coronaviruses replicate in the cytoplasm as the other ssrna (+) viruses. for alpha-and beta-coronaviruses, they are originated in highly metabolized mammals, such as bats and rodents. after being spilled over to alpacas, cows, civets, camels, or pigs, they can also infect humans and frequently cause sars and mers. gamma-and delta-coronaviruses mainly infect birds, but they sometimes infect mammals. molecular phylogenetic trees well support this idea of classifications. among several genes on the genome of interest, an evolutionary biologist tends to focus on a functionally conserved but sequentially diverged gene. this is because the role for the critical gene is conserved in various species, while it is rapidly evolving, indicating that a diverging force acts on the gene, e.g., by co-evolution such as symbiosis, evolutionary arms race, or others. of the cov genes, this review has centered on structural gene s and extra genes orf /orf that encode s and accessory proteins, respectively. importantly, the most frequently observed hotspots for recombination are within s gene and upstream region of orf gene. orf of sars-cov is assumed to be acquired from sarsr-cov by recombination, and is positively selected ( ). s protein contains the receptorbinding domain (rbd) critical for infection, and orf /orf proteins function viral species-specifically, e.g., by prescribing the virulence (orf ), anti-interferon activity (orf /orf ) or others. because orf /orf are different between sars-cov and sars-cov- , they might contribute to the difference in their virulence ( ) . additionally, s and orf genes are positively selected in civet sarsr-covs ( ). since rna viruses are easy to mutate and coronaviruses have high potentials for recombination, we can easily see the track of mutations and evolutions of the viruses, especially for sars-cov and mers-cov. rna recombination by rna-dependent rna polymerase with a low fidelity is widely observed and is supposed to shape current viruses by rearranging their genomes or disseminating functional modules ( ) . for coronaviruses such as mouse hepatitis virus (mhv), secondary structures of rna genome are responsible for highly dynamic spike structures ( ) . these might be an evolutionary cradle from "rna world, " still function in the living organisms nowadays. the non-processive replicase-driven template switching mechanism proposed among coronaviruses ( ) thus is a suitable model for the evolution of rnabased replicating system. among many viruses, coronaviruses (recombination frequency for the total genome in vivo is %) and picornaviruses are champions of the rna recombination frequency ( ) . let us move to the evolution of coronaviruses (figure ). in detail for each gene of the viruses noticed, orf is wellknown for viral evolution and the accompanied increase of virulence observed during the late onset of sars. for the rbd in s, sars-cov utilizes ace as a cellular receptor for infection, and mers-cov utilizes dpp as the receptor. the receptor recognition is important for infection process for the viruses. different use of the receptors among the coronaviruses is due to their sequences/structures of rbd. nonetheless, because of the common nature of rbd ( ) , it can be a promising target for development of novel antiviral compounds and antibody therapies for these viruses. however, it is also true in some cases, viruses went beyond the arms race and ingeniously evolved to counteract the clinical strategy. for example, the strategy is applicable for sars-cov wiv strain but not for shc and hku . hku is remarkable for its truncated form of rbd. for mers-cov, cells expressing suboptimal bat species-derived variant of dpp force the viruses to accumulate mutations in the viral spike during passages, resulting in enhanced viral entry merely with two amino acid mutations ( ) . the type of adaptation phenomena in virus evolution is testable either in clinical medicine or in vitro evolution system. thus, to consider the origin of new pathogens and the prevention of their transmission to humans, and control of the viruses, not only studies on sars-cov, mers-cov, and sars-cov- , but also those on their relatives sarsr-covs and mersr-covs are recommendable for bats tracked for the ecology and evolution. we better understand the interaction networks among viruses of the evolution and diversification by their detailed comparison (figure ) . the review mentions yunnan sarsr-covs might be the origins of the sars-covs, as symbionts, while domestication activity for mammals affects acquisition of pathogenicity to humans [refer also to banerjee et al. ( ) ]. both fieldworks and experimental biology are required to understand the viruses concomitantly with predicting or preventing potential outbreaks. sa and aa conceived the idea. sa wrote the manuscript. tk, nd, mn, and aa reviewed the manuscript. tk depicted figure . all authors approved its submission genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding genome composition and divergence of the novel coronavirus ( -ncov) originating in china a novel coronavirus from patients with pneumonia in china severe acute respiratory syndrome (sars) coronavirus orf protein is acquired from sars-related coronavirus from greater horseshoe bats through recombination sars-cov- and covid- : the most important research questions new insights into the mechanisms of rna recombination generation of coronavirus spike deletion variants by high-frequency recombination at regions of predicted rna secondary structure rna recombination in animal and plant viruses functional assessment of cell entry and receptor usage for sars-cov- and other lineage b betacoronaviruses adaptive evolution of mers-cov to species variation in dpp bats and coronaviruses we thank ms. fumie nishina (kansai medical university, osaka, japan) and ms. kazuko yoshida (tokushima university, tokushima, japan) for editorial assistance. key: cord- -nblmshni authors: savva, athina; roger, thierry title: targeting toll-like receptors: promising therapeutic strategies for the management of sepsis-associated pathology and infectious diseases date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: nblmshni toll-like receptors (tlrs) are pattern recognition receptors playing a fundamental role in sensing microbial invasion and initiating innate and adaptive immune responses. tlrs are also triggered by danger signals released by injured or stressed cells during sepsis. here we focus on studies developing tlr agonists and antagonists for the treatment of infectious diseases and sepsis. positioned at the cell surface, tlr is essential for sensing lipopolysaccharide of gram-negative bacteria, tlr is involved in the recognition of a large panel of microbial ligands, while tlr recognizes flagellin. endosomal tlr , tlr , tlr , tlr are specialized in the sensing of nucleic acids produced notably during viral infections. tlr and tlr are favorite targets for developing anti-sepsis drugs, and antagonistic compounds have shown efficient protection from septic shock in pre-clinical models. results from clinical trials evaluating anti-tlr and anti-tlr approaches are presented, discussing the challenges of study design in sepsis and future exploitation of these agents in infectious diseases. we also report results from studies suggesting that the tlr agonist flagellin may protect from infections of the gastrointestinal tract and that agonists of endosomal tlrs are very promising for treating chronic viral infections. altogether, tlr-targeted therapies have a strong potential for prevention and intervention in infectious diseases, notably sepsis. sepsis is one of the leading causes of death worldwide. incidence of severe sepsis is increasing and mortality rates remain significantly high despite early care management ( ) . moreover, more than % of survivors develop long-term functional disabilities and cognitive impairments ( ) . the surviving sepsis campaign is a global initiative incepted in early s with the aim to improve sepsis diagnosis and treatment in order to enhance the awareness of sequelae and to decrease high mortality rates associated with sepsis . in collaboration with many countries in europe and the united states, the surviving sepsis campaign suggests evidencebased guidelines and bundles. the most recent guidelines recommend acute resuscitation of septic patients, administration of antibiotics and support of organ failure. yet, no treatment targeting the underlying mechanism of sepsis is actually available ( ) . recombinant human activated protein c (rhapc, xigris®, eli lilly), the only drug specifically registered for sepsis, has recently been withdrawn from the market following the negative results from the prowess-shock study that did not show reduction in mortality at or days in patients with septic shock ( ) . it is generally admitted that sepsis results from a dysregulated host response to an initial insult, characterized by inflammation mediating tissue damage and organ failure and an immune suppression state responsible for the development of secondary infections ( ) ( ) ( ) . the immune response to an infection is initiated by the sensing of microbial structures through families of receptors collectively called pattern recognition receptors (prrs). the most well-described families comprise toll-like receptors (tlrs), nucleotide binding oligomerization domains (nods)-like receptors (nlrs), c-type lectin receptors (clrs, such as dectin- , dectin- , dc-sign), rig-i-like receptors (rlrs, rig-i, and mda ), and intra-cytosolic dna sensors. prrs are expressed by innate immune cells like dendritic cells and macrophages. the binding of microbial ligands to prrs promotes the release of mediators, among which cytokines, that initiate and regulate the inflammatory response necessary to eliminate invasive pathogens and coordinate the development of the adaptive immune response ( , ) . innate immune cells are also triggered by damage (or danger)associated molecular patterns (damps), known as alarmins. damps are endogenous components commonly released by injured or stressed cells, such as nucleic acids, histones, uric acid crystals, atp, cytochrome c, s molecules, and hmgb . damps are primarily sensed through the nlrp inflammasome, which controls the secretion of il- β and il- ( ) . the concept of prrs sensing microbial-associated molecular patterns (mamps) and discriminating self from non-self molecular structures was proposed by janeway ( ) . two major cornerstone discoveries largely confirmed janeway's concept. the first one was the demonstration of the essential antifungal role of www.frontiersin.org the toll protein in drosophila ( ) . the second one arose from the positional cloning linking lps (commonly called endotoxin) unresponsive phenotype of c h/hej and c bl/ sccr strains of mice to missense and null mutations of the toll-like receptor (tlr ) gene ( ) . the importance of these discoveries and of the role of dendritic cells as central regulators of innate and adaptive immunity has been acknowledged by the nobel prize in physiology or medicine attributed to bruce a. beutler and jules a. hoffmann "for their discoveries concerning the activation of innate immunity" and to ralph m. steinman "for his discovery of the dendritic cell and its role in adaptive immunity" ( ) . toll-like receptors belong to the most studied family of prrs, due to their central role in host defenses and involvement in a number of pathological processes that include sepsis. tlrs are type i trans-membrane proteins composed of an extracellular leucine-rich repeat (lrr) domain involved in ligand recognition, a trans-membrane domain, and a toll-interleukin receptor (tir) domain involved in signaling ( , ) . about functional human tlrs (tlr - ) and functional mouse tlrs (tlr - , tlr - ) have been described, each one being involved in the sensing of distinct microbial products ( table ) . expressed at the cell surface, tlr detects lps from gram-negative bacteria. tlr shuttles to late endosome to induce alternative signaling following lps sensing. tlr as heterodimers in association with either tlr or tlr (and possibly tlr ) senses a variety of microbial products, such as lipopeptides, lipoproteins, peptidoglycan, porins, β-glucan, glycosylphosphatidylinositol (gpi) anchors, and glycoproteins from gram-positive bacteria, gram-negative bacteria, mycoplasma, mycobacteria, fungi, parasites, and viruses. tlr senses flagellin of bacterial flagella. tlr , tlr , tlr , and tlr are strategically expressed in endosomal compartments to recognize microbial nucleic acids: double-stranded rna (dsrna) by tlr , single-stranded rna (ssrna) by tlr and tlr , and unmethylated cpg motif containing dna by tlr ( table ) . it is therefore not surprising that endosomal tlrs have been primarily involved in host defenses against viruses, whereas tlr , tlr , and tlr - have been mainly involved in host response to bacterial and fungal infection. tlrs cooperate with other molecules to recognize microbial ligands. for example, tlr requires cd , cd , and dectin- for the recognition of peptidoglycan, lipopeptides, and β-glucan, respectively. of note, tlrs are also triggered by damps released by injured or stressed cells during infection ( ) ( table ) . tlr activation enables pathogen elimination by promoting bactericidal activity of leukocytes, and maturation and function of antigen presenting cells, thus orchestrating the development of adaptive immune responses ( ) . the signaling pathways resulting from tlr triggering engage adaptors that are recruited by tir/tir domain interactions ( table ) : myeloid differentiation primary response gene ( ) (myd ), tir domain-containing adaptor protein (tirap, also known as mal), tir domain-containing adaptor inducing interferon (ifn)β (trif), and trif related adaptor molecule (tram) ( ) . myd is essential for signaling through all tlrs except tlr and is involved in early nuclear factor-κb (nf-κb) and mitogen-activated protein kinases (mapks) activation and pro-inflammatory gene expression. tirap serves as a bridge to recruit myd to tlr and tlr . trif initiates myd independent ifn regulatory factor (irf ) and late nf-κb activation involved in the production of type i ifns and ifn-inducible genes ( , ) . trif is recruited to the cytoplasmic domain of tlr and, in late endosome, through tram that bridges trif to tlr . a fifth tir domain-containing adaptor, sterile α-, and armadillo-motif containing protein (sarm) acts as a negative regulator of tlr and tlr signaling. sarm interacts with trif and inhibits the induction trif-dependent genes. the signaling pathways activated downstream tlrs have some redundancy. yet, the engagement of multiple tlrs, especially myd and trif-dependent tlrs, have synergistic effects on host responses ( , ) . intracellular cross talk between signaling pathways may also occur when different families of prrs are involved. for example, dectin- synergizes with tlr and tlr and increases cytokine production through canonical and noncanonical nf-κb pathways ( , ) . moreover, a single mamp can be detected by different prrs. this differential sensing is primarily depending on the localization of the mamp. indeed, flagellin is sensed by tlr expressed at the cell surface and by the naip /nlrc (also known as ipaf) inflammasome when localized in the cytoplasm ( ) ( ) ( ) . similarly, peptidoglycans stimulate membrane tlr and intra-cytosolic nod /nod dependent cell activation ( ) . interestingly, some cpg and non-cpg oligodeoxynucleotides directly stimulate and polarize t-cells through tlr and myd -independent mechanisms ( ), possibly through intracellular dna sensors. recently, hagan et al. and kayagaki et al. demonstrated non-canonical tlr -independent recognition of intracellular lps through an uncharacterized receptor ( , ) . this unconventional mode of lps sensing activates caspase- -dependent il- β secretion and sensitizes mice to endotoxic shock. all these observations indicate that the host has evolved different strategies to sense invading microorganisms. ideally, all possible interactions should be characterized and/or anticipated, so that the effect of treatment application can be predicted and/or translated. this mandates carefully planned experiments that represent real-life conditions and a detailed knowledge of compound's mode of action. obviously, the redundancy of microbial sensing pathways should be taken into consideration when developing or applying targeted-treatment strategies to a single prr. experimental animal models and human clinical studies support a crucial role for tlrs in infectious diseases. the first evidence came from the observation that tlr defective c h/hej and c bl/ sccr mice are hyporesponsive to lps and susceptible to otherwise non-lethal infection with escherichia coli and salmonella typhimurium. subsequent studies with mice knockout in tlrs or tlr adaptor molecules have demonstrated the importance of the tlr pathway in host defenses. for example, tlr knockout mice are highly susceptible to infections by staphylococcus aureus and streptococcus pneumoniae ( ) . more recently, human association studies have linked polymorphisms affecting tlr expression or tlr structure with an augmented propensity to develop infections ( ) ( ) ( ) ( ) . the discovery of tlrs and their involvement in innate immune responses has attracted much interest into the development of drugs for controlling infections and improving sepsis management. this field of research has been very dynamic, and ( ) . these two particular aspects of the tlr-targeting field will not be addressed in this review. herein, we will review the most popular agonist (tlr , tlr , tlr , tlr , tlr ) and antagonist (tlr , tlr , tlr , tlr ) agents used in pre-clinical and clinical models of acute and chronic infections, including sepsis. relevant registered clinical trials are listed in table . frontiers in immunology | microbial immunology lps is the main pro-inflammatory molecule anchored in the outermembrane of gram-negative bacteria ( ) . neutralization of bacterial lps, inhibition of its recognition by host cells or inhibition of signaling downstream lps binding to its receptor has long been considered a promising approach for the treatment of severe sepsis and septic shock. interestingly, endotoxemia is prevalent in septic patients, not only in those with gram-negative infection. indeed, translocation of viable bacteria and lps from the gastrointestinal tract has been proposed to participate in the pathophysiology of sepsis. tlr was identified years ago as the signal-transducing molecule of the lps receptor complex ( ) , which also comprises md- and cd . thus, tlr is regarded as a primary target for treating sepsis ( ) . tlr expression is increased in human monocytes of healthy volunteers challenged with lps ( ), as well as in patients with sepsis ( ) . moreover, polymorphisms in the tlr gene have been associated with gramnegative sepsis ( , ) . in the following sections, we present the most advanced tlr antagonists developed for the treatment of sepsis. strategies to inhibit lps-mediated toxic effects have been initiated years before the discovery of tlr ( ) and the unraveling of the crystal structure of the tlr -md- -lps complex ( ) . lipid a, the toxic moiety of lps, is highly conserved among endotoxins and constitutes an ideal therapeutic target ( ) . e , developed by eisai research institute of boston (andover, ma, usa), was the first-generation lipid a antagonist derived from rhodobacter capsulatus endotoxin. e conferred protection in experimental models of endotoxemia and lethal infection with e. coli ( ) . the protective effect likely occurred through the binding of e to the tlr -md- complex and the inhibition of the interaction between lps and tlr -md- ( ) . e also blocked endotoxin response in human healthy volunteers challenged intravenously with lps ( ). e development went through phase clinical trial, but was stopped due to issues of bioavailability. a second-generation lps antagonist drug candidate developed by eisai is eritoran tetrasodium (known as eritoran or e ), a synthetic lipid a analog of rhodobacter sphaeroides ( ) . eritoran blocked lps-induced cytokines in vitro and in experimental animal models ( ) ( ) ( ) . in a phase clinical trial enrolling healthy volunteers challenged with lps, eritoran inhibited pro-inflammatory cytokine production and diminished clinical symptoms of sepsis, including fever, chills, tachycardia, and headache. additionally, creactive protein levels and white blood counts were significantly decreased ( ) ( ) ( ) ( ) . the only adverse event observed was a dosedependent phlebitis, due to the fact that high doses of eritoran were used to achieve stable activity of the drug over time. a phase randomized control trial recruiting critically ill septic patients as assessed by the acute physiology and chronic health evaluation ii (apache ii) score disclosed a trend toward decreased mortality in the eritoran treated group ( ) . phase access (a controlled comparison of eritoran and placebo in patients with severe sepsis) clinical trial for severe sepsis started in , and results were published in . about patients were treated with eritoran and patients with placebo within h after the onset of the first organ dysfunction. unfortunately, analyses did not reveal reduced all-cause mortality in primary and secondary end-points (i.e., days and year mortality) ( ) . eisai (tokyo, japan) waived to submit eritoran to marketing authorization for the treatment of severe sepsis in january , based on preliminary results of the access trial. several reasons may account for the lack of efficacy of eritoran ( ) ( ) ( ) . for instance, patients were not enrolled or monitored based on the circulating levels of lps, questioning about the appropriateness of inclusion criteria. it is also possible that eritoran would be more efficient if administrated rapidly, before septic shock is underway, pointing the early and aggressive sepsis management as a possible interfering factor. other factors to take into account include the heterogeneity of patients for genetic background, underlying diseases, inflammatory and immune status, sepsis severity, infectious agent, and site of infection. as mentioned earlier, intracellular lps sensed in a tlr -independent manner sensitizes mice to endotoxic shock ( , ) . this non-canonical lps detection may have limited the efficacy of the anti-tlr strategy. moreover, upon infection, innate immune cells will likely sense several mamps via several tlrs and non-tlr prrs. for example, gram-negative bacteria express mamps that may trigger redundant inflammatory pathways through tlr (lipopeptides), tlr (lps), tlr (flagellin), tlr (ssrna), and tlr (bacterial dna). all these observations suggest that blocking one single pathway may be insufficient to interfere with the deleterious cascade of events observed in sepsis. the positive side of the access trial failure was a rethink of the design of sepsis clinical trials ( ) ( ) ( ) . clearly, a drug like eritoran should be tested in selected patients and treatment efficacy examined and adjusted according to predefined appropriate biomarkers (such as lps blood levels and genetic polymorphisms affecting the tlr pathway). a rigorous approach combining the power of "omics" technologies would allow the selection of homogeneous cohorts and the follow-up of the response to treatment, both of which are mandatory for the successful development of anti-sepsis drugs. another anti-sepsis agent that exhibited promising therapeutic properties is tak- [ethyl-( r)-[n -( -chloro- -fluorophenyl) sulfamoyl] or resatorvid] from takeda pharmaceutical company (osaka, japan). tak- was originally characterized as a suppressor of nitric oxide (no) and cytokine production by lpsstimulated macrophages and during endotoxic shock in mice ( ) . tak- binds to cysteine in the intracellular domain of tlr , thereby inhibiting both myd -dependent and myd independent pathways activated by lps ( ) . when administered in conscious guinea pigs following lps challenge, tak- significantly improved septic shock symptoms, decreasing hmgb systemic levels, and increasing survival in a dose-dependent manner ( ) . tak- also increased survival rates from to % and improved organ dysfunction when co-administered with antibiotics in a mouse model of cecal ligation and puncture (clp). no effect on circulating bacterial counts was observed ( ) . a double-blind, randomized, placebo-controlled trial was initiated with tak- ( ) . inclusion criteria comprised symptoms of severe sepsis accompanied with either shock and/or respiratory frontiers in immunology | microbial immunology failure. the study was stopped prematurely due to failure to achieve significant decrease of systemic cytokine levels at stage of the analysis ( ) . a phase clinical study was designed but never launched based on business decision and not due to safety or efficacy concerns. antibodies directed against tlr or the tlr -md- complex have been generated and showed promising results in several pre-clinical studies. we engineered a soluble chimeric protein composed of the n-terminal and central domains of mouse tlr (amino acid - ) fused to the fc domain of human igg ( ) . the chimeric molecule was used to generate high titer anti-mouse tlr rabbit polyclonal antibodies. the anti-tlr antibodies powerfully inhibited nf-κb and mapk activation and cytokine production by lps-stimulated cells in vitro. the antibodies also hampered cytokine production and protected mice from lethal endotoxemia when administered both prophylactically and therapeutically h after lps. prophylactic administration of anti-tlr antibodies blunted tnf production and strikingly increased survival in e. coli sepsis, from % in the control antibody group to % in the anti-tlr group. even more impressive, anti-tlr therapy initiated as much as h after the onset of infection in a model of e. coli peritoneal infection improved survival from to % ( ) . our studies demonstrate that anti-tlr antibodies are efficient as adjunctive therapy for e. coli sepsis, with a window of clinical application comprising prophylactic and therapeutic intervention opportunities. several anti-tlr monoclonal antibodies have been produced. the group of miyake (university of tokyo, japan) reported in the generation of mts , the first rat monoclonal antibody specific of the mouse tlr -md- complex ( ) . mts was shown to inhibit lps-induced nf-κb activation and tnf production by macrophages. e is a rat monoclonal antibody produced by novimmune sa (geneva, switzerland) that reacts with the tlr -md- complex ( ). e inhibited lps-induced cell activation, and protected mice from lethal endotoxemia when injected up to h after lps challenge. moreover, administration of e at the time of surgery improved the outcome of mice with colon ascendens stent peritonitis, a model of polymicrobial abdominal sepsis. finally, the rat monoclonal antibody a , that recognizes both mouse and human tlr -md- complexes, conferred protection in a model of e. coli sepsis, but not salmonella enterica, sepsis ( ) . although some tlr inhibitors have entered clinical and pre-clinical trials, others remain in the developmental stage. lps-trap-fc antibodies (comprising the extracellular domain of mouse tlr fused with md- and linked to human igg fc) dose-dependently decreased il- release by macrophages, opsonized gram-negative bacteria, and enhanced phagocytosis and complement-mediated bacterial killing ( ) . cell-penetrating peptides comprising the translocating segment of drosophila antennapedia homeodomain fused with bb loop sequences of tlr (i.e., tlr -bb peptides) inhibited lps-induced nf-κb and mapk activation and cytokine production ( , ) . further studies will be required before advancing these products toward the clinical level. altogether, the experimental data reported above provided strong support for the concept of tlr -targeted therapy for gram-negative sepsis. in the gloomy context following the withdrawn of rhapc and eritoran from the sepsis field, it is hopeful that ni- has entered clinical development. ni- is an anti-tlr monoclonal antibody produced by novimmune able to block tlr dimerization and tlr -mediated signaling triggered by lps and endogenous and chemical ligands of tlr . data from pre-clinical studies in models of arthritis, respiratory inflammation, and organ injury have highlighted the potential favorable action of this agent . a phase clinical study is currently recruiting participants to evaluate drug safety and tolerance in healthy volunteers before and after ex vivo and in vivo lps challenge. pharmacokinetics and pharmacodynamics will also be assessed. results from these studies are eagerly awaited. albeit less well characterized, tlr has been implicated in the sensing of non-bacterial microorganisms such as viruses and fungi. tlr recognizes o-linked mannan from candida albicans, and human studies have linked asp gly tlr polymorphism with susceptibility to bloodstream candidiasis and pulmonary aspergillosis ( ) ( ) ( ) . in a model of disseminated infection with c. albicans, c h/hej tlr -deficient mice exhibited a -fold increased fungal load in the kidneys, which was associated with reduced production of the chemokines kc and mip- and an impaired recruitment of neutrophils ( ) . treatment with hta , an anti-human tlr mouse monoclonal antibody ( ), interfered with neutrophil-mediated protection against c. albicans invasion and cell injury in an in vitro epithelial model of oral candidiasis ( ) and inhibited tnf production by human pbmcs stimulated with aspergillus hyphae ( ) . it is still unclear whether targeting tlr may be beneficial in the context of fungal infections. a more clear yet unexpected picture has arisen from viral infection studies. reactive oxygen species (ros) produced by the nadph oxidase generates oxidized host phospholipids that stimulate tlr and the production of cytokines involved in acute lung injury ( ) . using a mouse model of lethal infection with influenza, the group of stephanie vogel (university of maryland, baltimore, ma, usa) reported that eritoran significantly increases survival in a dose-dependent manner even when administered days after viral challenge. lung pathology and clinical symptoms were improved while viral titers and influenza-induced cytokine gene expression in lung homogenates were decreased compared to the placebo-treated group. these data suggest that the therapeutic effect of eritoran in a more practical timing of severe sepsis treatment remains substantial ( ) . they also suggest that, despite the failure of eritoran in the access trial, new therapeutic potentials might still emerge for this agent. toll-like receptor has been implicated in the recognition of an amazingly broad spectrum of microbial ligands originating from bacteria, fungi, viruses, and parasites ( ) . this property is at least partly due to the fact that tlr forms heterodimers with tlr , tlr , and possibly tlr ( , , ) . the biological relevance of tlr homodimers is controversial. indeed, some ligands have been reported to trigger cells through tlr independently of tlr and tlr . yet, only tlr /tlr and tlr /tlr heterodimers have been successfully crystallized ( , ) . tlr represents an interesting target for numerous conditions, but clinical development of tlr -targeting drugs has been less extensive than that of tlr . t . is a tlr neutralizing mouse monoclonal antibody. t . blocked pam csk lipopeptide (a tlr /tlr -ligand)stimulated nf-κb nuclear translocation and mapk phosphorylation in vitro. in models of pam csk -induced toxic shock and microbial challenge with a high inoculum of heat-inactivated bacillus subtilis, t . prevented lethal shock-like syndrome and increased survival when administered h before or up to h after infection ( ) . furthermore, t . used in combination with the a anti-tlr /md- antibody and antimicrobial therapy protected mice from sepsis caused by s. enterica and e. coli ( ) . intracellular antibodies, i.e., intrabodies, have been designed to block the intracellular translocation of tlrs from the endoplasmatic reticulum to the cell surface. αt ib is a functional anti-tlr scfv intrabody comprising the variable domains of the heavy and light chains of t . linked together by a synthetic (gly ser) amino acid sequence. αt ib bound intracellularly to tlr and led to retention and accumulation of tlr inside the endoplasmatic reticulum. adenovirus-mediated expression of αt ib in raw . macrophages and mouse bone marrow derived macrophages inhibited tlr surface expression and tlr -ligand-driven tnf production ( ) . these data suggest for a therapeutic potential of t . or αt ib in microbial infections. many studies have attempted to elucidate the pathogenesis of acute kidney injury associated with sepsis, which involves mechanisms similar to those occurring during ischemia/reperfusion ( ) . damps released during infection are detected through tlr by immune cells recruited to the ischemic tissue and/or by cells of the ischemic tissue itself, amplifying the inflammatory response and inducing injury upon reperfusion ( , ) . blocking tlr under these conditions may be cytoprotective ( ) . opn- is a humanized anti-tlr igg monoclonal antibody [derived from opn- ( ) developed by opsona therapeutics (dublin, ireland)]. opn- reduced tlr -driven pro-inflammatory cytokine production through blocking of tlr / and tlr / mediated signaling. in a porcine model of myocardial ischemia/reperfusion injury, pretreatment with opn- or administration of opn- h after ischemia was associated with a % decrease in infarct size ( ) . results from a first in human phase trial evaluating safety, tolerability, pharmacokinetics, and pharmacodynamics of ascending doses of opn- given intravenously in healthy adult subjects have just been released ( ) . tlr occupancy and inhibition of il- secretion induced by heat-killed listeria monocytogenes were assessed in whole blood collected up to days after treatment with either the antibody or placebo. opn- was well tolerated, with no significant toxicity even at the highest dose tested. impressively, opn- at doses of . and mg/kg occupied % of tlr molecules expressed on monocytes collected and days after challenge, respectively. il- release was inhibited in a parallel manner. these results suggest that treatment with opn- could provide short-term protection against ischemia/reperfusion and be adjusted to confer long-lasting blockage in the case of tlr -mediated chronic diseases ( ) . a phase trial assessing safety, tolerability, and efficacy of opn- in kidney transplant patients has been initiated (nct ). new techniques are continuously implemented to facilitate the identification of therapeutic targets for adjunctive treatment in sepsis. immunoprecipitation with systematic evolution of ligands by exponential enrichment (selex) was developed to screen and identify high-affinity dna and rna molecules that bind to tlr and could be used to detect other molecules influencing tlrdriven activity. a most promising candidate, ap- , was shown to interact with tlr , thereby obstructing ligand binding to the receptor and inhibiting tlr -ligand-induced nf-κb activity and il- and il- production in thp- and hek cells ( ) . cellpenetrating tlr -bb peptides have been generated and shown to interfere with tlr -ligand-induced activation of nf-κb and mapk and cytokine production ( ) . whether these compounds will undergo clinical evaluation is unknown. toll-like receptor is an endosomal prr that senses dsrna typically produced during viral infection ( ) . experimental models comparing tlr wild-type and tlr knockout mice revealed either a protective role (west nile virus, encephalomyocarditis virus, poliovirus, coxsackievirus, murine cytomegalovirus, herpes simplex virus), a deleterious role (west nile virus, influenza a virus, phlebovirus), or no influence (lymphocytic choriomeningitis virus, vesicular stomatitis virus, murine cytomegalovirus, reovirus) of tlr on anti-viral responses ( ) . therefore, tlr agonists and antagonists might be efficient adjunctive therapies for viral infections depending on the context. in the following sections, we describe the development of synthetic dsrna tlr agonists (see tlr agonists) and of synthetic ssdna tlr antagonists and anti-tlr neutralizing antibodies (see tlr antagonists). the dsrna synthetic analog polyinosinic:polycytidylic acid [poly(i:c)] is a potent immunostimulant. for clinical development, poly(i:c) was stabilized with polylysine and carboxymethylcellulose (poly-iclc) (hiltonol, oncovir, washington, dc, usa) and used to generate poly(i:c u) (rintatolimod, tradename ampligen, hemispherx biopharma, philadelphia, pa, usa) by substituting an uridylic acid at a molar ratio of : in the synthesis of the polycytidylic acid strand ( ) . poly(i:c) and its derivatives have been tested in several clinical trials as adjuvants for vaccines (for both infectious diseases and cancer) and complement to haart (highly active anti-retroviral therapy) in human immunodeficiency virus (hiv) infected patients, topics that we do not discuss here. poly(i:c u) is highly specific for tlr and, unlike its parental molecule poly(i:c), does not require melanoma differentiationassociated protein (mda , a cytosolic prr for viruses), for the induction of the signaling cascade leading to type i ifn production ( ) . poly(i:c u) has some anti-viral activity against hiv, hepatitis b virus (hbv), coxsackie b virus, and several flaviviruses. results from animal models of lethal respiratory viral infection by severe acute respiratory syndrome coronavirus (sars-cov) and punta toro virus highlighted the favorable impact of intranasal treatment with poly(i:c) or poly(i:c u) on survival and viral loads in infected mice ( , ) . the mode of action of poly(i:c) in the respiratory tract was linked to the induction of caspase-mediated apoptosis and ros, which are involved in the cleavage and shedding of soluble tnf receptor blocking tnf bioactivity ( ) . interestingly, poly(i:c) protected against bacterial infections in the respiratory tract as well as in the central nervous system (cns). in a mouse model of pseudomonas aeruginosa pneumonia secondary to clp, intranasal administration of poly(i:c) improved immune activation and lowered bacterial load in the lungs compared to the untreated animals ( ) . corroborating results showing enhanced phagocytosis and killing of e. coli by microglial cells suggest that tlr activation is crucial for the immune response of cns against invading pathogens ( , ) . these data support the development of tlr agonists as adjuvant therapies to prevent or reduce the severity of respiratory tract infections caused by viruses and possibly bacteria. in connection with that particular field, a phase safety, tolerability, and pharmacokinetic trial of nasally applied poly-iclc in human volunteers is ongoing and will explore immune activation markers. a phase / clinical trial is assessing the immunogenicity and safety of flumist®(live attenuated influenza vaccine, med-immune, gaithersburg, md, usa) intranasal influenza vaccine administered with and without a poly(i:c u). more recently, tlr antagonists have been developed taking into consideration that tlr over-activation by viral dsrna may have detrimental consequences in some situations. indeed, it has been reported that administration of poly(i:c) in mice prior to intratracheal challenge with s. pneumoniae impaired bacterial clearance and increased mortality. excessive production of type i ifn was involved in this phenomenon ( ) . single-stranded dna oligonucleotides (ssdna odns) efficiently competed with dsrna for binding to tlr , thus inhibiting cytokine production and costimulatory molecule expression by epithelial cells, pbmcs, and dendritic cells ( , ) . the efficacy of ssdna odns was demonstrated in cynomolgus macaques, where intranasal injection of ssdnas odns inhibited poly(i:c)-induced cytokine production in nasal secretions ( ) . in addition to microbial ligands, tlr senses damps released from injured tissue during inflammation, for example rna from necrotic cells, promoting an excessive inflammatory response. interestingly, administration of a tlr neutralizing antibody to mice reduced cecal damage induced by gut ischemia and improved survival of animals with polymicrobial sepsis when the antibody was given and h after clp surgery ( ) . collectively, these data demonstrate that tlr works as an endogenous sensor of necrosis and a regulator of the immune response, pointing to receptor modulation as a possible adjuvant therapy for sepsis. work remains to be done to clearly delineate the precise role of tlr in viral and bacterial infections and to appraise the benefit afforded by tlr agonistic or antagonistic strategies for infectious diseases, especially septic shock. a single tlr agonist has had clinical development, namely cblb . flagellin is the only ligand of tlr described to date. detailed structural basis of flagellin recognition by tlr has been obtained through crystallographic analyses, unraveling a unique mode of interaction between the two molecules as depicted from stoichiometry, ligand arrangement, and binding interfaces ( ) . the role of structural constraints for induction of the nf-κb signaling cascade downstream tlr was supported by structure-guided mutagenesis and deletion analyses on cblb (entolimob), a therapeutic agent derivative of s. enterica flagellin implemented by cleveland biolabs (buffalo, ny, usa). cblb is currently tested in a phase trial in late stage cancer patients (nct ). several clinical trials have investigated the safety and adjuvant efficacy of recombinant flagellin in a number of vaccine settings (against influenza virus, helicobacter pylori, campylobacter, yersinia pestis, west nile virus, etc) ( ) . cblb plays a protective role against radiation-induced tissue injury, probably by suppressing apoptosis, attenuating ros generation, and promoting tissue regeneration ( ) . these properties could explain the beneficial effect of this agonist in a murine model of acute ischemic renal failure when administered min after reperfusion ( ) . highlighting the favorable role of flagellin against tissue damage, results from two mouse studies suggested that protection and repair of the intestinal mucosa that serves as a first line defense barrier is the key mode of action of flagellin. in the first study, flagellin was shown to induce the expression of regiiiγ, a c-type lectin with bactericidal activity, and to restrict small intestine colonization with vancomycin-resistant enterococcus (vre) in animals inoculated with vre via oral gavage ( ) . in the second study, treatment with flagellin reduced intestinal epithelium destruction induced by dextran sodium sulfate (a chemical used to induce severe acute colitis) and increased survival of mice inoculated with s. typhimurium by oral gavage ( ) . these data suggest that flagellin or tlr agonists may represent attractive tools for treating pathologies that injure the intestinal tract, including severe sepsis. no tlr antagonists have been reported. toll-like receptor agonists tested in clinical trials are synthetic cpg oligodeoxynucleotides (cpg odns) among which cpg , imo- , sd- , and cpg that mimic unmethylated cpg dinucleotide-rich sequences enriched in microbial dna. cpg odns are powerful immunostimulants, exploited for their adjuvant properties in vaccines against infectious diseases (flu, malaria, hiv infection, pneumococcal and meningococcal diseases) and cancer (melanoma, leukemia, glioblastoma, and colorectal, prostate and breast cancer) ( ) . the adjuvant properties of cpg odns have been used to enhance www.frontiersin.org the phagocytosis and the killing of bacteria (s. typhimurium and s. pneumoniae) by phagocytic cells ( , ) . interestingly, a recent study showed that cpg odn given h prior to clp surgery prevented clp-induced cardiac dysfunction in mice. the authors proposed that targeting tlr could be a useful approach for the management of cardiovascular dysfunction in severe sepsis patients ( ) . therapeutic strategies focusing on tlr -mediated immunomodulation are currently being implemented for chronic viral infections, such as chronic hepatitis c (hcv). plasmacytoid dendritic cells are the main cells producing type i ifn and are therefore considered to play an important role in viral infections. tlr agonists stimulate plasmacytoid dendritic cells to produce large amounts of type i ifn, especially ifnα, which is the backbone of therapy for hcv. indeed, ifnα powerfully inhibits viral replication and promotes innate and adaptive host immune responses. moreover, ifn production appears to be impaired in plasmacytoid dendritic cells of hcv patients ( , ) . cpg was originally developed under the trade name actilon by coley pharmaceuticals (wellesley, ma, usa), a company recently incorporated by pfizer (new york city, ny, usa). cpg has undergone two phase studies with promising results. a phase a study for drug safety and pharmacokinetics conducted in healthy volunteers revealed well tolerated immunostimulatory effects without serious adverse events even when using high doses of cpg ( ) . cpg was also tested in hcv patients, infected with genotype hcv. cpg was administered subcutaneously to four randomized groups at different doses twice per week for weeks alone or in combination with pegylated ifnα and ribavirin. the tlr agonistic effect of cpg was associated with the induction of ifnγ and ifnα and the decrease of viral loads. the only serious adverse events were urticaria and pruritis, without manifestation of respiratory complications ( ) . a phase study enrolling non-responders genotype hcv patients has been completed, but results have not been released yet. imo- , manufactured by idera pharmaceuticals (cambridge, ma, usa), has undergone two phase trials: one for dose estimation and one for safety, pharmacokinetics, and pharmacodynamics, enrolling and treatment-naïve genotype hcv patients, respectively. imo- administration dosedependently decreased viral loads, increased the production of anti-viral cytokines and chemokines especially ifnα, and activated nk and t-cell responses ( , ) . a phase trial was planned, but in april the company postponed its initiation. the decision was made based on histological data from a -week non-clinical toxicology study of imo- in rodents and non-human primates . preliminary analyses suggested evidence of atypical lymphocytic proliferation, although no adverse events were reported in humans. thorough analysis results are pending. a phase b study sponsored by dynavax (berkeley, ca, usa) investigated the safety and efficacy of sd- in chronic hcv. sd- was administered as monotherapy or in combination with ribavirin to chronically infected, treatment-naïve, genotype hcv patients. results released in indicated that sd- was well tolerated and safe without any serious adverse events. the drug had significant anti-viral activity based on dose-dependent anti-viral response, with % of patients at the highest dose showing more than one log reduction in viral load, and increased expression of type i ifn-dependent anti-viral genes (ip- , mcp- mx-b, isg- k). these data comfort results from in vitro studies showing that sd- stimulated human pbmcs to produce -fold higher levels of both ifnα and ifnλ in comparison with first-generation tlr agonists ( ) . bacterial dna released during infection is a mamp, and exuberant activation of tlr may participate to the sepsis pathophysiology. hence, drug inhibitors of tlr may have therapeutic potential in human sepsis. as a proof of concept, administration up to h after surgery of a single dose of an inhibitory cpg odn blocking tlr signaling protected mice from polymicrobial sepsis following clp ( ) . hmgb proteins are essential for triggering nucleic acid receptor-mediated innate immune responses. hmgb-binding non-immunogenic-odns have been designed to inhibit hmgb-mediated pathologies. a non-immunogenic odn termed ism odn was tested in a mouse model of endotoxemia. impressively, % of mice treated with ism odn survived up to h after lps challenge, while all mice from the control group died within h ( ). these data argue for a possible use of non-immunogenic-odns in therapeutic interventions. antimalarial drugs such as chloroquine are well known for their anti-inflammatory properties in autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus ( ) . chloroquine blunted cytokine production and protected mice from toxic shock induced by cpg odn and lps. chloroquine down-regulated the expression of both tlr and tlr , suggesting that it acts at multiple levels to inhibit inflammation ( , ) . additionally, when administered h after clp in elderly mice treated with fluids and antimicrobials, chloroquine significantly improved survival, strengthened renal function and protected from multiple organ dysfunction ( ) . these results support clinical evaluation of chloroquine in patients with severe sepsis, especially those presenting with acute renal failure. based on its anti-inflammatory activity and because it inhibits endosomal acidification which are important for cell infection by viruses, chloroquine is also tested in multiple trials for prevention and/or treatment of viral infections (hiv, influenza, dengue, chikungunya). yet, the mode of action of chloroquine is multi-factorial and not only through tlr . toll-like receptor and are closely related tlrs well known for their capacity to recognize ssrna from viruses such as hcv, hbv, hiv, influenza virus, herpes simplex virus, epstein-barr virus, vesicular stomatitis virus, papilloma virus, respiratory syncytial virus, and sendai virus. in agreement, tlr is primarily expressed in plasmacytoid dendritic cells. more recently, tlr and tlr were shown to sense bacterial rna released within phagosomal vacuoles ( ) . tlr and tlr triggering induces potent antiviral immune responses characterized by the production of type frontiers in immunology | microbial immunology i ifns and nf-κb-dependent cytokines. tlr / agonists are primarily developed for treating viral diseases, but also as adjuvants for cancer and infectious disease vaccines. imiquimod (aldara, originally developed by m pharmaceuticals, maplewood, mn, usa) is the only tlr agonist marketed for anti-viral treatment, i.e., external ano-genital warts caused by human papilloma virus. numerous tlr / agonists are in clinical development, like cl , isatoribine, ana , ana , pf- ( a), r- (resiquimod), and gs- . although therapeutic strategies for hcv have evolved in the recent years, quest of new immunomodulatory targets remains mandatory. treatment with new agents such as protease inhibitors appears to be efficient but presents with issues of resistance in the long run ( ) . moreover, current protocol therapy with ifnα provides replenishment with a specific subtype of this cytokine. however, use of tlr agonists is able to induce a variety of ifn subtypes, possibly providing a more radical and integrated anti-viral activity ( ) . finally, administration of tlr / agonists may overcome the adverse events caused by ifnα, like the suppression of granulocyte colony stimulating factor (g-csf) leading to neutropenia. indeed, cl reversed ifnα-mediated inhibition of g-csf production by pbmcs obtained from hcv patients and healthy volunteers ( ) . cl also restored defective cytokine production by myeloid dendritic cells from hiv patients ( ) . isatoribine (anadys pharmaceuticals, san diego, ca, usa) was one of the first guanosin analog selective tlr agonists to be implemented and tested on humans ( ) . intravenous administration of isatoribine to hcv patients during a day treatment plan resulted in reduced plasma concentration of hcv rna, regardless virus genotype. adverse events comprised dose-dependent joint pain, decreased white blood cells, and platelets counts, insomnia, and headache ( ) . the development of an oral prodrug that could lack the detrimental effects of isatoribine, especially in the gastrointestinal tract, pointed to a new candidate, ana . preliminary results from a study conducted with ana were promising. oral administration of ana presented with elevated plasma levels of isatoribine at a concentration able to reduce hcv rna in the plasma of infected patients ( ) . unfortunately, anadys pharmaceuticals and novartis (basel, switzerland) announced in discontinuation of drug development due to unacceptable toxicity in pre-clinical animal studies ( ) . subsequent elaboration of an oral prodrug of isatoribine by anadys pharmaceuticals led to the generation of ana . ana exhibits efficient induction of endogenous type i ifns. a double-blind, placebo-controlled study was conducted in patients with chronic hcv, either treatment-naïve or relapsed from ifn-based therapy. interestingly, ana was safe and well tolerated and presented only with grade and adverse events. moreover, ana dose-dependently decreased hcv rna levels ( ) . in another study, repeated treatment with ana was associated with transient decrease of myeloid and plasmacytoid dendritic cells and increased levels of ifnα and ip- in the blood of patients achieving a reduction in the viral load, suggesting an impairment in ifnα production in the case of non-responders ( ) . pf- , formerly known as a, is a tlr agonist generated by pfizer and implemented so that repeated low doses of the drug would be accompanied with the benefits of the agonistic activity without adverse events. a proof of concept study was conducted to evaluate safety and tolerability of the drug and to determine pharmacokinetics and pharmacodynamics. twentyfour healthy volunteers received orally increasing doses of pf- . pf- induced immune response in a dosedependent and frequency-related manner. however, two of the subjects exhibited severe lymphopenia along with flu-like symptoms and hypotension ( ) . in an attempt to decide on the future perspectives of this compound, a model was used to predict the safety and efficacy of pf- in hcv patients. this model exploited clinical results from the former study in healthy volunteers along with those reported from the use of cpg . further optimization will be required before entering the drug in a phase study ( ) . other tlr / agonists have reached phase trials, but demonstrated lack of efficacy and serious adverse effects. when monocytes from hiv patients were stimulated with resiquimod, il- secretion was augmented while tnf production was decreased compared to the control group. additionally, hiv replication in cultured monocytes was inhibited ( ) . these promising in vitro results were reproduced in a phase trial that enrolled patients with herpes simplex virus type . topical application of resiquimod protected from viral lesion spreading ( ) . however, a phase trial disclosed lack of efficacy of the drug and, although a phase study for the treatment of hcv demonstrated decreased viral loads, adverse side effects similar to those resulting from ifnα treatment were of serious concern ( ). two phase clinical trials for the safety and pharmacokinetics of a novel compound, gs- (gilead sciences, foster city, ca, usa), are currently enrolling treatment-naïve and viral suppressed hbv patients respectively, while another one enrolling hcv patients has been recently approved. gs- is a tlr agonist tested originally in hbv infected chimpanzees. drug administration was associated with reduced viral loads both in plasma and liver, probably through enhanced apoptosis of hepatocytes ( ) . testing of ascending dosages of the drug in healthy volunteers presented with flu-like adverse events at a dose of - mg, but cytokine induction was achieved even at administration of mg pointing to a promising adjunctive treatment for chronic viral infections ( ). all the above data illustrate the efforts devoted to the development of tlr / agonists for treating viral pathologies. taking into account that bacterial rna triggers tlr and tlr pathways, one may speculate that tlr / agonists would impact on bacterial sepsis. unfortunately, there are almost no data available on that subject. in one report, intravenous injection of r- prior to sepsis induction using the colon ascendens stent peritonitis model increased cytokine release and bacterial clearance, but the effect on survival was not reported ( ) . finally, considering that excessive tlr activation induced by viruses or bacteria may have www.frontiersin.org detrimental effects for the host, it might be of interest to generate tlr / antagonists to counteract overwhelming immune activation in conditions of severe infections. targeting tlrs is a promising field for sepsis management and infection control. tlr agonists are widely used to optimize vaccine efficacy, taking advantage of their powerful adjuvancity. whether tlr agonists and antagonists will have such success for treatment therapies, especially for sepsis, has to be established. agonists of tlr and tlr - yielded very promising results for treating viral infections. only few studies tested antagonistic drugs. mainly for historical reasons and because of their ligand specificity, tlr and to a lesser extent tlr are the favorite targets for developing antisepsis drugs. this domain of research has monopolized important resources, but unfortunately many drugs tested in animal models have not entered human studies. those that proceeded, like eritoran and tak- , did not achieve primary endpoint goal and/or were accompanied with manifestation of adverse events. animal models provide invaluable information about the role of tlrs and the mechanism underlying infection pathogenesis, but lack complexity in terms of comorbidities and host response compared to human disease ( ) . so, how to explore more efficiently new treatment strategies? improving the design of clinical studies is mandatory. we should discriminate those patients that could benefit from therapy based on their genotype (for example affecting tlr pathways) and the expression of the targeted molecule, and select homogeneous, well-described population bearing the same underlying conditions and disease severity ( ) ( ) ( ) ) . ideally, sepsis studies should use specific biomarkers to help patient enrollment and weigh treatment efficacy in realistic conditions ( ) . the failure of the recent trials in patients with severe sepsis has taught us valuable lessons regarding patient selection, time of intervention, and follow-up ( ) ( ) ( ) . blocking one single mediator may also not be sufficient to interfere with sepsis. developing sepsis-specific tlr-targeted therapies for patients is a path strewn with obstacles, but an exciting and promising area of research. these studies offer new possibilities for prevention and intervention in infectious diseases, but also for other conditions, such as cancer, allergy, asthma and autoimmune diseases either directly or through improvement of vaccines. benchmarking the incidence and mortality of severe sepsis in the united states long-term cognitive impairment and functional disability among survivors of severe sepsis surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock drotrecogin alfa (activated) in adults with septic shock epigenetics in sepsis: targeting histone deacetylases sepsis: something old, something new, and a systems view harmful molecular mechanisms in sepsis regulation of adaptive immunity by the innate immune system toll-like receptors and their crosstalk with other innate receptors in infection and immunity inflammasomes and their roles in health and disease approaching the asymptote? evolution and revolution in immunology the dorsoventral regulatory gene cassette spatzle/toll/cactus controls the potent antifungal response in drosophila adults defective lps signaling in c h/hej and c bl/ sccr mice: mutations in tlr gene bridging innate and adaptive immunity newly described pattern recognition receptors team up against intracellular pathogens dampening inflammation by modulating tlr signalling toll-like receptors and the host defense against microbial pathogens: bringing specificity to the innate-immune system renal-associated tlr mediates ischemia/reperfusion injury in the kidney the family of five: tir-domain-containing adaptors in toll-like receptor signalling pattern recognition receptors and inflammation synergy and cross-tolerance between toll-like receptor (tlr) -and tlr -mediated signaling pathways selected toll-like receptor agonist combinations synergistically trigger a t helper type -polarizing program in dendritic cells syk kinase is required for collaborative cytokine production induced through dectin- and toll-like receptors dectin- directs t helper cell differentiation by controlling noncanonical nf-kappab activation through raf- and syk cytosolic flagellin requires ipaf for activation of caspase- and interleukin beta in salmonella-infected macrophages innate immune recognition of bacterial ligands by naips determines inflammasome specificity the nlrc inflammasome receptors for bacterial flagellin and type iii secretion apparatus signalling pathways and molecular interactions of nod and nod cpg and non-cpg oligodeoxynucleotides directly costimulate mouse and human cd + t cells through a tlr -and myd -independent mechanism cytoplasmic lps activates caspase- : implications in tlr -independent endotoxic shock noncanonical inflammasome activation by intracellular lps independent of tlr how important are toll-like receptors for antimicrobial responses? innate immunogenetics: a tool for exploring new frontiers of host defence human genetic susceptibility to infectious disease genetic variation in toll-like receptors and disease susceptibility targeting toll-like receptors: emerging therapeutics? phase trial of eritoran tetrasodium (e ), a toll-like receptor antagonist, in patients with severe sepsis effect of eritoran, an antagonist of md -tlr , on mortality in patients with severe sepsis: the access randomized trial a randomized, double-blind, placebo-controlled trial of tak- for the treatment of severe sepsis phase b dose-secalation study of sd- , a novel therapeutic tlr- agonist, in treatment-naive chronic hepatitis c patients a phase , multi-center, randomized, placebo-controlled, dose-escalation study of imo- , a tlr agonist, in hepatitis c-nonresponders imo- plus ribavirin gives substantial first-dose viral load reductions, cumulative anti-viral effect, is well tolerated in naive genotype hcv patients: a phase trial chloroquine for influenza prevention: a randomised, double-blind, placebo controlled trial chloroquine use improves dengue-related symptoms imiquimod . % cream applied daily to treat anogenital warts: combined results from women in two randomized, placebo-controlled studies imiquimod % cream for external genital or perianal warts in human immunodeficiency virus-positive patients treated with highly active antiretroviral therapy: an open-label, noncomparative study first-line therapy for human cutaneous leishmaniasis in peru using the tlr agonist imiquimod in combination with pentavalent antimony randomised clinical trial: anti-viral activity of ana , an oral inducer of endogenous interferons acting via tlr , in chronic hcv potent immune activation in chronic hepatitis c patients upon administration of an oral inducer of endogenous interferons that acts via toll-like receptor the innate immune response, clinical outcomes, and ex vivo hcv antiviral efficacy of a tlr agonist (pf- ) innate immune sensing and its roots: the story of endotoxin toll-like receptor modulation as a strategy to treat sepsis expression of tumour necrosis factor receptor and toll-like receptor and on peripheral blood leucocytes of human volunteers after endotoxin challenge: a comparison of flow cytometric light scatter and immunofluorescence gating increased expression of tolllike receptor- and - on leukocytes from patients with sepsis the structural basis of lipopolysaccharide recognition by the tlr -md- complex bacterial endotoxin: molecular relationships of structure to activity and function lipopolysaccharide interaction with cell surface toll-like receptor -md- : higher affinity than that with md- or cd a lipid a analog, e , blocks the endotoxin response in human volunteers with experimental endotoxemia eritoran tetrasodium (e ) treatment for sepsis: review of preclinical and clinical studies inhibition of endotoxin response by e , a novel toll-like receptor -directed endotoxin antagonist toll-like receptor antagonist (e ) prevents the chronic airway response to inhaled lipopolysaccharide physiologic, biochemical, and imaging characterization of acute lung injury in mice pharmacokinetics of e , a lipopolysaccharide antagonist, in patients with impaired hepatic function extended in vivo pharmacodynamic activity of e in normal volunteers with experimental endotoxemia safety, pharmacokinetics, pharmacodynamics, and plasma lipoprotein distribution of eritoran (e ) during continuous intravenous infusion into healthy volunteers continuous pharmacodynamic activity of eritoran tetrasodium, a tlr antagonist, during intermittent intravenous infusion into normal volunteers sepsis studies need new direction recombinant human activated protein c as a therapy for severe sepsis: lessons learned? trial watch: sepsis study failure highlights need for trial design rethink discovery of novel and potent small-molecule inhibitors of no and cytokine production as antisepsis agents: synthesis and biological activity of alkyl -(n-substituted sulfamoyl)cyclohex- -ene- -carboxylate analysis of binding site for the novel small-molecule tlr signal transduction inhibitor tak- and its therapeutic effect on mouse sepsis model the novel selective toll-like receptor signal transduction inhibitor tak- prevents endotoxaemia in conscious guinea-pigs combination of imipenem and tak- , a tolllike receptor signal transduction inhibitor, improves survival in a murine model of polymicrobial sepsis protection from lethal gram-negative bacterial sepsis by targeting toll-like receptor cutting edge: cell surface expression and lipopolysaccharide signaling via the toll-like receptor -md- complex on mouse peritoneal macrophages tlr /md- monoclonal antibody therapy affords protection in experimental models of septic shock tlr -induced ifn-gamma production increases tlr sensitivity and drives gramnegative sepsis in mice lipopolysaccharide-trap-fc, a multifunctional agent to battle gram-negative bacteria cutting edge: differential inhibition of tlr signaling pathways by cell-permeable peptides representing bb loops of tlrs targeting tlr signaling by tlr toll/il- receptor domain-derived decoy peptides: identification of the tlr toll/il- receptor domain dimerization interface immunity to fungal infections interplay between candida albicans and the mammalian innate host defense genetic susceptibility to candida infections the role of toll-like receptor (tlr) and tlr in the host defense against disseminated candidiasis md- , a molecule that confers lipopolysaccharide responsiveness on toll-like receptor human epithelial cells establish direct antifungal defense through tlr -mediated signaling involvement of cd and toll-like receptors in activation of human monocytes by aspergillus fumigatus hyphae identification of oxidative stress and toll-like receptor signaling as a key pathway of acute lung injury the tlr antagonist eritoran protects mice from lethal influenza infection the role of tlr in infection and immunity human tlr is a functional receptor, expressed by b cells and plasmacytoid dendritic cells, which activates gene transcription through myd crystal structure of the tlr -tlr heterodimer induced by binding of a tri-acylated lipopeptide recognition of lipopeptide patterns by toll-like receptor -toll-like receptor heterodimer antagonistic antibody prevents toll-like receptor -driven lethal shock-like syndromes generation of anti-tlr intrabody mediating inhibition of macrophage surface tlr expression and tlr -driven cell activation searching for mechanisms that matter in early septic acute kidney injury: an experimental study myocardial ischemia/reperfusion injury is mediated by leukocytic tolllike receptor- and reduced by systemic administration of a novel anti-toll-like receptor- antibody ischaemia-induced up-regulation of toll-like receptor in circulating monocytes in cardiogenic shock treatment with opn- , a humanized anti-toll-like receptor- antibody, reduces myocardial ischemia/reperfusion injury in pigs randomized, double-blind, placebo-controlled, dose-escalating phase i, healthy subjects study of intravenous opn- , a humanized anti-tlr- antibody identification and characterization of oligonucleotides that inhibit toll-like receptor -associated immune responses toll-like receptor, rig-i-like receptors and the nlrp inflammasome: key modulators of innate immune responses to double-stranded rna viruses antiviral responses induced by the tlr pathway structural requirements of the ri n-rc n complex for induction of human interferon essential role of mda- in type i ifn responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus tlr is essential for the induction of protective immunity against punta toro virus infection by the double-stranded rna (dsrna), poly(i:c u), but not poly(i:c): differential recognition of synthetic dsrna molecules intranasal treatment with poly(i*c) protects aged mice from lethal respiratory virus infections double-stranded rna induces shedding of the -kda soluble tnfr from human airway epithelial cells via tlr -trif-rip -dependent signaling: roles for dual oxidase -and caspasedependent pathways tlr agonist improves survival to secondary pneumonia in a double injury model the viral tlr agonist poly(i:c) stimulates phagocytosis and intracellular killing of escherichia coli by microglial cells microglia recognize doublestranded rna via tlr poly i:c enhances susceptibility to secondary pulmonary infections by gram-positive bacteria single-stranded oligonucleotides can inhibit cytokine production induced by human toll-like receptor singlestranded dna oligonucleotides inhibit tlr -mediated responses in human monocyte-derived dendritic cells and in vivo in cynomolgus macaques tlr is an endogenous sensor of tissue necrosis during acute inflammatory events structural basis of tlr -flagellin recognition and signaling flagellin as an adjuvant: cellular mechanisms and potential an agonist of toll-like receptor has radioprotective activity in mouse and primate models tlr agonist inhibits acute renal ischemic failure bacterial flagellin stimulates toll-like receptor -dependent defense against vancomycin-resistant enterococcus infection flagellin treatment protects against chemicals, bacteria, viruses, and radiation cpg still rocks! update on an accidental drug tlr activation in dendritic cells enhances salmonella killing and antigen presentation via involvement of the reactive oxygen species toll-like receptor stimulation enhances phagocytosis and intracellular killing of nonencapsulated and encapsulated streptococcus pneumoniae by murine microglia the toll-like receptor ligand, cpg oligodeoxynucleotide, attenuates cardiac dysfunction in polymicrobial sepsis, involving activation of both phosphoinositide kinase/akt and extracellular-signal-related kinase signaling rational design of new cpg oligonucleotides that combine b cell activation with high ifn-alpha induction in plasmacytoid dendritic cells a class c cpg toll-like receptor agonist successfully induces robust interferon-alpha production by plasmacytoid dendritic cells from patients chronically infected with hepatitis c safety, pharmacokinetics and immune effects in normal volunteers of cpg (actilon), an investigational synthetic toll-like receptor agonist phase b, randomized, double-blind, dose-escalation trial of cpg in patients with chronic hepatitis c virus toll-like receptor inhibition reduces mortality in polymicrobial sepsis suppression of immune responses by nonimmunogenic oligodeoxynucleotides with high affinity for high-mobility group box proteins (hmgbs) mechanism of endosomal tlr inhibition by antimalarial drugs and imidazoquinolines chloroquine inhibits proinflammatory cytokine release into human whole blood chloroquine protects mice from challenge with cpg odn and lps by decreasing proinflammatory cytokine release chloroquine and inhibition of toll-like receptor protect from sepsisinduced acute kidney injury detection of prokaryotic mrna signifies microbial viability and promotes immunity telaprevir and pegylated interferon-alpha- a inhibit wild-type and resistant genotype hepatitis c virus replication in patients stimulation of interferon and cytokine gene expression by imiquimod and stimulation by sendai virus utilize similar signal transduction pathways interferon-alpha suppressed granulocyte colony stimulating factor production is reversed by cl , a tlr / agonist tlr /tlr activation restores defective cytokine secretion by myeloid dendritic cells but not by plasmacytoid dendritic cells in hiv-infected pregnant women and newborns molecular basis for the immunostimulatory activity of guanine nucleoside analogs: activation of toll-like receptor isatoribine, an agonist of tlr , reduces plasma virus concentration in chronic hepatitis c infection discovery of ana : an oral prodrug of the tlr- agonist isatoribine masked oral prodrugs of toll-like receptor agonists: a new approach for the treatment of infectious disease use of modelling and simulation techniques to support decision making on the progression of pf- , a tlr agonist being developed for hepatitis c r- triggers the expression of tlr / and suppresses hiv replication in monocytes topical resiquimod . % gel decreases herpes simplex virus type genital shedding: a randomized, controlled trial oral resiquimod in chronic hcv infection: safety and efficacy in placebocontrolled, double-blind phase iia studies gs- , an oral agonist of toll-like receptor- , induces prolonged suppression of hepatitis b virus in chronically infected chimpanzees safety, pharmacokinetics and pharmacodynamics of gs- , an oral toll-like receptor agonist stimulation of tlr prior to polymicrobial sepsis improves the immune control of the inflammatory response in adult mice the search for effective therapy for sepsis: back to the drawing board? sepsis: time to reconsider the concept biomarkers in sepsis we would like to thank all our collaborators that participated in our studies. thierry roger is supported through grants from the swiss national science foundation ( _ , _ , _ , and _ ), an interdisciplinary grant from the faculty of biology and medicine of the university of lausanne (switzerland) and the european com the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. frontiers in immunology | microbial immunology key: cord- -j ay jkf authors: entrican, gary; lunney, joan k.; wattegedera, sean r.; mwangi, william; hope, jayne c.; hammond, john a. title: the veterinary immunological toolbox: past, present, and future date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: j ay jkf it is well-recognized that research capability in veterinary species is restricted by a lack of immunological reagents relative to the extensive toolboxes for small rodent biomedical model species and humans. this creates a barrier to the strategic development of disease control solutions for livestock, companion animals and wildlife that not only affects animal health but can affect human health by increasing the risk of transmission of zoonotic pathogens. there have been a number of projects aimed at reducing the capability gaps in the veterinary immunological toolbox, the majority of these focusing on livestock species. various approaches have been taken to veterinary immunological reagent development across the globe and technological advances in molecular biology and protein biochemistry have accelerated toolbox development. while short-term funding initiatives can address specific gaps in capability, they do not account for long-term sustainability of reagents and databases that requires a different funding model. we review the past, present and future of the veterinary immunological toolbox with specific reference to recent developments discussed at the international union of immunological societies (iuis) veterinary immunology committee (vic) immune toolkit workshop at the th international veterinary immunology symposium (ivis) in seattle, usa, – august . the future availability of these reagents is critical to research for improving animal health, responses to infectious pathogens and vaccine design as well as for important analyses of zoonotic pathogens and the animal /human interface for one health initiatives. the development of novel tools and technologies has been fundamental to the advancement of basic and applied immunology across species. the rate of progress of immunological reagent development for veterinary species has been much slower than that for humans and small rodent biomedical model species, and has impacted research capability in those species ( ) . historically, however, innovations in surgical procedures in veterinary species have resulted in major stepchanges in our understanding of the ontogeny, compartmentalization and function of the immune system. for example, bursectomy in chickens shed new light on mechanisms of b cell development and immunoglobulin production ( ) , in utero thymectomy of lambs revealed the importance of the thymus for lymphocyte development ( ) and lymphatic cannulation of sheep revealed that lymphocyte subsets differ between blood, afferent and efferent lymph ( ) . these ground-breaking experiments were feasible, in part, due to the size of the species under investigation, particularly for the technique of lymphatic cannulation due to the diameter of lymphatic vessels in ruminants ( ) . however, this momentum in veterinary immunological studies was not maintained; the vast majority of technological innovations and discoveries in immunology in the past years have been made in mice. the development of congenic mice, differing at a single histocompatibility locus, was a fundamental technological innovation in immunology that led to mice being the primary species of choice for research. that pioneering work of george snell and the later capability of genetically manipulating congenic mice has allowed immunologists to ascribe functions to genes, molecules and cells with high precision ( ) . the development of monoclonal antibody (mab) technology using congenic mice subsequently created almost boundless opportunities for research in basic and translational immunology ( ) . the availability of mabs that could phenotype cells and detect cytokines by elisa underpinned the discovery of two distinct cd +ve t-cell subsets in congenic mice ( ) . the subsequent th /th paradigm provided a fundamental framework for investigating immune activation and regulation that has expanded far beyond those original two subsets. current capability now extends to multi-parametric analyses such as simultaneous fourteen-color flow cytometry that can identify functionally-relevant cd +ve t-cell subsets in human blood ( ) . mass cytometry (cytof) methods using panels of well over conjugated antibodies are now allowing for even deeper analysis of single cell expression, offering new insights into cellular subsets and their differentiation ( , ) . such technologies cannot usually be applied directly to different species since molecular differences in immunological orthologs result in low cross-reactivity of reagents across species ( ) as affirmed by a recent comparison of reactivity of immune protein reagents for other species with swine orthologs ( ) . thus, reagent development needs to be evaluated on a caseby-case basis. gaps in capability for veterinary species are often prioritized based on the extensive mouse and human immunological toolboxes. the expansion of the toolboxes has revealed substantial differences in the ways that humans, mice and veterinary species respond to disease and highlighted to need for studying different species in their own right ( , ) . there have been coordinated efforts to evaluate species crossreactivity of anti-human cd antigen mabs through the animal homologs section of the human leukocyte differentiation antigen (hlda) workshops: for horses ( ) , dogs ( ) , pigs ( ) , and ruminants ( ) . in an effort to generate greater international co-ordination for immune reagent characterization activities, the international union of immunological societies (iuis) veterinary immunology committee (vic) supported a toolkit workshop ( ) . it is almost years since the last published review of the veterinary immunology toolbox from the iuis vic toolkit workshop at the th ivis in tokyo, japan ( ) . here, we review progress over the past decade by reporting on the iuis vic toolkit workshop at the th ivis in seattle, usa in and take a forward look to the future of the veterinary immunology toolbox. the success of the hlda workshops was based on good co-ordination, high-quality work and collective effort by the veterinary immunology community, as well as results from past species-specific cd workshops supported by iuis vic. common standards were applied to the distribution and evaluation of anti-human cd reagents being assessed in different laboratories and the collective generated data being reviewed centrally. the outcome was an evidence-based assessment for the activity of species cross-reactive mabs, with affirmation that only a limited number of mab directed against human cd antigens actually cross-react with other animal species ( ) . these results instilled confidence in the performance of those reagents and promoted their uptake by the research community and industry, including companies that market and sell veterinary immunological reagents. although the hlda workshops were primarily focused on evaluation of species cross-reactive antibodies, they played an important role in informing of capability gaps and therefore the prioritization of reagents for future development. a major step-change in the way veterinary immunological reagent development was supported came with the inception of a uk immunological toolbox funded by the biotechnology and biological sciences research council (bbsrc) and the scottish executive environment and rural affairs department (seerad) in . this was unique as it united several laboratories within a single project to take a collective multispecies approach to immunological reagent development. this was followed by the veterinary immune reagent network (virn) funded by united states department of agriculture (usda)/national institute of food and agriculture (nifa) in the us in . both projects included the creation of databases listing available veterinary immunological reagents, which will be discussed later. they also expanded the emphasis from mab anti-cd antigens to expression of immune proteins (cytokines and chemokines) and protein reactive mabs. the us project included direct collaboration with commercial partners to express these immune proteins. the us and uk projects worked together under a memorandum of understanding (mou) to avoid duplication of effort. this mou was created in the absence of a mechanism for joint international funding by the respective national agencies. the structure, priorities and achievements of these projects has been published previously ( ) . a key output from these initiatives was an increased recognition of the importance of coordinated, complementary approaches to reagent prioritization and development. their success has also been reflected by continued support for reagent development initiatives by funders seeking to build on the significant benefits from their original investments, with the assertion that long-term sustainability is essential. the funding for veterinary immunology reagent development has changed over the past years, moving from the multispecies models of the uk immunological toolbox and us virn, to single-species projects. with the exception of ruminants, there is very little species cross-reactivity of veterinary reagents, highlighting that the genes involved in immune responses are amongst the most rapidly evolving in vertebrate genomes ( , ) . however, this does not diminish their potential as disease models. bbsrc and usda/nifa have supported reagent development projects for ruminants, swine, horses, aquaculture species and poultry in the past years (box ). a barrier to formal international collaboration was lifted in when usda/nifa and bbsrc launched a pilot call to support animal disease research of strategic importance to both the us and uk which included the development of veterinary immunological reagents for agriculturally-relevant animal species. the swine toolkit was a landmark first transatlantic veterinary immunology reagent project funded under this initiative in (box ). although we have focused here on projects funded specifically to develop reagents and supporting technologies, this is not intended to ignore the veterinary immunological reagent development that is conducted within disease-driven projects, networks and within strategic programmes of government research institutes across the globe. the challenge is in capturing the outputs of these diverse activities. the websites of commercial reagent suppliers and peer-reviewed publications are sources of validated information on reagent activity. however, they do not capture everything, a particular gap being the paucity of "negative" data when reagents are found to be non-functional or where repeated attempts fail to generate specific antibodies. these are very valuable data as they can potentially prevent the duplication of wasted effort. the solution lies in community engagement for the sharing of knowledge on reagent availability and performance. workshops such as those hosted by iuis vic toolkit are a focal point for international information exchange, but they do not have the facility to capture, store and disseminate information at a detailed level. it has been recognized for many years that a major unmet need in veterinary immunology is the lack of centralized, non-commercial, searchable reagent databases ( ) . the original uk immunological toolbox ( ) ( ) ( ) ( ) ( ) ( ) ( ) and the us virn ( - ) both created lists of reagents but the databases were not sustainable beyond the term of funding. this is not surprising as curation is timeconsuming, requiring expert knowledge of immunology and information technology input to create web-based interfaces. this also highlighted the problem of sustainability when there is reliance on short-term funding for reagent development projects. finding solutions to these problems has been the focus of several recent workshops as discussed below. one exception to this has been the usda agricultural research service (ars) supported porcine translational research database (ptrd, http://tinyurl. com/hxxq ur) ( ) . the current landscape of the veterinary immunology toolbox has been shaped by new funding approaches to facilitate reagent development while also addressing the complex issues of database construction, collection and validation of data, and sustainability of the database and biobanks of the reagents listed therein. this report summarizes the outcomes of several international workshops where these various elements have been considered. before summarizing those outcomes, it is worth reviewing the scope of the toolbox in terms of species coverage and knowledge of immunological capability within those species. in the broadest sense, the concept of a veterinary immunological toolbox encompasses a broad range of livestock, companion animal, biomedical model and wildlife species. there has been progress in reagent development across all of those species in the past years which has been presented at various meetings and workshops. we have identified a number of published articles where reagent availability for different species have been reviewed. for the purposes of the toolbox, livestock species can largely be regarded as belonging to one of four major groupings, namely swine ( , ) , ruminants ( , ) , poultry ( ) ( ) ( ) , and aquaculture ( , ) . companion animals include horses ( , ) , cats ( ) , and dogs ( ) . as previously discussed, mice are the most common small-animal biomedical model for human ( ) . however, rabbits ( ) and ferrets ( ) are also popular small-animal biomedical models for human disease. there is interest in expanding the immunological toolboxes for wildlife species, for example buffalo ( ) and badgers ( ) due to their potential to act as reservoirs for economically-important livestock diseases. there is also interest in developing immunological reagents for marine mammals such as dolphins ( ) . in addition, although camelid species are not often regarded as a major target host species for disease studies, they have come to the fore with heightened awareness of mers-cov and the potential to reduce zoonotic transmission by investigating vaccine-induced responses in camels ( ) . importantly, camels make a unique technological contribution to the immunological toolbox via the production of nanobodies ( , ) . to date, the concept of the veterinary immunological toolbox has largely (but not exclusively) focused on reagent development for livestock species due to their strategic relevance for funders with a stake in livestock health, food safety and global food security. in the period between the last published review of the iuis vic toolkit workshop at the th ivis in tokyo ( ) and the iuis vic toolkit workshop at the th ivis in seattle, there have been several key meetings whose outcomes are directly relevant to the current status and future directions of the toolbox and merit discussion here. the first was at the th ivis in milan, italy in when bbsrc and the global strategic alliances for the coordination of research on the major infectious diseases of animals and zoonoses (star-idaz) supported a vaccinology https://www.pirbright.ac.uk/news/ / /bill-melinda-gates-foundation-funds-development-pirbright%e % % s-livestock-antibody-hub. to study cattle, pig and poultry antibody responses at high resolution to expand the understanding of protective immunity in those species and that can also be used as models for a range of human infectious diseases. usda/nifa: cattle ( - ): https://portal.nifa.usda.gov/web/crisprojectpages/ -immune-reagents-for-ruminants-with-primary-focus-on-bovine-specific-reagents.html. to develop, and make commercially available, mab reagents needed to elucidate cattle immune mechanisms by focusing on cd antigens, cytokines, and chemokines and relevant assays. https://portal.nifa.usda.gov/web/crisprojectpages/ -us-veterinary-immune-reagent-network.html. to clone, express, develop mab reagents specific for ruminants, swine, poultry, equine and aquaculture species, sharing methods across species. work with commercial partner to market expressed proteins for use by veterinary immunology community. workshop. the lack of immunological tools and reagents was recognized as a major barrier to progress. this can be seen in the subsequent bbsrc veterinary vaccinology strategy (https:// bbsrc.ukri.org/about/reviews/scientific-areas/ -veterinaryvaccinology-strategy/) and the creation of the bbsrc uk veterinary vaccinology network (vvn). in , bbsrc vvn hosted a workshop to discuss the toolbox initiatives in the uk and us with specific relevance to the aims and objectives of the newly-formed global challenges research fund (gcrf) international veterinary vaccinology network (ivvn). a full report is available on the bbsrc vvn website (http://www.vetvaccnet.ac.uk/publications/veterinaryimmunology-toolbox-meeting-uk-veterinary-vaccinologynetwork). at this workshop, the pirbright institute and the roslin institute at the university of edinburgh announced plans for a new uk immunological toolbox project. the combined project would be underpinned by core institute funding from the bbsrc, with additional support from the bbsrc gcrf tools and resources (https://www.immunologicaltoolbox.co. uk/about/funders). this project is addressing major gaps in capability and sustainability. the first of these is the creation of a publicly accessible, searchable database of veterinary immunological reagents to be accessed via a dedicated website. a follow up meeting was held at the vvn conference in stirling in early (https://www.vetvaccnet.ac.uk/news/ / / uk-veterinary-vaccinology-network-conference- -report) to discuss in more detail the focus of the website and new reagent development. it was agreed by the community that a key driver for the website would be the facility for researchers to submit information on reagent performance and request reagent production where gaps exist. it was discussed that the primary focus of new reagent development should be around t cell and b cell subsets to help dissect in more detail pathogen and vaccine responses. as well as new reagent development the toolbox aims to exploit new technologies to translate current hybridoma stocks into gene blocks via sequencing and create a recombinant antibody pipeline, express recombinant proteins (including cytokines and chemokines), build multiplex platforms and develop high-throughput screening systems for new antibodies. these sequences act as the template from which the constant region can be switched between different species while maintaining target specificity. a toolkit workshop was held at the th european veterinary immunology workshop (eviw) conference in utrecht, netherlands in . although this conference was organized under the auspices of the european veterinary immunology group (evig), as opposed to iuis vic, the iuis vic toolkit committee took a leading role in the organization of the toolkit workshop. notably, the toolkit workshop was structured to reflect four newly-formed major livestock groupings (swine, ruminants, poultry, aquaculture) of iuis vic toolkit which were announced for the first time at this meeting. the leaders of the species groups represented their respective areas at the workshop. they are listed on the iuis vic webpage and can be contacted by members of the community who are seeking information or looking to engage in reagent development for each of those areas (https://iuis.org/committees/vic/). the workshop covered the major projects in europe and the us on reagent development, including a presentation on the plans for the new uk immunological toolbox. in the panel discussion, there was broad international support for the approaches being taken within the new toolbox project and recognition of the complementary work being supported by usda/nifa in all of the target species (box ). this meeting cemented the requirement for community engagement in the website to provide and maximize information exchange about the availability and performance of reagents and the focus on the generation of novel antibodies and methods to distinguish t and b cell subsets. this particular area will be advanced by the development of a new livestock antibody hub centered at the pirbright institute which aims to improve both animal and human health globally by translating research outcomes in livestock diseases (box ). a core aim of this antibody hub is to develop tools, techniques and reagents for livestock research that bring the research capability to the same level as that for humans and mice. the iuis vic toolkit workshop at the th ivis in seattle was the forum for the international launch of the pirbright/roslin uk immunological toolbox website and the associated database (http://www.immunologicaltoolbox.co.uk). this database was built around the original information collated during the - bbsrc seerad-funded uk immunological toolbox and is therefore skewed toward three of the four major livestock groupings (swine, ruminants and poultry). however, aquaculture species, companion animals and now major animal pathogens are also included, and as the community engages the amount of information will increase. the main aim of the website is to collate reagent information and act as a centralized source to increase information exchange but is not the only source for any particular species. for example, the usda porcine translational research database (http://tinyurl.com/hxxq ur) is considered a very wide ranging and valuable community resource and cannot be duplicated but information is shared with the uk immunological toolbox via mutual awareness and direct communication. the uk immunological toolbox database contains data on reagents that are held in research laboratories, and also those available commercially, which immediately raises questions on the quality and reproducibility of reagents from different sources. the standardized production, evaluation and storage of commercially-available reagents would be expected to reduce batch-to-batch variation, whereas the same reagent produced and stored in different research laboratories is likely to have more variability due to the different conditions. when reagents are listed on the uk immunological toolbox website there will be information on their specificity and performance, preferably supported by peer-review publication wherever possible. there is also a facility for registered users to provide feedback on performance to add to the available information. such information will be checked before posting against the user's identification. it was emphasized that such a database can be as complete and useful as the community wants it to be. the website and database will be curated centrally, but the community has to take collective ownership by submitting reagents and information on their performance. it is pleasing to see that this is already happening. the toolbox website also serves as a reference point for non-veterinary immunologists looking to expand their choice of biomedical models and facilitate comparative immunology research ( ) . finally, several new opportunities were identified during the open discussion at the iuis vic toolkit workshop in seattle. these included the unique opportunity to salvage and store "orphan" mabs via the sequencing technology within the uk immunological toolbox. the preservation of sequences does not incur the high costs associated with maintaining hybridoma cells in liquid nitrogen. in addition, the sharing of sequences circumvents many of the logistical and financial issues involved in the shipment of live cells, particularly across international borders. as we enter the third decade of the st century, the "one health" agenda has never been more important. the development of solutions for controlling infectious diseases in livestock, companion animals and wildlife not only has direct benefits for the target species but can reduce disease transmission across species, including zoonotic transmission, thereby reducing the wider global disease burden ( ) . close contact between different animal species and between animals and humans is a risk for zoonotic disease, which can be difficult to manage in low and middle income countries (lmics) ( ) . given the importance of livestock to lmics, the veterinary immunological toolbox provides economic and health benefits by underpinning animal vaccine development. the quality of toolbox reagents and associated information in the uk immunological toolbox database are paramount. evidence-based validation and standardization of new technologies is essential to generate confidence in performance and encourage uptake by the community. there remain major capability gaps in multi-analyte protein technologies for veterinary species. the development of such technologies is technically challenging, but entirely feasible with the appropriate resources and effort. the key to success is in working together. the single-tube technology that simultaneously identifies functionally-relevant cd +ve t-cell subsets in human blood was developed and validated through the collective efforts of the multiple partners in the euroflow and periscope consortia ( ) . multiparametric technologies are extremely powerful; one way of expanding the flexibility of the relatively limited range of antibodies in veterinary species is the ability to efficiently conjugate small amounts of antibodies with different labels for defining immune correlates. the identification and quantification of immunological correlates of protection are aspirational goals for the development of safe and effective vaccines ( , ) . however, with the exception of anti-virus neutralizing antibodies, immunological correlates of protection tend to be multifactorial rather than singular, particularly in the case of cell-mediated protective immunity requiring not only cell subset identification but appropriate cytokine co-expression. the solution to identifying such correlates lies in the application of a range of multi-plex technologies that all detect multiple analytes at the genetic, protein, and cellular level, so called "systems vaccinology" ( ) . we are also moving into an era of high dependency on computational infrastructure as the data generated by such complex studies require specialized programmes for full analysis. hence, collective approaches are becoming increasingly important if we are to maximize our potential to develop and adopt complex technologies in the future. the importance of genomic information and alternate expression systems such as pichia pastoris, insect and mammalian cells has meant wider availability of species-specific immune proteins. the veterinary immunology community has a long history of working together for collective good, such as the hlda workshops, international cd workshops, toolkit committees, collaborative funding initiatives and the immunological toolbox. in doing so we need to maintain a global perspective and consider technologies that create solutions for animal diseases across borders. one example is the antibody sequencing technologies of the new uk immunological toolbox. in addition to the advantages described earlier, this technology offers particular cost-effective and sustainability benefits for the transfer and storage of reagents to lmics where veterinary immunology research is being conducted. in parallel to sequencing, expressing and engineering mabs, companies and research groups all over the world are adapting single b cell sequencing technologies to a range of host species. these technologies often rely to some extent on existing reagents to identify b cells (including antigen specific b cells) but are generally very adaptable to any given species and synergise well with existing mouse recombinant antibody expression methods. these methods are providing a completely new route to identifying antibodies against specific epitopes on pathogens as well as other foreign immunizing antigens. these antibodies can be used as reagents, including mapping complex epitope landscapes to inform structural vaccinology approaches to increase efficacy, and may also be used as therapeutics. antibodies are now a primary therapeutic goal of many companies for a range of human diseases. cats and dogs are not only a profitable target market for immunotherapeutics, they provide value data on in vivo mab function ( ) . although the cost of such treatments is currently prohibitive for food producing species, large animal models and speciesspecific reagents can have a very important role in testing manufacture, delivery and efficacy of mabs as part of the one health approach. the impact of veterinary immunology research will ultimately be measured by the development, or contribution to the development, of disease-control solutions including diagnostic tests, vaccines and genetic-based strategies. the range of vaccine-delivery platforms is rapidly expanding, including improved adjuvants, vector-based delivery systems and genetic vaccination with dna and rna. although viralvectored vaccines are successfully deployed in humans and companion animals ( ), public safety concerns remain regarding their use food animals ( ) . the immunological toolbox can be applied to safety and efficacy studies in livestock, thereby informing on the benefit-risk ratio that would be impossible to do at the same scale in humans or primates. animal genetics can provide insights into responses to infection and vaccination which can be translated into livestock breeding programmes ( , ) . breeding programmes require several generations to observe population effects and conclusive proof for the effect of a specific genotype on immune status requires functional evidence, hence reliance on the toolbox. new gene-editing technologies such as crispr now allow very targeted approaches to livestock production ( ) . this is the future of livestock farming and the immunological toolbox not only has a role to play in the identification of genes to be targeted, but it will also be important for defining subsequent immune function, including potential off-target effects. genome editing is also creating the opportunities to engineer species to act as better models for human diseases alongside or in addition to genetically defined and tailored breeds, such as scid pigs and mhc homozygous pigs ( , ) . for example, pigs are emerging as a very powerful model to predict human influenza vaccine responses but to achieve the maximum benefit of such models a complete toolkit is required ( ) . gene editing is already providing pig organs for future human xenotransplantation, a biomedical application that has helped drive reagent development in pigs ( , ) . the veterinary immunological toolbox is very broad in its scope and has evolved from multiple efforts across the globe. in the broadest sense, the toolbox incorporates livestock, companion animals, wildlife and biomedical animal species. each is important in its own right, but all are collectively important for the one health agenda and for controlling existing and emerging diseases that infect different animal populations and have zoonotic potential. as human populations expand, there is a need to protect food security without compromising food safety. disease prevention and control results in improvements in animal health and welfare, which not only has economic and ethical benefits but can also address concerns for climate change by making food production more efficient. basic immunology underpins these approaches, from vaccine design to understanding the effects of gene editing. the immunological toolbox website and associated searchable database provides a new focal point for information and knowledge exchange for the veterinary immunology community. the key to future success is global collective working facilitated by networks such as national immunological societies, eviw, ivvn, american association of veterinary immunologists (aavi), and iuis vic toolkit committee. veterinary immunology committee toolkit workshop : progress and plans effect of early bursectomy on germinal centre and immunoglobulin production in chickens the growth and development of lambs thymectomized in utero lymphocyte subsets show marked differences in their distribution between blood and the afferent and efferent lymph of peripheral lymph nodes a road less travelled: large animal models in immunological research a methods paper that led to much more continuous cultures of fused cells secreting antibody of predefined specificity two types of murine helper t cell clone. i definition according to profiles of lymphokine activities and secreted proteins age distribution of multiple functionally relevant subsets of cd + t cells in human blood using a standardized and validated -color euroflow immune monitoring tube use of mass cytometry to profile human t cell exhaustion immune monitoring using mass cytometry and related high-dimensional imaging approaches advanced model systems and tools for basic and translational human immunology porcine cytokines, chemokines and growth factors: update genomic responses in mouse models poorly mimic human inflammatory diseases an in-depth comparison of the porcine, murine and human inflammasomes; lessons from the porcine genome and transcriptome report of the first international workshop on equine leucocyte antigens monoclonal antibodies that define canine homologues of human cd antigens: summary of the first international canine leukocyte antigen workshop (claw) summary of the first round analyses of the second international swine cd workshop cross-reactivity with bovine cells of monoclonal antibodies submitted to the th international workshop on human leukocyte differentiation antigens a current perspective on availability of tools, resources and networks for veterinary immunology summary of the animal homologue section of hlda exploiting ovine immunology to improve the relevance of biomedical models the porcine translational research database: a manually curated, genomics and proteomics-based research resource expressed gene sequence and bioactivity of the ifngamma-response chemokine cxcl of swine and cattle development of new immune reagents for swine health, vaccine and disease studies contributions of farm animals to immunology the long view: a bright past, a brighter future? forty years of chicken immunology pre-and post-genome marek's disease in chickens: a review with focus on immunology development and characterization of monoclonal antibodies specific for chicken interleukin- and their neutralizing effects in chicken primary monocytes perspective on the development and validation of ab reagents to fish immune proteins for the correct assessment of immune function standardized imgt r nomenclature of salmonidae igh genes, the paradigm of atlantic salmon and rainbow trout: from genomics to repertoires the development of equine immunity: current knowledge on immunology in the young horse transcriptome analysis of immune genes in peripheral blood mononuclear cells of young foals and adult horses cats are not small dogs: is there an immunological explanation for why cats are less affected by arthropod-borne disease than dogs? parasites vect immunotherapy for dogs: running behind humans the wide utility of rabbits as models of human diseases moving forward: recent developments for the ferret biomedical research model characterization of leukocyte subsets in buffalo (bubalus bubalis) with cross-reactive monoclonal antibodies specific for bovine mhc class i and class ii molecules and leukocyte differentiation molecules immunological responses of european badgers (meles meles) to infection with mycobacterium bovis identification of monoclonal antibodies cross-reactive with bottlenose dolphin orthologues of the major histocompatibility complex and leukocyte differentiation molecules humoral immunogenicity and efficacy of a single dose of chadox mers vaccine candidate in dromedary camels a toolbox of nanobodies developed and validated for use as intrabodies and nanoscale immunolabels in mammalian brain neurons the therapeutic potential of nanobodies the uk veterinary immunological toolbox website: promoting vaccine research by facilitating communication and removing reagent barriers quantitative outcomes of a one health approach to study global health challenges global patterns of zoonotic disease in mammals a threshold method for immunological correlates of protection erratum to: a threshold method for immunological correlates of protection systems vaccinology and big data in the vaccine development chain anti-nerve growth factor monoclonal antibodies for the control of pain in dogs and cats multivalent and multipathogen viral vector vaccines working with gm vaccines: engaging the public genes controlling vaccine responses and disease resistance to respiratory viral pathogens in cattle host genetics of response to porcine reproductive and respiratory syndrome in nursery pigs on-farm livestock genome editing using cutting edge reproductive technologies the major histocompatibility complex homozygous inbred babraham pig as a resource for veterinary and translational medicine cd epsilon(+) cells in pigs with severe combined immunodeficiency due to defects in artemis t and b cell immune responses to influenza viruses in pigs the role of genetically engineered pigs in xenotransplantation research tolerance in xenotransplantation all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. our collective thanks to star-idaz, bbsrc vvn, gcrf ivvn, and aavi for supporting workshops and iuis for supporting the th ivis/iuis vic immune toolkit workshop. key: cord- -tpv authors: chatterjea, devavani title: teaching immunology as a liberal art date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: tpv a complex, rapidly evolving biomedical field that is of critical relevance to human health and well-being, immunology provides important and substantive opportunities to practice and teach the central tenets of a liberal arts curriculum. it's one of those "end of semester" days in december-i am looking forward to wrapping up the term, the familiar mix of exhaustion and anticipation in my bones. the junior and senior biology majors in my immunology survey course at an undergraduate liberal arts college in the midwest are setting up their immunology-themed art presentations. a pile of "plushies"-giant stuffed fabric white blood cells decorated with their known surface markers invites tactile exploration, and an impromptu game of toss. an immune cell synapse wired with leds lights up in series as "activation" switches are flicked on. students flock to the edible displays. a towering croquembouche "lymph node" of choux pastries invites them to pull out individual ones to taste-flavored with different fillings, the pastries represent the different cells in a lymph node. as the puffs get eaten, the spun sugar matrix of the tower loses shape, much as a lymph node matrix would without resident cells. the hematopoiesis cookie table is a hit. the student who set it up explains how a basic set of ingredients is flexibly transformed into different kinds of cookies-at which points commitments to certain final products occur and when and how steps become irreversible; class-mates sample some of the finished products and take turns building cookies of different "lineages" with nuts, fruit, chocolate chips, bits of candy sparking a spontaneous discussion about food allergies, routes of exposure and safe handling practices. a student clears their throat and the hum of chatter subsides. a self-described "non-artist, " they have chosen instead to deliver a "testimony to congress" to advocate for robust funding for immunological research inspired by the advocacy of members of the american association of immunologists ( ). as standin lawmakers, we listen attentively to the evidence-based arguments for the importance of basic immunology research for a healthy society. there are tough but respectful questions on animal research ethics, a plausible timeline for a universal flu vaccine and the structural inequities of access to cutting edge cancer therapies such as car-t cells. after the q&a, students read each other's artist's statements, take turns trying to sit on the fold-out monocyte chair without falling, and play with the stick and string co-stimulation maze which can only be solved with manipulations in the correct sequence! over years ago, i was an undergraduate in an immunology class, irresistibly drawn to the discipline despite the confounding maze of nomenclatures, the alphabet soup of transgenic tcr names and the flood of cell types and molecules that went over my head. through graduate and post-doctoral work, my love for the field endured and i realized that i wanted an undergraduate liberal arts curriculum to be the canvas for my immunology teaching and research. i don't think that, at the time, i could have foreseen a class period quite like the one i just described: students making the material their own in inventive and surprising ways, going confidently into the heart of foundational and cutting-edge concepts and using their intellectual and practical engagement with the material to connect their study of immunology with their lives. teaching and learning immunology as a liberal art together with my students has been transformative for all of us. macalester college is an urban small undergraduate liberal arts college with ∼ , students− % students of color, % international, and % first-generation. biology is one of the top majors. i teach an immunology survey course with laboratory for undergraduates who have taken cell biology and genetics. though the human immune system is the primary focus of the course, we study amoeba, social insects, bacteria, plants, and jawless fish to better understand the evolution of protective responses. students write multipledraft review papers with graphical abstracts, volunteer with the immune deficiency foundation, present art/performance works and write weekly reflective essays connecting their immunology learning to other parts of their academic or personal lives. my immunological methods course is embedded in my research program investigating the connections between environmental toxins, allergic responses, and chronic pain; students participate in scientific conversations and critique scaffolded by preparatory writing assignments, map metaarguments from sub-fields of published literature, cooperatively design, and execute experiments, and write a collaborative scientific paper. i use my upper-level seminar courses-neuroimmunology and cancer immunology to teach more advanced students about public communication of science. in our college's first year course program i offer semester-long immunology-themed courses: aids/influenza/malaria -ancient pathogens in a brave new world explores the persistence and reemergence of infections and inflammatory diseases in vulnerable populations around the world and bodies on fire centers on the global pandemic of inflammatory diseases. these courses do not have pre-requisites and are structured around connecting patient/physician memoirs, popular science books, and science journalism with the scientists and scientific discoveries they describe and typically ask students to explore these connections through writing, movement, and art. historian william cronon describes the essence of liberal education as "gaining the power and the wisdom, the generosity, and the freedom to connect"-through the acts of listening, reading, writing, talking, solving puzzles, seeking complex truths, seeing other perspectives, working in a community and being willing to both lead and follow in honest and imaginative ways ( ) . structurally, a liberal arts education connects the natural and physical sciences, humanities, social sciences, quantitative thinking, and artistic inquiry. even as they engage deeply with methods and analyses in particular areas of study, students learn to appreciate different ways of making meaning of our world with tools from different disciplines. they learn to recognize and interrogate the societal structures and deep assumptions that drive the ways in which such bodies of knowledge are constructed within and across academic disciplines. immunology is a perfect fit for a liberal arts education. while traditional practices such as variolation and uses of immunomodulatory foods and botanical medicines have existed for thousands of years in societies around the globe, the constructs of cellular and circulating immune mechanisms have been articulated in the context of academic biomedicine only as recently as the late s. and within these years, paradigms have been swiftly proposed, critiqued, modified and transformed into an ever more complex and nuanced understanding of immunity ( ). concepts of preservation of self over "non-self " have morphed into understandings of danger, disruption, repair, and memory embedded deep within cell lineages, epigenetic imprints and tissue architectures. mechanisms once described more bluntly as "killing pathogens" are now understood as highly regulated, selective, tunable responses to commensal and non-commensal microbes that constitute the multi-species ecosystems of multicellular hosts. while the immune system gives us critical protection for survival, virtually every global health concern from emerging infections, allergies and asthma, autoimmunity, chronic pain, and other psychiatric, cardiovascular, and metabolic imbalances are all fueled by these same mechanisms of inflammation, shifted by context to become harmful and pathological. author chimamanda ngozi adichie, in her ted talk "the danger of a single story, " warns that assuming a single story about a people leads to dangerous misconceptions, and learning to listen for the many different stories is essential for cross-cultural understanding ( ) . immune responses, with their double-edged nature, provide a natural set of case studies in the importance of "many stories." immune responses demand careful contextual analyses, and to study them closely is to learn to grapple with complexity and uncertainty-an essential skill in today's rapidly changing, connected yet divergent world. another advantage of studying immunology is its immediate personal and social relevance. students only have to look at their own bodies, experiences of well-being and illnesses, and their environments for applications of what they learn. for many students, one immunology-related class might be their only sustained experience with the discipline, but the lessons they draw from it have the potential to remain relevant and useful in their lives. as a powerful example of this, i have observed my neuroimmunology students particularly resonate with learning about the role inflammation plays in mental health. students on college campuses are experiencing anxiety and depression at unprecedented levels, and managing neurological diagnoses while removed from their families and support systems ( ) . understanding the roles of pathological inflammation intertwined with these mental health conditions, exploring the connections of stress, diet, and rest to these neuro-inflammatory pathways are empowering for students; appreciating the "bodily" basis of psychological challenges appears to make them seem more tractable. while these lessons do not take the place of the counseling and/or psychiatric support they or their peers need and receive, i have observed that students do find this scientific parsing of the mind-body connection to be of practical use. many immunology students are drawn to careers in biomedical research and its applications in the practices of medicine and/or public health. immunological researchdiscovery, translational, academic, clinical, industrial-and its applications in drug development, medical technologies, and public health interventions are at once scientific and social endeavors. countering anti-vaccine movements, crafting community, and government public health responses to disease outbreaks, regulating environmental toxicants in food, water, and air all contain important immunological arguments at their core. being able to understand and speak the language of immunology and tell its stories to specialist as well as general audiences so they can be truly heard is an important skill for students to practice. iteratively learning to read the often dense and technical immunological literature and synthesizing and communicating these findings in their own written and spoken words is both preparation for future work in biomedical fields and a core tenet of a liberal arts education-the importance of listening, reading, speaking, arguing, and writing. these skills are not unique to the study of immunology, but immunology offers undergraduates and their professors in a liberal arts context a rich and pragmatic field within the biomedical sciences in which to practice them. students in my courses and research laboratory write literature reviews, give talks and present posters on their research at conferences, and collaborate with me on writing papers and grant proposals for scientific audiences. however, they also write white papers and reflective essays connecting their learning in immunology to other disciplines, prepare educational materials for community organizations, teach secondary school students and mentor younger peers and, in doing so, practice translating the technical jargon of scientific communication into information that their audiences need and can use. a spacious liberal arts education makes room for multidisciplinary training, provides opportunities for immersive learning and community engagement and asks students to connect their learning to the world in different ways, giving them opportunities to make this complicated and compelling field their own. the perceived "difficulty" of immunology can be deconstructed in this more permissive, integrative environment to allow creative strategies for making meaning and finding purposeful engagement with the subject. immune systems are synergistic wholes of interconnected parts continuously stirred up by new discoveries that complicate existing knowledge and demand new ideas and interpretations; this has been so since paul ehrlich sketched his intricate visions of cells shedding neutralizing anti-toxins and butted heads with ilya metchnikoff 's cheeky but utterly prescient observation that immunity might just look like hungry amoeba out to forage ( ). in the last two decades, our view of the immune system has been transformed by newly discovered innate cell subsets, the regulation of immunity by microbial and viral symbionts, the control of immune responses by metabolic switches, and the realization that all cells, not just the ones that we recognize as immune cells, participate in and regulate immune responses of multicellular organisms. this framework of synergistic interactions and multi-factorial outcomes can provide our students with maps and metaphors useful beyond immunology, for broader understandings of complex social and planetary processes. the precarious balance of protective vs. harmful immune responses is a mirror of the collateral costs of inequities, statesanctioned violence, and xenophobia in our societies. chronic inflammation and accompanying adverse health outcomes are materially correlated with lower socio-economic status, lack of access to nutritious foods, stressful living conditions and unstable access to healthcare ( ) . that any immune response takes a toll on the living tissue it is trying to protect from real or imagined threats parallels the effect that xenophobic, reactive intolerance, and unregulated violence can have on a community or society at large. just as our own cells and those of our commensal symbionts maintain a collaborative understanding that we disrupt at our peril, our local and global communities are sensitive to the behavior of individuals and cooperation between the diverse populations who live in them. tolerance, balance, homeostasis, repair are technical terms with specific immunological meanings that are just as relevant to our social fabric as they are to our understanding of healthy and disease states of our bodies. and likewise, jingoistic militarized language about the immune system vanquishing pathogens can echo intolerant social rhetoric. the nuance and care required to understand and modulate immune responses and their outcomes serve as object lessons in how we speak and act as individual and collectives in social and political arenas. an immunological framework can also be applied to the relationship of humans with our planet as a whole. humaninduced climate change has driven our planet and its inhabitants to a perilous state of imbalance, with rapid rise in temperature and sea levels, catastrophic weather events, heat stress, food shortages, displacement of peoples, biodiversity loss, emerging pathogens (such as sars-cov ), and exacerbation of poverty and conflict, all of which create negative health outcomes for those who are most vulnerable and have the least access to resources. the united nations intergovernmental panel on climate change ( ) advocates for immediate, massive, and collective action to mitigate this crisis if we are to survive. our students are joining their climate activist peers-greta thunberg, isra hirsi, xiye bastida, and others in climate strikes and actions to emphasize the urgency of the situation. the literal health effects of climate change are, and will be marked by inflammatory processes in our individual bodies, and sharp increases in global disease burdens; it is as if the entire planet is in a state of chronic inflammatory distress. everything is connected and what we do individually, and collectively, to our bodies and to our world comes back to us. teaching about our immune systems in integrative, socially relevant ways can help our students make meaningful connections between the content of their learning and the larger global context in which they live. in her book teaching to transgress ( ), feminist author and educator bell hooks says: to educate as the practice of freedom is a way of teaching that anyone can learn. that learning process comes easiest to those of us who teach who also believe that there is an aspect of our vocation . . . is not merely to share information but to share in the intellectual and spiritual growth of our students. in information-dense, rapidly evolving fields of study, it is natural to feel overwhelmed by the responsibility to share information as accurately and comprehensively as possible before our limited time with any one group of students comes to a close. i am grateful that immunology-the beautiful, maddening, messy field that it is-keeps me humble and honest about the work i really want to do with my students and the way in which i want to do it. it resolutely refuses to be told as a "single story" and any arcane details memorized for exams are known to have modest shelf lives in any case. so with each passing year, i am challenged to re-imagine how i can best help my students be as prepared as possible to hear and understand all of the immunological stories that have not been written yet-to be able to know the workings of their future bodies and minds a little better, to understand and appreciate why a pandemic coronavirus can ravage one body it infects and leave another unscathed, to be able to use these stories to build healthy lives and communities, and make new discoveries. in her more recent book, teaching community: a pedagogy of hope ( ), bell hooks says: "it is imperative that we (teachers) maintain hope even when the harshness of reality suggests otherwise." i take these words to heart. much of western biomedical science has been built around concepts of illness rather than wellness and i wonder whether it is simply too overwhelming to keep coming back to narratives and mechanisms of morbidity, dysfunction, and imbalance. here again, the spaciousness of a liberal arts framework allows both instructors and students to be more open to leavening the difficult topics with moments of beauty and fun. psychologist alison gopnik has demonstrated that children who "pretend play, " in elaborate, unreal scenarios with the aid of language, props and gestures, are able to respond correctly to counterfactual questions about a novel real-world causal relationship ( ) . while the evolutionary imperative for play may well be to develop robust cognitive functions, children play because it is a lot of fun. the paradox of play is that in order to be able to reach a variety of practical benefits in the long run, one must be somewhat less concerned with immediate accomplishments of goals in the short run. eating a cardamom and orange cream-filled choux bun pulled out of a patisserie "lymph node" might not immediately seem central to learning about immune systems but it is delicious and it distills the joy of learning and sharing in a way that sticks in our brains and hearts-both my students' and mine. a liberal arts education with its emphasis on connective and integrative inquiry aims to be transformative, to crack the world open a little bit wider for every student with every course of study, every class, every discipline. but it is not only the student who is transformed, it is also the teacher. teaching immunology as a liberal art has made me a more curious, capable and happier immunologist than i had known i could be. the original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author. the goals of a liberal education immunology's coming of age the danger of a single story who world mental health surveys international college student project: prevalence and distribution of mental disorders microbe hunters reissued socioeconomic status and inflammation: a meta-analysis the intergovernmental panel on climate change. global warming of . c teaching to transgress: education as the practice of freedom teaching community: a pedagogy of hope the power of possibility: causal learning, counterfactual reasoning and pretend play the author confirms being the sole contributor of this work and has approved it for publication. i thank my students at macalester college for being my collaborators on this journey since , and my fellow immunologists at liberal arts institutions for their inspiring conversations and support. the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © chatterjea. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -cxz hf q authors: matarazzo, laura; casilag, fiordiligie; porte, rémi; wallet, frederic; cayet, delphine; faveeuw, christelle; carnoy, christophe; sirard, jean-claude title: therapeutic synergy between antibiotics and pulmonary toll-like receptor stimulation in antibiotic-sensitive or -resistant pneumonia date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: cxz hf q bacterial infections of the respiratory tract constitute a major cause of death worldwide. given the constant rise in bacterial resistance to antibiotics, treatment failure is increasingly frequent. in this context, innovative therapeutic strategies are urgently needed. stimulation of innate immune cells in the respiratory tract [via activation of toll-like receptors (tlrs)] is an attractive approach for rapidly activating the body's immune defenses against a broad spectrum of microorganisms. previous studies of the tlr agonist flagellin in animal models showed that standalone tlr stimulation does not result in the effective treatment of pneumococcal respiratory infection but does significantly improve the therapeutic outcome of concomitant antibiotic treatment. here, we investigated the antibacterial interaction between antibiotic and intranasal flagellin in a mouse model of pneumococcal respiratory infection. using various doses of orally administered amoxicillin or systemically administered cotrimoxazole, we found that the intranasal instillation of flagellin (a dose that promotes maximal lung pro-inflammatory responses) induces synergistic rather than additive antibacterial effects against antibiotic–susceptible pneumococcus. we next set up a model of infection with pneumococcus that is resistant to multiple antibiotics in the context of influenza superinfection. remarkably, the combination of amoxicillin and flagellin effectively treated superinfection with the amoxicillin-resistant pneumococcus since the bacterial clearance was increased by more than -fold compared to standalone treatments. our results also showed that, in response to flagellin, the lung tissue generated an innate immune response even though it had been damaged by the influenza virus and pneumococcal infections. in conclusion, we demonstrated that the selective boosting of lung innate immunity is a conceptually advantageous approach for improving the effectiveness of antibiotic treatment and fighting antibiotic-resistant bacteria. bacterial infections of the respiratory tract constitute a major cause of death worldwide. given the constant rise in bacterial resistance to antibiotics, treatment failure is increasingly frequent. in this context, innovative therapeutic strategies are urgently needed. stimulation of innate immune cells in the respiratory tract [via activation of toll-like receptors (tlrs)] is an attractive approach for rapidly activating the body's immune defenses against a broad spectrum of microorganisms. previous studies of the tlr agonist flagellin in animal models showed that standalone tlr stimulation does not result in the effective treatment of pneumococcal respiratory infection but does significantly improve the therapeutic outcome of concomitant antibiotic treatment. here, we investigated the antibacterial interaction between antibiotic and intranasal flagellin in a mouse model of pneumococcal respiratory infection. using various doses of orally administered amoxicillin or systemically administered cotrimoxazole, we found that the intranasal instillation of flagellin (a dose that promotes maximal lung pro-inflammatory responses) induces synergistic rather than additive antibacterial effects against antibiotic-susceptible pneumococcus. we next set up a model of infection with pneumococcus that is resistant to multiple antibiotics in the context of influenza superinfection. remarkably, the combination of amoxicillin and flagellin effectively treated superinfection with the amoxicillin-resistant pneumococcus since the bacterial clearance was increased by more than -fold compared to standalone treatments. our results also showed that, in response to flagellin, the lung tissue generated an innate immune response even though it had been damaged by the influenza virus and pneumococcal infections. in conclusion, we demonstrated that the selective boosting of lung innate immunity is a conceptually advantageous approach for improving the effectiveness of antibiotic treatment and fighting antibiotic-resistant bacteria. keywords: flagellin, toll-like receptor , antibiotic, resistance, streptococcus pneumoniae, pneumonia, superinfection introduction pneumonia constitutes a major cause of death, morbidity and health resource use worldwide. the main causative agents identified in adult patients hospitalized for community-acquired pneumonia (cap) are viruses (in - % of cases, the most common being rhinovirus, influenza and coronavirus) and bacteria ( - % of cases, with a marked predominance of streptococcus pneumoniae infections) ( ) ( ) ( ) . when faced with overt clinical signs of bacterial pneumonia, the standard of care is antibiotic treatment. the combination of a constant rise in antibiotic resistance in recent decades with a decline in the discovery of new drugs has led to an increase in treatment failure and mortality ( ) . in , the world health organization's global action plan highlighted the urgent need to control the emergence of antibiotic resistance ( ) . given this context, a number of new anti-infectious treatment strategies are being developed. the modulation of innate immunity [by targeting immune receptors, such as toll-like receptors (tlrs)] is a promising approach ( , ) . indeed, innate immunity is highly conserved in evolution, and this system constitutes the first line of defense against invading pathogens. moreover, innate immunity triggers a broad range of antimicrobial defense mechanisms and immune cells-thereby greatly reducing the risk of resistance in the pathogens. moreover, activation of tlr signaling has been associated with a favorable outcome in infections with antibiotic-resistant bacteria or colonization resistance by such pathogens ( ) ( ) ( ) . these observations support that stimulation and effector activities of innate immunity are not influenced by the antibiotic resistance mechanisms carried by bacteria. flagellin is the main protein component of the bacterial flagellum and is a natural agonist of tlr ; the latter is expressed at the surface of a many different cell types, including mucosal epithelial cells and immune cells such as dendritic cells, macrophages, and lymphocytes ( ) . various studies in animal models have highlighted the antimicrobial potency of flagellin against a wide variety of bacterial infections [such as intestinal infections caused by salmonella enterica, enterococcus faecium, clostridium difficile, and escherichia coli ( , ( ) ( ) ( ) , respiratory infections caused by pseudomonas aeruginosa and s. pneumoniae ( , ) ], and viral and fungal infections ( ) ( ) ( ) . although most studies have demonstrated the protective effect of flagellin administered before or during exposure to a microbial pathogen, the protein's immunostimulatory efficacy in therapeutic context has not been extensively characterized. using a mouse model of s. pneumoniae lung infection, we recently demonstrated that combination treatment with mucosally administered flagellin and an orally or intraperitoneally administered low-dose (i.e., subtherapeutic) antibiotic is more effective than the antibiotic alone (i.e., with a lower bacterial load in the lung, and a lower mortality rate). furthermore, the combination treatment was also effective in a model of post-flu pneumococcal superinfection ( ) . the effectiveness of these combination therapies depends on tlr signaling as demonstrated using tlr -deficient animals and tlr -mutated recombinant flagellin ( ) . our studies highlighted that the airway epithelium is the main tlr -specific signaling compartment ( ) ( ) ( ) . taken as a whole, these observations are the first to highlight the added value of respiratory delivery of flagellin as an immunomodulatory biologic for the adjunct treatment of bacterial pneumonia (i.e., in addition to the standard of care). our working hypothesis was that simultaneous treatment with an antibiotic and intranasal, i.e., respiratory flagellin constitutes a "double hit" against the pathogen. a combination of two drugs may result in independent actions or specific (i.e., additive, synergistic, or antagonistic) effects that define the biological outcome ( ) ( ) ( ) . an interaction between two drugs is considered to be synergistic when the measured effect of the combination treatment exceeds the predicted cumulative value of the two components given separately. synergy increases treatment efficacy, and is expected to limit the emergence of drug resistance. furthermore, synergy allows the physician to decrease the dose level or the frequency of dosing, which thereby dampens adverse drug reactions and may even enable the rehabilitation of neglected drugs. conversely, an antagonistic combination treatment has a smaller effect than the predicted cumulative value of the two components given separately. most studies of potentially synergistic antimicrobial agents are performed in in vitro systems such as bacterial cultures, using checkerboard assays and increasing doses of each drug ( , ) . unlike antibiotics that directly affect the bacteria, immunomodulatory biologic activity requires sentinel cells for detection, downstream signaling and thus the production of antimicrobial effectors and the recruitment and/or activation of innate immune cells. at present, there are no comprehensive in vitro models of this complicated physiological system. in the present study, we quantified the nature and magnitude of the interactions between antibiotics and intranasal instillation of flagellin with regard to antibacterial effectiveness in a murine model of s. pneumoniae respiratory infections. furthermore, we wanted to assess the efficacy of this novel therapeutic strategy against infection with antibiotic-resistant bacteria, which represents major public health issues today. to this aim, we investigated the combination's effect on antibiotic-resistant s. pneumoniae in a relevant model of post-flu pneumococcal pneumonia, and characterized the immune response induced by the flagellin-mediated protection. serotype s. pneumoniae (sp ; clinical isolate e ) was obtained from the national reference laboratory-ministry of health, uruguay ( ) . serotype s. pneumoniae (sp ; strain ) was provided by the institut pasteur (paris, france); it is a multidrug-resistant clinical isolate from a human bronchial secretion, and is resistant to amoxicillin (amx), cefotaxime, doxycycline, erythromycin, chloramphenicol, streptomycin, and cotrimoxazole (sxt). working stocks were prepared as described previously ( , ) . briefly, fresh colonies grown on bloodagar plates were incubated in todd hewitt yeast broth (thyb) (sigma-aldrich, saint-louis, mo) at • c until the od nm reached . - . units. cultures were stored at − • c in thyb + glycerol % (vol./vol.) for up to months. for infection, working stocks were thawed and washed with sterile dulbecco's phosphate-buffered saline (pbs, gibco, grand island, ny) and diluted to the appropriate concentration. the number of bacteria (as colony forming units [cfus] ) was confirmed by plating serial dilutions onto % sheep blood agar plates. female balb/cj mice, female swiss mice, and male c bl/ j mice ( - weeks old) (janvier laboratories, saint berthevin, france, or envigo, huntingdon, uk) were maintained in individually ventilated cages and handled in a vertical laminar flow cabinet (class ii a , esco, hatboro, pa). all experiments complied with institutional regulations and ethical guidelines (c - , institut pasteur de lille; protocol ). prior to intranasal infection, the mice were anesthetized via the intraperitoneal injection of . mg ( mg/kg) ketamine plus . mg ( mg/kg) xylazine in µl of pbs. for primary infections with sp , - × cfu were inoculated intranasally in µl pbs, as described previously ( ) . the influenza infection model was developed in our laboratory on the c bl/ j mice ( , ) . the sp pneumococcal superinfection model was therefore performed in these animals. briefly, mice were first infected intranasally with µl pbs containing plaque-forming units (pfus) of the pathogenic, murine-adapted h n influenza a virus strain scotland/ / , as described previously ( , ) . seven days later, animals were infected intranasally with cfu of sp in µl pbs. for the determination of bacterial counts in lung and spleen, mice were sacrificed at selected times via the intraperitoneal injection of . mg of sodium pentobarbital in µl pbs. tissues were collected and homogenized with an ultraturrax homogenizer (ika-werke, staufen, germany), and viable counts were determined by plating serial dilutions onto blood agar plates and incubating them at • c for - h. the recombinant flagellin flic − came from s. enterica serovar typhimurium flic and was produced with an histidine tag, as described previously ( , ) . the protein flic − was certified to be immunologically active in reporter cells and in mouse assays, and the residual lipopolysaccharide concentration was determined to be < pg per µg of flagellin ( ) . the treatments' effects on s. pneumoniae lung infection were quantified as the percentage bacterial growth (% growth ), corresponding to the ratio of the mean bacterial load in the lungs of infected, treated mice to the load in infected, nontreated (control) mice. for example, the effect of treatment a was calculated as follows: % growth[a] = (mean cfu [a] /mean cfu [control] ) × . the predicted additive effect (or predicted % growth ) of a combination treatment was calculated as described previously ( ) . briefly, the predicted % growth of a treatment combining compounds a and b is the product of the experimentally defined % growth values for each standalone treatment (predicted% growth[a+b] = % growth[a] × % growth [b] ). if the experimental % growth for the combination treatment is lower or higher than the predicted % growth , then the two drugs are synergistic or antagonistic, respectively. when the experimental and predicted % growth values are identical, the two drugs' effects are additive. total lung rna was extracted with the nucleospin rna plus kit (macherey-nagel, duren, germany) and reverse-transcribed with the high-capacity cdna archive kit (applied biosystems, foster city, ca). the cdna was amplified using sybr greenbased real-time pcr on a quantstudio k pcr system (applied biosystems). relative mrna levels ( − ct ) were determined by comparing first the pcr cycle thresholds (c q ) for the gene of interest and the reference genes actb and b m ( c q ), and then the c q values for infected mice treated with the amx+flagellin combination treatment and with amx alone (control group) ( cq). all the primers used in the study (listed in table ) were validated for efficacy. bronchoalveolar lavage (bal) fluid samples were obtained after intratracheal injection of × ml of pbs supplemented with % fetal calf serum (fcs). lungs were perfused with pbs, excised and finely minced then digested in a solution of rpmi medium (gibco) containing mg/ml collagenase viii (sigma-aldrich) and µg/ml dnase i (sigma-aldrich) for min at • c. after washes, red blood cells were removed using a lysis solution (pharmlyse, bd bioscience). lung cell homogenates were then suspended in a % percoll gradient and centrifuged at , rpm without brake at room temperature for min. the cell pellets were washed with pbs supplemented with % fcs and cells were filtrated before antibody labeling. bal and lung cells were stained with anti-cd -allophycocyanin-cyanine (clone f ), anti-cd b-brilliant violet (clone m . ), anti-siglecf-alexafluor (clone e - ), anti-ly c-peridinin chlorophyll protein-cyanine . (clone hk . ), anti-ly gphycoerythrin (clone a ), anti-cd c-phycoerythrin-cyanine (clone hl ), and ccr -brillant violet (clone sa g ) antibodies. dead cells were excluded from the analysis using propidium iodide. the antibodies were purchased from bd biosciences (san jose, ca) or biolegend (san diego, ca). data were collected on a bd lsr fortessa and analyzed with the bd facsdiva software. concentration of ccl , cxcl , cxcl , il- , il- β, and tnf was determined in bal fluids and lung homogenates by enzymelinked immunosorbent assay (elisa kit from ebioscience, r&d systems or becton dickinson). bal fluids were obtained by intratracheal injection of × ml pbs supplemented with protease inhibitors (roche). lungs were perfused with pbs and collected in t-per reagent (pierce) supplemented with protease inhibitors and debris were eliminated by centrifugation. all samples were stored at − • c. the results were described as the mean ± standard error of the mean (sem) or the median (range), as indicated. intergroup differences were analyzed using the mann-whitney test and the log rank test. all analyses were performed with prism software (version . , graphpad software, la jolla, ca). the threshold for statistical significance was set to p < . . in earlier research, we had shown that the intranasal administration of a combination of flagellin flic − and low-dose antibiotics improved the therapeutic outcome of lung infection with the antibiotic-susceptible sp [minimum inhibitory concentration (mic) amx = . µg/ml] ( ) . given the difficulty of performing in vitro checkerboard assays with immunomodulators, we therefore sought to evaluate the nature of antibiotic-flagellin interactions in vivo. we first defined the dose of flagellin that promoted saturating immune responses in sp -infected mice (figure ) . intranasally administered flagellin was associated with the production of various innate immunity-related components, including chemokines (cxcl , cxcl , and ccl ), inflammatory cytokines (il- β and il- ), and antimicrobial peptides (s a ), along with the recruitment of neutrophils to the airways ( , , , , , ) . mice were treated simultaneously with oral amx ( . mg/kg) and intranasal flagellin flic − (at doses of . µg to mg/kg, i.e., ng to µg per animal). immune responses were analyzed by monitoring the lung transcription of inflammatory genes associated with tlr signaling and by comparing mrna levels to animals that received amx alone. the results showed that doses from to µg per animal saturated the upregulation of transcriptional response for cxcl , cxcl , ccl , il b, and il genes. ultimately, the dose of . µg of flic − was selected as a saturating immunostimulatory dose in the context of pneumococcal infection and lung inflammation. the next set of experiments was designed to characterize the therapeutic interaction between intranasal flagellin flic − and oral amx. mice were infected with sp and treated h later with either a single intranasal instillation of flagellin ( . µg), a single intragastric administration of suboptimal amx doses of µg ( . mg/kg) or µg ( . mg/kg) or the combination treatment. to define the treatments' efficacy, lung bacterial counts were measured at h post-treatment. the results showed that flagellin alone had mostly no antibacterial effect, whereas and µg doses of amx alone were, respectively, associated with -and -fold smaller bacterial loads, relative to untreated mice (figure a) . the combination treatment (amx + flic − ) induced a -fold relative decrease in bacterial counts for µg of amx and a -fold relative decrease for µg of amx-showing that amx-flagellin combination treatment is more effective than the corresponding dose of amx or flagellin as monotherapy (figure a) . the nature of the interactions between flagellin and antibiotics was further analyzed by comparing the bacterial growth upon treatment. the % growth values for the combination treatment ( . % for flagellin + amx µg and . % for flagellin + amx µg) were much lower than the corresponding predicted % growth values for additive effects, calculated as % growth[amx] × % growth[flagellin] ( . % for flagellin + amx µg and . % for flagellin + amx µg) (figure b) . this experiment indicated strong synergy between the two compounds. similar experiments were carried out with the combination of the antibiotic sxt and flagellin (figures c,d) . the antibiotic sxt was administered intraperitoneally at doses of and mg ( and mg/kg, respectively). flagellin ( . µg) significantly improved the therapeutic outcome of sxt treatment, as evidenced by cfu counts in the mice's lungs h after administration of the treatments ( figure c) . the experimental % growth values for the combination treatment were lower than the corresponding predicted % growth values ( vs. . % for sxt mg, and . vs. . % for sxt mg)-reflecting a synergy between flagellin and sxt ( figure d ). taken as a whole, these results show that antibiotics + flagellin had a strong synergistic effect on pneumococcal lung infection in mice. furthermore, the synergy seems to be independent of the type of antibiotic, since it was observed with a compound that inhibits bacterial cell wall (amx) and a pair of compounds that inhibits folic acid synthesis (sxt). next, we looked at whether the combination treatment's effect on an antibiotic-sensitive s. pneumoniae strain was also exerted on antibiotic-resistant bacteria. to this end, a mouse model of infection with a sp strain that is resistant to a wide range of antibiotics including amx (mic amx = µg/ml, i.e., -fold higher than for sp ) was developed. we found that the sp strain failed to induce a lethal infection and other signs of disease (weight loss) in naïve mice-even at high doses of challenge ( or bacteria per animal) (figures a,b) . given that the influenza virus infection increases susceptibility to bacterial infections even after it has been eliminated ( - ), sp infection was assessed in mice that had already been exposed to the virus. briefly, mice were infected first with an intranasal, sublethal dose of h n virus ( pfu) and then infected days later with cfu of sp . this bacterial superinfection induced significant weight loss and was % lethal (figures a,b) . the bacterial counts increased gradually over time, and reached cfu per lung h post-infection ( figure c) . sp was also detected in the spleen-indicating a translocation and systemic dissemination of the bacteria-from h post-infection onwards (figure c ). in conclusion, the antibiotic-resistant sp strain induced effective pneumonia when animals had been previously exposed to experimental flu. in order to test the efficacy of an antibiotic+flagellin combination treatment in the post-influenza sp superinfection model, mice were treated with amx alone, flagellin flic − alone, or a combination of both compounds h after the bacterial infection ( figure a ). due to the high level of amx resistance, the doses of antibiotic used were µg ( mg/kg) and µg ( mg/kg). using this regimen, the serum concentration levels of amx in naïve animals were expected to be close to × mic and × mic, respectively (professor charlotte kloft, personal communication). flagellin treatment alone decreased bacterial counts in the lungs by . -fold, whereas amx treatments decreased bacterial counts by . -fold (for a dose of µg) and . -fold (for a dose of µg). when amx was combined with flagellin, bacterial counts were of , -and , -fold lower for the and µg doses of antibiotic, respectively. these results show a significant therapeutic advantage for the combination treatment, relative to standalone amx or flagellin treatments ( figure b) . we also determined cfu counts in the spleen; both amx and amx + flagellin treatments (either with or µg of the antibiotic) were able to prevent systemic dissemination of the infection (data not shown). comparison of % growth for the observed effect of the combination treatment vs. predicted additive effect ( . vs. . % for amx µg, and . vs. . % for amx µg) demonstrated the synergy of the combination in the context of superinfection and antibiotic resistance ( figure c) . after two administrations of treatments and h after sp superinfection, the flagellin + amx combination was found to significantly improve the survival of mice, relative to standalone treatments ( figure d) . these data strongly suggest that flagellin + amx have synergistic therapeutic effects to control the antibiotic-resistant pneumococcal infections in relevant pathophysiological contexts. since infection by influenza virus induces major changes in lung integrity and immune cell populations, we investigated the immunomodulatory impact of flagellin on post-flu respiratory infections by the antibiotic-resistant sp strain. to this end, c bl/ mice were infected with influenza a virus at day and then challenged with antibiotic-resistant sp at day . treatments with oral amx ( µg) combined or not with intranasal flagellin ( . µg) were administered h after sp infection. lungs were collected h post-treatment for transcriptional analysis using rt-qpcr assays, as described in figure . we observed that despite the superinfection, flagellin still enhanced the transcription of cxcl , cxcl , ccl , il b, il , and s a genes, i.e., surrogate markers of tlr mediated lung stimulation ( figure a) . we next quantified the cytokine/chemokine production after h of treatment both in the bal fluids and lung protein extracts. delivery of flagellin in the lung of amx-treated pneumococcal superinfection significantly increased levels of ccl , cxcl , cxcl , and tumor-necrosis factor (tnf) both in the lungs ( figure b ) and in the bal fluids ( figure c ) in amx+flagellin-treated mice compared with amx+pbs-treated mice. we also observed increased il- production in both compartments although it was not statistically significant. production of il- β (or pro-il- β) was detected only in the lung tissue and was increased in flagellin-treated mice. finally, we used flow cytometry to evaluate immune cell populations in bal fluids and lung tissue collected h posttreatment. the analysis showed that the neutrophil counts were higher in mice having receiving the combination treatment (i.e., tlr stimulation and amx) than in mice having receiving amx alone both in the lung tissues ( figure d ) and the bal fluids ( figure e) . interestingly, the innate response to combination treatment was also detectable in blood since the production of the inflammatory mediators were significantly augmented at h (for il- , ccl , cxcl , and cxcl ) and h (ccl and cxcl ) compared to amx alone treatment (supplementary figure ) . the blood cytokine production then diminished to an undetectable or very low level at h. thus, these observations showed that the mucosal delivery of flagellin does not induce sustained systemic inflammation. overall, the innate immune response to flagellin was effectively stimulated in the context of the influenza immunological imprinting, the superinfection challenge, and the antibiotic treatment. our present results demonstrated the synergistic efficacy of a combination of an antibiotic (amx or sxt) and the local administration of the immunomodulatory biologic flagellin against respiratory infections caused by s. pneumoniae. of note, the efficacy of combined antibiotic + flagellin treatment, previously demonstrated in inbred balb/c and c bl/ mice by porte et al. ( ) , was here extended to outbred swiss mice, showing genetic background independence of the protection. remarkably, flagellin was able to trigger lung innate immune responses in the context of inflammation (i.e., airways damaged by bacterial pneumonia and flu). immunostimulation in the lung was a dose-dependent process that was saturating by microgram-per-animal levels of flagellin. the synergy appeared to be independent of the antibiotic dose level and the antibiotic's target, since amx acts on the bacterial cell wall and sxt inhibits dna synthesis. the present study is also the first to have demonstrated that stimulating innate immunity can treat severe pneumonia induced by antibiotic-resistant pathogenic bacteria; this may open up new avenues for the treatment of pneumonia in the context of growing antimicrobial resistance. it has been demonstrated that intranasal administration of flagellin activates tlr -dependent local innate responses with broad-spectrum antibacterial activity ( , , , , , ) . the pulmonary response includes the production of various antimicrobial peptides (i.e., cathelicidin antimicrobial peptide and the β-defensins), cytokines (tnf, il- β, and il- ), and chemokines (i.e., ccl , cxcl , cxcl , cxcl , and cxcl ). this cytokine and chemokine production is in line with the observed recruitment of phagocytes (and especially neutrophils) in the lung following the intranasal administration of flagellin to naïve mice ( , , ) . flagellin intranasal administration specifically triggers tlr -mediated transcription in the lungs from to h after a pneumococcus infection or from to days after an influenza infection ( ) . here, we demonstrated that the lung innate immune signature induced by intranasal instillation of flagellin is still effective in a highly inflammatory context with associated lung damage (pneumococcal postinfluenza superinfection), and is not influenced by antibiotic treatment (figure ) . interestingly, earlier reports indicated that influenza infections promote the partial but sustained desensitization of tlr-mediated lung innate responses and a reduction in tlr expression ( ) . our observations demonstrate that, in the physiopathological context of superinfection, flagellin is still able to trigger sufficient levels of innate defense and exert synergy with antibiotics (figure ) . airway epithelial cells have been identified as an important component for detection of flagellin and tlr signaling at homeostasis ( , ) . these sentinel cells not only sense danger signals introduced in the conducting airways but also produce factors to directly impair the colonization and growth of pathogens or indirectly mobilize phagocytic and immune cells to clear infection. more generally, airway epithelium tlr signaling represent a key driving force in antibacterial defense ( ). recently, anas et al. demonstrated an essential contribution of epithelial signaling in the respiratory tract in response to flagellin in the context of infection with pseudomonas aeruginosa ( ) . our data showed that several antimicrobial peptides (s a ), cytokines (il- β and tnf), and chemokines (ccl , cxcl , and cxcl ) that were associated to epithelial responses are also upregulated after the administration of the combination treatment in the post-flu superinfection model, suggesting that the epithelium is also an important flagellin-specific driving force in the lung damaged by viral and bacterial infections. targeting epithelium is a serious benefit for immunostimulation since it allows reducing the dose and bypassing systemic adverse effects. our data contribute to highlight the therapeutic potential of the association of two drugs with distinct modes of action: an antibiotic with a direct effect on bacteria, and a tlr specific stimulator of innate immunity with indirect antibacterial activity mobilizing both multiple phagocytic host cells and various antimicrobial factors such as antibacterial peptides, and chemokines and cytokines that mobilize and activate immune cells. besides pathogen killing, the multitargeting of innate immunity by flagellin could impact on bacterial fitness and thereby increase susceptibility to the antibiotic. the innate immune response induced by tlr signaling may also modify the distribution of antibiotic in lung tissues while the antibiotic, by damaging the pathogen, could also enhance the immune signaling. in addition, the pharmacokinetics of the antibiotic and the immunostimulator, i.e., a short-term dose-dependent effect for the antibiotic, and an immediate and long-lasting impact of the immunostimulator due to cell mobilization, are likely complementary. finally, flagellin, by modulating innate immunity in the respiratory tract, has been shown to enhance the mucosal and systemic adaptive immunity ( , ) . such property may be of interest to elicit anti-pathogen immune memory and prevent recurrent/relapse infections. as an opportunistic bacterium, s. pneumoniae frequently colonizes the upper respiratory tract and thus represents lungs were collected h after treatment, and homogenized. after rna extraction, expression levels of selected genes were then analyzed using rt-qpcr assays. the relative expression level for each gene is expressed against that of the reference genes actb and b m and the reference condition amx+pbs (arbitrarily set to a value of ). the data represent the mean ± sem. lungs (b) and bal fluids (c) were collected h after treatment and cytokine (continued) figure | and chemokine levels were measured by elisa. data from amx+flagellin-treated and amx+pbs-treated mice were compared in a mann-whitney test and are represented as individual values and mean. lungs (d) and bals (e) were collected h after treatment. lungs and bal cell suspensions were stained using a mixture of antibodies specific for surface markers before flow cytometry analysis. neutrophils were defined as cd + cd b + ly g + cells after exclusion of dead cells and alveolar macrophages (cd + siglecf + cd c + cells) from the analysis. numbers of neutrophils in the lung parenchyma (d) and bal fluids (e) are shown for individual animal and the line represents the mean. data from amx+flagellin group were compared to those of amx+pbs group in a mann-whitney test. statistical significance is indicated as follows: *p < . , and **p < . . the prime cause of bacterial-associated cap ( ) . however, other microorganisms can cause cap and healthcare-associated pneumonia; they include gram-positive bacteria such as staphylococcus aureus, gram-negative bacteria like p. aeruginosa, klebsiella pneumoniae, haemophilus influenzae, mycoplasma (m. pneumoniae) and intracellular bacteria (legionella pneumophila) ( ). the diagnosis and treatment of cap is complicated by the broad variety of causative agents, and the progression of antibacterial resistance. in this context, immunomodulators such as flagellin are of great interest because they activate a large number of antimicrobial immune mechanisms. indeed, flagellin has already demonstrated its ability to protect against various pathogens including gram-negative and gram-positive bacteria ( , ( ) ( ) ( ) ( ) ( ) ) . furthermore, our present results showed that the therapeutic synergy between antibiotic and intranasal flagellin is independent of the antibiotic's mechanism of actionsuggesting that flagellin can potentially be combined with various antibiotics for a wide range of clinical situations. the synergistic effects of the combined therapy have been determined to be independent of capsule antigenicity (serotype or ) of pneumococcus, suggesting that the general innate immune protecting mechanisms triggered by flagellin could potentially be effective against a large variety of serotypes. given the progression of antibiotic resistance, a model of infection by antibiotic-resistant bacteria would constitute an important tool for developing alternative anti-infectious approaches. we first attempted to develop such a model in immunocompetent animals. the multidrug-resistant clinical isolate of pneumococcus sp was unable to induce a lethal infection, even at high doses. acquisition of antibiotic resistance is often associated with a loss of bacterial fitness ( ) , which might explain the sp 's very low virulence in naïve mice. it is now becoming clear that many cases of bacterial pneumonia result from co-infections or consecutive infections (especially influenza virus infections) ( ) . as shown by figures d,e, , influenza virus infection creates a favorable environment for colonization and invasion by the low-virulence antibioticresistant pneumococcus sp strain. our data demonstrated that the flagellin+amx combination treatment effectively reduces the bacterial burden caused by the sp strain in the lung, and improves the survival rate among treated mice. our proof-of-concept findings may be transposable to the clinic for patients with co-infections and superinfections, which are relevant physiopathological causes of hospitalization and complicated pneumonia. antibiotics constitute the current standard of care for bacterial pneumonia, and the growing threat of antibiotic resistance is a major public health concern. when defining the dosing regiments of antibiotics used to treat a patient, the physician must take account of the antibiotic' pharmacokinetic and pharmacodynamic characteristics. the relationship between in vivo exposure to the drug and in vitro susceptibility of the bacteria conditions not only the treatment's clinical outcome (i.e., clearance of the infection) but also adverse effects or drug toxicity ( ) . thus, the maximum dose of antibiotic that can be administered to a patient may not be enough to totally clear highly resistant bacteria. our data suggest that the antibacterial efficacy of these antibiotic dose levels can be synergistically enhanced by the effect of flagellin on lung innate immunity. taken as a whole, the present results suggest that the selective boosting of innate lung immunity by flagellin improves the therapeutic outcome of antibiotic treatment. in humans, this approach might be a useful generic alternative to the treatment of bacterial pneumonia, thereby reducing the antibiotic dose and regimen as well as the emergence of antibiotic resistance. moreover, such strategy promotes multitarget inhibition through multiple innate immune effectors that should be more resistant to the development of resistance and may restore some antibacterial activity to antibiotic in the context of antibiotic resistance. characterization of flagellin's contribution to the lung antibacterial defenses at the molecular and cellular level and the protein's synergy with antibiotics is likely to open up new avenues for the immunotherapy of respiratory tract infections. all experiments complied with institutional regulations and ethical guidelines (c - , institut pasteur de lille; protocol apafis # - ). the protocols were validated by the ethical committee for animal experiments (comitéd'éthique en expérimentation animale-nord-pas-de-calais ceea ). lm performed all animal, rt-qpcr, and flow cytometry experiments. fc, rp, dc, and cf provided lm with technical assistance. fw analyzed the bacterial species and antibiotic resistance. lm, cc, and j-cs designed experiments and wrote the manuscript. j-cs and cc supervised the experimental work as a whole. the study was funded by inserm, institut pasteur de lille, université de lille, inserm-transfert (grant: copoc innatebiotic r es), and the era-net joint programming initiative on antimicrobial resistance and the agence nationale de la recherche (grant anr- -jamr- - ). lm is a fellow of the innovation pharmaceutique et recherche program. aetiology of lower respiratory tract infection in adults in primary care: a prospective study in european countries community-acquired pneumonia requiring hospitalization among u.s. adults dynamics of lung defense in pneumonia: resistance, resilience, and remodeling overcoming the current deadlock in antibiotic research global action plan on antimicrobial resistance targeting toll-like receptors: promising therapeutic strategies for the management of sepsis-associated pathology and infectious diseases modulating immunity as a therapy for bacterial infections bacterial flagellin stimulates toll-like receptor -dependent defense against vancomycin-resistant enterococcus infection vancomycinresistant enterococci exploit antibiotic-induced innate immune deficits tlr- activation enhances il- -mediated colonization resistance against vancomycin-resistant enterococcus compartmentalized antimicrobial defenses in response to flagellin flagellin treatment protects against chemicals, bacteria, viruses, and radiation toll-like receptor stimulation protects mice from acute clostridium difficile colitis cutting edge: tlr -/-mice are more susceptible to escherichia coli urinary tract infection mucosal administration of flagellin protects mice from streptococcus pneumoniae lung infection flagellin stimulates protective lung mucosal immunity: role of cathelicidin-related antimicrobial peptide viral infection. prevention and cure of rotavirus infection via tlr /nlrc -mediated production of il- and il- recombinant tlr agonist cblb promotes nk cell-mediated anti-cmv immunity in mice flagellin-induced expression of cxcl mediates direct fungal killing and recruitment of nk cells to the cornea in response to candida albicans infection a toll-like receptor agonist improves the efficacy of antibiotics in treatment of primary and influenza virus-associated pneumococcal mouse infections radioresistant cells expressing tlr control the respiratory epithelium's innate immune responses to flagellin airway structural cells regulate tlr -mediated mucosal adjuvant activity indirect toll-like receptor -mediated activation of conventional dendritic cells promotes the mucosal adjuvant activity of flagellin in the respiratory tract analysis of drug combinations: current methodological landscape assessment and modelling of antibacterial combination regimens when does plus equal ? a review of antimicrobial synergy testing comparison of techniques for measurement of in vitro antibiotic synergism activation of type innate lymphoid cells and interleukin secretion in the lungs during streptococcus pneumoniae infection interleukin- is produced by invariant natural killer t lymphocytes during influenza a virus infection: potential role in protection against lung epithelial damages potential role of invariant nkt cells in the control of pulmonary inflammation and cd + t cell response during acute influenza a virus h n pneumonia invariant nkt cells modulate the suppressive activity of il- -secreting neutrophils differentiated with serum amyloid a deletion of flagellin's hypervariable region abrogates antibody-mediated neutralization and systemic activation of tlr -dependent immunity synergy testing of fda-approved drugs identifies potent drug combinations against trypanosoma cruzi both influenza-induced neutrophil dysfunction and neutrophil-independent mechanisms contribute to increased susceptibility to a secondary streptococcus pneumoniae infection influenza a inhibits th -mediated host defense against bacterial pneumonia in mice influenza and bacterial superinfection: illuminating the immunologic mechanisms of disease the co-pathogenesis of influenza viruses with bacteria in the lung mucosal administration of flagellin induces innate immunity in the mouse lung flagellin promotes myeloid differentiation factor -dependent development of th -type response lung epithelial cells: therapeutically inducible effectors of antimicrobial defense lung epithelial myd drives early pulmonary clearance of pseudomonas aeruginosa by a flagellin dependent mechanism understanding the pneumococcus: transmission and evolution antibiotic resistance and its cost: is it possible to reverse resistance? clinical applications of population pharmacokinetic models of antibiotics: challenges and perspectives we thank dr. aneesh vijayan for technical assistance in the production and quality control of recombinant proteins. we also thank the bicel flow cytometry core facility and the animal facility at the institut pasteur de lille for technical assistance. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material key: cord- -ffao vka authors: mellors, jack; tipton, tom; longet, stephanie; carroll, miles title: viral evasion of the complement system and its importance for vaccines and therapeutics date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: ffao vka the complement system is a key component of innate immunity which readily responds to invading microorganisms. activation of the complement system typically occurs via three main pathways and can induce various antimicrobial effects, including: neutralization of pathogens, regulation of inflammatory responses, promotion of chemotaxis, and enhancement of the adaptive immune response. these can be vital host responses to protect against acute, chronic, and recurrent viral infections. consequently, many viruses (including dengue virus, west nile virus and nipah virus) have evolved mechanisms for evasion or dysregulation of the complement system to enhance viral infectivity and even exacerbate disease symptoms. the complement system has multifaceted roles in both innate and adaptive immunity, with both intracellular and extracellular functions, that can be relevant to all stages of viral infection. a better understanding of this virus-host interplay and its contribution to pathogenesis has previously led to: the identification of genetic factors which influence viral infection and disease outcome, the development of novel antivirals, and the production of safer, more effective vaccines. this review will discuss the antiviral effects of the complement system against numerous viruses, the mechanisms employed by these viruses to then evade or manipulate this system, and how these interactions have informed vaccine/therapeutic development. where relevant, conflicting findings and current research gaps are highlighted to aid future developments in virology and immunology, with potential applications to the current covid- pandemic. the complement system is a heat-labile component of native plasma involving both extracellular and cell surface membrane-associated proteins which form a major constituent of the innate immune response. the whole system is comprised of over proteins which have the potential to react via an enzymatic cascade in response to recognition of various stimuli, such as pathogenassociated molecular patterns (pamps) and abnormal or damaged host cells. activation of the complement system typically occurs via three distinct target recognition pathways (the classical, lectin, and alternative pathways) which converge at a single point; the cleavage of complement component (c ) . each pathway has its own unique protease zymogens to recognize and respond to different antigens, but all pathways primarily work to: opsonise pathogens, lyse pathogens and infected cells, regulate the inflammatory response, and enhance the clearance of immune complexes and cell debris ( , ) . in the context of viral infections, the complement system has been shown to exhibit numerous antiviral mechanisms via direct neutralization of both enveloped and non-enveloped viruses, and/or the promotion of other immune responses. the complement system can directly neutralize virus particles through opsonisation ( ) , membrane attack complex (mac) formation on the virion ( ), mac formation on virus-infected cells ( ) , or targeting of intracellular viral components for proteasomal degradation ( ) . other immune responses may also be modulated by the complement system to promote viral clearance, including: the regulation of inflammation/chemotaxis ( ) , the induction of the antiviral state ( ) , and the enhancement of adaptive immune responses specific to viral antigens ( , ) . the effectiveness and outcome of this response can vary depending on the infectious agent and host genetic variability. the classical complement pathway is typically activated when hexameric c q proteins bind to the fragment crystallisable (fc) ch -domains of antigen-bound igm and/or igg immune complexes ( - ). the binding affinity of c q to igg is dependent on the igg isotype with the greatest affinity for igg- , then igg- , a weak association with igg- , and no interaction with igg- ( ) . however, for downstream activation and complement lysis activity, the response is more efficient following igg -c q interactions rather than igg -c q interactions ( ) . c q is also a versatile pattern recognition molecule. in absence of antibodies, c q can directly bind to apoptotic cells ( ) or proteins on the cell-surface membrane of some pathogens, such as human immunodeficiency virus (hiv) ( ) and dengue virus (denv) ( ) . c q can also bind other host plasma proteins such as c-reactive protein ( ) , fibronectin ( , ) , decorin ( ), lactoferrin ( ), pentraxin- ( ), and serum amyloid p component ( ) . the c q molecule is an assembly of six heterotrimers, each containing three chains (c qa, c qb, and c qc) with a central collagenous stem and a globular head at the c-terminus. in native plasma, the c q molecule forms a calcium-dependent complex with two c r and two c s serine proteases to form the c complex ( ). ligand recognition and binding via the c q molecule in the c complex induces a conformational change and autoactivation of the c r s tetramer to activate the classical complement pathway ( , ) . activated c s cleaves complement proteins c and c into active fragments c b and c a, along with an inactive fragment (c b), and a proteaseactivated receptor (par) /par ligand (c a) which increases endothelial cell permeability ( ). non-covalent binding of c b and c a forms the classical pathway c convertase, c bc a, responsible for cleavage of c into c a (anaphylatoxin) and c b (active component of the convertase). the newly formed c bc ac b complex is a c convertase formed from either the classical or lectin complement pathway activation, which cleaves the c molecule into c a (anaphylatoxin) and c b. c proteolysis and the successive steps are then the same for each of the three complement pathways -c b is deposited onto the activating surface and subsequent, irreversible binding of c , c , c , and multiple copies of c to permeate the cell surface membrane and form the mac ( , , ) . all three complement pathways are summarized in figure . the lectin complement pathway follows the same enzymatic cascade as the classical pathway but is distinct in the antigens and proteases required for its activation. the lectin pathway is activated in response to invading pathogens via direct binding of pamps by various c q-like lectins, complexed with mannosebinding lectin (mbl)-associated serine proteases (masps)- / / . these c q-like activators are mbl, ficolin- (m-ficolin), ficolin- (l-ficolin), ficolin- (h-ficolin), and collectin- (cl- ) ( - ). in humans, mbl is typically present as a trimer, tetramer, pentamer, or hexamer and these oligomeric structures influence its carbohydrate binding properties ( , ). each monomeric subunit in the complex is a homotrimer with each polypeptide containing a cysteine-rich region at the n-terminus, followed by a collagen-like domain, a neck region, and a carbohydrate recognition domain capable of binding specific sugars present on pathogenic surfaces i.e., n-acetyl-d-glucosamine and dmannose ( , ). similar to mbl, multimeric ficolin complexes are assembled through homotrimer subunits with cysteine-rich n-terminal segments which form interchain disulphide bonds, followed by collagen-like regions, but they are unique in their ability to bind distinct carbohydrates via their c-terminal fibrinogenlike domains ( - ). ficolin- is predominantly synthesized in monocytes and granulocytes where it can be found present on their surface or extracellularly in native human plasma. ficolin- is synthesized in the liver and secreted into the bloodstream where it can bind to various acetylated structures and sugars via three inner binding sites ( ). ficolin- is synthesized in the liver and lungs and is present in native plasma at a higher concentration than ficolins- / , although less is known about its functional capabilities ( ) . collectin- (cl- ) can form heterotrimeric complexes with collectin liver (cl- ) in serum and can also associate with masps to activate the lectin complement pathway ( ) . once one or more of these lectins have complexed with masp- and bound their specified target, masp- then cleaves c and c to form the c convertase (c bc a complex). following the proteolytic cleavage of c , the lectin complement pathway follows the same catalytic process as the classical pathway (figure ) ( ) . the roles of masp- and masp- in this pathway are relatively ambiguous ( , ). masp- is capable of cleaving complement component c and, to a much lower extent, components c and c ( , ), whilst masp- may have a negative regulatory role of the lectin pathway through downregulation of masp- cleavage activity ( ) . the alternative pathway does not require the specific proteinprotein or protein-carbohydrate interactions seen with the other two pathways. under normal physiological conditions, ∼ % of c per hour undergoes spontaneous hydrolysis as figure | overview of the complement system following activation via antigen (classical and lectin pathways) or spontaneous hydrolysis (alternative pathway). complement activation eventuates in formation of the membrane attack complex (mac) and the cleavage products regulate inflammation (c a and c a) and cell-mediated immunity (c a, c c, c d, c a). the internal thioester bond is cleaved to produce c (h o). this process is augmented in the presence of various surfaces which lack complement regulatory proteins, as electrostatic and/or hydrophobic interactions adsorb c to the surface to induce conformational changes ( , ) . c (h o) can then bind factor b to induce another conformational change, as factor b is then cleaved into two components by factor d: ba (which dissociates from the complex) and bb (which remains bound in the complex). the protein complex c bbb is the alternative pathway c convertase, which is stabilized through the binding of properdin to produce c bbbp and can cleave further c molecules through the serine protease activity of fragment bb. the alternative pathway therefore has the potential to both activate and enhance complement activity through an amplification loop; cleaved c components produce c convertases which cleave further c molecules ( , ) . cleavage of c also yields c a and c b, where c b remains bound in the complex to form the alternative pathway c convertase, c bbbpc b, and c a acts as an anaphylatoxin. the rest of the complement cascade is then identical for all pathways (figure ) ( , ) . although complement activity typically occurs via the three pathways described, less conventional mechanisms of activation and immune modulation can occur, and have been discussed in a recent review ( ) . typically, properdin is described as a positive regulator of the alternative pathway through stabilization of the c and c convertases. but its functions extend beyond this, including complement activation via direct antigen recognition of invading pathogens and apoptotic/necrotic cells ( ) ( ) ( ) , and as a potential ligand for nkp -mediated natural killer cell activation and subsequent secretion of x-chemokine ligand ( ) . similarly, c and its cleavage products are often described as extracellular components, yet they can have intracellular signaling roles to eliminate pathogens, alter cytokine signaling profiles and induce th responses ( , ( ) ( ) ( ) . most complement proteins are primarily synthesized in the liver and secreted into the bloodstream; this process is rapidly upregulated during infection ( ) . complement proteins can also be produced by epithelial cells ( ) , endothelial cells ( ) , and circulating immune cells such as dendritic cells, granulocytes, macrophages, and monocytes ( , ) . local production of complement also occurs in immune privileged sites including the brain ( ), eyes ( ) , and testis ( ) . to regulate this activity and prevent damage to healthy cells and tissues, many regulatory proteins are primarily expressed as either soluble plasma proteins or cell-surface receptors (figure and table ). complement regulatory proteins may be unique to a certain pathway or influence the downstream activity of all three pathways. factor h, factor i, and properdin are unique to the alternative pathway. factor h is both a soluble and cellsurface membrane regulator ( ) which accelerates the decay of the c convertase (c bbb) to reduce complement deposition ( ) , and it functions as a factor i cofactor to cleave c b and c b components ( ) . properdin is a positive regulator of the alternative pathway which stabilizes the c convertase (c bbb) and promotes its association with further c b molecules ( ) . the c -inhibitor (c -inh) is a negative regulator of all three pathways. c -inh competes with factor b to limit activation of the alternative pathway ( ) , inhibits c r and c s to prevent classical pathway activation ( ) , and inactivates masp- and masp- to prevent lectin pathway activation ( ) . further proteins are required to regulate the downstream complement activity. carboxypeptidase-n/r regulates the anaphylatoxin activity of c a and c a via cleavage of their arginine residues ( ) . c binding protein ( ) , clusterin ( ) , and vitronectin/s protein ( ) are all soluble proteins which prevent the complete assembly of the mac. cd /membrane cofactor protein ( , ) , cd /decay-accelerating factor ( , ), and cd /protectin ( ) are ubiquitously expressed on the surface of host cell surface membranes to protect the cell from complement deposition. one of the key functions of the complement system is to assist in the killing and containment of invading pathogens, including bacteria ( ), fungi ( ) , protozoa ( ) , and viruses ( ) . previous reviews have discussed various evasion mechanisms adopted by viruses to dysregulate or evade this complement activity ( ) ( ) ( ) ( ) . this knowledge may then be exploited and has previously identified novel methods for vaccine and frontiers in immunology | www.frontiersin.org therapeutic intervention-an area which has not been extensively discussed in the previous reviews. the antiviral mechanisms of complement have been divided into four main sections in this discussion; physiologically however, each section is not exclusive as they work together to form a complete system. briefly, complement deposition on a virion can block interactions with host cell receptors, aggregate virus particles, signal intracellularly to induce an antiviral state, and enhance phagocytosis ( , , ) . this can lead to formation of the mac and lyse lipid membranes of enveloped viruses ( ) or lyse infected host cells expressing viral antigens ( ) . activation of the complement system also produces pro-inflammatory anaphylatoxins (c a, c a, and putatively c a) which can enhance phagocytosis and in some cases, worsen disease symptoms ( ) . lastly, these processes can enhance the adaptive immune response to viral antigens, induce a th response ( ) , modulate treg and th responses ( ) , prolong b-cell memory and significantly increase antigen-specific antibody titres ( ) . many of these functions may be evaded or manipulated by different viruses (shown in figure ) and such examples are provided throughout. all three complement pathways can lead to virus opsonisation and complement deposition following activation. the outcome of this response largely depends on the infectious agent and could enhance viral infection, suppress viral infection, or be dysregulated by the expression of some viral proteins. the mbl protein of the lectin pathway can interact with numerous viral antigens and have varying effects on neutralization or viral enhancement. mbl can directly bind the ebola virus (ebov) glycoprotein (gp). high doses of mbl, relative to other complement proteins, can enhance ebov-gp pseudotyped virus infection into primary human macrophages and human monocyte-derived macrophage cell lines ( ) . however, mbl opsonisation of the ebov-gp can neutralize ebov-gp-pseudotyped virus by preventing cell interactions via dc-sign ( ). mbl has also been successfully used as a rescue therapy in % of mice when administered at supraphysiological levels, h post-lethal challenge with a mouse adapted ebov strain ( ) . so, in the context of ebov infection, the effects of mbl appear to be dependent on the cellular mbl has also been shown to bind the hiv- protein, gp . this interaction was sufficient to neutralize cell-line adapted hiv infection of cd + h lymphoblasts ( ) . a later study reported a similar finding, although much higher concentrations of mbl were required to achieve the same level of neutralization ( µg/ml rather than µg/ml of mbl), and these findings were not replicated when using hiv primary isolates or other cell lines for infection. in the later study, mbl was shown to be sufficient for virus opsonisation but not neutralization ( ) . this highlights an important consideration for in vitro studies when investigating complement and pseudovirus interactions, as small method variations can yield significantly different results. where possible, in vivo experiments can help validate this work and address possible discrepancies. further possible implications of mbl during hiv infection have been shown in a study of single nucleotide polymorphisms (snps). snps in the mbl gene which result in low serum concentrations of mbl were associated with increased risk of hiv infection and poorer prognosis following aids diagnosis ( ) . downstream from mbl binding, complement components are deposited on hiv virions which increase viral uptake and internalization into dendritic cells (dcs). both complementopsonised and complement-free hiv binding was reduced through the blockage of c-type lectins, integrins and cd . however, the use of individual blockers showed that complement-opsonised hiv utilized β -and β -integrin for binding and uptake, whereas complement-free hiv utilized β and β -integrin ( ) . a similar observation has been reported for herpes simplex virus (hsv)- during the infection of human dcs. complement deposition and interactions with complement receptor (cr ) enhanced hsv- infection of immature dcs and increased the production of new virus particles, whereas complement with the use of neutralizing antibodies significantly reduced infection ( ) . this highlights another important point with regards to in vitro investigations of complement and viral infection. plasma is often heat-inactivated for use in cell culture to overcome concerns of complement-mediated cytotoxicity. consequently, investigations of virus-host cell interactions may overlook important complement-mediated interactions that would normally be present during infection. the varied effects of mbl opsonisation during viral infection have also been described for severe acute respiratory syndrome coronavirus (sars-cov). multiple studies have shown the potential for mbl to bind immobilized sars-cov or the sars-cov spike protein ( , ) . this interaction was shown to be dependent on a single n-linked glycosylation site of the spike protein and this binding could prevent spike protein interactions with dc-sign but not the angiotensinconverting enzyme (ace ) receptor or cathepsin-l ( ). ip et al. showed that mbl binding to immobilized sars-cov could also inhibit sars infection into fetal rhesus kidney cells and enhance deposition of c ( ) . however, leth-larsen et al. did not observe any interactions between mbl and sars-cov spike protein in their study ( ) . similar to hiv, several studies have found a significant difference of mbl snps associated with lower or deficient mbl serum levels in sars patients compared to healthy chinese population control groups ( , ) , and a reduction of mbl protein concentrations in sars patient sera ( ) . however, one other study observed no significant correlation of mbl-deficient snps in sars patients compared to healthy chinese population control groups ( ) . the role of mbl in sars-cov infections appears conflicted but could be significant. as later discussed, the downstream effects of complement activation do significantly influence symptoms of coronavirus infections. other complement proteins and downstream products of its activation can opsonise virus particles. for denv and west nile virus (wnv), neutralization of the virions occurs in a c and c dependent manner following mbl binding. for wnv, neutralization was achieved independent of downstream c and therefore did not require formation of the mac ( ). for simian virus (sv ), complement-mediated neutralization is predominantly achieved through c deposition and the formation of virion aggregates, rather than virion lysis. for the closely related mumps virus (muv) however, the opposite effect is observed with few aggregates formed but greater susceptibility to complement lysis ( ) . similarly, complement activation in the presence of influenza a virus causes virion aggregation and opsonisation of the hemagglutinin receptor. although to achieve neutralization, igm antibodies and activation of the classical pathway is required ( ) . for chandipura virus (chpv), the alternative pathway and factors c , c , and factor b were required for complementmediated virus neutralization in absence of c or antibodies ( ) . a different study utilized antibodies to observe classical pathway activation and reported that c q, c , and c were essential components for neutralization, but this was independent of factor b and c ( ) . the discrepancy of the importance of factor b for chpv neutralization could depend on the presence of antibodies and the classical pathway. as mentioned previously, complement opsonisation of virions can enhance infection through interactions with complement receptors on host phagocytic cells ( , ) . however, some complement proteins can have a protective intracellular function as well, which is independent of cell-type ( ) . enveloped viruses may naturally evade the intracellular functions of complement, as the protein deposition would occur on the lipid membrane. so, for viral entry via membrane fusion or endocytosis, it is expected that the complement-opsonised viral envelope would be left on the host cell surface membrane or endosome plasma membrane. this has been demonstrated in vitro using respiratory syncytial virus (rsv), an enveloped virus which enters the cell via membrane fusion, where complement intracellular signaling was absent following infection ( ) . the intracellular immune function of complement has a better-defined role for non-enveloped viruses, although the area of intracellular complement immunity is still relatively new ( ) . in a c -dependent manner and independent of downstream complement activity, c deposition on the capsid of non-enveloped human adenovirus has been shown to contain the virus within the endosome, by blocking the fiber shedding and protein vi exposure mechanisms required for capsid disassembly ( ) . the use of an adenovirus type vector (adv) also showed that intracellular sensing of complement could inhibit infection and degrade the virus particle ( ) . a comparison of complement-coated adv to adv only, showed that intracellular c signaling induced the activation of proinflammatory cytokines (ifn-β, il- , il- β) through nf-κb, interferon-regulatory factor (irf), and activating protein- (ap- ) transcription factor activation. intracellular c sensing was shown to be mitochondrial antiviral-signaling protein (mavs)dependent, and independent of pamps and pattern recognition receptors. sensing of complement-coated adv also targeted the virion for degradation by valosin-containing protein (vcp) and the proteasome. c -mediated signaling could induce an antiviral state in previously uninfected cells, as the supernatant from complement-coated adv infected cells was able to protect uninfected hela cells from infection with interferonsensitive sindbis virus. lastly, some viruses have evolved evasion mechanisms to overcome the complement-mediated intracellular immune response. rhinoviruses and polioviruses were shown to inhibit the intracellular c complement signaling mechanism through the expression of a cytosolic c protease to degrade c ( ) . discussed in more detail below, some viruses encode proteases which enhance degradation of the c convertase to prevent further complement deposition or mac formation. this can protect the virion from complement opsonisation and viral lysis. following complement deposition and opsonisation, the complement cascade can progress to assembly of the mac. mac formation can perturb and lyse lipid membranes of enveloped viruses or destroy infected cells expressing viral antigens to reduce viral load ( , , , ) . again, viral proteins can be expressed to dysregulate and evade this response. zika virus (zikv) can lead to classical pathway activation via formation of antigen-antibody complexes or through direct binding of c q. for zikv derived from insect cell lines, this interaction resulted in mac formation and a reduction of viral titres in vitro. however, zikv derived from human cell lines were more resistant to complement mediated neutralization ( ) . zikv and other flaviviruses (including yellow fever virus (yfv), denv, and wnv) express and secrete the non-structural protein (ns- ) to regulate complement activity. the ns protein has a wide variety of functions in complement regulation which include: antagonism of c ( ), recruitment of host c binding protein ( ) , recruitment of host factor h ( ), recruitment of host vitronectin and inhibition of c polymerisation ( ) . however, the denv ns protein is also capable of complement activation and the resulting soluble c b- complexes have been found to correlate with disease severity in patients with dengue shock syndrome ( ) . this discrepancy was addressed with the possibility that relative igm, c and soluble ns concentrations in plasma, at different sites of infection, could influence the extent of inhibition and therefore have varied effects on complement activity ( ) . similarly, nipah virus (niv) exhibits factor i-like activity, either through acquisition of factor i host protein or through inherent protease activity. unlike soluble factor-i, niv exhibits no capacity for c b cleavage and showed no significant cleavage of c b with a cd cofactor, despite its integration in the niv lipid membrane. however, niv is capable of c b cleavage into ic b with factor i cofactors (factor h and soluble cr ) to protect against virus neutralization ( ) . chikungunya virus (chikv) also exhibits factor i-like activity, likely of viral origin and dependent on host factor h concentrations, to cleave c b into inactive ic b and resist complement-mediated neutralization ( ) . muv, sv , and hiv- can all recruit host cell cd into the viral lipid membranes during the budding process to protect from complement deposition and neutralization ( , ) . hiv- also incorporates glycosyl phosphatidylinositol-anchored cd and cd for further protection from complement mediated neutralization ( ) . conversely, complement deposition has been shown to enhance hiv- infectivity into peripheral blood mononuclear cells through interactions with complement receptors ( , ) . this highlights the complexity of complement and viral interactions with dualistic mechanisms, which has previously been reviewed in the context of hiv- infection ( ) . infected host cells which present viral antigens on the cell surface membrane can activate the classical pathway, as the antigens bind igm/igg to induce complement dependent cytotoxicity (cdc). the infected cell is then lysed via the mac in an attempt to reduce viral load. for influenza a virus infection, complement-dependent lysis (cdl) monoclonal antibodies can cross-react with h and h hemagglutinin subtypes for broader protection than neutralizing monoclonal antibodies ( ) . similarly, broadly neutralizing anti-hiv- antibodies can bind the viral envelope protein expressed on infected primary lymphocytes to initiate complement deposition. the deposition does not result in a rapid lytic effect but neutralizes viral spread to further cells ( ) . for hsv- and hsv- , the glycoprotein c (gc)- is expressed to protect virions and infected cells from complement mediated neutralization. the gc- protein binds c , c b, and c c to prevent subsequent binding of c or properdin. modification of gc- on hsv infected cells can therefore increase their susceptibility to antibody neutralization and cdc ( , ) . some of the cleavage products from complement activation can function as anaphylatoxins and have broader immune regulatory functions. primarily, cleavage products c a and c a can be generated via all three pathways and act as potent immune regulators, whilst c a is generated via the classical and lectin pathways only ( ) . the role of c a as an anaphylatoxin is disputed as it currently has no known anaphylatoxin receptor associated with its activity ( ) . however, it does function as an effector protein that is derived from complement activation, which enhances endothelial cell permeability and increases stress fiber formation via par and par ( ). the roles of c a and c a are better described as anaphylatoxins, with the latter demonstrating higher stability and broader biological activity. c a recruits neutrophils to the site of inflammation and both c a and c a can recruit: eosinophils, fibroblasts, macrophages, mast cells, and monocytes ( ) ( ) ( ) ( ) ( ) ( ) . these two anaphylatoxins demonstrate a large functional overlap but each have their own discrete functions. to varying degrees, both are capable of stimulating the production of pro-inflammatory mediators from monocytes and macrophages via inflammasome-caspase- activation ( , ) . both can induce the degranulation of mast cells ( ) ( ) ( ) ( ) , basophils ( ) ( ) ( ) , and eosinophils ( ) . both induce respiratory bursts in eosinophils ( ) and neutrophils, although only c a shows chemotactic activity for neutrophils whereas c a may actually prevent neutrophil mobilization from the bone marrow ( ) . further, only c a can stimulate respiratory bursts in macrophages ( ) . the activity of c a and c a is mediated via binding to two main g-protein coupled receptors; c ar or c ar, respectively ( ) . a secondary, non-g-protein coupled receptor (c l ) has been shown to bind c a and potentially regulate its biological functions in vitro, although its primary functions are not yet clear ( ) . these receptors are widespread across different cell types including both myeloid cells and non-myeloid cells (e.g., astrocytes, microglia, hepatocytes, endothelial and epithelial cells) to produce various biological functions dependent on the cell type ( , ) . c a and c a activity is further regulated by the enzyme carboxypeptidase-n. carboxypeptidase-n cleaves the carboxy terminal arginine amino acid of these anaphylatoxins to generate products with greatly reduced (c a-desarg) or absent (c a-desarg) anaphylatoxin activities ( ) . the cleavage product c a-desarg retains some chemotactic activity to recruit distant immune cells ( , ) whilst c a-desarg can function as a hormone for lipid metabolism ( ) . beyond their roles in chemotaxis, c a and c a have been associated with: the induction of smooth muscle contraction ( ) , regulation of vasodilation ( ) , an increase in vascular permeability ( ) , and the production of various cytokines including il- β, il- /cxcl- , ccl , il- , tnfα ( , ) . during viral infection, excessive complement activation leading to a strong pro-inflammatory response is often associated with more severe disease symptoms. the negative impact of complement activation has been associated with more severe symptoms during sars-cov and mers-cov infections. infection of c deficient mice with sars-cov revealed that the loss of complement activity resulted in milder disease outcomes ( ) . compared to the wild-type, the c deficient mice showed: no significant weight loss, improved respiratory function, reduced lung pathology, and lower levels of inflammatory cytokines and chemokines ( ) . proteomic analysis has shown that a product of complement activation, c c α chain, was significantly higher in sars-patient sera compared to non-sars patient sera ( ) . similarly, increased concentrations of c a and c b- were observed in sera lung tissues of hdpp transgenic mice challenged with mers-cov. the subsequent use of a c ar antibody to prevent c a functional activity resulted in reduced tissue damage and a lower viral load ( ) . cytotoxic effects of complement may also occur post-sars-cov infection. autoantibodies elicited -month after infection against epithelial and endothelial cells can mediate complementdependent cytotoxicity and enhance lysis against a cells and human placenta endothelial cells ( ) . in patients with severe denv infection and dengue shock syndrome, overactivity of the alternative pathway has been reported with increased levels of ns , c a, and sc b- in pleural fluids, which likely contribute to the symptoms of increased vascular permeability ( , ) . in denv infected cells, indicators of the alternative complement pathway are upregulated, with a relatively higher concentration of factor b to factor h proteins and increased cell surface c b deposition ( ) . in mice infected with ross river virus (rrv), complement activation products have been identified in serum and inflamed tissues. similar observations have been made in the synovial fluid of rvv-infected patients. in c knockout mice, the signs of severe disease and tissue damage from rvv infection were diminished compared to wild-type, which suggests complement promotes rrv-induced inflammation ( ) . rrv infected cells express the viral e protein which is glycosylated with n-linked glycans. e n-linked glycans are antigens for mbl and can activate complement via the lectin pathway, which results in greater inflammation and tissue damage during rvv infection ( , ) . complement activation also plays an important role in linking the innate and adaptive immune responses. this interaction can enhance the production of antigen-specific antibody titres and shape the t-cell response to target viral pathogens more efficiently. the importance of complement in the regulation of t-cell immunity has previously been reviewed ( , ) . cognate and co-stimulatory interactions (cd -, and cd -cd , and cd -cd ligand) between antigen presenting cells (apcs) and t-cells results in the local production of c , factor b, factor d, and c . receptors c ar and c ar are also upregulated on the t-cell surface whilst production of decay accelerating factor (daf) is down-regulated. the local production of complement components from immune cells enables signaling via c ar and c ar in an autocrine and paracrine manner. complement c can also be processed intracellularly, or internalized as c (h o) from the alternative pathway, to increase pro-inflammatory cytokine expression from t-cells and recycled back to the t-cell surface ( , ) . a major component of c cleavage on the t-cell surface is ic b. tcell membrane bound ic b binds to cr (and possibly cr ) on monocyte derived dcs to enhance t-cell proliferation ( ) . absence of c ar and c ar leads to: reduced complement protein and receptor regulation, lack of co-stimulatory molecule expression, impaired cytokine production (il- , il- , and il- ), an induction of an itreg cell response, and suppression of t-cell proliferation ( ) ( ) ( ) . activation of both c ar and c ar on dcs by their respective anaphylatoxins (c a and c a) can mediate the production of il- , il- , the il- receptor, and tgf-β to promote tcell differentiation into antiviral th and th subsets ( ) . induction of the th response also depends on c ar and cd activation on t-cells via their t-cell derived ligands ( ) . in mice infected with influenza a virus, inhibition of the c ar lead to a reduction in influenza-specific cytotoxic cd + t-cells ( ) and c deficiency lead to increased viral titres and delayed viral clearance ( ) . c is also required for the production of antigen-specific cd + t-cell responses during lymphocytic choriomeningitis virus infection in mice ( ) . during hcv infection, the hcv core protein can interact with gc qr on host immune cells and suppress the t-cell response. this interaction inhibits t-cell proliferation in a dosedependent manner to downregulate cd activation and reduce the production of ifn-γ and il- from t-cells ( ) ( ) ( ) . hcv core protein interaction with gc qr on monocyte-derived dcs inhibits il- production and promotes th cytokine production to limit differentiation into th cells ( ) . hcv core protein interaction with gc qr on b-cells has a differential response to the one observed on t-cells and dcs, as it increases cell surface costimulatory and chemokine receptor expression and enhances b-cell proliferation ( ) . furthermore, the hcv core protein exhibits intracellular functions, as it can suppress the t-cell factor- transcription factor required for c promoter activity regulation. this reduces c mrna and protein levels which are required for complete mac assembly ( ) . as discussed previously, complement activation can result in c deposition on the surface of virions. c and its cleavage products can interact with the b-cell receptor and b-cell coreceptor complex (cr /cd ligated with cd and cd ) to lower the b-cell activation threshold by several orders of magnitude. this can dramatically increase antibody titres, modulate the proliferation of mature b cells, and protect the b-cells from cd -mediated elimination ( , ) . immune complexes coated in c and c cleavage products covalently interact with complement receptors on follicular dcs (fdcs). the c -coated immune complexes on fdcs are then presented to b-cells in the germinal center for optimal b-cell responses, including: antibody production, somatic hypermutation, class switching, and affinity maturation ( , ) . fdcs can then retain the c -coated complexes within the lymphoid for extended periods of time to generate memory b-cells and promote survival ( ) . alternatively, some aspects of the complement system can suppress certain responses of adaptive immunity: stimulation of cr on dcs can suppress the release of inflammatory cytokines ( ) and c q-differentiated dcs demonstrate an increased phagocytic capacity but reduced expression of cd , cd , and cd required for t cell activation ( ) . it is apparent that the complement system has important implications for virus neutralization and development of the adaptive immune response. as our knowledge of virus and complement interactions improves, this can inform novel approaches for intervention and the development of therapeutics and vaccines. one such example is the use of rupintrivir against rhinoviral infections. rhinoviruses encode a cytosolic c protease which cleaves intracellular c to avoid the intracellular mechanisms of complement, mentioned previously. rupintrivir inhibits the viral cytosolic c protease to increase susceptibility of the virus to intracellular complement immunity ( ) . similarly, the use of fab fragments could prevent the c inhibition of human adenovirus vector for its use in adenoviral gene therapy to promote efficient transgene delivery ( ) . due to the multifaceted and complex immune functions of the complement system, direct manipulation of complement would need to be carefully considered. inhibition of the complement system could increase susceptibility to other diseases, whilst overstimulation could result in autoimmunity and damage to host cells. a method of complement stimulation through inhibition of the cd regulator has been proposed for the treatment of latent hiv- infection in cells. the use of provirus stimulants and a cd inhibitor showed a dose-response effect of cell sensitization to antibody-dependent cell-mediated lysis and reduced viral load. aside from the target cells, no significant non-specific cytolytic effects were observed in vitro. cd protects host cells from complement activity, is ubiquitously expressed, and so its inhibition has the potential to damage host cells ( , ) . deletion of cd in mice did not have a lethal outcome, however absence of the complement regulatory protein did lead to intravascular haemolysis and thrombosis ( ) . treatment in the context of hiv- infection would be short-term however ( ) and could be an exception for an otherwise incurable disease. similar approaches have been considered for other lifethreatening diseases such as cancerous conditions ( , ) . methods of complement inhibition have also demonstrated therapeutic benefit. mentioned previously, excessive complement activation is associated with more severe outcomes of mers-cov and sars-cov infections. use of a c ar antibody to block the pro-inflammatory effects of c a in mers-cov infected hdpp -transgenic mice resulted in: lower concentrations of proinflammatory cytokines, reduced viral replication in lung tissues, reduced lung and spleen tissue damage, and a reduction of viral antigen and microglia activation in the brain ( ) . excessive complement activation and similar lung pathology during sars-cov infection has also been observed in h n influenza cases, where the use of c ar and c ar antagonists reduced signs of acute lung injury and viral load in h n -infected mice ( ) . a novel coronavirus, sars-cov- , has recently emerged and is the causative agent of covid- -an acute self-limiting disease which has the potential to progress to severe disease and death. symptoms of severe disease involve major alveolar damage, wide-spread lung inflammation, and progressive respiratory failure ( , ) . the pathological features of lung, liver, and heart tissue in a severe case of covid- greatly resembled those seen in sars-cov and mers-cov infections which are complement-mediated ( ) ( ) ( ) ( ) ) . mbl has been shown to activate complement via binding to sars-cov spike protein in some studies and this could translate to the sars-cov- spike protein, which contains n-linked glycosylation sites that are targets for mbl ( , ) . thus, the widespread lung inflammation observed in severe cases of covid- could be exacerbated by excessive complement activation. furthermore, viral infections with similar lung pathology to covid- have demonstrated therapeutic benefit with the administration of complement inhibitors targeting c a/c ar or c a/c ar. this has been shown for h n ( ) , h n ( ) , and mers-cov ( ) infections. so, it seems plausible that the lung inflammation in severe cases of covid- is exacerbated by excessive complement activation and this pathologic inflammation could be attenuated through use of complement inhibitors. clinical trials are currently being conducted with the use of a c a inhibitor, the monoclonal antibody ifx- , which has proven to be well tolerated in clinical trial participants and aims to reduce inflammation whilst preserving mac formation ( ) . as sars-cov- is an enveloped virus ( ) , it is possible that mac formation could have some beneficial antiviral effects. therefore, a c a inhibitor such as ifx- (inflarx) may be favorable mechanistically over a c inhibitor such as eculizumab (soliris), which is also considered for use in clinical trials (nct ) ( ) . the ifx- monoclonal antibody targets a specific conformational epitope of the c a molecule to block its anaphylatoxin activity, whilst c b and downstream complement activity are preserved ( ) . eculizumab is a monoclonal antibody which targets the c molecule to prevent cleavage into c a and c b, and therefore inhibits all downstream complement activity ( ) . the distinction between the two antibodies is the preservation of mac activity which could be relevant against the enveloped sars-cov- , although the effects of complement lysis and whether it occurs on sars-cov- is not yet known. because the efficacy and safety of eculizumab is already well characterized ( ) , it is logical that this would take precedence over lesser-known options for urgent clinical trials. the use of eculizumab has already proven beneficial for treatment of severe cases of covid- , which shows that complement is partly responsible for the symptoms in severe cases ( ) . it would be interesting to compare the effects of preserving the mac during infection with the enveloped sars-cov- , as it may offer an antiviral, as well as an anti-inflammatory, effect. but it is also possible that coronaviruses have an intrinsic evasion mechanism, perhaps similar to the ones described in this review, to avoid the lytic activity of the mac. another important consideration could be the stage of infection for implementing complement inhibitors: maintaining complement activity may have a beneficial impact early on in infection for virus neutralization and the development of adaptive immunity, and intervention may only be required to treat excessive inflammation in severe cases. the complement system has several important considerations for vaccine development, one example being its involvement in antibody dependent enhancement (ade). ade is commonly observed when non-neutralizing antibodies are present following initial priming of the immune system. non-neutralizing antibodies can still bind the viral target with the potential to cross-link with fc receptors, or activate complement and interact with complement receptors, to enhance viral infection of host cells ( ) . ade is more commonly observed to be fc receptor-mediated, however complement-mediated ade has been reported for hiv- ( ), mers-cov ( ) , and ebov ( ) . but complement activation can have a positive effect against viral infections in the presence of some non-neutralizing antibodies. use of the non-neutralizing influenza virus m extracellular vaccine in mice required functional c to confer protection and induce effective humoral and cell-mediated immune responses ( ) . a similar effect has been reported for monoclonal antibodies against human cytomegalovirus (hcmv). following the use of gb/mf hcmv vaccination in humans, the immune sera had enhanced neutralization potency toward hcmv in the presence of complement. certain hcmv monoclonal antibodies rely on complement for viral neutralization, which appears distinct from cdc or virolysis, and is likely the result of blocking virus-host interactions ( ) . complement activity has also been implicated for optimal protection with non-neutralizing antibody mab- g against crimean-congo haemorrhagic fever virus infection in adult mice ( ) . in flavivirus infections, the mechanism of ade is predominantly shown to be fc mediated ( ) . complement has been shown to augment antibody-mediated neutralization of wnv in vitro ( ) and the addition of c q has been shown to lower the antibody concentrations required for wnv neutralization in vitro, which correlated with protective effects observed in vivo ( ) . c q was also shown to mediate effects of ade from flavivirus infections in a subclass specific manner, whilst mbl, factor b, or c depletion had no significant effect ( ) . although igg subclasses are known to bind c q with varying avidities, the mechanism to explain this effect on ade has not been identified. this could highlight the importance of selecting the right antibody subclass when considering monoclonal antibody therapies. in general, vaccines which effectively engage the complement system may gave rise to a more potent, virolytic serological response. for hiv vaccination in macaques, the presence of complement augmented virus neutralization and complement-mediated neutralizing antibody titres correlated with vaccine-mediated protection ( ) . other approaches have modified vaccines to utilize aspects of the complement system for increased antigen immunogenicity, such as complement component c d. c d is an end-stage cleavage product from c activation which interacts with cr on b-cells, t-cells, and fdcs. when bound to an antigen, c d can dramatically reduce the b-cell activation threshold for a stronger, more antigen-specific antibody response ( , , ) . cr on fdcs interacts with ic b, c d, and c dg to enhance antibody titres and promote long-term b-cell memory development ( ) . c d also bears t-cell epitopes so even with a lack of cr expression, the peptide can be internalized and presented on hla ii molecules to autoreactive t-helper cells and enhance antibody responses ( , ) . c d does not interact with other components of the complement system and so the associated risks are reduced, however a large enough reduction in the b-cell activation threshold could potentially lead to antibody-mediated autoimmune responses. c d has been used as a vaccine adjuvant against several different viruses. dna vaccines encoding the envelope glycoprotein of porcine reproductive and respiratory syndrome virus were more effective at increasing antigen specific neutralizing antibody titres, ifn-γ levels, and il- levels when engineered with gene copies encoding the cr binding site of c d in the same plasmid construct ( ) . similarly, use of hepatitis e virus peptide (hev-p ) for dna vaccination in mice had enhanced anti-hev-p antibody titres and avidity when fused with three tandem c d copies as genetic adjuvants ( ) . c d has also been used as genetic adjuvant for dna vaccines against newcastle disease virus and hiv- for increased efficacy and higher, longer-lasting antibody titres ( , ) . fusion of c d to target antigens is another approach for the development of safer, more immunogenic dna vaccines. coupling of c d to the secretory form of influenza virus haemagglutinin in mice provided an effective and safer mechanism for mucosal vaccination compared to the use of other adjuvants i.e., cholera toxin b subunits and escherichia coli labile toxin ( ) . so, the use of c d as an adjuvant can help to overcome the low immunogenicity associated with dna vaccines, whilst maintaining their safety. many of the viruses discussed can activate complement, resulting in beneficial and/or detrimental effects on its survival. in the examples where viral-mediated complement activation has been more extensively studied, a viral mechanism is often identified which protects the virus from certain antiviral functions, such as the acquisition of cd , cd , cd to protect from mac formation or the expression of a regulatory protein to inhibit the complement cascade at various points. for the viruses which have been shown to activate complement but do not have a clear evasion/regulatory mechanism, such as mers-cov ( ), sars-cov ( , ) , ebov ( ), and possibly sars-cov- , it is plausible that the mechanism simply has not yet been identified. the viruses which activate complement would consequently trigger the downstream antiviral effects, both intracellularly and extracellularly. therefore, it seems plausible that these viruses would utilize a mechanism, similar to the ones described in this review, to evade this antiviral activity and promote their survival. if such a regulatory protein or process is identified, then these may present as possible antiviral targets, similar to the targeting of the rhinovirus c protease with rupintrivir ( ). the complex interplay between viruses and the complement system can have profound implications for protection via innate immunity and the development of effective adaptive immunity. the effects of the complement system can vary between viral infections, and even during the different stages of the same viral infection, so a clear understanding of these mechanisms is important to improve efficiency of vaccine/therapeutic development whilst mitigating risk. such developments can also be applied for non-viral pathogens (including bacteria, fungi, protozoa) and to broader, more systemic functions of the complement system including: interferon signaling ( , ) , metabolism ( ), brain development ( ) , and the coagulation system ( ) . components of the complement system form an ancient aspect of innate immunity in vertebrates ( ) and even some invertebrates ( , ) . therefore, many animals which act as viral hosts or reservoirs for zoonoses also have an active complement system for targeting pathogens i.e., bats ( ) , cows ( ) , deer ( ) , pigs ( ) , rabbits ( ) , and rats ( ) , which the virus may have to overcome to avoid possible antiviral activity. further viral mechanisms of complement regulation may therefore exist which have not yet been identified and the plasticity of viral genomes could result in the emergence of novel protein regulatory functions. identifying these novel interactions could be important for the development and augmentation of vaccines and therapeutics or even the possibility of utilizing viralderived regulatory proteins as therapeutic complement inhibitors in other diseases ( ) . the benefits from understanding complement mechanisms in viral diseases may have relevance for the current sars-cov- outbreak. previous research has demonstrated the impact of the complement system in coronavirus infections and other diseases, and this knowledge has led to the consideration of several complement inhibitors as therapeutics for severe cases of covid- . jm wrote the manuscript and designed the tables and figures. tt, sl, and mc provided guidance and revised the manuscript. all authors contributed to the article and approved the submitted version. the complement system the complement system: history, pathways, cascade and inhibitors mannosebinding lectin binds to ebola and marburg envelope glycoproteins, resulting in blocking of virus interaction with dc-sign and complementmediated virus neutralization active human complement reduces the zika virus load via formation of the membrane-attack complex glycoprotein c of herpes simplex virus type prevents complementmediated cell lysis and virus neutralization intracellular sensing of complement c activates cell autonomous immunity complement anaphylatoxins (c a, c a, c a) and their receptors (c ar, c ar/cd ) as therapeutic targets in inflammation proliferation of resting b cells is modulated by cr and cr human t cell derived, cell-bound complement ic b is integrally involved in t cell activation novel evasion mechanisms of the classical complement pathway dissection of c q capability of interacting with igg timedependent formation of a tight and only partly reversible association human monoclonal igg isotypes differ in complement activating function at the level of c as well as c q direct binding of c q to apoptotic cells and cell blebs induces complement activation human immunodeficiency virus type activates the classical pathway of complement by direct c binding through specific sites in the transmembrane glycoprotein gp c q binding to dengue virus inhibits infection of thp- and cellular inflammatory responses evidence that complement protein c q interacts with c-reactive protein through its globular head region fibronectin binds to the c q component of complement new functional ligands for ficolin- among lipopolysaccharides of hafnia alvei heteromeric complexes of native collectin kidney and collectin liver are found in the circulation with masps and activate the complement system proteolytic activities of two types of mannose-binding lectin-associated serine protease natural substrates and inhibitors of mannan-binding lectin-associated serine protease- and− : a study on recombinant catalytic fragments masp- and its association with distinct complexes of the mannanbinding lectin complement activation pathway c adsorbed to a polymer surface can form an initiating alternative pathway convertase the tick-over theory revisited: is c a contactactivated protein? the amplification loop of the complement pathways quantitative modeling of the alternative pathway of the complement system the central role of the alternative complement pathway in human disease membrane attack by complement: the assembly and biology of terminal complement complexes new insights into the immune functions of complement properdin can initiate complement activation by binding specific target surfaces and providing a platform for de novo convertase assembly the complement protein properdin binds apoptotic t cells and promotes complement activation and phagocytosis properdin binds to late apoptotic and necrotic cells independently of c b and regulates alternative pathway complement activation complement factor p is a ligand for the natural killer cell-activating receptor nkp intracellular complement activation sustains t cell homeostasis and mediates effector differentiation human complement c deficiency: th induction requires t cell-derived complement c a and cd activation a c (h ) recycling pathway is a component of the intracellular complement system on the functional overlap between complement and anti-microbial peptides pulmonary alveolar type ii epithelial cells synthesize and secrete proteins of the classical and alternative complement pathways the endothelium is an extrahepatic site of synthesis of the seventh component of the complement system characteristics and biological variations of m-ficolin, a pattern recognition molecule, in plasma production of complement components by cells of the immune system selective expression of clusterin (sgp- ) and complement c qb and c during responses to neurotoxins in vivo and in vitro chronic low level complement activation within the eye is controlled by intraocular complement regulatory proteins differential expression of complement regulatory proteins decay-accelerating factor (cd ), membrane cofactor protein (cd ) and cd during human spermatogenesis novel collectin/c q receptor mediates mast cell activation and innate immunity identification of human cd as the phagocytic c q receptor (c qrp) by expression cloning murine cd (c qrp) contributes to the removal of apoptotic cells in vivo but is not required for c q-mediated enhancement of phagocytosis cd is rapidly shed from the surface of human myeloid cells and the soluble form is detected in human plasma activation of human neutrophils by c a and c a c a and c a are chemotaxins for human mast cells and act through distinct receptors via a pertussis toxin-sensitive signal transduction pathway c a and c a stimulate chemotaxis of human mast cells interleukin and granulocyte/macrophage-colony-stimulating factor render human basophils responsive to low concentrations of complement component c a the human c a receptor is expressed on neutrophils and monocytes, but not on b or t lymphocytes expression of a functional anaphylatoxin c a receptor by astrocytes activated human t lymphocytes express a functional c a receptor local production and activation of complement up-regulates the allostimulatory function of dendritic cells through c a-c ar interaction the anaphylatoxin c a receptor expression on human m macrophages is down-regulated by stimulating the histamine h receptor and the il- receptor differential expression of complement receptors on human basophils and mast cells. evidence for mast cell heterogeneity and cd /c ar expression on skin mast cells human t cells express the c a receptor and are chemoattracted to c a up-regulation of c a receptor expression and function on human monocyte derived dendritic cells by prostaglandin e c a regulates nkt and nk cell functions in sepsis the c q and collectin binding site within c q receptor (cell surface calreticulin) role of surfactant proteins a, d, and c q in the clearance of apoptotic cells in vivo and in vitro: calreticulin and cd as a common collectin receptor complex distribution of the α -macroglobulin receptor/low density lipoprotein receptor-related protein in human tissues direct interaction between cd and c q expression of complement receptors and on follicular dendritic cells is necessary for the generation of a strong antigen-specific igg response tumor-promoting phorbol esters stimulate c b and c b' receptor-mediated phagocytosis in cultured human monocytes complement receptor expression on neutrophils at an inflammatory site, the pseudomonas-infected lung in cystic fibrosis differential effects of the stimulation of complement receptors cr (cd ) and cr (cd ) on cell proliferation and intracellular ca + mobilization of chronic lymphocytic leukemia b cells complement receptor type (cr , cd ) expression on peripheral t lymphocytes: both cd -and cd -positive cells express cr the binding of immune complexes by the erythrocyte complement receptor (cr ) complement receptor type (cr , cd ) is a receptor for c q role of complement receptor (cr ; cd ) on epithelial cells: a model for understanding complement-mediated damage in the kidney identification of the membrane receptor for the complement fragment c d by means of a monoclonal antibody t lymphocyte expression of complement receptor (cr /cd ): a role in adhesive cell-cell interactions and dysregulation in a patient with systemic lupus erythematosus (sle) complement receptor ligation of dendritic cells suppresses their stimulatory capacity cr is the dominant phagocytotic complement receptor on human dendritic cells crig: a macrophage complement receptor required for phagocytosis of circulating pathogens the multiligand-binding protein gc qr, putative c q receptor, is a mitochondrial protein c q-mediated chemotaxis by human neutrophils: involvement of gclqr and g-protein signalling mechanisms chemotaxis of human monocyte-derived dendritic cells to complement component c q is mediated by the receptors gc qr and cc qr analysis of the interaction between globular head modules of human c q and its candidate receptor gc qr c l , a nonsignaling c a binding protein membrane cofactor protein of complement is present on human fibroblast, epithelial, and endothelial cells membrane cofactor protein (cd ) protects cells from complement-mediated attack by an intrinsic mechanism soluble forms of membrane cofactor protein (cd , mcp) are present in plasma, tears, and seminal fluid in normal subjects the cd -jagged interaction is critical for human t h immunity complement inhibitor is a regulator of the alternative complement pathway human factor h and c b-binding protein serve as factor i-cofactors both encompassing inactivation of c b and c b regulation of complement activation by c-reactive protein: targeting of the inhibitory activity of c b-binding protein analysis of c and the c binding protein in the mrl/lpr mouse c binding protein bears i antigenic determinants inactivation of c a and c a octapeptides by carboxypeptidase r and carboxypeptidase n identification of the complement decay-accelerating factor (daf) on epithelium and glandular cells and in body fluids decay-accelerating factor must bind both components of the complement alternative pathway c convertase to mediate efficient decay decay-accelerating factor regulates t-cell immunity in the context of inflammation by influencing costimulatory molecule expression on antigen-presenting cells human protectin (cd ), an , - , mw complement lysis restricting factor, inhibits c b- catalysed insertion of c into lipid bilayers cd functions as a signal-transducing molecule for human t cell activation alternative roles for cd post-transcriptional cd gene silencing by sirnas induces enhanced human t lymphocyte response to tumor cell lysate-loaded dcs clusterin, the human apolipoprotein and complement inhibitor, binds to complement c , c beta, and the b domain of c complement factor h: using atomic resolution structure to illuminate disease mechanisms insights into the effects of complement factor h on the assembly and decay of the alternative pathway c proconvertase and c convertase factor h as a regulator of the classical pathway activation role of human factor i and c b receptor in the cleavage of surface-bound c bi molecules factor i-dependent inactivation of human complement c b of the classical pathway by c b/c b receptor (cr , cd ) and membrane cofactor protein (mcp, cd ) complement and bacterial infections: from molecular mechanisms to therapeutic applications activation of the complement system by cryptococcus neoformans leads to binding of ic b to the yeast human c -inhibitor suppresses malaria parasite invasion and cytoadhesion via binding to parasite glycosylphosphatidylinositol and host cell receptors a human serum mannose-binding protein inhibits in vitro infection by the human immunodeficiency virus virus complement evasion strategies complement and viral pathogenesis viral-derived complement inhibitors: current status and potential role in immunomodulation complement evasion strategies of viruses: an overview. front microbiol natural antibody and complement mediate neutralization of influenza virus in the absence of prior immunity complement lysis activity in autologous plasma is associated with lower viral loads during the acute phase of hiv- infection complement-dependent lysis of influenza a virus-infected cells by broadly cross-reactive human monoclonal antibodies the role of anaphylatoxins c a and c a in regulating innate and adaptive immune responses c a receptor-deficient dendritic cells promote induction of treg and th 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selectively recognized by lung surfactant protein d and activates macrophages association between mannose-binding lectin gene polymorphisms and susceptibility to severe acute respiratory syndrome coronavirus infection influence of fcgammariia and mbl polymorphisms on severe acute respiratory syndrome direct complement restriction of flavivirus infection requires glycan recognition by mannose-binding lectin differential mechanisms of complement-mediated neutralization of the closely related paramyxoviruses simian virus and mumps virus alternative pathway of complement activation has a beneficial role against chandipura virus infection complement-mediated neutralization of a potent neurotropic human pathogen, chandipura virus, is dependent on c q cutting edge: productive hiv- infection of dendritic cells via complement receptor type (cr , cd b/cd ) intracellular complement -the complosome -in immune cell regulation complement c prevents viral infection through capsid inactivation antagonism of the complement component c by flavivirus nonstructural protein ns binding of flavivirus nonstructural protein ns to c b binding protein modulates complement activation west nile virus nonstructural protein ns inhibits complement activation by binding the regulatory protein factor h inhibition of the membrane attack complex by dengue virus ns through interaction with vitronectin and terminal complement proteins vascular leakage in severe dengue virus infections: a potential role for the nonstructural viral protein ns and complement a novel factor i activity in nipah virus inhibits human complement pathways through cleavage of c b a factor i-like activity associated with chikungunya virus contributes to its resistance to the human complement system human immunodeficiency virus type incorporates both glycosyl phosphatidylinositol-anchored cd and cd and integral membrane cd at levels that protect from complement-mediated destruction the paramyxoviruses simian virus and mumps virus recruit host cell cd to evade complement-mediated neutralization complement-mediated enhancement of hiv- infection in peripheral blood mononuclear cells the good and evil of complement activation in hiv- infection anti-hiv- antibodies trigger non-lytic complement deposition on infected cells mechanism of complement inactivation by glycoprotein c of herpes simplex virus c a: an anaphylatoxin in name only chemotactic responses of human peripheral blood monocytes to the complement-derived peptides c a and c a des arg c a is a chemotaxin for human eosinophils but not for neutrophils. i. c a stimulation of neutrophils is secondary to eosinophil activation the chemoattractant receptor-like protein c l binds the c a des-arg /acylation-stimulating protein a new biologic role for c a and c a desarg: regulation of tnf-alpha and il- beta synthesis cholesterol crystals induce complement-dependent inflammasome activation and cytokine release differential effects of the complement peptides, c a and c a des arg on human basophil and lung mast cell histamine release the effect of human anaphylatoxins and neutrophils on histamine release from isolated human skin mast cells complement peptides c a-and c a-induced mediator release from dissociated human skin mast cells the c a receptor on mast cells is critical for the autoimmune skinblistering disease bullous pemphigoid anaphylatoxin-induced histamine release with human leukocytes: studies of c a leukocyte binding and histamine release chronic myelogenous leukemia-derived basophilic granulocytes express a functional active receptor for the anaphylatoxin c a the degradation product of the c a anaphylatoxin c adesarg retains basophil-activating properties degranulation from human eosinophils stimulated with c a and c a c a activates reactive oxygen radical species production and intracellular calcium transients in human eosinophils the receptor for complement component c a mediates protection from intestinal ischemia-reperfusion injuries by inhibiting neutrophil mobilization ap- activation through endogenous h o generation by alveolar macrophages regulation by complement c a and c a anaphylatoxins of cytokine production in human umbilical vein endothelial cells expression of the receptor for complement c a (cd ) is up-regulated on reactive astrocytes, microglia, and endothelial cells in the inflamed human central nervous system the receptor for complement anaphylatoxin c a is expressed by myeloid cells and nonmyeloid cells in inflamed human central nervous system: analysis in multiple sclerosis and bacterial meningitis human c a and c a desarg anaphylatoxins have conserved structures, in contrast to c a and c a desarg inflammatory properties of human c a and c a des arg/ in mast cell-depleted human skin the role of complement factor c in lipid metabolism expression of the complement anaphylatoxin c a and c a receptors on bronchial epithelial and smooth muscle cells in models of sepsis and asthma the anaphylatoxins c a and c a are vasodilators in the canine coronary vasculature in vitro and in vivo vascular permeability changes induced by complement-derived peptides complement activation contributes to severe acute respiratory syndrome coronavirus pathogenesis serum proteomic fingerprints of adult patients with severe acute respiratory syndrome blockade of the c a-c ar axis alleviates lung damage in hdpp -transgenic mice infected with mers-cov autoantibodies against human epithelial cells and endothelial cells after severe acute respiratory syndrome (sars)-associated coronavirus infection dengue virus induces increased activity of the complement alternative pathway in infected cells complement contributes to inflammatory tissue destruction in a mouse model of ross river virus-induced disease mannose binding lectin is required for alphavirusinduced arthritis/myositis ross river virus envelope glycans contribute to disease through activation of the host complement system complement regulation of t cell immunity decayaccelerating factor modulates induction of t cell immunity locally produced complement fragments c a and c a provide both costimulatory and survival signals to naive cd + t cells absence of signaling into cd + cells via c ar and c ar enables autoinductive tgf-β signaling and induction of foxp + regulatory t cells complement c a receptor is essential for the optimal generation of antiviral cd + t cell responses complement component c promotes t-cell priming and lung migration to control acute influenza virus infection complement component is required for optimal expansion of cd t cells during a systemic viral infection interaction between complement receptor gc qr and hepatitis c virus core protein inhibits t-lymphocyte proliferation hcv core protein interaction with gc q receptor inhibits th differentiation of cd + t cells via suppression of dendritic cell il- production differential regulation of socs- signalling in b and t lymphocytes by hepatitis c virus core protein hepatitis c virus suppresses c complement synthesis and impairs membrane attack complex function the influence of immune complexbearing follicular dendritic cells on the igm response, ig class switching, and production of high affinity igg the generation of memory cells. i. the role of c in the generation of b memory cells immune modulation of human dendritic cells by complement provirus activation plus cd blockage triggers antibody-dependent complementmediated lysis of latently hiv- -infected cells blockage of cd function restores activities of neutralizing and nonneutralizing antibodies in triggering antibody-dependent complement-mediated lysis of hiv- virions and provirus-activated latently infected cells targeted deletion of the cd gene causes spontaneous intravascular hemolysis and hemoglobinuria in vivo targeting of human neutralizing antibodies against cd and cd to lymphoma cells increases the antitumor activity of rituximab neutralization of complement 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life asl napoli nord experience antibody-dependent enhancement of viral infections. dyn immune activ viral dis mechanism for complement-mediated, antibody-dependent enhancement of human immunodeficiency virus type infection in mt cells is enhanced entry through cd , cd , and cxcr chemokine receptors enhanced inflammation in new zealand white rabbits when mers-cov reinfection occurs in the absence of neutralizing antibody antibody-dependent enhancement of ebola virus infection complement c plays a key role in inducing humoral and cellular immune responses to influenza virus strain-specific hemagglutinin-based or crossprotective m extracellular domain-based vaccination complement enhances in vitro neutralizing potency of antibodies to human cytomegalovirus glycoprotein b (gb) and immune sera induced by gb/mf vaccination gp -targeting monoclonal antibodies protect adult mice against lethal crimean-congo hemorrhagic fever virus infection structural insights into the mechanisms of antibody-mediated neutralization of flavivirus infection: implications for vaccine development complement activation is required for induction of a protective antibody response against west nile virus infection c q reduces the stoichiometric threshold for antibody-mediated neutralization of west nile virus c q inhibits antibody-dependent enhancement of flavivirus infection in vitro and in vivo in an igg subclass specific manner complement-mediated virus infectivity neutralisation by hla antibodies is associated with sterilising immunity to siv challenge in the macaque model for hiv/aids c d of complement as a molecular adjuvant: bridging innate and acquired immunity complement activation and complement receptors on follicular dendritic cells are critical for the function of a targeted adjuvant novel function of complement c d as an autologous helper t-cell target c d adjuvant effects are mediated through the activation of c d-specific autoreactive t cells construction and immunogenicity of dna vaccines encoding fusion protein of murine complement c d-p and gp gene of porcine reproductive and respiratory syndrome virus fusion of c d molecule with neutralization epitope(s) of hepatitis e virus enhances antibody avidity maturation and neutralizing activity following dna immunization enhancement of antibodies to the human immunodeficiency virus type envelope by using the molecular adjuvant c d immune effect of newcastle disease virus dna vaccine with c d as a molecular adjuvant protection against influenza virus infection by intranasal administration of c d-fused hemagglutinin complement, interferon and lupus complement component regulates ifn-α production by plasmacytoid dendritic cells following tlr activation by a plant virus-like nanoparticle complement-mediated regulation of metabolism and basic cellular processes complement in the brain interaction between the coagulation and complement system evolution of the complement system: from defense of the single cell to guardian of the intravascular space sea urchin coelomocytes specifically express a homologue of the complement component c the role of complement in cnidarian-dinoflagellate symbiosis and immune challenge in the sea anemone aiptasia pallida specific alterations in complement protein activity of little brown myotis (myotis lucifugus) hibernating in white-nose syndrome affected sites the complement in milk and defense of the bovine mammary gland against infections complement-mediated killing of borrelia garinii-bactericidal activity of wild deer serum genetic association of the porcine c complement component with hemolytic complement activity inhibition of the alternative pathway of nonhuman infant complement by porin b contributes to virulence of neisseria meningitidis in the infant rat model key: cord- -tdswzj r authors: freitas, andré ricardo ribas; donalisio, maria rita title: excess of mortality in adults and elderly and circulation of subtypes of influenza virus in southern brazil date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: tdswzj r purpose: in the elderly population, the influenza infection and its clinical complications are important causes of hospitalization and death, particularly, in longer-lived age. the objective of this study is to analyze the impact of influenza virus circulation on mortality in the elderly and adults, in years with different predominant virus strains. methods: we performed a time trend study to evaluated excess of mortality for pneumonia and influenza, respiratory disease, and all-causes in southern region of brazil, from to . after considering other models, we opted for serfling regression. excess of death rates per , inhabitants were analyzed in specific age groups ( – , – , – , ≥ years) and by year of occurrence. mortality information were taken from brazilian mortality information system and etiological data were accessed in sentinel virological surveillance database, getting the weekly positivity of the immunofluorescence tests for influenza a (h n , h n ), and b. results: in southern brazil, there is an evident seasonal pattern of all death outcomes among different age groups in the dry and cold season (april–september). the highest excess mortality rates occurs among older, particularly in years of circulation of influenza ah n , especially among people ≥ years, in and —years of great severity of influenza activity. after , with the introduction of the pandemic influenza ah n , we observed a lower impact on the mortality of the elderly compared to < years. discussion: a cross reactivity antibody response from past exposure probably provided protection against disease in the elderly. despite not controlling for comorbidities, climate, and vaccination, for the > years, ratio of respiratory diseases excess mortality rates between ah n ( ) and severe year of h n ( ) shows protection in the pandemic year and great vulnerability during ah n virus predominance. conclusion: the reduced immune response to infection, and to vaccination, and presence of comorbidities recommend a special attention to this age group in brazil. besides medical assistance, the timeliness of vaccine campaigns, its composition, and etiological surveillance of respiratory diseases are some of the preventive and public health measures. introduction human influenza viruses can cause diseases through many direct and indirect pathological effects. consequences are destruction of infected cells, release of cytokines leading to fever, malaise, damage to respiratory epithelium and pulmonary parenchyma, and pneumonia. it includes secondary bacterial infections because of tissue damage and exacerbation of preexisting comorbidities such as cardiovascular and renal diseases, diabetes, or chronic lung disease ( ) ( ) ( ) . the rates of hospitalization and mortality associated with influenza are higher among patients with chronic diseases, children under year and after years of age ( , ) . with the aging population in recent decades, the raw number of hospitalizations and deaths related to pneumonia and influenza tends to increase ( ) , this phenomenon has been observed also in brazil ( , ) . however, the impact and severity of influenza virus circulation depend in part, on the strain that predominates in the season each year. due to the lack of laboratory confirmation, influenza-associated morbidity and mortality are often classified as pneumonia, other respiratory diseases, or other causes. given the difficulty of directly measuring influenza morbidity and mortality, time series models are used to elucidate disease patterns in various age groups. trends are usually determined by means of statistical inference, based on seasonal coincidence of the occurrence of certain diseases or death and laboratory confirmation of the viral circulation ( , ) . different approaches, with and without the quantification of the proportion of viral isolates, can produce average estimates of excess deaths associated with the circulation of certain viral variants ( ) ( ) ( ) . viral surveillance data, hospitalization, or death indicators are particularly useful for the study of influenza in the tropics, as seasonality may be less evident ( ) ( ) ( ) . serfling regression has been used to analyze excess of mortality related with respiratory virus circulation ( , ( ) ( ) ( ) . despite some limitations ( ) , the inclusion of sinusoidal terms in weekly regression may reduce spurious correlation between influenza occurrence and death ( , ) . it is particularly useful when no other covariables are available, and with small samples of viral sentinel surveillance data ( ) . poisson regression and the generalized linear model (glm) can produce more specific estimates and support adjustments for variables (temperature, humidity, comorbidities, other circulations of viruses), although they require a more robust and consistent virological surveillance and cannot be used for pandemics ( ) . in brazil, surveillance for influenza syndromes was implemented in , monitoring the occurrence of respiratory viruses (influenza a and b, parainfluenza , , and , respiratory syncytial virus, adenovirus). the brazilian ministry of health provides vaccination coverage annually since for seniors and some risk groups, with vaccine coverage of the elderly population at around % in southern brazil, the region with the highest coverage of the country. despite the adequate coverage, protective titers after vaccination (hi ≥ ) are consistently lower with poorer cell mediate and antibody responses in the elderly comparing to adults ( ) . considering the vulnerability of the elderly to influenza virus infection, and the lack of studies on its repercussion in brazil, the objective of this study was to analyze the impact of different strains of influenza a virus circulation. we analyzed particularly the most predominant variants (ah n and ah n ) on excess of mortality in the adults and elderly of different age groups in a region with marked seasonality of respiratory diseases in brazil. this is a time trend study to evaluated excess of mortality from to in southern region of brazil (states of paraná, santa catarina e rio grande do sul), total area is , , km , population is , , inhabitants with subtropical climate (köppen-geiger classification cfa). we choose these states for analysis because of the consistent seasonal pattern of influenza, as well as the availability and quality of etiological data from the virological surveillance system in that region. for the mortality rates of specific age groups ( - , - , - , and ≥ years) and death causes, we took data from brazilian mortality information system. causes are classified according to international causes of death icd- revision, pneumonia, and influenza (icd j to j . ), respiratory diseases (icd j to j ), and all-cause (excluding external causes of mortality). we obtained population of each year and age group from instituto brasileiro de geografia e estatística-ibge from the census- , and population estimates for the following years. etiologic information of flu-like syndrome was accessed in database of the national sentinel virological surveillance system. it has data from sentinel units distributed in all regions of the country-north ( units), northeast ( units), southeast ( units), south ( units), and central west ( units). surveillance is performed through the systematic collection of weekly samples of nasopharyngeal secretions from patients who present flu-like syndrome. reference laboratories process samples by using indirect immunofluorescence (iif), with tests for influenza a and b, parainfluenza , , and , respiratory syncytial virus, and adenovirus. a portion of the samples is submitted to polymerase chain reaction tests to identify the virus genotype. we calculated the laboratory positivity indicator using weekly positive results of iif divided by the total of weekly valid tests, i.e., excluding the results within inadequate samples (not enough biological material, improper storage, incorrect material in the sample) or inconclusive results (no valid results). influenza vaccination coverage (%) of southern region from to was obtained from brazilian national program of immunization data base (datasus). the criteria used to define the period of increase of influenza activity was when the positivity of the samples tested exceeded twice the annual mean of the weekly positivity of samples processed by surveillance, during two consecutive weeks. in the year , we consider the period officially recognized by the brazilian ministry of health as epidemic by the influenza ah n pmd strain, due to irregularity of the sample collection by the sentinel surveillance system at the end of epidemic. we calculated the weekly mortality rates by age group using the number of deaths per group of causes divided by the estimated population in the middle of the year multiplied by , . we constructed a serfling cyclical regression model ( ) for weekly data applied to each age group and causes of death (pneumonia and influenza, respiratory diseases, and all causes), as seen in others studies ( , ) , to estimate baseline of predicted deaths in the absence of influenza epidemics. to fit regression, we used period of years (from to ), excluding the weeks of epidemics periods. a cyclical linear regression was adjusted with the equation: where y is the mortality rate, β is the coefficients of regression, t is time in weeks, and t and t are variables for adjusting the secular trend of the disease. we used of sine and cosine for adjust of annual and semiannual periodic components. after adjusting a linear regression and define the expected mortality rate, we delimited % upper confidence limit of the baseline as the reference threshold in the absence of influenza epidemics. we calculated the excess of deaths as the observed mortality minus the expected mortality in the periods when mortality was above % of the confidence interval during epidemics periods. we also present ratios of excess mortality rates among years of predominant circulation of influenza strains ah n (mean and years of severity), ah n pre-pandemic, and ah n postpandemic for each age group. for data compilation, we used microsoft office excel , and for statistical analysis, spss for windows, version . . results table shows the proportion of positivity of the iif nasopharyngeal samples and the annual prevalence of strains of influenza in the period. before , the year of entry of the pandemic strain ah n pmd , there was a predominance of influenza ah n in the years to . after , there is alternation of strains in the southern brazil. annual elderly vaccination coverage in southern region is high and homogeneous, around %, and even higher in the recent years. there is an evident seasonal pattern of deaths from pneumonia and influenza, respiratory diseases, and all-causes among the elderly in different age groups in the dry, cold months (april-september) in southern region (figure ) . we note a progressive increase in the rates of excess deaths (of all outcomes) with increasing age, especially among those older than years. in the pre-pandemic years with dominance of the ah n strain, the excess of mortality rates associated with influenza were relatively low, compared to years of prevalence of ah n strain ( table ) . among those over years, the ratio of excess mortality rates between and the years with dominium of h strains was less than one. this ratio suggests that this age group was spared in the pandemic. however, in years of predominance of strain h , excess of mortality rate of all causes in this group were . per , (corresponding to , obits), , and . times greater than the same rate in years of circulation of h n in pre-and post-pandemic period, respectively. among adults ( - years), we observe a large excess of deaths rates during the pandemic ( obits), which correspond to . excess deaths from all causes, and excess mortality from respiratory diseases associated with viral infection in every , individuals of the age group. the ratio between excess mortality rates due to pneumonia/influenza in the pandemic year ( ) and the mean rate of the period was times higher among the youngest ( table ) . rates of excess mortality by pneumonia and influenza and respiratory diseases are lower than all causes in all age groups, but particularly high in older than years ( table ) . the results highlight the great vulnerability of elderly to influenza ah n , especially among older than years in severe years of influenza activity, like and . the study also shows the lower impact of influenza ah n pdm in this age group compared to younger. risk of dying among the elderly in years of circulating ah n influenza has been reported in several parts of the world ( , , , ) ; however, in brazil, there are no recent estimates available. few studies analyze the circulation and impact of influenza in tropical and subtropical regions ( , , ( ) ( ) ( ) . influenza b virus is also associated with severe disease ( ); however, this variant did not circulate with intensity during the study years in brazil. although the elderly are the most vulnerable group to viral respiratory infections, we found relative small excess of deaths in years of circulating ah n pre pandemic ( and ) . study comparing excess deaths from respiratory diseases in the elderly in latin america shows stable rates (mean of . per , inhabitants) in southern brazil between and (prepandemic flu a-h n ), although higher in brazil than in other countries ( ) . in the usa and in european countries, influenza seasons dominated by subtype ah n are typically associated with mortality two to three times higher than in seasons with predominance of ah n (prior to pandemic strain ) and of influenza b viruses ( , , , ) . when all causes of death are studied, the overall mortality associated with influenza among elderly exceeds that observed in younger age group. it should be considered that all causes mortality is a non-specific measure and a distant outcome of influenza infection. however, it is difficult to determine which group of causes of death could better characterize the influenza burden in mortality. by choosing only the respiratory causes, we may underestimate clinical complications of pulmonary viral infection (e.g., cardiovascular). therefore, in this study, we analyzed all causes, respiratory, and pneumonia and influenza deaths. the unfavorable evolution of infection in the elderly is possibly due to the prevalence of comorbidities, deficiencies in defense mechanisms, and poor antibody response to vaccination, as cellmediated and humoral responses limit severity of disease ( ) . patients with chronic diseases are more susceptible to infection due to decline of the immune function through inflammatory mechanisms, hindering the mucosal barrier, and the adaptive and innate immunological defense mechanisms ( ) . the immune response to infection in the elderly tend to be delayed and weak, with prolonged inflammatory responses, which involves different types of host reaction, mainly to clearance virus. the exacerbations of these mechanisms may induce immunemediated pathology causing tissue damage ( ) . cytokine high serum levels of il- , tnf-a, ifn-g and sil- r, chemokines ip- , mcp- , and monokine induced by ifn-g (mig), are associated with severe clinical cases and lung damage ( ) . immunological abnormalities in people with diabetes, chronic respiratory diseases, cardiopathy, or other chronic diseases have increased risk of severe infection and bad prognosis ( ) . for example, there is the consistent association of influenza infection with cardiovascular mortality, particularly acute myocardial infarction ( ) . in part, it is attributed to altering endothelial function due to an acute inflammatory and procoagulant stimulus during viral infection ( , ) . clinical complications of diabetes triggered by influenza infection cause impairment of leukocyte function and increase post-infection colonization rates resulting in poor prognosis in the elderly ( , ) . in young people and adults, in , the emerging influenza ah n strain had a notable impact on the mortality of people up to years in various parts of the world, including brazil ( , , , ) . excess mortality of individuals aged - years in the state of são paulo, brazil was identified during the pandemic ah n virus ( ) . pregnant women adults with metabolic conditions, including obesity, chronic respiratory disease, and other chronic diseases were significantly associated with severe acute respiratory syndrome and the lethality in brazil ( ) . our study showed a . -fold higher rate of mortality from pneumonia and influenza in adults ( - years) in the pandemic year ah n than the average of years with predominance of ah n circulation in southern region. in addition to the clinical severity and the large portion of the affected population, pandemics affect age groups in different ways ( ) . while only % of deaths from seasonal influenza occur among those under years of age, in the pandemics of , - , and , this proportion was , , and %, respectively ( ) . therefore, pandemics tend to affect a larger proportion of young people than seasonal influenza. in this study, higher rates of death due to pneumonia, influenza, respiratory, and all causes were observed among those aged - years in . one explanation for the higher mortality observed among the youngest is that they would be more prone to the situation known as "cytokine storm, " i.e., a dysfunctional overproduction of cytokines that would lead to diffuse damage to the respiratory tract with severe and potentially lethal systemic repercussions ( ) . viral replication and production of inflammatory mediators seem to be involved in the pathogenesis of infection with influenza a h n pmd , hindering the clearance of virus in lung tissue and leading to pathologic lesions ( ) . another explanation for the lower mortality in the elderly is that they were exposed previously to antigens of the pandemic virus. hancock et al. ( ) suggested a cross-reactive antibody response to pandemic ah n . similarities between ah n antigen from and were detected. this last virus strain has not circulated since ( ) , when the ah n strain was displaced by ah n (asian flu). at that time, ah n viral circulation occurred mainly in children, the current elderly of . the emergence of the ah n strain in the pandemic year (hong kong flu) affected several age groups. this new strain resulted from a large genetic mutation (shift) recombining virus material of the circulating ah n with the avian h , of asian origin, resulting in the new variant ah n ( ) . in - , under selective pressure an antigenic small mutation (drift), resulted in a/fujian/ / (h n ) a strains emerged after a "jump" in genes evolution of hemagglutinin and neuraminidase proteins of virus surface ( , ) . the circulation of the fujian strain had a great impact on the mortality from pneumonia in several parts of the world in - and - ( ) and in brazil ( ) . in , a new drift resulted in influenza ah n detected in south brazil ( ) also affecting hospitalizations and deaths in various parts of the world ( ) . we observed high rates of excess mortality in the elderly, in the years of and . limitations of this study refer mainly to the ecological analysis of pooled data. we did not analyze individual information regarding comorbidities and history of vaccination that could be important confounders influencing mortality ( ) . we just had the overall annual vaccination coverage which were in general, around % in the period. estimates of the number of deaths (all causes, respiratory, and pneumonia-influenza) supposedly related to influenza may be inaccurate in inferring the impact of respiratory viruses. correlations in time series studies may produce spurious associations, especially between all causes of death and influenza infection, due to the distance between cause and outcome, and to multiple components of the obits. serfling addresses part of this limitation by introducing sinusoidal terms in equation, since non-influenza mortality is not expected to coincide exactly with sinusoidal pattern ( , ) . moreover, excess mortality of pneumonia, respiratory diseases, and all causes can be considered as an alert to surveillance of viral respiratory diseases, such as a sentinel indicator to be investigated ( , ) . although all causes mortality is a non-specific indicator, it does not underestimate the complications of chronic diseases associated with influenza ( ). despite the influenza component in all causes mortality is small, the indicator can be considered an indirect measure, a warning, useful in epidemiological monitoring. another limitation is the lack of robust etiologic data from virological surveillance in the years - , which could lead to imprecision in the analyses; however, the data on the predominance strains in the southern region are reliable, and influenced the composition of the vaccine of each season. considering the option for the analysis model, serfling linear regression may produce different estimates when compared with other models (poisson, arima, and glm) ( , ); poisson and arima models produce higher mortality estimates than serfling, and serfling higher than glm, especially among the elderly ( , , ) . we chose serfling model because we do not have robust virological surveillance data, before , and the study period includes a pandemic year ( ) . besides, in this study, we did not analyze climatic variables (minimum temperatures and relative air humidity) that could also interfere with viral transmission and increase the impact of the disease, particularly in the elderly. in conclusion, probably previous exposures to influenza ah n in the past influenced the mortality of brazilian elderly in , despite the vulnerability of this age group to clinical complications. for the > years, we observe higher excess mortality rates (of all outcomes) in severe year of ah n circulation ( , ) . it is also 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viruses collected from young children in uberlandia, brazil-from virus influenza detectados no estado do rio grande do sul durante center for disease control and prevention. influenza activity -united states and worldwide, - season the impact of influenza epidemics on mortality: introducing a severity index deaths averted by influenza vaccination in the us during the seasons / through / authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -elom nx authors: yip, tsz-fung; selim, aisha sami mohammed; lian, ida; lee, suki man-yan title: advancements in host-based interventions for influenza treatment date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: elom nx influenza is a major acute respiratory infection that causes mortality and morbidity worldwide. two classes of conventional antivirals, m ion channel blockers and neuraminidase inhibitors, are mainstays in managing influenza disease to lessen symptoms while minimizing hospitalization and death in patients with severe influenza. however, the development of viral resistance to both drug classes has become a major public health concern. vaccines are prophylaxis mainstays but are limited in efficacy due to the difficulty in matching predicted dominant viral strains to circulating strains. as such, other potential interventions are being explored. since viruses rely on host cellular functions to replicate, recent therapeutic developments focus on targeting host factors involved in virus replication. besides controlling virus replication, potential targets for drug development include controlling virus-induced host immune responses such as the recently suggested involvement of innate lymphoid cells and nadph oxidases in influenza virus pathogenesis and immune cell metabolism. in this review, we will discuss the advancements in novel host-based interventions for treating influenza disease. influenza is a major acute respiratory infection that causes mortality and morbidity worldwide. two classes of conventional antivirals, m ion channel blockers and neuraminidase inhibitors, are mainstays in managing influenza disease to lessen symptoms while minimizing hospitalization and death in patients with severe influenza. however, the development of viral resistance to both drug classes has become a major public health concern. vaccines are prophylaxis mainstays but are limited in efficacy due to the difficulty in matching predicted dominant viral strains to circulating strains. as such, other potential interventions are being explored. since viruses rely on host cellular functions to replicate, recent therapeutic developments focus on targeting host factors involved in virus replication. besides controlling virus replication, potential targets for drug development include controlling virus-induced host immune responses such as the recently suggested involvement of innate lymphoid cells and nadph oxidases in influenza virus pathogenesis and immune cell metabolism. in this review, we will discuss the advancements in novel host-based interventions for treating influenza disease. keywords: host factors, influenza, cytokines, metabolism, immunomodulation introduction influenza remains a source of public health concern. influenza a virus (iav) has been the cause of historical noxious pandemics, such as the spanish flu h n , asian flu h n , hong kong h n flu , and more recently the pandemic of h n (swine flu). influenza also causes seasonal epidemics and outbreaks with high morbidity and mortality rates such as the h n outbreak in india ( , ) . the error-prone nature of the viral rna polymerase (rdrp) and virus' capacity for genetic re-assortment (antigenic drift and shift) result in the viral components' susceptibility to mutations, allowing the viruses to evade the immune system and increases their resistance to control strategies. currently, influenza vaccination and two classes of antiviral drugs-m ion channel blockers (amantadine and rimantadine) and neuraminidase (na) inhibitor (oseltamivir, zanamivir, and peramivir)-and the novel treatment option using polymerase inhibitor (favipiravir) are considered as mainstays in influenza infection treatment and control. the use of influenza vaccinations remains challenging due to antigenic drifts and shifts, with seasonal variation of new circulating species. production of vaccine is time consuming with efficacy concerns, especially in the case of pandemic. variations in vaccine efficacy caused by age should be aware, with studies suggesting that vaccineconferred protection may not be optimal in certain age groups ( ) . the disadvantages of using the conventional antiviral drugs have also been a concern. significant levels of resistance to both classes of drugs have been repeatedly reported ( , ) . high level of resistance (up to %) to m blockers has been reported in h n virus strain in american isolates ( ) . resistance has also been reported in h n virus ( ) . iav resistance to na inhibitors has also become an increasingly prevalent concern, with the recent highly fatal outbreak of influenza a(h n )pdm in india associated with oseltamivir drug resistance ( , ) . in addition, a large cluster of influenza a(h n )pdm viruses in japan was found to have increased oseltamivir and peramivir drug resistance ( ) . there is an urgent need to search for alternative targets to treat influenza virus infections, including non-viral targets such as host cellular factors; which are promising as viruses rely on the host machinery for replication. while host immune response is intended to confer a degree of protection against the infection, an impaired or exaggerated host immune response could be detrimental-iav h n and h n virus infection was reported to exaggerate aberrant cytokine release, resulting in a cytokine storm that caused accelerated host death ( ) ( ) ( ) . many recent studies have focused on the investigation of targeting host factors to control virus replication as well as modulate immune response, which we have previously evaluated ( ) . in this review, we will discuss the latest studies (in the past years) on the investigation of novel host-based approaches with potential for influenza treatment. the replication cycle of iav can be grossly divided into four different stages: ( ) entry, ( ) genome nuclear import, ( ) replication and protein synthesis, and ( ) genome nuclear export, apical transport, assembly, and budding. as an obligate intracellular pathogen, iavs are heavily dependent on host machinery for replication and propagation. to this extent, studies employing genome-wide rna interference (rnai) to screen for host factors involved in iav replication cycle have been performed ( , ) and an increasing number of approaches targeting these host factors to control iav replication have been investigated. entry of iav into the host cell is divided into several steps ( , ) . first, hemagglutinin (ha) on the surface of iav binds to the terminal α-sialic acid on the host cell receptor. this induces the internalization of the viral particle by clathrin-dependent, caveolin-, and clathrin-independent endocytosis ( ) . macropinocytosis was revealed as an alternative entry pathway for iav ( ) , which subsequently enters the canonical endocytic pathway ( , ) . the vesicle-containing viral particle forms an early endosome (also known as sorting endosome), which matures into a late endosome as the endocytic pathway progresses. a gradual decrease in intraluminal ph from ph . to . , mediated by v-atpase proton pump ( ) , takes place as the endosome matures ( , ) . this ph drop in the endosomal lumen induces a conformational change in ha, which is activated by proteolytic cleavage to generate ha and ha from precursor molecule ha ( , ) . this conformational change triggers the fusion of the viral envelope with the endosomal membrane, releasing the viral genome into the cytoplasm. acidification of the endosome causes the subsequent acidi fication of viral lumen via the iav m proton channel ( ) , which in turn promotes the dissociation of m layer from both the viral envelope ( ) and the viral ribonucleoprotein (vrnp) complex ( ) . interestingly, a sharp decrease in ph from neutral to an acidic ph of . as utilized by acid bypass has been observed to be sub-optimal for viral replication. it is hence proposed that a gradual decrease in endosomal ph is necessary for sequential reduction in viral stiffness, dissociation of m from the np in the vrnp complex, destabilization of m layer from the viral envelope, and the eventual conformational change of the ha for the release of viral genome and proteins to the cytoplasm from late endosome ( ) . proteolytic cleavage of ha to ha /ha is an important step in iav replication. this cleavage relocates ha , converting previously uncleaved ha to a metastable conformation that induces membrane fusion at acidic ph ( ) . inefficient cleavage and activation of ha leads to low infectivity ( ) . as identified proteins encoded by the viral genome do not possess proteolytic properties, the virus is dependent on host protease for the cleavage of ha. this provides a potential target to control iav infection. ha is commonly cleaved by trypsin-like proteases at the single arginine residue at position . human airway epithelium serine proteases hat and tmprss were identified as the host factors for cleavage at this residue ( ) . aprotinin, purified from bovine lung ( ) , is a protease inhibitor with a long history of clinical use as an antifibrinolytic agent in cardiac surgery ( ) . its potential as an anti-iav drug has been recognized for over a decade ( ) and has been shown to reduce the infectivity of a broad spectrum of iav strains ( , ) both in vitro ( ) and in vivo ( ) . once withdrawn from the western drug market due to its association with mortality ( ), aprotinin has been approved as a locally administered, small-particle aerosol drug for the treatment of iav infection in russia ( ) . however, side-effects associated with the systemic administration of aprotinin raises the need for an alternative protease inhibitor for use in treatment of iav infections. camostat, a serine protease inhibitor, was reported to demonstrate anti-iav potential in mice dating back to ( ) , but little to no research has been conducted to develop it into an anti-iav treatment. it was revisited and proven to be one of the most efficient serine protease inhibitors for the inhibition of iav replication in primary human tracheal epithelial cells in vitro when tested compounds were used at similar molarities ( ) . at present, camostat is widely administered for the treatment of liver fibrosis, chronic pancreatitis, and cancer ( , ) , making it a highly promising candidate for drug repurposing. despite the lack of association between camostat and increased mortality (as with aprotinin), reports of camostat potentially inducing acute eosinophilic pneumonia ( ) warrants the need for careful consideration and further research into the repositioning of drugs from the same class. highly pathogenic iav, such as the h and h subtypes, are reported to have ha cleavage sites rich in basic residues ( ) . the polybasic nature of the cleavage sites provides multiple targets for a broad spectrum of proteases, including the more ubiquitously expressed intracellular proteases such as furin ( ) . this increased protease spectrum could be utilized by these viruses for the activation of ha prior to viral budding, allowing for evasion of potential inhibition by exogenously administered serine protease inhibitors. furthermore, an in vivo study utilizing mice treated with a single protease inhibitor prior to infection with h virus bearing a polybasic cleavage site showed poor efficacy despite good results were obtained for infection with h n virus bearing single cleavage site ( ) , suggesting strain specificity in using serine protease inhibitors to treat iav infections. endosomal acidification is required for the release of iav genome (in the form of a vrnp complex) into the cytoplasm ( ) . research has shown that an increase in endosomal ph during the early phases of infection could inhibit iav infection in vitro ( ) , bringing to light the possibility of controlling iav infection through the prevention of endosomal acidification. the v-atpase inhibitor bafilomycin a , when used at high concentrations ( - nm) has been proven to inhibit iav replication through the efficient suppression of v-atpase ( , ) . however, prominent cytotoxicity to host cells was also observed at such concentrations ( ) . interestingly, lower concentrations ( . nm) of bafilomycin a lack inhibitory effects on v-atpase attenuated iav replication due to disruption of endosomal trafficking. thus, bafilomycin a is suggested to exert its antiviral function via distinct mechanisms at differing concentrations. diphyllin, isolated from the plant cleistanthus collinus, is a natural compound able to induce a v-atpase inhibitory effect ( ) . in contrast to bafilomycin a , diphyllin is well-tolerated in vitro without inducing obvious cytotoxic effects ( ) . most notably, diphyllin is found to effectively inhibit replication of viral strains resistant to amantadine and/or oseltamivir ( ) . since drug resistance to these widely administered antivirals is of major public health concern ( ), diphyllin is regarded as a promising antiviral against drug-resistant iav strains. the release of iav genomic material during replication requires the fusion of the endosomal membrane with the viral envelope. since cholesterol plays a major role in controlling the fluidity of the lipid bilayer in cells, it is hence suspected to have a role in the infection cycle of iav. interferon-induced transmembrane proteins (ifitms) are proteins expressed in many vertebrates (including humans) and are found on the plasma membrane, the membranes of early and late endosomes, as well as on lysosomes ( , ) . while humans express ifitm , ifitm , ifitm , ifitm , and ifitm , only ifitm , , and are both immune-related as well as interferon (ifn)-inducible ( ) , and have been observed to restrict the replication of different viruses, including iav ( ) . studies suggest that ifitms limit viral infection by reducing membrane fluidity and hence restrict the hemifusion (the mixing of lipid bilayer without the release of viral content) of viral and endosomal membranes ( ) , probably via the disruption of cholesterol homeostasis of late endosomes, where viral fusion and genome release conventionally take place ( ) . a recent study using rnai also demonstrated that cholesterol homeostasis can be regulated via acid phosphatase (acp )-mediated niemann-pick c activity and impaired the membrane fusion of iav and influenza b virus (ibv) ( ) , further suggesting the importance of controlling cholesterol homeostasis in the release of viral genome to cytoplasm. on the contrary, later studies suggest that ifitm exerts its antiviral activity in a cholesterol-independent manner, showing that an increase in cholesterol composition of late endosomal membranes fail to inhibit viral membrane fusion ( ) . in addition, studies suggested the accumulation of cholesterol level in the late endosome does not inhibit the iav genome release into cytoplasm ( , ) . with the modulation of cholesterol levels in host endosomal membrane as a mean to inhibit iav host cell entry is still under debate, further studies are required before clear conclusions can be drawn. by comparing the mirna profiles of the iav-permissive hek t cells and (less permissive) hela cells, mirna- a has been identified as a negative regulator for iav infection via the inhibition of archain (arcn , also known as δ-copi) ( ). arcn is a subunit of the copi complex that is required for intracellular trafficking and endosome function ( ) , depletion of which has been reported to inhibit iav infection ( ) . despite impaired iav internalization caused by arcn depletion via sirna ( , ), it was not able to recapitulate through acute inhibition of copi complex by pharmaceutical means ( ) . it is hypothesized that the long-term (lasting days) perturbation on arcn by rnai affected the general endosomal trafficking network, a phenomena which cannot be recapitulated by acute pharmaceutical inhibition to block iav infection ( ) . the potential of targeting arcn for iav treatment deserves further investigation, despite the favorable results from rnai studies. nuclear import of vrnp complexes from the cytoplasm following fusion of the viral and the endosomal membrane is required for replication to take place ( ) . an early study suggested that vrnp complexes could be transported to the periphery of the nucleus ( ), while recent studies report that vrnp complexes utilize the importin-α-importin-β (impα-impβ ) system for nuclear import ( , ) and lacking of importin-α , in an importin-α knockout mouse model were found to be resistant to iav infection ( ) . ivermectin has long been clinically administered for the treatment of parasitosis ( ) , but has recently come to attention as a potential inhibitor of impα/β ( ) . ivermectin inhibition of impα/β has shown to inhibit the replication of rna viruses such as dengue virus and hiv- ( ) . ivermectin was recently tested for the inhibition of iav in vitro, with nuclear import of vrnp complex (of both wild-type and antiviral mxa escape mutant) efficiently inhibited ( ) . given ivermectin's longstanding record of clinical applications and fda-approved status, repurposing of this drug for the treatment of iav should be considered, especially while under threat of pandemic iav outbreak. following the import of the vrnp complex into the nucleus of the host cell, rdrp uses the vrna as a template to synthesize mrna or crna. synthesized crna remains in the nucleus for new vrna generation, while mrna is exported out of the nucleus for translation. viral protein products are either transported to the cell surface via golgi (in case of ha and na) or imported back into the nucleus to bind with vrna, forming new vrnp complex ( ) . numerous host factors are involved in this process and hence could be possible targets for therapeutic intervention. out of the eight genome segments of iav, the m and ns segments are well known for undergoing splicing to generate at least two different mrnas per individual segment ( , ) . cdc -like kinase (clk ) is a kinase which regulates alternative splicing of pre-mrna ( ) . inhibition of clk by the chemical tg or knockdown of clk is shown to cause a decrease in m mrna generation and disrupt downstream m protein expression, prominently reduced iav propagation ( ) . clypearin and corilagin were both found to be potent anti-iav compounds, with a higher therapeutic index than tg in vitro ( ) . clypearin is isolated from herbs used by chinese medicine practitioners for treating respiratory tract diseases during replication, viral mrna is exported from the nucleus to cytoplasm, where protein synthesis takes place. human rna polymerase ii activity is found to be correlated with iav replication through the inhibition of nuclear export of certain viral mrnas, such as m mrna ( ) . cyclosporine a (csa) is a fda-approved drug with immunomodulatory functions ( ) that has been found to have an anti-iav effect in both cyclophilin a (cypa)-dependent and -independent manners ( ) . the cypa-dependent effect was found to correlate with nuclear export of vrnp complex (see targeting nuclear export complex). the cypa-independent effect caused inhibition of host rna polymerase ii. csa is a prospective drug candidate for treatment of iav infections with a relatively high barrier for development of intrinsic drug resistance, as opposed to commonly used antivirals ( ) . nuclear rna export factor (nxf ) is a host factor that has been identified to be involved in the nuclear export of iav mrna. the knockdown of nxf in hek t cells revealed prominent viral mrna nuclear retention in host cell nucleus ( ) . protectin d (pd ), an endogenously produced lipid in the respiratory tract, has been identified to have potent anti-inflammatory and antiviral effects ( ) . pd production was notably found to be reduced in the lungs of iav-infected mice. therapeutic administration of pd was shown to significantly reduce iav mrna expression, lower lung viral titer, as well as improve survival of iav-infected mice. mechanistic studies revealed attenuated cytoplasmic translocation of viral mrna with such treatment. a decrease in recruitment of viral transcripts to nxf was observed while nuclear export of host rna remained largely unaffected, suggesting a role of pd in regulating nxf in nuclear export of viral rna. natural pd expression in the human airway makes this an ideal candidate for novel therapeutics in the treatment of iav infection. the eukaryotic initiation factor- a (eif a) family plays an important role in protein translation ( , ) . eif a impairment has been proven to be related to antiviral activity in a broad spectrum of rna viruses in vitro ( ) , with inhibition of iav mrna translation ( ) . the eif a inhibitors, silvestrol and pateamine a were demonstrated to arrest viral protein synthesis, thus blocking viral genome replication in vitro ( ) . although both silvestrol and pateamine a caused high cytotoxicity at the concentration required effective for iav inhibition, drugs targeting mrna translation for various diseases have been approved by fda or are under active development ( ) . as such, inhibition of iav infections by disrupting mrna translation may well be a therapeutic approach in the future. post-translational modifications during protein maturation ensure proper function of proteins, with proteins of iav no exception. nitazoxanide, a fda-licensed drug used to treat enteritis, was found to be effective in controlling iav infection by interfering with ha n-glycosylation as well as intracellular trafficking in host cell and eventually led to a reduction in viral budding ( ) . despite the mechanism of nitazoxanide being presently unknown, its ability to inhibit replication of numerous viruses [iav, respiratory syncytial virus, coronavirus, hepatitis b virus, and many others ( ) ] suggests that it may act on host machinery. the drug has also been proven in vitro to inhibit the propagation of many circulating strains of human iav, including those resistant to oseltamivir or zanamivir ( ) . nitazoxanide has a high barrier of resistance to iav ( ) and other viral strains resistance to neuraminidase inhibitors ( ), making it a very promising therapeutic target for iav treatment. the drug is currently under phase iii clinical trials ( ). in the later stage of viral replication, viral rnas of iav packed with rdrp and np (known as vrnp complexes) are exported from the nucleus ( ), assembled ( ) , and transported to the plasma membrane [apical in polarized cells ( ) ] for budding. novel targets for influenza treatment frontiers in immunology | www.frontiersin.org july | volume | article exportin (xpo , also known as crm ) is well known for its function in the nuclear export of protein ( ) and rna, including viral rna ( ) . similar to hiv ( , ) , iav viral rna does not directly bind to xpo but is instead held together by several viral proteins. the viral nuclear export protein (nep, or previously known as ns ) and the vrnp complex have been proposed as the nuclear export complex ( ) . cellular xpo has been proven to be crucial in the nuclear export of the vrnp complex, with early studies using leptomycin b (lmb), a potent xpo inhibitor, revealing that in vitro inhibition of xpo led to nuclear retention of vrnp complex ( , ) . however, lmb was deemed unsuitable for development as a potential drug in the phase i clinical trial due to observed cytotoxic effects ( ) . verdinexor (also known as kpt- ) is a new bioavailable selective inhibitor of xpo . it has been shown to be effective against different strains of iavs both in vitro and in vivo as prophylactic and therapeutic treatments ( , ) . it is worth mentioning that delayed administration of verdinexor at day post-infection was still deemed beneficial, with reduced viral load in vivo ( ) . this suggests a prolonged therapeutic time window when compared to the mainstay antiviral drugs such as oseltamivir, where recommended administration is at the early stage of infection (within h of symptom onset) ( ) . currently, verdinexor has passed the phase i clinical study trials, suggesting that it does not pose severe cytotoxic effects as lmb does. in addition, a recent report demonstrated that a new drug, dp -e , which binds and inhibits the function of xpo , can suppress iav replication in vitro ( ) further strengthens the concept of iav intervention by targeting xpo . viral m protein is crucial in assisting the nuclear export of vrnp complex. it was commonly suggested that m protein links vrnp complex to viral nuclear export protein nep which interacts with xpo for nuclear export ( ) . thus, viral m protein may serve as a target to inhibit nuclear export of vrnp. as previously mentioned (see inhibition of mrna export), csa inhibits iav replication via both cypa-dependent and -independent mechanisms. a recent study using a transgenic mice overexpressing cypa showed greater resistance to iav challenge ( ) . in the cypa-dependent mechanism, csa enhances the binding of cypa to m protein ( ) , increases the self-association of m , and hinders m nuclear import ( ) . csa also promotes the cypa-dependent degradation of viral m protein ( , ) . csa seems to be a promising drug to inhibit the nuclear export of vrnp complex by inhibiting viral m protein stability and function. recently, cd , a tetraspanin (defined by four transmembrane domains with conserved residues) that is expressed abundantly in lungs and interacts with integrins has been implicated in the regulation of iav replication in vitro and in vivo ( ) . knockdown of cd in primary human nasal epithelial cells resulted in the nuclear retention of host xpo , viral np, nep, and m proteins, with an increased survival rate observed in iavinfected cd knockout mice. co-immunoprecipitation assays suggest that cd interacts with viral np, m , and nep proteins ( ) ; however, the exact domains involved in interaction and the mechanism of cd function in nuclear export remain unclear. given that a small molecule inhibitor for cd is now under development ( ) , more data revealing the role of cd in iav infection and subsequent use in targeting cd as anti-iav therapy is anticipated. during iav infection, raf/mek/erk signaling cascade is activated, while the inhibition of mek by u , probably mediated via myosin (light chain) ( ), a known motor protein, impairs the nuclear export of vrnp complexes ( ) . suppressing iav replication by inhibition of raf/mek/erk signaling cascade has been illustrated both in vivo ( ) and in vitro ( ) . the replication of ibv ( ) as well as borna disease virus ( ) was shown to be inhibited by u , suggesting the versatility of this approach in controlling infection by different viruses. despite being effective when administered locally to lungs via aerosol, u has little effect when administered orally ( ) . another mek inhibitor, ci- (also known as pd ) was shown to have high potency against iav in vitro ( ). ci- has completed phase ii clinical trials as an anti-tumor drug, with the application of ci- as a potential anti-iav drug candidate recently revisited. unlike u , ci- is orally bioavailable and oral administration of ci- at h post-infection protected % of the iav-infected mice, while the oseltamivir-treated group experienced a % death rate ( ) . oseltamivir is known to be effective only when administered in the early stages of iav infection. this suggests the potential use of ci- as an agent used in iav treatment due to its potentially longer therapeutic time window than mainstay antivirals. formyl peptide receptor (fpr ) located at the host cell surface was identified as an erk stimulator ( ) . antagonizing fpr promoted the survival of iav-infected mice ( ) . furthermore, fpr antagonists have been described to possess antiviral activity against not only iav but also ibv infection ( ) , promoting the idea that antagonizing fpr to suppress raf/mek/erk signaling cascade could potentially be a novel approach for the treatment of a broad spectrum of influenza viruses. after the nuclear export of the vrnp complexes, host cell's intracellular transport mechanism is required to deliver vrnp complexes to the host plasma membrane for the assembly of viral rnas and proteins at the final stage of viral replication. among the various vesicular compartments found in a cell, the rab a + endosomes are known to recycle endocytosed membrane proteins and lipids to the plasma membrane for membrane homeostasis ( ) , a property utilized by many rna viruses, including iav ( , ( ) ( ) ( ) . iav progeny virus production was found to be significantly reduced in rab a + knockdown human cell lines ( ) . furthermore, vrnp complex plasma membrane transport perturbation was observed in rab a knockdown cells ( , ) ; in cells expressing deletion mutant of rab family interacting proteins ( ) ; as well as cells treated with chemicals to interfere microtubule ( ) . direct interaction of vrnp complex with rab a has also been verified ( , ) , demonstrating the dependence of vrna complex transport on rab a + vesicles and the microtubule network during viral replication. since rab a proteins do not confer any mobile properties to the vesicle, molecular motors such as kinesins are required for the active transportation of vesicles through cytoskeletons. kif a, a kinesin- family member, was recently identified as a molecular motor for plasma membrane transportation of vrnp-loaded rab a + vesicles ( ) . kif a knockdown was found to reduce progeny virus production. overexpression of a mutant form of kif a lacking in motor capacity resulted in disruption of the plasma membrane distribution of vrnp complex during later stages of infection. this data suggest that the apical transport of viral components via rab a or kif a could potentially serve as therapeutic targets against iav infection. further examination is merited. tubulin acetylation and deacetylation affects microtubule stability ( ) . histone deacetylase (hdac ) was found to deacetylate α-tubulin, one of the subunits of microtubule ( ) . a study has demonstrated that hdac is involved in iav replication ( ) . inhibition of hdac by tubacin or knockdown of hdac gene resulted in an increase of progeny virus production with vrnp complex redistributed toward the periphery of infected cells. in addition, transportation of ha to the plasma membrane for viral budding was also found to be inhibited by hdac . this data suggests that activation of hdac by its stimulant could be a potential approach to anti-iav therapy, despite hdac stimulants still being under development. while several studies have suggested iav transmission between cells through apical membranes ( ) and intercellular connections ( ) , virus budding from cell membranes remains the major route for transmission of viruses to uninfected cells. na is responsible for the cleavage of sialic acid to prevent the interaction between ha and the host cell during viral budding. besides, viral na, viral ha, m as well as m , are also suggested to play an important role in the initiation of the budding process ( , ) . in section "controlling cholesterol homeostasis, " we discussed the involvement of host cholesterol in viral membrane fusion and viral genome release to cytoplasm. recent studies have demonstrated that host cholesterol may also play an important role in viral budding. it was demonstrated that overexpression of annexin a (anxa ), a phospholipid binding protein, could lead to a decrease in cholesterol levels within the golgi apparatus and plasma membrane ( ), ultimately causing a reduction in egression of progeny virion from infected cells ( ) . this reduction could be reversed by the addition of exogenous cholesterol ( ) . similar to anxa overexpression, addition of a hydrophobic polyamine, u a, could reduce cholesterol level in plasma membr ane, also inhibited viral replication ( ) . since iav is assumed to bud from lipid rafts (cholesterol-rich plasma membrane domains) ( ) , it was demonstrated that anxa overexpression or u a treatment could hinder progeny virus production by lowering the cholesterol content in the plasma membrane. this hypothesis was strengthened through recent studies resolving the cholesterol-binding site of viral m protein, suggesting that iav m clustering (which provides membrane curvature for scission) is mediated by cholesterol ( ) . a recent report utilizing two different fda-approved cholesterol-lowering drugs, gemfibrozil and lovastatin, stated that there was reduction in stability and infectivity of progeny virus compared to that replicating within cholesterol-sufficient host cells ( ) . taken together, this data suggests that controlling cellular cholesterol content would be an effective alternative with drugs available for repurposing iav treatment. further in vivo works are needed to confirm this hypothesis. the gi-type g-protein coupled receptor α -adrenergic receptors (α -ars) have been recently identified as a key host factor involved in iav replication ( ) . apical transport of the viral protein ha is inhibited by low intracellular camp level after stimulating the α -ar-mediated signaling. in vitro stimulation of α -ar by its agonist clonidine inhibits iav replication. therapeutic administration of clonidine reduced pulmonary edema and improved survival rate of iav-infected mice. development of a new antiviral targeting the α -ar-mediated signaling seems promising and deserves further investigation. although targeting host factors for viral interventions generally provides a better resistance barrier, emergence of resistance may still arise ( ) . therefore, combined use of interventions targeting both virus and host factors have been recommended to reduce opportunities for viral development of resistance. one such example would be the combined administration of na inhibitor (oseltamivir) alongside an anti-host factor [such as v-atpase inhibitor diphyllin ( ) , ha maturation inhibitor nitazoxanide ( ) , fpr antagonists ( ) , and xpo inhibitor verdinexor ( ) ]. while further direct assessment for the ease of emergence of escape mutants between single and combinatory use of drugs is required, the synergistic effects of a combined, multi-drug approach observed thus far highly suggest an increased effectiveness over a single-drug approach. table summarizes novel host targets regulating iav replication. compared to rnai, small molecular chemicals remain the best choice as drug candidates due to their fast acting and easy-todeliver properties. although small molecular chemicals targeting certain host factors aforementioned have yet to be developed, their rnai-identified involvement in the iav replication cycle provide leads for the development of new iav interventions. the immune system aims to protect the host from infection and clear the pathogen once an infection occurs. in addition, the complex networks formed between the host physiology and the immune system co-operatively shape the disease outcome; modulations on the networks could alleviate disease severity in iav infections. the immunological responses elicited by iav infection has been reviewed in detail ( ) ( ) ( ) . at the initial stage of iav infection, the respiratory epithelial cells are the primary target for infection. once the infection is initiated, the recognition of infection is accomplished via the detection of pathogen-associated molecular patterns (pamps) by pattern recognition receptors (prrs) (see toll-like receptors), and lead to the expression and secretion of different cytokines and chemokines, such as il- , il- , tumor necrosis factor (tnf)-α, and ccl as well as type i and iii ifns. as sentinel cells, alveolar macrophages could also be infected, inducing cytokines and is the main source of type i ifns ( , ) . type i ifns are known inducer for the upregulation of death receptor , which is the receptor for tnfrelated apoptosis-inducing ligand (trail), in lung pneumocytes ( ) . il- and ccl produced by both epithelial cells and macrophages act as chemoattractants for neutrophils and monocytes, respectively. neutrophils are one of the earliest immune cells being recruited to the site of infection ( ) with transmigration of neutrophils carry out by adhesion molecules, such as cd a, cd b, and cd ( ) . in addition to the antiviral activity of neutrophil-released reactive oxygen species (ros), defensin and pentraxin ( ) , uptaking iav by neutrophils could also help in controlling viral propagation as these cells do not support replication of iav ( ) . besides controlling viral replication, neutrophils also play an important role in guiding the migration of iav-specific cd + t-cells in the infection site by secreting and leaving a trail of cxcl ( ) . infiltrated monocytes will, however, differentiate into macrophages or dendritic cells (dcs). the monocytes-derived macrophages are reported to be a permissive host for iav production ( ) , sustaining inflammation by producing cytokines in a magnitude larger than that of the resident alveolar macrophages. the monocyte-derived dc as well as the resident airway cd c low b + plasmacytoid dc (pdc) and two types of conventional dcs (cd + cd b low and cd − cd b hi ) acquire the antigen of the invading pathogen through either direct infection or up-taking infected dead cells ( ) . in the presence of type i ifns, dcs mature when encountering pamps from invading pathogen ( ) . depending on the sub-cellular localization of the antigen, cytosolic and endosomal antigen will be loaded onto major histocompatibility complex (mhc) class i and ii molecules respectively ( ) . once mature, dcs migrate from the infection site to the draining lymph nodes via the interaction of ccr and ccl /ccl ( , ) for antigen presentation via mhc class i and ii to naïve cd + and cd + t-cells, respectively ( ) ( ) ( ) ( ) . interestingly, monocytesderived dcs that engulfed the infected dead cells are poor antigen presenters for cd + t-cells and require the transfer of intact mhc class i/peptide complex to lymph node-resident cd α + dcs which are the most efficient antigen-presenting cells to cd + t-cells ( ) . in addition to antigen presentation, pdc are well known for their high ability in type i ifns production to limit viral propagation ( ) . within the lymph node, naïve cd + t-cells are activated by the dcs, differentiate and clonal expand into cytotoxic t-lymphocytes (ctls) with the aid of various cytokines, including ifn-γ, il- , type i ifns, and il ( , ) , and the help from activated cd + t helper cells ( ). differentiated ctls downregulate their lymph node homing receptor ccr and upregulate ccr and cxcr for the migration to the site of infection. within the site of infection, ctls control viral replication by targeting and inducing apoptosis of virus-infected cells via the secretion of perforin and granzymes as well as the ligation of death receptors on the infected cells by tnf, fas ligand, and trail. on the other hand, cd + t-cells are activated by the presentation of mhc class ii/ antigen complex by dcs, with co-stimulatory receptors such as cd expressed on the t-cells and the ligand for cd (cd and cd ) expressed on dcs playing an important role ( ). activation of cd + t-cells lead to differentiation into different effector cells subsets, including the classical th and th , and the more recently identified regulatory t cells, follicular t helper cells, th , and th subsets ( ). th cells regulate to the differentiation of ctls as mentioned whereas th cells contributes to the activation of b-cells through cd l. within the pregerminal center of the lymph node, the follicular t helper cells interact with antigen-primed b-cells and promote their proliferation. antigen-primed b-cells differentiates into plasmablast and undergo antibody class-switching in the germinal center ( ) . detailed functions of regulatory t cells, follicular t cells, th , and th cells are discussed elsewhere ( , ). plasmablasts enter the blood-stream, are recruited to the inflamed tissue, and terminally differentiate into plasma b cells which specialize in the production of antibody for pathogen neutralization, opsonization, and antibody-dependent cell-mediated cytotoxicity, etc. memory t-and b-cells are also developed during the maturation process, and has been discussed and reviewed elsewhere ( ) ( ) ( ) ( ) . a schematic diagram showing a summary of the immune response after iav infection has been illustrated in figure . the yin and yang theory is always used to describe the importance in balancing the host immune response. in the light of this theory, the treatment strategy aims to suppress the overwhelming activation of the host immune response and in reverse to compensate any unfavorable suppression. although adaptive immune responses are important in viral clearance, the immediate innate immunity play an important role in the early control of an infection, and conversely, is a major factor for disease severity due to immunopathology. dysregulated immune responses caused by viral infections have been implicated in severe disease development ( , ) , such as acute lung injury (ali). ali in its most severe form, known as acute respiratory distress syndrome (ards), is reported to be the most prevalent cause of mortality in iav-infected patients ( ) . studies suggested that iav strains could be associated with either over-activating (human infection by avian h n and h n ) ( , ) or suppressing (h n , h n ) ( ) immune response. recent history has seen the outbreak of iav pandemics of varying severity takes place at the cost of millions of lives. one such example would be the deadly spanish flu of , which claimed the lives of - million of the million people infected worldwide. the pathological examination of lung sections from mice infected with reconstituted iav virus revealed necrotizing bronchiolitis and severe alveolitis in tissue, with neutrophils observed as the predominant inflammatory cell type present ( ) , suggesting neutrophil involvement in the pathogenesis of iav infection. the majority of immune cells in blood circulation are neutrophils; of which they are among the first innate immune cells recruited to the site of infection ( ) . neutrophils characteristically control microbial infections by generating bactericidal ( ) neutrophil extracellular traps (nets), consisting of granule proteins, histones, and decondensed chromatin ( ) . both protective and destructive role of neutrophils in iav infections have been described. the contrasting role of neutrophils could be explained by factors such as viral strain and viral dose used in different experimental setup, etc. the protective role of neutrophils was observed when mice infected with a low, non-lethal dose of iav h n strain hkx displayed neutrophil-mediated viral clearance via phagocytosis ( , ) . depletion of neutrophils has found to enhance viral load in the iav-infected animals ( ) . on the contrary, this protective nature is disputed due to the association of neutrophil-generated nets. extensive net formation was observed in mice infected with pr , an iav strain highly pathogenic to mice ( ) . histones and myeloperoxidase within the net induce cell death of lung epithelium and endothelium ( ) , leading to the loss of integrity of the alveolarcapillary barrier, a characteristic of ali. yet, while histones have been shown to suppress iav replication in vitro ( ) , in vivo study demonstrated that there was increase in lung inflammation and damage in iav-infected mice treated with histones ( ) . interestingly, co-treatment of lethally infected mice with anti-histone antibody and oseltamivir resulted in an increase in animal survival when compared to infected mice groups treated solely with oseltamivir ( ) . in agreement with the in vitro and in vivo data, it has been reported that net produced by cultured neutrophils from patient with h n and severe h n infection increased alveolar epithelial cell permeability ( ) leading to ali. more importantly, plasma net level positively correlated with the disease severity index (including higher acute physiology and chronic health evaluation ii score) and multiple organ dysfunction syndrome ( ) , further demonstrating the detrimental role of net in the pathogenesis of severe iav infections. studies have demonstrated the involvement of superoxide dismutase and myeloperoxidase in netosis, the formation of net ( ) . the presence of anti-myeloperoxidase antibody as well as the superoxide dismutase inhibitor (detc) significantly reduced netosis. finally, tetrahydroisoquinolines ( ) and a panpeptidylarginine deiminase (pad) inhibitor, named cl-amidine ( ) have been suggested to inhibit netosis. despite it has been reported that during iav h n infection, pad knockout mice displayed only slight improvement in weight loss and a slight prolonged but no end-point survival advantage was observed compared to wt mice ( ) , based on the extensive findings presented above, targeting net to prevent ali in the severe case of iav infection, including the highly pathogenic avian iav, remain promising and may warrant further investigation. innate lymphoid cells are cells of lymphoid lineages that do not express antigen-specific b-or t-cell receptors ( ) . similar to t-helper cells, they are classified into subsets by their ability to produce type (th ), type (th ), and type (th and th ) cytokines. previous studies confirmed the involvement of ilcs of group linage (ilc ) in iav infection and airway inflammation ( , ) . on the positive side, during the recovery phase of iav infection, ilc expresses amphiregulin which promote airway epithelium repair ( , ) , thus facilitating the recovery of the infected lung. on the other hand, in response to il- produced by macrophages, dcs, and nkt cells, ilc secretes il- and il- and induce airway hyper-responsiveness. recruitment of eosinophils by il- to the lung also mediates airway inflammation ( ) . since eosinophilia is a characteristic of allergic asthma and influenza is a major cause for morbidity and mortality in asthma patients ( ) , it will be of particular interest to investigate the role of ilc in iav infection, particularly in asthma patients. ilc s have been initially described as immature nk cells residing in the liver and share many phenotypic similarities with nk cells ( ) . it was recently appreciated that tissue-resident ilc s other than the previously recognized nk cells are the major early source of the antiviral ifn-γ at the primary site of various viral infection, including iav ( ) . interestingly, ifn-γ was found to suppress ilc activity and reduce il production which exacerbates disease severity during influenza a(h n ) pdm infection ( ) . this data may highlight a link between ilc and ilc and suggesting ilc can suppress ilc activity via ifn-γ production during iav infection. with ilcs finally identified, functions of these cells and their role in immune response to tumors and pathogen infections have been massively investigated in recent years. type i ifns, prostaglandin i , corticosteroids, and testosterone have been reported to suppress ilc activity ( , ) . in addition to il- , the epithelial cytokines il- , thymic stromal lymphopoietin, as well as the lipid mediator prostaglandin d were found to activate ilc ( ) . the therapeutic potential of these ilc activators and suppressors is yet to be deduced. with more and more studies demonstrating the involvement of ilc in iav infection, the interplay between different ilc subtypes in iav infection would, therefore, be an interesting area to explore and modulate the ilc activity may be a future approach to combat iav infection. reactive oxygen species, generated by specialized enzymes such as nadph oxidases, are released during iav infection ( ) . the nadph oxidase family consists of enzymes containing different catalytic subunit named nox - and dual oxidase (duox) and . ros have been reported to display both beneficial (limiting viral replication) and detrimental (promoting ali) effects in the course of iav infection. interestingly, the protective or destructive effect of ros is dependent on the enzyme of which the ros is generated ( ) . dual oxidase and are found to be host-protective ( , ) . in vitro, ros generated by nuclear duox indirectly regulates the splicing of iav mrnas via the nuclear speckle-associated splicing complex ( ) . in addition to altering viral mrna splicing, ros generated by doux has been attributed to the production of ifn-λ, an important anti-iav ifn. in response to iav infection, increased viral mrna replication was observed when duox was silenced in vitro ( ) . increased viral replication was also observed in mice with doux silenced ( ) , further depicting the protective role of doux in iav infection. unlike doux, nox activation could be harmful to host. iav infection was reported to induce nox -dependent endosomal ros production ( ) . ros could target the conserved cys on toll-like receptor (tlr) , and inhibit tlr -mediated type i ifn expression during a mild iav h n infection in vivo ( ) . iav-infected mice treated with specific nox inhibitor, cholestanol-conjugated gp ds-tat, were found to have reduction in endosomal ros production, restored tlr activity, and displayed a decreased viral load ( ) . in addition to nox , nox dependent ros production has also been reported to activate mapk/erk signaling ( ) , enhancing the export of vrnp complex, thus increasing viral replication (see targeting the raf/ mek/erk pathway). nox knockdown resulted in a reduction of viral replication in vitro ( ) . targeting the different nadph oxidase isoforms, instead of scavenging ros should be considered as the therapeutic approach for iav infection, as doux-mediated ros production is beneficial ( , ) , while nox and nox are harmful during iav infections ( , ) . finally, ns (not to be confused with iav ns protein) has been demonstrated to be a nox inhibitor, which could inhibit the activity of nox , nox , and nox . a study demonstrated that ns suppresses iav-induced nox and significantly inhibits iav virus replication ( ) . besides cholestanolconjugated gp ds-tat and ns aforementioned, apocynin, a phagocytic nox inhibitor as well as ros scavenger ( ) ( ) ( ) , has been demonstrated to ameliorate hyper upregulation of cytokines induced by iav infection through socs and socs in vitro ( ) and reduce peri-bronchial inflammation and viral titer in vivo ( ) . interestingly, ebselen, another nox inhibitor and glutathione peroxidase mimetic, could reduce inflammatory status measured in bronchoalveolar lavage fluid (balf) of mice pre-exposed to cigarette smoke and subsequently infected with iav ( ) . taken together, these reports highlight the potential use of nadph oxidases inhibitors and ros scavengers to treat iav infections. dysregulated cytokine production has been associated with the elevated mortality rate observed in severe iav infections ( , ) . as such, the immunomodulation of cytokines are regarded as promising therapeutic tactics. recent advancements developed with this approach will be highlighted in the following section. tumor necrosis factor has two main functions during viral infection-it activates nf-κb, inducing the expression of cytokines responsible for the host immune response; and induces apoptosis through activation of a signaling cascade involving tradd, fadd, and caspase , , , and ( ) ( ) ( ) . tnf is known to be highly upregulated in iav-infected hosts, especially in hosts infected with highly pathogenic iav ( , ) . however, it is both protective and counter-protective functions associated with tnf that makes it a target in the treatment of iav. the protective role of tnf is observed during infection by low pathogenic iav, where extrinsically derived tnf is responsible for attenuating tissue-damaging cd + t-cell response ( ) . in addition to recruiting monocytic cells to the infection site, cd + t-cells response was observed to deteriorate lung pathology ( ) and damage healthy, non-infected lung epithelial cells ( ) upon iav infection. furthermore, tnf deficiency has been associated with an increased detection of il- and il- in balf ( ) , which promote the survival of and proliferation of cd + t-cells ( , ) and subsequent tissue damage. exacerbated lung pathology caused by the upregulation of the monocyte chemoattractant protein- was observed in tnf −/− mice infected with sub-lethal dose of iav ( ) . in addition, decreased cd + t-cell contraction due to enhanced expression of the anti-apoptotic protein bcl- was observed in sub-lethally iav-infected tnfdeficient mice when compared to wt mice ( ) . as a whole, there is substantial evidence supporting the protective role of tnf in iav infection. on the other hand, the correlation of tnf with pulmonary edema has been well-documented ( ) . tnf has been observed to stimulate the expression of cxcl in alveolar epithelial cells in a transgenic mice model resembling extensive iav infection in lung tissue, causing alveolar damage, lung edema, and hemorrhage ( ) . in addition to lung edema, tnf has also been reported to correlate with iav-associated encephalopathy ( , ) . however, it is notable that despite iav-associated encephalopathy, direct invasion of the central nervous system is rare ( ) , suggesting that iav-associated encephalopathy could instead be a result of peripheral infection. furthermore, tnf has been shown to increase the permeability of the blood-brain barrier (bbb) ( , ) , contributing to neural damage ( ) . these studies further support an anti-tnf approach as a potential therapy for severe iav infection. at present, etanercept, an anti-tnf drug administered in the treatment of rheumatoid arthritis, is the only tnf inhibitor (or even tnf directed treatment) tested for iav treatment. etanercept has been shown to protect against the in vivo lethal infection of mice with a highly virulent, mouse-adapted iav strain ( ) , with observations made of an increased survival rate with decreased morbidity, expression of the proinflammatory cytokine il- , lung injury, and edema ( ) . the protective role of il- was demonstrated in mice challenged with sub-lethal iav infection. il- -deficient mice displayed exacerbated pulmonary damage ( , ) and lung injury due to an observed decline in the survival of alveolar type ii cells and alveolar epithelial cells ( ) . iav suppresses the anti-apoptotic mcl- and bcl-xl expression, causing cell death of neutrophils which are critical in viral clearance ( ) . addition of il- restored the expression of mcl- and bcl-xl in vitro and is considered as the underlying mechanism for the observed survival advantage of wt mice over il- knockout mice during mild iav infection. il- has also been shown to induce the proliferation of lung il- + regulatory t cells and il- , which act to limit excessive proliferation of cd + t-cells and subsequent cd + -inflicted damage. this would hence prevent the tissue damage observed in lung immunopathology ( ) . despite the apparent protective role of il- , high levels of il- in serum or cerebrospinal fluid have been reported in severe neurologically complicated iav cases, with il- used as a marker for prognosis ( - , , ) . the role of il- in regulation of bbb permeability was reported ( ) , with potentially detrimental neurological complications. as such, the suppression of hyper-induced il- as a form of therapy in severe iav infection should be considered. one such option is the anti-il antibodybased drug tocilizumab, which is currently administered clinically for the treatment of rheumatoid arthritis. however, study on the usage of this drug to treat hyper upregulation of il- due to severe iav infection has yet to be conducted. on the other hand, in a case of h n virus-induced ards, the use of an extracorporeal cytokine hemoadsorption device to remove cytokines including tnf and il- from the bloodstream ( ) has showed beneficial to the patient ( ) . more research is required to confirm whether the removal or neutralization of il- could be a potential therapy for severe iav infections. the activation of cd + t-cell is crucial for viral clearance. it should, however, be tightly regulated to limit cd + t-cell inflicted host cell damage. il- mediates il- induction ( ) . il- acts to suppress cd + t-cells and reduce morbidity through il- and regulatory t-cells ( ) . much like other immunomodulatory approaches, the timing for applying il- should be carefully assessed. compared to placebo-treated iavinfected group, early administration of il- to iav-infected mice in fact led to poorer viral clearance, increased morbidity, and deteriorated lung histopathology, while il- administration during the recovery phase ( - days post-infection) accelerated recovery and improve lung immunopathology ( ) . notably, il- could also suppress th responses and increases susceptibility to secondary s. aureus infection ( ) . therefore, co-administration of antibiotics should be considered when utilizing il- as potential iav treatment. both type i and iii ifns have antiviral properties, with viruses counteract ifns to gain an advantage for their propagation. the iav viral protein ns inhibits the production of ifns by antagonizing irf- , a key transcriptional factor for ifns. this prevents the processing of cellular pre-mrnas (including those for ifns) and directly interacts with retinoic acid-inducible gene (rig)-i receptors, which are critical in innate sensing, to suppress ifn production during infection ( , ) . in addition to inhibiting ifn expression, the induction of socs inhibits ifns signaling by suppressing cytokine signaling has been documented ( ) . the recognition of ′ triphosphate on viral rna by rig-i receptor is shown to induce the expression of socs , which in turn represses type i ifns expression ( ) . due to ifns being a key contributor to antiviral immune response, an impairment of type i or iii ifn production may cause the escalation of otherwise mildly pathogenic iav infection into a life-threatening one ( ) . while type i ifn has been demonstrated to inhibit iav replication in vitro ( ) ; the in vivo administration of type i ifn in animal models only displayed effectiveness in a prophylactic capacity. a lowered viral titer was detected in the nasal wash of test animals. however, host susceptibility to iav infection remained unchanged ( ) . notably, this protective effect is only conferred by an optimal dose of type i ifn of low to moderate amounts ( - units per mice daily); with higher dosages ( , - , units per mice daily) shown to increase morbidity ( ) . in addition, clinical trials demonstrated that prophylac tic administration of type i ifn reduced disease severity and lowered susceptibility to iav in males and participants aged or above ( ) . despite relatively successful results seen in the prophylactic use of ifns, its therapeutic use is of greater clinical relevance. mice treated with type i ifn post-iav infection showed a successful reduction in lung iav titer but displayed increased morbidity and mortality in comparison to vehicle-treated mice ( ) . a possible explanation for this phenomenon is the induction of excessive inflammatory response and trail-dr -mediated epithelial cell death by type i ifn ( ) , which accounts for the observed lung pathology in iav-infected animals treated with type i ifn ( ) . in addition, downregulation of γδ t-cells by type i ifn has been correlated with increased susceptibility to secondary s. pneumoniae infection ( ) , further arguing against the potential use of type i ifns for the treatment of iav infection. in comparison to type i ifns, the administration of type iii ifns may provide advantages in the control of iav replication ( , , ) without the risk of previously reported type i ifns-mediated immunopathologic side-effects ( , , ) . however, a recent study aiming to stimulate ifns signaling through the systematic administration of rig-i ligand post-iav infection demonstrated that type i, but not type iii ifns signaling is important in conferring protection during fatal iav infection in vivo ( ) . though, this study did not measure the production of type i and iii ifns as well as any changes in viral load with respect to ifnar or ifnlr knockout. in addition, while human immune cells are not primary targets in iav infection, they could be susceptible to iav and become efficient host cells for virus replication. they are reported to possess a subpar response to type iii ifns ( ) ; leading to the preliminary conclusion that solely using type iii ifn as treatment may not be feasible. as such, reports suggesting the use of type iii ifns over type i ifns as a front-line therapeutic agent to counter iav infections may require further investigation. the inhibition of cox- by selective inhibitors, nimesulide and celecoxib, was previously demonstrated to suppress the hyper upregulation of pro-inflammatory cytokines induced by highly pathogenic avian iav ( ) ( ) ( ) . in addition, the use of zanamivir in tandem with a specific cox- inhibitor was shown to increase the survival rate of mice lethally infected with avian h n iav, when compared to mice treated solely with zanamivir ( ) . activated cox- regulates downstream prostaglandin production. one such example is pge , a major type of prostaglandin recently demonstrated to play an important role during iav infection. pge was significantly upregulated in response to iav infection, leading to the inhibition of antiviral type i ifn production in macrophages and the subsequent increase in virus replication ( ) . the use of chemicals ah and gw x to antagonize pge downstream signaling molecules ep and ep respectively, was shown to induce antiviral type i ifn production. the in vivo treatment of mice lethally challenged iav with both ep and ep antagonists significantly improved the survival rate. a recent study demonstrated the ability of a modified tcm decoction to reduce peg production and subsequent morbidity in mice lethally challenged with iav. improved lung pathology was observed ( ) . the long history of clinical tcm use supports the clinical feasibility of peg inhibition as an option to treat severe iav infections. pattern recognition receptors on host cells sense specific pamps present on the viral surface or generated during replication. prrs can be broadly divided into two classes by their function or location. when defined by location, prrs are classified into groups-membrane-bound (tlrs and c-type lectin receptors), cytosolic (rig-i-like and nod-like receptors), and secreted (collectins and pentraxins) ( ) . significant research has been conducted on prrs with regards to iav infection. tlrs and rig-i receptors have been extensively studied for their major roles in eliciting host immune responses (cytokine and ifn expression) during iav infection ( ) ( ) ( ) . rig-i receptors have been investigated for their functional relevance to iav infection and targeting these receptors as a form of iav treatment has been extensively reviewed ( ) ( ) ( ) . this section will cover recent research on tlrs and the targeting of different tlrs to treat iav infection. humans have been identified to express tlr - , while mice have been identified to express functional tlr - as well as tlr - ( ) . most tlrs-with the exception of tlr utilize myd as an adaptor protein during signal transduction. tlr utilizes trif as an adaptor. tlr is known for its ability to utilize either myd or trif, with the choice of adaptor dependent on its sub-cellular location ( ) . different tlrs, such as tlr , , and ( ) as well as tlr , tlr , and most recently tlr ( ) , have been revealed to play a role in the orchestration of host immune responses contributing to iav pathogenesis. with tlr being an exception ( ) ( ) ( ) , tlr activation largely causes the release of pro-inflammatory cytokines, with hypercytokinemia leading to ali as a major cause of mortality in severe iav infections. in addition to dysregulated cytokine release, excessive production of ros has been associated with ali development. in fact, lung injury during severe pulmonary infections, such as iav and sars, could be caused by oxidative stress ( ) . iav infection activates nadph oxidase that subsequently produces oxidized papc, an endogenous phospholipid. the oxidized papc serves as an agonist for tlr , activating a tlr -trif-traf -nf-κb signaling cascade to eventually trigger the release of il- , ultimately inducing the onset of ali. in addition to oxidized papc, the induction of endogenous protein s a upon intracellular prr ddx recognition of iav subsequently induces the activation of tlr , further contributing to iav-induced mortality ( ) . since tlr has been proven to be important in ali induction (and hence iav-related mortality), manipulating the stimulation and antagonism of tlr could potentially reduce the severity of iav infections. eritoran (e ) is a specific tlr antagonist initially purposed for the treatment of sepsis, but a failed a phase iii clinical trial due to improved patient care in the placebo group prevented its eventual use in sepsis treatment ( ) . in vivo administration of eritoran in mice lethally infected with iav resulted in improved clinical score, lung pathology results, and reduced viral titer. delayed administration of eritoran, at day after infection beyond the recommended therapeutic time window (within h after the first display of clinical symptom) for use of oseltamivir ( ), also demonstrated a significant benefit to infected mice compared to non-treated group, suggesting a prolonged therapeutic time window for iav treatment when compared to mainstay antiviral drug treatment. a newer and structurally simpler specific tlr antagonist, fp ( ), alongside a newly developed decoy peptide r that has been shown to disrupt tlr , , , and signaling via tirap, has been shown to protect mice from lethal iav infection ( ) . these results support the potential use of tlr antagonism as a means to treat severe iav infection. the suppression of other tlr signaling pathways-such as blocking tlr -mediated signaling through the use of an anti-tlr antibody, significantly protected against lethality when administered on day and post-iav infection ( ) . a study also demonstrated that h n -infected tlr knockout mice had better survival than h n -infected wild-type mice, which is evident through the significantly faster regaining of body weight post-infection, lower viral titer in the lung, and fewer pathological changes in the lung ( ) . an increasing number of tlr antagonists are now under development ( , ) , alongside several other agents also shown to have effects on tlrs. polysaccharides isolated from r. isatidis, a traditional chinese medicinal herb used to treat iav infection, have recently been shown to inhibit pro-inflammatory cytokines such as il- and ccl- in vitro by down-regulating upstream tlr expression ( ) . menk, an endogenous protein expressed in the adrenal medulla, was shown to both prophylactically and therapeutically increase the survival rate while reducing viral-caused lung pathology and viral titer in mice lethally challenged with iav ( ) . this was determined to be caused by the downregulation of tlr . these results suggest the potential of down-regulating tlr expression in the treatment of iav infection. the above-mentioned data suggest modulation of tlr signaling or expression as a promising approach in treating severe influenza disease and deserves immediate investigation. table summarizes new immunomodulatory approaches to combat iav infections. it is well documented that patients with diabetes mellitus have a greater tendency to develop severe iav infection than healthy patients ( ) . hyperglycemia increases susceptibility of the host to iav infection via viral uptake, through the promotion of v-atpase assembly ( ) and immunosuppression ( ) . in addition, viruses rely on host metabolism to perform essential functions during replication ( ) ( ) ( ) ( ) . these processes exert a large energy demand on the host within a very short period of time ( ) ; energy of which is supplied by and is dependent on host metabolism. iav viruses have been reported to modify the metabolic state of the host. for example, increased c-myc-dependent glycolysis and glutaminolysis has been demonstrated in infected cells ( ) . the changes in glucose and glutamine metabolism were reversed upon the addition of bez , which inhibited the iav-mediated c-myc induction. administration of bez days prior to infection and up to days post-infection was shown to decrease lung viral titer and improve the survival rate in iav-infected mice. small molecules such as clotrimazole and α-mangostin that target lipid metabolism have also been demonstrated to suppress iav replication in vitro ( ) . in addition to being important for generating energy and biosynthesis, recent research demonstrates that cellular metabolism affects immune cell function. dysregulated immune responses observed in many diseases are associated with specific metabolic configurations. viruses, influenza inclusive ( ) , were found to induce drastic alterations in metabolic levels and programs ( ) . macrophages in infected hosts were observed to have marked differences in the krebs cycle, a key metabolic pathway. this is of significance due to the role of macrophages, which are immune cells critical in the pathogenesis of many inflammatory diseases ( , , ) . in activated macrophages, succinate, a krebs cycle intermediate, was found to possess inflammatory signal. accumulation of succinate generates ros, leading to subsequent activation of hypoxia-inducible factor α and the induction of cytokines such as il- β ( ) . a recent study identified the ability of itaconate, another krebs cycle-derived metabolite, to block the production of inflammatory factors. this prevented inflammation, protecting mice from lethal levels of inflammation that can occur during infection ( ) . this data suggest the critical roles of krebs cycle intermediates in regulating cytokine profiles and inflammation. metabolites generated by innate immune cells in distinct configurations could have different roles beyond that of bioenergetics, with functions in signaling regulation, transcription, and orchestrating innate immune responses. despite the lack of research conducted thus far on the application of immunometabolic approaches to influenza treatment, the prospect of manipulating immune responses by modulating immune cell metabolic state is promising. further research should focus on the identification of metabolites for modulation of immune cell function with substantial improvement of therapeutic strategies to treat iav disease. latest advancements in high-throughput technologies, e.g., meta bolomics is a useful approach to systematically investigate the changes of metabolic mechanisms during iav infections. identification of important metabolites involved during iav infection should be a new approach by modulating the host metabolism for interventions. multiple host-based intervention strategies against influenza have been developed or are under development. while approaches targeting host machinery required for virus replication seem to be promising thus far, additional research is needed to determine the effect of modulating host immune response on influenza treatment. this is increasingly important, since targeted host factors may play distinct roles in response to infection by different influenza viral strains ( ) , making the management of influenza through solely targeting a single specific host factor is difficult. host-based interventions offer obvious advantages over conventional antivirals, such as a higher barrier to drug resistance ( , , ) due to greater genetic stability of host factors than the mutation-prone nature of viral components. in addition, administration feasibility is a key factor to consider the usage of drugs. the mainstays of antivirals for iav infections, the na inhibitors, and m blockers, are recommended to be administered within h of symptom onset for optimal antiviral activity. this short treatment window may not be fully fulfilled in a clinical setting. novel host-based interventions were reported to have therapeutic time windows longer than this conventional timeframe ( , , , ) , even up to days post-infection ( ) , providing a clear clinical advantage over na inhibitors and m blockers. in addition, hypercytokinemia and ards could contribute to disease severity and mortality in instances of severe influenza infection, with virustargeting antivirals providing little to no alleviation of such complications. since host immune response is indispensable in host defense against invading pathogens, the use of immune-modulators to suppress detrimental effects while retaining beneficial protection of the host remains challenging. the timing and dosage of medication administration would be critical in determining the drug effectiveness in influenza treatment. targeting virus-induced metabolic changes to restore host normal metabolism may be a new direction to combat influenza disease. further research in the immunometabolism field, along side studies on modulating immune response to infectious disease by altering host metabolic processes; would create a new direction for future research and is expected to yield significant discoveries that may provide new therapeutic options in the treatment of iav infections. smyl conceptualized the work. il and asms drafted some review sections and tfy and smyl wrote the manuscript. influenza a(h n )pdm outbreak detected in inter-seasonal months during the surveillance of influenza-like illness in pune avian influenza a (h n ) virus infections in humans across five epidemics in mainland china age and influenza-specific pre-vaccination antibodies strongly affect influenza vaccine responses in the icelandic population whereas disease and medication have small effects global transmission 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dual role of tnf in pulmonary edema adam -mediated processing of tnf-α expressed by antiviral effector cd + t cells is required for severe t-cell-mediated lung injury serum and cerebrospinal fluid cytokine profile of patients with pandemic h n influenza virus-associated encephalopathy tumor necrosis factor-alpha, interleukin- beta, and interleukin- in cerebrospinal fluid from children with prolonged febrile seizures. comparison with acute encephalitis/encephalopathy detection of influenza virus rna by reverse transcription-pcr and proinflammatory cytokines in influenza-virus-associated encephalopathy tumour necrosis factor-alpha affects blood-brain barrier permeability and tight frontiers in immunology | www acute liver failure increase of tumor necrosis factor-alpha in the blood induces early activation of matrix metalloproteinase- in the brain sepsis-associated encephalopathy: the bloodbrain barrier and the sphingolipid rheostat inhibition of the inflammatory cytokine tumor necrosis 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intervention metabolic effects of influenza virus infection in cultured animal cells: intra-and extracellular metabolite profiling krebs cycle rewired for macrophage and dendritic cell effector functions succinate is an inflammatory signal that induces il- beta through hif- alpha itaconate is an anti-inflammatory metabolite that activates nrf via alkylation of keap key: cord- - nqmjdek authors: zhong, jixin; rajagopalan, sanjay title: dipeptidyl peptidase- regulation of sdf- /cxcr axis: implications for cardiovascular disease date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: nqmjdek dipeptidyl peptidase- (dpp ) is a ubiquitously expressed protease that regulates diverse number of physiological functions. as a dipeptidase, it exerts its catalytic effects on proteins/peptides with proline, alanine, or serine in the penultimate (p ) amino acid residue from the amino terminus. the evidence to date supports an important effect of dpp in catalytic cleavage of incretin peptides and this perhaps represents the main mechanism by which dpp inhibition improves glycemic control. dpp also plays an important role in the degradation of multiple chemokines of which stromal cell-derived factor- (sdf- , also known as cxcl ) is perhaps an increasingly recognized target, given its importance in processes, such as hematopoiesis, angiogenesis, and stem cell homing. in the current review, we will summarize the importance of dpp -mediated enzymatic processing of cytokines/chemokines with an emphasis on sdf- and resultant implications for cardiovascular physiology and disease. identified as a new dipeptide naphthylamidase in ( ) and subsequently found to be identical to t cell activation antigen cd , ada-binding protein, mouse thymocyte-activating molecule, and rat liver membrane glycoprotein gp ( ) . dpp consists of a short n-terminal intracellular domain ( residues), a -residue-long transmembrane α-helix domain ( amino acids), and a large c-terminal extracellular domain. the c-terminal extracellular domain is responsible for its catalytic activity and binding to a number of ligands, such as ada and matrix proteins ( ) . the catalytic activity of dpp depends on its dimerization and glycosylation of specific residues ( ) . dpp can also assemble into tetramers on the cell surface, which may involve the linkage of dimers located on the surface of two different cells, enabling it to function as a cell-cell communication molecule. in addition to its membrane-bound form, dpp also circulates as a soluble form in the plasma, which lacks the cytoplasmic and transmembrane domain with preserved catalytic activity. soluble dpp (sdpp ) is a homodimer with a molecular weight range of - kda ( ), but can form higher molecular weight assemblies migrating as -kda complexes ( ) . whether sdpp is cleaved from the membrane or is secreted is unclear. for instance, studies investigating viral liver infection suggested that sdpp is shed from membrane-bound dpp ( ) . sdpp has, however, also been detected in the lumen of secretory granules in pancreatic α cells and in the exocytic secretory lysosomes of natural killer cells ( , ) . sdpp is commonly elevated in many disorders, such as solid tumors, reactive airways disease, hepatitis c, type diabetes, and obesity ( , , ) . tissue distribution and cell specific expression and phenotype of dpp −/− mice dipeptidyl peptidase- is widely distributed throughout the body ( table ) , with particularly high expression on the apical surface of endothelial and differentiated epithelial cells. bone marrow cells, brush border of the small intestine, proximal tubular cells, and glomerular cells in the kidney express high levels of dpp as well ( ) . dpp is present on endothelial cells and fibroblasts throughout the body. among hematopoietic cells, dpp is expressed at the highest level on t cells with lower levels in monocytes and dendritic cells ( ) . dpp expression increases potential function serves as an adipokine mediating obesity-induced metabolic syndrome ( ) adipose tissue macrophage and dendritic cells enhances t cell inflammation and obesity-induced insulin resistance ( ) t cells promotes t cell activation by providing co-stimulatory signaling ( , ) endothelial cells regulates endothelial function and vascular tone ( , ) epithelial cells expressed in the epithelial cells in the kidney, lung, and gi tract. mediates mers-cov infection in the lung ( ), kidney fibrosis ( ) , diabetic nephropathy ( ), intestinal growth ( ) hepatocytes involved in lipogenesis ( ) and liver damage ( ) as monocytes differentiate into antigen-presenting cells as well as during t cell activation ( , ) . dpp is expressed at high levels in kidney, spleen, lung, pancreas, and prostate ( ) . mice lacking the gene encoding dpp are refractory to the development of obesity and hyperinsulinemia and demonstrate improved post-prandial glucose control ( , ) . mice deleted for dpp are fertile and appear healthy. only slight decrease of body weight in dpp −/− mice was observed compared to wild types. they have normal fasting blood glucose level, but shows reduced glycemic excursion after an oral glucose challenge ( ) . increased intact insulinotropic form of glp- and circulating insulin were seen in dpp −/− mice after oral glucose stimulation ( ) . pair feeding and indirect calorimetry studies indicate that reduced food intake and increased energy expenditure accounted for the resistance to high fat diet-induced obesity in the dpp −/− mice. ablation/ deletion of dpp is associated with improved metabolic control with improved insulin sensitivity, reduced pancreatic islet hypertrophy, and protection against streptozotocin-induced loss of β cell mass and hyperglycemia ( ) . pharmacological inhibition of dpp enzymatic activity improves glucose tolerance in wildtype but not in dpp −/− mice. interestingly, dpp inhibitor's also improve glucose tolerance in glp r −/− mice, indicating that dpp contributes to blood glucose regulation by controlling the activity of glp- as well as additional substrates ( ) . dipeptidyl peptidase- has been shown to be able to cleave a number of chemokines and cytokines, including sdf- , granulocyte-macrophage colony-stimulating factor (gm-csf), granulocyte colony-stimulating factor (g-csf), interleukin- (il- ), erythropoietin (epo), regulated on activation normal t-cell expressed and presumably secreted (rantes, also known as ccl ), macrophage-derived chemokine (mdc, also known as ccl ), eotaxin (also known as ccl ), monokine induced by ifn-γ (mig, also known as cxcl ), ifn-γ-induced protein- (ip- , also known as cxcl ), and interferon-inducible t-cell α chemoattractant (itac, also known as cxcl ) ( table ). the regulation of cytokine levels through catalytic cleavage may influence their levels in tissue domains. the differential contribution of dpp in the regulation of each of these cytokines is obviously dependent on the levels of expression of dpp , which may vary depending on the tissues, the cells predominantly expressing the cytokine of interest and the disease context. additionally, circulating or cell free dpp may also contribute to catalytic activity. dpp -mediated truncation of rantes abolishes the chemotactic activity to monocytes but not to t cells ( ) . eotaxin (ccl ) is an eosinophil chemotactic protein and has been shown to be involved in allergic responses. dpp truncation of eotaxin inactivates its chemotactic activity for eosinophils. dpp -truncated eotaxin shows impaired binding and signaling through ccr . in addition, truncated eotaxin suppresses calcium signaling and chemotaxis of intact eotaxin ( ) . dpp inhibition by either genetic deletion or pharmacological inhibition, enhances eotaxin-induced mobilization of eosinophils into the blood and recruitment into the injury/injection site ( ) . mdc (ccl ) is a chemoattractant for monocytes, dendritic cells, nk cells, and chronically activated t lymphocytes ( , ) . dpp -truncated mdc displays preserved chemotactic activity toward monocytes, but less potency toward lymphocytes and dendritic cells ( ) . cxcr interacts with mig (cxcl ), ip (cxcl ), and itac (cxcl ), all of which are targets of dpp . cleavage of those chemokines by dpp attenuates their chemotactic activity, with the cleaved products serving as endogenous antagonists for cxcr binding ( ) . dipeptidyl peptidase- is also involved in the inactivation of multiple colony-stimulating factors (csfs) and thus regulates hematopoietic stem cells (hscs) and hematopoietic progenitor cell (hpc) function. the proliferative action of gm-csf, g-csf, il- , and erythropoietin (epo) on hpcs is enhanced in dpp knockout mice or by pretreatment with a dpp inhibitor ( ) . catalytic inhibition of dpp or dpp deficiency promotes the engraftment of hscs and hpcs after bone marrow transplantation in mice ( ) . dpp -truncated csfs may suppress the activity of their respective full-length csf via antagonism ( ) . stromal cell-derived factor- is an -kda peptide that is encoded by cxcl ( ) . it is a chemoattractant for t lymphocytes, bone marrow stem cells [such as hsc, endothelial progenitor cell (epc), and mesenchymal stem cells (mscs)], endogenous cardiac stem cells (cscs), and adipose-derived regenerative cells ( ) ( ) ( ) . there are several isoforms of sdf- (sdf- α-ζ), resulting from alternative splicing of its mrna ( ) . among these isoforms, sdf- α is the best described. sdf- α is expressed in many tissues, including bone marrow, heart, liver, kidney, thymus, spleen, skeletal muscle, and brain ( , ( ) ( ) ( ) ( ) . in the cardiovascular system, sdf- α is expressed in stromal cells, endothelial cells, and cardiomyocytes ( , ) . sdf- is typically inactivated by exopeptidases, such as dpp , matrix metalloproteinase (mmp)- , and - ( ) . unlike cleavage of sdf- by dpp at position - , mmps cleave sdf- at position - , leading to the loss of its binding activity to cxcr ( ) . the relative contribution of each of these peptidases in regulation of sdf- levels is unclear. cxcr is an alpha-chemokine receptor specific for sdf- and belongs to a family of g-protein-coupled receptors. cxcr is expressed on a range of progenitor cells (including hematopoietic, endothelial, and cscs) and thus is important for cell migration and organ development during embryogenesis ( , , ) . mice deficient for either cxcr or sdf- display abnormal b-lymphocyte, hepatic, and cardiac (ventricular septal defects) development, and die in utero ( ) ( ) ( ) . loss-of-function cxcr mutations in humans also causes impaired neutrophil mobilization and b-cell lymphopenia ( ) . in addition to cxcr , cxcr has also been suggested to be an important receptor for sdf- ( , ) . however, the relative contribution and interactions of cxcr and cxcr is not fully elucidated. the involvement of cxcr in cardiovascular disease, if any, is also not yet known ( ) . dpp may also play a more general role in regulating csf activity and stem cell homing ( ) . it was previously believed that disruption of the interaction between cxcr receptor expressed by hematopoietic progenitors and sdf- expressed by bone marrow stromal cells is sufficient to detach anchored progenitors from their bone marrow niches, leading to their rapid mobilization to the peripheral blood. amd (also termed plerixafor) inhibits sdf- -mediated migration in vitro by blocking the chemokine binding to its major receptor cxcr ( ) . amd mobilizes immature progenitor cells from the bone marrow into the blood and has been approved for clinical mobilization in lymphoma and multiple myeloma patients undergoing autologous transplantation. when combined with g-csf, amd synergistically augments mobilization of progenitor cells, with increased in vitro migration to sdf- gradients and facilitates repopulation of transplanted non-obese diabetic/severe combined immunodeficient mice ( ) . amd has recently been shown to directly induce sdf- release from cxcr + human bone marrow osteoblasts and endothelial cells, with sdf- release from these cells into the circulation, representing a pivotal mechanism essential for steady-state egress and rapid mobilization of hpcs ( ). the sdf- /cxcr axis has been shown to be critical in tissue repair in multiple organ systems, including the eye, heart, kidney, liver, brain, and skin. specific to the heart, the sdf- /cxcr axis has been shown to be essential for cardiogenesis ( , ) . sdf- is now well known as a key regulator of stem cell migration to sites of tissue injury ( , ) . sdf- was first identified by askari et al. as a key regulator of stem cell migration to ischemic cardiac tissue ( ) . cd + stem cells express the sdf- receptor cxcr at high levels ( , ) . during myocardial infarction, sdf- levels are elevated h after infarction and return to baseline at day and further reduced to a low level thereafter ( ) . overexpression of sdf- in ischemic cardiomyopathy by either engineered cellbased or plasmid-based approach improved cardiac function in rats via enhancing stem cell homing and promoting revascularization of the infarct area ( , ) . therefore, the ability to express sdf- locally is believed to enhance the vasculogenic potential of adult cardiac progenitor cells ( ) . however, the enhancement of endogenous stem cell-based repair appears to be blunted due to the short half-life of sdf- at the time of acute myocardial infarction owing to its degradation by proteases ( ) . as a major enzyme mediating the degradation of sdf- , dpp may represent a potential target for improving stem cell homing with stem cell-based therapy. preservation of sdf- by dpp inhibition has been shown to promote stem cell repopulation and homing to ischemic tissues. dpp inhibitors diprotin a or val-pyr, enhance chemotaxis of hscs and hpcs and greatly increase homing and engrafting capacity of hscs ( , ) . pretreatment of hsc with dpp inhibitor diprotin a, enhanced their repopulation ability in lethally irradiated mice ( ) . enhancement of engraftment of human cd + cord blood cells with dpp inhibition has also been observed in xenogeneic mouse recipients (nod/scid or nod/scid/beta null ) ( , ) . pretreating either donor cells in vitro or recipients in vivo is able to enhance the engraftment of stem cells ( , ) . in a lung transplantation model, systemic dpp inhibition by vildagliptin increases sdf- levels in plasma, spleen, and lung, accompanied by a significant increase of stem cells in the lung grafts. dpp inhibitor-treated mice also shows less alveolar edema compared with untreated recipients ( ) . liebler showed that dpp inhibition enhances sdf- /cxcr axis and increased the retention of human bone marrow-derived cells in the injured lungs of immune deficient mice by % ( ) . in addition to sdf- , dpp inhibition also enhances bone marrow engraftment by preserving g-csf and gm-csf. both g-csf and gm-csf are substrates for dpp , with inhibition of dpp promotes bone marrow engraftment not only through sdf- but also csf-dependent mechanisms ( ) . g-csf and gm-csf in turn may also increase the expression of dpp on cd + cells, which results in their decreased responsiveness to sdf- ( ). angiogenesis and vasculogenesis are an immensely complex process that requires the coordinated action of a multitude of cells, transcription factors, and cytokines working in concert in a precisely choreographed manner. it is widely believed that these processes can be recapitulated in the adult through the participation of a progenitor cell population of which epcs are perhaps the best described and widely believed to be important building blocks for the assembly of functional vasculature in adults. while the origins of epcs are still controversial, what is clear is that these cells have the capacity to differentiate into mature endothelial cells ( ) . implantation of ex vivo-expanded epc has been shown to improve neovascularization of injured tissues in animal models ( ) ( ) ( ) . sdf- plays a pivotal role in the trafficking and homing of epcs to ischemic tissues ( ) ( ) ( ) . sdf- levels increase in plasma and ischemic tissue shortly after ischemic injury, in response to hypoxia which upregulates hif- α ( ) . hif- α upregulates sdf- , by binding to the promoter of sdf- and initiating its transcription ( ) . ex vivo priming with sdf- , enhances the proangiogenic potential of epc as evidenced by improved blood flow recovery when transplanted into a nude mouse model of hind-limb ischemia ( ) . disease states, such as diabetes associated with upregulation of dpp , may represent prototypical conditions associated with defective homing and integration of epc's owing to rapid degradation of sdf- ( ) . kanki et al. reported that sdf- could be cleaved by dpp- in both plasma and ischemic heart tissue ( ) . shih demonstrated an improvement in epc number and endothelial nitric oxide synthetase (enos) expression after dpp inhibition by mk- ( ) . transient engineered cell-based or plasmid-based overexpression of sdf- in ischemic cardiomyopathy has been shown to improve cardiac function in animal models ( ) . in a study that compared the effects of sdf- overexpressed on mscs alone or mesenchymal stem cells engineered to overexpress sdf- (msc-sdf) on cardiac function in lewis rats after acute myocardial infarction, tail vein infusion of msc and msc-sdf- , day after acute myocardial infarction, led to improved cardiac function by echocardiography by . and . %, respectively, compared with saline controls. the beneficial effects of msc-sdf transplantation were suggested to be mediated through preservation rather than regeneration of cardiac myocytes within the infarct area ( ) . cardiac progenitor cell and cxcr expression on cardiac myocytes are required for further local trophic effects of msc ( ) . the mechanism of action of sdf- overexpression in myocardial infarction and heart failure are likely multifactorial, including both systemic and direct trophic effects. an important effect of sdf- is its effect on the recruitment of cscs to the infarct and infarct border zone ( ) . delivery of mscs engineered to overexpress sdf- at the time of acute myocardial infarction has been shown to lead to improvement in cardiac function ( ) . the myocardial repair initiated by endogenous stem cell appears blunted because of the natural short-term expression of sdf- at the time of acute myocardial infarction. in light of these effects in regulation of sdf- , dpp inhibition has been suggested to be of potential benefit in cardiovascular diseases, such as myocardial infarction and peripheral artrerial disease. in combination with g-csf, dpp inhibition augments myocardial regeneration and improves cardiac function after myocardial infarction in mice ( , ) . in combination with cxcr overexpression, diprotin a treatment has shown to improve myocardial function and repair of infarcted myocardium ( ) . a bioengineered protease-resistant form of sdf- has shown greater potency in promoting blood flow recovery after hind-limb ischemia ( ) and improving cardiac function as well as capillary density in the infarcted heart ( ) . dual injection of g-csf and sitagliptin resulted in the mobilization of progenitor cells and relieved the symptom of end-stage heart failure in a -month-old boy ( ) . protease-resistant forms of sdf- display an enhanced potency in improving blood flow in experimental peripheral artery disease and myocardial infarction ( , ) . it has been shown that parathyroid hormone treatment after myocardial infarction improves dpp in chemotaxis and cardiovascular disease frontiers in immunology | www.frontiersin.org survival and myocardial function with potential involvement of enhanced homing of bone marrow-derived stem cells. huber et al. demonstrated that parathyroid hormone serves as a dpp inhibitor and increases cardiac sdf- level, which in turn enhances cxcr + bone marrow-derived stem cell homing to ischemic heart and attenuates ischemic cardiomyopathy after infarction ( ) . haverslag showed sdf- preservation by dpp inhibitor increases monocyte extravasation and thus accelerating perfusion recovery without detrimental side effects on plaque stability in atherosclerosis-prone apoe −/− mice ( ) . figure depicts modulation of sdf- levels in the myocardium by dpp inhibition and enhancement of myocardial angiogenesis by dpp levels. in a porcine model of hf, delivery of a plasmid sdf- with an endomyocardial injection catheter demonstrated safety at doses up to mg while improving cardiac function and vasculogenesis up to days post-injection at doses of . and mg ( ) . in a phase i dose escalation study with months follow-up in ischemic cardiomyopathy, subjects in new york heart association class iii heart failure, with an ejection fraction ≤ % on stable medical therapy, were enrolled to receive , , or mg of plasmid sdf- via endomyocardial injection. the primary end points for safety and efficacy were at and months, respectively. the primary safety end point was a major adverse cardiac event while efficacy end points were changes in quality of life, new york heart association (nyha) class, -min walk distance, single photon emission computed tomography, n-terminal pro-brain natruretic peptide, and echocardiography at and months. the primary safety end point was met. at months, all of the cohorts demonstrated improvements in -min walk distance, quality of life, and nyha class ( ) . stromal cell-derived factor- plasmid treatment for patients with heart failure (stop-hf) was a phase ii, double-blind, randomized, placebo-controlled trial to evaluate safety and efficacy of a single treatment of plasmid sdf- delivered via endomyocardial injection to patients with ischemic heart failure. the primary endpoint was a composite of change in min walking distance and minnesota living from heart failure questionnaire from baseline to months follow-up. the primary endpoint was not met (p = . ). for the patients treated with psdf- , there was a trend toward an improvement in left ventricular ejection fraction at months (placebo vs. vs. mg Δlvef: − vs. − . vs. . %, p = . ). patients in the first tertile of ef (< %) that received mg of psdf- demonstrated a % increase in ef compared with a % decrease in placebo (Δlvef = %, p = . ) at months ( ) . although the reasons for the overall failure are currently unclear, the differential benefit in those with advanced left ventricle dysfunction raises the possibility of differential mechanisms that would be operational in more advanced patients. these include the possible overexpression of cxcr in cardiac myocytes in the infarct border leading to a negative inotropic state ( ) . the transient overexpression of sdf- in ischemic cardiomyopathy has been shown to lead to long-term down-regulation of cardiac myocyte cxcr expression, re-recruiting the contractile function of the border zone ( ) . patients with greater left ventricle dysfunction are likely to have a greater volume of myocardial tissue under stress; therefore, a greater demonstrable response to sdf- overexpression. another important reason could be that the upregulation of dpp in the border zone of the infarct or around ischemic areas may have resulted in rapid degradation of sdf- limiting the efficacy of such an approach. since both the phase i and phase ii studies were performed in the absence of dpp inhibition, it could be speculated that the results may have been different if the trials had been performed either in the presence of a dpp inhibitor. figure | dipeptidyl peptidase- inhibition in modulation of sdf- and myocardial angiogenesis: expression of dpp increases in myocardial infarction. suppression of dpp enzymatic activities by pharmacological inhibitors preserves sdf- , which results in an enhanced homing of cxcr + progenitor cells from bone marrow to infarcted tissues. cxcr , chemokine (c-x-c motif) receptor ; dpp i, dpp inhibitor; mi, myocardial infarction; sdf- , stromal-derived factor- . dpp in chemotaxis and cardiovascular disease frontiers in immunology | www.frontiersin.org due to the importance of sdf- /cxcr axis in the stem cell and progenitor cell survival and function, understanding this axis and molecules that modulate their production and action will be of utility for the treatment of cardiovascular disease. there are a number of clinically approved drugs, including dpp inhibitors and parathyroid hormone, which have the ability to enhance sdf- /cxcr responsiveness and may improve the outcome of cardiovascular diseases. several recent large scale clinical trials have indicated that unlike most other oral anti-diabetic drugs that promote cardiovascular disease, dpp inhibitors are safe from cardiovascular standpoint despite lack of evidence showing beneficial effect ( ) ( ) ( ) . to what extent sdf- /cxcr axis contributes to this effect requires further investigation. this work was supported by grants from aha ( sdg and post ) and mid-atlantic nutrition obesity research center (norc pilot & feasibility program) to dr. zhong. a new dipeptide naphthylamidase hydrolyzing glycyl-prolyl-beta-naphthylamide pharmacology, physiology, and mechanisms of action of dipeptidyl peptidase- inhibitors dpp in cardiometabolic disease: recent insights from the laboratory and clinical trials of dpp inhibition serum high molecular weight dipeptidyl peptidase iv (cd ) is similar to a novel antigen dppt-l released from activated t cells a prediction of dpp iv/cd domain structure from a physico-chemical investigation of dipeptidyl peptidase iv (cd ) from human seminal plasma similar increased serum dipeptidyl peptidase iv activity in chronic hepatitis c and other viral infections specific localization of membrane dipeptidase and dipeptidyl peptidase iv in secretion granules of two different pancreatic islet cells natural killer cell cytotoxicity: how do they pull the trigger? dipeptidyl peptidase is a novel adipokine 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monocyte recruitment and chemotaxis acute dpp- inhibition modulates vascular tone through glp- independent pathways molecular basis of binding between novel human coronavirus mers-cov and its receptor cd linagliptinmediated dpp- inhibition ameliorates kidney fibrosis in streptozotocin-induced diabetic mice by inhibiting endothelial-to-mesenchymal transition in a therapeutic regimen diabetic nephropathy amelioration by a low-dose sitagliptin in an animal model of type diabetes (zucker diabetic fatty rat) stimulation of intestinal growth and function with dpp inhibition in a mouse short bowel syndrome model glucagon-like peptide- reduces hepatic lipogenesis via activation of amp-activated protein kinase serum dipeptidyl peptidase- activity in insulin resistant patients with non-alcoholic fatty liver disease: a novel liver disease biomarker cd /dipeptidyl peptidase iv differentially regulates the chemotaxis of t cells and monocytes toward rantes: possible mechanism for the switch from innate to acquired immune response cd /dipeptidyl-peptidase iv down-regulates the eosinophil chemotactic potency, but not the anti-hiv activity of human eotaxin by affecting its interaction with cc chemokine receptor inhibition of cd /dipeptidyl peptidase iv enhances ccl /eotaxin-mediated recruitment of eosinophils in vivo human macrophage-derived chemokine (mdc), a novel chemoattractant for monocytes, monocyte-derived dendritic cells, and natural killer cells stcp- (mdc) cc chemokine acts specifically on chronically activated th lymphocytes and is produced by monocytes on stimulation with th cytokines il- and il- truncation of macrophage-derived chemokine by cd /dipeptidyl-peptidase iv beyond its predicted cleavage site affects chemotactic activity and cc chemokine receptor interaction amino-terminal truncation of cxcr agonists impairs receptor signaling and lymphocyte chemotaxis, while preserving antiangiogenic properties stromal cell-derived factor- retention and cardioprotection for ischemic myocardium dipeptidylpeptidase negatively regulates colony-stimulating factor activity and stress hematopoiesis stromal derived factor α: a chemokine that delivers a two-pronged defence of the myocardium the chemokine sdf- is a chemoattractant for human cd + hematopoietic progenitor cells and provides a new mechanism to explain the mobilization of cd + progenitors to peripheral blood implantation of adipose-derived regenerative cells enhances ischemia-induced angiogenesis role of the sdf- -cxcr axis in stem cellbased therapies for ischemic cardiomyopathy sdf- α as a therapeutic stem cell homing factor in myocardial infarction trafficking of normal stem cells and metastasis of cancer stem cells involve similar mechanisms: pivotal role of the sdf- -cxcr axis molecular cloning and characterization of a murine pre-b-cell growth-stimulating factor/stromal cell-derived factor receptor, a murine homolog of the human immunodeficiency virus entry coreceptor fusin the pleiotropic effects of the sdf- -cxcr axis in organogenesis, regeneration and tumorigenesis effect of stromal-cell-derived factor on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy structural localization and expression of cxcl and cxcr in rat heart and isolated cardiac myocytes matrix metalloproteinase activity inactivates the cxc chemokine stromal cell-derived factor- defects of b-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the cxc chemokine pbsf/sdf- the chemokine receptor cxcr is essential for vascularization of the gastrointestinal tract function of the chemokine receptor cxcr in haematopoiesis and in cerebellar development mutations in the chemokine receptor gene cxcr are associated with whim syndrome, a combined immunodeficiency disease cxcr : a new sdf- -binding receptor in contrast to normal cd + progenitors is functional and is expressed at higher level in human malignant hematopoietic cells the role of sdf- -cxcr / cxcr axis in the therapeutic effects of hypoxia-preconditioned mesenchymal stem cells for renal ischemia/reperfusion injury rapid mobilization of functional donor hematopoietic cells without g-csf using amd , an antagonist of the cxcr /sdf- interaction human progenitor cells rapidly mobilized by amd repopulate nod/scid mice with increased frequency in comparison to cells from the same donor mobilized by granulocyte colony stimulating factor rapid mobilization of hematopoietic progenitors by amd and catecholamines is mediated by cxcr -dependent sdf- release from bone marrow stromal cells sdf- -enhanced cardiogenesis requires cxcr induction in pluripotent stem cells cardiogenic induction of pluripotent stem cells streamlined through a conserved sdf- /vegf/ bmp integrated network sdf- in myocardial repair the chemokine sdf- activates the integrins lfa- , vla- , and vla- on immature human cd (+) cells: role in transendothelial/stromal migration and engraftment of nod/scid mice mechanical and electrical effects of cell-based gene therapy for ischemic cardiomyopathy are independent plasmid-based transient human stromal cell-derived factor- gene transfer improves cardiac function in chronic heart failure heterogeneity in sdf- expression defines the vasculogenic potential of adult cardiac progenitor cells modulation of hematopoietic stem cell homing and engraftment by cd cell surface peptidase cd / dipeptidylpeptidase iv regulates cxcl /stromal cell-derived factor- alpha-mediated chemotaxis of human cord blood cd + progenitor cells amd and cd modulate mobilization, engraftment, and survival of hematopoietic stem and progenitor cells mediated by the sdf- /cxcl -cxcr axis inhibition of cd in human cord blood cd + cells enhances their engraftment of nonobese diabetic/severe combined immunodeficiency mice cd inhibition on cd + or lineage-human umbilical cord blood donor hematopoietic stem cells/hematopoietic progenitor cells improves long-term engraftment into nod/scid/beta null immunodeficient mice diprotin a infusion into nonobese diabetic/severe combined immunodeficiency mice markedly enhances engraftment of human mobilized cd + peripheral blood cells cd /dpp- inhibition recruits regenerative stem cells via stromal cell-derived factor- and beneficially influences ischaemiareperfusion injury in mouse lung transplantation retention of human bone marrow-derived cells in murine lungs following bleomycin-induced lung injury upregulation of cd peptidase downregulates the functional chemotactic response of cd +cd -human cord blood hematopoietic cells isolation of putative progenitor endothelial cells for angiogenesis sphingosine- -phosphate stimulates the functional capacity of progenitor cells by activation of the cxcr -dependent signaling pathway via the s p receptor psgl- -mediated activation of ephb increases the proangiogenic potential of endothelial progenitor cells stromal cell-derived factor- effects on ex vivo expanded endothelial progenitor cell recruitment for ischemic neovascularization a novel mechanism for endothelial progenitor cells homing: the sdf- /cxcr -rac pathway may regulate endothelial progenitor cells homing through cellular polarization statin and stromal cell-derived factor- additively promote angiogenesis by enhancement of progenitor cells incorporation into new vessels sdf- involvement in endothelial phenotype and ischemia-induced recruitment of bone marrow progenitor cells comp-ang stimulates hif- α-mediated sdf- overexpression and recovers ischemic injury through bm-derived progenitor cell recruitment ex vivo priming of endothelial progenitor cells with sdf- before transplantation could increase their proangiogenic potential nitric oxide cytoskeletal-induced alterations reverse the endothelial progenitor cell migratory defect associated with diabetes mk- , a dipeptidyl peptidase- inhibitor, improves neovascularization by increasing both the number of circulating endothelial progenitor cells and endothelial nitric oxide synthetase expression sdf- expression by mesenchymal stem cells results in trophic support of cardiac myocytes after myocardial infarction myocardial cxcr expression is required for mesenchymal stem cell mediated repair following acute myocardial infarction synergy between cd /dpp-iv inhibition and g-csf improves cardiac function after acute myocardial infarction granulocyte colony-stimulating factor treatment plus dipeptidylpeptidase-iv inhibition augments myocardial regeneration in mice expressing cyclin d in adult cardiomyocytes genetically manipulated progenitor cell sheet with diprotin a improves myocardial function and repair of infarcted hearts proteaseresistant stromal cell-derived factor- for the treatment of experimental peripheral artery disease first treatment of a child suffering from severe ischemic cardiomyopathy with g-csf and sitagliptin parathyroid hormone is a dpp-iv inhibitor and increases sdf- -driven homing of cxcr (+) stem cells into the ischaemic heart cd inhibition enhances perfusion recovery in apoe-/-mice an open-label dose escalation study to evaluate the safety of administration of nonviral stromal cell-derived factor- plasmid to treat symptomatic ischemic heart failure changes in ventricular remodelling and clinical status during the year following a single administration of stromal cell-derived factor- non-viral gene therapy in chronic ischaemic heart failure patients: the stop-hf randomized phase ii trial cxcr modulates contractility in adult cardiac myocytes saxagliptin and cardiovascular outcomes in patients with type diabetes mellitus alogliptin after acute coronary syndrome in patients with type diabetes effect of sitagliptin on cardiovascular outcomes in type diabetes key: cord- -vrkdtrfz authors: roberts, ceri a.; durham, lucy e.; fleskens, veerle; evans, hayley g.; taams, leonie s. title: tnf blockade maintains an il- (+) phenotype in human effector cd (+) and cd (+) t cells date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: vrkdtrfz cd (+) and cd (+) effector t cell subpopulations can display regulatory potential characterized by expression of the prototypically anti-inflammatory cytokine il- . however, the underlying cellular mechanisms that regulate expression of il- in different t cell subpopulations are not yet fully elucidated. we recently showed that tnf inhibitors (tnfi) promote il- expression in human cd (+) t cells, including il- (+) cd (+) t cells. here, we further characterized the regulation of il- expression via blockade of tnf signaling or other cytokine/co-stimulatory pathways, in human t cell subpopulations. addition of the tnfi drug adalimumab to anti-cd -stimulated human cd (+) t cell/monocyte cocultures led to increased percentages of il- (+) cells in pro-inflammatory il- (+), ifnγ(+), tnfα(+), gm-csf(+), and il- (+) cd (+) t cell subpopulations. conversely, exogenous tnfα strongly decreased il- (+) cell frequencies. tnf blockade also regulated il- expression in cd (+) t cells upon antigenic stimulation. using time course experiments in whole peripheral blood mononuclear cell (pbmc) cultures, we show that tnf blockade maintained, rather than increased, il- (+) cell frequencies in both cd (+) and cd (+) t cells following in vitro stimulation in a dose- and time-dependent manner. blockade of il- , ifnγ, il- r, or cd /cd -mediated co-stimulation did not significantly regulate il- expression within cd (+) or cd (+) t cell subpopulations. we show that tnf blockade acts directly on effector cd (+) t cells, in the absence of monocytes or cd (+) cd (high)cd (low) regulatory t cells and independently of il- , resulting in higher il- (+) frequencies after days in culture. il- /il- r blockade reduced the frequency of il- -expressing cells both in the presence and absence of tnf blockade. addition of recombinant il- alone was insufficient to drive an increase in il- (+) cd (+) t cell frequencies in -day cd (+) t cell/monocyte cocultures, but resulted in increased il- expression at later time points in whole pbmc cultures. together, these data provide additional insights into the regulation of il- expression in human t cells by tnf blockade. the maintenance of an il- (+) phenotype across a broad range of effector t cell subsets may represent an underappreciated mechanism of action underlying this widely used therapeutic strategy. tnf blockade maintains an il- + phenotype in human effector cd + and cd + t cells ceri a. roberts cd + and cd + effector t cell subpopulations can display regulatory potential characterized by expression of the prototypically anti-inflammatory cytokine il- . however, the underlying cellular mechanisms that regulate expression of il- in different t cell subpopulations are not yet fully elucidated. we recently showed that tnf inhibitors (tnfi) promote il- expression in human cd + t cells, including il- + cd + t cells. here, we further characterized the regulation of il- expression via blockade of tnf signaling or other cytokine/co-stimulatory pathways, in human t cell subpopulations. addition of the tnfi drug adalimumab to anti-cd -stimulated human cd + t cell/monocyte cocultures led to increased percentages of il- + cells in pro-inflammatory il- + , ifnγ + , tnfα + , gm-csf + , and il- + cd + t cell subpopulations. conversely, exogenous tnfα strongly decreased il- + cell frequencies. tnf blockade also regulated il- expression in cd + t cells upon antigenic stimulation. using time course experiments in whole peripheral blood mononuclear cell (pbmc) cultures, we show that tnf blockade maintained, rather than increased, il- + cell frequencies in both cd + and cd + t cells following in vitro stimulation in a dose-and time-dependent manner. blockade of il- , ifnγ, il- r, or cd /cd -mediated co-stimulation did not significantly regulate il- expression within cd + or cd + t cell subpopulations. we show that tnf blockade acts directly on effector cd + t cells, in the absence of monocytes or cd + cd high cd low regulatory t cells and independently of il- , resulting in higher il- + frequencies after days in culture. il- /il- r blockade reduced the frequency of il- -expressing cells both in the presence and absence of tnf blockade. addition of recombinant il- alone was insufficient to drive an increase in il- + cd + t cell frequencies in -day cd + t cell/monocyte cocultures, but resulted in increased il- expression at later time points in whole pbmc cultures. together, these data provide additional insights into the regulation of il- expression in human t cells by tnf blockade. the maintenance of an il- + phenotype across a broad range of effector t cell subsets may represent an underappreciated mechanism of action underlying this widely used therapeutic strategy. keywords: tumor necrosis factor, anti-tnf, tnf inhibitors, adalimumab, interleukin- , cd + t cell polarization, cd + t cell polarization, il- regulation introduction the treatment of immune-mediated inflammatory diseases has improved considerably over the last years with the advent of biological therapeutics. tnfα was the first cytokine to be fully validated as a therapeutic target in ra ( ) . tnfα inhibitors (tnfi) have revolutionized treatment of ra and have been used in over a million patients worldwide ( ) . despite the good clinical response observed in many patients, tnfα blockade does not offer a curative treatment; approximately one-third of patients do not respond and loss of efficacy is frequently observed ( ) . importantly, it is currently not possible to predict which patients will respond to tnfi therapy. in addition, in some inflammatory diseases such as sjögren's syndrome ( , ) and multiple sclerosis ( ), tnfi have not shown clinical efficacy. furthermore, and paradoxically, some patients treated with tnfi develop de novo autoimmune diseases ( ) . these observations indicate that the underlying mechanisms relating to tnf blockade in humans are incompletely understood and require further exploration. the effects of tnfi are more wide-ranging than simply neutralizing the biological activity of soluble and membrane-bound tnfα (mtnfα). for example, by binding mtnfα, anti-tnf mabs can mediate cell death by complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity ( ) ( ) ( ) ( ) . tnfα inhibitors have also been shown to affect downstream cytokine pathways (il- , il- , and il- ) ( ), modulate apc function ( ) , and promote regulatory t cell (treg) expansion ( ) ( ) ( ) although opposite findings regarding the latter have been reported ( ) ( ) ( ) ( ) . recent data from our laboratory demonstrated that tnf blockade promotes il- expression in human cd + t cells ( ) . it was shown both cross-sectionally and longitudinally that inflammatory arthritis patients on tnfi therapy have an increased frequency of peripheral blood (pb) il- + cd + t cells. these in vivo findings were reproduced in vitro by coculturing cd + t cells from healthy donors with autologous cd + monocytes and anti-cd mab, in the presence of different tnfi drugs (adalimumab, infliximab, etanercept, or certolizumab) ( ) . furthermore, we showed an increase in the percentage of il- co-expressing il- + cd + t cells, suggesting that otherwise pro-inflammatory cells displayed anti-inflammatory potential. indeed, re-sorted tnfi-exposed il- + cd + t cells secreted increased levels of il- , which was biologically active and could modulate markers of monocyte activation ( ) . although il- + cd + t cells are recognized as an important cell population in inflammatory disease, other cd + t cell subsets also contribute to inflammation ( ) ( ) ( ) ( ) , as well as cd + t cells which can also be potent producers of proinflammatory cytokines ( ) ( ) ( ) ( ) ( ) . in this study, we therefore investigated in vitro whether tnf blockade regulates il- expression in other pro-inflammatory cytokine-producing t cell subsets, whether blockade of other cytokines or t cell activation pathways also drives il- expression, and how tnf blockade may manifest its il- -regulating effect on t cells. peripheral blood samples were obtained from healthy adult volunteers. peripheral blood mononuclear cells (pbmcs) were isolated by density gradient centrifugation using lymphoprep™ (axis-shield, oslo, norway). cd + monocytes and cd + t cells were isolated by magnetic-activated cell sorting (macs) according to the manufacturer's instructions (miltenyi biotec, bergisch-gladbach, germany), and purity was confirmed by flow cytometry. monocytes (average purity %) were isolated by positive selection using anti-cd microbeads. cd + t cells were isolated via negative depletion (average purity %), and in some experiments, cd ro + cd + t cells were subsequently enriched by positive selection using cd ro microbeads (average purity %). in some experiments, cd + t cells were sorted to very high purity (> %) and part of the cells depleted of cd + cd high cd low tregs by facs-sorting after labeling cells with cd percp cy . (sk ), cd pe (m-a ), cd alexa fluor (a d ) mabs (all from biolegend, cambridge, uk). the study was approved by the bromley research ethics committee ( /q / ), and written informed consent was obtained from all participants. cells were cultured at °c with % co in culture medium [rpmi medium supplemented with % heat-inactivated fetal bovine serum (lot# f k, south american origin)] and % penicillin, streptomycin, and l-glutamine (all life technologies, carlsbad, ca, usa). freshly isolated bulk cd + or memory (cd ro + )-enriched t cells ( . × ) and cd + monocytes ( . × unless otherwise indicated) were cocultured with ng/ml anti-cd mab (clone okt , janssen-cilag ltd., high wycombe, uk). cocultures were incubated at °c with % co for days. in some experiments, macs-isolated cd + t cells were cultured alone with anti-cd /cd stimulation. anti-cd (okt ) was coated onto culture plates at . µg/ml in pbs for a minimum of h at °c. wells were then washed three times with pbs before adding cd + t cells in culture medium. anti-cd (clone cd . ; bd biosciences) was added to the well at a final concentration of µg/ml. in some experiments, whole pbmcs were cultured with ng/ml anti-cd mab for up to days. where indicated, the following recombinant cytokines, neutralizing antibodies, or other reagents (from r&d systems unless otherwise indicated) were added at the start of the culture period: recombinant human (rh) tnfα ( ng/ml; biosource, camarillo, ca, usa), rhil- ( - ng/ml), rhil- ( ng/ml; rhil- used to generate data in figure s in supplementary material from peprotech, rocky hill, nj, usa), neutralizing/blocking antibodies to ifnγ (clone , µg/ml), il- (clone , µg/ml), il- r (polyclonal, µg/ml), il- (clone , µg/ml), il- r (clone , µg/ml), and il- (polyclonal, µg/ml). the following isotype control antibodies were used at an assay-appropriate concentration: human igg (abcam, cambridge, uk), mouse igg , mouse igg a, mouse igg b, goat igg (all r&d systems). clinical grade biologics were purchased anti-tnf maintains il- + cd + /cd + t cells . antibodies to detect cd and/ or cd were added intracellularly, to allow for staining of these markers following our t cell culture conditions. stained cells were acquired using a facscantoii or lsrfortessa (bd biosciences); in most experiments, , t cell events were recorded. the gating strategies used to analyze intracellular cytokine expression in cd + t cells are described in figure s in supplementary material. all flow cytometry data were analyzed using flowjo software (version . . or , tree star, inc., ashland, or, usa). il- was measured in cell culture supernatants using the il- elisa max kit (biolegend), according to manufacturer's instructions. il- was measured in cell culture supernatants using the duoset human il- elisa (ebioscience), according to manufacturer's instructions. microwell absorbance was read at nm using a wallac microplate reader (perkin elmer, waltham, ma, usa). concentrations of the analytes were determined based on the standard curve included on each plate. statistical testing was performed with graphpad prism . or . (graphpad, san diego, ca, usa). data sets were tested for normality using the d' agostino and pearson omnibus normality test, followed by statistical significance testing using the appropriate tests as indicated in figure legends. data sets with n values < were tested non-parametrically. p values < . were considered statistically significant. tnf blockade regulates il- expression in human cd + and cd + t cells to investigate whether tnf blockade regulates il- expression in different pro-inflammatory cytokine-producing cd + t cells, we isolated cd + t cells from pb of healthy donors and cocultured the cells with cd + monocytes and anti-cd mab ( ng/ml) in the absence or presence of the anti-tnf mab adalimumab ( µg/ml), as previously described ( ) . after days, cells were restimulated with pma and ionomycin in the presence of golgistop for h and stained for cytokine expression (gating strategy shown in figure s in supplementary material). in agreement with our previous data ( ) , tnf blockade led to a significant increase in il- + cells within total cd + t cells as well as in the il- + cd + t cell subset. in addition, we found a strong increase in il- -expressing cells within ifnγ + , tnfα + , gm-csf + , and il- + cd + t cell populations (figures a,b) . tnf blockade did not alter the frequencies of ifnγ + , tnfα + , or gm-csf + cd + t cell populations but did induce a modest increase in il- + cd + t cells, as previously shown ( ) , and in most donors in il- + frequencies, although this did not reach statistical significance ( figures s a, b in supplementary material). addition of an isotype control human igg mab did not promote il- expression in cd + t cells ( figure s in supplementary material). contrary to the effects of tnf blockade, addition of rhtnfα to cocultures of cd + cd ro + t cells, monocytes and anti-cd mab led to a striking decrease in the percentage of il- + cells within total cd + t cells and within il- + , ifnγ + , and tnfα + cd + t cell subsets (gm-csf + and il- + cd + t cells were not tested) ( figure c ). tnfα addition did not significantly alter the frequencies of il- + , ifnγ + , or tnfα + cd + t cell subsets ( figure s c in supplementary material). since pro-inflammatory cytokine-expressing cd + t cells also contribute to immune-mediated inflammatory diseases ( ) ( ) ( ) , we investigated whether tnf blockade can regulate il- expression in cd + t cells. we adapted the culture system to stimulate whole pbmc with anti-cd mab ( ng/ml) in the absence or presence of a dose range of adalimumab ( . - µg/ml). after days, the cultures were restimulated with pma/ionomycin in the presence of golgistop and cytokine expression was assessed within either the cd + or the cd + t cell populations. significant increases in the percentages of il- + cells within both cd + and cd + t cell populations were observed, including in the il- + and ifnγ + subpopulations (figure ) . the regulation of il- expression by tnf blockade was dose responsive in both cd + and cd + t cell subsets, with significantly increased il- + frequencies following culture with either or µg/ml adalimumab ( figure c ). these doses reflect figure | tnf blockade promotes, while tnfα impairs il- expression in pro-inflammatory cd + t cell subsets. cd + t cells (open symbols) or cd + cd ro + t cells (filled symbols) were cocultured with autologous cd + monocytes and anti-cd mab ( ng/ml) in the absence or presence of the anti-tnf mab adalimumab ( µg/ml) (a,b) or rhtnfα ( ng/ml) (c). after days, cells were restimulated and assessed for intracellular cytokine expression as described in the methods. cd + t cells were gated as described in figure s in supplementary material. (a,b) representative dot plots (a) and cumulative data (b) showing the percentages of il- + cells within total cd + t cells (n = ), or within il- + cd + (n = ), ifnγ + cd + (n = ), tnfα + cd + (n = ), gm-csf + cd + (n = ), or il- + cd + (n = ) cells after culture in the absence or presence of anti-tnf. data were analyzed using wilcoxon matched-pairs signed rank test. (c) cumulative data showing the percentages of il- + cells within total cd + cd ro + t cells or within il- + (n = ), ifnγ + (n = ), or tnfα + (n = ) cd + cd ro + t cells after culture in the absence or presence of rhtnfα. data were analyzed using paired t test. each connecting line represents an individual donor (*p < . , **p < . , ***p < . , ****p < . ). figure | tnf blockade regulates il- expression in cd + and cd + t cells in a dose-dependent manner. (a,b) freshly isolated peripheral blood mononuclear cells were cultured with anti-cd mab ( ng/ml) in the absence (control) or presence (anti-tnf) of adalimumab ( µg/ml). after days, cells were restimulated and assessed for intracellular cytokine expression as described in the methods. cells were gated on live single cd + cells and il- expression was assessed within cd + (a) or cd + (b) t cell subsets. representative dot plots show the percentages of il- + cells within total cd + or cd + t cells, or within il- + or ifnγ + subsets after culture in the absence or presence of anti-tnf. (c) cells were cultured as described above in the presence of increasing doses of adalimumab ( - μg/ml). box-whisker plots represent data from n = individual donors; whiskers show minimum to maximum values. data were analyzed by friedman test with comparison to control by dunn's multiple comparisons test (*p < . , **p < . , ***p < . , ****p < . ). the clinically effective trough levels for adalimumab in human serum following treatment ( , ) . in agreement with data from the cd + t cell/monocyte cocultures, addition of rhtnfα ( ng/ml) to anti-cd -stimulated pbmc led to a reduction in il- + cell frequencies, in both cd + and cd + t cells (n = , data not shown). since these in vitro studies of t cell responses to tnf blockade all relied on monoclonal anti-cd stimulation, we tested whether tnf blockade could still elicit increased il- + t cell responses in the context of a more physiological antigenic stimulation. pediacel is a pentavalent vaccine consisting of purified diphtheria toxoid, purified tetanus toxoid, acellular pertussis vaccine, inactivated poliovirus, and haemophilus influenzae type b polysaccharide. revaxis is a booster vaccine containing purified diphtheria and tetanus toxoids and inactivated poliovirus. each vaccine was added to cd + t cell/monocyte cocultures ( : , dilution) in the presence or absence of tnf blockade. after days, antigen-stimulated cd + t cells cultured in the presence of adalimumab demonstrated elevated il- + frequencies as compared to cells that were not exposed to tnf blockade ( figure s in supplementary material). together these data demonstrate that in vitro tnf blockade provided at a physiological concentration and in a physiological setup can promote il- expression in cd + and cd + t cells, including in subsets expressing pro-inflammatory cytokines. tnf blockade maintains il- expression in cd + and cd + t cells thus far, assessment of cytokine expression following in vitro tnf blockade was carried out after days of culture. time course experiments were performed to assess the kinetics of il- expression in cd + and cd + t cells. pbmc were cultured with anti-cd mab in the absence or presence of adalimumab, and the frequencies of il- expressing cells were analyzed - h after stimulation. the data show that early after tcr stimulation ( - h) the frequencies of il- expressing cells within cd + and cd + t cells increased rapidly irrespective of the presence of tnf blockade. however, at later time points (from to h), higher frequencies of il- + cells were sustained within cd + and cd + t cells in the presence of tnf blockade (figure ) . these experiments suggest that tnf blockade maintains, rather than directly induces, an il- + phenotype in cd + and cd + t cells following tcr stimulation. blockade of tnfα, but not il- , ifnγ, il- r, or cd /cd -mediated costimulation, regulates il- in human cd + t cells our previous work demonstrated that in addition to adalimumab, other tnfα inhibitors (etanercept, infliximab, or certolizumab) as well as tnfr / blocking mabs were capable of increasing frequencies of il- -expressing il- + cd + t cells ( ) . we investigated whether blockade of additional pro-inflammatory pathways could promote il- expression in cd + t cells. blockade of il- a did not enhance the frequencies of il- + cells in any of the cd + t cell populations tested (figure a) . blockade of ifnγ did not affect the percentage of il- + cells within total cd + t cells, or within ifnγ or tnfα + subpopulations, but led to modestly increased frequencies of il- + expressing cells within the il- + population, although this effect was much weaker than the effect of tnf blockade in parallel cultures ( figure a) . addition of tocilizumab (il- r blockade) or abatacept (ctla -ig, which blocks cd /cd -mediated co-stimulation), both of which are biologic drugs routinely used in the clinic to treat rheumatoid arthritis, did not increase il- + frequencies in cd + t cells, il- + , ifnγ + , or tnfα + cd + t cell subpopulations (figure b) . to determine whether blockade of these pathways might regulate il- expression with different kinetics to tnf blockade, il- + frequencies were analyzed within both cd + and cd + t cells at different time points in anti-cd -stimulated pbmc cultures exposed to these antibodies or drugs. il- + cd + and il- + cd + t cell frequencies were not regulated at any time point by blockade of il- , ifnγ, il- r, or cd /cd -mediated co-stimulation ( figure s in supplementary material). blockade of il- r in cd + t cell/monocyte cocultures resulted in a significantly increased proportion of il- + cells within total cd + t cells and within il- + , ifnγ + , or tnfα + subpopulations ( figure b) . however, this effect was not replicated in either cd + or cd + t cells in whole pbmc cultures ( figure s in supplementary material), indicating that the capacity of il- blockade to regulate il- expression may be dependent on the in vitro culture conditions. together these data indicate that il- expression in cd + t cells and cd + t cells can be regulated by blocking tnfα signaling, but not by blocking ifnγ, il- , il- r, or cd /cd -mediated co-stimulation, at least in vitro. since cd + monocytes are major producers of tnfα, we explored whether the presence of monocytes was required for the effects of tnf blockade on regulating il- . cd + t cells were sorted to a very high purity (> %) and stimulated with platebound anti-cd and soluble anti-cd mab in the absence of monocytes or placed in coculture with monocytes and anti-cd mab, with or without addition of anti-tnf mab. in the cd + t cell only cultures, tnf blockade still brought about increased il- + cell frequencies within the total cd + population and also within il- + , ifnγ + , or tnfα + subpopulations, similar to the increased il- + frequencies observed in cd t cell/monocyte cocultures (figures a,b) . analysis of supernatants from anti-cd /cd -stimulated cd + t cells by elisa confirmed that levels of secreted il- were increased during culture in the presence of tnf blockade (figure c) . one potential interpretation of the observed increased frequencies of il- + cells following tnf blockade could be that there is an expansion of a pre-existing population of tregs. our previous work indicated that in three donors, macs-depletion of cd + cd + cells did not impair the anti-tnf-mediated increase in il- + frequencies within il- + cd + t cells and that no increase in foxp + cell frequencies was apparent upon tnf blockade ( ) . to investigate this further, macs-isolated cd + t cells were facs sorted to very high purity (> %) to yield either total cd + t cells or effector cd + cd -cd + t cells (teff; depleted of cd high cd low treg). these cells were then cultured with anti-cd /cd mabs in the absence of monocytes, or placed in coculture with monocytes and anti-cd mab, in the absence or presence of adalimumab. our data show that tnf blockade still resulted in increased frequencies of il- -expressing cells in effector t cells in the absence of cd high cd low tregs (figure ). together these data indicate that tnf blockade regulates il- expression directly in cd + effector t cells and does not require the presence of cd + monocytes or cd + cd high cd low tregs. (a) cd + t cells (n = ) or cd ro + enriched cd + t cells (n = ) were cocultured : with autologous monocytes in the presence of anti-cd ( ng/ml), without (control) or with anti-tnf mab (adalimumab, μg/ml) or neutralizing antibodies to ifnγ or il- ( μg/ml) or the appropriate isotype control antibodies (migg a and migg b, respectively). (b) cd ro + enriched cd + t cells (n = ) were cocultured : with autologous monocytes in the presence of anti-cd ( ng/ml), without (control) or with anti-tnf mab (adalimumab, μg/ml), tocilizumab ( µg/ml), abatacept ( µg/ml) or anti-il- r -blocking ab ( μg/ml). (a,b) after days, cells were restimulated and assessed for intracellular cytokine expression as described in the methods. data show frequencies of il- + cells within the total cd + t cell population or within il- + , ifnγ + , or tnfα + cd + t cell subpopulations. data were analyzed by wilcoxon matched-pairs signed rank test with comparison to the appropriate control condition (ns p > . , *p < . , **p < . , ***p < . , ****p < . ). in support of our findings that tnf blockade can exert its effects on cd + t cells in the absence of monocytes, we did not find evidence for a role of the monocyte-derived anti-inflammatory mediator il- ( ) in mediating il- expression in the context of tnf blockade. this was demonstrated by the findings that tnf blockade did not induce il- secretion, rhil- enhanced il- secretion but did not result in increased percentages of il- + cd + t cells, and il- blockade did not impair the increased il- + cd + t cell frequencies brought about by tnf blockade ( figure s in supplementary material). il- partly contributes to tnf blockade-mediated il- regulation finally, we explored whether modulation of il- expression in cd + t cells by tnf blockade was dependent on il- signaling itself. neutralizing antibodies against il- and il- r were added to cd + t cell/monocyte cocultures stimulated with anti-cd mab, in the presence or absence of tnf blockade, and intracellular cytokine expression was assessed after days. blockade of il- signaling in the absence of adalimumab led to significantly reduced il- + frequencies within total cd + t cells and within il- + , ifnγ, or tnfα + subpopulations ( figure a) . when adalimumab and il- /il- r blocking mabs were added in combination to the cocultures, a decrease in il- + cd + t cell frequencies was observed as compared to the condition treated with adalimumab alone; however, an increase was still observed as compared to il- /il- r blockade alone. these data suggest that pathways additional to il- signaling could play a role in the regulation of il- expression by tnf blockade. indeed, when we tested whether addition of ) showing frequencies of il- + cells within the total cd + t cell population or within il- + , ifnγ + or tnfα + cd + t cell subpopulations. (c) after days in culture but prior to re-stimulation, supernatants were harvested from cd + t cell only cultures (n = ) and analyzed by enzyme-linked immunosorbent assay for il- secretion. data were analyzed by wilcoxon matched-pairs signed rank test (*p < . , ****p < . ). roberts et al. anti-tnf maintains il- + cd + /cd + t cells frontiers in immunology | www.frontiersin.org february | volume | article exogenous il- ( ng/ml) could drive il- expression in cd + t cell/monocyte cocultures, we found that this by itself was not sufficient to increase il- + cell frequencies in total cd + t cells, or within il- + , ifnγ + , or tnfα + subpopulations. addition of rhil- even decreased the percentage of il- + cells in total cd + t cells and tnfα + cd + t cells (figure b) . the biological activity of the recombinant il- was validated by culturing freshly isolated monocytes with , , or ng/ml rhil- for h and confirming that rhil- addition resulted in increased cd and cd and reduced hla-dr expression by flow cytometry, in accordance with previous studies ( , ) (n = , data not shown). we next investigated the regulation of il- in response to rhil- in both cd + and cd + t cells using whole pbmc cultures assessed for il- expression over a -day time course (figure s in supplementary material). we found that at earlier time points ( - h) , rhil- addition reduced il- + cd + t cell frequencies (n = , p = . , wilcoxon matched-pairs signed rank test), while at later time points ( - and - h), rhil- slightly increased il- + cd + t cell frequencies (n = , p = . , wilcoxon matched-pairs signed rank test). il- + cd + t cell frequencies remained very low throughout, regardless of rhil- addition. together, these data indicate that il- signaling contributes in part to tnf blockade-mediated regulation of il- expression in cd + t cells, but that other factors also play a role. the effects of exogenous il- on regulating il- expression in cd + t cells appear to be dependent on both time and in vitro culture conditions. here, we show that t cell stimulation in the presence of tnf blockade maintains the proportion of cells expressing the antiinflammatory cytokine il- . this phenomenon is observed in total cd + and cd + t cells as well as within a variety of pro-inflammatory cytokine-expressing (il- + , ifnγ + , tnfα + , gm-csf + or il- + ) subpopulations. tnf blockade regulates il- expression whether cd + t cells are stimulated in the presence or absence of monocytes, and when an antigenic stimulus is used in place of monoclonal anti-cd stimulation. we found that blockade of il- , ifnγ, il- r, or cd /cd -mediated costimulation did not regulate il- expression in cd + or cd + t cell subpopulations. blocking antibodies to il- r resulted in increased il- + cd + t cell frequencies when added to cd + t cell/monocyte cocultures, but this effect was not replicated in whole pbmc cultures. one explanation could be that additional cell types are present in pbmc cultures that can produce or respond to il- . neither cd + cd high cd low tregs nor il- was required for tnf blockade to exert its il- -promoting effects. our data confirm and extend our previous work showing that tnf blockade promotes il- expression in cd + t cells ( ) and support other findings in the literature. a small study of ra patients found that frequencies of il- + pbmc were increased following treatment with infliximab ( ) . however, in the three patients studied, it was not confirmed which cell subset(s) expressed il- . ebert later showed that supernatants from cultures of either monocytes (adherent pbmc) or t cells (non-adherent, cd − cd − hla-dr − pbmc), contained increased il- following exposure to infliximab in vitro ( ) . antiga et al. demonstrated increased percentages of il- + cd + t cells in t cell blasts isolated from skin lesions of psoriasis patients, following treatment with etanercept ( ), and pb il- + cd + t cells were also increased in posterior uveitis patients following treatment with a p tnfα receptor fusion protein ( ) . earlier studies assessing mouse transgenic t cells ( ) or human cd + t cell clones ( ) also found that tnf blockade led to increased il- production in cell culture supernatants. in further support of our data, boks et al. showed that blockade of tnfα during in vitro priming of naïve cd + figure | il- expression in cd + t cells is inhibited by il- blockade, in both the absence and presence of tnf blockade. (a) cd ro + enriched cd + t cells were cocultured : with autologous monocytes in the presence of anti-cd (okt , ng/ml), with or without anti-tnf mab (adalimumab, μg/ml), with or without neutralizing antibodies to il- and il- r (both μg/ml). after days, cells were restimulated and assessed for intracellular cytokine expression as described in the methods. box-whisker plots show frequency of il- + cells within the total cd + t cell population or within il- + , ifnγ + or tnfα + subpopulations; whiskers show minimum to maximum values. data were analyzed by repeated measures anova, with comparison between selected conditions by sidak's multiple comparison test (n = ). (b) cd ro + enriched (filled symbols, n = ) or bulk (open symbols, n = ) cd + t cells were cocultured : with autologous monocytes and stimulated with anti-cd mab ( ng/ml), with or without recombinant human il- (rhil- ; ng/ml). after days, cells were restimulated and assessed for intracellular cytokine expression as described in the methods. data show frequencies of il- + cells within the total cd + t cell population or within il- + , ifnγ + , or tnfα + subpopulations and were assessed by wilcoxon matched-pairs signed rank test (ns p > . , *p < . , **p < . , ***p < . , ****p < . ). t cells with tolerogenic dcs favored the development of il- + cd + t cells ( ) . thus, tnfi-mediated regulation of il- in cd + t cells has been indicated by a growing number of in vitro and ex vivo studies. the presence of il- -expressing cd + t cells has been reported in several mouse models of infection, including acute influenza infection ( ) , chronic mycobacterium tuberculosis infection ( ) , and coronavirus-induced encephalitis ( ) . furthermore, il- + cd + t cells have been found in patients with hiv ( ) and chronic hepatitis c infection ( ) . il- expression in cd + t cells can be induced by il- , either alone ( ) or in combination with il- ( ) , or dexamethasone ( ) . however, to the best of our knowledge, this is the first report showing that tnf blockade can regulate il- expression in cd + t cells. il- can stimulate il- production by various effector cd + t cell subsets ( , , ) . our data suggest however that the capacity of tnf blockade to affect il- expression in cd + t cells is not dependent on il- . in agreement with other work on human t cells ( , ) , we found that addition of recombinant il- to anti-cd /cd -stimulated cd + t cells resulted in increased il- secretion, although this was not strictly dosedependent. however, in contrast to these other studies, which used naïve cd + t cells, our analysis of intracellular cytokine expression did not demonstrate increased il- + cd + t cell frequencies after days in culture with il- . this is possibly anti-tnf maintains il- + cd + /cd + t cells frontiers in immunology | www.frontiersin.org february | volume | article due to different kinetics of il- expression in a bulk cd + t cell population. it is of note that although in cd + t cell/monocyte cocultures il- blockade led to reduced il- + frequencies in both the presence and absence of tnf blockade, il- addition by itself was not sufficient to drive il- expression in cd + t cells. these data are in line with previous work showing that il- is necessary but not sufficient to enhance development of il- -producing tr cells following in vitro differentiation in the presence of vitamin d plus dexamethasone ( ) . however, when added to anti-cd -stimulated whole pbmc cultures, rhil- did slightly increase il- + cd + t cell frequencies after and days in culture, indicating that the regulation of il- expression in cd + t cells is dependent on in vitro culture conditions. in inflammatory diseases, such as ra, the normal balance of immune regulation is disturbed and t cell-derived proinflammatory mediators can contribute to disease pathogenesis in an uncontrolled manner. this study indicates that a potentially immunoregulatory il- + phenotype is broadly maintained in effector t cells following exposure to tnf blockade, which may represent an underappreciated mechanism of action for this widely used therapeutic strategy. we previously provided evidence that the il- secreted by il- + cd + t cells following tnf blockade is biologically active ( ) ; however, further work is required to investigate whether the increase in il- + frequencies across different t cell subpopulations is functionally relevant in the context of inflammatory disease. additional insights into the underlying molecular mechanisms via which tnf blockade maintains il- expression could identify potential targets for novel therapeutic strategies. author contributions cr, ld, and vf designed and performed experiments and analyzed and interpreted data. lt and he conceived the study, contributed to experimental design, and interpreted data. cr and lt wrote and revised the manuscript. all authors edited the manuscript. development of anti-tnf therapy for rheumatoid arthritis anti-tnf biologic agents: still the therapy of choice for rheumatoid arthritis direct comparison of treatment responses, remission rates, and drug adherence in patients with rheumatoid arthritis treated with adalimumab, etanercept, or infliximab: results from eight years of surveillance of clinical practice in the nationwide danish danbio registry inefficacy of infliximab in primary sjögren's syndrome: results of the randomized, controlled trial of remicade in primary sjögren's syndrome (tripss) lack of efficacy of etanercept in sjögren syndrome correlates with failed suppression of tumour necrosis factor α and systemic immune activation the university of british columbia ms/mri analysis 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diminishing the antigen-presenting capacity of monocytes via downregulation of class ii major histocompatibility complex expression human monocytes express cd , which is upregulated by il- and identical to p infliximab and the tnf-α system etanercept downregulates the th pathway and decreases the il- +/il- + cell ratio in patients with psoriasis vulgaris anti-tnfα therapy modulates the phenotype of peripheral blood cd + t cells in patients with posterior segment intraocular inflammation the role of tnf alpha and related cytokines in the development and function of the autoreactive t-cell repertoire chronic exposure to tumor necrosis factor (tnf) in vitro impairs the activation of t cells through the t cell receptor/cd complex; reversal in vivo by anti-tnf antibodies in patients with rheumatoid arthritis inhibition of tnf receptor signaling by anti-tnfα biologicals primes naïve cd + t cells towards il- + t cells with a regulatory phenotype and function effector t cells control lung inflammation during acute influenza virus infection by producing il- clonal expansions of cd + t cells with il- secreting capacity occur during chronic mycobacterium tuberculosis infection highly activated cytotoxic cd t cells express protective il- at the peak of coronavirus-induced encephalitis hiv-specific il- -positive cd + t cells are increased in advanced disease and are associated with decreased hiv-specific cytolysis intrahepatic virus-specific il- -producing cd t cells prevent liver damage during chronic hepatitis c virus infection il- induces a suppressive il- -producing cd + t cell population via a cdkn a-dependent mechanism cytokine-induced il- -secreting cd t cells represent a phenotypically distinct suppressor t-cell lineage glucocorticoids drive human cd (+) t cell differentiation towards a phenotype with high il- and reduced il- , il- and il- production suppression of autoimmune inflammation of the central nervous system by interleukin secreted by interleukin -stimulated t cells a dominant function for interleukin in generating interleukin -producing anti-inflammatory t cells il- is a key regulator of il- and il- production by human cd + t cells il- induces the differentiation of tr -like cells from human naive cd + t cells via the phosphorylation of stat and stat vitro generation of interleukin -producing regulatory cd + t cells is key: cord- - zwkfzab authors: scala, stefania; pacelli, roberto title: fighting the host reaction to sars-cov- in critically ill patients: the possible contribution of off-label drugs date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: zwkfzab the severe acute respiratory syndrome coronavirus (sars-cov- ) is the etiologic agent of the coronavirus disease (covid ). the majority of infected people presents flu like symptoms and among them – % develops a severe interstitial pneumonitis (ip) that may eventually evolve in acute respiratory distress syndrome (ards). ip is caused by the viral glycoprotein spike (s) binding to the angiotensin converting enzyme (ace ) expressed on the surface of alveolar pneumocytes. the virus is recognized by the “pattern recognition receptors” (prr) of the immune cells that release cytokines activating more immune cells that produce a large number of pro-inflammatory cytokines, tissue factors and vasoactive peptides. affected patients might develop the “cytokine storm syndrome,” a fulminant and fatal hypercytokinaemia with multiorgan failure. in patients infected by sars-cov- increase in t-helper (th ) cytokines (il- and il ) are reported in addition to the t-helper (th ) cytokines (il b, ifnγ, ip , and mcp ) previously detected in other coronavirus infections. cytokines and other molecules involved in immune response and inflammation are conceivable therapeutic targets for ip and ards, improving symptoms and decreasing intensive care unit admissions. to this aim off label drugs may be used taking into consideration the window timing for immunosuppressive drugs in virus infected patients. some off label therapeutic options and preclinical evidence drugs are herein considered. in a report on more than thousands patients of the chinese province of hubei, the majority of infected symptomatic people presented flu like symptoms (mainly fever and cough), with - % of patients developing a severe interstitial pneumonitis (ip) that could evolve in acute respiratory distress syndrome (ards). the case fatality rate in the whole population resulted . % ( and %, for patients older than and , respectively). in critical patients % of case fatality rate was registered ( ) . ip is caused by the attack of the virus against the alveolar pneumocytes (aps) through the binding of the viral glycoprotein (spike, s) to the angiotensin converting enzyme (ace ) expressed on the surface of the aps ( ) . the virus enters in the host target cells through receptor-mediated endocytosis and quickly replicates; virus release in the extracellular space occurs through either budding or cell death. in the extracellular space the virus is recognized by the prr of immune cells ( ) . this process contributes to the virus elimination through an amplification cascade in which the immune cells produce a large number of pro-inflammatory cytokines, tissue factors, and vasoactive peptides. these molecules reach the blood vessel wall causing a burst of nitric oxide, damages to the blood vessels and to the coagulation system ( ) . among the most involved cells, macrophages play an important role in acute lung injury, which identify pathogen-associated molecular patterns (pamp) and trigger innate immunity ( , ) . macrophages secrete a large number of inflammatory mediators and cytokines, such as tumor necrosis factor-alpha (tnf-α), interleukin- beta (il- β), interleukin- (il- ), inducible nitric oxide synthase (inos), and macrophage migration inhibitory factor (mif). tnf-α can directly damage cells of the pulmonary vascular endothelium, increasing capillary endothelial permeability, causing pulmonary edema, predicted by il- level ( ). progression to acute respiratory distress syndrome (ards) is based on the acute onset of lung inflammation, determined by monocyte/macrophage polarization and function. during active infection, inflammatory monocytes/macrophages (imms), and resident macrophages undergo marked phenotypic and functional changes, from m proinflammatory (classically activated) to m inflammatory-resolving macrophages, with a dynamic continuum through discrete categories. during acute infection, monocytes/macrophages often display a phenotype of classically activated macrophages that mediate antiviral host defenses but also promote lung injury by producing nitric oxide (no), reactive oxygen species (ros), il- , il- , and il- and tnf-α. simultaneously, some macrophages may become m macrophages alternatively activated, exerting anti-inflammatory function and regulating wound healing by producing matrix metalloproteinases (mmps), growth factors, and anti-inflammatory cytokines, particularly tgf-β. pro-inflammatory macrophages diminish at the removal of stimulus ( ) ( ) ( ) . evidence of a cytokine storm has been found in severe pneumonitis linked to coronavirus infection ( ) . previously, in patients with sars, il b, il , il , ifnγ, ip , and mcp were found to be increased ( ) . in patients with mers, ifnγ, tnfα, il , and il were shown to participate in the severity of the pneumonitis ( ) , and an elevated inflammatory innate immune response has been shown in the lower respiratory tract. although those cytokines were elevated, down-regulation of genes encoding inflammatory th and th molecules was noted ( ) . interestingly, in patients infected by sars-cov- , there is an increase in il β, ifnγ, ip , and mcp , probably leading to activated t-helper- (th ) cell responses, and increased production of t-helper- (th ) immunosuppressive cytokines, such as il and il ( ) . in particular, a significant increase in il , il , il , g-csf, ip , mcp /ccl , mip a, and tnfα was noted in patients requiring admission to the intensive care unit (icu) compared to patients with a milder disease. as the infiltrate of monocytes, neutrophils, lymphocytes, and macrophages are the cellular actors of the inflammatory response ( ) , chemokine ligands and receptors play an important role in driving immune cell migration and homing ( ) . these cytokines may explain the observation of reduced levels of circulating lymphocytes. peripheral blood examinations on admission in the majority of patients with covid- displayed lymphopenia, elevated infection-related biomarkers (i.e., procalcitonin, erythrocyte sedimentation rate, serum ferritin, and c-reactive protein) ( ) and several elevated inflammatory cytokines (i.e., tumor necrosis factor (tnf)-α, interleukin (il)- r and il- ). patients with more severe cases had higher leukocyte and neutrophil count, lower lymphocyte count and higher neutrophil-to-lymphocyte ratio (nlr) ( ) . lymphocyte subsets showed that the total number of b cells, t cells and nk cells was significantly decreased in patients with covid- , more significantly so in severe cases. in particular, t cells (t helper, t suppressor, and tregs cells) were mostly affected by sars-cov- . in addition, recent evidence in sars-cov infection suggests that seroconversion may also play a role in lung injury. a detrimental role of early appearance of antispike (s)-igg was demonstrated during sars-cov infection in a macaque model ( ) . despite markedly reducing virus titers, anti-s-igg caused lung injury during the early stages of infection, impairing the wound-healing macrophage response and tgf-β production, while promoting pro inflammatory cytokine il- , mcp production, and inflammatory macrophage accumulation ( ) . interestingly, in sars patients who died in hong kong during the outbreak, the anti-spike (s) glycoprotein neutralizing antibodies appeared significantly before and reached a higher titer than in patients surviving ( ) . consistently, preexisting serum antibodies, derived by exposition to influenza seasonal strains, may recognize but fail to neutralize, the new pandemic strain and were found to associate with worse clinical severity during the influenza pandemic ( , ) . the inflammatory status together with pulmonary edema and respiratory failure define the clinical picture of the ards associated with covid- ( ) . the most compelling emergency that the health system faces in this epidemic is the shortage of critical care units. the saturation of intensive care units (icu) precludes the rescue of patients who might be saved, increases covid- lethality rate and worsens the prognosis for other pathological conditions requiring icu admission. the severe ip or ards of the covid- requires ventilator support and can kill infected people averaging in weeks from the appearance of the first symptoms ( , ) . therapy in use for hiv and other viral disease have been empirically administered without much benefit ( ) , while promising experimental antiviral drugs such as remdesivir and chloroquine, an old antimalarial drug with in vitro activity on the viral infection, are currently in clinical trials ( , ) . in the absence of specific validated approaches, and waiting for a vaccine, a clinical empirical rational management is needed. another reasonable approach would be drugs targeting the host immune-inflammatory reaction. methylprednisolone, although somewhat controversial, seems to be overall useful in these patients ( ) , while dexamethasone has been shown to be useful in patients with ards of different etiologies ( , ) . cytokines and the other molecules involved in the immune response regulation and inflammation are conceivable targets to improve ip and ards lung injury. to this aim off label drugs may be used considering the timing for immunosuppressive drugs in virus infected patients. unfortunately, the time window is not univocally defined and data may derive from clinical studies. several therapeutic options that could be rapidly translated to clinical trials are available. some of them are listed below. tocilizumab is an anti-il receptor antibody (roactemra, roche) approved to treat moderate to severe rheumatoid arthritis (ra). tocilizumab has been used to counteract the side effects of immune checkpoint inhibitors and car-t therapy in cancer bearing patients ( ) and, recently, to antagonize the host reaction in patients affected by ards linked to covid ( ) . at today covid- national management guidelines of chinese health authorities include the use of tocilizumab for severe pneumonia. a preliminary report on critical cases of covid- suggests efficacy of the treatment with faster recovery and lower risk of death for treated patients, while no toxicity was associated with the reported administration schedule (one or maximum two doses) ( ) . timing of administration seems to be crucial as tocilizumab may be more efficient if administered earlier than actual use ( ) . anakinra is an interleukin- receptor antagonist (il- ra) previously evaluated in clinical trials for ra patients. il- beta/il- alpha are two stimulating cytokines of monocyte-macrophage cells acting upstream of the inflammatory signaling pathway induced by inflammasome, thus anakinra should block the cytokine storm. in a small open-label study, anakinra has been tested as agent preventive of mechanic ventilation in patients hospitalized for moderate-severe covid- . amelioration of oxygen flow and blood inflammation markers was described without significant toxicity ( ) . in clinically moderate and severe covid- patients preliminary evidence reported high levels of three cytokines, cxcl , ccl and il- , rather than il- , ( ). in chronic use anakinra could determine reaction at the site of injection and infection as the main side effects ( ) . emapalumab is a fully human igg monoclonal antibody that binds free and receptor-bound interferon-γ. emapalumab is approved by the us fda for the treatment of haemophagocytic (hlh) ( ) a rare disorder characterized by pathologic immune activation and hyperinflammation that eventually damage multiple organs. a prospective study has shown a good safety profile of emapalumab in pediatric and adolescent patients affected by hlh, with the infection susceptibility being the major threat ( ) . blocking ifn γ activity could counteract the host immune hyper-reaction to sars-cov- . mycophenolic acid has been used as immunosuppressant agent in pemphigus as a corticosteroid-sparing agent and in kidney transplant patients to avoid rejection. it inhibits inositol monophosphate dehydrogenase, that causes depletion of guanosine and deoxyguanosine nucleotide pools impairing the activity of b and t lymphocytes. the drug has also been demonstrated to inhibit mrna expression of pro-inflammatory cytokines tnf-α, il- , and il- β ( ) . mycophenolic acid has been shown to have activity in vitro against zika virus replication ( ) and coronavirus through a non-competitive inhibition of mers-cov papain-like protease ( ). urinary infections, diarrhea, and leukopenia are the side effects more often described ( ) . anti-tnfα agents used in autoimmune diseases, such as ra and ulcerative colitis, in principle, may have a role in treating severe respiratory syndrome of covid- . infliximab is a monoclonal antibody targeting tnf alpha while etanercept is a receptor fusion protein (human igg -fc plus soluble p tnf alpha extracellular domain). tnf-α is a proinflammatory cytokine produced by macrophages, lymphoid cells, endothelial cells, cardiac myocytes, adipose tissue, and brain cells such as microglia and astrocytes. its receptors are widely expressed and tnf-α plays a key role in immunological defense processes such as inducing fever, inhibiting viral replication during infections, and leading to a permanent growth arrest in cancer ( , ) . toxicity profile includes augmented risk of infections ( ) . proteasomal system regulates different cell functions, among which nuclear factor kb (nf-kb) key transcription factor for innate and adaptive immunity ( ) . bortezomib inhibits proteasome and it is used in the treatment of myeloma and mantle cell lymphoma. it has been shown to have antiviral activity against herpes virus, targeting viral entry, replication, and assembly ( ) . another proteasome inhibitor, vr , possess powerful antiinflammatory activity reducing il- in synovial cells from ra patients, and improving lps-induced acute lung injury by decreasing neutrophil migration, tnf-α secretion, and tissue inflammation in a mice model ( ) . the doselimiting toxicity of proteasome inhibitors is the peripheral neuropathy ( ) a clinically relevant complication, which negatively impacts the quality of life of multiple myeloma survivors ( ) . pandemic viruses decrease type i interferon (ifn) abundance ( ) . in humans different types of poly-adenosine ′diphosphate (adp)-ribose polymerase (parp) are recognized. parps transfer adp ribose from nicotinamide adenine dinucleotide (nad+) to targeted proteins achieving a post translational modification called adp-ribosylation, generally in response to stress conditions such as dna damage, heat shock and viral attack ( ) . parp is an adp ribosyl-transferase that inhibits interferon type i (ifn-i) antiviral activity. ifn-i is a key component of the immune response against viral pathogens that induces the expression of several genes (interferon stimulated genes -isgs) with diverse antiviral properties ( ) . parp inhibitor, rucaparib has been shown to restore the activity of ifn-i against different viruses in a murine model ( ) . there is evidence that zikv infection triggers type i ifn production by host cells, zikv is sensitive to the antiviral activity of ifn and ifn i seems crucial also in sars-cov- infection ( , ) . parp inhibitors are used in subgroup of patients with breast or ovarian cancer. toxicity is mainly hematological ( ) . peroxisome proliferator-activated receptor gamma (pparγagonists, rosiglitazone and pioglitazone, are drugs in clinical use for diabetes ( ) . insulin resistance amplifies inflammation, associated with an increase in c-reactive protein, il- , and tnf-α ( ) and produces a pro-coagulant state with increased fibrinogen and plasminogen activator inhibitor, (pai- ) ( ). pioglitazone, in clinical studies on diabetic patients, was able to reduce the plasma level of different inflammatory factors among which cpr, il- , il- , and tnf-α ( ). thus, it is of great interest that pioglitazone can produce an anti-inflammatory effect also on lung inflammation and fibrosis ( ) . considered the excellent tolerability, pparγ agonists may be tested for amelioration of virus induced lung injuries. plerixafor is a cxcr antagonist used for stem cell mobilization in patients undergoing autologous stem cell transplantation. cxcr -mediated inflammatory responses is based on the efficient chemotaxis function of inflammatory cells, such as neutrophils, lymphocytes, and monocytes ( ) . in murine models of acute lung insufficiency cxcr expression was significantly increased in macrophages sorted from bronchoalveolar lavage fluid and receptor downregulation reduced il- and tnf-α. administration of amd significantly attenuated the influx of inflammatory cells to the airway and reduced the levels of il- , il- , and il- in an murine asthmatic model either in the lavage fluid and lung homogenate through attenuation of the th ( ), cell population. no adverse events have been described for a single injection of plerixafor ( ). fingolimod, a sphingosine- -phosphate (s p) receptor agonist is approved for the treatment of multiple sclerosis (ms). s p is mainly expressed in vascular endothelial cells and lymphocytes in lung tissue. s p agonists (cym- and rp- ) have been reported to protect mice from death caused by severe influenza infection, attenuating cytokine production and inhibiting infiltration of innate immune cells. in a mouse model of h n pandemic influenza, the s p receptor agonist significantly inhibited synthesis of il- α, il- β, il- , il- , mcp- , tnf-α, and gm-csf, and reduced deaths from lethal infections by more than %. in addition the combination of oseltamivir can reduce mouse mortality by % ( ) . recently a multiple sclerosis (ms) patient in treatment with fingolimod that was diagnosed with covid- reported a favorable outcome ( ) . as reported, the toxicity profile even for long term use, is reassuring ( ) . in conclusion, while specific antiviral therapies are in rapid development (remdesivir, chloroquine, vaccine), controlling the powerful inflammatory response causing severe ip or ards is a reasonable approach. agents that are available now to improve the lung injuries due to the host reactions and reduce the lethality of the disease are badly needed, and some are already in clinical studies. drugs targeting multiple cyto/chemokines involved in sars-cov- ip are available for trial or for off-label use, but close attention is needed to the schedule of administration, considered the immunosuppressive action of these drugs. to this aim rapid identification of prognostic factors in the peripheral immune profile may support therapeutic approach. careful clinical studies are warranted. ss and rp both equally contributed in designing and realizing the manuscript. the continuing -ncov epidemic threat of novel coronaviruses to global health -the latest novel coronavirus outbreak in wuhan, china emerging coronaviruses: genome structure, replication, and pathogenesis severe acute respiratory syndrome coronavirus (sars-cov- ) and coronavirus disease- (covid- ): the epidemic and the challenges characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention angiotensinconverting enzyme (ace 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plerixafor enables safe, rapid, efficient mobilization of hematopoietic stem cells in sickle cell disease patients after exchange transfusion dissecting influenza virus pathogenesis uncovers a novel chemical approach to combat the infection covid- infection in a patient with multiple sclerosis treated with fingolimod benefitrisk profile of sphingosine- -phosphate receptor modulators in relapsing and secondary progressive multiple sclerosis the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © scala and pacelli. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -zr zx pv authors: hoo, regina; nakimuli, annettee; vento-tormo, roser title: innate immune mechanisms to protect against infection at the human decidual-placental interface date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: zr zx pv during pregnancy, the placenta forms the anatomical barrier between the mother and developing fetus. infectious agents can potentially breach the placental barrier resulting in pathogenic transmission from mother to fetus. innate immune responses, orchestrated by maternal and fetal cells at the decidual-placental interface, are the first line of defense to avoid vertical transmission. here, we outline the anatomy of the human placenta and uterine lining, the decidua, and discuss the potential capacity of pathogen pattern recognition and other host defense strategies present in the innate immune cells at the placental-decidual interface. we consider major congenital infections that access the placenta from hematogenous or decidual route. finally, we highlight the challenges in studying human placental responses to pathogens and vertical transmission using current experimental models and identify gaps in knowledge that need to be addressed. we further propose novel experimental strategies to address such limitations. the human placenta is the temporary extra-embryonic organ that is present only during pregnancy and is the anatomical boundary between the mother and fetus. it has a range of functions including transport of nutrients and gases, and hormonal production ( ) . the placenta forms a physical, selective barrier between the maternal and fetal circulations, preventing transfer of pathogens. the uterine mucosal lining, the endometrium, is transformed into the decidua during early pregnancy ( ) . a range of innate immune mechanisms can respond to pathogens in both the decidua and the placenta ( , ) . the maternal-fetal interface is a protective barrier against pathogens, but some pathogens can transfer from the mother to fetus by different routes and cause fetal infection ( , ) . vertical transmission during pregnancy can occur on distinct boundaries between the mother and the fetus: (i) the intervillous space (ivs), where placental villi is in direct contact with the maternal blood, (ii) the implantation site or decidua basalis, where maternal cells are in direct contact with the invading fetal trophoblast, and (iii) the fetal membranes, which are in direct contact with the uterine cavity ( ) . defense mechanisms in the cervix, such as the production of mucus and antimicrobial peptides (amp), limit ascending infection from pathogens present in the lower genital tract, that otherwise may access the uterine cavity ( ) . however, some pathogens can escape antimicrobial strategies at the cervix and ascend to the uterus, where they can bypass the fetal membranes and lead to the inflammation of the membranes-also known as chorioamnionitis-and infection of the amniotic fluid ( , ) . pathologic and immune features of chorioamnionitis and intra-amniotic infection are generally associated with bacterial invasion and inflammation [refer to ( , ) for a comprehensive review on these mechanisms]. here, we focus on infections and innate immune mechanisms at the uterine-placental interfacecases (i) and (ii) (figure ) . infections at the uterine-placental interface are commonly associated with viruses, parasites and few bacteria ( table ) . viral pathogens such as human cytomegalovirus (hcmv), zika (zikv), and rubella virus are the most common vertically transmitted pathogens through the decidual-placental interface ( table ) ( , ) . non-viral pathogens, such as toxoplasma gondii and listeria monocytogenes, can cross the placental barrier via cell-to-cell transmission ( table ) ( , ) . fetal infection can result in various forms of congenital anomalies in humans ( table ) . understanding the pathogenic mechanisms used by infectious agents is central to preventing vertical transmission and controlling infection during pregnancy. how the innate immune cells and mechanisms in the placenta and the uterus recognize and respond to protect both the fetus and mother remains controversial due to technical and ethical constraints. however, there are several different models currently used to interrogate the uterine-placental interface in pregnancy. firstly, mice are frequently used as a pregnancy model for infection. although the murine models have provided important insights into the pathogenesis of various infection agents in the context of pregnancy, there are still limitations with this approach. the anatomy of placentation, length of gestation, and use of inbred strains, make extrapolation to humans problematic ( , ) . secondly, a range of human trophoblast and choriocarcinoma cell lines are used as in vitro models for infection with pathogens. in contrast to the first trimester trophoblast in vivo, these cell lines do not recapitulate normal human trophoblast characteristics such as expression of the human leukocyte antigen (hla) class i and methylation of elf ( , ) . thirdly, human primary placental explants are frequently used. the syncytium dies rapidly in these cultures and it is virtually impossible to standardize the types of villi sampled ( ) . therefore, these in vitro experimental factors should be taken into careful consideration when interpreting studies of infection of trophoblast. in this review, we cover the innate immune features of the decidual-placental interface throughout gestation. we identify the gaps in knowledge and highlight the limitations of current studies and experimental models. finally, we discuss novel experimental strategies for understanding how infection affects pregnancy in humans. the trophoblasts of the placenta are the barrier between fetal and maternal tissues. they are derived from the trophectoderm, the outer layer of the blastocyst that forms an inner mononuclear layer with an outer primary syncytium following implantation ( ) . the trophoblast in contact with the maternal cells can be: (i) syncytiotrophoblast (sct), a single layer multinucleated, syncytial layer formed by fusion of the underlying villous cytotrophoblast (vct), and (ii) extravillous trophoblast (evt), that invade from the cytotrophoblast shell and anchoring villi into the transformed maternal endometrium, the decidua ( ) . the function of evt is to transform the uterine spiral arteries so that maternal blood is delivered to the intervillous space at low pressure. the arteries are surrounded by interstitial evt that destroys the smooth muscle cells of the arterial media, known as "fibrinoid" change ( , ) . subsequently, endovascular evt (eevt) moves down the spiral arteries from the placentadecidua boundary ( ) . these eevt form a plug of cells, limiting surges of arterial blood from damaging the delicate villi. evt invasion transforms the arteries to support optimal regulation of blood flow into the placenta during fetal development ( ) . the plugs dissipate between and weeks of gestation when the full hemochorial circulation is established ( ) . maternal blood then flows into the ivs, and establishes direct contact with the sct allowing for proper nutrient and gas exchange between the mother and the fetus. hofbauer (hb) cells are fetal macrophages of the human placenta ( ) . hb cells can be detected in the placental villous stroma as early as weeks post-conception and are present throughout pregnancy ( , ) . they are likely to have a variety of functions including control of villous remodeling and differentiation, hormonal secretion, and trophoblast turnover ( , ) . several lines of evidence have led to the postulation that hb cells may have a role in infection during pregnancy. hb cells with zikv viral particles detected intracellularly have been shown ( , ). human immunodeficiency virus (hiv- ) has also been detected in hb cells from first trimester infected placenta ( ) . whether the hb cells can serve as a reservoir or limit virus replication is still unknown. isolated hb cells from healthy term placenta show elevation of pro-inflammatory cytokines such as il- , mcp- , ip- , and ifn-α upon in vitro infection with zikv ( ) . hb cells from the first trimester placenta are also permissive for zikv infection and replication ( ) . however, this must be interpreted with caution because in vitro culture of hb cells do not entirely recapitulate the complexity of villous stromal microenvironment, such as presence of hormone and growth factors, all of which will influence the function and activity of hb cells ( ) . the sct is the barrier between maternal blood and the placental core as it separates the ivs from the underlying fetal villous stroma. blood-borne pathogens such as viruses and parasites can potentially be transmitted through the sct barrier (figure ) . how can pathogens cross the sct barrier and the vct to infect the villous stroma? although the sct is an efficient barrier due to its stiff, highly dense actin cytoskeleton network and continuous membrane ( ), the syncytium undergoes continuous breaks or gaps and dynamic repair processes ( ) . breaks in the syncytium could potentially lead to transmission of pathogens into the underlying vct. our recent work showed that a novel population of maternal macrophages (m ) is associated with the sct in early pregnancy and might be involved in repairing the breaks in the syncytium ( ). it is intriguing that m macrophages infected with intracellular pathogens could possibly gain access to the underlying vct via the syncytial breaks (figure ) . only a few viral entry receptors on the sct are described. notably, the sct lacks expression of zikv entry receptors, axl, and tyro ( ) and the hcmv entry co-receptor integrin α/β ( ) . this is further supported by the transcriptomic expression of viral receptors in placental cells ( , , ). expression of surface receptors commonly used by zikv such as axl and hcmv such as nrp and pdgfra are lowly expressed by the sct ( ) . in addition, there is minimal co-expression of ace , the receptor gene for human severe acute respiratory syndrome coronavirus (sars-cov- ), and tmprss , the viral spike protein serine protease gene ( , ) . in line with this, there is no conclusive and direct evidence of vertical transmission of sars-cov- in a placenta from a healthy individual. there are some reports showing sars-cov- is predominantly localized at the sct of the second trimester placenta ( , ) and can lead to severe inflammatory infiltrate in the ivs ( ) . however, these findings are presented in a very small number of patients with severe disease or pre-existing pregnancy complications ( , ) . alternative transplacental mechanisms have been postulated at the syncytial barrier. neonatal fc receptor (fcrn) is expressed on the apical surface of the sct and functions to selectively figure | toll-like receptors and potential inflammatory response at the sct-blood interface. predominant tlrs found in the human placenta from early and term pregnancies. tlr and tlr are expressed in human placenta sct, vct, and in hb cells. infiltration of infected maternal blood, infected immune cells, or release of pathogenic determinant such lipopolysaccharide (lps), peptidoglycan, or parasite materials such as hemozoin or gpi (glycosylphosphatidylinositol) into the ivs will activate tlr-mediated signaling, leading to the production of a wide range of cytokines and chemokines. severe infection is characterized by massive immune cell infiltration including monocytes and neutrophils from systemic circulation and overproduction of inflammatory cytokines upon tlr activation. this may lead to sct inflammation and damage. sct also secretes antimicrobial peptides as innate immune mechanisms. figure is created by biorender.com. frontiers in immunology | www.frontiersin.org transport maternal igg ( ) . fcrn could be exploited by certain viruses to enter the placenta including zikv, hiv- , and hcmv ( , , ) . transferrin receptor (tfr ) is expressed on the apical end of the sct, and functions as the primary iron transporter into the basal side of the sct to provide sufficient iron stores into fetal circulation ( ) . tfr has been associated with viral entry into a broad host cell range, including hepatitis c virus ( , ) suggesting a possible mechanism of viral transport across the sct barrier. some pathogens, although unable to cross the sct barrier, can still adhere to the syncytium and cause further pathology. for instance, plasmodium falciparum infected red blood cells can bind with high affinity to chondroitin sulfate a expressed on the sct, resulting in local inflammation, syncytial breaks, and damage ( ) ( ) ( ) . although the sct is an effective barrier to most pathogens, local inflammation, tissue damage, and fcrn or tfr -mediated viral entry at the sct can potentially allow pathogen to breach the syncytial barrier, giving opportunity for transmission from maternal blood into placental villi (figure ). during the first trimester of pregnancy, fetal evt invades deeply into the uterus. the decidua basalis, the region located at the implantation site, is populated at this time by a distinctive subset of innate lymphocytes, decidual natural killer cells (dnk), which constitute up to % of leukocytes. we have identified three major populations of dnk by single-cell rna-sequencing with unique phenotypes and functions in early pregnancy ( ). in addition, there are populations of decidual macrophages (dms) (∼ %), conventional dendritic cells (dcs) and small proportions of t cells (∼ - %), whereas b cells, plasma cells, mast cells, and granulocytes are virtually absent ( ) (figure ) . the proportion of immune cells will vary throughout pregnancy, with an increase in the proportion of t cells at term ( ) . systemic infections will reach all organs including the decidua. whether pathogens can also access the decidua via the cervix is still unclear. chlamydia trachomatis, a common sexuallytransmitted intracellular bacteria, was detected in glandular epithelial cells and unidentified decidual cells in decidual biopsies ( ) . this suggests the possibility of infections ascending and spreading from cell-to-cell from the lower genital tract into endometrial glands and vascular endothelium. the decidua basalis is in close contact with fetal cells and the maternal vasculature (figure ) . first trimester dms and decidual stromal cells are susceptible to zikv infection and replication ex vivo ( ) . hence, infection could possibly spread from infected maternal immune and non-immune cells at the decidua, into uninfected vct in the columns of the anchoring villi, and finally into the fetal compartment. however, this is likely to be limited to certain microorganisms which are capable of cell-to-cell spread, have an intracellular host niche, and are able to escape host innate defense mechanisms (table ) . hcmv, the most common cause of congenital infection, is mostly reported to infect from the decidua ( , ) . women with primary hcmv infection and first pregnancy are more likely to transmit the virus to their fetus, compared to multiparous women with previous infection and demonstrable antibodies ( ) ( ) ( ) . low affinity maternal antibodies against hcmv correlate with higher viral loads detected in the decidua, whereas patients with intermediate to high neutralizing antibodies have minimal viral replication ( ) , suggesting that maternal immunity against hcmv reduces risk of vertical transmission. hcmv protein was also detected in a range of cells within the decidua including endothelial, decidual stromal cells, dcs and macrophages ( , ) , suggesting that that infected maternal leukocytes could initiate transmission through contact and infection of endothelial cells that line decidual blood vessels. despite the evidence of decidual infection, the mechanism of vertical transmission for hcmv is still in debate. dnks have been proposed to play a protective role against hcmv infection through several mechanisms including modulation of their cytotoxic effector function ( ) and the interactions between the killer-cell immunoglobulin receptors (kirs) expressed by dnk and hla molecules expressed in the infected cells ( , ) . activating kir ds by dnks has been demonstrated to be more cytolytic against hla-c hcmv-infected maternal decidual stromal cells ( ) . similar cytotoxic response was also observed when peripheral blood nk cells expressing kir ds were exposed to hcmv-infected fibroblasts ( ) . hence, this implies that in the decidua, dnks are capable of eliminating harmful infection depending on the combination of kir/hla interactions between dnk and infected cells. dnks are also able to control hiv- infection in vitro through production of ifn-γ ( ) . the role of dnk in controlling viral infection may protect against potential risk of vertical transmission from the decidua. pattern recognition receptors (prr) are encoded in the germ-line and recognize specific, conserved pathogen-associated molecular patterns (pamps). these include gram-negative bacteria lipopolysaccharide (lps), gram-positive bacteria lipoteichoic acids, lipoprotein, dna, rna, glucans, and peptidoglycans ( , ). pathogen recognition is not only an essential component of the innate immune response against infection, but also plays an important role in bridging the innate and adaptive systems by toll-like receptors (tlr) activation of antigen presenting cells by up-regulation of major histocompatibility complex (mhc) and co-stimulatory molecules ( ) . tlrs, the most studied family of prr, are type i transmembrane proteins with large extracellular domains containing leucine-rich repeats that are expressed at the cell surface or intracellularly ( ) . each tlr recognizes distinct pamps, leading to the activation of the transcription factor nf-κb and/or the interferon-regulatory factor (irf) family, and the production of a wide range of cytokines and chemokines, including type i ifns ( , ) expression of tlrs is dynamic and changes in response to different pathogens and cytokines ( ) . tlr (which recognizes bacterial proteoglycan) and tlr (which recognizes bacterial lps) are the most well-studied, with immunohistochemical evidence of expression in healthy primary sct at term ( ) ( ) ( ) . in contrast, in the first trimester, tlr and tlr proteins are expressed in vct and evt, but minimally in sct ( , ) (figure ) . there is therefore variation in tlr and tlr expression in the different trophoblast lineages across pregnancy. why and how such dynamic regulation of tlr expression occurs during gestation requires further investigation in a broader range of human placental samples (different donors, gestation stages, genetic background, sampling regions). it is likely that alteration in cytokines profiles in the microenvironment as pregnancy progresses ( ) may result in the variation in the expression of tlrs in the placenta. current evidence is only limited to in vitro tlr / stimulation studies using placental explants and primary first trimester trophoblast cells, which drives the expression of figure | toll-like receptors and potential inflammatory response at the decidua. predominant tlrs found in the human placenta from early and term pregnancies. tlr and tlr are expressed in evt. dm and dnk also express a wide range of tlr families, where stimulation of tlr agonists lead to the production of a variety of cytokines and chemokines. infiltration of infected cells and release of pamps in the decidua, which will activate tlr-mediated signaling. overproduction of inflammatory cytokines at the decidua may lead to local inflammation. figure is created by biorender.com. pro-inflammatory cytokines il- , il- , tnf-α, and ifn-γ ( , , ) . tlr and tlr proteins are expressed in hb cells, confirmed by co-expression of cd in healthy term placentas ( ) . in early pregnancy, our findings indicate that only tlr but not tlr transcripts are expressed in steady-state hb cells ( ) (figure ) . enhancement of il- and il- secretion upon stimulation of isolated first trimester hb cells with tlr agonist, lps ( ), does suggest a role for tlrs on hb cells in bacterial recognition and placental inflammation during early pregnancy. hb cells are postulated to have a role in viral replication ( , ), however evidence on the expression and function of viral nucleic acid sensing receptors tlr , tlr , tlr , and tlr in hb cells is lacking. our findings show that tlr , which recognizes viral single-strand rna (ssrna) ( ) is expressed in steady-state hb cells (figure ) ( ) . other tlrs have also been shown to be expressed in decidua cells. dms and dnks isolated from first trimester pregnancies show steady state level expression of tlr - transcripts and respond to a broad range of pamps, including heat-killed bacteria, microbial membranes, and nucleic acids ( ) . stimulating primary dms with these pamps produces high levels of tnf-α, il- β, il- , il- , il- , il- , and il- ra, whereas dnks secrete il- , il- , and ifn-γ ( ) . this study suggests that, in addition to the physiological roles of dms and dnks in accommodating the uterus for placentation, dms and dnks may play a role in pathogen recognition and antimicrobial response via activation of tlr signaling (figure ) . the extent to which subsets of dms or dnks population ( ) are critical for tlr-mediated response at the decidua is currently unknown. in malaria endemic populations, single nucleotide polymorphisms (snps) within the tlr coding and tlr promoter regions are associated with variation in disease severity and parasitemia control ( , ) . in the case of pregnancy malaria, primiparous infected mothers with common tlr and tlr polymorphic variants are correlated with severe complications such as low birth weight and maternal anemia ( ) . this highlights the importance of studies involving large cohorts of individuals which include genotyping from pregnant mothers living in malaria endemic regions (see section on "challenges and future perspective"). animal models have also been used to study the functional role of tlr signaling, particularly for pathogens that are intracellular at some stage of their life cycle ( table ) . tlr and tlr are strongly activated by malaria parasite pamps such as glycosylphosphatidylinositol (gpi), dna, and hemozoin ( , ) (figure ) . in a mouse model of placental malaria, tlr , and myd signaling activation resulted in placental expression of pro-inflammatory markers, such as il- and tnf-α ( , ) . these studies also demonstrated that malaria parasite infection and inflammation in the mouse placenta lead to reduced fetus growth rate and disorganization of the vascular space in the placenta ( , ) . however, tlr-mediated inflammation and pathology in the human placenta upon malaria infection is unknown and remains to be further investigated. studies of congenital toxoplasmosis are also currently limited to animal models. tlr and tlr are associated with recognition of t. gondii's infection in mice ( ) . engagement of the t. gondii ligand by tlr and tlr at the sct-blood or in the evt-decidua compartments is plausible, although there is still no direct evidence for such host-parasite interaction in humans. tlr has a role in controlling t. gondii infection in mice ( , ) , however in humans tlr is a pseudogene and is not expressed ( ) . cytosolic prrs play an important role in fighting against viral infection by eliciting host type i interferons (ifn) antiviral response through recognition of single and double stranded rna (ssrna and dsrna) ( , ) . examples of prrs are the cytosolic retinoic acid-inducible gene-i-like (rig-i) and the melanoma differentiation-associated protein (mda ) receptors, both expressed in the sct and vct of term placenta ( ) . in the human placenta, there is limited information on the function of rig- and mda , but they may play a crucial role in recognizing a variety of rna viruses, including zikv and dengue virus ( ) . the nucleotide binding oligomerization domain-like receptors (nod-like receptors; nlr) recognizes intracellular pathogen products which have entered into the host cytoplasmic compartment ( ) . both nod- and nod- receptors, which are known to detect intracellular bacterial peptidoglycans ( ) , are expressed in the sct in the first trimester and term placentas ( , ) . the nlr pyrin-containing and proteins (nlrp and nlrp ) form the major inflammasome complexes, which contribute to activation of inflammatory caspases and pathogen clearance ( , ) . activation of nlrp and aim inflammasomes, together with high expression of il- r, il- β, and caspase- was recently shown in the placental tissue of mothers infected with p. falciparum with significant pathology ( ) . in a murine model of intra-amniotic inflammation induced by bacterial lps, tissue sections from the decidua basalis region expressed high levels of nlrp , but negligible caspase- activation suggesting a possible non-canonical activation of the nlrp inflammasome ( ) . our analysis shows that decidual dm expresses high levels of nlrp transcript at steady state compared to other cell types ( ) (figure ) , thus dm may play a role in nlrp -mediated pathogen recognition during early pregnancy. amp secreted by epithelial and immune cells are small peptides that bind and destroy most groups of pathogens-bacteria, yeasts, fungi, and viruses ( ) . in addition to direct killing of pathogens, amps can rapidly modulate innate host immune responses by recruiting myeloid cells and lymphocytes to the site of infection and mediating activation of tlr ( , ) . the human placenta expresses high levels of β-defensins, a family of broad spectrum antimicrobial peptides which participate in direct bactericidal and anti-viral activity ( ) . specific subtypes of β-defensins (hbd- , , and ) are expressed in scts ( ) , suggesting these amps can target potentially bacterial or viral infection from the maternal blood. recognition of pamps by prrs during infection leads to production of pro-inflammatory cytokines that can aid in clearing the pathogen ( ) . studies on the direct role of proinflammatory cytokines on the placenta in the case of infection is limited. inflammatory mediators can directly influence infection outcome and fetal development, but they can also cause damage to the placenta if produced in excess ( ). amongst the proinflammatory cytokines associated with uterine-placental infection during pregnancy, the antiviral ifn are the most well-characterized. ifns are secreted by a variety of cell types as the first line of defense against viral infection ( ) . type i ifns, including ifn-α and ifn-β, are potent antiviral cytokines. ifn-α and ifn-β bind to the ifnar / receptor and lead to expression of ifn stimulated genes (isgs), which control virus infection through a variety of mechanisms ( ) . loss of ifnar in the placenta leads to vertical transmission and fetal mortality in murine herpesvirus- (mhv ) infected mice ( ) . in the mouse model of zikv infection, type i ifn-mediated signaling is essential for the control of viral replication in the placenta, but can also lead to significant placental pathology and fetal mortality ( , ) . the mechanism of type i ifn-mediated placental pathology has been recently elucidated. ifn-induced transmembrane (ifitm) protein, which normally blocks viral entry into host cells, impairs syncytin-mediated fusion of vct to form sct, leading to aberrant placental development ( ) . type ii ifn, ifnγ, predominantly produced by nk and cd + t cells is crucial in controlling parasitic infection, such as t. gondii in mice ( , ) . however, elevated levels of ifnγ in response to t. gondii infection can lead to pathological effects during pregnancy including fetal demise ( , ) . severe placental pathology and fetal death have also been associated with elevation of ifnγ during pregnancy in a murine model of malaria ( ). hence, proper regulation of type i and ii ifn-mediated signaling at the uterine-placental interface is crucial in limiting pathogen replication, whilst preserving a balanced environment for normal placental development ( ) . type iii ifn, ifnλ, are constitutively secreted by the human sct, which presumably confers antiviral effects against zikv infection ( ) ( ) ( ) . tryptophan metabolism by ido indoleamine , -dioxygenase (ido) is a host intracellular enzyme which metabolizes the amino acid tryptophan ( ) . ido has been associated with maternal immunoregulation during pregnancy ( ) . it also plays a key role in the control of bacterial and viral replication, through limiting the bioavailability of tryptophan ( ) . ido also inhibits the replication of several parasitic pathogens including t. gondii in human fibroblasts ( ) and leishmania spp in human macrophages ( ) . mouse infection with l. monocytogenes showed that ido is elevated in an ifn-γ-dependent manner in stromal cells of the metrial gland and decidua basalis; a crucial process to resolve bacterial infection in the mouse placenta ( ) . our findings also show ido expression is enriched in epithelial glandular and dc cell type in the first trimester decidua ( ) (figure ) . the presence of ido in decidua suggests that the enzyme might have a central role in limiting parasitic, viral, and bacterial replication, thus preventing their spread to the fetus. research on how the human placenta safeguards itself against infections is challenging due to obvious logistical and ethical issues in obtaining tissue from early in gestation (box ). although animal experimental models have provided important insights relating to the immune responses to pathogenic infection, major differences between human and animal placentas must be considered ( , ) . likewise, differences between strains of pathogens adapted for mice compared with human clinical isolates should be taken into account as this may lead to variation in pathogenesis and cellular response. one such example is the use of mouse cmv, which is unable to cross the mouse placental barrier, unlike the hmvc counterpart which can be transmitted transplacentally in humans ( ) . therefore, all data obtained from studies of infection in pregnant animals needs careful interpretation and consideration prior to translation to clinical infection in humans. inherent properties of trophoblast cell lines, primary cultures or explants vary between donors, and are likely to be confounded by the area of the placenta that is sampled and as well as stage of gestation ( ) . for instance, villous placental explants will vary depending on the types of villi sampled and the presence of box | perspective of vertical transmission and innate immune function during pregnancy and infection. a variety of maternal infections can lead to vertical transmission ( table ) . the exact mechanisms these pathogens use to escape host defense and cross the placental barrier into the fetal compartment are not entirely known. experimental models that recapitulate infection of the human placenta and thus vertical transmission are challenging to set up. more data and representative experimental models are needed to answer these questions: (i) how do different pathogens escape or modulate the maternal-fetal host innate immune barrier (ii) why do some pathogens lead to congenital infection but not others? studying infected human placentas will be essential in understanding this but access to these samples is difficult especially in low and middle-income countries (lmic) where maternal infection is particularly prevalent (who, maternal mortality index ). despite evidence of expression in primary placental tissue, functional studies on important innate immune features such as tlrs, amps, rig-i, mda , nlrs, and ido during infection and pregnancy are lacking. understanding how different cell types at the uterine-placental interface (hb cells, dnks, and dms) respond to pathogen challenge is essential, but remains under-researched. a critical obstacle is to also extrapolate the protective and pathological mechanisms of cytokines from mouse to human infection. therefore, systematic comparison of the innate immune effector mechanisms across gestation, in the placenta and decidua from natural human infection vs. healthy pregnancy, will provide a more accurate representation in clinical settings. attached decidual tissue ( ) . caution is therefore needed when interpreting data using these experimental models. to overcome such limitations, population-based cohort studies of women with infection during pregnancy with extensive tissue sampling should be performed. these need to include and focus on lmic where infection is still a major cause of maternal and fetal mortality and morbidity. cohort studies and epidemiological surveillance on maternal infections can offer significant insights into disease pathogenesis and accelerate clinical interventions ( ) . collaborations between clinicians and researchers for population-based cohort collection and sample processing will be instrumental to achieving this goal. biological samples such as blood or placenta collected from controls and infected pregnant individuals could be stored and cryopreserved retrospectively. to capture the overall heterogeneity of infected and non-infected placenta samples, sampling, and biobanking criteria of different regions of placenta should be considered ( ) . protocols are now available to use frozen tissue processed for single-cell/nuclei and spatial genomics ( , ) . hence, application of single-cell "omics" on infected vs. healthy human placental and decidual samples will enable us to evaluate cellular heterogeneity in response to infection. the capacity to detect transcripts specific to host or pathogen mrna from the same tissue using in situ nucleic acid hybridization methods will provide direct quantification of infection burden and identification of potential target host cells within the same tissue ( ) . recent advances in spatial transcriptomics methods have also allowed gene expression signatures to be quantified and resolved from individual tissue sections ( ) . combination of these emerging technologies with new methods to integrate single-cell and spatial data computationally ( ) will provide an unbiased approach to characterize and profile the transcriptome of individual cells in situ from the placenta and decidua in response to infections. we anticipate that high-throughput datasets generated from cohort sampling studies will unravel novel cell states and tissue spatial localization associated with placental infections and inflammation. this will also allow us to better characterize not only the innate immune response or makers of infection, but also other adaptive immune states in the human placenta (box ). the use of in vitro models will also further define host responses to infection. the recent generation of human trophoblast stem cells (htscs) ( ) and three-dimensional ( d) trophoblast organoids ( , ) offer a great opportunity to study infections in early pregnancy where the access to first trimester placental samples is a concern. more importantly, the htscs and trophoblast organoids fulfill the criteria characteristic of human first trimester trophoblast in vivo ( ) . both htscs and trophoblast organoids can differentiate in vitro into sct and evt with appropriate media ( , ) allowing infection experiments on both the major trophoblast subpopulations present at the two major sites of contact between maternal and fetal cells. sequencing of both host and pathogen transcriptomes from infected trophoblast at single-cell resolution will also advance our understanding on host-pathogen interactions in placentas ( , ) . further refinement of the trophoblast organoid and htscs culture system is needed to address key biological questions unanswered by current models. these include studying the effect of infection on cellular crosstalk between trophoblast and other primary placental cells such as hb cells, or decidual cells in culture, such as dnk or decidual stromal cells. adaptation of crispr/cas genome editing technology for the trophoblast organoids or htscs will offer novel insights into essential host genes required for vertical transmission and placental defense mechanisms in humans. major maternal and fetal complications as a result of infection are still a concern, especially in lmic with highest prevalence reported in countries of sub-saharan africa (who, maternal mortality index ). profound limitations on current study models and ethical regulations on studying human placenta have significantly delayed the development of therapies and vaccines for maternal-fetal infection. how vertical transmission occurs and how the uterine-placental innate immune system reacts to infection remain as major unresolved questions. revolutionary advances in single-cell genomics, imaging, computational, and stem cell biology methods are currently underway to study the molecular and cellular mechanisms of human diseases. therefore, it is now an exciting time to apply these transformative technologies to comprehensively address fundamental questions on host-pathogen interaction at the human uterine-placental interface. rh, an, and rv-t wrote and edited the manuscript. all authors contributed to the article and approved the submitted version. rh and rv-t were supported by wellcome sanger core funding (wt ). an was supported by a sanger international fellowship, a nurture fellowship (d tw ) and a group leader award supported through the deltas africa initiative ( /z/ /z). the deltas africa initiative is an independent funding scheme of the african academy of science (aas), alliance for 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recapitulate the developmental program of the early human placenta scdual-seq: mapping the gene regulatory program of salmonella infection by host and pathogen single-cell rna-sequencing we would like to thank ashely moffett for the useful discussions and critical review of the manuscript. we are also very grateful to sarah aldridge, loren gibson, damiana alvarez, carlos talavera-lopez, and anna arutyunyan for their insightful comments and corrections. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © hoo, nakimuli and vento-tormo. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - qur isb authors: gardinassi, luiz g.; souza, camila o. s.; sales-campos, helioswilton; fonseca, simone g. title: immune and metabolic signatures of covid- revealed by transcriptomics data reuse date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: qur isb the current pandemic of coronavirus disease (covid- ) has affected millions of individuals and caused thousands of deaths worldwide. the pathophysiology of the disease is complex and mostly unknown. therefore, identifying the molecular mechanisms that promote progression of the disease is critical to overcome this pandemic. to address such issues, recent studies have reported transcriptomic profiles of cells, tissues and fluids from covid- patients that mainly demonstrated activation of humoral immunity, dysregulated type i and iii interferon expression, intense innate immune responses and inflammatory signaling. here, we provide novel perspectives on the pathophysiology of covid- using robust functional approaches to analyze public transcriptome datasets. in addition, we compared the transcriptional signature of covid- patients with individuals infected with sars-cov- and influenza a (iav) viruses. we identified a core transcriptional signature induced by the respiratory viruses in peripheral leukocytes, whereas the absence of significant type i interferon/antiviral responses characterized sars-cov- infection. we also identified the higher expression of genes involved in metabolic pathways including heme biosynthesis, oxidative phosphorylation and tryptophan metabolism. a btm-driven meta-analysis of bronchoalveolar lavage fluid (balf) from covid- patients showed significant enrichment for neutrophils and chemokines, which were also significant in data from lung tissue of one deceased covid- patient. importantly, our results indicate higher expression of genes related to oxidative phosphorylation both in peripheral mononuclear leukocytes and balf, suggesting a critical role for mitochondrial activity during sars-cov- infection. collectively, these data point for immunopathological features and targets that can be therapeutically exploited to control covid- . the current pandemic of coronavirus disease has affected millions of individuals and caused thousands of deaths worldwide. the pathophysiology of the disease is complex and mostly unknown. therefore, identifying the molecular mechanisms that promote progression of the disease is critical to overcome this pandemic. to address such issues, recent studies have reported transcriptomic profiles of cells, tissues and fluids from covid- patients that mainly demonstrated activation of humoral immunity, dysregulated type i and iii interferon expression, intense innate immune responses and inflammatory signaling. here, we provide novel perspectives on the pathophysiology of covid- using robust functional approaches to analyze public transcriptome datasets. in addition, we compared the transcriptional signature of covid- patients with individuals infected with sars-cov- and influenza a (iav) viruses. we identified a core transcriptional signature induced by the respiratory viruses in peripheral leukocytes, whereas the absence of significant type i interferon/antiviral responses characterized sars-cov- infection. we also identified the higher expression of genes involved in metabolic pathways including heme biosynthesis, oxidative phosphorylation and tryptophan metabolism. a btm-driven meta-analysis of bronchoalveolar lavage fluid (balf) from covid- patients showed significant enrichment for neutrophils and chemokines, which were also significant in data from lung tissue of one deceased covid- patient. importantly, our results indicate higher expression of genes related to oxidative phosphorylation both in peripheral mononuclear leukocytes and balf, suggesting a critical role for mitochondrial activity during sars-cov- infection. collectively, these data point for immunopathological features and targets that can be therapeutically exploited to control covid- . keywords: covid- , transcriptomics, inflammation, metabolism, sars-cov- , sars-cov, influenza, oxidative phosphorylation introduction the outbreak of coronavirus disease (covid- ) , first recognized in wuhan, china, rapidly became a pandemic of major impact not only on global public health but also on economy and social well-being ( ). sars-cov- infection results in clinical outcomes ranging from asymptomatic status to severe disease and ultimately, death ( ) . understanding of the molecular mechanisms underlying the pathology of covid- is required to design effective therapies and safe vaccines. in this context, current investigations have been devoted to biochemical characterization and cellular phenotyping in patients to development of animal models of covid- ( ). transcriptomics of peripheral blood cells has been a powerful tool to characterize human immune responses to diverse pathogens, including respiratory viruses ( - ). gene expression profiling by different analytical platforms and sample types revealed that covid- patients exhibit: (i) activation of humoral immunity, hypercytokinemia, apoptosis ( ) , and dynamic toll like receptor (tlr) signaling ( ) in peripheral leukocytes; (ii) induction of interferon stimulated genes (isgs), chemokines and inflammation in the lower respiratory tract ( , , ) . of importance, the results and interpretation of these data were based on single-gene-level analyses, in which significance of quantitative changes of each gene are calculated separately and they are latter submitted to pathway enrichment analysis. however, the statistical power and sensitivity to identify pathways, or gene modules (computational gene networks), associated with disease phenotypes can be enhanced by the use of non-parametric rank-based tests such as the robust positional framework gene set enrichment analysis (gsea) ( ) . moreover, interpretation of transcriptional changes during covid- has been primarily evaluated using canonical pathways that do not often reflect human responses. therefore, we propose alternative strategies to analyze and interpret transcriptomics data, which provide novel insights into immune and metabolic responses during covid- . datasets used in this study included public transcriptomes available at the genome sequence archive (gsa) or human gsa in national genomics data center, beijing institute of genomics (big), chinese academy of sciences for rna-seq data related to sars-cov- infection (cra and hra ); gene expression omnibus (geo) for rna-seq data related to sars-cov- infection (gse ) and microarray data related to sars-cov- infection (gse ) or influenza a virus (iav) infection (gse , gse , gse , gse , gse , gse , gse , gse , gse , gse ); and arrayexpress for nanostring ncounter data related to sars-cov- infection (e-mtab- ). deseq -normalized counts were used for the rna-seq dataset cra ( ), while raw read counts for the rna-seq datasets gse ( ) or hra ( ) were treated and normalized to log counts per million with edger package for r ( ) . normalized data was acquired for nanostring ncounter e-mtab- ( ) . normalized microarray datasets were acquired with omicc platform ( ) . detailed information about the datasets used in this study are described in table . data were analyzed with the positional framework gene set enrichment analysis (gsea) ( ), using pre-ranked mode, , permutations and weighted enrichment statistics. the blood transcriptional modules (btms) ( ) and metabolic pathways annotated in the kyoto encyclopedia of genes and genomes (kegg) database ( ) were used as gene sets. to construct the network of btms from peripheral blood mononuclear cell (pbmc) transcriptomes, genes were preranked by the wald test statistics score calculated with deseq package comparing each gene in covid- patients and healthy controls, as described ( ) . btms detected with a false discovery rate (fdr) adjusted p < . were then linked by the number of genes shared between two gene modules. to perform the btm-driven meta-analysis between respiratory viruses, gene lists from each dataset were preranked by log fold change of experimental samples over healthy controls. gene modules significantly associated with at least % of the datasets were selected by a nominal p < . for pbmcs and whole blood. the datasets were not merged at the single-gene-level. each dataset was composed by a different number of genes and samples, and different types of samples ( table ). the output of the gsea provides a normalized enrichment score (nes) for each btm associated with each dataset. the nes was then compared between datasets selected at the determined cut-off (p < . ). to enforce confidence in the enrichments, we also retained only the btms that were associated with at least % of the datasets, independently of infection, sample type and regulation. metabolic pathways from kegg database were selected by a fdr adjusted p < . for pbmcs from covid- patients. for balf datasets (cra and hra ), genes were also pre-ranked by log fold change of experimental samples over healthy controls and used as input in pre-ranked gsea. btms and kegg metabolic pathways were selected by relaxed significance (nominal p < . ) and consistent up-or downregulation in both datasets. for lung biopsies (gse ), one sample from covid- patients shows a distinct read count profile and was considered an outlier as described ( ) . the remaining sample was used to perform single sample gsea, in which genes were pre-ranked by log fold change of the experimental sample over healthy controls. networks were visualized and generated with cytoscape v . . ( ) . heat maps were generated with the package gplots for r and hierarchical clustering with the package amap for r, using euclidian distance metric and ward linkage. the bubble plots were generated with the package ggplot for r. graphpad prisma v. was used to perform t-tests on nanostring ncounter data and generate bar plots. to evaluate the robustness of our approach, validate previous findings and obtain novel perspectives into immune responses to sars-cov- infection, we constructed a modular transcriptional network of pbmcs from covid- patients. genes were pre-ranked by the wald test statistics score calculated with deseq package [ [, and used as input in pre-ranked gsea. we interpreted the dynamics in gene expression of covid- patients using the alternative tool to conventional pathways, the btms, which were particularly devised to evaluate human immune responses ( ) . to ensure maximal confidence, we applied a conservative statistical cutoff (fdr adjusted p < . ) to select significant btms ( figure a) . the transcriptional network captured several cellular characteristics of sars-cov- infection in peripheral blood, including t and nk cell ( figure d ) cytopenia ( ) , and upregulation of cell cycle or genes associated with plasma cells and immunoglobulins ( ) . in addition, our approach also detected increased signals of monocytes (figure b) , dendritic cells ( figure c ) and of the mitochondrial respiratory electron transport chain in sars-cov- infection (figure a) , suggesting a critical role of metabolic pathways for the immune response of covid- patients. to gather further insights on host responses to sars-cov- infection, the modular transcriptional signature of covid- patients was compared to that of individuals infected with sars-cov- or iav. for this, we analyzed additional public transcriptome datasets, spanning over samples from human pbmcs or whole blood. gene lists from each dataset were preranked by the log fold changes relative to healthy controls and used as input in pre-ranked gsea. the statistical cutoff was established at nominal p < . , whereas only bmts present in at least % of datasets are shown (figure a) . independently of the cohort, technology to quantify gene expression (rnaseq or microarray) and type of sample (pbmcs or whole blood), we observed a core transcriptional response that is comparable between infections caused by sars-cov- , sars-cov- , and iav. this core response includes modules of cell cycle and proliferation, monocytes and dendritic cells. indeed, the module m (dendritic cells) was upregulated in almost all datasets. of interest, sars-cov- and iav infections also induced significant reduction of peripheral t lymphocytes and nk cells. datasets from iav infection induced activation of type i interferon/antiviral responses or rig- like receptor signaling, while only sars-cov- induced significant association to one module, antiviral ifn signature. data from a different cohort of patients and analytical platform also demonstrated that several genes involved in type i interferon/antiviral responses were not significantly altered in whole blood of covid- patients (figure b) . we also evaluated btms that were uniquely associated to the transcriptomes from covid- patients, which showed enrichment in immune-related modules and heme biosynthesis (figure c) . data indicates an upregulation of heme biosynthesis in pbmcs from covid- patients ( figure d ). because immune responses are tightly connected to metabolic programs ( , - ), we explored metabolic pathway enrichment with the kegg database. in addition to porphyrin metabolism, which shares significant proportion of genes with btm m (heme biosynthesis ii), our analysis confirmed the upregulation of glycolysis and gluconeogenesis ( ), and detected other pathways such as tricarboxylic acid (tca) cycle, oxidative phosphorylation, tryptophan metabolism, glycan degradation, nucleotide metabolism and galactose metabolism (figure e ). because the lung is the primary site of infection and failure of this organ is a severe complication of sars-cov- infection, we also evaluated immune and metabolic signatures in the lower respiratory tract of covid- patients. for that, we performed a btm-driven meta-analysis of transcriptomes from samples of bronchioalveolar lavage fluid (balf) ( ) . using a relaxed statistical cutoff (nominal p < . ), there were nine significant btms and three kegg metabolic pathways that were consistently up or downregulated among both datasets ( figure a) . btms reflect upregulated networks of chemokines and neutrophils, as well as reduced expression of genes related to dendritic cells, monocytes, and t cell activation. we also found consistent upregulation of the modules related to chemokines ( figure b ) and neutrophils ( figure c ) in lung tissue data from one covid- patient. few metabolic pathways were consistently regulated between the balf datasets, including the upregulation of oxidative phosphorylation and downregulation of fructose and mannose metabolism and other glycan degradation ( figure a) . none of these metabolic pathways were significantly enriched on the sample of lung tissue. here, we used a robust modular transcriptomics approach that captured significant changes of cellular patterns in peripheral blood of covid- patients, including t lymphopenia and reduced numbers of nk cells ( ) . several hypothesis have been formulated to explain the lymphopenia during covid- , including t cell infection by sars-cov- ( ), or t cell exhaustion ( ) . in addition, we identified upregulated expression of chemokines and neutrophils in the lung tissue and balf of covid- patients that support an immunopathological role for these granulocytes ( ) . these data are in line with findings by zhou et al. ( ) , which also suggest higher proportion of neutrophils, activated dendritic cells and activated mast cells via cell deconvolution of balf transcriptomes. interestingly, our data suggest increased proportion of monocytes and dendritic cells in the circulation, but not in the balf. using single-cell rna-seq, some studies demonstrated that dendritic cells are indeed reduced in the balf ( ) and there are significant phenotypical alterations of monocytes from covid- patients compared to healthy controls ( ) . we demonstrated that compared to sars-cov- or iav, sars-cov- infection fails to induce significant type i interferon responses in pbmcs (figure a) or whole blood (figure b) , which corroborates the low concentrations of type i interferon in the circulation of covid- patients ( , , ) . these findings contrast with induction of isg expression in both lung tissue ( ) and balf ( ) of covid- patients, while recent studies indicate that type i and iii interferons negatively affect the lung epithelium during viral infections ( , ) . the transcriptional response of peripheral leukocytes reflects the systemic adaptations to the inflammatory environment imposed by sars-cov- infection, whereas type i interferon signaling in peripheral leukocytes might affect immunity in other organs such as the kidneys ( ) . importantly, recent data suggest an improvement of patients with uncomplicated covid- treated with interferon-alpha b ( ) . we expect that several factors will contribute to differences in transcriptional profiles of larger cohorts of covid- patients, especially those bearing comorbidities associated with severe disease. higher expression of angiotensin-converting enzyme (ace ) has been suggested as a potential mechanism of susceptibility of individuals with comorbidities associated with covid- ( ) . however, severe disease and death also occur after infection of otherwise healthy individuals, indicating that a series of mechanisms account for the severity of covid- . upregulated expression of genes that coordinate heme biosynthesis has been described in sepsis secondary to pneumonia and suggest a protective mechanism against oxidative stress ( ) . hypoxia also modulates the expression of genes coding for proteins that coordinate heme biosynthesis ( ) . we hypothesize that excessive heme accumulation could amplify pro-inflammatory cytokine production ( , ) or cause intravascular coagulation ( ) and promote pathology during covid- . strikingly, we observed the modulation of several metabolic pathways in pbmcs and balf, while oxidative phosphorylation was the only significant metabolic pathway overlapping in both compartments. this suggests a critical role for mitochondrial activity during covid- . many metabolites composing the pathways identified in the current study have been quantified via metabolomics of plasma or serum from covid- patients ( , ) . mass spectrometry measurements revealed the modulation of pathways such as tca cycle and fructose and mannose metabolism ( ) , tryptophan metabolism, glycolysis and gluconeogenesis and others ( ) . metabolomics analysis of human pbmcs infected with iav showed activation of tryptophan metabolism and glycolysis, whereas glucose consumption via hexosamine biosynthesis underlies the cytokine storm promoted by iav infection ( ) and could also affect covid- . taken together, this study demonstrates unappreciated inflammatory networks and metabolic pathways that are associated with covid- . publicly available datasets were analyzed in this study. this data can be found here: genome sequence archive (gsa) or human gsa in national genomics data center, beijing institute of genomics (big), chinese academy of sciences for rna-seq data related to sars-cov- infection (cra and hra ); gene expression omnibus (geo) for rna-seq data related to sars-cov- infection (gse ) and microarray data related to sars-cov- infection (gse ) or influenza a virus (iav) infection (gse , gse , gse , gse , gse , gse , gse , gse , gse , gse ); and arrayexpress for nanostring ncounter data related to sars-cov- infection (e-mtab- )/b. lg: selected the data, performed data analysis, interpreted the results, and wrote the manuscript. cs, hs-c, and sf: interpreted the results and critically reviewed the manuscript. all authors contributed to the article and approved the submitted version. pandemic fear" and covid- : mental health burden and strategies clinical characteristics of , covid- patients: a meta-analysis infection and rapid transmission of sars-cov- in ferrets integrative metabolomics and transcriptomics signatures of clinical tolerance to plasmodium vivax reveal activation of innate cell immunity and t cell signaling blood transcriptional profiling reveals immunological signatures of distinct states of infection of humans with leishmania infantum transcriptomics in human challenge models. front immunol transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood 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recognition type i and iii interferons disrupt lung epithelial repair during recovery from viral infection multiorgan and renal tropism of sars-cov- interferon-alpha b treatment for covid- ace expression is increased in the lungs of patients with comorbidities associated with severe covid- genetic signature related to heme-hemoglobin metabolism pathway in sepsis secondary to pneumonia hypoxia decreases the expression of the two enzymes responsible for producing linear and cyclic tetrapyrroles in the heme biosynthetic pathway characterization of heme as activator of toll-like receptor hemolysis-induced lethality involves inflammasome activation by heme excess of heme induces tissue factordependent activation of coagulation in mice proteomic and metabolomic characterization of covid- patient sera plasma metabolomic and lipidomic alterations associated with covid- o-glcnac transferase promotes influenza a virus-induced cytokine storm by targeting interferon regulatory factor− the authors thank the administrative and technical support provided by the iptsp -ufg. we thank all authors that contributed to the generation of the datasets used in the study. we also thank dr. key: cord- -mkz z o authors: hou, dongni; chen, cuicui; seely, eric john; chen, shujing; song, yuanlin title: high-throughput sequencing-based immune repertoire study during infectious disease date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: mkz z o the selectivity of the adaptive immune response is based on the enormous diversity of t and b cell antigen-specific receptors. the immune repertoire, the collection of t and b cells with functional diversity in the circulatory system at any given time, is dynamic and reflects the essence of immune selectivity. in this article, we review the recent advances in immune repertoire study of infectious diseases, which were achieved by traditional techniques and high-throughput sequencing (hts) techniques. hts techniques enable the determination of complementary regions of lymphocyte receptors with unprecedented efficiency and scale. this progress in methodology enhances the understanding of immunologic changes during pathogen challenge and also provides a basis for further development of novel diagnostic markers, immunotherapies, and vaccines. introduction the adaptive immune system is composed of b and t cells that form a highly selective guard against evolving pathogens. the foundation of the adaptive immune response is based on the enormous diversity of t and b cell antigen receptors that can recognize epitopes from a near infinite number of different internal and external antigens. this profound diversity of t (tcrs) and b cell receptors (bcrs) is generated by v-d-j gene recombination of the tcr/bcr locus and subsequent somatic hypermutation and class-switching recombination of b cells after antigen stimulation. thus, study of the immune repertoire, portrayed as the antigen-specific information within lymphocytes, has been a key to understanding the response of adaptive immunity during infection. despite extensive efforts using traditional techniques, analysis of the immune repertoire with high resolution has remained difficult. several sequencing strategies, for example, sanger sequencing, have been implemented to determine cdna segments encoding variable regions of immunoglobulin (or tcrs) ( , ) . however, these low-throughput techniques lack the power to provide a broad picture of the full immune repertoire. during the past two decades, however, technical advances in high-throughput sequencing (hts), also known as next-generation sequencing (ngs), along with evolving bioinformatic and statistical tools, have provided a new approach capable of analyzing the immune repertoire at the single sequence level. these methods create an unprecedentedly high-resolution picture of the immune repertoire and also provide massive data that cover each lymphocyte from the sample, in theory, dispensing with limitation of sequencing quantity ( ) . considering the extremely important role of the adaptive immune system in defending against infectious agents, hts has great potential to aid in the discovery novel infectious agents and also offers new approaches for antibody or vaccine development. in this review, we introduce the implementation of hts to the study of the immune repertoire and review the associated bioinformatic tools required for data processing and analysis. we then focus on the success of this technology in facilitating the exploration of infection-related immune repertoires for clinical diagnosis, treatment, and prevention. amazing diversity makes the immune system the most effective system to fight against a broad scope of disease causing pathogens. this repertoire is generated by a complex series of genetic events ( ) . for t cells, the variable region of each tcr chain consists of three complementary determining regions (cdrs) and four frame regions (frs) . cdrs are the variable portion of the receptor and determine the antigen specificity. while cdr and cdr are formed by variable (v) gene, cdr is generated by random selection and recombination of variable (v), diversity (d), and joining (j) gene segments in the heavy chain (v and j region gene segments in light chain) ( , ) (figure ) . thus, cdr is the most diverse component of a receptor, which binds mhc molecules and (or) antigens. construction of the tcr with an alpha chain and a beta chain is also a process that contributes to receptor diversity. the formation and revision of the t and b cell lymphocyte receptor repertoire is a highly dynamic process. the number of each lymphocyte clone changes dramatically and depends on cell specificity and the history of antigen exposure. when encountering exogenous antigens, t cells that express receptors capable of binding to a specifically compatible peptide-mhc (pmhc) complex will expand, resulting in a massive population of antigen-specific t cells that initiate the adaptive immune response ( ) ( ) ( ) ( ) ( ) . this antigen-driven proliferation process of t cells is distinct between cd + and cd + t cells after initial antigenic stimulus. although these two types of t cells show comparable protein expression, proliferation rate, and transcriptome features after days of non-infective stimulation, subsequent division of t cells differently depends on continuous existence of self-pmhc complexes. cd + t cells proliferate in a limited pattern, and its subsequent response requires persistent stimulation from antigen-presenting cells. cd + t cell is "programed" to extensive expansion after short stimulation even when transferred into antigen-free hosts ( ) . the post-antigen stimulation response of b cells is more complicated because it is accompanied by somatic hypermutation and class-switch recombination that offer additional diversification of the b cell repertoire ( ) . somatic hypermutation is the process of introducing point mutations at cdr , cdr , cdr , and fr to produce b cells of higher affinity to target antigens. these higher affinity clones are then selected and expanded, which is called affinity maturation. additionally, in class-switch recombination, the gene loci encoding the c region of bcrs are excised and replaced by a series of new constant gene segments, resulting in functional differences of igg, ige, or iga that participate in different immune mechanisms during pathogen elimination. traditional strategies for studying the immune repertoire prior to hts, many strategies were developed to explore postinfection immune repertoires ( ). immunoscope spectratyping has been used to investigate tcr/bcr repertoires since the s ( , ) . in this technique, using one (for b cell) or more (for t cell) v or j gene specific primer pairs, the length of cdr can be determined ( ) . cdr length in healthy population shows a bellshaped pattern, indicating a polyclonal repertoire. however, the unusual peaks in infected patients imply a perturbed oligoclonal repertoire with clonal expansion. as such, cdr spectratyping provides robust information on the complexity and stability of circulating t/b cell repertoires and insights into the immune repertoire after infection ( ) ( ) ( ) ( ) ( ) . even if it is relatively easy and cheap, the nucleotide sequences of cdr remain obscure, and the extent of heterogeneity within a particular cdr length cannot be assessed. detailed nucleotide sequences of gene segments encoding the variable region can be determined by traditional dna sequencing techniques such as sanger sequencing ( , ) . flow cytometry and cdr spectratyping help to isolate t/b cells of interest, which complements weaknesses of sanger sequencing in quantity limitation. single-cell sequencing is able to identify sequences of several b cells that produce monoclonal antibodies specific to certain virus, which contributes greatly to analyzing genetic features of the antibodies in the process of antibody discovery ( , ( ) ( ) ( ) . after collecting peripheral blood samples, the b cells or memory b cells are isolated and immortalized to produce antibodies. according to the hai titers and neutralizing titers determined by elisa, the virus-specific b cells can be identified, which helps to narrow b cell candidates for sequencing. these functional test-based antibody discovery strategies are successful but laborious. despite this, these strategies are well designed for targeted antibody searching; however, it is insufficient for creating a high-resolution picture of the human immune repertoire. high-throughput sequencing has recently become a novel and powerful tool to investigate the immune repertoire. the depth and comprehensiveness of high-throughput immune repertoire sequencing are greater than ever, and the enormous sequencing data of disease-specific tcr/bcr clones provide great potential for the revealing dynamic changes in clonality during infectious states. establishing a lymphocyte repertoire database starts from sample collection from carefully selected populations and isolation of interested t cell or b cell subgroups. due to the well-acknowledged heterogeneity of tcrs and bcrs between individuals, longitudinal studies tracking dynamic alterations in certain population help to reduce difficulties in data interpretation at unraveling the infection course. classification of subgroups of t cell and b cells, e.g., naive and memory t/b cells, cd +, and cd + t cells, is necessary if distinct behavior of these subgroups is considered in detail. the methodology of library preparation and amplification need careful design since it affects accuracy of the ultimate sequencing data. due to the difference of v and j gene segments, a common primer does not apply to sequencing of cdr . multiplex pcr is capable to amplify multiple loci simultaneously and, however, is likely to introduce bias. it is because of non-specific amplification, primer-dimer formation, and uneven reaction conditions. more precise and quantitative multiplex pcr may be achieved through primer concentration adjustment and bias filtering using amplification bias among the templates as controls ( ) . another alternative pcr method is ′race pcr, which provides a less biased pcr library using primers that bind downstream of the variable domain ( ) . sequencing techniques are evolving continuously to be deeper and more precise, and there are three prevalent hts platforms available today. the comparison of mechanisms, sequencing depth, and other critical features of each platform is shown in table . the illumina and roche platforms have been most commonly used during immune repertoire analysis. the outputs of each platform must be analyzed with caution because of notable platform-specific sequencing error ( ) ( ) ( ) ( ) . insertions and deletions of nucleotides, resulting from imperfect interpretation of homopolymeric stretches, are considerable for the roche platform ( ) , while substitution errors are predominant in illumina platform ( , ) . the overall error rate of illumina platform is lowest while that of ion torrent is highest among the three ( ) . in an attempt to correct sequencing errors, three algorithms are most commonly used including k-mer spectrum, multiple sequence alignment, and suffix tree ( ) . based on ( ) . pcr and sequencing errors inevitably result in overestimate of repertoire diversity. the common statistical strategy for both pcr and sequencing error removal is eliminating low abundance and low-quality sequences (with low phred score), but it leads to a great loss of sequencing information. to rescue these sequences, low-quality cdr sequences with no more than three low-quality nucleotides can be mapped to "core clonotypes" derived from high-quality sequences with allowed mismatches at low-quality position. then, the low-abundance core clonotypes are merged with the high-abundance core clonotypes with less than three allowed mismatches at v (≤ mismatches), d (≤ mismatches), and j (≤ mismatches) gene segments to correct pcr errors. this integrated algorithm based on sequence quality abundance could efficiently correct artificial errors and avoid information loss, thus providing more reliable estimation of repertoire diversity ( ) . unique molecular identifiers that label each starting molecule help to reduce both pcr and sequencing errors ( , ) . combined with this experimental strategy, molecular identifier groups-based error correction (migec) corrects pcr and sequencing errors more efficiently than other quality-and frequency-based strategies ( ) . determination of the v-d-j gene segments from which the cdr s are rearranged, as well as identification of point mutations, is often achieved using the immunogenetics database (http:// www.imgt.org) ( ) , despite the controversies about its validity ( ) ( ) ( ) . new v-d-j gene annotation tools based on various algorithms are reported, such as igblast ( ), ihmmune-align ( ) , and decombinator ( , ) ( table ). in addition, many integrated bioinformatic tools (mitcr, lymanalyzer, change-o, etc.) for data processing are developed recently ( - ) ( table ) , which provides various statistical approaches for diversity estimation, repertoire comparison, clustering analysis, and somatic hypermutation analysis ( table ) . despite these tools, standardized bioinformatic analysis and visualization strategy is lacking, which remains the main obstacle for comparison of researches from different investigators. high-throughput sequencing techniques have revolutionized the study of the immune repertoire. utilizing hts, many important insights into mechanisms of immune response have been gained. it is also the cornerstone for potential clinical applications of repertoire analysis, including identification of diagnostic biomarkers, design of therapeutic antibodies, and development of new vaccines. estimating the diversity of a tcr/bcr repertoire is necessary for estimating the theoretical size of the repertoire and for tracking changes in clonal populations during the clinical course of infection. several different methods may be used to describe the diversity of lymphocyte repertoires at different levels -vdj recombination diversity ( ), simpson diversity index ( , ) , and some non-parametric methods. decrease in the overall diversity of the immune repertoire have been observed after various antigen exposures, including hiv, influenza, and human herpes virus, which implies expansion of particular t/b cell clones ( , , , ) . our group compared changes in the diversity of the tcr beta chain and bcr heavy chain after h n virus infection. interestingly, these results show that the diversity of the bcr heavy chain starts to increase weeks after h n infection, while the tcr beta chain repertoire continues to contract. in addition, a more diverse bcr repertoire and a less diverse tcr beta chain repertoire in convalescent phase correlate with improved prognoses, implying differences in the response process of humoral and cellular immunity. the immune response to vaccination has been used as an ideal model for antibody repertoire research due to the convenience drawing blood samples at well-defined time points ( ) . studies using vaccines, such as influenza and tt, have revealed dynamic changes in the size and diversity of antibody repertoires before and after antigen stimulation ( , , , ) . comparison of post-vaccination responses suggests divergent repertoire properties among individuals, different age groups, and successive immunization of the same individual with different influenza vaccines (tiv and laiv) ( , , ) . the maximum clonal response has been found to occur days after vaccination, but the magnitude of response varies between individuals despite an identical immune challenge, which may be influenced by previous exposure, age, and other concurrent immune responses. in addition, study of b cell memory can be achieved by repeated sequencing of samples taken from the same individual in separate immune responses to an antigen. vollmers and colleagues identified a group of b-cell clones as a recall response to two substantially different vaccine compositions, implying the possibility of identifying cross-specific antibody using repertoire analysis ( ) . immune responses recorded by sequencing data have also been useful in testing the role of adjuvants in eliciting broad-spectrum antibodies. wiley and co-workers tested the immune response of mice immunized with malaria vaccine by analyzing igg repertoires. they found that tlr agonist used as adjuvant increases the diversity of igg variable region, which is related to improved ability of the antibodies to recognize a broad spectrum of epitopes ( ) . these studies exemplify a new level of details assessing vaccine response and pioneered hts implications in vaccine design. in infected patients, antigen-specific t/b cell repertoires form in response to antigen exposure in both circulation and peripheral tissues. immune repertoire sequencing provides broad information including crucial antigen-specific clones, which have the potential to halt the spread of pathogens ( ) . diagnostic marker discovery using sequencing data relies on these antigen-specific clones with stereotyped features in the post-infection repertoires. these features are assessed at different levels such as gene rearrangement, identical or similar cdr sequence overlap, and certain cdr s length. after influenza h n vaccination, the dominant clonotype of ig heavy chains has the same v-j gene rearrangement, cdr length, and somatic mutation position in cdr and cdr with previously reported influenza antibodies ( ) . however, in this study, the convergent dominant sequence is only found in one individual. further researches in a broader population including non-dominant sequences are needed. a more successful example is reported in ig repertoires related to dengue virus infection. using cross validation and other approaches, stereotyped cdr sequences or cdr lengths that have high prevalence in the acute dengue samples are found to be specific to acute dengue infection, which are absent or of low prevalence in healthy and post-convalescent population ( ) . identification of pathogen-specific sequences also helps in differential diagnosis between infectious and non-infectious diseases. according to dziubianau et al., comparing pbmcs-derived t cell clonotypes specific to a given virus with t cells from different origins (allograft-derived and urine-derived lymphocytes) provides a new methodology for differential diagnosis of two post-transplant complications -bkv-associated nephropathy and acute cellular rejection, which shows a glimpse of applications of t cell sequencing in diagnosis ( ) . in addition, a recent study searched sequencing data for cdr amino acid motifs that have been reported to be specific for a particular pathogen and succeeded in identifying cdr sequences identical or similar to these motifs in post-vaccination volunteers ( ) . according to these results, interestingly, it is low frequency sequences that possess the probability of becoming promising biomarkers instead of the dominant ones. however, immune responses show dramatic differences in cdr sequences responding to same pathogens across individuals and age groups. this intrinsic divergence between individuals is the major obstacle in finding "public" sequences as optimal biomarkers. nevertheless, we hold a promise for the application of hts data in differential diagnosis because it provides a large number of candidate sequences for biomarker investigation. instead of using single biomarkers such as psa or afp in diagnosis, a combination panel of selected sequences may establish a pathogen-specific sequence library for diagnosis, which holds the potential of unprecedented sensitivity and specificity. recombinant monoclonal antibodies have great potential in the treatment of specific infections. in recent years, several strategies, including phage display libraries, single b cell expression, and b cell immortalization, have been used to discover antibodies against specific antigens ( ) . hts of the antibody repertoire, combined with subsequent bioinformatic tools or traditional screening tools, has facilitated the identification of antigenspecific sequences ( ) ( ) ( ) ( ) . the methodology predicting antigen specificity completely from analysis of bcr sequences has not been possible yet, albeit it has considerable potential in immune repertoire studies. nevertheless, there have been many efforts made in mining the hts data for functional antibodies ( ) . a relatively direct method for identifying such sequences is based on the similarity of amino acid sequence to previously reported antibodies. researchers have successfully found sequences of high identity with the broadly neutralizing antibodies and strain-specific antibodies from established antibody repertoires of patients with influenza infection or vaccination ( ) . some of these sequences have proven to have neutralizing activity, validating the potential of deep sequencing-based antibody identification. success of this work also suggests the possibility of monoclonal antibody synthesis without cell cloning for treatment ( ) . furthermore, another method using the frequency rank of heavy chain and light chain sequences to predict the function of antibody sequences has been reported successful in mouse models ( ) . exciting work by kwong and coworkers demonstrates the feasibility of identifying neutralizing active clones through bioinformatic analysis from hiv patients ( , ) . they established several steps for interrogating variants of known neutralizing antibody classes from hiv-infected patients, with or without previous knowledge if the patient had antibodies belonging to this family. first, the heavy or light chain sequences, derived from the germline ighv or iglv gene same as template antibody, are isolated from a new donor. then, these sequences were compared with the germline gene for "divergence" and the template antibody sequence for "identity, " generating a contour plot called "divergence/identity plot. " the sequences segregate into clusters in the plot, from which the high divergence and high identity sequences were selected as candidates for neutralizing antibodies. this process is called "grid-based strategy. " then, the germline sequence, candidate sequences, and template antibody sequences of the same class are merged to build a phylogenetic tree rooted by the template sequence, which is called "cross-donor analysis. " the heavy chain sequences from new donor clustered in the subtree of the template sequences are then expressed with template light chain to generate antibodies. most of these sequences have neutralizing activity to hiv- . the outstanding efficiency of this method was also demonstrated when compared to sequence-based strategy and prevalence-based strategy ( , ) . lu et al. also validated this phylogenetic method in identifying functional anti-staphylococcus aureus antibodies ( ) . these strategies start from previously reported antibody sequences. however, such antibody sequences are not always available, especially during poorly characterized viral infections such as h n . pairing the heavy and light chains as an integrated antibody has been another challenge for hts-based immune repertoire analysis. in most cases, researchers only focus on the heavy chain, which causes a critical loss of antibody integrity and leads to problems in following synthesis of artificial monoclonal antibody. two strategies have been reported to correctly pair the heavy chain and light chain sequences based on the frequency or evolution models. reddy and colleagues ( ) have pioneered pairing based on the frequency ranks, using plasma cells isolated from bone marrow of immunized mice and matching the two chains of similar rank order. monoclonal antibodies expressed in this way did show antigen specificity. due to the linkage of heavy chain and light chain as an integrated protein, their evolution undergoes the same enzymatic mutation process, and they evolve together to bind the same antigen with high affinity. based on this theoretical foundation, phylogenic analysis has been used as another method to compare the evolutionary topography of the heavy chain and light chain after bioinformatic identification of transcripts related to a known hiv neutralizing antibody ( , ) . reconstituted novel antibodies consist of phylogenetically matched chains showing similar neutralizing function but less auto-reactivity compared to the mismatched ones. several groups have recently achieved advances in the technology of paired sequencing of antibodies. single-cell pcr has been utilized to create a two-dimensional bar-coded primer matrix to link two chains of the bcr ( ) . using this technique, busse and coworkers analyzed paired sequences of over , b cells in one experiment and accomplished subsequent antibody gene cloning and expression. at the same time, turchaninova and coworkers performed pioneering research in emulsion-based technology for sequencing antibody repertoires of paired chains ( ) . they used water-in-oil emulsions for cell-based overlap expansion rt-pcr, although its yield was relatively low yield. another high-throughput paired sequencing method by dekosky et al. used micro well plates to isolate b cells and magnetic beads to capture mrnas ( ) . very recently, dekosky's group combined and improved these previous techniques, and developed a costeffective and efficient methodology to establish a more precisely paired repertoire ( ) . predicting t cell specificity based on tcr heterodimer sequence is more difficult than antibodies because of the highly variable nature of each of the components of the tcr-peptide-mhc complex ( ) . due to the challenges posed by the highly variable cdr loop of the tcr and the complexity of predicting protein-protein interactions ( , ) , experimental functional tests for mining antigen-specific t cells might be a more fruitful approach ( ). recent advances in hts-based antibody sequencing may provide the biggest benefit for the field of vaccine development. over the years, efforts to elicit protective immune responses to hiv by immunization have not been successful. during acute viral infections, high-affinity neutralizing antibodies develop in just weeks. however, generating effective broadly neutralizing antibodies during chronic infections, such as hiv, takes significantly longer time. furthermore, the neutralizing power of these antibodies is often variable due to impairment of the host immune function, unusual features of env, and co-evolution of the virus in response to the host antibody response ( , ) . deep sequencing analysis has identified rare variants of known hiv-neutralizing antibodies and has elucidated the ontogeny of these neutralizing antibodies ( , , , ) . these findings have cast a light on antibody guided vaccine development. in following studies, the hts-based phylogenetic strategy greatly facilitated study in co-evolution of neutralizing antibodies and virus mutants ( ) . combined with long-term follow-up studies, these results illustrate how mutations in some sites allow the virus to escape some neutralizing antibodies, and how the virus, with the help of secondary neutralizing antibodies, becomes sensitive to the neutralizing antibody ( , , ) . these studies suggest a promising pathway to elicit broadly neutralizing antibodies by sequential immunization with selected immunogens ( , ) . furthermore, structure studies of the neutralizing antibody family provide candidates for future vaccine designs ( ) . high-throughput sequencing has been a breakthrough technology for the study of the immune repertoire and has already had a profound effect on our knowledge of the immune systems physiology during health and disease. in particular, hts has transformed our understanding of immune repertoire formation during infection, malignancy, and autoimmunity. advances in this filed of research will rely on progress of similar laboratory techniques. collection of more precise sequencing data can be anticipated with consistent improvement of sequencing techniques and error correction strategies. an increasing number of researchers have shown interest in this area in recent years, and this has provided vast quantities of data that could provide answers to important existing questions. these data repositories should be effectively utilized. establishing a public database and collecting deep sequencing data for collective collaboration will facilitate information exchange and the investigation of the varieties of repertoires across gender, age, race, and healthy state. in addition, development of standardized bioinformatic tools will be indispensable for harnessing hts output. although the expected potential of immune repertoire studies in clinical use is enormous, more work remains to be done to incorporate the observed dynamic changes and sequence signatures with clinical features and outcomes. questions remain about how the severity of certain infections is related to alterations of the immune repertoire response and various manifestations of cdr sequences, and how to predict abundance of protective immunoglobulins or t cell from a given sequence library. in terms of therapeutic discoveries, identification and production of functional antibodies and t cells will promote the development of passive immune therapies and vaccines. traditional and recently reported large-scale screening strategies may contribute greatly to this process. advances in hts of the immune repertoire during health and disease will provide comprehensive views of the adaptive immune response in very near future and will open the door to more rationale immunotherapy for infection. dh was involved in study design, wrote the first draft of the manuscript, conducted the literature search, reviewed the abstracts, performed the analysis, and contributed to the final draft; cc and sc were involved in study design and reviewed the abstracts. es revised the manuscript. ys designed and supervised the study, revised the final draft, and contributed to the analysis. all authors have read and approved the final manuscript. analysis of the b-cell progenitor compartment at the level of single cells tools to therapeutically harness the human antibody response sequence analysis of t-cell repertoires in health and disease immunology: the roots of 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variable domain sequence analysis tool ihmmune-align: hidden markov model-based alignment and identification of germline genes in rearranged immunoglobulin gene sequences decombinator: a tool for fast, efficient gene assignment in t-cell receptor sequences using a finite state machine soda : a hidden markov model approach for identification of immunoglobulin rearrangements model for comparative analysis of antigen receptor repertoires personalized, sequencing-based immune profiling spurs startups preparing unbiased t-cell receptor and antibody cdna libraries for the deep next generation sequencing profiling change-o: a toolkit for analyzing large-scale b cell immunoglobulin repertoire sequencing data immunexplorer (imex): a software framework for diversity and clonality analyses of immunoglobulins and t cell receptors on the basis of imgt/highv-quest preprocessed ngs data lymanalyzer: a tool for comprehensive analysis of next generation sequencing data of t cell receptors and 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analysis by next generation sequencing allows complex differential diagnosis of t cell-related pathology identification of antigen-specific b cell receptor sequences using public repertoire analysis selecting and screening recombinant antibody libraries synthetic antibodies designed on natural sequence landscapes construction of a rationally designed antibody platform for sequencing-assisted selection shaping of human germline igh repertoires revealed by deep sequencing deep sequencing of phage display libraries to support antibody discovery the application of next generation sequencing to the understanding of antibody repertoires monoclonal antibodies isolated without screening by analyzing the variable-gene repertoire of plasma cells de novo identification of vrc class hiv- -neutralizing antibodies by next-generation sequencing of b-cell transcripts mining the antibodyome for hiv- -neutralizing antibodies with next-generation sequencing and phylogenetic pairing of heavy/light chains identifying functional anti-staphylococcus aureus antibodies by sequencing antibody repertoires of patient plasmablasts somatic populations of pgt - hiv- -neutralizing antibodies identified by pyrosequencing and bioinformatics single-cell based high-throughput sequencing of full-length immunoglobulin heavy and light chain genes pairing of t-cell receptor chains via emulsion pcr high-throughput sequencing of the paired human immunoglobulin heavy and light chain repertoire in-depth determination and analysis of the human paired heavy-and light-chain antibody repertoire beyond model antigens: high-dimensional methods for the analysis of antigen-specific t cells cdr loop flexibility contributes to the degeneracy of tcr recognition deconstructing the peptide-mhc specificity of t cell recognition combinatorial hla-peptide bead libraries for high throughput identification of cd (+) t cell specificity broadly neutralizing antibodies and the search for an hiv- vaccine: the end of the beginning human antibodies that neutralize hiv- : identification, structures, and b cell ontogenies focused evolution of hiv- neutralizing antibodies revealed by structures and deep sequencing co-evolution of a broadly neutralizing hiv- antibody and founder virus cooperation of b cell lineages in induction of hiv- -broadly neutralizing antibodies maturation and diversity of the vrc -antibody lineage over years of chronic hiv- infection b-cell-lineage immunogen design in vaccine development with hiv- as a case study structural repertoire of hiv- -neutralizing antibodies targeting the cd supersite in donors key: cord- -gs c fy authors: schreiber, gideon title: the role of type i interferons in the pathogenesis and treatment of covid- date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: gs c fy type i interferons (ifn-i) were first discovered over years ago in a classical experiment by isaacs and lindenman, who showed that ifn-is possess antiviral activity. later, it became one of the first approved protein drugs using heterologous protein expression systems, which allowed its large-scale production. it has been approved, and widely used in a pleiotropy of diseases, including multiple-sclerosis, hepatitis b and c, and some forms of cancer. preliminary clinical data has supported its effectiveness against potential pandemic pathogens such as ebola and sars. still, more efficient and specific drugs have taken its place in treating such diseases. the covid- global pandemic has again lifted the status of ifn-is to become one of the more promising drug candidates, with initial clinical trials showing promising results in reducing the severity and duration of the disease. although sars-cov- inhibits the production of ifnβ and thus obstructs the innate immune response to this virus, it is sensitive to the antiviral activity of externally administrated ifn-is. in this review i discuss the diverse modes of biological actions of ifn-is and how these are related to biophysical parameters of ifn-i–receptor interaction and cell-type specificity in light of the large variety of binding affinities of the different ifn-i subtypes towards the common interferon receptor. furthermore, i discuss how these may guide the optimized use ifn-is in combatting covid- . type i interferons (ifn-i) are a family of cytokines that bind the type i interferon receptor, constituted of two transmembrane subunits, ifnar and ifnar ( figure ). the two receptors are constituted of an extracellular domain, which binds ifn-i, a transmembrane helix and an unstructured intracellular domain (icd) that binds jaks and stats ( , ) . jak is associated with ifnar and tyk with ifnar . stat and stat (and maybe also other stats) were found to be constitutively bound to the icd of ifnar ( ) ( ) ( ) . binding results in close proximity of the intracellularly associated jaks, jak and tyk , resulting in their activation through cross phosphorylation ( figure ) ( , ) . this also results in receptor phosphorylation, which role is still under debate ( , ( ) ( ) ( ) . the phosphorylated stats dissociate from the receptor and form homo and hetero dimers, which are transported to the nucleus, where they serve as transcription factors for a large number of genes. the most prominent effects are associated with stat /stat heterodimerization, which together with irf form the interferon-stimulated gene factor (isgf ), which bind a distinct group of target genes harboring the interferonstimulated response elements (isre). in addition to this, ifn-i drives stat /stat and stat /stat homodimerization, the formation of a stat /irf binary complex and more ( , ( ) ( ) ( ) (figure ). this leads to the transcription activation or suppression of over , genes, which drive a wide range of innate and adaptive immune functions. these, in turn respond against various pathogens, act as important regulators in tumor immunity and have a role in pathophysiology and autoimmune diseases ( , ( ) ( ) ( ) ( ) ( ) ( ) . stat knockout cells still activate a stat /stat response mediated by irf , while stat knockout cells activate a stat /irf -induced response ( ) . surprisingly, no change in the gene induction relative to wildtype cells was observed in stat knockout hela cells, despite the strong ifn-i-induced phosphorylation of stat . however, as ifn-i responses are cell-type specific, a stat /stat induced response may still be found in other cells than hela. due to this wide range of physiological responses, ifn-i has provided therapeutic benefits for multiple diseases, including multiple sclerosis, some cancers and viral diseases (hepatitis b and c) ( ) ( ) ( ) . due to the efficient activation of antiviral activities by ifn-is, most viruses have contemplated mechanisms to avoid its actions ( ) ( ) ( ) . for example, the ebola virus, which outbreak in central africa killed tens of thousands of people ( , ) , avoids ifn-i activity by producing the vp protein that binds the karyopherin alpha nuclear transporter. thereby, it inhibits the nuclear transport of phosphorylated stat , rendering cells refractory to ifn-is. another example of viral mechanisms that evolved to eliminate ifn-i functions in inducing innate immunity is given by the sars corona virus, where both the production of ifnb and the ifn-i induced signaling are attenuated. recently, a more infective version of sars has emerged, sars-cov- (which causes the covid- disease). covid- cases have been first reported by the end of in china, and rapidly became a world-wide epidemic with unprecedented consequences ( , ) . sars-cov- seems to have originated from horseshoe bats. similar virus strains that circulate in bats in hubei province in china may in the future cause further new zoonotic outbreaks ( ) . sars-cov- has % homology to the sars-cov virus that also spread from china in ( ) . sars-cov- proved to be much more infectious compared to the original sars virus, resulting in a global epidemic. as ifn-i drives strong antiviral activities, the mechanisms sars-cov and sars-cov- combat ifn-i activities has been a matter of intense research, with at least proteins being identified to counteract ifn-i functions in the sars-cov virus ( ) . in addition, ifn-is were implicated in contributing to the severity of the cytokine storm, which is a major complication of sars-cov and sars-cov- and can lead to respiratory distress syndrome (ards) and death ( , ) . in this review i will describe our current knowledge on the involvement of ifn-is in the development of the covid- disease, and how this relates to the different activities associated with type i interferons. type i interferon receptors are found on all cell types, and are a major component of the innate immune system. human type i interferons include similar ifnas with % homology between them and single ifnw, k, ϵ and b, with lower homology ( - %). all of them bind the receptor complex, composed of ifnar and ifnar at the same proximal location ( , , ) . despite structural similarities among the ternary ifn-i-ifnar -ifnar complexes, ifn-is drive a range of different activities, dependent on the cell type and the interferon subtype ( ) . this apparent paradox has major implications for understanding the role of ifn-i in health and disease and its varied applications as a drug against a pleiotropy of diseases. ifn-i signaling is initiated by binding of ifn-i to its receptor. it has been suggested that cytokine receptors are pre-associated, with ligand binding activating signaling through the induction of conformational changes ( ) . however, more recent singlemolecule receptor tracking on life cells has clearly shown that for many of the cytokines, its role is to bring the receptors into close proximity, which drives signaling ( ) . this seems to be the case also for ifn-i induction, as shown both using single receptor tracking and mutational analysis ( figure ) ( , ) . while structurally, the ternary ligand-receptor complex seems to be the same for all ifn-is, the binding affinity differs by many orders of magnitude. the tightest binding ifn-i is ifnb, which binds ifnar with nm affinity and ifnar with subnanomolar affinity. the different ifna subtypes bind ifnar with . to µm affinity and ifnar with to nm affinity, with ifna being the weakest binding ifna ( , ) . even weaker binding was measured for ifnϵ, with~ -fold reduced affinity relative to ifna proteins ( ) . interestingly, ifnϵ is constitutively expressed by the reproductive tract epithelium and is regulated by hormones during the estrus cycle, reproduction, menopause and by exogenous hormones. thus, its mode of action is different from other ifn-is ( ) . these large differences in binding affinity between ifn-i subtypes were suggested to result in major differences in biological activity. to obtain a better insight into the molecular mechanisms of their actions, ifna was engineered to cover the whole range of binding affinities of natural ifn-is to both the high affinity (ifnar ) and low affinity (ifnar ) receptor chains ( ) . these studies have shown that indeed, the binding affinity to both receptors is a major determinant of ifn-i activity ( ) . using both natural and engineered ifn-is has shown that even weak binding ifn-is activate the cellular antiviral program at very low (pm) concentrations ( ) . moreover, the antiviral program was activated in all cell-lines tested. despite the -fold higher affinity of ifnb over ifna towards binding ifnar receptors, its potency to elicit an antiviral response is similar. for example, in wish cells (originally thought to be of amniotic origin, but later found to be a hela (cervix cancer) contaminant) the ec for antiviral activity of ifna is . pm, while the ec for ifnb is . pm ( ) . wish cells have been extensively used to characterize ifn-i activity, including for definition of ifn-i unit activity. an upper limit for antiviral potency was further verified by engineering an ifna variant, yns-a -tail, with fold tighter binding to ifnar and -fold tighter binding to ifnar in comparison to ifna (thereby surpassing the receptor binding affinity of natural ifnb). still, the ec for antiviral activity is only -fold lower in comparison to ifna ( , ) . conversely to antiviral activity, ifnb is much more potent in activating the antiproliferative program relative to ifna , a result that was also verified using the ifna variant, yns-a tail ( ) . the ec for antiproliferative activity on wish cells is nm for ifna , pm for ifnb and pm for yns-a -tail. a similar increase in antiproliferative potency was observed also for ovcar and hela cells. interestingly, while antiviral activity was observed in all cell lines tested, some cell lines were not susceptible to ifn-i induced antiproliferative activity (for example t d and k ), independent on the concentration and subtype of ifn-i ( ) . to better understand the molecular basis for this finding, ifn-i induced gene expression was monitored using various ifn-i subtypes or engineered mutants on the background of different cell-lines. these experiments showed that low concentrations of weaker binding interferons activate the expression of mostly antiviral genes. higher concentrations of interferons activate also other genes, many of them related to immune-modulation ( ) . examples for such genes are chemokines such as cxcl and , which are involved in chemotaxis of t cells and natural killer cells, induction of apoptosis, regulation of cell growth and more. we gave the term of "robust" for the common ifn-i induced program (including its antiviral activity) and "tunable" for the other programs induced by ifn-is, which include between others antiproliferative and immunomodulatory activities ( ) . further investigations into these two programs has shown that cells with low receptor numbers activate only the robust program, and that not all cell types execute the tunable program, conversely to the robust program that is common to all cells ( ) . tighter binding ifn-is at higher concentrations are essential for the activation of the tunable program. genes upregulated by the robust program are mostly classical antiviral genes, such as mx and mx , oas and , pkr, ifit , and , isg , and many more. figure a according to string and go analysis, the commonly upregulated genes have a strong antiviral signature. the top go terms (fdr < − ) are response to type i interferon, innate immune response, response to virus, defense response and immune system process. it is interesting to note that antiviral genes constitute most of the upregulated genes common to all cell lines. antiviral genes are also the majority of upregulated genes in k and t d cells. conversely, ovcar and hela cells have many unique upregulated genes, many of them related to immunomodulatory functions, cell cycle, apoptosys and more. ifn-i. the diagram shows that genes are commonly upregulated by all cell-lines. figure b shows string protein interaction analysis of these common genes. clearly, these form a tightly interacting mesh of gene products. gene ontology analysis shows these genes to have an extremely high signature for antiviral activity and ifn-i activation. promoter analysis of common isgs has shown them to be driven by the classical isre promoter sequence ( ) . conversely, for tunable genes no clear promoter sequence was identified. the exact mechanism of how tunable genes are upregulated by ifn-i is thus not yet fully understood. from an immunological point of view, ifn-is have three major functions: . to activate an antiviral state in infected and neighboring cells that limits spread of infection. . modulate innate immune responses, including antigen presentation and natural killer cell functions while restraining pro-inflammatory pathways. . activating the adaptive immune system for the development of high-affinity antigen-specific t and b cell responses ( ) . as ifn-is are highly active molecules, their expression and signaling potency is highly regulated. opposing augmenting and suppressive signals are induced by host factors. suppressive pathways include ifn-i activation of usp , an isg that suppresses signal transduction by reducing the ability of ifn-is to form an active receptor complex ( , ) . a second inhibitory mechanism is the induction of socs and socs , which kir domain block the substrate binding groove on jak, thereby inhibiting stat phosphorylation ( ) . a third mechanism is by rapid endocytosis and subsequent lysosomal degradation of activated ifnar complexes ( , ) resulting in reduced receptor numbers ( figure ). it has been demonstrated that a mutant in ifnar (s a and s a in human and mouse respectively), which fails in ifnar endocytosis through blocking its ubiquitination result in high incidence of inflammation ( , ) . at the transcriptional level, ifn-i response can also be regulated by mir- , which is highly induced by pattern recognition receptors and inflammatory signaling, and suppresses the expression of over genes. between them genes related to the interferon pathway. it was shown that mir- -deficient cd (+) t cells had enhanced type i interferon signaling and were more susceptible to interferon's antiproliferative effect ( ) . high basal ifn-i levels are implicated in various immunological diseases, such as systemic lupus erythematosus and more ( , , ) . however, ifn-i has also anti-inflammatory effects, as best demonstrated by their ability to suppress multiple-sclerosis ( ) . it is important to note that beneficial results in treating multiplesclerosis were observed only for ifnb but not for ifna treatment ( ) . to see whether this relates to the higher receptor binding affinity of ifnb, we established a transgenic mouse harboring the human interferon-receptors extracellular domains fussed to the mouse intracellular domains and compared the severity of eae in a mice model upon treatment with ifna , ifnb and the high-affinity engineered ifn-yns-a -tail. we found that the ifn-yns-a -tail had the strongest suppressive effect on the development of eae ( ) . the effect was further enhanced by pasylation of ifn-yns-a -tail, which extends it plasma half-life by -fold. interestingly, we found a tight relation between the increased levels of expression of pd-l in mice and the severity of the disease. these data show that tight binding ifn-is induce preferential anti-inflammatory responses, at least in this ms mouse model. another example for the immunosuppressive activity of ifn-i was shown for lcmv infection, which induces consistent ifn-i production including the immunosuppressive factors il- and pd-l ( ) . in addition to the above, interferons contribute to inflammasome activation through several different mechanisms, including caspase- expression and the ifn-i inducible gbp protein expression, which was reported to have an important role in caspase- activation and pyroptotic cell death ( ) . ifn-is have important roles in protecting the lung from spread of respiratory viruses. in addition to their direct role, ifn-is have also been found to be critical in initiating lung inflammatory responses, by inducing recruitment and activation of immune responses, which have to be kept under control. ifn-is have been shown to result in the production of chemokines such as ccl and cxcl , which play important roles in the recruitment of monocytes/macrophages, t cells, nk cells, and dcs, therefore directly influencing inflammation in the lung ( ) . this varied effect of type i ifns on t cells is partly dependent on the different stats induced by type i ifns. in the absence of ifn-is, the detection of accumulating viral rna and downstream processing of the signal is compromised, leading to viral spread and also to reduced inflammation in the lung. interestingly, there is an age-related reduction of ifn-i production and isg induction after viral infection, which may be related to the higher susceptibility of elderly population to lung infections ( ). viruses have developed many strategies to interfere with the synthesis of ifn-is or the ifn-i induced responses. one of them, is the stimulation of turnover of the interferon receptors. among other viruses implicated in accelerating the turnover of ifnar are ebv, herpes simplex virus, hepatitis c and b viruses, vesicular stomatitis virus and the sars coronavirus ( , ) . sars-cov has been shown to suppress ifn-i responses in the host through multiple mechanisms. a subdued ifn-i response diminishes antigen presentation and reduces the antiviral adaptive th- immune response. ifn-is communicate between cells against pathogens and have a critical role in the immune system, such as activating natural killer (nk) cells and macrophages. in addition, ifn-is cause flu-like symptoms, which are observed in various diseases. these symptoms may have a role in alerting a person of his/her sickness, in order to limit disease-spread to other individuals. in sars-cov and mers-cov, the induction of ifnb is suppressed altogether. this dampening approach is highly associated with the disease severity and increased mortality ( ) . in the lethal cases of sars-cov or mers-cov infections, the increased influx of inflammatory cells is always observed. in a mouse model of sars-cov infection, imbalance in ifn-i and inflammatory cells were shown as the main cause of fatal pneumonia ( ) . in addition to these, sars-cov implements strategies to evade the immune response by antagonizing ifn-i induced signaling pathways. the orf protein blocks the expression of stat activated genes ( ) . sars-cov and mers-cov encode papainlike protease (plp) that is able to impede the immune response function ( ) . in addition, sars-cov interacts with isg and antagonizes the ifn-i-mediated antiviral response ( ) . the mers-cov orf b antagonizes the antiviral ifnb production by inhibiting irf and irf ( ) . also sars-cov inhibits activation of irf / , slowing ifnb production upon infection ( ) . while irf is expressed in many different cell types, plasmacytoid dendritic cells are the only cells constitutively expressing irf ( ) . ifn-i treatment has been studied against mers-cov and sars-cov in numerous experiments, both in vitro and in vivo, and in combination or not with lopinavir/ritonavir, ribavirin, remdesivir, corticosteroids, or ifng. while ifna and b were efficient in vitro and in certain animal models, their success in humans was less convincing [for review see, ( , ) ]. it should be noted that reduction in ards mortality (not related to sars) was also found to be at best marginal upon treatment with ifn-i ( ) . still, one has to consider that mice studies have shown the timing of ifn-i administration to be critical, with positive effects being observed if ifn-i was administered shortly after infection. conversely, ifn-i failed to inhibit viral replication and resulted in unwanted side-effects when administered later in the disease circle ( , ) . these include elevated lung cytokine/chemokine levels, vascular leakage, and impaired virus-specific t cell responses. it is interesting to note that a knockout of the ifn-i receptor in mice resulted in its protection from lethal sars-cov infection. these findings have major implications on how to treat humans against sars and mers, and could have affected the outcome of the clinical studies. the covid- pandemic started in december in wuhan, china. by the summer of , thirty million cases were reported worldwide, with over , fatalities. as covid- is closely related to the sars-cov virus, the interest in the effect of interferons on its disease progression, and its potential as a drug was immediate. disease progression of covid- goes through a number of stages. the initial stage, which last from to days (usually - days) from infection is asymptomatic. a certain proportion of patients never produce any symptoms (the percentage of those is under debate, but a range of - % is most likely). of those who develop symptoms, they are mostly mild ( % of those who develop symptoms). from the remaining %, about half will develop severe symptoms, which require hospitalization in intensive care units. the mortality rate, from those developing symptoms is % to %. the numbers given above are average, and change dramatically with age. at young age most of the infected people will be asymptomatic, while over the age of about % will have symptoms. moreover, as the age progresses, symptom severity increases ( ) . the major complication of severe infection is pneumonia, which can develop into acute respiratory distress syndrome (ards). in addition, covid- has been linked to cardiovascular sequelae, such as myocardial injury, arrhythmias, cardiomyopathy and heart failure, acute kidney injury, neurological complications, and acute ischemic stroke ( ) . developing severe symptoms and death is strongly related to background conditions. the strongest relation is to age, with the risk to people under being very small, while the risk peaks for people over the age of . in addition, chronic kidney disease, chronic obstructive pulmonary disease, immunocompromised state, obesity, heart conditions and type diabetes are linked to higher incidents of sever disease ( ) . cov- is presumed to infect people mostly though inhalation of viral particles, which can be airborne, in droplets or otherwise through infection through touching infected surfaces. the spike protein on the cov- surface binds to the human ace protein, which serves as its receptor ( figure ) . the homotrimeric spike glycoprotein is made from s and s subunits. its binding and subsequent cleavage by the host protease tmprss results in the fusion between cell and viral membranes and cell entry ( ) . blocking the ace receptors by specific antibodies voids viral entry ( ) ( ) ( ) . interestingly, cov- receptor-binding domain (rbd) exhibited significantly higher binding affinity to ace than the sars-cov rbd, which was speculated to relate to the higher infectivity of covid- in relation to sars. after membrane fusion, the virus enters through the endosomal pathway and the viral rna is released into the host cell. the viral rna is then translated into viral polyproteins, which are cleaved into small products by viral proteases (papain-like protease [plpro] and the main protease [mpro]). viral proteins and genome rna are subsequently assembled into virions in the er and golgi and then transported and released out of the cell. the exact mechanism of viral self-assembly is still under intense investigation ( , ) . investigating ace and the viral entry-associated protease tmprss expression levels in lung tissue and trachea has shown that tmprss is expressed in both tissues, while ace is predominantly expressed in a transient secretory cell type ( ) . in addition, ace and tmprss co-expressing cells were found within lung type ii alveolar cells (which also release pulmonary surfactant), enterocytes, and nasal goblet secretory cells ( ) . using single-cell rna-sequencing, ace and tmprss were found to be highly expressed also in the nasal goblet and ciliated cells ( ) . the inhaled virus likely binds to epithelial cells in the nasal cavity and starts replicating. the virus propagates and migrates down the respiratory tract along the conducting airways, and a more robust innate immune response is triggered. for about % of the infected patients, the disease will be mild and mostly restricted to the upper and conducting airways. unfortunately, about % of the infected patients will progress to more severe disease and will develop pulmonary infiltrates and some of them will develop ards ( ). like many other viruses, also sars-cov and sars-cov- have evolved mechanisms to reduce their exposure to ifn-i. in both viruses, mechanisms to block the production of ifnb were identified. while the antiviral potency of ifn-is on sars-cov is moderate, sars-cov- seems to be highly sensitive to ifn-i. this is evident by the significant reduction in viral replication observed following ifn-i treatment at both and h postinfection ( ) . in sars-cov- -infected cells, ifn-i results in elevated stat levels and isg production (in contrast to sars-cov infected cells). this raises the question of why the innate immune system fails to combat sars-cov- ? the apparent answer to this is in the inhibition of ifnb production by proteins of the sars-cov- virus. within cells, rna viruses are sensed by the innate immune system through three major classes of pattern recognition receptors (prrs): toll-like receptors (i.e. tlr- , - , - ), rig-i-like receptors (rlrs), and nod-like receptors (nlrs) ( ) . to identify the molecular mechanisms that block ifnb production through activation of irf / , several research groups transfected cells individually with all the cov- viral genes and with either rig i, mda , or mavs ( , ) . among the cov- proteins transfected to cells, they identified nsp and orf as competent suppressors of ifnb. yuen et al. also identified nsp and , while lei et al. identified nsp , nsp and the m protein as potent inhibitors of the mavs pathway, leading to inhibition of ifnb production ( figure ) . orf was between the strongest suppressors of ifnb production in both studies. orf was also the only sars-cov- gene suppressing the activity of an interferon-stimulated response element (isre) promoter in both studies. lei et al. also identified nsp and nsp as potent inhibitors of the induction of an isre promotor. in another study, li et al. showed that the viral orf , orf , and nucleocapsid proteins were strong inhibitors of ifnb production, and through this of the ifn-i innate immune response ( ) . in this study, orf and orf also inhibited induction of transcription an isre promotor driving a luciferase as reporter, following ifnb treatment. in addition to the above-mentioned sars-cov- genes, orf b was implicated by konno et al. as being a potent antagonist towards ifn-i production ( ) . an interesting civet in this study is the finding that a natural variant, with a longer orf b reading frame increased disease severity in two patients. in light of the much higher than expected coding capacity of the sars-cov- genome, where many more proteins than genes were identified ( ), we may find even more proteins and peptides being involved in eliminating the innate immune response, including through inhibition of ifn-i activities. another mechanism by which sars-cov- inhibit antiviral functions of the cell is thought the activity of the papain-like protease (plpro), which is essential for viral polyprotein processing. this gene was found to preferentially cleave the ubiquitin-like modifier interferon-stimulated gene (isg ), figure | sars-cov- has multiple effects on the immune system, including inhibition of ifnb production, which results in isgs not to be produced, cd + and cd + exhaustion and increased levels of pro-inflammatory proteins (tnfa, il , nf-kb). currently, the most promising drugs against covid- include ifn-is, antiinflammatory and antiviral drugs, protease inhibitors, antibodies, sars-cov -ace (receptor) binding inhibitors and more. which is an ifn-i induced gene with strong antiviral activity ( ) . this represents another layer of attenuation of ifn-i responses by sars-cov- and is similar to the mechanism previously identified for sars-cov ( ) . inhibition of ifnb production by cov- got further confirmation from measuring the levels of different cytokines in sars-cov- -infected patients. an integrated immune analysis, including immune cell analysis, whole-blood transcriptomics and cytokine quantification on covid- patients at to days after disease onset has shown an impaired ifn-i response that is a result of low ifn-i levels ( ) . this, in turn results in the low production of interferon-stimulated genes. conversely, high levels of il and tnfa were measured ( figure ) ( , ) . this is in contrast to what is seen in patients infected with highly pathogenic influenza viruses. the high production of pro-inflammatory cytokines and low production of ifn-is during sars-cov- infection suggests effective activation of nf-kb but not irf and irf ( ) . impaired ifn-i production during severe covid- may also lead to an imbalance in the pro-inflammatory versus pro-repair functions of airway macrophages. this was indeed seen in severely ill patients with covid- . other innate immune cells such as natural killer (nk) cells are also regulated by ifn-is during coronavirus infection. severe covid- is associated with exhaustion of cd + and cd + t cells ( ) , which may be a result of deficient ifn-i production, as ifn-is promote survival of t cells. an important issue to consider is that early production of ifn-is promote efficient t cell responses, while a delayed response may inhibit t cell proliferation or their exit from lymphoid organs and thus cause their functional exhaustion. indeed, t reg cell counts in covid- patients inversely correlate with disease severity ( , ) . interestingly, transcriptomic analysis of blood, lung, and airways of cov- -infected patients showed that while ifnb was indeed not highly expressed in either, a number of ifnas were highly upregulated in the lung and airways but not in blood ( ) . moreover, a clear ifn-i-induced gene expression profile was also detected for lung and airways, but not for blood (pbmcs). a similar finding of elevated ifna but not ifnb, during covid- infection was also found by wei et al. ( ) . in this study, the elevated ifn-i response was restricted to the stage in the disease were patients were in intensive care. in another study of patients, of whom did not produce ifn-i, those patients had higher viral load, required more aggressive medical intervention and their time of stay in the intensive care unit was longer that ifn-i producing patients ( ) . pdcs are the most rapid and abundant ifn-i producers. pdcs express tlr and tlr which are important in sensing viruses. the response of pdcs to viruses, particularly ifn-i production, is significantly impaired with ageing while secretion of all other pro-inflammatory cytokines was comparable to that of younger individuals ( ) . this may relate to the master regulator for ifn-i production, irf , which expression, phosphorylation and nuclear translocation decreases with age. in addition, local neutrophil-mediated inflammation is increased with age, while cytotoxicity of nk cells induced by type i ifn-is decreases in aged mice ( ) . in addition to age, other factors were also associated with reduced interferon responses. one of them is obesity, which is related to impaired ifna and ifnb responses, which may relate to inadequate response of obese people against viral infections ( ) . clinical trials of using ifn-i for treating corona viruses has a long history. already in , intranasal human ifna was given both before and after corona virus challenge, a strain that is causing common cold. the incidence of colds, the severity of symptoms and signs, and virus replication were all reduced in subjects receiving interferon as compared with those given placebo ( ) . for sars-cov, no randomized placebo-controlled trials have been performed to test the efficacy of ifn-is, however, comparing the clinical outcome of patients treated with ifn-a (infacon- ) with patients at different locations (not a control group) that were not treated, has suggested clinical benefits ( ) . these studies have raised the hope that ifn-i may be a potent drug also against covid- . this hope was further exuberated by the observation that externally administrated ifn-i induced a strong antiviral response, much more than that observed for sars-cov ( ) . while some of the sars-cov- proteins may affect isg production (most notably, orf and , see above), the main defense of sars-cov- against ifn-i innate immunity seems to be the prevention of ifnb production, which can be substituted by external administration. a major problem in assessing the efficiency of ifn-i against covid- is the lack of a good small animal model. while such models are now under development, they are still not perfect. in a recent study, mice were infected with a replication-deficient adenovirus containing human ace , and then infected with sars-cov- . these mice developed pneumonia, severe pulmonary pathology, and high-titer virus replication in lungs. to test the role of ifn-i in disease development, ifnar ko mice were infected with sars-cov- , showing higher viral titer over time. next, the mice were treated prior to infection with poly i:c, a strong inducer of ifn-i. this resulted in significantly diminished clinical disease and induced more rapid virus clearance ( ) . these results suggest that at least in a mice model, ifn-i may benefit disease recovery. due to the lack of a good animal model, and the availability of clinically approved ifn-i therapies, multiple clinical studies have been conducted administrating different subtypes of ifn-is using different routes of administration (for summary see table ). in a preventive study, nasal drops of ifna were given to , healthy medical staff in shiyan city hospital, hubei province for days to prevent sars-cov- infections. none of them developed serious side effects or was infected with cov- . while the study lacked a control group from the same city, overall in hubei province , medical staff were diagnosed with covid- ( ) . the study thus gives an indication that ifn-i may help in preventing infection for high risk medical personal. to test the benefit of subcutaneous injection of ifnb on early stage patients, an open clinical trial was conducted with patients, were assigned to the combination of lopinavir, ritonavir, ribavirin, and three doses of million international units of ifnb, while the control group of patients were given all the above except ifnb. the median number of days from symptom onset to start of study treatment was days. patients given also ifnb had a significantly shorter median time from the start of treatment to negative nasopharyngeal swab ( - days) in comparison to the control group ( - days) . moreover, ifnb reduced viral load and number of significantly ill patients relative to the control group, this without significant side-effects ( ) . in a medical study on the effects of treatment with ifna b in a cohort of confirmed covid- patients, some of the participants were given nebulized ifna b with or without arbidol while others were given only arbidol. treatment with ifna b with or without arbidol reduced the duration of detectable virus in the upper respiratory tract and reduced duration of elevated blood levels of il and c-reactive protein, which are inflammatory markers ( ) . while the study did not include a standard care group, and all patients recovered, it still provides an indication of ifn-i efficiency. the efficiency of ifnb a subcutaneously injected three times weekly for weeks for treatment of severe covid- was tested in a randomized clinical trial. all the patients (including the control group) received standard of care, including a range of other medicines (hydroxychloroquine, antibiotics, antiviral medicine and more). while the clinical response was not significantly different between the ifnb and the control groups, the -day overall mortality was significantly lower ( % vs. %) in the ifnb treated group ( ) . in a retrospective study of patients receiving ifna through inhalation, alone or in combination with other drugs at a relative early versus late stage of the infection, it was found that those receiving ifna at an early stage had a significantly lower rate of mortality. in contrast, late interferon therapy increased mortality and delayed recovery ( ) . the study suggests a relation between the time of ifn-i treatment and its efficiency. synairgen, a uk-based company, performed a controlled clinical trial of inhaled ifnb on patients and reported that compared with placebo the odds of developing severe disease during the treatment period decreased by % for hospitalized patients receiving sng , and that patients who received sng were more than twice as likely to recover from the virus during the treatment period versus those randomized to placebo. these are between the best results achieved so far in curing covid- . more clinical trials are now under way to evaluate ifn-i efficiency, but clearly the initial trials have been encouraging. moreover, due to the many years of experience in treating patients with ifn-is, the availability of the drug and its relatively modest cost make it an excellent candidate for mass treatment, once approved. however, critical questions remain concerning the use of ifn-is for covid- and other diseases ( figure ) . these questions relate to the optimal ifn-i subtype, drug-concentration, duration of treatment, mode of treatment and at which frequency should it be given. ample experience exists with subcutaneously administration, which is almost the only route ifn-is were used in the clinic. here, non-modified ifn-is are usually administrated two to three times weekly, while pegylated ifn-is are administrated once per week or less. injection of ifn-is will result in a systemic response, where ifn-is were shown to have antiviral functions as well as pro and anti-inflammatory functions. contrary, if given by inhalation, it will directly target the epithelial, and thus replace the ifnb, which production is inhibited by the virus. administration as nasal drops of ifna may be an excellent prophylactic method for people at high risk. ideally, these questions could be answered using animal models. the problem is that the disease in those is not equivalent to that observed in humans. due to the severity of the disease and the high proven safety of ifn-is, more clinical trials on humans, testing the many open questions related to its best mode of administration may be the fastest way forwards. the subtype to use is another important question. for multiple-sclerosis, ifnb has been used for many years ( ) , as it seems to provide a better anti-inflammatory response than ifnas. this may relate to its higher binding affinity to the interferon receptors, as has been demonstrated using a tight binding ifna mutant (yns-a tail), which binding affinity even surpasses that of ifnb [see above ( ) ]. for combating viral disease, most notable hepatitis c, ifna has been most commonly used ( ) , which was later replaced by pegylated (long plasma half-life) ifna ( ) . also, for cancers ifnas were mostly used ( ) . a good clinical explanation of why specific ifn-i subtypes were used is often missing, and decisions of which interferon to use may often relate to availability rather than to efficacy. moreover, due to the specie specificity of ifn-is, one cannot deduce from mouse experiments, which ifn-i to use in humans, as the data are not transferable ( , ) . the main difference between ifnas and ifnb is that the later has a stronger potency to induce antiproliferative and immunomodulatory responses (tunable), while ifna will provide a cleaner antiviral response (robust) without the additional responses associated with ifnb. the open question is which is desired for covid- treatment, where complications arise from the exuberated immune response. another, important parameter is the time of intervention by ifn-i, in early or late-stage covid- disease. in a recent study in mice it has been shown that prolonged 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recovery from viral infection the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © schreiber. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -h skaq authors: li, conglei; li, june; ni, heyu title: crosstalk between platelets and microbial pathogens date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: h skaq platelets, small anucleate cells circulating in the blood, are critical mediators in haemostasis and thrombosis. interestingly, recent studies demonstrated that platelets contain both pro-inflammatory and anti-inflammatory molecules, equipping platelets with immunoregulatory function in both innate and adaptive immunity. in the context of infectious diseases, platelets are involved in early detection of invading microorganisms and are actively recruited to sites of infection. platelets exert their effects on microbial pathogens either by direct binding to eliminate or restrict dissemination, or by shaping the subsequent host immune response. reciprocally, many invading microbial pathogens can directly or indirectly target host platelets, altering platelet count or/and function. in addition, microbial pathogens can impact the host auto- and alloimmune responses to platelet antigens in several immune-mediated diseases, such as immune thrombocytopenia, and fetal and neonatal alloimmune thrombocytopenia. in this review, we discuss the mechanisms that contribute to the bidirectional interactions between platelets and various microbial pathogens, and how these interactions hold relevant implications in the pathogenesis of many infectious diseases. the knowledge obtained from “well-studied” microbes may also help us understand the pathogenesis of emerging microbes, such as sars-cov- coronavirus. platelets are the second most abundant cells in human blood circulation ( , ) . anucleate platelets are found only in mammals; in lower vertebrates, cells involved in hemostasis and blood coagulation are nucleated and termed thrombocytes ( , ) . under physiological conditions, thrombopoietin (tpo) predominantly produced by the liver, via binding to the tpo receptor c-mpl on megakaryocytes, is the major regulator of megakaryocyte differentiation and megakaryopoesis ( ) ( ) ( ) . historically it is known that platelets are produced from their precursor megakaryocytes in the bone marrow of mammals ( , , ) . however, recent research surprisingly uncovered that platelets could also be generated by megakaryocytes in the lung of mice ( ) , although further validation is required in both murine and human studies. additionally, the relative contribution of lung-generated platelets to total circulating platelets and whether they possess different function is still unclear ( ) . in extension to their traditional roles in haemostasis and thrombosis ( ) ( ) ( ) ( ) , recent studies suggest that platelets are also involved in many other physiological and pathophysiological processes, such as innate and adaptive immunity, angiogenesis, atherosclerosis, and tumor progression ( , , ( ) ( ) ( ) ( ) ( ) ( ) ( ) . we have previously compiled a comprehensive overview of the importance of platelets in modulating immune responses ( ) . in this review article, we mainly focus on the bidirectional interplay between platelets and microbial pathogens and the significant impact it has on the host responses. infectious diseases are unresolved challenges to human health, and remain as one of leading causes of morbidity and mortality worldwide, especially in resources-limited countries (https://www.who.int/news-room/fact-sheets/detail/the-top- -causes-of-death). microorganisms encounter platelets when they enter the mammalian blood circulation. platelets can directly bind to many pathogens (e.g., bacteria, viruses, and parasites), or pathogen-igg immune complexes via fc receptors expressed on platelets ( ) ( ) ( ) . this platelet-pathogen interaction has functional consequences on both platelets and pathogens (figure ). reduced levels of circulating platelets are commonly observed in patients with infectious diseases, and the underlying mechanisms vary depending on specific pathogens ( , , , ) . in addition, it has been demonstrated that reduced platelet counts in patients or mice are associated with increased susceptibility of the host to infections ( ) ( ) ( ) ( ) . sepsis is a life-threatening inflammatory syndrome caused by a dysregulated host response to infection ( ) , and it has been demonstrated that sepsis altered the transcriptional and translational profiles of platelets in both humans and mice ( ) . although the evolutionary pressure to drive the pathogens to develop various strategies to target platelets is not wellunderstood, one possibility is that platelets may protect the host from certain invading pathogens. platelet adhesion, activation and aggregation at the damaged vessel endothelium are critical for bleeding arrest ( ) ( ) ( ) ( ) . platelet surface glycoprotein receptor, gpibα, via interacting with von willebrand factor (vwf; anchored on collagen in the injured vessel wall), initiates platelet adhesion, particularly under the high shear conditions ( , , , ) . the gpibα-vwf interaction is also critical for endovascular growth of occlusive thrombi at sites of arterial stenosis where blood flows with wall shear rates that may exceed , s − , corresponding to shear stresses exceeding , pa ( ) . the glycoprotein gpiibiiia (αiibβ integrin), can also contribute to platelet adhesion under the lower shear conditions. this abundant platelet integrin is essential for both fibrinogen-dependent and fibrinogenindependent platelet aggregation ( , ( ) ( ) ( ) ( ) . interestingly, in addition to the platelet accumulation (platelet adhesion and aggregation, the first wave of haemostasis), we recently found that the plasma fibronectin can rapidly deposit onto the injured vessel wall and mediate a "protein wave of hemostasis, " which occurs even earlier than the first wave of haemostasis ( , ) . platelets may release their plasma fibronectin content from α granules and contribute to this protein wave of hemostasis, which is likely a compensatory mechanism for heamostasis in fibrinogendeficient mice and humans since their platelet fibronectin levels increase - -fold ( , , ) . notably, activated platelets can promote the cell-based generation of thrombin that markedly enhances the blood coagulation (the second wave of haemostasis) leading to the generation of polymerized fibrin ( , , ) . thus, platelets contribute to all three waves of haemostasis, which may directly or indirectly affect the dissemination of micropathogens in vivo. it has been well-understood that deficiencies in platelet adhesion/aggregation or the coagulation system are linked with various bleeding disorders ( , , ) . however, inappropriate formation of platelet plug may lead to thrombosis, and thrombosis in the cerebral or coronary arteries is the major cause of morbidity and mortality worldwide ( ) ( ) ( ) . moreover, it has been recognized that thrombus formation in the placenta can lead to fetal loss during pregnancy in several disease conditions ( ) , such as antiphospholipid syndrome ( , ) . as platelets contain both pro-inflammatory and antiinflammatory molecules, platelets can interact with many immune cells (e.g., dendritic cells, neutrophils, and lymphocytes), which can shape both innate and adaptive immunity ( , , , , ) . in addition, platelets are involved in the development of lymphatic vessels, the critical network facilitating immune cell trafficking and surveillance ( , ) . platelets achieve this via the binding of platelet c-type lectin-like receptor to podoplanin on lymphatic endothelial cells, leading to the separation of lymphatic vessels from blood vessels during embryonic development ( ) ( ) ( ) . platelets contribute to the host innate immunity in various ways. platelets express the functional pathogen recognition receptors, such as toll-like receptors (tlrs) (tlrs - in human platelets and tlrs - in murine platelets), and nodlike receptor ( , , , ) . platelets contain many proinflammatory molecules (e.g., cd and serotonin), cytokines (e.g., il- ) and chemokines (e.g., ccl , cxcl , and ccl ), and antimicrobial factors (e.g., kinocidins and defensins) in their granules ( , ) . in addition, platelets express several functional chemokine receptors, such as ccr , ccr , and cxcr ( ) . platelets can also shed microparticles that are capable of transporting inflammatory molecules (e.g., cd l and il- ) to inflammatory cells ( , ) . interestingly, platelets also contain multiple anti-inflammatory molecules and cytokines, such as transforming growth factor-β (tgf-β) ( ). it has been shown that platelet-derived tgf-β diminishes the anti-tumor activity of natural killer (nk) cells ( , ) . platelets also modulate adaptive immune response of the host. activated platelets express cd l on their surface, which plays a key role in supporting antibody isotype switching (e.g., from igm to igg) and enhancing cd + t cell function ( , ) . platelets exert their direct effects on microbial pathogens by either binding them and sequestering them thereby limiting their systemic dissemination or by directly eliminating them, and indirect effects by shaping the subsequent host immune response to these invaders. reciprocally, many invading microbes can alter platelet count or/and function, and impact the host auto-and alloimmune response to platelet antigens in several immune-mediated diseases. upon platelet activation, p-selectin is translocated from the α-granule to the platelet surface ( ) . p-selectin, via interacting with peripheral node addressin on high endothelial venules and p-selectin glycoprotein ligand- on lymphocytes, mediates the rolling and recruitment of lymphocytes to peripheral lymph nodes ( ) . and platelet-derived tgf-β was shown to inhibit the cytotoxic t cell response in the tumor microenvironment ( ) , and might improve function of regulatory t cells ( ) . tgf-β is a key factor in iga isotype switching ( ) . since iga plays an important role in controlling the homeostasis of gut microbiota ( ) , and preventing pathogen invasion at mucosal sites ( ) , it remains to be investigated whether platelet tgf-β contributes to the production of intestinal iga. in addition, tgf-β is critical for the differentiation of regulatory t cells under non-inflammatory conditions, in both mice and humans ( ) ( ) ( ) . since platelets contain many pro-inflammatory molecules, and reduced platelet counts in patients or mice are linked with the host's susceptibility to infections ( , , , , ) , it suggests that platelets may protect the host from certain microbial infections. platelets are involved in the early detection of invading microorganisms and are actively recruited to sites of infection ( , , ) . review of recent literatures shows that platelets exert their direct effects on microbial pathogens by either binding them and sequestering them thereby limiting their systemic dissemination or by directly eliminating them ( figure ). platelets also have indirect effects on microbial pathogens by shaping the subsequent innate and adaptive immunity of the host to these invaders (figure ). in the context of staphylococcus aureus (s. aureus) infection, platelets bind s. aureus and use the pseudopods to encapsulate the bacteria ( ) . this ability of platelets to collect and bundle bacteria [e.g., s. aureus, escherichia coli (e. coli) and listeria monocytogenes (l. monocytogenes)] may trap these bacteria, limit their dissemination within the bloodstream and present them to phagocytes ( , ) . moreover, α-toxin derived from s. aureus stimulated human platelets to release β -defensins, which significantly retarded the growth of two strains of s. aureus isolated from patients with sepsis ( ) . in addition to pathogen trapping, platelets can kill certain pathogens. plasmodium. falciparum is the most common species that cause malaria in humans. in the infected host, plasmodium invades red blood cells in the bloodstream and replicate until erythrocytes burst. it has been demonstrated platelets can bind plasmodium-infected erythrocytes and directly kill plasmodium inside red blood cells both in vitro and in vivo ( , ) . subsequent studies revealed that the chemokine platelet factor (also known as cxcl ) released from platelets plays a key role in this platelet-mediated parasite destruction ( , ) . in addition, platelets can secrete many antimicrobial factors including defensins to inhibit the growth of bacteria and viruses ( ) . notably, human platelets and megakaryocytes express the antiviral immune effector molecule: interferoninduced transmembrane (ifitm ) ( ) . it has been recently demonstrated that viral infections (e.g., influenza and dengue viruses) upregulated the expression of ifitm on platelets and figure | effects of platelets on microbial pathogens. the direct effects of platelets on microbial pathogens include pathogen encapsulation and elimination. platelets also exert the indirect effects on microbial pathogens by shaping the innate and adaptive immune responses of the host against these invaders. megakaryoctyes, eliciting rapid antiviral immunity, and that megakaryocytes were capable of limiting viral infections in both megakaryocytes and hematopoietic stem cells via secretion of type i interferons ( ) . however, it is important to note that some viruses [e.g., dengue virus, human immunodeficiency virus (hiv), and hepatitis c virus (hcv)], which can be actively engulfed by platelets and induce platelet activation through tlr signaling, may also utilize platelets to disseminate through the entire body of host ( ) ( ) ( ) ( ) . therefore, the protective role of platelets against viruses may be context-dependent. in addition to the direct effects on pathogens, platelets can shape the host immune responses to invading pathogens and the involved mechanisms are summarized as follows (figure ) : platelets can utilize the functional pattern recognition receptors expressed on their surface to sense the intravascular pathogens, and release various chemokines (e.g., ccl , cxcl , and ccl ) to recruit leukocytes to sites of vascular invasion ( ) . in addition, activated platelets use cd l to trigger the inflammatory reaction on cd -expressing vascular endothelial cells, leading to increased expression of the adhesion molecules (e.g., vascular cell adhesion molecule and intercellular adhesion molecule ) and secretion of proinflammatory cytokines (e.g., ccl ) by endothelial cells ( ) . this phenotypic alteration of vascular endothelial cells may further promote the recruitment of leukocytes at sites of infection ( , ) . activated platelets can also directly interact with leukocytes, forming platelet-leukocyte conjugates, and this interaction is largely mediated by p-selectin on activated platelets and pselectin glycoprotein ligand on leukocytes ( ) . the plateletleukocyte triggers the activation of leukocytes and their increased expression of β and β integrin, leading to enhanced adhesion of leukocytes to vascular endothelial cells ( ) . in addition, activated platelets already deposited at sites of infection can act as docking platforms for leukocyte recruitment ( ) . more importantly, activated platelets deposit chemokines cxcl and ccl on the surface of vascular endothelial cells, instructing the extravasation of leukocytes at sites of infection ( , ) . in the liver, the tissue-resident macrophages, kupffer cells, play a key role in the innate defense against blood-borne pathogens. wong et al. showed that kupffer cells act as docking platforms for both bacteria and platelets. platelets formed aggregates around the bacteria that are bound to kupffer cells, and promoted kupffer cell-mediated phagocytosis of these bacteria ( ) . during gram-negative bacterial infections, platelets actively contribute to nets formation ( , ) . platelet tlr is capable of detecting intravascular tlr ligands [e.g., lipopolysaccharide (lps)], inducing platelet binding to neutrophils. this tlr dependent platelet-neutrophil interaction results in robust neutrophil activation and production of nets, which are dna-based structures capable of capturing and eliminating microbes from the bloodstream ( , ) . platelet depletion in vivo significantly impairs nets formation and bacterial clearance ( , ) . antigen acquisition by dendritic cells is critical for generation of the cytotoxic cd + t cell response against intracellular pathogens ( ). verschoor et al. found that platelets could actively bind l. monocytogenes in the circulation and shuttle this subset of gram-positive bacteria to splenic cd α + dendritic cells, enhancing anti-bacterial cd + t cell expansion ( ) . in addition to affecting antigen presentation, platelets have been shown to promote the polarization of th and th cells, and modulate the balance of regulatory and nonregulatory t cells ( , ) . furthermore, platelet-derived cd l alone is sufficient to induce igg isotype switching against adenovirus ( ) , but it remains to be investigated whether platelet cd l also promotes antibody class switching to other immunologobulin isotypes (e.g., iga), since antibody class switching to different isotypes involves distinct dna repair pathways ( ) . conversely, platelet antimicrobial responses may be detrimental to the host if they are dysregulated. for example, it has been reported that nets formation contributed by platelets that were activated by microbial derived products could cause the injury to blood endothelial cells due to the many proteases contained within nets ( ) , which can directly act as a scaffold and stimulus for thrombus formation ( ) . as mentioned above, reduced platelet count is a common feature with some infectious diseases, and the underlying mechanisms vary depending on specific pathogens ( , , ) . considering the important role of platelets in the regulation of host immunity, it is not surprising that various pathogens target platelets in the course of infections. many invading pathogens can directly or indirectly target platelets in the host, altering platelet function or/and count (figure ) ; in addition to these alterations, it has been shown that viral infections (e.g., dengue and influenza viruses) and sepsis can markedly alter the platelet transcriptome ( , ) . furthermore, microbial pathogens impact the host autoimmune and alloimmune response to platelet antigens in several immunemediated diseases, such as immune thrombocytopenia, and fetal and neonatal alloimmune thrombocytopenia ( - ) (figure ) . the interaction between microbial pathogens and platelets can lead to alteration of platelet function (i.e., platelet activation and apoptosis) (figure ) . activated platelets can trigger the coagulation system, leading to excessive clotting ( , ) , which may exacerbate the symptoms caused by microbial infections and thus may be detrimental to the host. the capacity to trigger platelet activation is a well-known feature for many pathogens. for example, lps purified from gramnegative bacterium e. coli, via interacting with tlr , induces platelet activation both in vitro and in vivo ( , ) , and direct interaction between e. coli and platelets has also been observed in vivo ( ) . dengue virus, which causes hemorrhagic fever in around % infected patients, directly bind platelets via multiple receptors (e.g., dc-sign, heparin sulfate proteoglycan receptors and tlr- ), and activate platelets, triggering the conformational activation of platelet αiibβ integrin, the translocation of pselectin to platelet surface and the release of pro-inflammatory molecules (e.g., il- β) ( , , ) . in addition, microbial infections cause the release of inflammatory cytokines in the host ( , ) , and these cytokines (e.g., tnf-α) were shown to enhance platelet activation in vivo ( ). and for some pathogens (e.g., influenza virus), anti-microbial antibodies form the immune complexes with pathogens and activate platelets via fc receptors ( , ) . it has been shown that the secreted products by s. aureus, such as α-toxin and staphylococcal superantigen-like , can directly activate platelets ( , ) . interestingly, the lipoteichoic acid secreted by s. aureus can inhibit platelet activation and aggregation ( ) . thus, the effects of microbial pathogens on platelet function is dependent on microbial strain or/and the microbial products. once activated, platelets undergo apoptosis ( ) . in addition, some pathogens (e.g., pathogenic e. coli and s. aureus) are found to directly induce platelet apoptosis through degradation of anti-apoptotic bcl-xl protein ( ) . platelet apoptosis induced by microbial pathogens (e.g., dengue virus) not only reduces mitochondrial potential, but also increases the surface exposure of phosphatidylserine that potentially triggers the activation of coagulation system ( , ) . figure | effects of microbial pathogens on platelets. many invading microbes can alter platelet function, leading to platelet activation or/and apoptosis. reduced platelet count is a common feature with some infectious diseases, and the underlying mechanisms include accelerated platelet clearance and impaired platelet production. in addition, microbial pathogens impact the host autoimmune (e.g., in itp) and alloimmune (e.g., in fnait) response to platelet antigens. vzv, varicella zoster virus. reduced platelets in the context of infectious diseases can be due to enhanced platelet clearance or/and altered platelet production (figure ) . as mentioned above, some microbial pathogens can activate the platelet and coagulation system, leading to thrombosis ( ) . exaggerated thrombus formation, especially within the setting of sepsis-associated disseminated intravascular coagulation, may excessively consume platelets, resulting in reduced circulating levels ( , , ) . secondly, platelet clearance may be enhanced through collateral stimulation of the immune system by some microbial pathogens [e.g., varicella zoster virus, hiv, hcv, and helicobacter pylori (h. pylori)] ( ) ( ) ( ) ( ) ( ) . for example, thrombocytopenia in children following varicella zoster virus infection first described antigenic mimicry for some microbial pathogens that encompass host generation of crossreactive antibodies to certain glycoproteins (e.g., gpiiia) on the platelet surface, resulting in accelerated platelet clearance ( ) . third, direct platelet-bound microbial products (e.g., lps) or inflammatory byproducts (e.g., c-reactive protein) could enhance antibody mediated phagocytic responses ( ) ( ) ( ) . microbial induced platelet clearance can also occur via removal of terminal sialic residues of the abundantly expressed platelet surface glycans. scavenging of host sialic residues by microbial pathogens increases immune evasion and assists in survival and dissemination ( , ) . direct cleavage of platelet sialic residues by pathogen-derived neuraminidase has been reported in bacterial, and parasitic infections ( ) . indirectly, pathogens could induce platelet desialylation mediated by platelet-derived neuraminidase, as was reported with dengue virus infection ( ) . by either mechanism, loss of terminal sialic residues not only leads to rapid platelet clearance via lectin receptors predominantly in the liver ( ) , but also potentiates platelets to hyperactivity contributing to pathological disseminated intravascular coagulopathy and thrombotic complications of sepsis ( ) ( ) ( ) . although, animal models and preliminary human studies demonstrate sialidase inhibitors or hepatic lectin receptor ashwell-morell inhibitors can ameliorate coagulopathies and thrombocytopenia in microbial infections ( , ) , other lectin receptors such as the recently identified kupffer macrophage galactose lectin receptor may also contribute ( ) . likely there are multiple and redundant receptor/ligand interactions that mediate clearance of desialylated or desialylation activated platelets. depending on the specific pathogens, there are several means by which invading microbes can negatively impact the platelet production by megakaryocytes in the bone marrow. for example, hcv can interfere with tpo production by damaging the liver tissue ( ) . some pathogens (e.g., dengue virus and hiv) can directly infect megakaryocytes or their precursors, or alter the bone marrow microenvironment, leading to the defective platelet production in bone marrow ( ) ( ) ( ) ( ) . however, it is important to note that inflammatory cytokines (e.g., tnf-α and il- ) induced by certain microbial infections are capable of enhancing platelet production by triggering acute emergency megakaryopoiesis ( , ) . thus, the impact of microbial infections on platelet production is context-dependent. in addition to the effects on platelet count and function, microbial pathogens impact the host auto-and alloimmune response to platelet antigens in several immune-mediated diseases, such as immune thrombocytopenia (itp), and fetal and neonatal alloimmune thrombocytopenia (fnait) ( - ) (figure ) . itp is an autoimmune disorder in which an abnormal immune response develops against one's own platelets, leading to autoantibody-induced platelet/megakaryocyte destruction and suppressed platelet production, and an increased risk of bleeding ( , , [ ] [ ] [ ] [ ] . in adult itp patients, detectable antibody reactivity against gpiibiiia and gpib/ix predominate ( - %) ( , , ) . however, it is uncommon for patients to possess single antibody specificities, other glycoprotein targets including gpv, gpiv, and gpia/iia are often detected ( ) ( ) ( ) . moreover, extensiveness of anti-glycoprotein antibody repertoire has been correlated with more severe disease ( ) . the antiplatelet antibodies not only accelerate platelet clearance mediated by splenic macrophages and hepatic kupffer cells ( , , ) , but also inhibit the development of bone marrow megakaryocytes and promote their apoptosis, thus inhibiting platelet production ( , , , , ) . in addition to anti-platelet autoantibodies, cytotoxic cd + t cells, and regulatory cd + t cells might also contribute to the pathogenesis of itp ( , , ( ) ( ) ( ) ( ) ( ) ( ) . cytotoxic cd + t cells were shown to directly lyse platelets, induce the apoptosis of platelets, and inhibit platelet production by megakaryocytes ( , , ) . it has been reported that the frequency or/and function of regulatory cd + t cells were defective in the circulation of itp patients ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , and interestingly, the tgf-β level was also reduced in these patients ( , , ) . it has been reported that peripheral deficiency of regulatory cd + t cells might be caused by their retention in the thymus in murine model of itp ( ) , although it remains to be investigated whether this mechanism is also present in itp patients. the therapies (e.g., steroids and b cell depletion) that improve platelet counts also restored the frequency or/and function of regulatory cd + t cells in the periphery ( , , , , ) , and the level of circulating tgf-β in itp patients ( , ) , although it remains to be investigated whether the improvement of regulatory t cells is due to changes in circulating tgf-β ( ). chronic infections (e.g., hiv, hcv, and h. pylori) can cause secondary itp, in which antimicrobial antibodies cross-react with platelets, leading to platelet destruction ( ) . acute infections have long been suspected as triggers that initiate the pathogenesis of primary itp, but in most acute itp cases, the specific pathogen could not be identified ( ) . retrospective studies suggested that infectious events (e.g., viral and fungal infections) precede the development of primary itp in around % itp patients ( ) , but future definitive studies are required to confirm the causal relationship between the specific pathogen(s) and initiation of primary itp, and to identify the underlying mechanisms ( , ) . furthermore, it has been demonstrated that infections during itp worsened the pathogenesis of primary itp and the therapeutic response to platelet transfusions, but the underlying reasons were unclear ( ) . since inflammation induced hemorrhage in thrombocytopenic mice ( ) , it is possible that inflammation associated with microbial infections may aggravate the bleeding risk in thrombocytopenic patients (e.g., itp). c-reactive protein is markedly upregulated during acute infections and inflammation ( ) , and it has been shown that c-reactive protein, via binding to platelet phosphorylcholine residues, enhanced the igg-mediated phagocytic responses against platelets and thereby thrombocytopenia, which has implications in the pathogenesis of both itp and fnait ( , ) . in addition to itp, infections also play an important role in the pathogenesis of heparin-induced thrombocytopenia, in which pathogenic antibodies to the complexes of platelet factor (pf ) and heparin develop post-heparin exposure, leading to lifethreatening complications of thrombocytopenia and thrombosis ( , ) . it has been demonstrated that pf bound to various bacteria, induces the generation of antibodies that could crossreact with the major antigen in pf /heparin complex, resulting in heparin-induced thrombocytopenia ( , ) . fnait results from the development of maternal alloantibodies targeting paternally derived antigens on fetal platelets during pregnancy, and these maternal antibodies cross the placenta and destroy fetal or neonatal platelets, leading to bleeding disorders ( - ). similar to itp, most of the reported fnait cases are characterized by maternal alloantibodies to platelet gpiibiiia ( ) ( ) ( ) ) . in contrast, there are very few reported cases of fnait with anti-gpibα complex antibodies ( ) ( ) ( ) ( ) ( ) , which is different from the to % prevalence of anti-gpibα antibodies in itp patients ( , ) . to gain new insights into the pathogenesis of fnait, our laboratory established animal models of fnait using β −/− and gpibα −/− mice, respectively ( , , , ) . we observed neonatal thrombocytopenia and severe bleeding disorders (e.g., intracranial hemorrhage) in the heterozygous pups from wild-type (wt) platelet immunized β −/− dams, which recapitulated fnait in humans ( , ) . in contrast, miscarriage unexpectedly occurred in most of the anti-gpibα-mediated fnait, which is far more frequent than that mediated by anti-β antibodies ( ) . besides miscarriage, maternal immune response against fetal platelet antigens caused intrauterine growth restriction to fetuses due to placental abnormalities in animal models of fnait ( ) . the roles of bacterial/viral infections in the pathogenesis of fnait were unclear. to test whether bacterial infection contributed to fnait, we utilized lps to mimic gram-negative bacterial infection, and co-administered it with low-dose wt platelet antigens to gpibα −/− and β −/− mice ( ). we found that lps co-administration significantly boosted the production of anti-gpibα and anti-β antibodies, and miscarriage occurred in most of these co-stimulated gpibα −/− and β −/− mice, while miscarriage infrequently occurred in the dams immunized with low-dose wt platelets alone. furthermore, we utilized poly i:c to mimic viral infections, and observed that coinjection of poly i:c and wt platelets also enhanced production of anti-gpibα antibodies in gpibα −/− mice and the severity of fnait ( ). however, it remains to be investigated whether live bacterial or viral infections indeed exacerbate the pathogenesis of fnait. overall, our data suggested that both bacterial and viral infections were likely to be involved in the pathogenesis of fnait in animal models, but it warrants further studies to test whether this is also the case for human fnait patients. the effects of microbial infections in the pathogenesis of fnait may be also translatable to another alloimmune thrombocytopenia: post-transfusion purpura, in which antiplatelet alloantibodies develop against transfused platelets from genetically distinct donors ( ). our understanding of platelet functions beyond haemostasis and thrombosis has dramatically expanded in the past years. accumulating evidence indicates that platelets play an important role in the host immunity against microbial infections, and future discoveries will undoubtedly uncover more versatile features of platelets. the interaction between platelets and microbial pathogens are bidirectional, as this interaction causes the biological consequences on both platelets and microbes (figure ) . the knowledge we obtained from these "well-studied" microbes may also help us understand the pathogenesis of emerging microbes, such as severe acute respiratory syndrome coronavirus (sars-cov- ). the sars-cov- infection causes the pandemic coronavirus disease in humans, but the pathogenesis of covid- is still largely unclear ( , ) . thrombocytopenia has been observed in around - % of covid- patients ( , ) , and two recent meta-analysis studies with covid- patients revealed that severe reduction in platelet counts might be a poor prognostic marker for this life-threatening disease ( , ) . importantly severely ill covid- patients exhibit profound hypercoagulable states ( , ) , and excessive clotting has been observed in severely ill covid- patients ( ) ( ) ( ) . is the thrombocytopenia in severe covid- cases caused by the platelet hyperactivities and consumption during micro-thrombi formation? do the hypercoagulable states synergize with platelet activation, which provide phosphatidylserine, propel the cellbased thrombin generation ( ) , and lead to thrombosis? do platelets release/synthesize their cytokines and contribute to the cytokine storm in covid- patients? do platelets contribute to the immune response against sars-cov- ? finally, are platelets friends or foes or able to switch their roles during sars-cov- infection? all these questions are important and warrant further investigations. overall, we believe that understanding the interactions between platelets and microbial pathogens will shed light on the pathogenesis of infectious diseases and that modulation of platelet-pathogen interactions could provide new therapeutic avenues. cl designed and wrote most of the paper. jl wrote and edited the manuscript. hn was the principal 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pathological findings of covid- associated with acute respiratory distress syndrome china medical treatment expert group for: clinical characteristics of coronavirus disease in china the association between severe covid- and low platelet count: evidence from observational studies involving participants thrombocytopenia is associated with severe coronavirus disease (covid- ) infections: a meta-analysis lupus anticoagulant and abnormal coagulation tests in patients with covid- covid- and thrombotic or thromboembolic disease: implications for prevention, antithrombotic therapy, and follow-up: jacc state-of-theart review prevalence of venous thromboembolism in patients with severe novel coronavirus pneumonia clinical pathology of critical patient with novel coronavirus pneumonia (covid- ) we would like to thank alexandra florescu and dr. jennifer gommerman for the inspiring discussion during manuscript preparation. jl was a recipient of a ph.d. graduate student fellowship from canadian blood services centre for innovation. cl was a recipient of the canadian institutes of health research postdoctoral fellowship. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.the reviewer rk declared a past co-authorship with one of the authors hn to the handling editor.copyright © li, li and ni. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - lzjhn j authors: tian, bin; zhou, ming; yang, yu; yu, lan; luo, zhaochen; tian, dayong; wang, ke; cui, min; chen, huanchun; fu, zhen f.; zhao, ling title: lab-attenuated rabies virus causes abortive infection and induces cytokine expression in astrocytes by activating mitochondrial antiviral-signaling protein signaling pathway date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: lzjhn j rabies is an ancient disease but remains endemic in most parts of the world and causes approximately , deaths annually. the mechanism through which the causative agent, rabies virus (rabv), evades the host immune response and infects the host central nervous system (cns) has not been completely elucidated thus far. our previous studies have shown that lab-attenuated, but not wild-type (wt), rabv activates the innate immune response in the mouse and dog models. in this present study, we demonstrate that lab-attenuated rabv causes abortive infection in astrocytes, the most abundant glial cells in the cns. furthermore, we found that lab-attenuated rabv produces more double-stranded rna (dsrna) than wt rabv, which is recognized by retinoic acid-inducible gene i (rig-i) or melanoma differentiation-associated protein (mda ). activation of mitochondrial antiviral-signaling protein (mavs), the common adaptor molecule for rig-i and mda , results in the production of type i interferon (ifn) and the expression of hundreds of ifn-stimulated genes, which suppress rabv replication and spread in astrocytes. notably, lab-attenuated rabv replicates in a manner identical to that of wt rabv in mavs−/− astrocytes. it was also found that lab-attenuated, but not wt, rabv induces the expression of inflammatory cytokines via the mavs- p /nf-κb signaling pathway. these inflammatory cytokines increase the blood–brain barrier permeability and thus enable immune cells and antibodies infiltrate the cns parenchyma, resulting in rabv control and elimination. in contrast, wt rabv restricts dsrna production and thus evades innate recognition by rig-i/mda in astrocytes, which could be one of the mechanisms by which wt rabv evades the host immune response in resident cns cells. our findings suggest that astrocytes play a critical role in limiting the replication of lab-attenuated rabv in the cns. rabies is an ancient disease but remains endemic in most parts of the world and causes approximately , deaths annually. the mechanism through which the causative agent, rabies virus (rabv), evades the host immune response and infects the host central nervous system (cns) has not been completely elucidated thus far. our previous studies have shown that lab-attenuated, but not wild-type (wt), rabv activates the innate immune response in the mouse and dog models. in this present study, we demonstrate that lab-attenuated rabv causes abortive infection in astrocytes, the most abundant glial cells in the cns. furthermore, we found that lab-attenuated rabv produces more double-stranded rna (dsrna) than wt rabv, which is recognized by retinoic acidinducible gene i (rig-i) or melanoma differentiation-associated protein (mda ). activation of mitochondrial antiviral-signaling protein (mavs), the common adaptor molecule for rig-i and mda , results in the production of type i interferon (ifn) and the expression of hundreds of ifn-stimulated genes, which suppress rabv replication and spread in astrocytes. notably, lab-attenuated rabv replicates in a manner identical to that of wt rabv in mavs−/− astrocytes. it was also found that lab-attenuated, but not wt, rabv induces the expression of inflammatory cytokines via the mavs-p /nf-κb signaling pathway. these inflammatory cytokines increase the blood-brain barrier permeability and thus enable immune cells and antibodies infiltrate the cns parenchyma, resulting in rabv control and elimination. in contrast, wt rabv restricts dsrna production and thus evades innate recognition by rig-i/mda in astrocytes, which could be one of the mechanisms by which wt rabv evades the host immune response in resident cns cells. our findings suggest that astrocytes play a critical role in limiting the replication of lab-attenuated rabv in the cns. introduction rabies is an acute encephalomyelitis. the hallmark of rabies is that the disease is almost always fatal once clinical signs develop ( , ) . the causative agent, rabies virus (rabv), is a negative-strand rna virus belonging to the genus lyssavirus in the family rhabdoviridae ( ) . rabv enters neurons from the neuromuscular junction closest to the site of infection. after a short incubation, rabv travels to the central nervous system (cns) through sensory or motor neurons. only slight tissue damage and neuroinflammation can be observed in the brains of rabid patients ( ) . in contrast, lab-attenuated rabv induces extensive inflammation and apoptosis, as well as increases in the expression levels of innate immunity-related genes in the cns of infected mice ( ) ( ) ( ) ( ) ( ) ( ) . these findings suggest that wt, but not lab-attenuated, rabv evades the host immune responses. the innate immune system is the first line of defense against viral invasion. viruses are usually confronted by various pattern recognition receptors (prrs), including toll-like receptors (tlrs) and retinoic acid-inducible gene i (rig-i) like helicases (rlrs) ( ) . tlr family members, such as tlr and tlr , are generally involved in recognizing negative-strand rna viruses ( ) . tlr binds double-stranded rna (dsrna), whereas tlr recognizes single-strand rna. the main sources of dsrna in infections with single-strand rna viruses are the replicative intermediates generated by viral rna-dependent rna polymerase ( ) . tlr and tlr initiate signaling though the adaptor molecules trif and myd , respectively. both signaling cascades triggered by these proteins lead to irf phosphorylation in the c terminal region at serine , which is critical for irf activation by two iκb kinases (tbk- and ikkε) ( , ) . activated irf homo-or hetero-dimerizes with irf and then translocates into the nucleus, to interacts with the creb binding protein cbp/p and stimulates the transcription of interferon (ifn)-β, as well as some ifn-stimulated genes (isgs) ( , ) . alternatively, rna viruses can be recognized by two rlrs, rig-i, and melanoma differentiation-associated protein (mda ), located in the cytoplasm ( , ) . rig-i solely senses short and blunt dsrna of negative-strand rna viruses containing ′ triphosphate rna in the panhandle region of their single-stranded genome ( ) . unlike rig-i, mda preferentially binds blunt-ended dsrna, with or without ′ triphosphate ( ) . rig-i and mda signaling is mediated through mitochondrial antiviral-signaling protein (mavs), which is also known as ips , visa or cardif ( ) . similar to tlr signaling, rlr signaling results in irf activation and nuclear translocation ( ) . several groups have attempted to identify the prrs that recognize rabv. prehaud et al. found that tlr mrna expression is upregulated following rabv infection in postmitotic human neurons ( ) . furthermore, enhanced tlr expression has also been observed in the cerebellar cortical tissues of rabies patients ( ) . the observation that tlr is upregulated following infection suggests that tlr plays a role in the innate recognition of rabv. faul et al. found that both rig-i and mda were responsible for inducing dendritic cell (dc) activation and type i ifn production upon rabv infection ( ) . mavs, the adaptor protein for both rig-i and mda , is essential for inducing innate immune responses in dcs. in another study, li et al. found that mice lacking tlr exhibited a phenotype associated with intermediate mortality rates between those of myd −/− and control mice, indicating that tlr may play an important role in controlling rabv infections. however, the role of tlr in rabv infection is not entirely understood ( ) . astrocytes are the most abundant glial cells in the cns and constitute the blood-brain barrier (bbb) along with endothelial cells and pericytes ( ) . astrocytes are generally involved in regulating the cns microenvironment and also play roles in neuronal metabolic support, synaptic transmission, and neurotropism. moreover, astrocytes participate in developing and maintaining the bbb and guiding neuronal migration during development. the roles of astrocytes in innate immunity and inflammation have been reported recently ( ) . astrocytes are the reservoir for many neuroinvasive viruses, such as human immunodeficiency virus, theiler's murine encephalomyelitis virus (tmev), john cunningham virus, and herpes simplex virus (hsv) ( ) ( ) ( ) ( ) . astrocytes have also been shown to play multiple roles in viral infections. specifically, they can increase bbb permeability ( ) by producing cytokines and degrading tight junction proteins ( ) . moreover, astrocytes have been shown to induce the innate immune response to produce ifn ( ) . an early report showed that both wt and lab-attenuated rabv could successfully infect primary astrocytes in the early stages of rabv infection ( ) . a recent study demonstrated that a rabv vaccine strain sad-l caused an abortive infection in astrocytes, but the detailed mechanism was not revealed ( ) . in this study, it was found that lab-attenuated rabv produces higher level of dsrna than wt rabv, which is recognized by rig-i/mda and results in the activation of mavs signaling pathway. following mavs activation, the production of isg and inflammatory cytokines helps to clear the lab-attenuated rabv from the cns. in contrast, wt rabv can maintain a persistent infection in astrocytes by evading the innate recognition. mouse bend. cells and vero cells were obtained from the american type culture collection (atcc; manassas, va, usa) and were maintained in dulbecco's modified eagle medium (dmem) supplemented with fetal bovine serum (fbs; gibco, carlsbad, ca, usa). mouse neuroblastoma (na) cells were maintained in rpmi- medium (thermo-fisher, usa) supplemented with % fbs. the drv-ah (drv) was isolated from a rabid dog in anhui province, china ( , ) . cvs-b c (b c), originated from cvs- virus by passage in bhk- cells ( ), has been used as a lab-attenuated rabv ( ) ( ) ( ) . both drv and cvs-b c were propagated in suckling icr mouse brains. all the viruses were manipulated under the standard biosecurity procedures made by the ministry of agriculture of china. a rabbit anti-ubiquitin protein c (ubc) polyconal antibody was purchased from abclonal technology (woburn, ma, usa). a rabbit anti-rig-i monoclonal antibody was purchased from enzo life technology (farmingdale, ny, usa), and rabbit polyclonal antibodies against irf , stat and occludin were obtained from santa cruz biotechnology (santa cruz, ca, usa). a rabbit antiphospho-irf monoclonal antibody was purchased from cell signaling technology (washington, dc, usa), and rabbit anti-ifit and irf monoclonal antibodies were purchased from abcam (cambridge, ma, usa). a rabbit polyclonal anti-claudin- antibody, a biotinylated goat anti-rabbit or mouse antibody, and an alexa fluor -conjugated goat antimouse or rabbit antibody were purchased from invitrogen (grand island, ny, usa). mouse monoclonal anti-zoluna occludens- (anti-zo- ) antibodies were obtained from sigma (st. louis, mo, usa), and mouse monoclonal anti-gapdh antibody was purchased from proteintech (wuhan, china). the mouse anti-rabv n and p monoclonal antibodies were prepared in our laboratory, and the fluorescein isothiocyanate (fitc)-conjugated anti-rabv nucleoprotein antibody used herein was obtained from female wt or mavs-knockout (mavs−/−) c bl/ mice ( - weeks old) were infected intracerebally (i.c.) with µl of drv-ah ( ffu), b c ( ffu), or mock infected with the same volume of dmem. at days postinfection (d.p.i.), the mice were euthanized with co when moribund, and their brains were collected for immunohistochemistry analysis. blood-brain barrier permeability was determined by measuring sodium fluorescein (naf) uptake as described previously with minor modifications ( ) . briefly, µl of naf ( mg/ml) was injected intraperitoneally (i.p.) into each mouse. after anesthetization, peripheral blood and brains of each mouse were collected. the fluorescence in serum and brain homogenate samples was analyzed by a spectrophotometer (biotek instruments, vt, usa) with excitation at nm and emission at nm. standards ( - , g/ml) were prepared to calculate the naf content. naf uptake into tissue is calculated as (μg of fluorescence spinal cord/mg of tissue)/(μg of fluorescence sera/ml of blood) to normalize values for blood levels of the dye at the time of tissue collection. to detect cd + cell in the brain, infected mice were anesthetized with ketamine-xylazine ( . ml/ g body weight), perfused with ml pbs, and then the brains were transferred into % neutral buffered paraformaldehyde (pfa) for at least h ( ) . briefly, the brain sections were antigenic recovered and blocked with donkey serum, incubated with primary antibodies overnight at °c, and then secondary antibodies was applied. pbs was treated as a negative control by replacing primary antibodies. sections were photographed and analyzed using an olympus bx microscope (tokyo, japan). primary mixed glial cell cultures were established as described previously ( ) . briefly, the brain cells of -to -day-old neonatal c bl/ mice were dissociated by repeated pipetting and then passed through a -nm nylon mesh (corning, ny, usa). the cells were subsequently washed once in cold pbs and cultured in dmem (with high glucose) supplemented with % fbs and % penicillin-streptomycin. the medium was changed on days , , and for the astrocytes and on day only for the microglia. on day , the flasks were shaken at rpm for h to remove any non-adherent cells (mainly microglia). the remaining adherent astrocytes were detached with trypsin-edta and then plated again for further experiments. the purity of the astrocyte cultures was greater than %. mouse neurons were obtained from embryonic mouse brains as previously described ( ) and then dissociated by repeated pipetting (approximately times) before being passed through a -nm nylon mesh. the cells were then washed once in cold pbs and cultured in dmem (with high glucose) supplemented with % fbs and % penicillin-streptomycin for h, after which the medium was replaced with serum-free neural-basal medium (invitrogen, carlsbad, ca, usa) supplemented with % b- (invitrogen, carlsbad, ca, usa). viral titers were determined by direct fluorescent antibody assay ( ) . na cells cultured in -well plates were inoculated with viruses diluted serial -fold and then incubated at °c for h. then the culture supernatant was subsequently removed, and the cells were fixed and stained with fitc-conjugated anti-rabv n antibodies. the antigen-positive foci were counted under a fluorescence microscope (zeiss, germany), and the viral titers were calculated as ffu per milliliter. all titrations were conducted in quadruplicate. confocal microscopy table | primers used for quantification of viral mrna, ifn-stimulated genes, chemokines, and cytokines. retinoic acid-inducible gene i (rig-i) f gcgtctcagtgcagcacatcatt qrt-pcr antibodies against dsrna, rabv n, rabv p, claudin- , occludin, zo- , or dapi. infected wt or mavs−/− mice were anesthetized with ketamine-xylazine ( . ml/ g body weight) and then perfused with pbs followed by % neutral buffered formalin, as described previously ( ) . three independent mouse brain samples were collected from each group and embedded in paraffin for coronal sectioning. the sections were subsequently stained with antibodies against gfap, map , rabv p, rabv n, or dapi. after being washed, the cells or sections were incubated with an alexa fluor -conjugated goat antirabbit or mouse secondary antibodies or an alexa fluor -conjugated goat antirabbit or mouse secondary antibodies for h at room temperature. staining was visualized with a nikon a confocal laser microscope system equipped with nis-elements imaging software (nikon, melville, ny, usa) and was quantified using fiji, an imagej distribution package manufactured by nih (http://imagej.net/introduction). mean fluorescence intensity (mfi) was quantified using the region of interest, which encompassed the entire cell to include the membrane, and background staining was quantified using three negatively stained regions per cell. these regions were subtracted from the total mfi. for the protein synthesis inhibition tests, primary astrocytes were pretreated with cycloheximide (chx) (invivogen, san diego, ca, usa) for h at a dose of µg/ml, infected with drv or b c at an moi of . and then continued to be incubated with chx for another h. for the tbk activation blockage assays, bx (invivogen, san diego, ca, usa) at a dose of µm was used to treat primary astrocytes. for the inflammatory pathway blockage assays, the primary astrocytes were pretreated with a p inhibitor (skepinone-l; sellek, houston, tx, usa), a jnk inhibitor (jnk inhibitor ix; sellek, houston, tx, usa), an nf-κb inhibitor (sc ; sellek, houston, tx, usa), or dmso as control at a dose of µg/ml for h. then the cells were infected with drv or b c at moi and incubated with the above inhibitors for h. the concentrations of tnf-α and il- in the supernatant of astrocytes were measured by the commercial elisa kits according to the manufacturer's instruction (abclonal technology, woburn, ma, usa). rna was isolated with trizol ® reagent (invitrogen), according to the manufacturer's instructions, and qrt-pcr was performed as described previously ( ) . briefly, ng of total rna (from either cells or tissue) was transcribed into cdna in a reaction mixture with a total of volume of µl using a superscript iii reverse transcription kit (toyobo). the reaction mixture comprised μl of cdna combined with µl of iq sybr green mix (biorad, hercules, ca, usa), µl of diethyl pyrocarbonatetreated water, and . µl of primer mix (the concentration of each primer was mm). the cdna was amplified using an iq icycler (bio-rad), and the cycle threshold (ct) values were recorded. the ct value was inversely correlated with the mrna concentration, and each ct unit represented a twofold change in the mrna concentration. basal mrna expression levels were expressed as Δ ct values and were normalized to β-actin mrna expression levels [ Δ ct ct (isg)/ Δ ct (β-actin)]. induced mrna expression levels were expressed as fold changes relative to mock-infection levels using the ΔΔct method. all the primer sequences are listed in (table ) . to quantify cellular rabv n rna levels, we transcribed the total rna using avian myeloblastosis virus reverse transcriptase xl (takara, kusatsu, japan) and a primer specific for the rabv n genomic sequence. a standard curve was generated from serially diluted plasmids carrying a rabv n gene and the copy numbers of n mrna were normalized to mg of total rna. the cells were lysed with ripa buffer containing protease inhibitors (roche), and the protein concentrations were measured using a dc protein assay kit (bio-rad). equal quantities of protein were resolved by or % sds-page and then transferred to polyvinylidene difluoride membranes (bio-rad), which were blocked with % nonfat milk before being incubated with primary antibodies against rig-i, phosphorylated irf (p-irf ), stat , ifit , rabv n, claudin- , occludin, zo- , or gapdh and then probed with the appropriate secondary antibodies. the blots were then visualized using ecl reagent (ge, pittsburgh, pa, usa) and detected under an intelligent dark box ii (ge, pittsburgh, pa, usa). the -gain pmt scan generated the optimal standard curve, and the results of this scan were analyzed using q-analyzer software for qam-cyt- (raybiotech). transendothelial permeability assay was performed according to the methods described previously ( ), with some modifications. b.end cells were grown on -μm-pore transwell filter inserts until they reached % confluency. the medium was then treated with uv-inactivated cell culture supernatants collected from rabv-infected astrocytes. after the cells had incubated for h, they were treated apically with dextran-fitc at a dose of . µg/ml for min. the samples were then removed from the lower chamber and subjected to fluorescence measurement, which were performed using a fluorimeter (biotek, winooski, vt, usa; the excitation wavelength was nm, and the emission wavelength was nm). the fluorescence values for the experimental cells were subsequently compared to corresponding values for a control cell monolayer. data are expressed as the mean and standard error of the mean (sem), and the significance of the differences between groups was evaluated by student's t test or one-way analysis of variance followed by tukey's post hoc test. the survival ratios were analyzed by log-rank (mantel-cox) test. the asterisks indicated statistical significance (*, p < . ; **, p < . ; ***, p < . ). graphs were plotted and analyzed using graphpad prism software, version . (graphpad software, la jolla, ca, usa). results comparison of the pathogenicity of drv-ah and cvs-b c in mice first, the pathogenicity of the wt rabv strain drv-ah (drv) and lab-attenuated strain cvs-b c (b c) used in this study were compared in a mouse model. c /bl mice were i.c. inoculated with ffu b c or drv and the development of rabies was observed. as expected, drv-infected mice displayed development of the diseases at d.p.i. and all moribund at d.p.i., earlier than b c-infected mice which all succumb to rabies at d.p.i. (figure a) . we found that b c replicated faster than drv at the early stage of the infection in the cns. to ensure that the viral load in the brains is similar, c /bl mice were i.c. inoculated with ffu b c or ffu drv. then the viral load in the mice brain were determined at , , , , , and d.p.i. the results showed that the genomic rna of drv was lower than that of b c at and d.p.i., but reached the same level as that of b c at and d.p.i. (figure b) . previous studies have shown that the lab-attenuated rabv infection enhances the bbb permeability and induces inflammation in the brain ( , ) . thus, the na-fluorescence (na-f) uptake of both strains at d.p.i. was measured, and the data showed that na-f uptake in b c-infected brain was significantly higher than that of drv or mock-infected brains ( figure c) . consistently, there were more cd + lymphocytes in b c-infected brains than drv-infected mouse brains (figures d,e) . all these results are consistent with the previous findings related to the cns inflammation and bbb permeability change during comparison of the pathogenicity between wt and lab-attenuated rabv ( , , ) . astrocytes play an important role in the induction of innate immunity in the cns. to investigate the role of astrocytes in the pathogenesis of rabv, we isolated primary astrocytes and neurons from suckling mice and infected them with drv or b c. the growth kinetics of both viruses in astrocytes was assessed by virus titration and immunofluorescence assay (ifa). the viral loads in the cell culture supernatants infected with b c quickly reached peak at d.p.i., and then gradually decreased until d.p.i. in contrast, the virus titer of drv was initially relatively low but subsequently increased steadily until the endpoint (figure a) . to be noted, the viral titer of drv in the cell supernatant maintained at a relatively low level than that of b c in astrocyte at and d.p.i., but n mrna transcription levels of drv was significantly higher than that of b c at and d.p.i. (figure c) . the ifa results showed that drv persistently replicated in astrocytes and large immunofluorescence foci could be observed at d.p.i., while no obvious immunofluorescence foci could be found in b c-infected cells ( figure d) . as a control, virus replication kinetics in primary neurons was also compared. it was found that viral titers of b c were always higher than those of wt rabv at the indicated time points (figure b) . the ifa results also showed that both drv and b c could efficiently replicate in neuron ( figure d) . to confirm these observations in vivo, c bl/ mice were i.c. infected with ffu b c or ffu drv. infected mice were euthanized when moribund and the brains were harvested for fluorescence ihc analysis. gfap was a well-known surface marker for astrocytes ( ) , and the gfap staining demonstrated drv could effectively infect astrocytes in the brains ( figure e) . however, few infected astrocytes could be observed in b cinfected mouse brains (figure e ). in contrast, neuron was intensively infected by both drv and b c ( figure f) . together, these results show that the b c causes abortive infection in astrocytes both in vitro and vivo. previous studies have demonstrated that rabv can be recognized by rig-i and mda , which share a common adaptor mavs in dcs ( ) . to assess innate immune responses in astrocytes, cells were infected with drv or b c at an moi of . and the expression of several proteins involved in the mavs signaling pathway, namely, rig-i, p-irf , stat and ifit (isg ), was measured by western blot. the ubiquitin ligase trim mediates lysine -linked ubiquitination of rig-i's n-terminal card domains is indispensable to induce type i ifn production and antiviral immunity ( ) . thus, the immunoprecipitation of rig-i was carried out and then resolved by western blot by using an anti-ubiquitin antibody. as expected, rig-i was much more robustly ubiquitinated in astrocytes infected with b c than that in astrocytes infected with drv. consistently, the expression levels of rig-i, p-irf , stat , and ifit in b c-infected astrocytes were higher than those in drv-infected cells ( figure a) . next, we attempted to determine which viral product (rna or protein) activates mavs signaling pathway in astrocytes. primary astrocytes were treated with chx, a protein synthesis inhibitor, to inhibit viral protein synthesis. it was found that b c and drv n transcription levels were similar between chxtreated and mock-treated astrocytes at h p.i. (figure b) . however, the expression levels of the genes involved in the mavs signaling pathway, namely, rig-i, mda , mavs, irf , ifn-β, stat , and ifit , were significantly upregulated by b c compared with drv in both chx-treated and mock-treated astrocytes, indicating that viral rna rather than proteins activates ifn pathway depending on mavs (figures c-i) . taken together, these data demonstrate that the lab-attenuated rabv, but not wt rabv, activates the mavs signaling pathway by viral rna, resulting in the production of ifn as well as isgs in astrocytes. retinoic acid-inducible gene i and mda activation is induced mostly by dsrna, which is produced during viral replication ( ) . to compare the amount of dsrna produced by wt and labattenuated rabv, primary astrocytes were infected with drv or b c at an moi of . . at and d.p.i., dsrna was stained with specific antibody and observed by a confocal fluorescence microscope. the results demonstrate that more dsrna is synthesized in b c-infected astrocytes than those in drv-infected cells ( figure a) . the dsrna intensity per cell in b c-infected cell was significantly higher than that in drv-infected cells (figure b) , considering the similar rabv intensity per cell (figure c) . the ratio of dsrna intensity to rabv intensity in b c-infected astrocyte was significantly higher than that in drv-infected cells ( figure d) . these results suggest that lab-attenuated rabv produces more viral dsrna than wt rabv during viral replication, ( ffu) . (e) at d.p.i., mice were euthanized, perfused with pbs and then fixed with % pfa. the brains were subsequently coated with paraffin, and the brain sections were stained with antibodies against gfap (red), rabv p protein (green), or dapi (blue). the white arrows indicate rabv-infected astrocytes. the scale bars represent µm. (f) the same brain sections as (e) were stained with antibodies against map (green), rabv p protein (red), or dapi (blue), then visualized under a confocal microscope. the scale bars represent µm. notably, b c titers were significantly increased in mavs−/− astrocytes compared with wt astrocytes (figures a,b) . moreover, the cell numbers of immunofluorescence plaques in mavs−/− astrocytes caused by b c infection were significantly more than those in wt astrocytes (figures e,f) . in contrast, mavs deficiency had no significant influence on drv replication and spread in astrocytes (figures a,e) . tbk is an iκb kinase downstream of the mavs signaling pathway and is critical for irf phosphorylation. treatment with the tbk specific inhibitor bx significantly increased b c titers in astrocytes (figures c,d) . to verify these observations in vivo, mavs−/− mice were i.c. infected with drv or b c, and virus infection of astrocytes was determined by ifa. we found that both drv and b c could efficiently infected mavs−/− astrocytes (figures g,h) . taken together, these findings suggest that mavs signaling significantly restricts the replication and spread of lab-attenuated but not wt rabv in astrocytes. recent studies have shown that tlr may be another innate immune molecule that recognizes rabv ( ) . thus, the role of tlr in rabv replication was investigated in astrocytes. astrocytes from tlr −/− and control mice were isolated and then infected with drv or b c at an moi of . . at different time points p.i., viral titers (figures a,b ) and the formation of viral immunofluorescence foci was analyzed (figures e,f) . the results demonstrated that tlr deficiency did not significantly affect the replication and spread of either drv or b c in astrocytes, suggesting that tlr does not play a role in containing rabv replication and spread in astrocytes. astrocytes are one of the major sources of inflammatory cytokines in the cns post viral infection, and these cytokines play an important role in regulating bbb permeability. to investigate rabv-induced cytokine production, primary astrocytes from wt and mavs−/− mice were prepared and infected with drv or b c at an moi of . . at indicated time points, the cell culture supernatants were harvested and analyzed with a cytokine array kit. the results showed that b c induced significantly higher levels of cytokine expression, namely, tnf-α, il- , il- β, ifn-γ, il- , and vegf expression, than drv in wt and mavs−/− astrocytes. the levels of cytokine expression in mavs−/− astrocytes were significantly lower than those in wt astrocytes, indicating that rabv-induced inflammatory cytokine production in astrocytes is dependent on the mavs signaling pathway (figures a-f) . the specific pathway through which rabv induces cytokine production was subsequently identified in astrocytes. a previous study demonstrated that rabv induces cytokine production in macrophages mainly through p , jnk, and nf-κb pathways ( , ) . thus, primary astrocytes were treated with p , jnk, and nf-κb pathway inhibitors skepinone-l, jnk ix, and sc , respectively, and the concentrations of tnf-α and il- in the cell supernatant were measured by elisa. the results showed that skepinone-l and sc caused greater reductions in tnf-α ( figure g ) and il- ( figure h ) protein levels than jnk ix in b c-infected astrocytes. none of these inhibitors significantly altered the expression levels of tnf-α and il- in drv-infected astrocytes. these findings suggest that lab-attenuated rabv induces cytokine expression in astrocytes mainly through the p and nf-κb pathways and that wt rabv suppresses cytokine production in astrocytes. our previous studies demonstrated that the chemokines/ cytokines induced by rabv infection are responsible for reducing tj protein expression and enhancing bbb permeability ( ) . to investigate the effect of these cytokines on bbb permeability, a mouse brain microvascular endothelial cell line b.end , was cocultured with uv-inactivated supernatants infected with drv or b c and collected them at different time points after infection. treatment with the supernatants from b c-infected astrocytes for h (figure a ) induced significant increase in dextran-fitc infiltration from to h p.i. notably, treatment with the supernatants from b c-infected mavs−/− astrocytes elicited significant increases in dextran-fitc permeability at h p.i. (figure b) . no significant increases in dextran-fitc permeability were observed after treatment with the supernatants from wt or mavs−/− astrocytes infected with drv. to gain an insight into the mechanisms by which labattenuated rabv enhances bbb permeability, the expression levels of the indicated tj proteins (claudin- , occludin, and zo- ) were assessed in astrocytes by western blot. it was found that the expression levels of claudin- and occludin were unchanged, while zo- was significantly reduced after the cells were treated with the supernatants from b c-infected wt astrocyte ( figure c) . however, this reduction was attenuated in cells treated with the supernatants from mavs−/− astrocytes infected with b c. no significant changes in zo- expression were observed in b.end cells treated with supernatants from wt or mavs−/− astrocyte infected with drv ( figure d) . similarly, ifa results showed that zo- expression was significantly decreased in b.end cells cocultured with the supernatants from b c-infected astrocytes collected at h p.i. (figure e) . zo- fluorescence intensity was only slightly decreased in b.end cells cocultured with the supernatants from b c-infected mavs−/− astrocytes collected at h p.i., indicating that cell-to-cell contact was not significantly decreased in these cells. moreover, no zo- degradation was observed in b.end cells cocultured with the supernatants from wt or mavs−/− astrocytes infected with drv. to be noted, no significant changes in claudin- and occludin expression were detected in either wt or mavs−/− astrocytes upon rabv infection (figures c,e) . together, these results illustrate that lab-attenuated, but not wt, rabv upregulates the production of inflammatory cytokines in astrocytes, resulting in zo- degradation in bmecs and subsequent increases in bbb permeability. furthermore, these results indicate that cytokine production is dependent on mavs signaling pathway. astrocytes play critical roles in host defense during viral infections of the cns ( ) . prr activation in astrocytes results in the expression of many immune mediators, including type i ifns and inflammatory cytokines ( , ) . during infections caused by pathogens for which glia are not susceptible targets, activation of the innate immune system caused by pathogen recognition in astrocytes may promote antiviral immune responses in susceptible neurons, as well as cns leukocyte trafficking ( , , ) . in an early report, primary murine, feline, and human astrocytes were infected with wt (srv) and lab-attenuated rabv (era) ( ), after which viral loads and replication were assessed by infectivity assay and immunofluorescence. the results showed that astrocytes can be infected by rabv, suggesting that astrocytes may play a role in viral spread and persistence and/or neuronal dysfunction ( ) . however, in that study, viral loads and replication were evaluated only at the early time point after infection ( d.p.i.) . in this present study, the growth of drv with that of b c, which were used as a pair of wt and lab-attenuated rabv in previous studies ( ) ( ) ( ) , was compared in a long-term experiment. surprisingly, we found that lab-attenuated rabv, but not wt rabv, caused abortive replication in astrocytes, a feature that may be associated with the ability of the virus to evade the innate immune response. productive infection of the astrocyte is critical for neurotropic pathogens to induce encephalitis. astrocytes sensed viral entry into the cns and mounted a type i ifn response, which rapidly restricts the virus after neuronal transport into the cns. previous studies demonstrated that tlr −/− astrocytes were more permissive to hsv infection and caused severe symptom of encephalitis and tissue damage, which was due to impaired type i ifn production in the absence of tlr ( ) . a recently work found that abortively infected astrocytes are the major producers of ifn-β after infection of the brain with diverse neurotropic viruses, including tmev, rabv, and vesicular stomatitis virus (vsv) ( ) . consistent with these studies, we also found that the abortive infection of lab-attenuated rabv in astrocytes was related to its ability to activate ifn signaling pathway. basal isg expression levels are an important determinant of susceptibility to viral infection. we have found that astrocytes have higher basal expression levels of the mrnas encoding isg proteins, such mda and stat , and other molecules crucial for recognizing viral invasion and creating an more antiviral environment than neurons ( ) , which may explain why lab-attenuated rabv activates the innate immune response to a degree sufficient to restrict viral replication in astrocytes but not in neurons. double-stranded rna is a viral product that plays an essential role in inducing innate immunity, which leads to the production of type i ifns and the activation of hundreds of isgs. early biochemical studies of viral replication suggested that most viruses produce dsrnas ( ) . however, in , weber et al. reported that dsrna could be detected by ifa in cells infected with positive-stranded rna viruses, but not with negative-stranded rna viruses ( ) . this notion was challenged by two other studies on dsrna production in cells infected with negative-stranded rna viruses ( , ) . moreover, a recent study demonstrated the dsrna formation in cells infected with several negative-stranded rna viruses, such as vsv, measles virus (mev), and influenza a virus, although the intensity of the staining of dsrna tended to be weaker in cells infected with negative-stranded rna viruses when compared with those infected with positive-stranded rna viruses ( ) . consistent with this finding, the production of dsrna was detected in both lab-attenuated and wt rabv-infected astrocytes, although lab-attenuated rabv produced significantly more dsrna in the cytoplasm than wt rabv. we attempted to investigate why lab-attenuated rabv produces more dsrna than wt rabv. pfaller et al. found that dsrna expression was much lower in cells infected with wt mev than in cells infected with a mutant mev whose c protein was knocked out which is known to control genome replication and transcription ( ) . similarly, takeuchi et al. observed dsrna formation in cells infected with sendai virus ( ) with c protein knocked out but not in cells infected with wt sev, indicating that the c protein limits or masks dsrna production ( ) . both mev and sev are within the family paramyxoviridae, thus it is possible that their c protein possess the similar function to subvert the production of dsrna. ebola virus protein vp adopts a unique strategy to mask key cellular recognition sites on dsrna ( ) . a recently study showed that the coronavirus endonuclease (endou) activity is the key to prevent early induction of dsrna. replication of endou-deficient coronaviruses is greatly attenuated in vivo and severely restricted in primary cells even during the early phase of virus infection ( ) . in this present study, by exchanging viral genes between wt and lab-attenuated rabv, we found that single n, p, and g of wt rabv could not suppress dsrna formation (data not shown). however, multiple viral proteins of rabv may work together to limit the production of dsrna. further studies are needed to elucidate the mechanism through which wt rabv restricts dsrna formation and thus evades recognition by the innate immune system in infected cells. the bbb, which is composed of specialized bmecs joined by tjs and ensheathed by astrocytes and pericytes, plays an important role in protecting the cns. our previous studies have shown that rabv does not infect bmecs, nor does it modulate tj protein expression in bmecs ( ) . however, brain extracts prepared from mice infected with lab-attenuated rabv but not wt rabv reduced tj protein expression in bmecs, indicating that the above enhancements of bbb permeability and reductions in tj protein expression are not caused by rabv infection. rather, they are caused by virus-induced inflammatory chemokines/ cytokines. the innate immune mechanisms that regulate bbb function in the setting of infectious diseases have been appreciated only recently. multiple inflammatory cytokines, including tnf-α, il- , il- β, ifn-γ, il- , and vegf, disrupt bbb and tj integrity in bmecs ( , , ( ) ( ) ( ) , and inflammatory cytokine signaling at the bbb during infection facilitates leukocyte trafficking into the cns, which is essential for the clearance of many pathogens ( , ) . our present study demonstrates that lab-attenuated rabv induces production of several inflammatory cytokines in astrocytes, especially tnf-α, il- , il- β, ifn-γ, il- , and vegf, which cause disruption of the bbb and tj integrity. our findings suggest that astrocytes play a critical role in regulating bbb permeability as a major source of cytokines during viral infection. furthermore, we found that the production of inflammatory cytokines in astrocytes by lab-attenuated rabv was dependent on mavs signaling pathway, underscoring the critical role of mavs signaling in defensing against rabv infection in cns. | the proposed model for the mechanism through which wild-type (wt) and lab-attenuated rabies virus (rabv) infect astrocytes. during infection, lab-attenuated rabv produces double-stranded rna (dsrna), which is recognized by retinoic acid-inducible gene i (rig-i)/melanoma differentiation-associated protein (mda ). activation of mitochondrial antiviral-signaling protein (mavs), the common adaptor protein for rig-i and mda , leads to enhanced production of interferon, as well as some ifn-stimulated genes, which limit rabv replication and spread. mavs activation stimulates the p and nf-κb signaling pathways and induces cytokine production in astrocytes. inflammatory cytokines promote blood-brain barrier (bbb) permeability, enabling peripheral inflammatory cells and antibodies to infiltrate into the central nervous system, thereby facilitating rabv clearance. in contrast, wt rabv prevents activation of the mavs signaling pathway by restricting dsrna production. conclusion based on the results, we propose the following model: labattenuated rabv produces dsrna recognized by rig-i, mda , or both, resulting in the activation of the mavs signaling pathway in astrocytes. ifn expression induces the transcription of hundreds of isgs to inhibit rabv replication in astrocytes and causes the abortive infection by lab-attenuated rabv. the inflammatory cytokines induced by lab-attenuated rabv enhance bbb permeability, enabling immune cells and antibodies to infiltrate the cns and facilitate rabv clearance. conversely, wt rabv restricts dsrna production and then evades recognition by the innate immune system, resulting in persistent viral replication in astrocytes (figure ). current status of rabies and prospects for elimination the cell biology of rabies virus: using stealth to reach the brain rhabdoviruses: rabies virus role of apoptosis in rabies viral encephalitis: a comparative 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negative regulator of tlr /trif-induced corneal inflammation by inhibiting activation of c-jun n-terminal kinase regulation of innate antiviral defenses through a shared repressor domain in rig-i and lgp involvement of the ubiquitin-like domain of tbk /ikk-i kinases in regulation of ifn-inducible genes recognition of ' triphosphate by rig-i helicase requires short blunt double-stranded rna as contained in panhandle of negative-strand virus structural basis of double-stranded rna recognition by the rig-i like receptor mda mavs forms functional prion-like aggregates to activate and propagate antiviral innate immune response induction of irf- and irf- phosphorylation following activation of the rig-i pathway virus infection switches tlr- -positive human neurons to become strong producers of beta interferon toll-like receptor (tlr ) plays a major role in the formation of rabies virus negri bodies rabies virus infection induces type i interferon production in an ips- dependent manner while 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organ tropism of murine coronavirus infection of pericytes in vitro by japanese encephalitis virus disrupts the integrity of the endothelial barrier regional astrocyte ifn signaling restricts pathogenesis during neurotropic viral infection influenza a virus ns targets the ubiquitin ligase trim to evade recognition by the host viral rna sensor rig-i overexpression of tumor necrosis factor alpha by a recombinant rabies virus attenuates replication in neurons and prevents lethal infection in mice rabies virus-induced activation of mitogen-activated protein kinase and nf-kappab signaling pathways regulates expression of cxc and cc chemokine ligands in microglia doublestranded rna is produced by positive-strand rna viruses and dna viruses but not in detectable amounts by negative-strand rna viruses measles virus c protein impairs production of defective copyback double-stranded viral rna and activation of protein kinase r production of il- , il- , ifn-gamma and ip- in human astrocytes correlates with alphavirus attenuation sendai virus c protein plays a role in restricting pkr activation by limiting the generation of intracellular double-stranded rna ebolavirus vp coats the backbone of double-stranded rna for interferon antagonism early endonuclease-mediated evasion of rna sensing ensures efficient coronavirus replication astrocytes produce and release interleukin- , interleukin- , tumor necrosis factor alpha and interferon-gamma following traumatic and metabolic injury cellular mechanisms of il- -induced blood-brain barrier disruption expression of interferon gamma by a recombinant rabies virus strongly attenuates the pathogenicity of the virus via induction of type i interferon the authors wish to thank the staff members of the animal facility at huazhong agricultural university for caring for the mice. this work was partially supported by the national natural science foundation of china ( and to zf and and to lz); the national program on key research project of china ( yfd to lz). key: cord- -w ne fn authors: schrumpf, jasmijn a.; van der does, anne m.; hiemstra, pieter s. title: impact of the local inflammatory environment on mucosal vitamin d metabolism and signaling in chronic inflammatory lung diseases date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: w ne fn vitamin d plays an active role in the modulation of innate and adaptive immune responses as well as in the protection against respiratory pathogens. evidence for this immunomodulatory and protective role is derived from observational studies showing an association between vitamin d deficiency, chronic airway diseases and respiratory infections, and is supported by a range of experimental studies using cell culture and animal models. furthermore, recent intervention studies have now shown that vitamin d supplementation reduces exacerbation rates in vitamin d-deficient patients with chronic obstructive pulmonary disease (copd) or asthma and decreases the incidence of acute respiratory tract infections. the active vitamin d metabolite, , -dihydroxy-vitamin d ( , (oh)( )d), is known to contribute to the integrity of the mucosal barrier, promote killing of pathogens (via the induction of antimicrobial peptides), and to modulate inflammation and immune responses. these mechanisms may partly explain its protective role against infections and exacerbations in copd and asthma patients. the respiratory mucosa is an important site of local , (oh)( )d synthesis, degradation and signaling, a process that can be affected by exposure to inflammatory mediators. as a consequence, mucosal inflammation and other disease-associated factors, as observed in e.g., copd and asthma, may modulate the protective actions of , (oh)( )d. here, we discuss the potential consequences of various disease-associated processes such as inflammation and exposure to pathogens and inhaled toxicants on vitamin d metabolism and local responses to , (oh)( )d in both immune- and epithelial cells. we furthermore discuss potential consequences of disturbed local levels of (oh)d and , (oh)( )d for chronic lung diseases. additional insight into the relationship between disease-associated mechanisms and local effects of , (oh)( )d is expected to contribute to the design of future strategies aimed at improving local levels of , (oh)( )d and signaling in chronic inflammatory lung diseases. vitamin d is a pleiotropic hormone that is well-known for its role in the regulation of calcium and phosphate homeostasis and bone mineralization. the vitamin d receptor (vdr) acts as the receptor for the active form of vitamin d, i.e., , dihydroxy-vitamin d [ , (oh) d], and is expressed in nearly all tissues and cell-types and regulates a large number of genes (∼ . - % of the total genome) ( , ) . as a result, vitamin d affects many additional processes including cell proliferation and differentiation, apoptosis, dna repair, ion transport, metabolism, cell adhesion, and oxidative stress responses ( , ) . vitamin d deficiency [serum -hydroxy-vitamin d [ (oh)d] < nmol/l; (oh)d is the main circulating form of vitamin d and its levels are used to assess vitamin d status in the clinic ( , ) affects more than % of the children and adults worldwide and is a major cause of bone diseases such as rickets and osteoporosis ( ) . increasing evidence has indicated that vitamin d deficiency is also associated with various other diseases such as cancer, cardiovascular disease, alzheimer's disease and muscle myopathy, as well as several immune-related diseases such as type diabetes, multiple sclerosis, inflammatory bowel disease (ibd), psoriasis and chronic inflammatory lung diseases including asthma, cystic fibrosis (cf), and chronic obstructive pulmonary disease (copd) ( ) ( ) ( ) ( ) . several studies have now shown that vitamin d deficiency is prevalent in copd patients and inversely correlated with lung function and severity of the disease ( ) ( ) ( ) ( ) ( ) . it is currently unknown whether vitamin d deficiency is a cause or consequence of copd, since many copd patients have low physical activity levels and spend most time indoors ( ) . there are however studies suggesting that low (oh)d levels are associated with development of copd, based on observed associations between polymorphisms in the vitamin d binding protein (vdbp), (oh)d serum levels and copd severity ( , , , ) . in addition, one study in mice showed that maternal vitamin d deficiency can impair lung -development, -structure andfunction in the offspring and suggests that even before birth, maternal (oh)d serum levels are important for a healthy lung development ( ) . this might be relevant, since associations have been found between lower childhood lung function and development of copd later in life ( ) . the link between maternal (oh)d status and asthma development is however much clearer, since two recent randomized controlled trials (rcts) have shown that maternal vitamin d supplementation reduces the risk of childhood asthma/recurrent wheeze ( ) . this might be explained by the fact that multiple vitamin d-regulated genes are transcriptionally active during alveolar maturation and a number of these genes are differentially expressed in asthma ( ) . additionally, this protective effect was linked to the gggenotype of the q functional snp rs , which is associated with lower expression of ormdl and increased sphingolipid metabolism ( ) . moreover, maternal circulating (oh)d levels affect the gut microbiota and can therefore indirectly modulate immune responses in the lung via the gutlung-axis ( ) . also later in life, optimal (oh)d levels remain crucial for keeping the lungs healthy. for example, heulens et al. showed that subacute and chronic cigarette smoke (cs) exposure decreased lung function and promoted early signs of emphysema and airway inflammation in vitamin d-deficient mice compared to vitamin d-sufficient animals ( ) . similarly in an elastase-induced copd mouse model, topical administration of vitamin d in the lungs counteracted alveolar damage and improved lung function ( ) . yet in humans, it is still unclear whether vitamin d status influences copd development and disease progression. taken together, these observations suggest an important role for vitamin d during fetal and childhood lung maturation, and indicate that sufficient (oh)d levels might contribute to protection against development of childhood asthma and possibly copd at older age. systemic levels of biologically active , (oh) d are tightly regulated to preserve sufficient levels of calcium (ca + ) and phosphate (po − ) for optimal bone mineralization, whereas in mucosal tissues locally produced (autocrine) , (oh) d levels and signaling can be elevated or decreased upon exposure to inflammatory mediators, pathogens or inhaled toxicants ( ) . this could be important, since the inflamed airway mucosa of patients suffering from chronic inflammatory lung diseases is constantly exposed to these disease-associated factors ( , , ) . impaired local levels of , (oh) d and vdr signaling might have consequences for disease pathogenesis and progression. dysregulated host defenses as found in patients with chronic inflammatory airway diseases include aberrant immune responses, altered microbiome composition, impaired epithelial barrier function, and aberrant secretion of host defense molecules ( ) ( ) ( ) . adequate , (oh) d levels may provide protection against these dysregulated processes by maintaining the integrity of the mucosal barrier and promotion of killing of pathogens (e.g., via the induction of the antimicrobial peptide [amp] hcap /ll- ) and via the modulation of both innate and adaptive immune responses ( , , ) . in this review, we first discuss the effects of these disease-associated factors on local synthesis and availability of , (oh) d and , (oh) d-induced responses in the lung mucosa. in the second part of the review we will describe the mechanistic links between vitamin d deficiency and the pathogenesis of chronic inflammatory lung diseases such as asthma, cf and copd, and discuss recent evidence related to the protective effects of vitamin d on copd and on copd exacerbations. vitamin d enters the circulation either via food intake (plantbased: vitamin d /animal-based: vitamin d ) or as a result of its synthesis in the skin by uvb radiation. it subsequently binds to the vdbp ( , ) , after which this complex is transported to the liver where it is converted by vitamin d- hydroxylases (cyp ri and cyp a ) into (oh)d. however, recent studies showed that also other cell types such as airway epithelial cells, keratinocytes, intestinal epithelial cells, and monocytes/macrophages express cyp ri and cyp a , and thus are able to (locally) convert vitamin d into (oh)d ( , ) . this inactive (oh)d needs to be converted into the active , (oh) d by -hydroxyvitamin d- α-hydroxylase (cyp b ) in the kidney and in other cells, including several immune-and epithelial cells ( ) ( ) ( ) ( ) ( ) ( ) ( ) . , (oh) d regulates expression of several genes by binding the nuclear vdr, which heterodimerizes with the retinoic acid receptor (rxr) to interact with vitamin d response elements (vdres) that are present on the promoter region of these genes ( , ) . vdr is most abundantly expressed in intestinal enterocytes, pancreatic islets, renal distal tubules and osteoblasts, but is also present at lower levels in most other tissues and several other epithelial-and immune cells ( ) ( ) ( ) ( ) ( ) . expression of vdr is classically regulated by , (oh) d, growth factors and hormones such as fgf- and pth, respectively, circulating calcium levels, bile acids, transcriptional co-activators/repressors, and genetic-and epigenetic modifications, which is tissue specific ( ) ( ) ( ) ( ) . , (oh) d regulates its own negative feedback by several mechanisms, including induction of expression of the catabolic enzymes -hydroxyvitamin d- -hydroxylase (cyp a ) and cyp a ( , ) . cyp a is expressed in most tissues and converts both (oh)d and , (oh) d into , (oh) d or , (oh) d and , , (oh) d or , , (oh) d, respectively (dependent on whether cyp a hydroxylates at c- or at c- ). these are further converted into metabolites that have been found to be excreted into the bile (summarized in figure ) ( , , ) . cyp a is mainly expressed in the liver and small intestines and contributes to the metabolic clearance of (oh)d and , (oh) d by converting (oh)d into β, (oh) d, and , (oh) d into frontiers in immunology | www.frontiersin.org , r, (oh) d or , s, (oh) d ( ). expression of both cyp b and cyp a in the kidneys is tightly regulated to maintain optimal ca + -and po − levels in the circulation, which are important for bone mineralization ( ) . in short, in response to low ca + levels, parathyroid hormone (pth) is secreted by the pituitary glands, which in turn reduces ca + excretion and reabsorption of po − ( ). pth further induces expression of cyp b and represses expression of cyp a in the kidneys ( ) . this will increase the levels of , (oh) d in the circulation, which promotes intestinal ca + and po − absorption ( ) . these elevated circulating ca + and po − levels will subsequently induce expression of fibroblast growth factor (fgf- ) in osteocytes and osteoblasts and impair secretion of parathyroid hormone (pth) by the parathyroid glands ( ). in the kidneys, fgf- suppresses expression of cyp b and induces expression of cyp a , thereby inhibiting the synthesis and promoting degradation of , (oh) d ( ). these complex mechanisms that explain how vitamin d and its metabolic enzymes maintain sufficient ca + and po − levels in the circulation are more extensively discussed by quarles et al. ( ) . in summary, it has become increasingly evident that the effects of vitamin d are not limited to homeostasis of ca + and po − and bone mineralization, because several extra-renal cells such as airway epithelial cells and immune cells express the vdr and are capable of converting circulating (oh)d into the active , (oh) d metabolite. local levels and activity of , (oh) d are in part determined by expression of vdr and the equilibrium between the vitamin d metabolic enzymes cyp b and cyp a . it is important to realize that mucosal expression of cyp a , cyp b and also vdr can be affected by several disease-associated inflammatory mediators, toxicants and pathogens, summarized in table . as a consequence of this, the local availability of , (oh) d and/or vdr signaling in tissues such as the inflamed airways of patients that suffer from chronic inflammatory airway diseases might be reduced. chronic lung diseases are characterized by airway inflammation and impaired respiratory host defense, which is illustrated by the increased susceptibility for respiratory infections and exacerbations ( , , ) . furthermore, exposure to inhaled toxicants such as cigarette smoke and air pollutants are associated with disease pathogenesis and exacerbations in copd, cf and in asthma patients ( ) ( ) ( ) . it would therefore be of great interest to investigate these effects on local , (oh) d levels and on , (oh) d-mediated respiratory host defense in the airway mucosa. studies in airway epithelial cells have shown that exposure to uv-inactivated non-typeable haemophilus influenzae (nthi) increased expression of the coga- a (colon cancer epithelial cell line) trophoblasts tnf-α; il- β; il- cyp a ↑ macrophages macrophages (derived from thp- ) macrophages (derived from thp- ) t cells t cell activators (anti-cd /anti-cd ; pha; pma/ionomycin) other hand, in the bronchial cell line beas- b expression of vdr was decreased after infection with respiratory viruses such as human rhinovirus (hrv) and respiratory syncytial virus (rsv) ( ) . collectively, these studies have shown in airway epithelial cells that respiratory viral-and bacterial infections can either promote or impair , (oh) d synthesis and responses. a local airway inflammatory milieu can also exert differential effects on , (oh) d synthesis and signaling, dependent on the type of inflammatory mediators that are predominantly present. we have shown in differentiated primary airway epithelial cells that th cytokines such as il- and il- , enhance expression of cyp b and expression of hcap /ll- upon (oh)d treatment, which suggests that a th -inflammatory environment, as found in allergic airway inflammation, increases the conversion of (oh)d into the active , (oh) d ( , ) . the observation that levels of both , (oh) d and hcap /ll- were increased in bronchoalveolar lavage (bal) after allergen challenge is in line with this proposed mechanism ( ) . this effect of th cytokines was in contrast to the effects (chronic) exposures to the proinflammatory cytokines il- β, tnf-α and il- a that strongly increased the expression of the (oh)d-and , (oh) d-degrading cyp a , even in absence of its inducer , (oh) d ( ). furthermore, shortterm exposures to tgf-β , a pleiotropic growth factor which is elevated in the lungs of copd, cf and asthma patients, also increases the expression of cyp a ( ) . as a consequence, , (oh) d-mediated expression of the amp hcap /ll- was impaired, which was likely the result of the enhanced degradation of both (oh)d and , (oh) d by this enzyme ( , ) . in addition to pathogens and cytokines, exposure to inhaled toxicants such as cigarette smoke (cs) and particulate matter (pm) may also alter expression or activity of vdr and cyp b . studies have demonstrated that cigarette smoking or exposure to cs extract (cse) decreases expression of cyp b and inhibited membrane bound (m)vdr translocation to the cell membrane in airway epithelial cells and a cells (an alveolar tumor cell line), respectively ( , , ) . this inhibition reduces the conversion of (oh)d to , (oh) d and , (oh) d-mediated gene expression as well as nongenomic actions of , (oh) d-membrane associated, rapid response steroid-binding (marrs)-signaling ( , , ) . this adverse effect of cigarette smoking on the synthesis and effects of , (oh) d in airway epithelial cells was recently confirmed in vivo by vargas buonfiglio et al. who demonstrated that vitamin d supplementation increased antimicrobial activity in apical surface liquid (asl) in the airway of healthy non-smokers, but not in smokers ( ) . on the other hand, exposure to pm increases the expression of both cyp b and vdr in airway epithelial cells, thereby possibly promoting the synthesis and effects of , (oh) d ( ). it is however important to consider that several retrospective and observational studies have demonstrated that air pollution is an independent risk factor for developing vitamin d deficiency ( ) . in conclusion, exposure to cs, tgf-β and presence of a proinflammatory milieu appeared to most strongly decrease local presence and signaling of , (oh) d in airway epithelial cells. igm/anti-cd /il- ) cyp b ↑ vdr ↑( ) whereas, various studies show that exposure to proinflammatory stimuli most likely affects local (oh)d and , (oh) dlevels and reduces the effects of (oh)d and , (oh) d in (airway) epithelial cells, the opposite appears to be the case for immune cells. in monocytes, macrophages and neutrophils, effects on , (oh) d synthesis and antimicrobial responses upon (oh)d treatment were generally enhanced by these proinflammatory stimuli as illustrated by increased expression of both vdr and cyp b ( , , ( ) ( ) ( ) ( ) . it is therefore tempting to speculate that this apparent increase in antimicrobial responses upon (oh)d treatment in immune cells in an inflammatory environment may serve as a second line of defense and compensate for the enhanced epithelial degradation of (oh)d and , (oh) d during inflammation. inhaled toxicants may also affect , (oh) d availability and responsiveness of immune cells. this is illustrated by two recent studies studying the effects of cigarette smoke on the human monocyte/macrophage-like cell line thp- . one study showed that treatment with cigarette smoke extract (cse) increased the expression of vdr without enhancing , (oh) d responses ( ), while the other study -that focused on the effects of benzo[a]pyrene (bap) (a component produced by cigarette combustion)-demonstrated that , (oh) dmediated cyp a expression was induced, which was found to further enhance degradation of , (oh) d ( ). in summary, proinflammatory stimuli generally increased the effect of (oh)d and , (oh) d on immune cells, whereas more studies are needed to fully determine the impact of exposure to cigarette smoke and other inhaled toxicants. whereas, these studies provide evidence that inflammation and inhaled toxicants may affect (oh)d and , (oh) d metabolism and responsiveness in epithelial cells and immune cells, it is not clear whether this has an impact on these events in lung tissue of patients with chronic lung diseases. although evidence is limited, we can speculate that levels of , (oh) d and responses are also affected by disease-associated factors in mesenchymal cells that are present in the lung mucosa. one study that showed in a bleomycin fibrosis model and in primary lung mouse fibroblasts that tgf-β reduced expression of the vdr might support this assumption ( ) . it is currently insufficiently studied whether exposures to disease-associated factors promote or impair levels of , (oh) d and responses in immune-, mesenchymal and epithelial cells combined to give a better reflection of the in vivo situation. interestingly, one study did already show that nasal cyp b -and , (oh) d-levels are both reduced in chronic rhinosinusitis (crs) patients with nasal polyps as compared to crs-patients without nasal polyps, whereas no difference was found in circulating , (oh) dlevels ( ) . since most other studies were performed in vitro using monocultures of epithelial cells or immune cells, more complex models are needed to delineate this. therefore, animal models or preferably more complex animal-free cell culture models using co-cultures or organs-on-chips models of primary fully differentiated epithelial cells, airway-derived fibroblasts or smooth muscle cells and immune cells could be considered in future studies. after discussing altered (oh)d and , (oh) d metabolism and responsiveness in the inflamed airway mucosa, it is important to consider the possible consequences of these inflammation-induced changes in the airway mucosa keeping in mind the pleotropic effects of , (oh) d that were introduced earlier. in several cells, tissues and organs, , (oh) d regulates multiple cellular processes that affect normal and malignant cell growth and differentiation ( , ) . , (oh) d displays furthermore protective effects on mucosal host defense by maintaining the integrity of the epithelial barrier, inhibition of epithelial-to-mesenchymal transition (emt), stimulating production of amps and modulating both innate-and adaptive immune functions ( , , ) . in addition, , (oh) d maintains both energetic and survival homeostasis in the mucosal epithelium through the modulation of stress and damage responses, including clearance of disturbing and stressful agents ( , ) (figure ). in chronic inflammatory lung diseases, epithelial barrier function is impaired, and as a consequence the susceptibility toward respiratory infections is increased ( ) . there is increasing evidence that , (oh) d promotes epithelial barrier integrity or protects against epithelial barrier destruction. in cells of the bronchial epithelial cell line hbe, , (oh) d inhibited csemediated reduction of the epithelial barrier and expression of ( ) . only the first study that used a more severe mouse model with higher levels of inflammation and edema found an effect of vitamin d on epithelial barrier function. since inflammation is detrimental for epithelial barrier integrity ( ) , it cannot be excluded that the main protective effects of , (oh) d on the epithelial barrier in the first study by shi et al. were in fact exerted through inhibition of inflammation rather than via direct induction of cell junction proteins. , (oh) d might also promote epithelial barrier function through its ability to increase expression of cystic fibrosis transmembrane conductance regulator (cftr) in airway epithelial cells ( ) . cftr maintains optimal asl-and mucus hydration, volume and ph that support mucociliary clearance and activity of amps ( ) . moreover, cftr is also affected in the airways of smokers and copd patients ( ) . in summary, these studies indicate that , (oh) d promotes both the integrity and function of the epithelial barrier and might additionally protect against epithelial damage by dampening inflammatory responses. the loss of epithelial barrier function with a decrease in epithelial polarization and cell-junction proteins and a gain of expression of mesenchymal markers is a hallmark of emt ( ) . emt is primarily involved in development, wound healing and stem cell differentiation, and tgf-β signaling plays a major role in this process ( ) . elevated tgf-β levels are found in the lungs of patients with chronic inflammatory lung diseases and this was associated with cigarette smoking, inflammation and fibrosis ( , ) . there are indications that , (oh) d counteracts various pathways leading to emt. in mouse models and in airway epithelial cell lines, vitamin d supplementation and , (oh) d, respectively, has been shown to inhibit emt and fibrosis, in particular when this process is induced by tgf-β ( , ( ) ( ) ( ) ( ) . in addition to maintenance of the epithelial barrier and inhibition of fibrosis as discussed in the previous paragraphs, vitamin d is also actively involved in respiratory host defense by a variety of mechanisms ( , ) . , (oh) d is an important inducer of amps, which are mostly cationic peptides that have a broad-spectrum antimicrobial activity, the ability to modulate immune responses and to promote epithelial wound repair and angiogenesis ( ) . hcap /ll- is likely to be the most prominent amp that is induced by , (oh) d and is expressed in several types of mucosal epithelial cells and immune cells such as monocytes and neutrophils ( , , ) . in macrophages and intestinal epithelial cells, , (oh) d also increases expression of human β-defensin- (hbd- ), whereas in keratinocytes expression of both hbd- and human β-defensin- (hbd- ) is increased by , (oh) d ( ) ( ) ( ) ( ) . collectively these data show that amps are modulated by , (oh) d in mucosal tissues, which could have impact on susceptibility to both bacterial and viral infections and on the composition of the microbiota, which will be discussed in the next section. diseases such as copd and asthma are characterized by chronic inflammation, a low-grade and prolonged inflammation that may result in destruction and aberrant repair of surrounding tissue by growth factors, proteases and cytokines that are released at the site of inflammation ( ) ( ) ( ) . cumulative data suggest that vitamin d exerts anti-inflammatory effects via its actions on both innate and adaptive immune responses. upon viral infection or exposure of pro-inflammatory stimuli such as poly(i:c) or pm, , (oh) d attenuates induced expression of cytokines and chemokines e.g., via inhibition of nuclear factor (nf)-κb or oxidative stress, respectively, in (airway) epithelial cells ( , , ) . furthermore, , (oh) d increases expression of the soluble decoy receptor for il- (sst ) by airway epithelial cells, which in turn inhibits the actions of the type alarmin il- ( ) . taken together, these findings suggest that on the one hand , (oh) d protects against infections by enhancing epithelial barrier function and production of amps, and on the other hand , (oh) d induces tolerance and dampens proinflammatory responses in various cell types of the airway mucosa. thereby, , (oh) d may prevent exaggerated inflammatory responses and further damage to the mucosal tissue, qualities that are very relevant in the context of chronic inflammatory (lung) diseases (figure ) . copd is considered to be a disease of accelerated aging lungs, underscored by markers of aging being increased in these patients partly as a result of oxidative stress ( ) . evidence that , (oh) d may protect epithelial cells from oxidative stress was provided by pfeffer et al. who demonstrated that , (oh) d increased expression of the antioxidant gene g pd in airway epithelial cells. furthermore, , (oh) d increased the ratio of reduced to oxidized glutathione and decreased the formation of -isoprostane after exposure to pm ( ) . the induction of klotho by , (oh) d might be another , (oh) d-mediated anti-aging mechanism ( ) . klotho is an anti-aging protein that is mainly expressed in the kidney, brain and in the lung by airway epithelial cells and exerts its protective effects through the inhibition of inflammation, insulin/igf- signaling and activation of forkhead transcription factor (foxo) signaling, which enables removal of reactive oxygen species (ros) ( ) ( ) ( ) . expression of klotho is impaired in the airways of smokers and further decreased in the airways of copd patients and in cultures of the bronchial epithelial cell line hbe after cse exposure ( ) . these studies suggest that , (oh) d may protect against aging via inhibition of oxidative stress and possibly via its ability to restore klotho expression (figure ) . however, direct evidence showing that , (oh) d indeed increases expression of klotho in airway epithelial cells is currently lacking. in addition to providing protection against oxidative stress and aging, data from studies using intestinal epithelial cells suggest that , (oh) d may also promote cellular survival via the induction of autophagy and reduction of apoptosis ( , ) . in chronic inflammatory lung diseases, aberrant activation of autophagy plays a role in disease pathogenesis ( ) . a recent study showed that club cells and autophagy-related proteins were both decreased in copd patients and that these proteins were important for club cell structure and function in airways ( ) . however, the effects of , (oh) d on autophagy in the airway mucosa of chronic inflammatory lung diseases are still unclear and need to be further evaluated ( ) . clearly vitamin d has pivotal actions in host defense that are relevant in the context of chronic inflammatory lung diseases, in which vitamin d deficiency may be prevalent. strategies to promote local levels of , (oh) d or use it as a treatment itself could be therefore of interest. here, we will discuss the latest clinical evidence accompanied with functional in vitro and animal studies that may explain the effects of vitamin d supplementation on typical hallmarks of chronic airway diseases. currently, inhaled corticosteroid (ics)-use with or without long acting bronchodilators is the most frequently used treatment for copd and asthma patients . however, the response to corticosteroids is not always effective in many copd patients and in patients with steroid resistant (sr)-asthma ( ) . there are several complex mechanisms that underlie the resistance to corticosteroids in both copd and sr-asthma that include but are not limited to genetic background, impaired glucocorticoid receptor binding, t helper type cell (th )-inflammation and oxidative stress (e.g., from air pollution or smoking) and decreased numbers of il- secreting regulator t cells (tregs), which normally prevent skewing toward th -inflammation ( ) . direct evidence of the ability of , (oh) d to reverse sr was provided by a study showing that ex-vivo stimulation https://goldcopd.org ( ). with , (oh) d promoted generation of il- -secreting tregs which restored sensitivity toward corticosteroids in cd + t cells that were derived from sr-asthma patients ( ) . a further potential treatment role of , (oh) d was elegantly illustrated by studies that showed that vitamin d deficiency is associated with decreased steroid responsiveness in asthmatics and by the fact that several potential underlying mechanisms of sr such as oxidative stress and th -mediated inflammatory responses could be reversed by vitamin d treatment ( , ( ) ( ) ( ) ( ) ( ) ( ) . interestingly, the corticosteroid dexamethasone was shown to increase expression of the (oh)d and , (oh) d degrading enzyme cyp a in renal cells and osteoblasts ( ) , which suggests a bidirectional interaction between corticosteroids and , (oh) d and could further limit , (oh) d levels for patients. additional research is needed to determine if vitamin d may also improve corticosteroid responsiveness in copd. exacerbations are a major burden for copd patients, they accelerate decline in lung function and frequently result into hospital admissions ( , ) . exacerbations are often triggered by pollutants or by bacterial-and/or viral infections ( , , ) . copd patients generally have lower serum (oh)d levels than age-and smoking-matched controls, which is associated with more and more severe exacerbations ( , ) . several in vivo and in vitro studies have provided evidence that explain the protective effects of vitamin d on exacerbations in copd patients and this will be discussed accordingly. first of all, pfeffer et al. showed that (oh)d and , (oh) d reduce the production of proinflammatory cytokines in part via the ability to enhance antioxidant responses in airway epithelial cells that were exposed to pm ( ) . this was also demonstrated in human dcs that were matured in presence of pm, where treatment with , (oh) d counteracted the expansion of proinflammatory il- a + and ifn-γ + th . cells ( ) . in line with this, bolcas et al., showed that vitamin d supplementation counteracted the development of airway hyperresponsiveness and accumulation of th /th cells in mice that had been repeatedly exposed to both diesel exhaust and house dust mite allergens ( ) . vitamin d could therefore exert a protective role in air pollution-triggered exacerbations. in addition to its protective effects against pollutants, there is also increasing evidence that , (oh) d may enhance clearance of respiratory viral infections that account for - % as underlying cause of exacerbations in copd patients ( ) . infections with respiratory viruses such as hrv, coronaviruses and to a lesser extend respiratory syncytial virus (rsv) and (para)influenza virus are present during exacerbations and may predispose the host toward secondary bacterial infections that can eventually lead to uncontrolled bacterial outgrowth, more severe exacerbations and neutrophilic inflammation ( , ) . two recent in vitro studies showed that acute exposure to relatively high doses ( - nm) of , (oh) d reduced hrv-infection in undifferentiated cultures of airway epithelial cells ( , ) . in those models, , (oh) d most likely interfered with viral replication by increasing expression of interferon-stimulated genes and expression of hcap /ll- , which has been shown to have direct antiviral activity ( , , ) . in fully differentiated airway epithelial cells, treatment with lower concentrations of , (oh) d ( nm) during epithelial differentiation had no effect on acute hrv infection ( ) . as for other viruses than hrv, both hansdottir et al. and telcian et al. showed that , (oh) d did not decrease rsv infection in airway epithelial cells, but did reduce virus-induced inflammatory responses ( , ) . in addition, two other studies reported in influenza (h n and h n )-infected a cells comparable findings ( , ) . moreover, inhibitory effects of , (oh) d on poly(i:c)-induced inflammatory responses were furthermore confirmed in primary airway epithelial cells hansdottir et al. and by our group ( , ) . up to now, the afore mentioned studies suggest that higher doses of , (oh) d might be protective against hrv-infections in undifferentiated airway epithelial cells only, whereas for other respiratory viral infections , (oh) d mainly reduces inflammatory responses without affecting viral clearance. however, more studies are needed, especially in differentiated airway epithelial cells using multiple hrv-serotypes that use different receptors for infection to verify if , (oh) d indeed is capable of promoting hrvclearance. there is more consensus about , (oh) d reducing virus-induced inflammatory responses and this may certainly help to alleviate the burden of exacerbations in copd ( , ) . in addition to viral infections, also bacterial infections are associated with copd exacerbations and account for ∼ % of all exacerbations ( ) . due to improved study design and sampling techniques from the lower airways using bronchoscopy in recent decades, the causative role of bacteria in copd-related exacerbations has become clear ( ) . this was additionally supported by sethi et al., who found that acquisition of a new strain of pathogenic bacterial species into the airways was linked to copd exacerbations ( ) . recent developments in assessing the airway microbiota using s rrna sequencing techniques further demonstrated that during exacerbations, the relative abundance of haemophilus, pseudomonas, and moraxella was increased and the microbial composition was shifted toward the proteobacteria phylum ( ) . the ability of , (oh) d to promote antibacterial activity was recently demonstrated in cultures of airway epithelial cells. in differentiated airway epithelial cells, we have shown that both (oh)d and , (oh) d treatment enhances epithelial expression of hcap /ll- and antibacterial activity against nthi, a gram-negative bacterium, which is associated with copd exacerbations ( , ) . in addition, yim et al. demonstrated that , (oh) d treatment increased expression of the amp hcap /ll- and killing of pseudomonas aeruginosa and bordetella bronchiseptica, which are both gram-negative bacteria ( ) . these observed antibacterial effects of , (oh) d on airway epithelium in vitro were recently confirmed in vivo by vargas buonfiglio et al. the authors demonstrated that vitamin d supplementation increased antimicrobial activity against the gram-positive staphylococcus aureus in asl in healthy non-smokers and was dependent on presence of hcap /ll- ( ) . in murine airways, studies showed no effects of , (oh) d on the expression of defb or mcramp (the murine homolog for camp) ( ) . this can be explained by the fact that both the promotors of mcramp and defb lack vdres, suggesting that mice might not be suitable for studying the role of , (oh) d in amp-mediated host defense in infection ( ) . indeed, niederstrasser et al. showed no effects of vitamin d deficiency on the susceptibility of mice to pulmonary infection with streptococcus pneumoniae or pseudomonas aeruginosa ( ) . however, in a recently developed mouse model by lowry et al., who transfected mcramp knockout mice with the human camp gene, topical vitamin d treatment increased expression of camp and promoted antibacterial effects on the mucosa of the skin ( ) . there are also multiple other murine studies that demonstrate protective effects of vitamin d on bacterial infections in the gut, indicating that , (oh) dmediated antibacterial effects are additional modulated by other mechanisms such as via enhancement of epithelial barrier integrity ( , ) . in conclusion, these observations show that , (oh) d promotes protection against pollutants and enhances clearance of viral-and bacterial infections (both gram-positive and negative bacteria) in combination with a dampening effect on exaggerated immune responses and these features might explain why vitamin d (deficiency) is linked to copd exacerbations. there are strong indications that modulation of immune responses and antibacterial activities by , (oh) d and/or , (oh) d-regulated amps as well as autophagy have implications for the composition of the microbiota at the epithelial mucosa of the airways and the gut ( ) . evidence for a role of amps in regulating the composition of the microbiota in the gut came from a variety of studies, including those showing that paneth cell-derived defensins may modulate the composition of the microbiome ( ). this notion is further supported by observations showing that many commensal gut bacteria are protected from killing by amps such as the , (oh) d-inducible hcap /ll- and hbd- , whereas pathogens are in general more sensitive ( ) . alterations in the gut microbiota have been linked to many diseases of the gut such as ibd but also with diseases affecting the lungs such as copd and asthma, implicating an important role for the so-called gut-lung axis ( , ) . the mechanisms that explain how gut microbiota affect lung health and disease are complex and include the production of short chain fatty acids (scfas). scfa have a wide range of effects on both immune and structural cells, and the effect of scfa produced in the intestine on lung immunity may in part be explained by modulation of myeloid cells in the bone marrow, which subsequently migrate to the airways and modulate local immune responses ( ) . microbiota that are diverse, rich and contain a higher abundance of scfaproducing species within these populations are considered to be associated with health ( ) . in the gut there is strong evidence that both vitamin d deficiency and/or supplementation affect composition of the adult and infant microbiota ( , ) , specifically in relation to disease ( ) . however, due to the limited number of rcts and small sample sizes, the precise effects on the microbiota and the mechanisms involved in this are still unclear ( ) . alterations in the lung microbiota are also observed in copd and asthma patients and are likely the result of environmental exposures, airway remodeling, infections and treatments such as the use of antibiotics. this may contribute to disease pathogenesis through altered epithelial innate and adaptive immune responses that damages the airway epithelial barrier and provokes further changes in the lung microbiome that accumulates with increasing disease severity ( , ) . to date only studies describe a possible influence of vitamin d on composition of the microbiota in the airways ( , ) . toivonen et al. showed an association between low serum (oh)d levels and reduced richness of the nasopharyngeal microbiota and bronchiolitis severity in patients with low (oh)d levels ( ) , whereas in another study vitamin d supplementation decreased the abundance of staphylococcus aureus, staphylococcus epidermidis and corynebacterium species in sputum samples in vitamin d-deficient cf patients compared to sufficient cf patients ( ) . in summary, there is evidence that alterations in the airway or gut microbiota can affect chronic airway disease and that these changes could be related to both vitamin d deficiency and/or supplementation. however, due to the limited number of rcts and small sample sizes more rcts are needed in larger patient populations. the above described protective and therapeutic possibilities of vitamin d, together with observations that many copd patients are vitamin d deficient, suggest that copd patients might benefit from vitamin d supplementation. as discussed elsewhere in this review, the link between circulating (oh)d levels and the number of exacerbations has been extensively studied ( ) . so far however, only rcts have investigated the effect of vitamin d supplementation in the context of copd: only out of rcts showed that vitamin d supplementation reduces the number of exacerbations ( ) ( ) ( ) ( ) . however, in a post-hoc analysis, selecting those patients that were vitamin d deficient, exacerbations were indeed reduced after vitamin d supplementation. jolliffe et al. summarized these rcts and performed a recent individual participant data meta-analysis and concluded that vitamin d supplementation is only protective against exacerbations in copd patients with baseline serum (oh)d levels < nmol/l ( ) . these important findings suggest that exacerbations in this specific subset of copd patients are connected to vitamin d deficiency and this part can be resolved with supplementation. in summary, the protective effects of vitamin d in patients suffering from copd are most prominent in those with vitamin d deficiency and this would indicate that serum levels (oh)d in these patients should always be determined before considering using vitamin d supplementation. since only rcts with relatively small patient populations have been conducted in both vitamin d-sufficient and -deficient copd patients, more rcts are needed, especially in vitamin d-deficient patients. currently, a multicenter rct is being conducted by rafiq et al. in a group of vitamin-deficient copd patients ( (oh)d < nmol/l), which may reveal whether vitamin d supplementation is indeed protective against exacerbations in this group ( ) . in addition to the effects of vitamin d supplementation in copd patients, the effects of vitamin d supplementation has also been extensively investigated in other lung diseases (which have associations with vitamin d deficiency) such as asthma, cystic fibrosis, upper respiratory tract infections. most rcts that investigated the effects of vitamin d supplementation were performed in acute respiratory tract infections (artis) and asthma. a recent meta-analysis that assessed the effects of vitamin d supplementation in rcts ( , participants) showed that indeed vitamin d supplementation was protective against atris and this effect was again more profound in patients with vitamin d deficiency (oh)d < nmol/l at baseline ( ) . a recent meta-analysis in asthma that included a total of randomized controlled trials ( , participants), indicated that vitamin d supplementation reduced the rate of asthma exacerbations and increased lung function, especially in patients with vitamin d insufficiency ( (oh)d < nmol/l) ( ) . interestingly, in asthma patients that were supplemented with vitamin d, the frequency of respiratory infections was reduced, and this effect was related to the increase of hcap /ll- ( ) . cf patients with vitamin d deficiency had a higher rate of exacerbations as compared to patients with sufficient (oh)d levels ( ) . however, only one recent multicenter rct was conducted and indicated that vitamin d supplementation did not affect the number of exacerbations in cf patients with serum (oh)d concentrations between and . nmol/l ( ) . in summary, the protective effects of vitamin d supplementation in patients suffering from copd, asthma or artis are most prominent in those with vitamin d deficiency and this would indicate the importance of establishing serum levels (oh)d in these patients as supplementation could reduce unnecessary aggravated disease pathology as a result of this deficiency. many drivers of copd pathogenesis such as chronic exposure to noxious particles and gases, which are present in cs and air pollution, proteolytic enzymes, cytokines and chemokines that are released by infiltrating inflammatory cells, are known to harm the epithelial barrier and cause aberrant remodeling of the airway epithelium with important functional consequences for e.g., host defense. a dysfunctional epithelial barrier increases the susceptibility toward bacterial and viral infections, which are important triggers of copd exacerbations and these exacerbations contribute importantly to disease progression. sufficient local levels of , (oh) d may provide partial protection against these effects by reducing the effects of oxidative stress induced by exposure to inhaled oxidants or those derived from recruited inflammatory cells. , (oh) d furthermore protects against impairment of epithelial barrier function by promoting the integrity of the epithelial barrier, and by modulating both innate and adaptive immune responses. protection against the detrimental effects of both bacterial and viral infections is provided by the ability of , (oh) d to promote of antiviral responses, induce expression of amps and modulate of inflammatory responses. taken together, these activities suggest that , (oh) d may provide protection against development and progression of copd, and against disease exacerbations. in addition, the local inflammatory milieu as well as the chronic exposure to noxious particles and gases, which are present in cs and air pollution, may negatively affect synthesis and signaling of , (oh) d. here we discussed recent in vitro studies that demonstrated that disease-associated factors such as inflammation and exposure to cs and air pollution could interfere with , (oh) d signaling and its degradation and activation by affecting expression of vdr, cyp a and cyp b , respectively. these findings indicate that , (oh) d levels and its activities on airway mucosa might be impaired especially in patients with copd with exposures to cigarette smoke and cytokines such as tnf-α, il- β, il- a and tgf-β . this suggests that even in patients with sufficient (oh)d serum levels the local activity of , (oh) d in the lungs can be improved. we have to start generating more information on both systemic and local , (oh) d levels and gene expression signatures related to (oh)d and , (oh) d metabolism or responses in copd (and other chronic inflammatory diseases that are related to vitamin d deficiency), both at baseline and after vitamin d supplementation. this information could lead to improved treatment strategies that enhance local efficacy of , (oh) d, using e.g., specific cyp a -inhibitors such as vid ( ) . alternatively, degradation by cyp a could be prevented by using , (oh) d analogs that are insensitive to cyp a -mediated degradation, such as sulfone and sulfoximine derivatives, that also act as a vdr agonist ( ) . a third option is to entail the use of combination treatment with vitamin d and anti-inflammatory or certain anti-fibrotic drugs that target cytokines/proteins that are known to potentially decrease local levels and signaling of , (oh) d by inducing expression of cyp a ( , , ) . when considering such strategies, it should be noted that these may enhance the calcemic side effects and lead to unwanted inhibition of the immune system. we therefore need to carefully analyze the preclinical in vivo and in vitro studies and balance the pros and cons of the different strategies. in conclusion, future studies in copd and but also in other chronic inflammatory diseases that are related to vitamin d deficiency, should be designed with more focus on assessing and improving local levels of , (oh) d. these new insights may lead to the development of new treatment strategies, such as those targeting cyp a to enhance local , (oh) d resulting in improved homeostasis and protection of the airway mucosa in patients with 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interest.copyright © schrumpf, van der does and hiemstra. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -uqxzds g authors: inatomi, takio; amatatsu, masaaki; romero-pérez, gustavo a.; inoue, ryo; tsukahara, takamitsu title: dietary probiotic compound improves reproductive performance of porcine epidemic diarrhea virus-infected sows reared in a japanese commercial swine farm under vaccine control condition date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: uqxzds g lactogenic immunity transferred to piglets after inoculation of a live vaccine to pregnant sows was proved limited to control porcine epidemic diarrhea (ped). hence, here we evaluated the efficacy of administration of a probiotic compound containing bacillus mesentericus, clostridium butyricum, and enterococcus faecalis together with a commercial live-attenuated ped vaccine (nisseiken ped live vaccine, nisseiken, tokyo, japan) to improve the health and reproductive performance of ped-infected sows. twenty pregnant sows in a ped-positive farm were equally divided into probiotics-administered (vp) and control (vc) sow groups. a commercial live-attenuated vaccine was injected as per the manufacturer’s instruction. the probiotic compound ( g/day) was orally administered to vp from weeks pre-parturition to days post-parturition (ppd ). vp had a significantly higher body weight at ppd than vc ( vs kg; p < . ). at day post-parturition (ppd ) ( . vs . kg/day) and ppd ( . vs . kg/day), milk produced by vp was significantly (p < . ) greater than that by vc. total immunoglobulin (ig)a and igg concentrations at day were significantly (p < . ) higher in whey of vp ( . and . g/dl, respectively) than in that of vc ( . and . g/dl, respectively). however, total igg concentration in whey of vp and vc at ppd and ppd did not differ. antibody titer was significantly higher at day in serum of vp than it was that of vc ( vs in geometric mean; p < . ). likewise, the antibody titer in whey of vp and vc was found to be similar at day ( vs in geometric mean; p = . ). consequently, vp had fewer days between weaning and return to estrus than did vc ( vs days; p < . ). moreover, piglets of vp had a significantly (p < . ) higher litter weight at birth ( , g/litter) and a lower mortality ( %) during suckling than those of vc ( , g/litter and %, respectively). in summary, probiotic-supplemented, ped-vaccinated sows were healthier, transferred ped-specific antibodies via colostrum to piglets, had greater litter weight at birth, and reduced mortality during suckling. introduction porcine epidemic diarrhea (ped) is an enteric disease that causes severe economic losses to the pig industry worldwide ( ) . ped was first recognized in the uk and quickly spread to other european countries ( , ) . in the following years, ped was detected in many asian countries, including japan ( ), where it re-emerged in and caused approximately , outbreaks in of prefectures ( ) . intriguingly, the ped strains emerged in asia are quite distinct from those previously reported ( ) , as they cause more deleterious effects on all pigs regardless of age ( ) . porcine epidemic diarrhea virus is an enveloped, singlestranded rna virus belonging to the group of the genus coronavirus ( ) . ped is characterized by watery diarrhea, dehydration, vomiting, anorexia, and reduced appetite in pigs of all ages. for newborn piglets, mortality caused by ped is close to % ( , ) , whereas for suckling piglets mortality can be as high as % ( ) . in addition, ped can seriously diminish the production performance of surviving animals ( ) . interestingly, the deleterious effects of ped on reproductive performance of gilts and sows depend on the pregnancy stage during which they contract the disease ( ) . for example, sows infected with ped during the first days of pregnancy have decreased farrowing rates, increased abortion rates, and more mummified fetus per litter, whereas females infected with ped during - days of pregnancy have more stillbirth piglets per litter ( ) . although depressed milk secretion has been previously reported in pedinfected sows ( ) , the extent of the effect of this viral infection on lactating sows, including passive immunity or the health status and survival rate of suckling piglets, is yet to be fully investigated. in recent years, vaccines have been developed in an attempt to eradicate ped ( ) ( ) ( ) ( ) . in theory, pre-parturition vaccination of sows putatively permits enteric sensitization to antigens, after which immunoglobulin (ig)a immune cells are transferred to the mammary gland and secrete antibodies ( ) . as a result, passive immunity against ped is putatively conferred by the sow to suckling piglets via colostrum and milk ( , ) . in fact, however, lactogenic immunity by live vaccines has been proven only somewhat efficacious in providing protection to piglets against ped ( ) . for example, ped infections have recently re-emerged in japan ( ), even though, following infection outbreaks, live vaccines were administered to pigs in this country. this lack of effectivity is likely due to either strains from live vaccines producing more infectious mutations in the wild ( ) or vaccine strain sequences having limited compatibility with those of wild types ( ) . it is, therefore, imperative to find antiviral agents that act as adjuvant to existing vaccines and help increase their effectivity. recently, a probiotic bacteria fused with ped virus core neutralizing epitope antigen was developed to use an anti-ped vaccine ( ) . in mice, oral administration was the effective strategy for this vaccine, targeting mainly mucosal dendritic cells in the intestine and stimulating ped virus specific immunity. in contrast, efficacy of this oral vaccine for pigs is still obscure, because no validation study for this species has been conducted yet. administration of probiotics such as lactic acid bacteria (lab) has been recognized as a viable alternative to antiviral medication for treating viral infections. for example, viable lactobacillus acidophilus and lactobacillus reuteri have been shown to protect experimental models against viral strains such as human rotavirus (hrv) by improving total intestinal iga-releasing cell immune responses, as well as total serum igm, and intestinal igm and igg titers ( ) . similarly, colonization of the gut of neonatal gnotobiotic pigs with probiotic strain such as lactobacillus rhamnosus strain gg and bifidobacterium animalis ssp. lactis bb- resulted in significantly lower fecal scores and reduced hrv shedding concentrations but increased intestinal iga hrv antibody concentrations and hrv-specific iga antibody-releasing cell numbers in infected animals ( ) . likewise, in our premises we demonstrated that a cell preparation of enterococcus faecalis strain ec- , which is a heat-killed lab, protected weaning piglets from rotavirus infection ( ) and stimulated luminal iga secretion in young calves ( ) and chicks ( ) . recently, sirichokchatchawan et al. ( ) demonstrated that live lab could reduce ped infectivity in vitro. these data highlight the potential that probiotics and immunogenics may have to enhance lactogenic immunity and the efficacy of live vaccines administered to farm animals. in a separate study, we demonstrated that a probiotic compound containing bacillus mesentericus, clostridium butyricum, and e. faecalis prevented colibacillosis in weaning piglets ( ) . we also reported that twofold concentration of total iga was detected in the ileum in piglets when this probiotic compound was administered. finally, we also reported in a different study that this threeprobiotic strain compound induced twofold concentration of total iga in the ileum of chicks affected by coccidian infection ( ) . the aim of the present study was to evaluate the efficacy of the aforementioned probiotic compound mixed with peptide-zinc to improve the health and reproductive performance of pedinfected lactating sows when administered along with a ped vaccine injection in japan. probiotic product bio-three pz (toa pharmaceutical co. ltd., tokyo, japan) containing a mixture of bacterial strains b. mesentericus to-a ( × cfu/g), c. butyricum to-a ( × cfu/g), and e. faecalis t- ( × cfu/g) in a peptide-zinc compound ( mg/g) was used in this study. the present work was carried out in a commercial swine farm in kyushu region of japan. the farm operates a farrow-to-finish business and has approximately a stock of sows (landrace x large white). for the present work, all sows were impregnated by duroc boars. experiments were carried out between april and may . sows ate commercial feed during gestation (shuton-b; minami nihon kumiai siryo, kagoshima, japan) or lactation (shuton-lactation; minami nihon kumiai siryo) period. the diet was free from intestinal microbiota modifiers, such as antimicrobials and probiotics. a preliminary enteropathogen survey of the farm showed that none of the following diseases were detected in sows and suckling piglets: rotavirus, transmissible gastroenteritis virus, clostridium . ± . . ± . < . perfringens, enterotoxigenic escherichia coli, salmonella sp., brachyspira hyodysenteriae, lawsonia intracellularis, or classical swine fever virus. detection of other pathogens such as porcine reproductive and respiratory virus and porcine circovirus type was positive, but not active during this study. the infection rate of these two viruses in the farm was constant at about %, according to the survey of randomly selected sows conducted every months. infection outbreaks of ped virus occurred in december in the region where the farm was located. as a consequence, an infection screening was conducted at the farm by an independent livestock hygiene center. the screening results showed that all sows used in the present study were indeed naturally infected with ped virus. the present experiment was approved by the ethical committee at inatomi animal hospital (approval number ). the experiment was carried out between may and june . twenty pregnant sows were equally divided and allocated to either a probiotic-administered group (vp) or a control group (vc) with a similar mean parity (= . ). six weeks before parturition to week after parturition, g/day of the probiotic compound was orally administered to sows in the vp group, whereas g/ day of a standard, probiotic-free diet was given to sows in the vc group. a commercial live-attenuated vaccine (nisseiken ped live vaccine, nisseiken, tokyo, japan) containing ped virus strain p- v (seed) was injected to all sows ( . tcid / head) and weeks before parturition. each sow fostered nine neonates throughout the experiment. some piglets died during the experiment. clinical signs included watery yellowish diarrhea and dehydration. an autopsy showed that their small intestine was thin and flaccid. a qualified clinical veterinarian (dr. takio inatomi) diagnosed that piglets died from ped infection. blood was collected from the jugular vein of sows days prior to parturition and and days post-parturition. milk secretion was determined by a general method described in the standard methods of evaluation of reproductive performance compiled by the japan's pork producers association. daily volume of milk secretion of all sows was measured at days and postparturition. a portion of milk was collected at days , , and post-parturition. in addition, the total body weight of neonates was repeatedly measured immediately prior to and after daily suckling. when piglets of the experimental sows died during lactation, healthy and similar-age foster piglets were introduced from other sows so that all piglets ingested a similar amount of maternal milk. protein and fat percentages in milk at days and post-parturition were determined by a milk analyzer (milkoscan™ ft ; foss, eden prairie, mn, usa). http://www.jp-nisseiken.co.jp/en/products/pdf/pig/ped_en.pdf. http://www.jppa.biz. whey was collected from milk after centrifugation ( , × g, min, °c). serum was collected from blood after centrifugation ( , × g, min; room temperature). total iga or g concentrations were measured by a commercial elisa kit (porcine iga or igg elisa quantitation set; bethyl, montogomery, tx, usa). the determination method was as previously described elsewhere ( ) . a portion of whey was sent to the nansatu livestock hygiene center (kagoshima, japan), and neutralized antibody titer against ped in whey and serum was determined by a general method as previously described elsewhere ( , ) . either the student's or the welch's t-test was applied to analyze differences between mean values in all parameters. values are shown as mean ± se. differences between mean values were considered significant at p < . and a tendency to be significant at p < . in all statistical analyses. all calculations were made using statcel (oms, tokyo, japan) as add-in application for microsoft excel ® (microsoft, seattle, wa, usa). the mean feed intake of sows is shown in table and probiotic compound had a significantly higher (p < . ) feed intake before ( . kg) and after parturition ( . kg) compared with sows vaccinated against ped virus but receiving no dietary probiotic supplementation ( . and . kg, respectively). in comparison with that of vc sows, the mean body weight of vp sows showed only a tendency (p < . ) to be greater as they approached parturition ( vs kg, respectively), but it was significantly higher (p < . ) by day post-parturition ( vs kg, respectively) (figure ). following parturition, milk produced by vp sows was found to be significantly greater (p < . ) than that by vc sows at days ( . vs . kg/day, respectively) and after parturition ( . vs . kg/day, respectively) ( figure ) . the protein percentage in milk of vp sows was significantly greater (p < . ) at parturition day ( . %) than that measured in milk of vc sows ( . %) (figure ) . however, when it was measured again at day postparturition, no difference in protein percentage was detected between milk samples of vp and vc sows ( . vs . %). when looking at the lactogenic immunity parameters, total iga concentration at day (parturition day) was significantly (p < . ) higher in whey of vp sows ( . g/dl) than in that of vc sows ( . g/dl), but afterward, total iga concentration only showed a tendency (p < . ) to increase in whey of vp but not vc sows, when it was measured at days ( . vs . g/dl, respectively) and post-parturition ( . vs . g/dl, respectively) (figure a) . similarly, the concentration of total igg was significantly (p < . ) greater in whey of vp sows ( . g/dl) at day when compared with that of vc sows ( . g/dl). however, unlike iga, the concentration of igg was the same in whey of both vp and vc sows, when it was measured at days ( . vs . g/dl, respectively) and post-parturition ( . vs . g/dl, respectively) ( figure b ). in addition, while the antibody titer was found significantly (p < . ) higher at day in serum of vp sows ( . in geographic mean) when compared with that of vc sows ( . ) , it was similar in serum of sows in both experimental groups days prior to parturition ( . vs . , respectively) and days post-parturition ( . vs . , respectively) ( figure c) . likewise, the antibody titer was found to be similar ( . vs . at day ; . vs . at day ) in whey of both vp and vc sows ( figure d ). reproductive performance the combined administration of probiotics and vaccine had a positive effect on reproductive performance. indeed, the days between weaning and return to estrus for most vp sows were fewer ( days) (p < . ) than those for vc sows ( days) ( figure ) . moreover, piglets farrowed by vp sows had a significantly (p < . ) higher weight ( , g/litter) ( figure a ) and a lower (p < . ) mortality percentage ( %) than those farrowed by vc sows ( , g/litter and %, respectively) ( figure b ). porcine epidemic diarrhea-vaccinated sows that were also supplemented with probiotics improved their feed intake, gained reasonable bodyweight during the period prior to parturition, and had only marginal weight loss by day post-parturition than did sows that were vaccinated but did not receive probiotics supplementation (figures and ) . moreover, those sows receiving both probiotic supplementation and vaccine administration produced more milk than did sows that were only vaccinated (figure ) . previously, alexopoulos et al. ( ) showed that supplementing feed with a probiotic compound containing bacillus licheniformis and bacillus subtilis spores not only increased the body weight of sows but also helped minimize their weight loss during the suckling period. likewise, kritas et al. ( ) reported that supplementing with b. subtilis c- resulted in higher feed consumption and better body condition of sows. thus, it is very likely that in the present study there were similar effects, in which probiotics indirectly promoted weight gain in sows by competing with pathogens in the gut and stimulating the immune system of the host and hence making the animal more resistant to infections ( ) . it is also possible that probiotics fought back the viral infection directly and decreased the viral load in sows and eventually in piglets as sirichokchatchawan et al. ( ) recently suggested when discussing the potency of probiotic lab to reduce ped infectivity in vitro. in addition, the peptide-zinc mixture added to the probiotic compound may have also contributed to enhance the immune response. indeed, zinc has been previously shown to help reduce diarrhea incidence and increase body weight in pigs by stimulating the immune response against viral infection ( ) . ultimately, healthier sows in the vp group consumed more feed, utilized better feed nutrients, and consequently gained more weight and produced more milk (figures and ) . in commercial swine farms, pregnant sows are inoculated with vaccines to trigger an immunoprophylactic reaction known as lactogenic immunity ( ) . lactogenic immunity protects piglets against infections via the suckling of colostrum and milk of vaccinated sows ( ) . nonetheless, in the present work, probiotics supplementation significantly improved the concentration of total iga and igg in the milk of sows than did ped vaccination alone, possibly resulting from stimulation of gut immunity by bacteria in the administered probiotic compound (figures a,b) . the sole administration of ped vaccine to sows has been shown to increase ped-specific igg levels in serum ( ) . interestingly, by administering the ped vaccine along with probiotics supplementation, we were able to trigger an even higher serum ped-specific antibody titer than did ped vaccination alone, very likely due to a greater stimulation of the immune system of sows. then, as our results show, these antibodies were mobilized to fight back ped infection before and after parturition (figure c) , which consequently also helped to stimulate ped-specific antibodies production in whey ( figure d) . finally, once piglets started to suckle, passive immunity against ped was putatively transferred from sows via colostrum and milk ( , ) , possibly inducing early maturation of gut immunity of piglets that lessened the infection, which in turn heightened the effect of vaccination ( ) . this ultimately resulted in the farrowing of piglets with a greater weight and a lower mortality percentage by sows receiving both probiotics and the ped vaccine (figure ) . viral infections can cause serious biological disturbances in sows including a higher recurrence of estrus ( ) , as well as abnormal tissue development such as ovarian cysts formation ( ) . these disorders can lead to low fertility ( ) . in addition, it is believed that a decrease in appetite and lower efficiency of nutrient utilization during the course of an infection can cause a lower body weight of sows which in turn cause a longer period before sows can return to estrus ( ) . nonetheless, it has been previously shown that b. subtilis c- supplementation helped reduce the number of days between weaning and return to estrus ( ) . furthermore, a probiotic compound containing the same bacteria used in this study improved the percentage of sows returning to estrus ( ) . in agreement with this, in the present work ped vaccination and probiotic administration to ped-infected sows considerably lowered the number of days of recurrence of estrus than did ped vaccination alone ( figure ). as previously mentioned, it is very likely that an enhanced microbiota permitted sows to eat more and utilized nutrients more efficiently, thus preventing malnutrition from causing a longer period between weaning and return to estrus ( ) . olanratmanee et al. ( ) found that ped infection caused a reduction in the body weight of piglets at birth. in the present study, piglets farrowed by sows vaccinated against ped and supplemented with probiotics had a greater mean litter weight at birth and a lower mortality percentage than did those farrowed by sows receiving ped vaccination but no probiotic supplementation (figures a,b) . however, unlike our study, kritas et al. ( ) found that supplementation of a single probiotic strain (b. subtilis c- ) to sows infected with pathogenic e. coli did not alter the body weight at birth of piglets. this apparent discrepancy may be due to in the present study and unlike that by kritas et al. a probiotic mixture was given to sows. indeed, it has been demonstrated both in vivo and in vitro that multistrain probiotic compounds are more effective at inhibiting pathogens than single-strain probiotics and that some strain mixtures are more effective than others ( , ) . in conclusion, we demonstrated that supplementation of a mixture of probiotics and a peptide-zinc compound enhanced the immune system of ped virus-infected sows. in addition, we also showed that probiotics were able to act as adjuvant to commercial live ped vaccines administered to pregnant sows. this experiment was approved by the ethical committee of inatomi animal hospital in japan. takio inatomi belongs to inatomi animal hospital informed consent to animal owners. improvement of ped symptom by probiotic administration frontiers in immunology | www.frontiersin.org december | volume | article author contributions ti designed and performed this experiment and contributed to the discussion and interpretation of data. ma supplied the experimental materials and contributed to the discussion and interpretation of data. gr-p wrote the manuscript. ri contributed to the discussion and interpretation of data and wrote the manuscript. tt designed this experiment, contributed to the discussion and interpretation of data, and wrote the manuscript. all authors have read and approved the final manuscript. references epidemiological factors associated to spread of porcine epidemic diarrhea in japan porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus molecular characterization of pig epidemic diarrhoea viruses isolated in japan from an outbreak of swine diarrhea of a newtype associated with coronavirus-like particles 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evaluation of a probiotic and its synergism with vaccination against coccidiosis in broilers the evaluation of secretion volume and immunoglobulin a and g concentrations in sow colostrum from anterior to posterior teats isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate field evaluation of the efficacy of a probiotic containing bacillus licheniformis and bacillus subtilis spores, on the health status and performance of frontiers in immunology | www sows and their litters reproductive performance of sows was improved by administration of a sporing bacillary probiotic (bacillus subtilis c- ) evaluation of probiotics as a substitute for antibiotics in a large pig nursery highdose dietary zinc oxide mitigates infection with transmissible gastroenteritis virus in piglets strategies for design and application of enteric viral vaccines pathological evaluation of reproductive system of porcine reproductive and respiratory syndrome virus-vaccinated and nonvaccinated anestrus sows and gilts body weight loss during lactation and its influence on weaning-to-service interval and ovulation rate in landrace and yorkshire sows in the tropical environment of thailand in vitro evaluation of single-and multi-strain probiotics: inter-species inhibition between probiotic strains, and inhibition of pathogens health benefits of probiotics: are mixtures more effective than single strains? key: cord- -asbt mcj authors: schulz, katharina s.; mossman, karen l. title: viral evasion strategies in type i ifn signaling – a summary of recent developments date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: asbt mcj the immune system protects the organism against infections and the damage associated with them. the first line of defense against pathogens is the innate immune response. in the case of a viral infection, it induces the interferon (ifn) signaling cascade and eventually the expression of type i ifn, which then causes an antiviral state in the cells. however, many viruses have developed strategies to counteract this mechanism and prevent the production of ifn. in order to modulate or inhibit the ifn signaling cascade in their favor, viruses have found ways to interfere at every single step of the cascade, for example, by inducing protein degradation or cleavage, or by mediate protein polyubiquitination. in this article, we will review examples of viruses that modulate the ifn response and describe the mechanisms they use. the mammalian immune system evolved to detect and fight viral infections effectively. the induction of type i interferon (ifn), predominantly ifn-α and ifn-β, forms the first line of defense. the type i ifn response consists of two parts. first, the cell produces type i ifn, when triggered by a viral stimulus. the ifn is then secreted and, in the second part of the response, it is sensed by the producing, as well as neighboring cells, resulting in the production of ifn-stimulated genes (isgs) [reviewed in ref. ( ) ]. viruses, which have coevolved with their host, develop strategies to counteract the signaling cascades of the innate immune system and ensure their replication. recently, several reviews were published, describing the innate immune evasion strategies of individual viruses or virus families, such as influenza virus ( , ) , phleboviruses ( ), herpes viruses ( - ), coronaviruses severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) ( ) , human immunodeficiency virus (hiv) ( , ) , as well as multiple rna viruses ( , ) . moreover, there are recent articles that review how viruses prevent detection by pathogen recognition receptors (prrs) ( , ) and how viruses modulate innate immune signaling by use of viral deubiquitinases ( ) . in this review, we will compare the different strategies viruses have developed to suppress innate immune signaling of individual components of the innate immune signaling cascade. due to the tremendous amount of data in this field, we will focus on recent discoveries. older studies were summarized in ref. ( , ) . ( ) ]. the most important viral markers for the innate immune system are viral nucleic acids. the detection of viral dna through the cgas-sting pathway and the counter measurements taken by viruses have been reviewed recently ( ) and are not part of this review. viral rnas, which are mostly double-stranded (ds-)rna, are recognized by three prrs: the endosomal toll-like receptor (tlr ), the cytoplasmic retinoic acid-inducible gene i (rig-i)like receptors (rlrs), and the nucleotide-oligomerization domain (nod)-like receptors (nlrs) ( ) . tlr and the rlrs are important for inducing the type i ifn response, whereas nlrs have been shown to regulate interleukin- β (il- β) maturation through activation of caspase- ( ) . the group of rlrs consists of rig-i, melanoma differentiation-associated gene (mda ), and laboratory of genetics and physiology (lgp ). the three receptors have a similar structure, all containing a caboxyterminal domain, which functions as a repressor domain (rd) in rig-i and lgp ( ) and a central helicase domain, but lgp lacks the caspase activation and recruitment domains (cards) that function in signaling [reviewed in ref. ( , ) ]. both the helicase and the carboxy-terminal domain are required for rna binding. rig- and mda- detect specific viral rna pamps, while lgp negatively regulates rig-i signaling and promotes rna binding to mda [reviewed in detail in ref. ( ) ]. in unstimulated cells, rig-i and mda- are kept in a repressed state due to phosphorylations on serine and threonine residues in the cards and carboxy-terminal domains ( , ) . upon binding of rna, both rig-i and mda- undergo conformational changes, resulting in release of their cards ( , ) . recruited phosphatases remove the phosphate residues, and e ubiquitin ligases attach lys -linked ubiquitin polymers onto the cards and c-terminal domain of rig-i, which are important for rig-i tetramerization ( ) ( ) ( ) ( ) ( ) . rna-bound rig- then interacts with - - ε, a mitochondrial trafficking protein, and the trim ubiquitin ligase, which together transport rig-i to the mitochondria ( ) . there the cards of rig-i or mda- interact with the card of the mitochondrial activator of virus signaling (mavs, also known as ips- , visa, and cardif), which is an essential signaling adaptor protein. the activation of mavs has recently been reviewed in detail in ref. ( ) . tlr interacts with trif, which serves as a molecular platform and forms physical interactions with several adaptor molecules ( ) . by interacting with upstream adaptors, trif undergoes conformational changes and recruits the downstream tnf receptor-associated factor (traf) and traf [reviewed in ref. ( ) ]. the kinase receptor-interacting protein- (rip- ) is part of both the signaling pathways downstream of tlr and rig-i. it can interact with trif to induce nfκb activation ( ) . moreover, the dsrna-activated tlr can recruit trif, rip- , and caspase- and induce apoptosis ( ) . also, rip- and its adaptor protein fas-associated protein with death domain (fadd) are part of the signaling cascade downstream of rig-i and mda- and involved in the activation of the transcription factors interferon regulatory factor (irf) and irf ( ) . traf serves as a linker between the upstream adaptor proteins (trif or myd for tlrs and mavs for rlrs) and the downstream signaling kinases tbk /ikkε or irak /ikkα. the recruitment of traf to the tlr or rlr signaling complexes activates the e ligase activity of traf , which then catalyzes its own k -linked ubiquitinylation. subsequent traf activates tbk /ikkε or irak/ikkα [reviewed in ref. ( ) ] (figure ) . viruses target rig-i directly or indirectly to block the type i ifn response. the phlebovirus toscana virus expresses a non-structural protein, which directly interacts with rig-i and induces its proteasomal degradation ( , ) . foot-and-mouth disease virus (fmdv) proteins l pro , c pro , and b increase the rig-i mrna expression but decrease the protein expression of rig-i. l pro and c pro both induce rig-i degradation, whereas the mechanism of how b reduces rig-i protein levels has not been solved yet ( ) . other viruses target rig-i indirectly. hepatitis b virus (hbv) induces mir a, which then posttranscriptionally inhibits the expression of rig-i and suppresses the production of type i ifn ( ) . the dengue virus ns protein binds to - - ε and prevents the translocation of rig-i to mavs. the binding site on ns is a highly conserved phosphomimetic motif, which was verified by generation of a virus containing a mutation in this motif ( ) . it has been proposed that in certain cell types rig-i requires sentinels, such as the protein ddx , which associates with rig-i and promotes the rig-i rna-binding activity ( , ) . other studies question ddx acting as a broadly active enhancer of antiviral responses ( , ) and instead suggest that ddx only functions in the antiviral response to specific viruses, such as hepatitis c virus ( ) . however, there are data indicating that influenza a virus and hepatitis c virus attenuate ifnβ-promoter activation by targeting the sentinel ddx . both viruses activate the epidermal growth factor (egf) receptor, which in turn phosphorylates ddx on tyr- and tyr- . this results in the attenuation of ddx -dependent rig-i activation. in addition, independent of its role as sentinel for rig-i viral rna recognition, ddx plays a role in viral rna degradation ( ) (figure ). mitochondrial activator of virus signaling is blocked by different viruses in various ways. the dengue virus protein ns a targets mavs, and the interaction prevents the binding of mavs to rig-i ( ) . the porcine reproductive and respiratory syndrome virus (prrsv) c-like protease ( clsp), by contrast, cleaves mavs in a proteasome-and caspase-independent manner at glu (e /g ). both cleavage products fail to activate the type i ifn response ( ) . likewise, the hepatitis c virus protein ns - a ( , ) , as well as the highly pathogenic porcine reproductive and respiratory syndrome virus (hp-prrsv) protein nsp ( ) have been shown to cleave mavs and block rlr signaling. the porcine epidemic diarrhea virus (pedv) also targets mavs in small intestinal epithelial cells (iecs). however, the exact mechanism has not been solved yet ( ) (figure ) . the sars coronavirus protein orf b not only influences antiviral signaling but also alters host cell mitochondria morphology by inducing degradation of the dynamin-like protein (drp ). mavs becomes concentrated into small puncta in the presence ( ) . in addition to mavs, also the levels of traf and traf are reduced by orf b. however, it is unlikely that traf and traf are targeted directly. more likely, they are degraded due to their interaction with mavs ( ) (figure ) . human t-cell lymphotropic virus type i (htlv- ) protein tax disrupts innate immune signaling in multiple ways: it binds to the rip homotypic interaction motif (rhim) domains of rip- and disrupts the interaction between rip- and rig-i or mda- and the activation of the type i ifn promoter. tax also binds to trif and thereby interrupts the tlr signaling cascade. finally, tax blocks the association between rip- and irf , which resulted in repression of the irf activity ( ) (figure ) . middle east respiratory syndrome coronavirus m protein interacts with traf and disrupts the interaction between traf and tbk , which ultimately leads to a reduced irf activation. for the interaction with traf , the n-terminal transmembrane domain of the mers-cov m protein is sufficient ( ) , similar to what has been shown for sars-cov before ( ) (figure ). triggering of the tlr -and rlr-signaling cascade results in the activation of the transcription factors nfκb and irf /irf . in its inactive state, the transcription factor nfκb is complexed with its inhibitor iκb ( ) . upon stimulation, iκb is phosphorylated by the iκb kinase (ikk) complex, which is composed of two catalytic subunits, such as ikkα and ikkβ, and a regulatory subunit, such as nfκb essential modulator (nemo) ( ) . the phosphorylation of iκbα induces its polyubiquitination through the e ubiquitin ligase β-transducin repeat-containing protein (β-trcp) and subsequent proteasomal degradation ( ) , allowing nfκb to translocate into the nucleus and induce the expression of target genes ( ) (figure ) . encephalomyocarditis virus (emcv) protein c cleaves traf family member-associated nfκb activator (tank), which inhibits traf -mediated nfκb activation, on gln . as a result, nfκb is activated and the unstable c-terminal fragment of tank is subjected to proteasomal degradation ( ) . also, other viruses express proteases that cleave tank, although on other residues, such as porcine reproductive and respiratory syndrome virus (prrsv) (tank is cleaved by nsp ), fmdv (protease c cleaves tank), and equine arteritis virus (eav) (tank is cleaved by nsp ). thus, tank seems to be a common target of several positive rna viral proteases ( ) (figure ) . several viruses have been shown to disrupt ifn signaling by cleaving nemo. pedv c-like protease, nsp , cleaves nemo at gln ( ), whereas the hepatitis a virus c protease ( c pro ) cleaves nemo at gln ( ) and the picornavirus fmdv protease c pro at gln , removing the c-terminal zinc finger domain from the protein ( ) . the human rotavirus has developed another way. its non-structural protein (nsp ) has been shown to inhibit the nfκb pathway by degrading β-trcp and consequently stabilizing iκb ( ) (figure ) . tank-binding kinase (tbk ) and inhibitor of κb kinase ε (ikkε) are classified as non-canonical serine/threonine kinases and are both able to induce irf and irf phosphorylation and subsequent dimerization ( ) ( ) ( ) ( ) . however, while tbk is constitutively expressed in most cell types, the expression of ikkε is more restricted ( ) . upon stimulation, tbk and ikkε are recruited by adaptor proteins to signaling complexes to be activated by phosphorylation on ser and both have been shown to be subjected to k -linked polyubiquitination [reviewed in ref. ( , ) ]. for tbk , k -linked polyubiquitination seems to be important for tlr-and rlr-induced ifn production, as ubiquitin chains might serve as a platform for the assembly of tbk signaling complexes. moreover, deubiquitinases are able to terminate the tbk -mediated pathway by cleaving the k linked ubiquitin chains [reviewed in ref. ( , ) ]. activated tbk /ikkε phosphorylates irf and/or irf in the cytosol at specific serine residues. this phosphorylation results in homo-or heterodimerization of irf and irf and nuclear translocation ( , ) . interestingly, while irf is constitutively expressed, irf is expressed at low levels in most cell types and expression is induced upon ifn signaling. therefore, in most cells, irf strongly enhances the production of ifn [reviewed in ref. ( ) ]. once phosphorylated irf and/or irf dimers have translocated into the nucleus, they bind to the transcription coactivator creb-binding protein (cpb)/p ( , ) . together with other factors, such as nfκb, they form the enhanceosome on the ifnβ promoter and induce the expression of type i ifn [reviewed in ref. ( ) ]. the viral proteins that target tbk act by either blocking activation of tbk by mavs or by inhibiting activation of irf by tbk . the mers-cov protein orf b blocks ifnβ production by binding to tbk and ikkε and suppressing the formation of a mavs/ikkε complex ( ) . in addition to inhibiting tbk /ikkε activation, orf b can also inhibit the production of ifnβ in the nucleus; however, the mechanism has not been solved yet ( ) . recently, two herpes simplex virus proteins have been shown to target tbk /ikkε and inhibit the phosphorylation of irf : icp ( ) and vp ( ) . also, dengue virus serotype non-structural proteins ns a and ns b, as well as the ns a and ns b proteins of other dengue viruses, inhibit the phosphorylation of tbk ( ) and pedv n protein has been shown to interact with tbk , hampering the association of tbk with irf and preventing the activation of irf activation ( ) . the human t-cell leukemia virus type oncoprotein tax has been shown to also interact with tbk . however, studies came to contradicting results on how that influences the production of ifnβ. while one group showed that tax activates tbk and the production of ifnβ ( ), another group showed that tax suppresses the ifn production by interaction with tbk ( ) . interestingly, when a recent study tested how the rabies virus p protein of street strains behaves compared to laboratory-adapted strains with regard to the induction of type i ifn, they found that both street strains and laboratory strains inhibit tbk -mediated signaling, but only the p protein of street strains also interacts with and inhibits ikkε-inducible irf dependent ifnβ expression ( ) (figure ) . interferon regulatory factor is targeted by many viruses to impair innate immune signaling. most viruses inhibit the phosphorylation and thereby also the dimerization and translocation of irf , such as the porcine deltacoronavirus ( ) or poliovirus ( ) . hepatitis e virus protein orf also suppresses irf phosphorylation, but in an indirect way. it activates the signal regulator protein α (sirp-α), which negatively regulates type i ifn induction ( ) . in contrast, porcine bocavirus (pbov) np protein does not affect irf expression, phosphorylation, or nuclear translocation. instead, it interacts with the dna-binding domain of irf and inhibits the dna-binding activity ( ) . a very interesting way of how to circumvent the host innate immune response was found when studying gammaherpesviruses kaposi's sarcoma-associated herpesvirus (kshv) and rhesus macaque rhadinovirus (rrv). they express several viral homologs to the irfs, called viral irfs (virfs). these virfs have found multiple ways to suppress type i ifn production. for kshv, different strategies have been reviewed in ref. ( ) . recently, the rrv virf r has been shown to interact with the transcriptional coactivator cbp in the nucleus, similar to the kshv virf . as a result, cbp cannot form a complex with the phosphorylated irf , and the ifn expression is not induced ( ) ( ) ( ) . interestingly, rrv r is the first virf for which an association with the viron could be shown. therefore, virf v can shut down the type i ifn response shortly after the cell was infected, rendering the cell more susceptible to infection ( ) . the pedv protein nsp also targets cbp. nsp induces cbp degradation in a proteasome-dependent manner and thereby interrupts enhanceosome assembly and the production of type i ifn ( ) (figure ) . for most of these interactions, the molecular mechanisms have not been unraveled yet. a protein that has been shown to interact with and induce proteasomal degradation of irf some time ago is classical swine fever virus (csfv) npro ( , ) . recently, the molecular mechanism has been published. irf and npro interact direct and form a soluble : complex. moreover, it was shown that npro interacts with the full-length irf , not with individual domains, and that npro binds the constitutively active form of irf in the presence of cpb. thus, npro interacts with both the monomer and the active irf dimer and likely targets both species for ubiquitinylation and proteasomal degradation ( ) . interferon regulatory factor is targeted by two human enteroviruses, such as enterovirus and enterovirus . they downregulate irf by cleaving it with their protease c, leaving the cleavage products unable to induce ifn expression. while enterovirus cleaves irf once at gln -ser ( ), enterovirus cleaves it twice, the cleavage sites being gln and gln ( ) . moreover, megalocytivirus, a dna virus that infects marine and freshwater fish, induces the expression of the host microrna pol-mir- , which then specifically suppresses the expression of irf ( ) (figure ) . the type i ifns act in an autocrine, paracrine, or systemic manner to stimulate antiviral responses. they are recognized by the ifnα/β receptor (ifnar), which consists of the subunits ifnar and ifnar expressed on virtually all cell types ( ) . the interaction of type i ifn with the receptor results in the phosphorylation and activation of the ifnar -and ifnar -associated tyrosine kinases tyrosine kinase (tyk ) and janus kinase (jak ), which then phosphorylate ifnar tyrosine residues, resulting in the recruitment and activation of signaling molecules, such as the signal transducer and activator of transcription (stat) family of transcription factors ( , ) . upon activation, stat and stat , together with irf , form the ifn-stimulated gene factor (isgf ), which then translocates into the nucleus to induce transcription of isgs [reviewed in detail in ref. ( ) ( ) ( ) ]. several viruses target ifnar to prohibit ifn binding and signaling. influenza virus induces the degradation of ifnar . hemagglutinin (ha) triggers the phosphorylation and ubiquitinylation of ifnar , thus promoting protein degradation ( ) . encephalitic flaviviruses, such as tick-borne encephalitis virus or west nile virus, inhibit ifnar surface expression. their protein ns binds the cellular dipeptidase prolidase (pepd), which is involved in ifnar maturation and accumulation, activation of ifnβ-stimulated gene induction, and ifn-dependent viral control. this interaction inhibits ifnar intracellular trafficking and glycosylation but does not promote ifnar degradation ( ) (figure ) . both stat and stat are targeted by many viruses to suppress isg induction. pedv induces stat ubiquitinylation and targets it for degradation in the proteasomes ( ) . ( ) . similarly, human metapneumovirus (hmpv) protein sh impairs stat expression, phosphorylation, and activation ( ) . simian varicella virus not only inhibits stat phosphorylation but also promotes degradation of irf in a proteasome-dependent manner through its protein orf ( ) . also, infectious bronchitis virus (ibv) inhibits phosphorylation and nuclear translocation of stat . however, despite detailed analyses, it is unclear which viral protein is responsible. it was, however, shown that the accessory protein a contributes to ibv resistance to type i ifn, although the target is unknown as well ( ) . in case of the human parvovirus b , it becomes evidently clear that both the virus and the immune system constantly evolve to prevail. while its protein ns suppresses stat phosphorylation, the immune system senses the protein and triggers the production of type i ifn ( ) . sftsv, an emerging tick-borne pathogen, developed multiple ways to prevent isg induction. the viral non-structural protein ns impairs stat expression, phosphorylation, and activation ( ) and interacts with stat and sequesters stat and stat into viral inclusion bodies, where they are trapped ( ) (figure ) . the jak-stat signal transduction pathway is negatively regulated by the suppressor of cytokine signaling (socs) family of proteins in form of a classical feedback loop ( , ) . some viruses induce the expression of socs to take advantage of this mechanism to minimize the induction of isgs. japanese encephalitic virus (jev) downregulates the expression of micro-rna mir- , which then results in upregulated socs levels ( ) . varicella-zoster virus (vzv) infection induces the expression of socs ( ) and respiratory syncytial virus (rsv) nonstructural proteins ns and ns induce upregulation of socs and socs , which also inhibited the induction of chemokines ( ) (figure ). viruses fully depend on the translation machinery of the host cell for replication. accordingly, they have evolved multiple ways to hamper host protein synthesis [reviewed in ref. ( ) ]. one way is to shut off host protein synthesis. for some time, it was thought that gamma-and deltacoronaviruses do not induce host shutoff, such as alpha-and betacoronaviruses do. however, a recent study showed that the infectious bronchitis gammacoronavirus induces host shutoff using its protein b. it seems like b is a functional equivalent of nsp , the host shutoff protein of alpha-and betacoronaviruses ( ) . viruses evolved to have various strategies to circumvent the innate immune response by blocking the production of type i ifn or the expression of isgs. while these diverse strategies may appear contradictory between viruses, several factors require consideration. for example, the use of clinical isolates versus viral evasion of interferon pathways frontiers in immunology | www.frontiersin.org november | volume | article laboratory-passaged strains could yield different results, particularly with rna viruses that rapidly accumulate mutations due to error-prone rna-dependent rna polymerases. moreover, the choice of cell line can greatly influence experimental outcomes, as many immortalized or transformed continual cell lines harbor mutations in critical innate immune signaling ( ) . likewise, the use of genetic knockout versus knockdown cell lines or organisms can influence experimental outcomes, as can the experimental procedures themselves, particularly when endogenous interactions are disrupted with the use of overexpression approaches. studying the mechanisms used by viruses to prevent an immune response is of great importance for the development of new strategies to limit the sequelae of viral infections. identification of key immune evasion proteins allows development of antivirals to target these proteins. alternatively, identification of key cellular antiviral pathways allows development of strategies to enhance these pathways to overwhelm incoming viruses. information on key immune evasion factors further facilitates the engineering of safe and effective vaccine strains and designing strategies to target new emerging viruses from the same or closely related family. ks and km conceptualized the scope of the review article. ks wrote the review with input from km. this work was supported by a postdoctoral fellowship from the deutsche forschungsgemeinschaft (schu / - ). work in the mossman laboratory on innate antiviral signaling is supported by the canadian institutes for health research. induction and function of ifnbeta during viral and bacterial infection functions of the influenza a virus ns protein in antiviral defense to conquer the host, influenza virus is packing it in: interferon-antagonistic strategies beyond ns phleboviruses and the type i interferon response the tiers and dimensions of evasion of the type i interferon response by human cytomegalovirus herpesviruses: interfering innate immunity by targeting viral sensing and interferon pathways evasion of host antiviral innate immunity by hsv- , an update middle east 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innate immunity ddx , a dexd/h box helicase, is a novel antiviral factor promoting rig-i-like receptor-mediated signaling ddx is involved in rig-i-dependent and independent antiviral responses, and its function is attenuated by virus-induced egfr activation a diverse range of gene products are effectors of the type i interferon antiviral response mouse superkiller- -like helicase ddx is dispensable for type i ifn induction and immunity to multiple viruses dengue virus subverts host innate immunity by targeting adaptor protein mavs porcine reproductive and respiratory syndrome virus c protease cleaves the mitochondrial antiviral signalling complex to antagonize ifn-beta expression hepatitis c virus protease ns / a cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity hepatitis c virus ns - a inhibits the peroxisomal mavs-dependent antiviral signalling response highly pathogenic porcine reproductive and respiratory syndrome virus nsp cleaves visa to impair antiviral responses mediated by rig-i-like receptors porcine epidemic diarrhea virus inhibits dsrna-induced interferon-beta production in porcine intestinal epithelial cells by blockade of the rig-i-mediated pathway sars-coronavirus open reading frame- b suppresses innate immunity by targeting mitochondria and the mavs/traf /traf signalosome pcbp mediates degradation of the adaptor mavs via the hect ubiquitin ligase aip oncogenic human t-cell lymphotropic virus type tax suppression of primary innate immune signaling pathways middle east respiratory syndrome coronavirus m protein suppresses type i interferon expression through the inhibition of tbk -dependent phosphorylation of irf suppression of innate antiviral response by severe acute respiratory syndrome coronavirus m protein is mediated through the first transmembrane domain a firm hand on nfkappab: structures of the ikappabalpha-nfkappab complex ikappab kinases: key regulators of the nf-kappab pathway inducible degradation of ikappabalpha by the proteasome requires interaction with the f-box protein h-betatrcp shaping the nuclear action of nf-kappab encephalomyocarditis virus c protease relieves traf family member-associated nf-kappab activator (tank) inhibitory effect on traf -mediated nf-kappab signaling through cleavage of tank porcine epidemic diarrhea virus c-like protease regulates its interferon antagonism by cleaving nemo hepatitis a virus c protease cleaves nemo to impair induction of beta interferon foot-and-mouth disease virus c protease cleaves nemo to impair innate immune signaling nsp of human rotaviruses commonly inhibits nf-kappab signalling by inducing beta-trcp degradation ikkepsilon and tbk are essential components of the irf signaling pathway triggering the interferon antiviral response through an ikk-related pathway regulation and function of ikk and ikk-related kinases involvement of the ubiquitin-like domain of tbk /ikk-i kinases in regulation of ifn-inducible genes ikappab kinase epsilon (ikkepsilon): a therapeutic target in inflammation and cancer regulation of tbk activity by optineurin contributes to cell cycle-dependent expression of the interferon pathway negative regulation of tbk -mediated antiviral immunity irfs: master regulators of signalling by toll-like receptors and cytosolic pattern-recognition receptors the irf family transcription factors in immunity and oncogenesis the irf family transcription factors at the interface of innate and adaptive immune responses direct triggering of the type i interferon system by virus infection: activation of a transcription factor complex containing irf- and cbp/p mechanism for transcriptional synergy between interferon regulatory factor (irf)- and irf- in activation of the interferon-β gene promoter middle east respiratory syndrome coronavirus orf b protein inhibits type i interferon production through both cytoplasmic and nuclear targets hsv- icp targets the tbk -activated sting signalsome to inhibit virus-induced type i ifn expression herpes simplex virus serine protease vp blocks the dna-sensing signal pathway by abrogating activation of interferon regulatory factor dengue virus ns proteins inhibit rig-i/mavs signaling by blocking tbk /irf phosphorylation: dengue virus serotype ns a is a unique interferon-regulating virulence determinant porcine epidemic diarrhea virus nucleocapsid protein antagonizes beta interferon production by sequestering the interaction between irf and tbk htlv- tax protein recruitment into ikkepsilon and tbk kinase complexes enhances ifn-i expression suppression of type i interferon production by human t-cell leukemia virus type oncoprotein tax through inhibition of irf phosphorylation contribution of the interaction between the rabies virus p protein and i-kappa b kinase to the inhibition of type i ifn induction signalling porcine deltacoronavirus (pdcov) infection suppresses rig-i-mediated interferon-beta production proteolysis of mda and ips- is not required for inhibition of the type i ifn response by poliovirus hepatitis e virus infection activates signal regulator protein alpha to down-regulate type i interferon porcine bocavirus np protein suppresses type i ifn production by interfering with irf dnabinding activity functional analysis of human herpesvirus -encoded viral interferon regulatory factor and its association with cellular interferon regulatory factors and p viral interferon regulatory factor of kaposi's sarcoma-associated herpesvirus (human herpesvirus ) binds to, and inhibits transactivation of, creb-binding protein a rhesus rhadinovirus viral interferon (ifn) regulatory factor is virion associated and inhibits the early ifn antiviral response suppression of type i interferon production by porcine epidemic diarrhea virus and degradation of creb-binding protein by nsp classical swine fever virus npro interacts with interferon regulatory factor and induces its proteasomal degradation the npro product of classical swine fever virus and bovine viral diarrhea virus uses a conserved mechanism to target interferon regulatory factor- pestivirus npro directly interacts with interferon regulatory factor (irf ) monomer and dimer cleavage of interferon regulatory factor by enterovirus c suppresses cellular responses c protease of enterovirus d inhibits cellular defense mediated by interferon regulatory factor pol-mir- , a teleost mirna upregulated by megalocytivirus, negatively regulates virus-induced type i interferon response, apoptosis, and cell cycle arrest the interferons and their receptors -distribution and regulation jak-stat signaling: from interferons to cytokines stats get their move on transcriptional regulation by stat and stat in the interferon jak-stat pathway stat and irf : beyond isgf . jakstat ( ) dynamic control of type i ifn signalling by an integrated network of negative regulators hemagglutinin of influenza a virus antagonizes type i interferon (ifn) responses by inducing degradation of type i ifn receptor flavivirus antagonism of type i interferon signaling reveals prolidase as a regulator of ifnar surface expression porcine epidemic diarrhea virus infection inhibits interferon signaling by targeted degradation of stat la piedad michoacan mexico virus v protein antagonizes type i interferon response by binding stat protein and preventing stats nuclear translocation human metapneumovirus small hydrophobic (sh) protein downregulates type i ifn pathway signaling by affecting stat expression and phosphorylation varicella viruses inhibit interferon-stimulated jak-stat signaling through multiple mechanisms infectious bronchitis coronavirus inhibits stat signaling and requires accessory proteins for resistance to type i interferon activity nonstructural protein (ns ) of human parvovirus b stimulates host innate immunity and blunts the exogenous type i interferon signaling in vitro suppression of type i and type iii ifn signalling by nss protein of severe fever with thrombocytopenia syndrome virus through inhibition of stat phosphorylation and activation disruption of type i interferon signaling by the nonstructural protein of severe fever with thrombocytopenia syndrome virus via the hijacking of stat and stat into inclusion bodies the role of suppressors of cytokine signaling (socs) proteins in regulation of the immune response regulation of the immune system by socs family adaptor proteins japanese encephalitis virus exploits the microrna- to regulate the expression of suppressor of cytokine signaling (socs) suppressor of cytokine signaling expression induced by varicella-zoster virus infection results in the modulation of virus replication respiratory syncytial virus nonstructural proteins upregulate socs and socs in the different manner from endogenous ifn signaling viral subversion of the host protein synthesis machinery infectious bronchitis coronavirus limits interferon production by inducing a host shutoff that requires accessory protein b deregulation of interferon signaling in malignant cells the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -dj fo sn authors: vignesh, ramachandran; shankar, esaki m.; velu, vijayakumar; thyagarajan, sadras panchatcharam title: is herd immunity against sars-cov- a silver lining? date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: dj fo sn nan the emergence of a novel coronavirus severe acute respiratory syndrome coronavirus- (sars-cov- ) in late and its wide global spread has led to millions of infections and substantial morbidity and mortality ( ) . coronavirus disease (covid- ) caused by sars-cov- infection can range from mild self-limiting disease to acute respiratory distress syndrome and death ( ) . with the who having reported , , confirmed cases and , deaths globally as of nd september , the covid- pandemic seems to show almost poor indication of abating ( ) . while the global scientific community is racing against time to strategize combating possibilities, with several vaccine trials, drug discoveries and validations underway, we still need a practical and sustainable solution to face the ongoing threat to global public health. the terminology "herd immunity", coined by topley and wilson in , later formed the basis for vaccines, applications and vaccination programs, especially against certain viral infections ( ). the concept of herd immunity refers to the indirect protection from infection conferred on susceptible individuals when a sufficiently large proportion of individuals immune to the infection exist in a population. herd immunity concept is generally applicable to those infections that are transmitted directly from person to person and for those humans serving as the reservoir of infection. history has shown that a significant drop in the number of cases and even eradication is rendered by vaccines, and herd immunity is achieved against infectious diseases like small pox, polio, measles, rubella, diphtheria, pertussis and mumps ( - ). the concept of herd immunity appears to be highly critical in the context of disease elimination programs because the said eradication of an infectious agent becomes possible in spite of the absence of an effectual vaccine. interestingly, natural herd immunity is a classical concept that has been successfully accomplished in instances like the h n influenza pandemic wherein no vaccine was available ( ) . going by this old school modus operandi, in the absence of a vaccine, building natural herd immunity against sars-cov- is theoretically considered feasible. however, letting an existing super infectious condition to run amok in the pretext of building up effective herd immunity might not be a smart strategy to end the pandemic. it requires judicious analyses to avoid the colossal burden it could inflict on the healthcare system and the surge in mortalities and associated complications. in a detailed classical analysis, fox et al. has listed down the following four conditions for effective herd immunity to ensue (i) the infectious agent must exist and be restricted to a single host, (ii) the transmission must occur primarily through direct contact, (iii) the infection must induce a robust and life-long immunity, and (iv) the cumulative or herd immunity is amplified if the population possesses a random mixing pattern ( ) . applying the aforementioned conditions to herd immunity against sars-cov- , though the infectious agent has been identified and believed to be zoonotic, we are yet to place a finger on the intermediate host ( ) . secondly, the transmission, of course, occurs through direct person-to-person contact ( ) . however, regarding the third condition, we have a paucity of data on the immune response elicited by sars-cov- in humans, till date ( ) and the questions remains about the long-lasting immunity for the exposed individuals. finally, though the entire human population is susceptible to covid- , the mixing of the pattern varies that is dependent on several societal factors and practices such as universal lockdown, mass quarantine, isolation, social distancing and public health preventive measures, particularly for those at risk ( ) . table listed the various infectious agents and their corresponding r values and herd immunity thresholds. while earlier studies have estimated the basic reproductive number (r ) of sars-cov- to be in the range of to , recent estimates place the r at . ( , ) . this indicates the highly infective nature of the virus, meaning that on an average each infected individual can give rise to about . newer infections and subsequently spread the agent on an exponential scale. assuming an r estimate of . , using the mathematical formula - /r , the herd immunity threshold for covid- would be~ . %, meaning that the incidence of infection will begin to decline once the proportion of individuals with acquired immunity to sars-cov- in the population exceeds . %. however, it remains to be noted that the estimate of the proposed threshold is only theoretical with the assumptions of a homogenous population and presence of uniform sterilizing immunity in the recovered patients. mathematical modeling studies have shown that disease-induced herd immunity threshold would be substantially lower than the values calculated by conventional formula due to the population heterogeneity ( , ) . furthermore, there are several instances of "super spreading events" reported from various countries, wherein a single patient goes on to infect far more number of people than an average patient does, based on estimated r value ( ) . several factors such as increased viral load, asymptomatic individuals, immune suppression and extensive social interactions have been implicated in these "super-spreading events" ( ) . studies point towards these "super-spreaders" as the reason for the heterogeneous propagation of sars-cov- across geographical locations and since these present with a relatively higher r value, they could potentially impact the dynamics of spread and lower the herd immunity threshold ( , ) . the whole concept of herd immunity against sars-cov- hinges on the assumption that an infection would generate sufficient, effective and protective long-lasting immunity. however, data are scarce if the acquired immunity developed by humans is sterilizing enough and whether it would stay long enough. earlier studies on survivors of sars-cov- and mers-cov infections have shown that the antibodies against sars-cov- persisted for nearly two years and antibodies to mers-cov lasted for almost years ( , ) . besides, studies have also demonstrated evidence of seroconversion within to days of disease onset among sars-cov- patients with covid- ( , ) . a study has reported the interesting finding of t cell reactivity to sars-cov- in about % of specimens collected between and before the viral emergence ( ) . this could reflect the circulating t cell memory to the seasonal coronaviruses- e, oc , nl , and hku . a study also suggests that sars-cov- -reactive antibody responses have been detected in unexposed individuals who are igg seropositive for oc and nl and this cross-immune reactivity mainly targets viral ab polyprotein and s proteins, these viral antigens have high sequence similarity with low pathogenic human coronaviruses and sars-cov- ( , ) . in addition, a study has also shown that these antibodies were particularly prevalent in children and adolescents ( ) . it is also important to note that sera from uninfected individuals exhibited variability in their reactivity, they are reactive with sars-cov- s and nucleoprotein but not with the s subunit or the receptor binding domain (rbd) of spike protein. since many studies from different geographical locations are documenting preexisting immunity to sars-cov- , it will be important to define specificities of these t and b cell immune response carefully to assess their association with covid- disease severity. this preexisting cross-reactive t and b cell immunity to sars-cov- may have wide implications as this could explain differential clinical outcomes in covid- patients, disease severity, vaccine development, and important in accessing herd immunity for sars-cov- viral infection/covid- disease. studies characterizing the sars-cov- -specific t cell responses suggest that there is marked activation of t cells in acute covid- patients ( ) ( ) ( ) . several studies have provided strong evidence for the importance of sars-cov- specific ctls, and t helper cells in mild and moderate patients compared to severe covid- disease ( , , ( ) ( ) ( ) . the effector memory cd t cell population is decreased in the severe patients compared to the recovered patients ( ) ( ) ( ) . similarly, the t cells in severe patients compared to convalescent patients express higher levels of exhaustion markers pd- and cd on the memory cd t cells with the signature pertaining to hyperimmune activation (hladr + cd + cd + ) with lower cd t cell response. in line with cd t cells, the effector memory cd t cell frequencies are also elevated in the covid- patients ( ) ( ) ( ) . interestingly, the recovered patients seem to have higher levels of activated circulating t follicular helper (tfh) population, indicative of recent antigen encounter and emigration from germinal center. similar to the pd- levels of cd t cells, the pd- levels were increased in the cd t cells. as a result of the decrease in the t cells, non-t cell frequencies that includes, macrophages, monocytes, neutrophils were observed to be elevated in severe covid- patients ( , ) . similar to t cells the b cell subpopulations are altered in the covid- disease and it has been shown that the frequency of plasmablast is highly elevated ( % of the circulating b cells) in the covid- patients ( ) . these blood plasmablast frequencies are shown to correlate with activated circulating tfh cells ( , ) . the proliferating b cells are markedly elevated in the covid- patients with activated phenotype, suggestive of altered b cell response during covid- disease. several studies have provided strong evidence for the importance of sars-cov- specific neutralizing antibodies in association with less disease severity in covid- patients ( , ) . sars-cov- specific antibodies are found in convalescent plasma and most recovered individuals have rbd-specific antibodies with potent antiviral activity ( , ) , importantly severe and critical patients are currently being treated with convalescent plasma therapy in many parts of the world ( , ) . overall, these data suggest that vaccines or therapeutic strategies that induce functional anti-viral sars-cov- specific t and b cell responses are important to curtail sars-cov- infection in vivo. there remain many unknowns and several other research questions unanswered. the degree of protection afforded by the antibodies against sars-cov- among the infected is still ambiguous, and strong evidence-based findings are awaited to know if the host responses generated would be of strong sterilizing or weak and waning immune responses. earlier studies on the coronavirus e, responsible for common cold have shown that the titers of antibodies produced were not sufficient to prevent reinfection in all the individuals studied ( ) . interestingly, similar pattern exists in sars-cov- infection and where there is a rapid decay of sars-cov- of antibodies in persons with mild symptoms and recovered patients ( , ) . similarly, as a recent study demonstrated the evidence of reinfection of sars-cov- by phylogenetically distinct sars-cov- re-infection, this may also suggest that the immunity generated during primary sars-cov- infection may be short lived and may not protect if reinfection happens by the second distinct virus ( ) . these results also suggest that sars-cov- may continue to circulate among human population despite herd immunity with natural infection or with vaccination. however, a recent study on monkeys has indicated the development of protective immunity against reexposure to sars-cov- (homologous) ( ) . hence, massive longitudinal monitoring of sars-cov- seroprevalence remains the need of the hour to determine the percentage of the population already infected and if reaching a herd immunity threshold is even feasible. it also remains logical to consider the viral factors such as the possibility of mutations and emergence of new strains of a virus, which can go on to make herd immunity futile ( ) . given the unavailability of a vaccine against sars-cov- , it is prudent to foresee the consequences of achieving the herd immunity threshold in epidemiological and immunological viewpoints. a recent population-based sero-epidemiological study from spain involving participants has revealed % seroprevalence of sars-cov- , thereby highlighting that the majority of the spanish population remains seronegative ( ) . figure represents this scenario of a population with only % seroprevalence of sars-cov- antibodies and the importance of achieving herd immunity threshold. similarly, seroprevalence among the community in los angeles county has been reported as . %, about . % in japan, and . % in hong kong ( ) ( ) ( ) . based on a serological survey conducted by the indian council for medical research (icmr) in may -june, across districts in india involving over , participants, the seroprevalence was found to be only . % ( ) . the lower rates of seroprevalence of sars-cov- antibodies reported from various geographical locations point in the direction that we are still a long way ahead of achieving herd immunity. according to a modeling study, assuming a scenario of achieving uniform herd immunity threshold of % and with an infection fatality rate of . % for sars-cov- , the estimate of the absolute number of deaths worldwide would easily exceed a whopping million ( ) . since these estimates are based on various assumptions and the real-life scenario could throw us curve balls of multiple factors influencing the outcomes, the fatality rate could even be higher. a recent modelling study has estimated that about one in five individuals worldwide would be at increased risk of severe covid- , upon infection with sars-cov- , owing to the underlying conditions. the study projects an alarming figure of about million people requiring hospital admission and substantial mortality rates ( ) . furthermore, in the case of highly populated and resourcestrapped countries, the increase in the number of infected cases could overwhelm the healthcare facilities and could lead to a shortage of essential medical services exacerbating further complications and deaths. thus, weighing on the immunological and epidemiological consequences, achieving natural herd immunity at the cost of severe morbidities and mortalities worldwide, in the absence of an effective and safe vaccine appears farfetched and seemingly an impracticable solution. with so many vaccines being tested in various phases of clinical trials, the availability of an effective vaccine seems to be the only way forward in this war against covid- . echoing dr. anthony fauci's statements, even when a vaccine with a modest efficacy is made available in near future, anti-science and anti-vaccine campaigns need to be counteracted and cooperation of the general public is needed to achieve an efficient and successful herd immunity against covid- ( ). rv, es, vv, and st led the writing of this opinion article. all authors contributed to the article and approved the submitted version. the views, opinions, assumptions, or any other information set out in this article are solely those of the authors and should not be attributed to anyone. the authors salute all the health care workers who are at the front lines of the covid- pandemic, helping patients and their families. frontiers in immunology | www.frontiersin.org september | volume | article an interactive web-based dashboard to track covid- in real time clinical features of patients infected with novel coronavirus in wuhan who. coronavirus disease (covid- ) dashboard. available at: https://covid . who.int/?gclid=cjwkcajw_qb braveiwavwq vvsmd-becu ak qlye rcmzf-muzkht fj phh wl hb fcqzsmhrocvoiqavd_bwe the spread of bacterial infection. the problem of herd-immunity herd immunity: history, theory, practice herd immunity": a rough guide vaccination and herd immunity: what more do we know? dynamics of population immunity due to the herd effect in the covid- pandemic herd immunity: basic concept and relevance to public health immunization practices a review of coronavirus disease- (covid- ) transmission routes of -ncov and controls in dental practice perspectives on therapeutic neutralizing antibodies against the novel coronavirus sars-cov- early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia herd immunity: understanding covid- high contagiousness and rapid spread of severe acute respiratory syndrome coronavirus a mathematical model reveals the influence of population heterogeneity on herd immunity to sars-cov- herd immunity thresholds for sars-cov- estimated from unfolding epidemics super-spreading events and contribution to transmission of mers, sars, and covid- identifying and interrupting superspreading eventsimplications for control of severe acute respiratory syndrome coronavirus do superspreaders generate new superspreaders? a hypothesis to 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covid- longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of sars-cov- infected patients deep immune profiling of covid- patients reveals distinct immunotypes with therapeutic implications reappearance of effector t cells is associated with recovery from covid- sars-cov- -specific t cell immunity in cases of covid- and sars, and uninfected controls humoral and circulating follicular helper t cell responses in recovered patients with covid- neutralizing antibody responses to sars-cov- in a covid- recovered patient cohort and their implications. medrxiv ( ) . . rapid generation of neutralizing antibody responses in covid- patients convergent antibody responses to sars-cov- in convalescent individuals potent neutralizing antibodies against multiple epitopes on sars-cov- spike convalescent plasma as a potential therapy for covid- effect of convalescent plasma therapy on time to clinical improvement in patients with severe and life-threatening covid- : a the time course of the immune response to experimental coronavirus infection of man rapid decay of anti-sars-cov- antibodies in persons with mild covid- longitudinal evaluation and decline of antibody responses in sars-cov- infection covid- reinfection by a phylogenetically distinct sars-coronavirus- strain confirmed by whole genome sequencing sars-cov- infection protects against rechallenge in rhesus macaques covid- : herd immunity and convalescent plasma transfer therapy prevalence of sars-cov- in spain (ene-covid): a nationwide, population-based seroepidemiological study seroprevalence of sars-cov- -specific antibodies among adults estimation of seroprevalence of novel coronavirus disease (covid- ) using preserved serum at an outpatient setting in kobe, japan: a cross-sectional study seroprevalence of sars-cov- in hong kong and in residents evacuated from hubei province, china: a multicohort study prevalence of sars-cov- infection in india: findings from the national serosurvey global, regional, and national estimates of the population at increased risk of severe covid- due to underlying health conditions in : a modelling study fauci says covid- vaccine may not get us to herd immunity if too many people refuse to get it the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © vignesh, shankar, velu and thyagarajan. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - trhh authors: frey, andreas; lunding, lars p.; ehlers, johanna c.; weckmann, markus; zissler, ulrich m.; wegmann, michael title: more than just a barrier: the immune functions of the airway epithelium in asthma pathogenesis date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: trhh allergic bronchial asthma is a chronic disease of the airways that is characterized by symptoms like respiratory distress, chest tightness, wheezing, productive cough, and acute episodes of broncho-obstruction. this symptom-complex arises on the basis of chronic allergic inflammation of the airway wall. consequently, the airway epithelium is central to the pathogenesis of this disease, because its multiple abilities directly have an impact on the inflammatory response and thus the formation of the disease. in turn, its structure and functions are markedly impaired by the inflammation. hence, the airway epithelium represents a sealed, self-cleaning barrier, that prohibits penetration of inhaled allergens, pathogens, and other noxious agents into the body. this barrier is covered with mucus that further contains antimicrobial peptides and antibodies that are either produced or specifically transported by the airway epithelium in order to trap these particles and to remove them from the body by a process called mucociliary clearance. once this first line of defense of the lung is overcome, airway epithelial cells are the first cells to get in contact with pathogens, to be damaged or infected. therefore, these cells release a plethora of chemokines and cytokines that not only induce an acute inflammatory reaction but also have an impact on the alignment of the following immune reaction. in case of asthma, all these functions are impaired by the already existing allergic immune response that per se weakens the barrier integrity and self-cleaning abilities of the airway epithelium making it more vulnerable to penetration of allergens as well as of infection by bacteria and viruses. recent studies indicate that the history of allergy- and pathogen-derived insults can leave some kind of memory in these cells that can be described as imprinting or trained immunity. thus, the airway epithelium is in the center of processes that lead to formation, progression and acute exacerbation of asthma. with more than million people affected bronchial asthma is one of the most common chronic inflammatory diseases worldwide ( ) . actually, out of deaths is associated with asthma and it causes annual direct medical (drugs, care, hospitalization) and indirect economic (productivity loss, early retirement) costs of about € billion for the eu ( ) and over billion for the united states ( , ) , making it a major burden for public healthcare systems ( ) . asthma is characterized by acute broncho-obstruction, in combination with additional symptoms such as cough, chest tightness, shortness of breath, and wheezing, which vary in extent and over time. these symptoms arise on the basis of chronic airway inflammation in response to a trigger, most commonly inhaled allergen(s), that causes airway hyperresponsiveness (ahr), airway remodeling and mucus hypersecretion ( ) . due to the complexity and variation of the symptoms along with its pathogenesis, asthma is nowadays described as a heterogeneous syndrome with distinct sub-or endotypes. however, the majority of asthma patients displays allergic inflammation of the airways, which can be classified by profiles of several characteristic mediators in "th high" or "th low" subtypes ( ) . thus, in sensitized individuals t helper (th ) cells orchestrate allergic inflammation by releasing a typical array of cytokines including interleukins (il)- , - , - , and - and granulocyte-macrophage colony stimulating factor (gm-csf). these mediators induce the production of allergen-specific immunoglobulin (ig) e, th cell development, goblet cell differentiation, submucosal gland activity, as well as recruitment, maturation, and activation of eosinophils and its precursors ( ) . activation of mast cells and eosinophils via ige-bound allergens results in their degranulation and, thus, in the release of a plethora of effector molecules and growth factors that on the one hand destroy airway tissue and on the other hand conduct its repair. chronic activation of these processes ultimately lead to signs of airway remodeling such as increased smooth muscle mass, subepithelial fibrosis, and epithelial desquamation, which in turn give rise to the pathological changes and clinical symptoms characteristic for asthma ( ) . allergic sensitization against aeroallergens represents the strongest factor predisposing for the development of asthma, indicating a hyperreaction of the immune system to be the central event within the pathogenesis of this disease. nevertheless, structural cells and particularly airway epithelial cells also appear to be of critical importance. this is not surprising since these cells represent the barrier that first encounters environmental stress factors like air pollutants, bacterial and viral pathogens, as well as allergens, and markedly contributes to their neutralization by a mechanism called mucociliary clearance (mcc). besides these barrier and cleaning functions airway epithelial cells also exert a number of immunological tasks interweaving the role of the epithelium with that of the above-mentioned cells of the immune system. here we aim to review these immune functions of the airway epithelium against the background of asthma pathogenesis. the main purpose of mucosae is to separate the body from its environment and therefore they are essential for the maintenance of the inner homeostasis. though this task is not commonly regarded as an "active" or "typical" immune function, it is absolutely central for the defense against allergens, pathogens and other harmful environmental factors. in order to fulfill this function the airway epithelium forms a continuous, selfcleaning barrier with a considerable resistance against biological, chemical or physical stressors ( ) . together with the physical barriers of the mcc and glycocalyx, this is achieved by three types of intercellular epithelial junctions that form the structural adhesion forces of the airway mucosa by linking the intracellular structures of the cytoskeleton of one epithelial cell to that of its neighbors. these junctions involve adherens junctions (ajs), hemidesmosomes, and tight junctions (tjs). ajs can appear as spots (adhesion plaques) or as bands encircling the cell (zonula adherens). in the junctional zone ajs interconnect the actin filaments of the adherent cells via homotypic transmembrane e-cadherin adhesions and anchor proteins like actinin, vinculin, and α-, β-, and p catenins, while adhesion plaques attach the cells to the extracellular matrix ( ) . similarly, hemidesmosomes are focal structures that form adhesive bonds between the cytoskeleton of epithelial cells and the lamina lucida, which is a part of the lamina propria. hemidesmosomes utilize integrin α β , plectin a and the tetraspanin cd connecting laminin and fibronectin of the extracellular matrix to the intermediate filaments of the cytoskeleton ( ) . in contrast, tjs form a multiprotein junctional complex called zonula occludens (zo) that in turn appears as the main regulator of the paracellular permeability. these complexes are formed by several transmembrane and cytoplasmic proteins that are attached to actin filaments of the cytoskeleton. the main components of tjs are claudins and occludins, proteins with four transmembrane domains, as well as so-called junctional adhesion molecules (jams) belonging to the immunoglobulin superfamily with only one transmembrane domain. these proteins are connected to actin filaments by cingulin and zo proteins , - , and - ( ) . in the airways of healthy individuals, the tjs of the zonula occludens and ajs of the zonula adherens constitute dense protein networks that interconnect the basolateral sides of epithelial cells in such a way that they prevent the paracellular passage of basically all molecules, including water, ions and proteins, as well as of pathogens or other inhaled particulate matter. several findings strongly indicate that in asthma patients the barrier function is impaired by epithelial disruption. for example, endobronchial biopsies revealed a fragile or even injured airway mucosa with partially or completely uncovered areas and detachment of columnar, ciliated cells ( ) . epithelial desquamation is further indicated by the presence of epithelial cells in bronchoalveolar lavage (bal) and of creola bodies (epithelial cell aggregates) in sputum of asthmatics ( ) . furthermore, bronchial biopsies of asthmatic subjects displayed patchy disruption of tjs ( ) and the expression of a number of proteins that are essential for the formation of tjs and ajs has been shown to be markedly reduced. among these proteins are α-catenin ( ), β-catenin ( ), occluding ( ) , zo- ( , ) , and e-cadherin ( , ) . the levels of the latter one in sputum also correlate with asthma severity ( ) . these data are further supported by in vitro studies where primary bronchial epithelial cells are kept in air liquid interface (ali) culture, a method that allows the cells to differentiate and form a pseudo-stratified epithelial monolayer largely resembling the physiological structure of the airway mucosa. once this structure has been established, in vitro barrier integrity can be assessed by measuring the transepithelial electrical resistance (teer), a characteristic that is indicative of the tightness of a cell layer ( ) . several studies showed that ali cultured airway epithelia from asthma patients display a decreased teer in comparison to epithelia derived from healthy controls ( , , ) . to date, three different factors are discussed to have a harmful impact on the barrier integrity of the airway epithelium in asthma pathogenesis: allergens themselves, viral infection, and (allergic) inflammation. according to the "protease hypothesis" allergens with an inherent protease activity are capable of cleaving the protein components of the aforementioned intercellular epithelial junctions so that the barrier function is disrupted and allergens can penetrate the airway mucosa on the paracellular route, which eventually could result in sensitization against them. accordingly, a considerable number of allergens has been tested in vitro for proteolytic potential and for an effect on epithelial barrier integrity. several studies provided evidence for a direct cleavage of e.g., occludin and zo- proteins by the major allergen from house dust mites (dermatophagoides), der p ( , ) . house dust mite extracts as well as der p have been shown to increase the permeability and to decrease teer of epithelial layers in vitro ( , , ) . comparable effects have been shown for extracts of the allergenic fungus alternaria alternata that reduced teer of human bronchial epithelial cells in vitro ( ) or the aspergillus fumigatus-derived alkaline protease (alp- ) ( ) . similarly, a variety of different pollen extracts has been investigated for their effect on the barrier integrity of epithelial cells in vitro. diffusates of italian cypress (cupressus sempervirens), orchard grass (dactylis glomerata), olive (olivia europaea), and scots pine (pinus sylvestris) have been shown to affect claudin- , e-cadherin, and occludin expression and thus to disrupt epithelial junctions in ali cultures of calu- cells, an effect which could be suppressed by protease inhibitors ( ) . japanese hop (humulus japonicus) extract also reduced expression of occludin in a comparable setting ( ) . another study provided evidence for proteolytic activity of giant ragweed (ambrosia trifida), kentucky bluegrass (poa pratensis), and white birch (betula pendula) as shown by reduced expression of claudin- , occludin, and zo- in calu- as well as in mdck cells ( ) . however, an inherent protease activity appears not to be the only way, by which allergens can impair the barrier integrity of the airway epithelium. cockroach, hdm, fungus, and mold extracts have also been shown to activate the protease-activated receptor (par-) and/or , which in turn leads to degradation of aj components ( , ( ) ( ) ( ) . the effect of viral infections on airway barrier function is even more pronounced than that of allergens. respiratory viruses cause junction dysfunction by different mechanisms: human rhinoviruses (hrv), respiratory syncytial virus (rsv), human metapneumovirus (hmpv), influenza and parainfluenza viruses bind to their entry receptor, which are typically protein or sugar structures expressed on the cellular surface for other purposes, leading to endocytosis of the virus. once the virus has been internalized, it uncoats and initiates the viral replication process, which has certain consequences for infected cells. on the one hand, the cell starts with the production of type i interferons (ifn) in order to slow down the internal virus replication and to activate the cellular immune response against the virus. in consequence, infected airway epithelial cells are killed by virus-specific, cytotoxic cd + t cells. on the other hand, the virus itself also kills epithelial cells, since it induces morphological alteration of the cells summarized as cytopathic effect (cpe). for hrv and influenza viruses, the cpe manifests in rounding and detachment of airway epithelial cells that are ultimately lysed by the virus in order to release freshly produced viruses. paramyxoviruses such as rsv and hmpv induce cell fusion so that four or more cells form typical syncytia ( , ) . additionally, at least hrv and rsv affect the barrier integrity of the airway epithelium by reducing the expression of epithelial junction proteins ( ) ( ) ( ) ( ) . it could be shown that hrv increases epithelial permeability by a reduction of occludin and zo- expression ( , ) . rsv also disrupts junctional complex structures by fostering the activity of protein kinase d (pkd) ( ). the antiviral immune response also includes the release of cytokines that directly affect epithelial barrier function as well. this is especially true for il- β, ifn-γ and tumor necrosis factor (tnf). these cytokines have been shown to support epithelial permeability and to decrease expression of claudins, jam, occludin, and zo- in several in vitro studies ( - ). in case of asthma, these effects are even more pronounced because of the allergic inflammatory response that already exists before the viral infection of the airway epithelium. hence, th type cytokines like il- and il- also increase barrier permeability by inhibiting the surface expression of β-catenin, e-cadherin, occludin, and zo- ( , ). in addition to cytokines, mast cell derived mediators also appear to have an effect on the barrier function of the airway mucosa. histamine for example has been shown to contribute to transient disruption of apical junctional complex integrity and thus to increase epithelial permeability in vitro ( ). allergens, viruses, and the inflammatory response to their exposure represent extrinsic factors that impair the barrier integrity of the airway epithelium. however, some studies suggest that epithelial cells of asthma patients inherently predispose for an increased permeability. as already mentioned above, airway epithelial cells that have been isolated from asthmatics and propagated in vitro to form an epithelial monolayer under ali culture conditions, display a decreased teer as compared to cells from healthy donors ( , ) . this observation indicates that the cellular properties leading to an increased barrier permeability are somehow imprinted within the cells. whether this is a matter of genetic predisposition encoded in epithelial stem cells or whether epithelial cells from asthma patients "remember" previous insults by epigenetic modifications that predispose for asthma development in later life remains elusive. besides the barrier function of the epithelium provided by the mere presence of the sealed cell layer itself, two additional barrier structures are "exported" onto the luminal surface by the airway epithelium, a static one dubbed glycocalyx or periciliary layer (pcl) and a mobile one termed mucus. the glycocalyx or pcl is a sponge/fleece-like, cell membraneanchored layer of glycolipids and glycoproteins -mainly mucins (see below) -that vertically stick out of the apical epithelial cell membrane. although mainly attributed to the gut epithelium where it can extend up to nm ( ) and to the vascular endothelium ( , ) a glycocalyx/pcl is also present throughout the airway epithelium even down to the alveoli ( ), again with heights up to nm in certain areas ( ) (figure ). this static glycoprotein and glycolipid coat stores water to control mucus hydration but also serves as a protective zone against the compression of the mucus lying above in order to allow persistent cilia beating for ongoing functionality of the mucociliary clearance (mcc; see below) ( , ). beyond that, the glycocalyx/pcl regulates receptor specificity by architectural means and prevents the progression of viruses through the occasionally patchy mucus layer. it has been shown that the height and density of the epithelial glycocalyx can determine whether a ligand-equipped nanoparticle may attach to its membrane receptor or not ( , ). in line with this, the inefficiency of adenovirus-mediated gene transfer into the airway epithelium was found to be caused by the membranetethered glycocalyx/pcl proteins that put a halt to the advance of the viral vectors ( , ). consequently, the susceptibility of the airway epithelium toward infection is at least to some extent controlled by the glycocalyx/pcl. very small viruses such as bocavirus (hbov ) and hrv, which are - nm in size ( , ) should readily advance through the pcl to the epithelial plasma membrane as has been observed with nanoparticles of the respective size ( ). consequently, those viruses should be able to luminally infect airway epithelial cells as long as their receptor is present on the apical side. little is known about receptor distribution in vivo but at least on cultured airway epithelial cells apical receptor expression and/or infectivity has been demonstrated for both viruses ( - ). larger particles of about nm and above, on the other hand, are efficiently blocked by the pcl ( ). hence, viruses such as rsv, hmpv, influenza and parainfluenza viruses, adenovirus or coronavirus, which are in this size range ( ) ( ) ( ) ( ) , should be hindered efficiently by the pcl to infect the host. yet, those viruses often are associated with respiratory infections and asthma exacerbations ( , ) . one possibility for them to infect the airway epithelium may be the presence of the viral receptor on structures that extend from the pcl such as the tips of the cilia. an example for this is chemokine receptor cx cr via which rsv can infect its host. in differentiated human airway epithelial cells this molecule is highly abundant on cilia ( , ) . another possibility is the preceding action of a door-opener such as hbov which may pave the way for further viral infections. hbov was shown to persist for several months in the human airway epithelium ( ) and causes pyroptotic cell death, epithelial cell hypertrophy, loss of cilia and disruption of the tight junction barrier ( , ). such a predamaged epithelial barrier may then readily fall victim to an influenza, parainfluenza or hpmv infection. with up to % of asthma exacerbations in small children found to be associated with hbov infection ( ) it may be worthwhile to further investigate possible coinfections with hbov in asthma exacerbation cases. in this context, it may also be of interest that the treatment of chronic inflammatory diseases of the airways such as asthma with corticosteroids (cs) seems to reduce the glycocalyx/pcl height on the alveolar epithelium ( ) thereby rendering the lung more susceptible to e.g., pneumocystis carinii infection. consequently, alleviating chronic inflammation in asthma with cs may make the patient more susceptible to certain infections, which in turn may enhance inflammation again, clearly a two-edged outcome of cs therapy in asthma. although the glycocalyx appears to be static on the architectural level, it may not be invariant in terms of its molecular composition. it was shown that lipopolysaccharide exposure could lead to heparan sulfate shedding from the airway epithelium thereby causing increased lung permeability ( ). allergen exposure of experimental animals resulted in different glycosylation patterns of the glycocalyx ( ), which may result in a deviant presentation of viral and bacterial receptors on the cell surface. in light of the above, the airway epithelial glycocalyx seems to play a so far underestimated but possibly important role in airway epithelial defense. whether or not the molecular composition figure | protection of epithelial surfaces by physical barriers. in the healthy state, ciliated cells (cc) form a tight epithelial layer where paracellular passage is prevented by sealing of lateral intercellular spaces with tight junctions (tj). the apical epithelial cell surface, including the cilia, is covered by a layer of membrane-anchored glycoproteins and glycolipids, the glycocalyx. the dense meshwork of glycostructures restricts access of luminal matter to the apical cell surface; depending on their size, larger pathogens can be cut off from their receptor if it is not present on cilia (inset). goblet cells (gc) secrete mucus, consisting of highly glycosylated mucins which absorb large quantities of water to form a viscous gel. the mucus -and any matter trapped within -is transported upward in the airway lumen by the coordinated beating of the cilia. bc, basal cells. in the asthmatic state, barrier functions can be compromised by partial disruption of tight junctions and gaps in the pcl/glycocalyx meshwork due to loss of cilia. mucus clearance is impeded by increased mucus viscosity and swelling of the gel matrix, and by disturbance of ciliar beating due to disorganization and dykinesia of cilia. of this static cell coat is different in asthma, remains to be investigated. while the role of the glycocalyx/pcl is still subject of debate, the importance of mucus in airway luminal defense is unchallenged. mucus is an unstirred discontinuous sheet of secreted mucous hydrogel which floats on top of the epithelium and is transported toward the oral cavity like the cargo on a conveyor due to the constant and coordinated beating of underlying ciliated cells ( ) . the mucus carried upward by this mucociliary clearance (mcc) mechanism can be swallowed or expectorated. as the mucus layer separates the airway lumen from the epithelium only objects that diffuse faster "vertically" toward the epithelial surface than the mucus is transported "horizontally" toward the oral cavity can reach the epithelial cell membranes. thus, only nanoscalar objects and smaller, like gases, water, salts and nutrients are able to reach the epithelial cells ( , ) . this way the mucus carpet provides a protective line of defense against pathogens, dust and other harmful objects that might be inhaled by an individual. in addition to that, the sticky texture of the mucus slows down airborne objects and further prevents their advance to the epithelial cell layer. in order to exert these functions properly, mucus requires a specific composition. mucus consists mainly of water, further components are salts, lipids and proteins. among those, antimicrobial proteins like lysozyme, immunoglobulins and antimicrobial peptides are major molecular scavengers distributed within the mucus layer ( ) ( ) ( ) . the characteristic viscous, elastic and sticky properties of the mucus are provided by a group of macromolecules named mucins ( , ) . to date, genes coding for mucins have been described ( ) . their protein products are secreted either to form mucus or remain immobile on the apical membranes of the airway epithelial cells where they become part of the glycocalyx/pcl. muc ac and muc b are the major secreted mucins in the airways. in addition, muc and muc are also part of airway mucus, albeit to a considerably smaller proportion, and thus belong to the family of secreted mucins ( ) . in contrast, muc , muc and muc are tethered to the cells of the airway epithelium (figure ) thereby contributing to the static luminal epithelial barrier, which resides underneath the mobile mucus layer ( , ) . the viscous and elastic properties of the mucous gel are primarily given by the secreted polymeric mucins muc ac and muc b ( ) . these mucins are highly o-glycosylated proteins enriched with amino acids like proline, serine or threonine ( ) . although both have a similar structure, muc b and muc ac differ in charge due to differential glycosylations ( ) . their production depends on cell type and site of production. in the upper airways, muc ac is produced by epithelial goblet cells while muc b is secreted from mucous cells in submucosal glands from secretory cells in the tracheal and bronchial epithelium. in the distal airways, muc b is also produced by secretory cells of the epithelium and seems to be the major mucin of this airway region ( , , ) . before secretion, polymeric proteins are stored in secretory granules in a compacted, dehydrated state. upon release, they switch to a hydrated form, which is necessary for the mucous gellike properties ( , ) . whether the two different mucins have different functions restricted to their site of production or whether the two mucins mingle to create a novel type of barrier structure is not clear yet. at least some studies analyzing airways of piglets have shown that muc b strands are becoming coated with muc ac to some extent after release at the epithelial surface ( , ) . a possible role of muc and muc has not been identified yet. howsoever, under healthy conditions, the viscous mucus traps noxious substances, which are then removed from the airway via cilial beating by the mcc ( ) . in asthma, the mcc is impaired leading to mucus plug formation which in turn results in the characteristic obstruction observed in asthmatics. this is already a feature of mild stable asthma and the dysfunction worsens during aggravation of asthma and in asthma exacerbations ( ) ( ) ( ) . one reason is an increased mucin content of the mucus thereby disturbing its regular composition. normally, the airways' mucus consists of ∼ % water and only ∼ % solid factors mainly mucins. in obstructive diseases, the amounts of mucins rise up to - % ( , ). due to its hygroscopic nature, this leads to acquisition of water from the underlying pcl/glycocalyx and shrinking of this static layer. the now protruding cilia either project into the mucus or get bend ( ). both effects impede passing on of the mucus to the next cell. loss and/or disorientation of cilia as it is typical for asthmatics will further disturb the "bucket chain"-like transport process ( ) . on the cargo side enhanced intermolecular crosslinking of mucus constituents by oxidative processes may further complicate forwarding. it will also increase mucus viscosity eventually leading to plug formation. oxidative intramolecular crosslinking of biomolecules is predominantly caused by cysteines whose thiol side chains can form disulfide bridges. all mucins are rich in cysteines, especially in their less glycosylated carboxy-and amino terminal regions. in the "normal" mucous gel of healthy individuals these cysteines are believed to be only moderately crosslinked, forming a lightly entangled network. in asthma, however, the degree of crosslinking and the density of the mucin network increases considerably ( , ) (figure ). increased oxidative stress appears to play an important role in this respect with eosinophils being the main suspects for oxidant production. the abnormally high levels of eosinophil peroxidase detected in the sputum of asthma patients may form an oxidative milieu. this would also bring the widely observed correlation between airway eosinophilia and airway obstruction into a causative relationship ( ) . lastly, the muc ac of asthmatics tends to tether to the epithelium, which also complicates mucus forwarding ( ) . in asthmatics not only the amount but also the composition of the mucus changes, especially the ratio of muc ac to muc b as well as the posttranslational modification of muc b. mucus from healthy individuals contains predominantly muc b, which is essential for the mcc and protection against pathogens ( ) ( ) ( ) . in asthma, the proportion of muc b relative to muc ac often decreased ( , ) along with the expression of a lowcharge form of muc b. consequently, there was a changed ratio between the two differently glycosylated forms of muc b ( , ) . the importance of muc b is indicated by muc bdeficient mice, which showed an accumulation of undesired substances e.g., bacteria, resulting in severe inflammation and airway obstruction ( ) . the ratio between muc b and muc ac changes dramatically in asthma because muc ac expression and protein production are substantially upregulated in asthmatic patients ( , , ) . especially patients with an eosinophilic type asthmatic phenotype showed a shifted ratio toward higher muc ac concentrations ( , ) . this is in line with the assumption that muc ac seems to be important for the defense against enteric nematodal and influenza infections ( , ) . the increased expression of muc ac seems to depend on substantially increased levels of il- . the il- signaling pathway activates the signal transducer and activator of transcription (stat ), which induces the expression of muc ac via various regulators ( ) and appears to be involved in ahr development ( , ) . several studies using in vitro systems with human epithelial cells or murine models validated this mechanism ( ) ( ) ( ) ( ) . furthermore, egfr, which is also overexpressed in asthma, also induces the expression of muc ac ( ) ( ) ( ) ( ) . this excessive production of mucins in the asthmatic airway epithelium leads to an increased volume of intracellular stored mucins, a mucus metaplasia ( ) . thus, a higher number of goblet cells compared to the healthy situation appears in case of asthma ( ) . it is not exactly understood, whether this switch from a muco-ciliary phenotype to a mucous metaplastic phenotype develops from goblet cell hyperplasia, metaplasia or both as reviewed before ( ) . in animal models, goblet cell metaplasia/hyperplasia arises from an increased expression of primarily il- , but also of il- and il- ( ) ( ) ( ) ( ) . these cytokines are highly upregulated in asthmatic individuals ( ) ( ) ( ) ( ) . one important factor for the development of the goblet cell metaplasia is notch regulated by il- ( ) . studies analyzing the function of sam-pointed domain containing ets transcription factor (spdef) highlighted its essential role in the development of goblet cell differentiation, hyperplasia and mucous metaplasia ( ) ( ) ( ) . therefore, spdef seems to inhibit the expression of forkhead box protein a (foxa ) which is an important negative regulator of genes associated with mucous metaplasia and goblet cell hyperplasia ( , , , , ) . thus, the physical barriers provided by the airway epithelial layer seem to be deeply disturbed in asthmatic individuals. although mucin is one of the most important barrier molecules in the airways its unbalanced overproduction is clearly detrimental to the desired outcome. mucus plugging impedes egress of the active luminal defense molecules necessary to eliminate invaders. although strong walls (tight junctionally sealed epithelial cell layer) surrounded by a glacis (pericilial layer/glycocalyx) and a moat filled with flowing liquid (mcc) are crucial to prevent invaders from entering a castle, active defenses are necessary to end the siege. this is of particular importance when the besieger can replicate and thus may increase continuously by number, as is the case when pathogenic bacteria colonize the luminal side of the airway epithelium. in order to get rid of a potential invader the airway epithelium possesses a battery of defense molecules, with which a potential microbial enemy can be attacked, destroyed or removed out of the airway lumen. prominent innate molecular scavengers are lysozyme, transferrin and antimicrobial peptides. lysozyme is produced in large amounts ( mg/day) by serous cells of the upper human airway epithelium ( ) and is able to destroy the polysaccharide capsules of many bacterial species. it has been shown that the production of lysozyme by serous cells residing in the serous glands of the upper airways is crucial for defending against bacterial airway invaders ( ) . once the polysaccharide capsule is destroyed or damaged, so called defensins or antimicrobial peptides may exert the lethal hit to the invader. defensins can form holes or pores into a bacterial cell membrane thereby killing a pathogen that aims to enter the body ( , , ) . in addition, lactoferrin is produced and secreted by serous cells ( ) , and transferrin is expressed by alveolar type i cells ( ) . these ferrins are iron-binding proteins, which deplete their environment from iron ions that are essential for the growth of a self-replicating organism ( , ) . consequently, the pathogen is starved out. besides this innate "rapid response team, " the polarized epithelium of the human airways is also able to transport and apically release immunoglobulins that carry a j-chain (joining chain) by using its poly ig receptor (pigr) ( ) ( ) ( ) that is expressed by all non-stratified epithelial cells (figure ) . only igm and multimeric iga are equipped with j-chains ( , ) . these two immunoglobulin classes not only circulate in the bloodstream but are also produced directly underneath the airway epithelium by b cells, given those lymphocytes express the j-chain ( , ) . functionally, igm can substitute for multimeric iga. for that reason iga-deficient individuals do not show a strong phenotype concerning susceptibility to infection. nevertheless, secreted iga (siga) outperforms igm in terms of mucosal protection as it usually displays a higher affinity toward its antigen and, more importantly, is able to crosslink with mucins upon target binding ( , ) . this way an incoming viral or bacterial pathogen becomes trapped in mucus and is removed from the airway surface via the mcc. the protective function of secreted iga has been demonstrated with various model systems, both for the gastrointestinal mucosa as well as for the airways, using passively administered monoclonal iga ( ) ( ) ( ) ( ) , injected hybridoma cells whose target specific, dimeric igas are then transported across the mucosae ("backpack tumor model") ( ) ( ) ( ) and by neutralization of preexisting mucosal iga immunity with mucosally administered anti-iga immunoglobulins ( ) . although adaptive multivalent target binding via its hypervariable regions is probably the main mode of protection in those models, siga is also able to bind in an innate manner to luminal pathogens via its carbohydrate components by presenting decoy structures that mimic target cell surface receptors ( ) . if both modes of repelling fail and a pathogen has nonetheless invaded an epithelial cell, dimeric iga may still be able to protect the infected cell, this time from inside. this is possible whenever the respective pathogen does not directly infect the cytosol of its target cell or inject its nucleic acids directly into the cytosol but rather uses an initial endocytosis step for infection. depending on the infected organelle, vesicles, which concurrently translocate iga toward the apical site, may fuse with the infected organelle, bind to the invader and carry it away into the lumen. in addition to this removal activity, mucus crosslinking and the tricking of pathogens by offering decoy receptors, siga also scavenges il- and thereby inhibits il- -driven neutrophil chemotaxis ( ) . in addition to these molecular interactions with a pathogenic target, iga also binds to numerous cell types that patrol at the airway epithelium. the most important cellular partner seems to be the eosinophil as this cell possesses a total of five different receptors for iga: fcαri (cd ), transferrin receptor (tfr) (cd ), pigr, asialoglycoprotein receptor (asgpr) and a receptor for secretory component (scr) with the integrin mac- (cd b/cd ) serving as a coreceptor for fcαri ( , ) . depending on the receptor addressed and the form of iga offered, i.e., soluble versus target-bound and cross-linked, eosinophils are either calmed down or activated ( ) ( ) ( ) . yet, eosinophils are not only manipulated by iga, they also influence iga production themselves ( , ) . thus, immunoglobulin a and eosinophils share a really intimate relationship. equipped with less receptors but still responsive to iga are neutrophils, dendritic cells, macrophages, basophils and even epithelial lining cells that express the transferrin receptor such as alveolar-type cells ( ) . an additional, so far unidentified receptor is present on m cells (microfold cells) ( , ) . m cells are a specialized epithelial lining cell type that is responsible for antigen sampling at mucosal surfaces and predominantly occurs in the epithelium above organized mucosa-associated lymphoid tissue ( , ) . this receptor senses the distance between two heavy chain domains in iga. thus, it is not able to bind iga , an iga subclass present only in primates. iga is different from iga in that it contains a mucin-like, highly glycosylated extension of its hinge region. it is believed that this hinge region also serves as a ligand for yet other iga receptors ( ) . if this holds true, subclass switching may be another adjusting wheel for iga function. the class switch from iga to iga depends on the presence of the cytokines april and baff which were shown to be produced by the epithelial layer itself, at least in case of the gut, upon bacterial stimulation ( ) . this way the microbiome as sparring partner of the epithelium comes into play as the true master of this adjusting wheel. in contrast to the gut where iga prevails, iga is the predominant iga subclass in the airways ( ) . this, however, does not imply that iga is of less importance for airway defense. iga simply may be the first class formed after pathogen challenge. those initial secretory iga (siga) responses are believed to be not very mature. upon pathogen challenge the human body apparently rapidly switches its current igm repertoire to iga even if most of such "first line of defense" iga are of low affinity ( ) . this can be regarded as just a "better than nothing" attempt; yet it creates a window of opportunity for the body to develop more powerful siga via affinity maturation. such optimized immunological scavengers are then able to block and eventually clear a microbial infection. thus, the iga system in which the transporting epithelium plays a key role is a complex defense machinery that combines innate with adaptive immune responses. it is therefore not surprising that the role of secretory iga attracted attention in asthma research in recent years. the role of siga in chronic inflammatory lung diseases is still ambiguous. some studies show that siga is necessary to maintain immune homeostasis, other reports claim that siga may play a detrimental role in asthma. a harmful effect of iga in asthma may be explained by its ability to activate eosinophils and neutrophils because both cell types play a central role in the pathogenesis and persistence of asthma ( ) . when iga is able to activate those cell types, this would readily lead to the hypothesis that in case of allergic asthma, allergen-specific iga is responsible for this activation upon allergen exposure. this assumption is supported by the finding that increased levels of both, allergen-specific ige and iga were observed in the airway mucosa of patients with atopic asthma and/or rhinitis ( ) ( ) ( ) ( ) ( ) , and it was shown that allergen-specific iga levels were positively correlated to eosinophil activation marker release after segmental lung challenge of asthmatic patients ( ) . yet, coincidence and correlation do not necessarily imply a causative relationship. in the abovementioned study, where a positive correlation of allergen-specific iga and eosinophil activation was observed, the non-allergic control patients also displayed allergen-specific iga in their airways; but in contrast, they did not have any allergen-specific ige as was the case for asthmatics. either so the allergen-specific ige was responsible for eosinophil activation in asthmatics or the eosinophils of asthmatics underwent some kind of imprinting or immune training that rendered them more sensitive to allergen-specific iga. with the expression of five different iga receptors on the eosinophil described so far ( ) , locked-in differences in iga receptor expression in eosinophils of asthmatics versus healthy individuals are not impossible. on the other hand, a coincidence of allergen-specific iga and ige does not necessarily imply a pathological role of iga either. it may still be the case that iga are beneficial to chronic airway inflammation, and the concomitant production of allergenspecific iga can also be interpreted as a rescue attempt of the body to counteract the allergen-specific ige. in fact, more evidence points toward a beneficial role of iga in asthma and other chronic airway inflammations. it was shown for instance that upon aging iga knockout mice tend to develop chronic airway inflammation that resembles chronic obstructive pulmonary disease (copd) in humans ( ) . a copd-like phenotype also develops in pigr knockout mice upon exogenous bacterial challenge ( ) , and it has been shown in the past that copd patients have an impaired pigr expression ( ) and reduced siga levels on the airway epithelium ( ) . recently a similar phenomenon was reported for asthma ( ) and rhinosinusitis ( ) . in the study of ladjemi et al., asthmatics show a reduced immunostaining of pigr in airway epithelia, with il- and il- being the suppressors of pigr formation in the airway epithelium. notably, there were no significant differences in the pigr gene expression rate among asthmatics and healthy individuals ( ) . thus, a posttranslational event such as proteolytic degradation of pigr in the epithelium may be responsible for the observed differences. a beneficial effect of allergen-specific iga in the airway lumen was highlighted by schwarze et al. ( ) . they showed that local application of a human monoclonal iga antibody directed against the ragweed allergen amb a attenuated the proinflammatory response to allergen inhalation in mice sensitized to amb a i, whereas a control iga against ovalbumin did not. notably, the instilled anti-ragweed iga induced the formation of amb a i-specific igg a in the animals upon allergen challenge which indicates a shift toward th . thus, iga residing in the airways may have an anti-allergic/anti-asthmatic immunomodulatory activity. this effect may be explained by the iga feedback loop, via which a secretory iga response is adjusted to current needs. in order to provide an optimal defense against luminal noxa luminal iga are continuously sampled at the epithelium and transported to the basolateral side, where it is inspected by immune cells whether it is loaded with antigen or not. if this is the case, an immune response is mounted or boosted ( ) . although this type of transcytotic event has been attributed primarily to m cells, the set-up of the ragweed-study rather precludes that route in this specific case in as much as a human iga against amb a i was used and this type of iga does not bind to murine m cells ( ) . however, with a plethora of iga receptors known, other epithelial cell types may have taken over the task. the iga-binding transferrin receptor, for instance, is expressed by type ii pneumocytes and was shown to transport transferrin conjugates to the basolateral site ( ) . in addition, similar to the gut, airway dendritic cells, which also carry iga receptors, send protrusions to the epithelial layer via which luminal antigen can be sampled ( , ) . yet, sampling antigen-loaded iga from the airway lumen and driving the airway immune response toward th requires the presence of iga in the lumen, which is reduced by the th micro-milieu in allergic asthma. this results in a vicious circle of a locked-in th environment where a lack of iga causes a further lack of iga. this is in line with clinical observations on asthmatic patients that suggests a critical role for iga in asthma pathogenesis. patients with selective iga deficiency tend to bronchial hyperresponsiveness ( ) and children that show a delay in maturation of iga production display atopic manifestations more often ( ) . moreover, immunotherapy against the respective aeroallergen result in higher specific mucosal iga levels along with lower skin prick test sensitivity ( ) or lower airway hyperreactivity ( ) . nevertheless, most of the above suggests a prominent role of siga or, more precisely, the lack thereof in the pathogenesis and chronicity of atopic asthma. whether a lack of siga also plays a prominent role in asthma exacerbations remains to be elucidated. in addition to all the homeostatic defense functions like the maintenance of barrier integrity, transcytosis, and the mucosal clearance the airway epithelium also plays a major role against inhaled materials by producing several defense proteins such as mucins, defensins, antimicrobial peptides, cytokines, and chemokines ( ) . thus, it contributes to local acute inflammatory reactions by regulating early inflammatory events via transcription and secretion of antimicrobial and pro-inflammatory proteins and by activating of mucin production ( , ) . consequently, it is also a critical player during sensitization processes, asthma pathogenesis and acute exacerbations of the established disease (figure ). inhaled pathogens that are not cleared by mcc are recognized by airway epithelial cells ( ) . equipped with a large number of prrs such as cytoplasmic nod like receptors (nlr) and transmembrane toll like receptors (tlr) that can respond to figure | inflammatory response of the airway epithelium during stable allergic asthma and exacerbation. during stable allergic asthma airway epithelial cells (aecs) release il- , il- and tslp supporting differentiation of t helper (th) cells that are activated by dendritic cells (dcs). th cells in turn secrete il- and gm-csf that together with aec-derived eotaxins, rantes and mcps regulate the production, maturation, recruitment and activation of eosinophils. local degranulation of eosinophils in the lung eventually leads to damage of the airway epithelium. in parallel, the th -type cytokines il- and il- induce goblet cell (gc) metaplasia in airway epithelium. during viral induced asthma exacerbations, several other additional factors lead to an aggravation of this inflammatory response. viral infection can be detected by the airway epithelium via pattern recognition receptors (prr). subsequently, aecs secrete on the one hand tarc, the main chemokine for the recruitment of th cells that amplifies the proinflammatory effects of th cells via release of il- , and on the other hand il- , which leads to the recruitment of neutrophils. local degranulation of neutrophils in the lung eventually leads to additional damage of the airway epithelium. viral infection of aecs also directly leads to damage of the airway epithelium. in summary, these conditions result in a markedly increased damage of the airway epithelium compared to the stable disease, which further impairs barrier integrity and leads to release of matrikines further amplifying the ongoing inflammation. damps and pamps aecs represent the first line of cells, which can respond to pathogens and other danger signals like cell stress and cell death in the lung ( ) . damps are molecules that are release from injured cells. their presence is a clear sign for the loss of homeostatic integrity of specific cell compartments or even whole cells. hence, they originate from the cytoplasm (s proteins, heat shock proteins, defensins, galectins, uric acid), the nucleus (high-mobility-group-protein (hmgb )), the endoplasmic reticulum (calreticulin), mitochondria (atp, mitochondrial dna, n-formylated peptides) or from the extracellular matrix (fibronectin, hyaluronan, versican) ( ) . airway epithelial cells are both, responder to and producer of damps ( , ) . the dna binding protein hmgb for example, set free during necrosis of one cell can be detected from nearby cells by binding to their receptor age (rage), which leads to activation of nuclear factor kappa b (nfκb) and thereby to the production of pro-inflammatory mediators and consequently the recruitment of immune cells. in turn, pamps are preserved molecules and structures from pathogens and toxins. they can originate from such different sources as bacteria, mycobacteria, viruses, fungi and parasites ( ) . pamps and damps activate signaling pathways resulting in the transcription and production of cytokines and chemokines. in brief, signal transduction through myd and myd independent mechanisms leads to the activation of nfκb, mitogen-activated protein (map) kinases, and interferon regulatory factor (irf) ( ) . based on the nature of the triggering pamps and damps and a possible preexisting inflammatory environment in the lung its signals can result in protective effects or pathological effects for the host organism ( ) . repeated cell stress and exposure to pathogens trigger chronic activation of prr pathways in airway epithelial cells that are highly active and play an important role in chronic airways diseases ( ) . activation of prrs by damps leads to a massive secretion of proinflammatory mediators like il- , cxcl , tnf that consequently entail infiltration of activated immune cells. some of these cells like dendritic cells (dc), lymphocytes and mast cells are also involved in the pathogenesis of asthma ( , , ) . after contact for example with hdm extracts, representing a major source of asthma associated allergens, tlr dependent activation of nfκb and protease induced injuries in airway epithelial cells lead to secretion of chemokines and cytokines like thymic stromal lymphopoietin (tslp), gm-csf, il- , and il- ( ) ( ) ( ) ( ) ( ) . this results in the activation and infiltration of dcs, innate lymphoid cells type (ilc ) and th cells ( ) ( ) ( ) . during infection with bacterial pathogens airway epithelial cells can sense bacterial cell wall components via tlr (recognizing e.g., lta), tlr (recognizing e.g., lps), nucleotidebinding oligomerization domain-containing protein (nod ) and nod (recognizing peptidoglycans) leading to activation of nfκb and subsequent immune responses and consequently to regulation of bacterial clearance ( , ) . nucleic acid patterns arising during viral infection can be sensed via tlr , tlr / , retinoic acid inducible gene i (rig- ), and melanoma differentiation-associated protein (mda- ) ( ) ( ) ( ) ( ) ( ) . in response to tlr activation airway epithelial cells can also produce antimicrobial peptides such as human β-defensin (hbd- ) after tlr activation ( , ) . the signals of different prrs like tlrs, nlrs, and rage cooperate to regulate cellular immune responses to cell stress, infection and inflammation, which can amplify or dampen their effects ( ) . airway epithelial cells are very potent producers of cytokines and chemokines. the presence of aggressors like toxins and pathogens leads to production and fast and early secretion of il- β, il- , tnf, cxcl , ccl , and ccl ( , ( ) ( ) ( ) . thereby, airway epithelial cells regulate and orchestrate local immunity by interacting with the recruitment of dcs, t-cells, and b-cells (ccl ), eosinophils (ccl ), and neutrophils (cxcl ). during viral infections they constitutively produce ifnβ to reduce viral replication and to support epithelial apoptosis ( ) . thereby, airway epithelial cells represent the frontline of antiviral defense mechanisms. as mentioned earlier, increased concentrations of proinflammatory cytokines like il- β, il- , il- , and tnf can directly lead to damage of the barrier function of the airway epithelium ( - , , ) . in allergic asthma airway epithelial cells are one of the main producers of proinflammatory cytokines and chemokines like il- , il- , tslp, ccl (rantes), ccl (mcp- ), ccl (tarc), ccl , and several eotaxins. all of these cytokines strongly direct or support the development of a th polarized inflammation ( ) . the chemokines ccl and ccl play a prominent role in the recruitment of th cells by binding to the ccr receptor, since activation of it is a key event for th cell specific chemoattraction ( , ) . as a highly potent producer of tslp the airway epithelium can create a local micro-milieu that supports and maintains a th polarized inflammation ( , ) . in response to different epithelial injuries, airway epithelial cells secrete so-called alarmins like tslp, il- , and il- that direct t helper cell differentiation toward an th phenotype ( ) . additionally, secreted gm-csf from airway epithelial cells leads to maturation and survival of eosinophils ( ) ( ) ( ) . both effects are supporting allergic inflammation in asthma. taken together the airway epithelium plays a major role for the recognition of pamps and damps in the lung. binding of these molecules to their respective receptors enables the airway epithelium to regulate pathways important for barrier function, mcc and local immune responses. functional disorders of the airway epithelium in the ability to answer the presence of pamps and damps favor the development of chronic airways diseases. viral infections and exposure to bacteria in early life modulate the acquisition of th and th immunity during further development and influence the responses to following exposures. these effects could play an even greater role in patients with asthma since they show disrupted mcc that could amplify the disease morbidity ( , ) . a cytokine induced th polarization of the epithelium in combination with a barrier dysfunction induced by the same cytokines augments barrier impairment, further infiltration of proinflammatory cells, and enhanced penetration of inhaled allergens, which can be described as a self-reinforcing mechanism that predisposes for the development and perpetuation of allergic asthma ( ) ( ) ( ) . consequently, it has been suggested that an abnormal programming of the airway epithelium in general paired with an impaired capability to produce anti-inflammatory mediators such as il- or α melanocyte stimulating hormone (α-msh) may be the origin of chronic inflammatory airway diseases ( , ) . viral infections of the airways is of critical importance for the pathogenesis of allergic bronchial asthma: on the one hand recurrent respiratory viral infections during early childhood represent one of the strongest factors increasing the risk for the development of asthma in later life ( ) ( ) ( ) ( ) ( ) . on the other hand such infections are by far the most common cause for acute exacerbation of already established asthma leading to acute aggravation of disease symptoms and necessitating increased medication, gp visits, and can lead to hospitalization and critical care measures under certain conditions ( ) . indeed, the airway epithelium is in the center of action during such an exacerbation, since it is not only the barrier that first comes into contact with viral pathogens, but its cells are also the target for their infection and the site of their replication. thus, viral infection of the airway epithelium does not only impair the barrier integrity as already mentioned before, but also triggers the release of chemokines, cytokines, alarmins, and matrikines of the epithelial layer that affect the pre-existing inflammatory response in the asthmatic airway, which largely contributes to the formation of an acute exacerbation. the viruses that have been implicated in asthma pathogenesis and especially the formation of acute exacerbation are hrv, rsv, influenza and parainfluenza viruses, human metapneumovirus, corona and adenoviruses, however, hrv infections appear to be the most common cause ( , ) . hrv is a non-enveloped, icosahedral virus, which belongs to the family of picornaviridae (genus enterovirus) and is subdivided into three clades (a, b, and c). the single-stranded positive rna genome of hrv is constituted of ca. nucleotides ( , ) . clades a and b, which comprise the most common serotypes, are further subdivided into a major and minor group. the major group utilizes the intracellular adhesion molecule- (icam- ) to bind to and transfect the host cell ( ) ( ) ( ) . the minor group hrv bind to the low density lipoprotein (ldl) receptor ( ) ( ) ( ) ( ) and are considered to be more infectious. clade c consists of serotypes ( ) , which all bind to the cadherin-related family member (cdhr ). all species of hrv have been shown to infect and replicate in airway epithelial cells ( ) . viral engagement with the specific receptor leads to transfection of the host cell (e.g., bronchial epithelial cells) and subsequently to a multitude of cellular responses. it is this cellular response that is believed to facilitate acute asthma exacerbation. the cellular response to the virus is initiated by the detection of single-stranded rna via tlrs - , - , and - , mad , and rig-i ( , ) . activation of these receptors ultimately leads to the secretion of cytokines such as il- , il- , il- , il- , chemokines like cxcl , ccl (eotaxin), ccl and anti-viral interferons of type i and iii ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the interferons have not only innate but also adaptive immune-system functionality to keep the viral infection locally at bay by mobilizing the adaptive immune response for effective viral clearance ( ) . furthermore, proinflammatory cytokines like il- β and il- are not specific for special types of immune response and thus, not only support the immune response against the invading virus but also promote the allergic immune response already established in the airways. consequently, augmentation of the allergic immune response results in acute amplification of tissue damage, mucus production, and mediator release, and therefore in acute symptom aggravation. especially, the interferon response is thought to be an early post-infection event. in asthmatics epithelial interferon responses are believed to be hampered and as a consequence antiviral responses lack sufficient clearance ( ) . not only interferons seem to be differently expressed in epithelial cells from asthmatics but also tslp, which promotes th responses ( ) . another critical cytokine elevated in humans after hrv infections is il- , which also augments th cell development ( ) . it is of note that viral infections not only initiate an immune response but also drive remodeling of the epithelial barrier and the subepithelial extracellular matrix (ecm). hence, hrv induces perlecan, collagen v, tenascin c and matrix-associated (ma-) vegf expression in an either tlr- or tlr- /- associated manner in vitro ( , ) . elevated expression of ma-vegf and tenascin c was replicated in a mouse model of hrv infection, in which also collagen i and fibronectin was found to be increased ( , ) . in addition, our group found human nasal epithelial cells infected with rv- in vitro to significantly downregulated genes associated with ecm receptor interaction and focal adhesion ( ) . after infection and viral replication, the release of a vast array of mediators is among the earliest responses. in tissue culture a pneumocyte cell line expresses large amounts of il- and ccl readily after h post-infection ( ) . also, the interferon response is thought to be an early post-infection event. in asthmatics epithelial interferon responses have been suggested to be hampered and as a consequence antiviral responses lack sufficient clearance ( ) . but not only interferons seem to be differently expressed in epithelial cells from asthmatics but also tslp, which promotes th responses ( ) . another critical cytokine elevated in humans after hrv infections is il- . in line with that, jackson et al. impressively showed, that the release of il- by bronchial epithelial cells induces il- , il- , il- , and gata expression in th cells. an effect, which could be entirely blocked by an antibody against the il- receptor ( ) . recently, active fragments from epithelial deposited ecm molecules have gained some recognition in asthma and asthma exacerbation. matrikines are a class of molecules derived from ecm proteins (e.g., via proteolysis) with different properties from the parent molecule ( ) ( ) ( ) . in , burgess et al. reported diminished levels of the collagen iv isoform alpha (col a ) in airways from asthmatic subjects. the non-collagenous domain of col a is referred to as tumstatin and a biologically active matrikine. treatment of mice with experimental allergic asthma with human recombinant tumstatin let to a significant reduction of hallmark disease features (e.g., airway hyperresponsiveness, ma-vegf, eosinophil influx, il ) ( ) . in another study, van der velden et al. identified an anti-angiogenic effect of tumstatin in a sheep model of asthma ( ) . further investigations revealed a novel active region in tumstatin (cp ), which significantly reduced neutrophil influx, mucus production in a mouse model of viral asthma exacerbation and reduced migrational speed and production of reactive oxygen species of neutrophils in vitro ( , ) . while the matrikine tumstatin conveys protection from experimental features of asthma and asthma exacerbation, a collagen i derived matrikine (pgp, acetylated-(ac) pgp) has been shown to be a more potent inducer of neutrophil chemotaxis then il- and is found to be increased in severe asthmatics, a group of patients prone to develop exacerbations ( , ) . albeit ecm derived matrikines follow a different kinetical pathway (deposited first, released during inflammation) as compared to cytokines and chemokines (de novo production after viral infection), they can serve as protective or aggravating factors in asthma exacerbations. in addition, the notion of epigenetic modification due to hrv infection in epithelial cells in asthma has gained attraction. mcerlean et al. infected nasal epithelial cells from asthmatics and found evidence of reproducible changes to the methylome ( ) . we confirmed these results, which may unriddle how asthmatic airway epithelial cells may be able to respond differently to the same stimuli as compared to healthy epithelial cells ( ) . first studies to investigate this effect in vivo are underway ( , ) . infection associated stimuli appear not to be the only factors that imprint mucosal immune reactions of the airway epithelium. in addition, the interaction between epithelial cells and leukocytes can lead to sustained alteration of respiratory epithelial cell biology. even though these cells are definitely not able to constitute an immunological memory, it becomes more and more obvious that especially epithelial cells somehow memorize their exposure to certain environmental factors and the following insults and thereby develop some kind of trained immunity. we are just at the beginning to understand these processes and questions of which parts of the epithelium are trained and of how long the training effects sustain in the mucosa remain to be answered. thus, respiratory epithelial cells are constantly exposed to many types of challenges, including pathogens, allergens and environmental pollutants. consequently, they are able to respond quickly and effectively to cellular damage such as the local cytokine production, lateral transport by ion exchanges, wide arrays of mucus compositions, secretion of antimicrobial peptides, and epithelial shedding. to date, it appears possible that different inflammatory environments as originated by for example typical th -or th -directed immune responses have a different impact on the biology of the respiratory epithelium and lead to some kind of e -or e -polarization of the respective epithelial cells ( ) (figure ) . there is in vivo evidence for the inhibitory role of ifnγ on asthma pathogenesis at the epithelial level indicating that type- responses counteract allergy ( , ) . the direct implication of airway epithelial cells was demonstrated by selective transgenic expression of the ifn-γ receptor on the airway epithelium and showed that ifn-γ inhibits mucus secretion, release of chitinases and eosinophilia independent of the activation of th cells ( ) . in turn, gata- inhibition causes an increase of t-bet und ifn-γ expression levels, leading to a dampened allergic phenotype ( ) . in addition, an increase in dna-methylation of ifn-γ was observed during allergic sensitization ( ) , while perinatal prevention of allergy mediated by acinetobacter does not show the anticipated drop in h acetylation in the ifn-γ promoter ( ) . the immunological consequence of epithelial differentiation becomes increasingly interesting, as sensitization, but also recovery processes and airway remodeling could open new options for intervention and prevention of lung damage. increasing evidence of mechanisms involving epithelial cytokine production such as ccl- , and the epithelium-derived alarmins tslp and il- are substantiating the current focus on the cross talk between airway epithelium and immune cells in allergy research. the role of tissue cells in the early phase of disease is largely unknown, but could provide important information about the pathologic development and could help to identify the causal relationships. however, bronchial epithelial cells are pre-committed to a type- (e ) or type- (e ) like phenotype. e epithelial cell activation by allergens takes place and their pro-inflammatory cytokines and chemokines induce inflammation and contribute to an epithelial type- response, so called "e response" with epithelial alarmins tslp, il- , ccl- , il- , and il- . local type- responses involve multiple cytokines such as il- , il- , il- , il- , il- , il- , and increase of eosinophils. a series of chemokines are produced and migration of inflammatory cells to the allergic tissues takes place. the activation of e.g., smooth muscle cells by adam lead to remodeling. bronchial hyperreactivity takes place leading to an enhanced susceptibility to bronchoconstriction. e epithelial cells respond to an infection releasing cxcl , cxcl , il and ccl , thus stimulating the local synthesis of ifn-γ, il- , il- , il- , il- , and tnf-α that present a wide range of antiviral activities, inducing up-regulation of mhc-i molecules and antiviral resistance in uninfected cells. neutrophils respond to the infection signals il- and ifn-γ by releasing pro-inflammatory cytokines, leading to the containment of the infection, rise of body temperature and to the recruitment of further phagocytic cells. however, it is unknown whether epithelial cells are influenced by il- prior or with entry into terminal differentiation. this early influence could imprint the offspring cells that populate the epithelial surface and therefore have major consequences for the physiology of the airways. il- was shown to have a major effect on the epithelium, as mice overexpressing this cytokine under the club cell-secretory protein (cc ) promoter show increased cellular infiltration, epithelial hypertrophy, mucus cell hyperplasia, secretion of gastric mucins and surfactant proteins (sp) a and b ( ) . while this model effectively demonstrated all hallmarks of experimental allergic asthma, it did not demonstrate whether il- itself is inducing differentiation of basal cells or whether secondary effects trigger epithelial differentiation. however, the differentiation effects could also be observed in human primary epithelial cells of the nose, where il- modulates the differentiation toward less ciliated and more secretory cells ( ) . to date, it is controversially discussed, whether il- and il- can also affect fully differentiated epithelial cells in air liquid-interphase cultures or whether this is only possible in immature submerged cultures ( , ) . however, during the epithelial differentiation process induced by air-liquid exposure, the addition of il- enhances expression of certain antimicrobial peptides ( ) and eicosanoids ( ) . furthermore, it was demonstrated that il- and il- , through inhibition of tlr expression and signaling (irf ), impair immune responses to hrv infection ( ) . this is in line with the finding that chronic house dust mite exposure in the airways not only causes a strong th -directed inflammation but also diminishes anti-rhinovirus responses and local ifn expression, particularly of epithelial ifn-λ ( ) . in line with this, transgenic il- expression in the lungs reduces cytotoxic t cell responses against influenza viruses ( ) . on the level of secreted factors, it was shown that cytokines such as wnt a (wingless-type mmtv integration site family, member a) or il- are expressed as response to il- stimulation only, while proteins with known pathological roles such as the il- induced protein ccl- or periostin were shown to be upregulated by il- and down-regulated in ifn-γ environment. these results were consistent when comparing upper and lower airway secretions, thus confirming nasal lining fluids as a proxy for the lower respiratory tract, particularly for epithelial type- biomarker like ccl- and il ( , ) . the e -related transcription factor network contained the e hub-transcription factors gata , nfe , meis , hey , and ahr: gata is well known as the master transcription factor of type- response in immune cells, however it was also shown to be expressed in airway epithelial cells. nfe was demonstrated to have a cytoprotective activity against epithelial cell injury by cigarette smoke, which could hint on a protective role in an il- dominated micromilieu ( ) . for meis , it has been demonstrated that its inactivation produces an increase in airway smooth muscle mass and a corresponding decrease in cartilage and suggesting an important role in allergic airway diseases. a loss of hox gene function, however, does not preclude airway repair, but regenerated epithelium displays goblet cell metaplasia and less scgb a -positive cells, demonstrating the essential role of hoxa for correct differentiation. this goblet cell metaplasia is further associated with increased notch signaling activity. consistent with these findings, expression levels of activated notch and the effector gene hey are in turn enhanced in patients with allergic disease ( , , ) . taken together, e polarization has a marked impact on the barrier and especially immune functions of the airway epithelium and at least supports impairment of the mcc and antiviral responses, both factors that are critical in asthma pathogenesis. in summary, the airway epithelium exerts a broad variety of immune functions that range from passive barrier over mcc, active production and transport of pathogen-neutralizing molecules to pathogen recognition and targeting as well as cytokine and chemokine release. the airway epithelium is usually the first tissue that is exposed to inhaled allergens, pathogens or pollutants. since it is able to react on this contact by inducing local inflammatory reactions, it is clearly a central part of the local immune response and bridges innate and adaptive immune functions against all types of harmful intruders entering the respiratory system. therefore, the airway epithelium is a key factor in asthma pathogenesis and plays a critical role in the development as well as in the progression and exacerbation of the disease: hence, a disturbed cellular barrier enables allergens to enter the body and to induce a sensitization reaction, which is widely regarded as the starting point of an asthma career. down the line, the protective mucus and pcl layers provided by the epithelium as physical barriers are compromised and the release of pathogen deterring molecules such as secretory iimmunoglobulins becomes impaired. as a consequence, this frontline toward invading pathogens can be breached and airway epithelial cells are infected and even destroyed by respiratory pathogens. a vicious cycle is started where barrier disturbance and infection promote each other. the latter, in particular with certain viruses, is also one of the strongest factors predisposing toward asthma development in early childhood. once asthma has been established, the airway epithelium responds to further viral infection with the release of manifold factors promoting not only the antiviral response but also augmenting the already present allergic inflammation and thus promoting acute asthma exacerbations. finally yet importantly, it is also impaired and polarized by products released from cells of the chronified allergic immune response in the airways, which leads to impairment of mucus production, mcc, and the antiinflammatory ifn-response in e -polarized airway epithelial cells. recent studies indicate that, in addition to this e polarization, also the history of allergy-and pathogen-derived insults does not only have a transient effect on the airway epithelium, but leaves some kind 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metaplastic differentiation of bronchial epithelial cells th cytokines impair innate immune responses to rhinovirus in respiratory epithelial cells no exacerbation but impaired anti-viral mechanisms in a rhinovirus-chronic allergic asthma mouse model local il- expression in the lung reduces pulmonary influenza-virus-specific secondary cytotoxic t cell responses biomatrix for upper and lower airway biomarkers in patients with allergic asthma early il- producing b-cells and coinciding th/tr shifts during three year grass-pollen ait the cytoprotective role of dj- and p nfe against human primary alveolar type ii cell injury and emphysema the loss of hoxa function promotes notch-dependent goblet cell metaplasia in lung airways current and future biomarkers in allergic asthma all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. this work was supported by the german center for lung reearch (dzl). key: cord- -qv orlv authors: mutti, luciano; pentimalli, francesca; baglio, giovanni; maiorano, patrizia; saladino, rita emilena; correale, pierpaolo; giordano, antonio title: coronavirus disease (covid- ): what are we learning in a country with high mortality rate? date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: qv orlv nan covid- has been declared a pandemic by the who ( ) . following the outbreak of the disease in china, italy was the first european country to be heavily struck ( , ) . initially, three covid- cases were reported in early february, which were all related to individuals who had traveled to china; then, on the th, a young man who had not traveled abroad presented with severe sars-cov- -induced pneumonia in lombardy, a region in the north of the country ( ) . over the next weeks, many patients in the surrounding areas were diagnosed with covid- , which was often severe, and another cluster was identified in the nearby region of veneto ( ) . there then followed an exponential increase in cases, mostly in the north, although the disease spread throughout the whole country, leading to the hypothesis that the virus had been circulating since january ( , ) . at that point, italy reached incidence and mortality rates that were amongst the highest in the world ( ) ( ) ( ) . many factors explain differences from other countries, including different application of detection tests, a larger elderly population, and different prevention policies and capacity to provide intensive care ( ) . while it is paramount to conceive preventive strategies and apply more effective early treatments, it is also crucial to understand the biological mechanisms underlying these fatal outcomes. in italy, the possibility of performing autopsies or post-mortem diagnostic studies on suspect, probable, or confirmed covid- cases has been intensively debated ( , ) ; however, postmortem pathological analysis of covid- patients in china has shown findings consistent with acute respiratory distress syndrome (ards) ( - ) (figure ). at present, the exact nature of the acute lung injury trigger is not yet fully clarified; however, it could be ignited by t cells overreacting to virus-specific epitopes, thus recruiting multiple cytokineactivated inflammatory cell lineages ( ) ( ) ( ) . other possibilities that deserve further experimental evidence include an exaggerated antibody-mediated response with complement activation and/or fcγ receptor-mediated leukocyte engagement and/or a hypothetical cytopathic effect of the virus ( , ) . the latter could explain the recently described microvascular damage leading to disseminated intravascular coagulation (manifested as thrombosis, thrombocytopenia, and gangrene of extremities), anti-phospholipid syndrome, and mimicry of vasculitis, which are described in both chinese cohorts ( ) and italian patients ( ) ( ) ( ) . in our experience, ≈ % of patients develop interstitial pneumonia, and a subset of these (≈ %) develop ards that, especially when so serious as to require invasive ventilation, is mostly fatal. the risk of ards rises with age, and almost all deaths regard patients with pre-existing chronic conditions ( , ) . pre-admission hypertension, in particular, has been reported as a key mortality risk factor ( ) . the risk of death further rises where there is a lack of ventilators or ventilation is refused, as described in xu et al. ( ) . moreover, an increasing number of clinical reports describe a biphasic behavior: a first phase where covid- -infected patients are completely asymptomatic, which lasts on average seven days, and a second phase where the patients present mild to moderate flu-like symptoms, anosmia, ageusia, and blind conjunctivitis, which may last - days ( , ) . a minority of patients who are unable to achieve complete virus coverage develop severe cardio-respiratory symptoms with radiological signs of pneumonitis, ards, and then multiorgan failure ( ) . the last phase occurs, on average, - days after infection. in the latter case, patients may test negative for covid genome research standard molecular tests. altogether, these clinical findings, as well as the available pathology studies, support the hypothesis of an inappropriate immunerelated inflammatory response to covid epitopes and consequent auto-antigen release and t-cell cross-presentation in the damaged alveolar tissue. consistently, recent results indicate that a systemic immune dysregulation that triggers auto-sustaining inflammatory lung damage, causing fatal respiratory-failure and consequent multiorgan-failure, is the main virus-related-death cause in patients who develop sars-cov- ( ). the culprit is the cytokine storm unleashed in this context by the infection and already described in cancer patients treated with cart or immunotherapy, including the "old" treatments with interleukins (il and il , in particular) and the newest anti-ctla- and or anti-pd- /pdl immunecheckpoint inhibitors. a greater risk of pneumonitis has already been recorded in chinese patients bearing a highfrequency of specific class-i and ii hla alleles associated with poor virus clearance and development of immune-related pneumonitis and other inflammatory-related autoimmune diseases ( ) . this viral-load-independent different response to the infection might depend on a genetic predisposition causing extreme and often lethal inflammatory reactions. given the inefficacy of steroids ( ), understanding the molecular features underlying such threatening immune-related events provides a strong rationale for using biological drugs for the early treatment of symptomatic patients, aimed at hampering the effects of the most relevant cytokines able to trigger an antibody response and acute inflammatory reaction, such as il and il α. to this purpose, abs against the il receptor, or drugs able to disrupt its downstream signals, can inhibit its function on specific inflammatory cell subsets. these agents have so far been promising in the clinical setting for curbing the inflammatory response to control the severe immune-related adverse events related to cart-therapy and immune-checkpoint blockade and autoimmune diseases, including juvenile rheumatoid arthritis, psoriatic arthritis, and ulcerative colitis, all related to particular hla class i and ii alleles, some of which, like class i b * and b * , might sustain both mitochondrial stress and cross-reactivity with several pathogens ( ) . therefore, while antiviral drugs help to contain viral replication, moabs to il in the early phase of respiratory involvement could control the risk of a fatal virus-inducedcytokine storm. a great effort should be made to recognize lung involvement as, at least theoretically, the earlier the treatment, the better the outcome will be, with il inhibitors being able to "nip in the bud" the inflammatory cascade and prevent the fatal permanent damage to the alveolar pneumocytes. on this basis, il inhibitors are currently being tested in china and italy in patients with respiratory failure, and other il inhibitors are also being considered. iatrogenic cues might also contribute to exacerbating the acute inflammatory lung injury triggered by the virus. most hospitalized patients in fact received oxygen either through intubation or mechanical or non-invasive ventilation ( ) ; however, oxygenation in ards patients with acute lung inflammation has been previously shown to interfere with the anti-inflammatory response induced locally by hypoxia through the activation of the adenosine a a receptor ( ) . similarly, in covid- , patients, oxygen therapy could worsen lung injury by weakening such anti-inflammatory pathways. consistently with this hypothesis, in a cohort of , patients hospitalized with covid- in the new york city area, mortality reached . % for those requiring mechanical ventilation ( ) . in lombardy, the intensive care unit mortality was %, and indeed, a large proportion of admitted patients required mechanical ventilation ( ) . these data support the possible use of adenosine agonists in patients presenting with ards (figure ) . identifying infected patients at higher risk of poor prognosis even without evident risk factors could represent an important step forward. in this direction, zhou et al. reported some predictive biomarkers of the severity of the infection ( ). nguyen and colleagues, in a preprint article, analyzed the sars-cov- proteome and identified a range of hla alleles potentially able to present (or not) viral epitopes. they suggest that individuals bearing hla-b * (which has the fewest predicted binding peptides for sars-cov- ) may be particularly vulnerable to covid- , whereas individuals bearing hla-b * (which has the greatest predicted capacity to present sars-cov- peptides) could exhibit cross-protective t-cell based immunity. the authors highlight that a thorough understanding of how hla variation correlates with covid- onset and outcome could help identify high-risk subjects ( ) . indeed, we have preliminary evidence that the prevalence of specific hla class i alleles across italian regions/provinces correlates with increased covid- incidence (correale p., mutti l., submitted for publication). if confirmed in wide case-control studies, the identification of hla alleles that are more permissive to viral infection would provide the first genetic explanation for the wide differences in covid- incidence rates among italian regions and also among nearby provinces with similar environmental factors. figure | host response and possible outcomes of sars-cov- infection. viral infection seems to occur mainly upon sars-cov engagement of angiotensin i converting enzyme (ace ), which acts as a functional receptor for the spike glycoprotein of the coronavirus. the hla genetic system acts as a key player in determining the anti-viral immune response. in particular, the ability of hla to trigger an adequate cytotoxic t-lymphocyte (ctl) response will result in viral clearance and host healing, along with the development of the igm, iga, and igg humoral response. conversely, an inadequate hla asset will result in an inefficient ctl response and, consequently, incomplete viral clearance. in this context, various factors underlie increased covid- severity, including an exaggerated ab response, complement activation, leukocyte-mediated antibody-dependent cell-mediated cytotoxicity (adcc), and t-cell-mediated inflammation, as discussed in the text. without a protective immune response, the virus is able to migrate, propagating into other ace -expressing tissues, while the damaged lung cells induce high inflammation, triggering the cytokine storm that represents the main cause of the acute respiratory distress syndrome (ards) and subsequent multiorgan failure. incomplete viral clearance can also lead to virus hiding in sanctuary sites and patient relapse with symptoms arising in new districts. in the purple boxes, different therapeutic approaches aimed at targeting either the virus or endogenous host players are represented. overall, understanding the role of pro-inflammatory cytokines certainly unravels a new battleground against the lethal clinical effect of codiv- infection; this, along with the identification of a high-risk autoimmune profile, including the genotyping of class i and ii hla, which have a key role in shaping the anti-viral immune response and th /th lymphocyte subset response (figure ) , and immune-profiling, could also help to prevent these dangerous evolutions of the disease ( ) . in particular, the isolation of genetically at-risk individuals, including healthcare workers, will inform future vaccination campaign priorities and clinical management strategies. the finding of healed patients retesting positive after an apparent complete virus clearance is a matter of intense debate in italy and worldwide. assuming that testing was reliable, various hypotheses are being considered, including viral mutation, although variation among sequences seems very low at present ( ) . a preprint study in rhesus macaques argues against a risk of re-infection ( ) . host inability to develop immunological memory with subsequent longterm protection is also being evaluated. interestingly, another preprint study identified specific sars-cov- neutralizing antibodies (nabs) in the plasma of patients who had recovered from infection and recorded that % of patients failed to develop high titers of nabs after covid- infection ( ) . another possibility is that newborn sars-cov- might hide in sanctuary sites, such as the ncs and/or testis, which are protected from both antiviral drugs and proficient immuno-effectors; this hypothesis is supported by the recent description of viral detection in the cerebrospinal fluid but not in the nasopharyngeal swab in a case report ( ) . overall, these distinct biological patterns of response to the virus should be taken into account for the design of new preventive and therapeutic strategies. lm, pc, and ag conceived the study. fp and gb evaluated current data. rs and pm studied hla involvement. pc conceived and fp sketched the figure. all authors contributed to manuscript writing and agreed with content. this work was supported by the sbarro health research organization (www.shro.org) and the commonwealth of pennsylvania. available online at case-fatality 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and human disease oxygenation inhibits the physiological tissue-protecting mechanism and thereby exacerbates acute inflammatory lung injury presenting characteristics, comorbidities, and outcomes among patients hospitalized with covid- in the new york city area human leukocyte antigen susceptibility map for sars-cov- could pd- /pdl immune checkpoints be linked to hla signature? the establishment of reference sequence for sars-cov- and variation analysis lack of reinfection in rhesus macaques infected with sars-cov- . biorxiv neutralizing antibody responses to sars-cov- in a covid- recovered patient cohort and their implications. medrxiv a first case of meningitis/encephalitis associated with sars-coronavirus- we are grateful to barbara colombo for the figure graphics. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © mutti, pentimalli, baglio, maiorano, saladino, correale and giordano. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -okvt fg authors: alberca, ricardo wesley; teixeira, franciane mouradian emidio; beserra, danielle rosa; de oliveira, emily araujo; andrade, milena mary de souza; pietrobon, anna julia; sato, maria notomi title: perspective: the potential effects of naringenin in covid- date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: okvt fg coronavirus disease (covid- ), caused by severe acute respiratory syndrome coronavirus- (sars-cov- ), was declared a pandemic by the world health organization in march . severe covid- cases develop severe acute respiratory syndrome, which can result in multiple organ failure, sepsis, and death. the higher risk group includes the elderly and subjects with pre-existing chronic illnesses such as obesity, hypertension, and diabetes. to date, no specific treatment or vaccine is available for covid- . among many compounds, naringenin (nar) a flavonoid present in citrus fruits has been investigated for antiviral and anti-inflammatory properties like reducing viral replication and cytokine production. in this perspective, we summarize nar potential anti-inflammatory role in covid- associated risk factors and sars-cov- infection. the respiratory diseases named coronavirus disease (covid- ) is generated by a respiratory infection with severe acute respiratory syndrome coronavirus- (sars-cov- ) ( , ) . due to the rapidl viral transmission, the disease was declared a pandemic by the world health organization a few months after the first diagnosed case ( , ) . besides the similar clinical manifestations to previous severe acute respiratory syndrome coronavirus- (sars-cov- ), sars-cov- infection presents a much lower death rate ( ). approximately % of patients progress to a severe covid- , developing mainly severe acute respiratory syndrome, with % needing assisted respiratory mechanic ventilation. coronavirus disease can progress to septic shock and multiple organ failure ( ) and exhibits a death rate of approximately % ( ). the sars-cov- can infect human cells by entry via the angiotensin-converting enzyme (ace ) receptor and transmembrane serine protease (tmprss ) ( ). although this process is wildly accepted, other possible infective routes are being explored such as antibody-dependent enhancement (ade) ( ) and via cd ( ). angiotensin-converting enzyme expression is one of the main explanations for the higher airway infection, as it is highly expressed in the respiratory tract such as epithelial cells of the alveoli, trachea, and bronchi, some bronchial glands and alveolar macrophages ( ). however, ace is also expressed in the ileo, kidney, adipose tissue, heart, brain, blood vessels, stomach, liver, and oral and nasal mucosas ( ), which could corroborate the systemic inflammatory profile in covid- . upon viral entry, the virus induces the host to increase the production and release of inflammatory cytokines, which can lead to greater immune activation and tissue damage ( ). hypothetically, the reduction of inflammation could aid covid- patients ( ). several compounds have been associated with antiviral and anti-inflammatory properties and could impact covid- development such as vitamin d ( ), vitamin e ( ), vitamin b ( ), omega- ( ), and flavonoids ( ). naringenin (nar) is an important natural flavonoid present in citrus fruits, like grapefruit ( . mg/ ml) and oranges ( . mg/ ml) ( ), with a high analgesic, anti-oxidant, anti-inflammatory, anti-tumoral, and anti-viral effect ( - ) (figure ) . the consumption of ml/kg of orange juice increases nar plasma levels from to µg/l h after ingestion ( ). the antiviral effect of nar has been studied in several viruses, such as dengue ( , ), hepatitis c ( ), zika ( ), chikungunya ( ), semliki forest ( ), herpes simplex and ( ), yellow fever ( ), and human immunodeficiency virus ( ). several in vitro studies have highlighted nar's antiviral effect in pre-infection and post-infection ( ). similar to other natural compounds, nar has extensively been investigated in in vitro, but has very limited results in in vivo models of viral infection ( , ) ( figure b) . nevertheless, the in vitro and in vivo anti-inflammatory potential of nar has been highlighted in several animal models, including respiratory syndromes ( , ) . in this perspective, we highlight the mechanism in which nar may present an important anti-inflammatory role in covid- . inflammation can be characterized by the regulation of proand anti-inflammatory mediators in resident cells and leukocytes recruited from the blood ( ). there are strong pieces of evidence of the role of nar under inflammatory conditions due to a wide range of mechanisms. the immunomodulatory properties of nar are associated with the regulation of key signaling pathways, like nuclear factor kappa-light-chainenhancer of activated b cells (nf-κb) ( ), pi k/akt ( ), and mitogen-activated protein kinases (mapk) ( ) in different cell types ( figure a) . macrophages are an important cell in the covid- pathology, being able to sense and respond to pathogens and produce inflammatory cytokines and chemokines ( ). in murine macrophages, nar can reduce inflammatory mediators production induced by lps, and in a murine endotoxemia model reduces the mortality rates from to % ( ) . murine macrophages infected with a gram-negative bacteria (chlamydia trachomatis) nar reduced the production of il- β, il α, il- , tnf, il- p , and il- in a dose-dependent manner ( ) . moreover, nar's anti-inflammatory effects have been demonstrated in vivo ( ) , in macrophage and ex vivo human whole-blood models, reducing il- β, il- , il- , and tnf upon lps stimulus to close to non-stimulated levels ( ) (figure ) . barnes et al. described that the cytokine storm developed by severe covid- patients is related to an exacerbation of neutrophil activation ( ) . it is clear the central role of neutrophils in covid- , as neutrophilia and neutrophil-tolymphocyte ratio in covid- patients is associated with disease severity ( , ) . lung biopsies have also identified an infiltration of neutrophils ( ) and the formation of neutrophil extracellular traps in covid- patients ( ) (figure a) . although some animals like cats, ferrets, mice, hamsters, and macaques can be infected by sars-cov- , the usage of animal models in covid- is currently limited ( ) . in an animal model of acute respiratory distress syndrome (ards), a syndrome with an increase in il- , tnf, and neutrophils in the lungs, nar supplementation can reduce neutrophils infiltration and oxidative stress, greatly reducing airway inflammation and lung injury ( ) . naringenin reduction of oxidative stress is partially mediated by a curb in the anion superoxide production ( , ) (figure ) . naringenin can suppress inflammatory molecules production through both transcriptional and post-transcriptional mechanisms ( ). in a lps-induced model of inflammation in a mouse model, nar suppressed tnf and il- production by macrophages and t lymphocytes without interfering in the toll-like receptor (tlr) cascade but by increasing intracellular cytokine degradation through lysosome-dependent mechanisms ( ). these data indicate a potential role in the control of inflammation and oxidative stress-related to airway inflammatory insults (figure a) . these anti-inflammatory and anti-oxidant effects are also described in chronic comorbidities like in diabetes mellitus ( , ) , dyslipidemia, hyperinsulinemia, and being overweight ( ) , which are all risk factors associated with severe covid- ( , , ) ( figure c) . in animal experimental models, nar was able to modulate different inflammation syndromes and at different sites, such as colitis ( ) , hepatitis ( ), obesity ( ), cancer ( ) , and acute respiratory syndrome ( ). this is particularly important in covid- , because sars-cov- infection induces a systemic inflammation and can infect many different organs including lungs, heart, liver, brain, kidneys, and the intestines ( ) . in addition, nar can promote lysosome-dependent cytokine protein degradation, which may be important in covid- ( , ) , considering the systemic and cytokine storm during severe covid- ( ) . in fact, nar-induced immunomodulation has been demonstrated in airway inflammatory disorders. in a murine asthma model, treatment with nar reduced airway hyperactivity and airway inflammation, with a reduction in the levels of il- and il- in bronchoalveolar lavage and serum ige levels as well improvement in lung function assay ( ) ( ) ( ) . overall, the treatment with nar reduced lung eosinophilia to similar levels to non-asthmatic group ( ) ( ) ( ) . in lung fibrosis induced by infection with mycoplasma pneumoniae, nar reduced autophagy-mediated airway inflammation and lung fibrosis ( , ) , and, in a chronic obstructive pulmonary disease (copd) model, nar was able to mitigate lung inflammation, reduce the expression of tgf-β, and increase glucocorticoid receptor expression (gcr) nar reduces viral entry, assembly, and replication via modulation of surface molecules, production of antiviral components, inflammatory molecules and/or direct interaction with viral components. (c) nar can influence the development and severity of many different diseases, in different organs, such as cancer, hepatitis, colitis, and severe acute respiratory syndrome. ( ) . naringenin anti-inflammatory effect was also verified in radiation-induced lung injury, reducing lung inflammation and il- β levels ( ) . the nar anti-inflammatory effect is thus not directly mediated to a type or type / immune response but a regulation of the immune response. studies have highlighted the increase in t regulatory cells and transforming growth factor-β after nar consumption via aryl hydrocarbon receptor-mediated pathway ( ) . nevertheless, the excessive regulation of the inflammatory response could impair anti-viral immune response, that has not been previously observed with nar supplementation. naringenin can also activate the interferon-stimulated response element and enhance ifn-i production via an increase in the expression of irf ( ) and increase nk cell activity via enhanced nkg d ligand expression ( ) . considering the crucial role of nk cells and ifn-i in the anti-viral immune response, nar may also contribute to the viral load control. overall, these previous studies demonstrated, in vivo and in vitro, that nar is a strong candidate as an adjuvant in reducing airway and systemic inflammation. two coronaviruses have been responsible for recent epidemics. in , the sars-cov- epidemic caused , cases, with deaths in countries ( ) ( ) ( ) . in , in the middle east, another coronavirus also caused severe acute respiratory syndrome, being named mers-cov ( ) . until , mers-cov had caused , cases, with associated deaths ( ) . clinical manifestation of sars-cov- and mers-cov is similar. patients report clinical symptoms such as fever, cough, body pain, headache, and less commonly, diarrhea and nausea ( ) . however, the need for intensive care and mechanical ventilation is greater in mers-cov than in sars-cov- ( , ) . similarly to mers-cov and sars-cov- , sars-cov- infection is mainly transmitted by respiratory droplets expelled from an infected person during sneezes or coughs ( , ) . severe acute respiratory syndrome coronavirus- surface glycoprotein spike (s protein) binds to ace on the surface of the host's cell surface. this invading process is the same used by sars-cov- ( ) . in comparison, mers-cov uses dipeptidyl peptidase (dpp ), a multifunction surface protein to entry into cells ( ) . dipeptidyl peptidase is mainly expressed on the kidney, intestine, liver, prostate, and activated leukocytes. dipeptidyl peptidase is expressed on the lower respiratory tract, glands located in submucosa of the upper respiratory tract, lung macrophages, and alveolar epithelial cells ( ) . after these coronaviruses (mers-cov, sars-cov- , and sars-cov- ) invade the host's cell, polypeptides are released from the polyproteins by proteolytic processing. the proteolytic process is mediated by papain-like protease (pl pro ) and chymotrypsin-like protease ( cl pro ). the cl pro cleaves the polyprotein to generate various non-structural proteins, crucial for viral replication ( , ) . due to the main role of cl in coronaviruses viral cycle, inhibitors of cl could potentially be used in covid- . flavonoid inhibition of the cl protease has been described in mers-cov ( ) and sars ( ), but nar was not among the flavonoids investigated. nevertheless, in silico analysis demonstrated that nar has the potential to inhibit sars-cov- cl pro ( ) . a recent study verified that sars-cov- and sars-cov- share . % of genetic similarity of cl, with only punctual mutations ( ), leading to the possible inhibition of cl by nar and other flavonoids. another possible mechanism is the inhibition of the twopore ionic channel (tpc and tpc ) ( ) . inhibition of tpc and tpc reduces mers-cov infectivity, intracellular traffic ( ) , and viral replication ( , ) . due to sars-cov- viral genome sequencing similarities with mers-cov and sars-cov- ( ), it is possible that similar mechanism of inhibition of tpc and tpc channel be effective in covid- , aiding in the reduction of viral replication ( ) .interestingly, nar can inhibit the activity of tpc and tpc both in humans and plants ( ) . nar is a hydrophilic substance with a higher affinity for the cytoplasmic membrane generating intracellular accumulation of nar ( ) . therefore, this affinity probably enhances intracellular signaling and the modulation of and tpc and tpc ( ). therefore, the tpc and tpc 's modulation by nar should be further investigated as a possible anti-coronavirus intervention. several reports of natural compounds with anti-sars-cov- potential are currently being investigated. substances that may compete with the ace receptor or reduces the ace expression may present an alternative or adjuvant therapy in covid- ( ) . in fact, nar consumption has been associated with a reduction in ace expression in the kidneys of rats ( ) and could bind directly to the ace receptor ( ) . however, nutritional interventions aiming to regulate sars-cov- entry receptor ace need to be carefully evaluated, as downregulating of ace could also lead to greater inflammation and lung damage ( , ) . previous reports demonstrated that the oral consumption of nar can reduce acute lung injury in a mouse model ( ) and reduce the production of proinflammatory cytokines ( ). this is extremely relevant, as a part of covid- lung injury can be classified as ards ( ) . coronavirus disease can also lead to cytokine storm, progress to septic shock, and cause death ( , ) . modulating the cytokine storm is thus a vital process for treating covid- . naringenin has been used in experimental models to regulate the production of il- and tnf ( ), cytokines that are increased in covid- and further increased in severe cases ( , ) . also in an animal model of septic shock, the consumption of nar has been demonstrated to reduce kidney damage via an increase in antioxidant enzymes ( ) . studies verified a direct role of nar in abrogating viral replication in human cells, before ( ) and after infection ( ). in sars-cov , in silico analysis demonstrated that nar has the potential to inhibit sars-cov- cl pro and consequently inhibit viral replication ( ) , which still needs to be further verified experimentally. the consumption of nar via citrus fruits ( ) or supplementation ( ) can rapidly increase circulating levels of nar and increase intracellular levels of nar ( , ) . an increase in the concentration of nar in plasma samples can be observed min oral consumption and peaking around h post-consumption ( ) . in addition, in vitro models have also demonstrated a long-term anti-viral benefit, even after discontinuation of supplementation with nar ( ), although there is little evidence of in vivo antiviral activity ( ). previous clinical trials with the consumption of ml/day for weeks of orange juice, rich in nar, has demonstrated an adjuvant effect in antiviral therapy ( ). the consumption of ml of grapefruit juice per day (containing approximately mg of nar) also improved cardiac-related measurements in post-menopause women ( ) . although nar is one of the most important naturaly occurring flavonoids, there is a lack of clinical trials and data on pharmacokinetic aspects, metabolic fate, and chemical stability that may limit the usage of this bioactive compound in humans ( ). a caveat of nar is the oral consumption. although widely accepted by patients, it could be a barrier in severe covid- patients. therefore, nar may be better applied as a prophylactic intervention or on the onset of sars-cov- infection. the possible effect of nar on the ace receptor also needs to investigated, as ace reduction could lead to greater inflammation ( , ) . naringenin is mostly absorbed in the small intestine ( ) , and differences in microbiota may thus also present an important inter-individual variable ( , ) . another caveat is the nar poor aqueous solubility and bioavailability; currently, the usage of liposomes, nanoparticles, and other formulations may present itself as a solution ( ) ( ) ( ) ( ) . furthermore, nar interactions with the cytochrome p (cyp) system need to be evaluated, as nar can affect drugmetabolizing enzymes and pharmacokinetic of important drugs that may be of regular use or specific in covid- patients ( ) ( ) ( ) . in conclusion, nar potential as an anti-inflammatory nutritional intervention has been demonstrated in many different diseases, such as sars-cov- and mers-cov. further investigations and clinical trials are needed to help understand the role of nar consumption in humans during a viral infection, especially in sars-cov- infection and covid- . all datasets generated in this study are included in the article/supplementary material. ra: conception, writing, and review. ft: writing, drawing, and review. db, eo, ma, ap, and ms: writing and review. all authors contributed to the article and approved the submitted version. what is covid- ? front young minds covid- : immunopathology and its implications for therapy covid- , ace , and the cardiovascular consequences the citrus flavonoid naringenin confers protection 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nanoparticles for biomedical applications -in vivo toxicological evaluations self-nanoemulsifying drug delivery system (snedds) of the poorly water-soluble grapefruit flavonoid naringenin: design, characterization, in vitro and in vivo evaluation formulation and evaluation of naringenin nanosuspensions for bioavailability enhancement enantiomers of naringenin as pleiotropic, stereoselective inhibitors of cytochrome p isoforms naringenin and interindividual variability in interaction of coumarin with grapefruit juice the fate of naringin in humans: a key to grapefruit juice-drug interactions? the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © alberca, teixeira, beserra, de oliveira, andrade, pietrobon and sato. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -swkcdvx authors: becerra-diaz, mireya; song, mason; heller, nicola title: androgen and androgen receptors as regulators of monocyte and macrophage biology in the healthy and diseased lung date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: swkcdvx androgens, the predominant male sex hormones, drive the development and maintenance of male characteristics by binding to androgen receptor (ar). as androgens are systemically distributed throughout the whole organism, they affect many tissues and cell types in addition to those in male sexual organs. it is now clear that the immune system is a target of androgen action. in the lungs, many immune cells express ars and are responsive to androgens. in this review, we describe the effects of androgens and ars on lung myeloid immune cells—monocytes and macrophages—as they relate to health and disease. in particular, we highlight the effect of androgens on lung diseases, such as asthma, chronic obstructive pulmonary disease and lung fibrosis. we also discuss the therapeutic use of androgens and how circulating androgens correlate with lung disease. in addition to human studies, we also discuss how mouse models have helped to uncover the effect of androgens on monocytes and macrophages in lung disease. although the role of estrogen and other female hormones has been broadly analyzed in the literature, we focus on the new perspectives of androgens as modulators of the immune system that target myeloid cells during lung inflammation. the immune system is essential for maintaining homeostasis within tissues and organs and protecting them against threats, such as harmful pathogens or cancerous transformation ( ) . it comprises both innate and adaptive components. the innate immune system is made up of the innate lymphoid (innate lymphoid cells [ilcs] , natural killer cells [nks] , and lymphoid tissue inducers [lti] ) and innate myeloid subsets ( , ) . the innate immune system consists of a network of immune cells and molecules that provide rapid, first-line defense against pathogens. in contrast, the adaptive immune response, made up of b and t lymphocytes ( ), takes days or even weeks to become established ( ) . innate immune cells express pattern recognition receptors that recognize unique and conserved pathogen-associated molecular patterns such as lipopolysaccharide (lps), viral ssrna, and fungal β-glucan ( ) . b and t cells have evolved to recognize a finer repertoire of self-and nonself-antigens that facilitate pathogen-specific actions, immunologic memory generation, and host immune homeostasis regulation ( ) . to accomplish this, the adaptive immune response involves a tightly regulated interplay between t and b lymphocytes and antigen-presenting cells of the myeloid lineage, such as dendritic cells (dcs), monocytes, and macrophages ( ) . myeloid cells arise from the bone marrow. the type and magnitude of the immune response is influenced by biological sex and age ( ) , and therefore differs between males and females. sex differences in the function of the immune system arise from both genetic (chromosomal) sex differences and differences mediated by the action of male and female sex hormones. because the concentration of sex hormones changes over the lifespan and throughout the course of the menstrual cycle in women, the function of the immune system also changes during different stages of life. innate myeloid immune cells, like other cell types, express sex hormone receptors and are responsive to sex hormones ( ) . sex hormones are synthesized from cholesterol through a defined enzymatic cascade, predominately in the gonads and the adrenal glands ( ) . sex hormones are also produced in other tissues, including the brain, placenta, mammary glands, liver, and adipose tissue ( ) ( ) ( ) . in addition to driving sexual development of egg and sperm production, sex hormones are responsible for the development of male and female secondary sexual characteristics, like breast development and growth of facial hair, that occur during puberty ( ) . androgens include testosterone, dihydrotestosterone (dht), androstenedione, androstenediol, and dehydroepiandrosterone (dhea), with dht being the most potent ( ) . the concentration of androgens in circulation is about seven-fold higher in adult men than in adult women ( , ) . estradiol and progesterone are the predominant female sex hormones ( ) synthesized by the ovaries and adrenal glands. both male and female sex hormones are bound to the plasma proteins, albumin and sex hormone binding globulin (shbg), and only a small percentage exists as free hormone ( - %). thus, the bioavailability of sex hormones is regulated by their biosynthesis and also the amount of albumin and shbg. importantly, sex hormones mediate not only anatomic differences between women and men but also direct sex differences in immune responses, leading to different risks for immunologic diseases ( ) . overall, women have a greater risk for autoimmune diseases (such as systemic sclerosis and systemic lupus erythematosus) ( ) , whereas men are more likely to die of infectious and parasitic diseases ( ) . moreover, men have a greater risk of non-reproductive cancers ( ) ( ) ( ) . both gender and sex are important mediators of these and other health and disease differences observed between men and women. while gender refers to the array of socially constructed roles, attitudes, personality traits, and behaviors, sex represents a biological characteristic of an individual ( ) , including the hormonal milieu and chromosome complement ( ) . in general, estrogens are considered to have proinflammatory properties and androgens are thought to have anti-inflammatory properties ( ) . in the united states ( ) and worldwide ( ) , relevant evidence highlights important epidemiologic sex differences in incidence, susceptibility, and severity of a number of diseases that affect the respiratory tract. in this review, we will focus on how male sex hormones, the androgens, modulate the response of myeloid cells in the lung and how this modulation impacts the outcome of different diseases of the lung. biological sex mediates differences in the incidence and pathophysiology of lung diseases. these differences arise from sex differences in the structure and function of the lung itself, and also in the immune cells that populate the lung and are recruited to it during inflammation. before birth, the female lung has several structural advantages over the male lung. surfactant is produced earlier, and, although the female lung is smaller, it has more alveoli per unit area. neonatal females have higher expiratory flow rates than do male neonates when corrected for size. thus, male sex is a major risk factor for the development of respiratory distress syndrome, bronchopulmonary dysplasia in neonates ( ) ( ) ( ) ( ) , and asthma in childhood ( , ) . in addition to the contribution of structural differences of the lung between the sexes, sex differences in lung function and lung diseases are also dependent on the action of sex hormones. we have summarized some broad concepts that define how testosterone and estrogen affect lung macrophage function and how this may contribute to the outcome of particular lung diseases in figure . as testosterone rises after puberty, the immunosuppressive effects of this hormone on protective immune responses to infectious diseases in males can worsen pulmonary disease. this would be exemplified by tuberculosis or influenza. some of these effects are a result of androgen effects on critical inflammatory macrophage functions although the effects on the adaptive immune system also have a significant contribution to the overall outcome. thus, testosterone appears to play a key immunoregulatory role in lung macrophages. testosterone's immunoregulatory properties also appear to be dependent on the amount of cellular expression of ar and on the concentration of the hormone. low concentrations of testosterone have been noted in patients with asthma, copd, and tuberculosis. low testosterone may also be linked to insufficient control of tissue-damaging inflammatory responses seen in copd and pulmonary fibrosis. estrogen tends to promote wound healing responses in macrophages. dysregulation of wound healing responses and overactive tissue remodeling macrophages in the lung could be broadly used to describe the th response in allergic asthma, which is worse in women. cancer could also be considered an aberrant wound healing response driven by m -like tumor associated macrophages. we have highlighted here how sex hormones contribute to changes in lung macrophage function that contribute to lung disease. however, it should be pointed out that not every sex difference in lung disease is due to direct effects on macrophages but on the broader coordinated immune response as a whole. figure | sex differences in lung diseases discussed in this review and how they may be connected to the effects of androgens (and estrogens) on inflammatory macrophages in the lung. asthma in boys than in girls. with the onset of puberty, male and female sex hormones and their effects on the structural cells of the lung and on the immune system contribute to the incidence of asthma ( , ) . the incidence and severity of asthma are greater in adult women than in adult men ( , ) and greater in female than in male mice ( , ) . female sex hormones, such as estrogen, appear to worsen asthma, although a straightforward correlation between amount of female sex hormone and asthma symptoms has not been concluded. androgens have multiple immunoregulatory and bronchodilatory functions and may contribute to, or be biomarkers for, better lung function in men ( ) . accordingly, serum testosterone is low in men with moderate to severe asthma ( ) ( ) ( ) . in one study, each ng/dl increase in serum testosterone correlated with a % ( % ci, %- %; p = . ) decrease in the likelihood of having asthma ( ) . on the other hand, high concentrations of testosterone and cyclic amp in sputum of asthmatic women during the luteal phase of the menstrual cycle were thought to play a role in premenstrual exacerbations ( ) . the idea that sex hormones may be a causal factor in asthma was significantly strengthened by a recent study of , adults that quantified serum sex hormones and asthma outcomes ( ) . that study showed that low testosterone in both women and men was associated with an increased incidence of asthma. the other interesting finding was that higher testosterone was protective against asthma in obese women. obesity is a risk factor for asthma ( ) ( ) ( ) . therefore, how high body mass index (bmi) and circulating sex hormones together affect asthma requires further investigation. another androgen, dehydroepiandrosterone (dhea), also known as androstenolone, is an endogenous steroid hormone and one of the most abundant circulating steroids in humans. it is a precursor for the synthesis of both testosterone and estrogen. dhea is sulfated at the c β position into dhea-s by the action of the sulfotransferase enzymes sult a and sult e in the adrenal glands. the amount of dhea-s in the circulation is ∼ - times those of dhea. dhea became of interest to the asthma field because women with severe asthma had very low concentrations of dhea-s ( ) and dhea-s concentration correlated with lung function ( ) . interestingly, dhea-s is suppressed by oral or inhaled glucocorticoids, the mainstay therapy for asthma ( ) . human dhea peaks at around age and then follows an age-dependent decline until they reach prepubertal concentrations. reduced secretion of dhea with age has been related to a number of age-associated conditions. replacement of dhea has been considered as a possible therapeutic that could activate protective responses in an aging immune system. dhea is known to downregulate th -inflammatory cytokines while upregulating il- synthesis ( , ) in concanavalin a-stimulated peripheral blood mononuclear cells from adult males with atopic dermatitis ( , ) . thus, it was hypothesized that it would be a useful treatment for atopic diseases including asthma and the results of the clinical trials for dhea in asthma patients show promise. the results are discussed in a later section titled "effects of androgen exposure on monocytes, macrophages in humans with lung disease". sex differences also have been reported in chronic obstructive pulmonary disease (copd), a heterogeneous, chronic, and progressive respiratory disorder that includes chronic bronchitis and emphysema ( ) . chronic exposure of the airways to insults, such as cigarette smoke, leads to epithelial cell injury, destruction of pulmonary capillary vasculature, acceleration of epithelial cell senescence, and airway remodeling. the loss of lung compliance ultimately leads to copd ( , ) . copd was previously thought to affect mostly elderly men, primarily because of the higher prevalence of smoking in men. however, as smoking rates increased in women, the number of copd cases in women exceeded that of men ( ) . these differences are not only based on gender, as women develop more severe copd with earlyonset disease (< years) and have greater susceptibility to copd with lower tobacco exposure ( ) . moreover, increasing age in female smokers leads to a faster annual decline in forced expiratory volume in the first second when compared to that of male smokers, even when they smoke fewer cigarettes ( ) . similarly, pulmonary fibrosis is another lung disease that manifests sex differences ( ) , with men being more affected than women ( , ) . it is characterized by destruction of the pulmonary parenchyma and deposition of extracellular matrix with alterations in phenotype of both fibroblasts and alveolar epithelial cells ( ) . the lungs are also the target of respiratory viruses such as influenza a ("flu"), respiratory syncytial virus, and coronaviruses, such as severe acute respiratory syndrome and the middle east respiratory syndrome. the viruses infect the airway epithelial cells and cause damage to the epithelial barrier by themselves or as a result of the immune response to the viral infection. sex differences have been noted in the immune response to influenza a virus and to the influenza vaccine. in general, women have a more robust protective immune response to influenza virus and vaccine than do men. although this elevated response is helpful in clearing virus, women of reproductive age also experience higher mortality and hospitalizations ( ) ( ) ( ) ( ) ( ) , possibly from collateral tissue damage to the lungs. the vigorous immune response in women also means that women experience more adverse events after vaccination ( ) . indeed, a systems biology approach identified that high testosterone was correlated with a blunted response to the flu vaccine in men ( ) . as testosterone wanes in elderly men, mortality increases ( ) . since the male immune response to the virus is also less robust, the incidence of seasonal flu is generally higher in men than in women in developed countries, according to the world health organization ( ). it is not yet known how fluctuations in sex hormones across the menstrual cycle and lifespan affect the immune system's response to the influenza virus in humans. mouse studies have revealed that estrogen is protective at high, but not low, concentrations ( , ) . on the other hand, testosterone replacement in gonadectomized or aged male mice enhanced survival rates ( ) . despite these findings in mouse models, studies examining the effect of sex hormones on cellular and molecular mechanisms in human immune cells during influenza infection are lacking. like influenza infection, tuberculosis (tb), a lung disease caused by mycobacterium tuberculosis, exhibits notable sex differences in the number of cases worldwide, with men being almost twice as frequently affected than women ( , ) . both sex and gender differences impact the incidence of tb. although tb affects less women than men in adulthood ( ) , women in their economically active years ( - years old) have a higher tb incidence compared to women in other age groups ( ) . this indicates that factors associated with gender, such as exposure to the bacteria, are important in this disease. however, because male predominance does not occur in children, this suggests that biological factors such as male sex hormones also play a significant role ( ) . this is supported by a study of medically castrated men who experienced a significantly smaller proportion of death from tb, . % compared to . % in intact men ( ) . understanding how androgens lead to the greater susceptibility of men to tb is critical, as tb is still one of the leading fatal infectious diseases worldwide and may also may favor the development of other diseases, such as lung cancer ( ) . lung cancer is a very complex disease that depends on a number of variants such as sex, gender, race, and socioeconomic status ( ) . the development of lung cancer is also related to environmental factors, such as pollution due to industrialization and urbanization ( ) . an additional gender-associated risk factor, significantly linked to developing lung cancer, is cigarette smoking ( ) . historically, more men develop lung cancer and suffer lung cancer-associated deaths compared to women ( ) . however, the incidence of lung cancer has changed notably in both women and men. in men, lung cancer incidence started to increase in the s and started to decrease in the early s, while in women, the mortality rates and incidence began to rise in the s ( ) . changes in smoking habits in the last several decades with a rise in the number of women who smoke correlate with an increase in the incidence of lung cancer in this demographic group ( ) . smoking is definitely a key factor in the development of lung cancer; however, recent studies show a higher incidence of lung cancer in young women compared to young men ( , ) , even when the prevalence of cigarette smoking among young women has approached but not exceeded that among men ( ) . this suggests that the higher incidence of lung cancer in women is not explained simply by gender differences in smoking habits: a deeper analysis of differences mediated by sex, such as greater sensitivity to tobacco smoke in women is warranted ( , ) . furthermore, men and women develop different specific types of lung cancer. malignant mesothelioma is more common in men, while women develop more adenocarcinoma ( ), particularly non-small cell lung cancer (nsclc) ( ) . women have a superior survival rate for lung cancer compared to men ( ) . tumor-associated macrophages are critical in tumor progression yet how androgens influence macrophage behavior in lung cancer and in responses to treatment must be addressed more deeply to develop better therapies and increase survival rates in men. the lungs are a primary interface with the external environment. the delicate structures needed for gas exchange make them susceptible to damage from invading pathogens and toxic molecules. some insults to the lung can lead to the development of chronic conditions such as allergic asthma. as a protective mechanism, alveolar macrophages clear the air space of infectious, toxic, or allergenic particles to maintain homeostasis in the alveoli. thus, alveolar macrophages have a dual function as inflammatory cells, phagocytosing and killing inhaled bacteria or viruses, and also as controllers of the inflammatory immune response, minimizing alveolar damage. resident alveolar macrophages are seeded embryonically from yolk sac and fetal liver monocytes ( ) ( ) ( ) . in asthma and other lung diseases, recruited alveolar macrophages derived from blood monocytes can turn into pathogenic cells, worsening the condition ( , ) . mouse alveolar macrophages are characterized by high surface expression of siglec f and produce tgfβ. tgfβ both supports am development ( ) and their maintenance of immune homeostasis by induction of tregs and suppression of b and t cell proliferation. another important function of am is the clearance of surfactant. am from male and female mice respond differently to surfactant protein a (sp-a) ( , ) . sp-a acts as an opsonin and is important in clearance of pathogens. sex differences in am responses to surfactant could affect bacterial clearance and regulate the production of proinflammatory mediators. the molecular mechanisms that mediate these differences and how sex hormones change this important am function is an open question. in the human lung, there appears to be more diversity in the subtypes of lung macrophages compared to mice. the main determinant of the frequency of subtypes of macrophages in humans appears to be their anatomical location within the lung. am are the predominant immune cells in the lung airways (bronchi and bronchioalveolar space). flow cytometric panels have employed hla-dr, cd , cd , and cd to differentiate between am, im and monocytes. human am were identified as large, highly autofluorescent cd -cd + cells that also express cd , cd , and marco ( , ) . there appear to be two populations of am distinguished by either high or low expression of cd . more recent approaches to characterize the macrophage populations in the lung involve single-cell transcriptomic analysis ( , ) . although macrophages show a large variation in the transcriptional phenotype, expression of marco, ccl , apoc , apoe, pparg, and mrc was found in macrophages from healthy donors ( , ), while chi l , marcks, il rn, pla g , mmp , and spp were highly expressed in macrophages from pulmonary fibrosis patients ( ) . thus, a second contributor to diversity is likely the activation state of the cells. there are no data that describe sex differences in human am responses and the effect of sex hormones on these cells. from our mouse and human mdm studies, we would predict that androgens augment the immune homeostatic functions of these cells in the male lung. further work is still needed to standardize characterization of the different subpopulations of human lung macrophage populations and their role in maintaining healthy lung function and in disease. interstitial macrophages (ims), are another macrophage population found in the lung. they are a minor population of monocyte-derived macrophages ( ) , which comprise - % of lung macrophages ( ) and are localized in the lung parenchyma ( ) . ims contribute to maintaining homeostasis through the spontaneous release of il- , a cytokine that dampens inflammation ( ) . ims can prevent the development of aberrant type allergic responses triggered by inhaled allergens ( ) and have been related to reduction of asthma ( , ) . different subpopulations of ims have been found in the lung; however, their characterization has not arrived at a consensus due to difficulties in their identification and isolation. in the mouse lung, different subpopulations of ims have been described based on the expression of surface markers. one report described three different subpopulations of ims based on the differential expression of proinflammatory cytokines, chemokine ligands, mhc-ii, cd c, cd , and lyve- ( ); other group identified two subpopulations, based on similar markers but including cx cr ( ) . moreover, ims subpopulations can be also described based on the different anatomic locations these cells populate inside the mouse lung parenchyma ( ) . further work is needed to better characterize and define the different im populations, as the different subtypes may have different functions during the inflammatory process. smaller in size than their am counterparts, human ims express more of the monocytic marker cd than am, perhaps suggesting their monocytic origin, and have lower expression of cd than human am. the responses of im to androgen will depend on their expression of ar which has not been measured. this will be a challenge due to difficulties in clearly identifying this population (and its subpopulations) from the monocytic, am and other myeloid populations in the lung. monocytes are produced in the bone marrow along with a number of other myeloid cells. myeloid cells originate from common pluripotent hematopoietic stem cells and represent the major subset of white cells in circulation ( ) . these cells comprise basophils, neutrophils, eosinophils, dcs, monocytes, and macrophages, among others ( ) . monocytes are released into circulation, then blood monocytes are recruited into inflamed tissue and can mature into macrophages or dendritic cells. there are two main subsets of mouse monocytes, "classical" or ly c high monocytes that originate directly from ly c + precursors, and "non-classical" or ly c low monocytes that derive from ly c high monocytes ( ). the origin of ly c low monocytes was demonstrated by sunderkotter, et al. by tracking the maturation of dii-labeled ly- c high monocytes into dii-labeled ly c low monocytes ( ) . this process depends on the transcription factor nr a , which regulates the development and survival of ly c low monocytes ( ). these two monocyte subsets mirror the human cd + classical and cd + non-classical monocyte populations, respectively ( ) . ly c high monocytes highly express the chemokine receptor cc-chemokine receptor (ccr ), whereas ly c low monocytes highly express cx cr ( ) . importantly, ccr expression is required for ly c + monocyte egress from the bone marrow into the circulation and entry into noninflamed and inflamed tissues ( ) ( ) ( ) from the blood ( ). as monocytes migrate into tissue, they mature into macrophages developing unique, tissue-dependent morphology and functions ( ) . they lose expression of ly c and gain expression of mhc class ii, becoming more efficient antigen-presenting cells ( ) . some authors have proposed the concept of "tissue monocytes, " which are monocytes that can enter non-lymphoid organs without obligatory differentiation into macrophages. therefore, monocytes are much more than simply precursors for macrophages. in human lungs, monocytes, which can be both beneficial and pathogenic in a variety of pulmonary diseases ( ) , are present at steady state ( ) . multiple-color cytometric analysis on cells obtained from different anatomical locations of the lung of healthy subjects (non-smokers with normal lung function and absence of disease or infection) revealed that while intermediate monocytes (cd + cd + ) are more frequent in the airways, classical monocytes (cd + cd − ) are more frequent in blood ( ) . moreover, the different monocyte subsets produced tnfα to different degrees upon stimulation with tlr ligands ( , , and / ). thus, the anatomic location where samples are obtained should be considered and reported when working with human bronchoscopies, as this may alter the type and abundance of monocytes and macrophages found. accurate identification of monocytes in the lung compartments in humans has been a challenge because monocytic "contamination" from the blood vessels ( , ) . overcoming this challenge, desch et al. performed a flow cytometric phenotyping study and identified two additional lung monocyte populations by analyzing lungs obtained from donors who died of non-pulmonary causes ( ) . cd + cd − cd c − cd a − intravascular monocytes were similar to cd + blood monocytes and cd + cd + cd c − cd a − monocytes were described as tissue "monocytes." these studies highlight that we are just at the beginning of understanding the complexity of lung monocyte subtypes and their functions depending on the inflammatory state of the lung. other myeloid populations, like dcs, occupy the lung parenchyma at steady state, and their relative numbers change during inflammation. we refer readers to previous excellent reviews in this journal that cover the importance of dcs in immune responses in the lung and how they are affected by sex differences. therefore, we will not discuss dcs here ( , ( ) ( ) ( ) ( ) ( ) . polarization is a very important effector characteristic observed in monocytes and macrophages. polarization refers to the change in phenotype and function of monocytes and macrophages as they are exposed to different inflammatory milieus or factors in the tissue microenvironment. to understand the effects of the differing inflammatory or tissue environments on monocyte-macrophage phenotype and function, researchers have used cytokines and other factors in vitro to mimic different inflammatory and tissue microenvironments. monocytes and macrophages stimulated with interferon-γ, lps, tnfα, interleukin (il)- , and granulocyte-macrophage colonystimulating factor promote a pro-inflammatory macrophage phenotype denoted as m polarization. the activation state was also known as "classical" activation. m -polarized macrophages mediate immunity to intracellular infections, such as viruses and bacteria, and they are generally considered tumoricidal ( ) ( ) ( ) ( ) . m macrophages accomplish these functions by inducing production of nitric oxide, reactive nitrogen intermediates, reactive oxygen species, and hydrogen peroxide ( ) ( ) ( ) . in contrast, activation of macrophages with il- or il- , as in extracellular parasitic infections and allergic reactions, leads to m polarization or "alternative" activation of macrophages ( ) . m macrophages produce inflammatory mediators and chemokines, such as chitinase-like proteins ( ), il- ( ) , ccl , ccl , ccl , and ccl , which activate th cells and promote eosinophil infiltration into the lungs ( , ) . in allergic asthma, a th -inflammatory response to inhaled allergens drives lung macrophages toward an m phenotype. increased number and percent of m macrophages have been correlated with asthma severity and a decline in lung function in humans and mouse models ( ) ( ) ( ) . similarly, m macrophages are the predominant subset seen in pulmonary fibrosis and are responsible for fibrogenesis ( ) . during copd, the number of macrophages in airways, lung parenchyma, bronchoalveolar lavage fluid, and sputum increases ( , ) . this increase may occur as a result of enhanced monocyte recruitment from circulation in response to chemokines such as ccl and cxc-chemokine ligand- , which are increased in the sputum and bronchoalveolar lavage fluid of patients with copd ( ) . unlike in allergic asthma and pulmonary fibrosis, macrophages in copd are polarized toward an m profile ( ) . in addition to affecting men and women differently, another commonality of copd is that macrophages both in the alveolar space and in lung tissue present an altered activation phenotype. different concentrations of cytokines (tnf-α, il- β, il- , il- , il- ) and chemokines (ccl , ccl , ccl , ccl , ccl , il- , cxcl , and cxcl ) are found comparing smokers to healthy subjects ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . thus the external provoking stimulus uniquely shapes macrophage phenotype and function. while the m /m designations are useful for in vitro studies with stimulation with defined cytokines, the in vivo phenotype of macrophages exists on a spectrum somewhere in between these two well-defined opposing phenotypes or does not fit the paradigm at all. for example, m and m markers can exist simultaneously within the same cell in some cases ( ) ( ) ( ) . the key factors dictating the macrophage phenotype or activation state are the stage of the immune response and the soluble factors and interactions in a particular tissue microenvironment. for example, the lung environment is rich in gm-csf, tgfβ, and pparγ and is critical for development of mature ams after birth in both mice ( , , ( ) ( ) ( ) ( ) ( ) and humans ( ) ( ) ( ) ( ) ( ) ( ) . furthermore, interactions between cd on type ii alveolar epithelial cells and cd r on the surface of the am deliver regulatory signals to the am to prevent proinflammatory signaling and macrophage activation ( ) . thus, macrophage nomenclature has evolved as our understanding of the phenotypes and functions of different types of tissue resident macrophages, recruited monocytes and monocyte-derived macrophages advances. in-depth studies of the effects of androgens and other sex hormones on tissue macrophage plasticity and phenotype have yet to be carried out. because androgens are lipophilic steroid hormones, they can easily diffuse across cell membranes without the need for receptor-mediated import ( ) . androgens in circulation are found mostly bound to sex hormone-binding globulin and albumin ( ) . free (unbound) steroid sex hormones can signal through two different mechanisms: the classical ar, located in the intracellular compartment, and the membrane, or nonclassical, ar ( ) . androgen binding to classical and nonclassical ars mediates genomic and non-genomic androgen effects, respectively ( ) . upon androgen binding, the classical ar undergoes a conformational change and dissociates from heat-shock and other chaperone proteins. an androgen-ar complex is formed that translocates to the nucleus, dimerizes, and binds to androgen responsive elements that modulate the transcription of target genes ( ) . importantly, it has been reported that the androgen-ar complex can also mediate nongenomic changes ( ) by causing calcium flux and by activating second messenger pathways including erk, akt, and mapk, at least in cell lines ( ) ( ) ( ) . whereas, genomic modulation may need hours or days ( ), non-genomic modulation can occur within seconds to minutes after androgen exposure, does not involve the complex binding to dna, and therefore does not affect transcription of target genes ( ) . dhea has no known unique receptor and is not a direct ar agonist. it affects immune function but, because it can interact with other sex hormones, it has been difficult to establish its mechanisms of action. most studies of androgen-ar complex-mediated gene expression have been carried out in the context of male reproductive tissue in prostate cancer (pca) ( ) ( ) ( ) . as previously discussed, immune cells are responsive to sex hormones, and almost all immune cells express sex hormone receptors ( ) . mouse monocytes, macrophages ( ) , and dcs ( ) express both classical and non-classical ars although the vast majority studies do not specifically dissect the role of the two types of ar on the outcomes being measured in the study. because recent literature has described how sex steroids modulate the functions of dcs ( , , ), we will not discuss it here. we will focus on the importance of androgen-ar regulation of monocyte and macrophage function and how androgen-ars modulate monocytes and macrophages in lung diseases. androgen receptor expression in mouse and human monocytes and macrophages is summarized in table . in general, the expression of the mrna and protein for classical ar has been assessed, often by non-quantitative means, and non-classical ars have not been measured. we have summarized the outcomes of many studies on mouse and human monocyte-macrophages responses in the presence of androgens in figure . in general, monocyte-macrophage exposure to androgen results in a reduction of pro-inflammatory responses (boxed and shaded in green). it is possible that the reduction in inflammation by androgen may be due to ar suppression of estrogen/erα-driven pro-inflammatory responses. ar was demonstrated to inhibit erα activity by binding eres in breast cancer cells ( ) . whether this indirect mechanism accounts for the broad immunosuppressive effects of androgens in normal untransformed immune cells is not known. in keeping with reduced pro-inflammatory responses, we found that androgen enhanced il- -induced m polarization of bone marrow derived and alveolar macrophages in vitro and macrophage-specific deficiency of ar diminished m polarization of lung macrophages in vivo ( ) . in some cases, however, inflammatory responses are increased by androgens (boxed and shaded in red). the different responses may be due to different types of tissue macrophages or experimental system. monocyte-macrophage responses are dependent on the concentration of the hormone, expression of ar, and upon the inducing stimuli to which the macrophage is exposed. the majority of in vitro studies examining the effects of androgens on monocytes and macrophages have not clearly acknowledged or separated the effect of androgen on membrane ars and nonclassical ar signaling from that of classical ars. therefore, we have to assume that the studies described in the section below are a result of classical ar activity unless explicitly investigated or stated. determining how non-classical ar signaling and androgen-independent activation of ar affects monocyte and macrophage function is a gap in our knowledge that must be addressed in future studies. androgens modulate the expression of proinflammatory molecules such as tnfα in mouse monocytes and macrophages. in , lai et al. ( ) demonstrated that lps-induced production of tnfα was decreased in bmm lacking classic ars. moreover, they found that ar, in the presence of dht, induced tnf-α promoter activity ( ) . on the other hand, several reports have suggested the contrary. in one study that used splenic macrophages from midline laparotomy trauma-hemorrhaged mice, dht suppressed tnf-α production from lps-stimulated cells ( ) . this effect was also observed in the mouse macrophage cell line j ( ) , in which testosterone inhibited tnf-α production. in addition, testosterone also decreased expression of the proinflammatory molecule nitric oxide in response to lps in the mouse macrophage cell lines raw . ( ) and j ( ) , but it enhanced the expression of il- in the latter. other molecules important in monocyte-macrophage functions are also affected by androgens. for example, the expression of ccr was enhanced in mouse monocytes by androgens and thereby enhanced chemotaxis ( ) . however, suppressing ar with sirna in prostate cells increased macrophage recruitment via ccl upregulation, which might promote prostate cancer ( ) . phagocytosis was increased by testosterone in rat peritoneal macrophages at − m but not at concentrations lower or higher than − m ( ). cytotoxicity of raw macrophages to the mouse prostate cancer cell line, tramp c , was enhanced by dht alone ( ) . this was attributed to enhanced expression of the m polarization markers, trail and tnf-α, in the macrophages. testosterone ( , , and nm) induced apoptosis in mouse bmm through fas-fasl ( ) and activation of caspase , and poly (adp-ribose) polymerase ( ) . in terms of m polarization of macrophages, we showed recently that in vitro exposure of bmm to dht prior to il- stimulation enhanced chi l and arg gene expression, as well as production of ym ( ) . androgen amplified the m phenotype by increasing il- -mediated m polarization. our results were similar to those found in response to il- in the raw cell line ( ) . this enhanced m macrophage polarization correlated with decreased tlr expression and sensitivity to a tlr -specific ligand observed in testosterone-treated raw cells ( ) . taken together, these observations suggest that androgens and ars can either promote or suppress inflammatory properties of mouse macrophages, depending on the external environmental conditions, ar expression, and concentration of hormone. overall, androgens are more likely to reduce polarization of m macrophages. this could represent an important mechanism by which inflammatory pathways are downregulated in males. the opposite effects seen in different inflammatory contexts highlight the need for a deeper and broader study of the androgen/armediated modulation of monocytes and macrophages, as these cells participate in both the initial and late phases of immune responses in a variety of diseases. most of the studies analyzing the role of ar have focused on prostate cancer, primarily in transformed cell lines ( - ) but macrophages are vital in cancer development and metastasis ( ) . furthermore, it is important to consider that opposing effects could result from differential activation of either classical or non-classical (arindependent) effects ( , ) which have been rarely studied to date. androgens affect a number of key monocyte and macrophage functions. studies of androgen receptor function in human monocytes and macrophages have focused primarily on the roles of male sex and sex hormones in promoting atherosclerotic foam cell formation ( ) and inhibiting cutaneous wound healing ( , ) . foam cells are a type of macrophage localized in the blood vessel walls where they engorge cholesterol ( ). foam cells exhibit enhanced inflammatory cytokine secretion and cause atheroma, contributing to cardiovascular disease ( , ). the effect of androgen on monocytes and macrophages in other immune-mediated human diseases where monocytes and macrophages play a role has been neglected. the degree of ar expression in monocytes and macrophages is likely the primary determinant of responsiveness, although most studies examining responses to androgens do not quantify ar expression (see table ). the expression or action of androgens on non-classical ars in human monocytes and macrophages has yet to be examined carefully. most studies assume that the outcomes that are measured are a result of the activity of classical ar. sex differences in ar content may also play a role in responsiveness. this fact highlights the importance of considering the sex of cells in all in vitro studies to accurately assess how sex hormones affect the responses of monocytes and macrophages. apoptosis was significantly greater in human thp- cells cultured for days with nm testosterone than in control cells or cells treated with estradiol (e ), owing to a reduction in proliferating cell nuclear antigen, induction of poly-adp ribose polymerase-cleaved, an increase in iκb-α, and a decrease in phosphorylated iκb-α ( ) . e , in contrast, promoted cell survival. other studies noted concentration-and time-dependent regulation of apoptosis in thp- cells, with an increase in the proto-oncogene bax and fas ( ) . androgen exposure inhibited proliferation of the human monoblastic leukemia cell line u , depending on the concentration and time of exposure ( ) . cell cycle arrest occurred at the g /m phase, although another study measured no effect of testosterone on pma-differentiated u cells ( ) . how testosterone regulates apoptosis and survival of untransformed primary human monocytes and mdms has not been well-studied. toxicity was observed when monocytes were differentiated into macrophages over days in the presence of . mg/ml androgen, but not at lower concentrations of the hormone ( ) . testosterone reduced the viability of monocytes from a healthy control and a patient with systemic lupus erythematosus in a concentration-dependent fashion ( , ) . these two studies highlight the importance of concentration in studies of sex hormones. an additional example is the finding that e enhances tnf-α secretion from antigen-stimulated t-cells at low concentrations and inhibits secretion at high concentrations ( ) . il- β-induced nf-κb activation is also inhibited at high but not at low e concentrations ( ) . hence, it is important to carry out in vitro studies of sex hormone responses over a wide range of physiologic concentrations of sex hormones. in general, androgens have a suppressive effect on proinflammatory cytokine expression in monocytes and mdms. this finding is consistent with the idea that the immune system of females produces cytokines in response to pathogens and insults more robustly than that of males. monocyte or mdm expression of tnf-α, il- β, il- , and il- is reduced in the presence of testosterone ( ) ( ) ( ) . many studies in this field have relied on human cell lines, such as thp- and u , with or without pma-induced differentiation into macrophages, and differentiated hl- cells, although primary monocytes and mdms have been used in a few cases ( , ) . another immunoregulatory function of testosterone is the upregulation and secretion of c inhibitor (c inh) from monocytes ( ) . c inh is a kda plasma protein whose main function is inhibition of the complement system to prevent spontaneous activation. thus, testosterone keeps complement activation in check. another mechanism by which testosterone limits inflammation is by decreasing the generation of reactive oxygen species generation from differentiated hl- cells. interestingly, the production of reactive oxygen species in response to zymosan, but not lps, was inhibited by testosterone ( ) . in terms of allergic immune responses, metabolism of arachidonic acid into inflammatory leukotrienes (lts) via the -lipoxygenase ( -lo) pathway is sex-dependent in human monocytes. pergola et al. ( ) reported that primary human peripheral blood monocytes from women synthesize more -lo product than do the same cells from men. α-dht ( nm) suppressed lt synthesis in female cells to the levels observed in males. erk activation by androgens reduced phospholipase d activity in monocytes and impaired -lo product formation by reducing active diacylglycerides. the other branch of arachidonic acid metabolism is the cyclooxygenase (cox) pathway, which generates prostaglandins. prostaglandin e (pge ), one of the most abundant cox products produced by the airway epithelium and smooth muscle ( , ) , can either stimulate or suppress immune cell function. testosterone reduced pge production in monocytes obtained from heparinized peripheral blood of healthy adults and incubated for h with lps ( ) . a few studies have examined the effect of dhea on human monocytes and macrophages. in the presence of lps, dhea induced il- and tnf-α production by primary human monocytes and il- and tnf-α production by thp- cells ( ) . in these experiments, dhea counteracted the effects of cortisol and the glucocorticoid receptor on lps-induced il- and tnf-α by inducing expression of the scaffolding protein rack (receptor for activated c kinase ) in thp- cells and primary human monocytes ( ) . rack is involved in multiple signal transduction cascades, including the mapk, protein kinase c, and src signaling pathways. rack shuttles proteins around the cell, anchors proteins at particular locations, and is involved in cell migration ( ) . in contrast, dhea added to alveolar macrophages lavaged from non-smoking asbestos workers significantly reduced superoxide anion release in vitro ( ) , consistent with its role in dampening th inflammation ( ) . therefore, the effect of dhea on monocytes and macrophages may be stimulus-dependent and needs more in-depth investigation. the formation of foam cells (lipid-filled macrophages) is generally associated with the pathogenesis of cardiovascular diseases, such as atherosclerosis. however, foam cells are also found in patients with silicosis ( ) and other fibrotic lung diseases ( ) and in tuberculosis. alveolar macrophages take up extracellular and intracellular lipids in response to inhaled silica, vaping products ( ) , and mycobacterium tuberculosis ( ) . furthermore, the metabolism of fatty acids by macrophages by β-oxidation for sustained energy production is a key feature of the functional phenotype of macrophages with a pro-resolving, tissue reparative (m ) phenotype. therefore, we have included how androgens modulate foam cell formation and lipid handling in macrophages as part of this discussion. macrophages from men and those exposed to testosterone favor the processes of lipid handling and foam cell formation, supporting evidence that atherosclerosis is a male-dominant disease when age is taken into account ( ) . atherosclerotic plaques composed of a number of different immune cells form in blood vessel walls. in advanced stages of atherosclerosis, macrophages in plaques take up oxidized low-density lipoprotein (ldl), creating foam cells. eventually, cholesterol crystals accumulate, trigger inflammation and plaque rupture. the role of sex in the inflammatory events of atherosclerosis has been reviewed elsewhere ( ) . in vitro studies have sought to ascertain how testosterone promotes these processes by utilizing primary mdms. in mdms from healthy men, androgen treatment was shown to upregulate genes involved with lipoprotein processing, transporter proteins, cell-surface adhesion, and other pathways, but none of these genes were upregulated in female macrophages ( ) . the marked sex specificity of androgen effects on human macrophage gene expression is most likely related to sex differences in mdm ar expression. similarly, treatment of mdms with modified and native ldl led to changes in expression of mrnas involved in homeostatic regulation of lipid metabolism, depending on the sex of macrophage donors ( ) . functionally, androgen-treated mdms from men but not women accumulate cholesteryl esters ( ) . male macrophages exhibit increased rates of lysosomal acetylated ldl degradation and upregulated expression of scavenger receptor class b type i ( ), increasing high-density lipoprotein ( )-induced cholesterol efflux. the expression of ar in monocytes/macrophages also upregulates lectin-type oxidized ldl receptor molecules that are involved in foam cell formation ( ) . however, corcoran et al. ( ) observed no effect of testosterone on cholesterol content or efflux from mdms of healthy male and postmenopausal female donors (age - years). because their study used healthy donors, it is possible that the absence of other health-related factors, such as smoking, poor health, and genetic risk factors for coronary heart disease in the healthy blood donors may have produced these results. chemotaxis of thp- cells was diminished when androgen receptor was knocked down by sirna suggesting a role for ar in migration of monocytes ( ) . the authors identified tnf-α as a key ar-regulated molecule important in monocyte migration. in contrast, a handful of studies have tested the effect of testosterone on primary human monocyte phagocytosis and migration, but no effect was found ( , ( ) ( ) ( ) . testosterone did not change the cytotoxic capacity of monocytes from male donors (age range - years) to lyse red cells sensitized with igg antibodies ( ) . most studies that have used mouse models to investigate sex differences in lung diseases have focused on the role of estrogen and estrogen receptors ( ) ( ) ( ) . the importance of androgen and ars in lung disease has been poorly studied. earlier studies were directed at modulating monocyte and macrophage functions unconnected to ar function, as years ago it was believed that mouse macrophages did not express classical ars ( ) . nevertheless, recent studies have examined sex differences in mouse models of allergic asthma, copd, and influenza. we and others have reported sex differences in mouse models of allergic lung inflammation ( , , , ) . some of the observed differences have been clearly attributed to the effect of androgens. we showed that dht reconstitution of castrated male mice reduced overall lung inflammation ( ) . a reduction of total serum ige and total immune cell recruitment to the lungs, specifically eosinophils, revealed the regulatory effect of androgens on several cell types. however, the unexpected enhancement of the production of the canonical m macrophage marker involved in eosinophil recruitment ( , ) , ym , by dht in alveolar macrophages ( ) showed that androgens have a regulatory or an activating effect depending on the cell type. we demonstrated that deletion of classical ars on monocytes and macrophages (ar flox lysmcre mice) resulted in reduced inflammation (less eosinophil recruitment to the alveolar space), along with less mucus production and lung cell infiltrate, despite no differences in serum testosterone level between arsufficient and ar flox lysmcre mice ( ) . this finding indicates the importance of androgens as modulators of m macrophage polarization and the critical role of these cells in allergic lung inflammation. other recent studies have shown that testosterone has an anti-inflammatory role in a mouse model of allergic lung inflammation induced by house dust mite but focused on other cell types in lung, such as th ( ) and ilc cells ( , ) . similarly, high concentrations of androgens in circulation have been related to a decrease in the expression of tnfα and other proinflammatory cytokines, such as il- and il- β, in rodent macrophage cell models and in human monocytes ( , , , , ) . how androgen and ars impact functions on ims still needs to be studied. at the time this review was written, no reports on ar expression in ims were found. however, we hypothesize that as ims are derived from blood monocytes ( ) , but once in the tissue they develop an intermediate size and phenotype between monocytes and am ( , ) , their expression of ar could be somewhere in between. therefore, androgen and ars could regulate the functions and activation of these cells. this requires further study, as ims are a constitutive macrophage population in the lung, and may play a role mediating sex differences in lung diseases. mouse models have also shown that sex differences affect copd. in , tam et al. ( ) reported that smokeinduced copd is characterized by small airway remodeling in female but not male mice and that ovariectomy before smoke exposure ameliorates the disease. another study focusing on α- antitrypsin deficiency, the leading genetic cause of emphysema, also uncovered a higher susceptibility of female mice for this condition ( ) . however, these studies did not determine if androgens mediate resistance to copd, or if the key to the observed sex differences is ovarian sex hormones. thus, the role that androgens play in copd and copd models remains unclear. mouse studies that have focused on sex differences in influenza showed that at moderate influenza virus a (iav) loads, morbidity, mortality, and the associated inflammatory response is greater in female than in male mice, but that mortality is similar at higher loads ( , , ) . the role of sex hormones was well-addressed in these studies. high levels of estrogen in estrogen-reconstituted female mice protected against lethal iav doses ( ), whereas the lower estrogen levels in intact females were associated with greater inflammatory responses and increased morbidity after infection. similar observations were made after progesterone replacement ( ) . in males, a decrease in androgen levels after castration increased morbidity and pathology upon iav infection, but replacement of testosterone or dht reduced morbidity, mortality, and inflammation ( , ) . these findings suggest that although estrogen may be protective or detrimental, depending on concentration, androgens may suppress inflammation in a broader way. gonadectomy studies in mice have been used to uncover the role of androgens in tb. similar to observations in castrated men, castrated male mice that displayed greater pro-inflammatory responses in the lung (more tnf-α, ifn γ, il- , inos, and il- ) than intact males. ifn-γ-activated macrophages (m macrophages) control of tb infection in both human and mouse ( ) . ovary removal in females did not impact susceptibility to tb ( ) , suggesting that testosterone is responsible for male susceptibility to tb. we previously reported that dht enhances m macrophage polarization through ar ( ) . therefore, we speculate that the greater male susceptibility to tb could be at least in part mediated by enhanced m responses that are poorly protective and decrease protective proinflammatory macrophage responses. formal studies to address this idea as well as how androgen effects on other key immune cell players in tb are needed. how androgens affect monocyte and macrophage biology in lung cancer models in mice has not been well-studied. monocytes and macrophages are important cellular players in tumorigenesis. tumor-associated macrophages (tams) can be classified into two phenotypes that are either pro-inflammatory and tumoricidal (m -like) or promote tumor growth and suppress anti-tumor immune responses (m -like) ( ) ( ) ( ) . as mentioned previously, sex hormones augment m macrophage polarization, thus, play an important role in lung carcinogenesis. the greater overall incidence of lung cancer in men could be explained by an enhanced m polarization by androgens ( ) . on the other hand, estrogen has been shown to induce tumor angiogenesis ( ) . estrogen signaling though the camp, mapk, and akt pathways with the consequent phosphorylation of erk and egfr signaling, along with the enhanced expression of cmyc and cyclin d, results in nsclc cell proliferation ( ) . mouse models must therefore address the role of androgens on monocytes and macrophage function in the establishment and progression of lung cancer in male and female animals. few studies have examined the effect of sex hormones on peripheral blood monocytes and lung macrophages from men and women with asthma or the other lung diseases we have discussed here. in women with asthma, dominance of m macrophages in airways and lung tissue has been documented ( ) and a connection between female sex and female sex hormones surmised. there is a paucity of literature regarding how introducing or depleting exogenous sex hormones (such as in female-to-male transgender individuals receiving testosterone supplementation or women with estrogen blockade) affects the function of blood monocytes and lung macrophages in men and women with asthma. most studies correlate concentrations of sex hormones with either inflammatory markers, such as cytokines or chemokines in serum and other fluids, or with lung function measurements. we will summarize below the small number of studies in which androgen concentrations were manipulated in humans and the effects on monocyte or macrophage function. hypogonadism in men refers to a deficiency in testosterone production from the testes that results from testicular, hypothalamic, or pituitary abnormalities. klinefelter's syndrome in men, which is a result of additional x-chromosomes (e.g., xxy), is the most common cause of hypogonadism. testosterone replacement therapy is the primary treatment option to restore physiologic testosterone levels, typically in the range of to ng/dl. in general, exogenously administered testosterone has a suppressive effect on the proinflammatory immune response from monocytes. for example, spontaneous production of proinflammatory cytokines (il- β, il- , and tnfα) ex vivo was reduced or completely absent in the monocytes and dcs from men with type- diabetes who had partial androgen deficiency and were treated for months with testosterone replacement. this suppression was maintained for more months after testosterone withdrawal ( ) . testosterone replacement therapy also is associated with a reduction or complete abrogation of spontaneous ex vivo production of inflammatory cytokines by antigen-presenting cells ( ) . on the other hand, the circulating monocytes from hypogonadal men treated with testosterone replacement therapy exhibited significantly upregulated expression of cd b at baseline compared to monocytes from healthy controls. this was also seen after stimulation with cpg oligodeoxynucleotides to mimic bacterial dna exposure ( ) . membrane expression of cd b, also known as lysosome-associated membrane protein (lamp) , is indicative of release of lysosome and/or phagolysosome contents into the extracellular medium, a mechanism that may be involved in killing and/or digesting target cells. these data suggest that testosterone increases the inflammatory function of these cells, an effect that would contrast with its typical role as an immunosuppressant. the immune system of individuals with klinefelter's syndrome provides unique insight into the genetic contribution of the x-chromosome and that of diminished testosterone to sex differences in different diseases. men with klinefelter's syndrome have an increased risk of developing autoimmune diseases, particularly those that are typically female-dominant, such as rheumatoid arthritis and systemic lupus erythematosus ( ) . as might be predicted due to the negative effect of lower concentration of testosterone on lung function, men with klinefelter's syndrome are more likely to be diagnosed with pulmonary diseases, such as copd and pneumonia ( ) . asthma is also reported in these individuals ( ) ( ) ( ) and it was successfully controlled with long-acting β-agonists and oral testosterone replacement in one case report ( ) . at the cellular level, however, cytokine production in stimulated whole blood from klinefelter's men was similar to that of women ( ) . these data suggest that the effect of the additional x-chromosome was more dominant than the reduction in circulating androgen in klinefelter's men. in the same study, however, purified monocytes showed the opposite response: cytokine production from the monocytes of healthy and klinefelter's men was similar and less robust than that from the monocytes of women. this observation led to the opposite conclusion-that androgen plays a more important role in monocyte cytokine production than does chromosomal complement. pcos is a disease characterized by hyperandrogenism, amenorrhea, and polycystic ovaries. the cystic folliclesovarian theca cells-produce testosterone that causes significant elevations in serum concentrations of testosterone, androstenedione, dhea, and dhea-s. in women with pcos, serum testosterone is in the range of - ng/dl ( - nmol/l) ( ), compared with a range of - ng/dl in healthy, ovulatory women ( ) . this endocrinopathy is associated with metabolic disorders, such as dyslipidemia, insulin resistance, metabolic syndrome, and cardiovascular complications. immune function is impaired in women with pcos, leading to increased secretion of autoantibodies and increased risk of type diabetes, asthma, and thyroid disease ( ) . because androgens downregulate the inflammatory responses that contribute to asthma, one might hypothesize that women with pcos would have less asthma. however, htet al. ( ) found that asthma prevalence was . % in women with pcos compared to only . % in women without pcos (p = . ). women both with and without pcos who had asthma tended to have a higher bmi than those without asthma ( ) . after multivariable analysis, the authors concluded that both pcos and high bmi were independently associated with asthma ( ) . it is therefore possible that testosterone contributes to the chronic inflammatory state that accompanies high bmi and that the metabolic dysfunction overpowers the protective effects of testosterone on asthma development. few cellular and molecular studies have endeavored to uncover mechanisms that explain the association between asthma and pcos. at the cellular level, circulating monocytes from women with pcos expressed the receptor for advanced glycation endproducts (rage) more strongly than monocytes from healthy control women ( ) . ages are involved in the pathogenesis of a number of chronic lung diseases, ranging from cystic fibrosis to asthma. rage can also bind other alarmins, such as the s a /a heterodimer (calprotectin) or the high-mobility group box (hmgb) protein. both of these ligands have been implicated in the pathogenesis of allergic asthma ( , ) , as they induce cell proliferation or apoptosis, inflammation, collagen synthesis, and cell migration in many different cell types. the concentration of age proteins and testosterone correlated positively, even after controlling for bmi and other metabolic function tests ( ) . taken together these two studies suggest that monocytes from women with pcos would be more responsive to rage ligands. this heightened responsiveness could promote cellular inflammatory responses that contribute to asthma pathogenesis. studies are needed to examine how the increased testosterone in women with pcos affects circulating monocytes and lung macrophages to increase asthma prevalence in this group. testosterone has been administered therapeutically for asthma. in an early study, asthmatic women were given testosterone either daily for days over weeks or daily for days over or more weeks. although the number of participants in the study was small, % saw improved symptoms, with % reporting no asthma attacks up to months later ( , ) . no studies have examined the effect of exogeneous testosterone administration on blood monocytes or lung macrophages in men and women with asthma. testosterone deficiency is also present in patients with copd ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . in a clinical study of exercise and testosterone injection in men with copd, the interventions did not significantly alter pulmonary function or blood gas variables ( ) . on the other hand, a retrospective study of two large cohorts of men who commenced testosterone replacement therapy within months of a copd diagnosis showed a . - . % decrease in hospitalizations, dependent on age ( ) . more work is needed to understand how testosterone and its signaling pathways can be harnessed to alleviate lung disease without affecting reproductive systems or having unwanted metabolic effects. asthmatic patients have decreased serum concentration of dhea and dhea-s ( ) ( ) ( ) . therefore, some clinical trials have tested whether dhea-s supplementation reduces asthma. men and women with poorly controlled moderate-to-severe asthma were given nebulized dhea-s for weeks. this treatment led to a statistically significant improvement in the asthma control questionnaire (acq) and trends toward better asthma symptom scores and more symptom-free days and nights ( ) . oral dhea for weeks improved lung function in asthmatic women with low dhea-s < µg/dl ( ) . however, neither of these clinical studies examined the cellular component of the disease pre-or post-intervention. dhea and dhea-s are also lower in patients with copd than in healthy controls, and copd leads to pulmonary hypertension (ph). dhea supplementation improved the -min walk test, pulmonary hemodynamics, and the diffusing capacity of the lungs for carbon monoxide of patients with ph-copd ( , ) . the therapeutic potential of dhea is currently being investigated in patients with ph in the ediphy (effects of dhea in pulmonary hypertension) trial. however, outcome measures of this trial do not include examination of the immune cells or the effect of dhea treatment on those cells. analysis of immune cell function would add important cellular mechanistic insight to these types of trials and help uncover some of the widespread effects of this hormone on the immune system. modulation of monocyte and macrophage function mediated by the interaction of androgen and ar has been examined mostly by correlative studies in humans following lifespan changes in sex hormones or using hormonal manipulation in mouse models of lung disease. most human-based reports are merely descriptive or correlative and do not consider variables such as age, bmi, and phase of the menstrual cycle as key modulators of circulating sex hormone concentrations. taking these factors into account should be encouraged if we are to gain a better understanding of the impact of sex hormones in health and disease. analyses of the function of immune cells from male and female healthy controls and patients with lung diseases are needed to unlock how sex hormones alter the biology of the innate and adaptive immune response. studying the role of sex hormones as modulators of the immune system is complex because they interact with other hormonal systems and with one another, and because of the nearly ubiquitous expression of sex hormone receptors in most cells of the body. males and females have all types of sex steroids, although in different circulating concentrations. in humans, changes in the concentration of sex steroids have implications for lung health and may contribute to disease by affecting the function of the immune system. female sex hormones have been more widely studied as immune system modulators than have androgens. more focus in the future must be directed to how androgens affect the immune system and the interaction between male and female sex steroids in immune function. historically, animal models have used only males as study subjects, leaving females aside out of concern for the variability in results introduced by sexually mature adult females with active estrous cycles. as a result, biomedical and preclinical research has neglected to reflect more than % of the world's population. this omission had some notable negative consequences: eight of ten drugs withdrawn by the fda between and had significant health risks to women ( ) . it was not until that the nih addressed this oversight with its requirement to include sex as a biological variable in all research studies ( ) . the practices of using only male animals, not clearly reporting the sex (and age) of the animals used, and mixing male and female results have obscured a proper understanding of how sex and sex hormones influence normal biology and that of disease states. moreover, many reports comparing sex as a variable lack strict controls on culture conditions in vitro, which can alter the results. for example, if investigators fail to appreciate that animal serum or ph indicators, such as phenol red, may act as a source of steroids or sex hormone receptor agonists and do not clearly report their use, the interpretation and reproducibility of the experiments can be diminished. we strongly advocate for the use of hormone-free serum or animal serum replacements (for human cell studies) and use of culture medium that does not contain sex steroid receptor agonists. moreover, rigorous experimentation should include careful and detailed reporting of cell culture conditions, donor sex and age for cell studies, accurate age and sex in animal work (adherence to arrive guidelines), and separate male and female results. here, we have highlighted the importance of sex hormones as modulators of monocytes and macrophages and the important role of these innate immune cells in lung diseases where sex differences are apparent. these cells are part of a larger response that includes the adaptive immune system as well as the structural cells of the lung that are all affected by the action of sex steroids. as such, how innate cells like monocytes and macrophages shape the pulmonary immune response and how they resolve lung inflammation differently in the male and female lung and in the presence of different sex steroids needs intensive study. uncovering the cellular and molecular mechanisms will be crucial for finding new ways to treat different lung diseases depending on the sex of the patient. mb-d, ms, and nh wrote and revised the manuscript, interpreted the literature, approved and are accountable for all aspects of the final version. all authors contributed to the article and approved the submitted version. funding nih (nhlbi) r hl (to nh). dept of defense cdmrp w xwh- - - (to nh). overview 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results of a -week, randomized, double-blind, placebo-controlled study dhea) improves pulmonary hypertension in chronic obstructive pulmonary disease (copd): a pilot study association of adrenal hormone metabolites and mortality over a -year follow-up in copd patients with acute exacerbation sex as an important biological variable in biomedical research sex as a biological variable: a -year progress report and call to action key: cord- -aqcllik authors: diao, bo; wang, chenhui; tan, yingjun; chen, xiewan; liu, ying; ning, lifen; chen, li; li, min; liu, yueping; wang, gang; yuan, zilin; feng, zeqing; zhang, yi; wu, yuzhang; chen, yongwen title: reduction and functional exhaustion of t cells in patients with coronavirus disease (covid- ) date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: aqcllik background: the outbreak of coronavirus disease (covid- ) caused by severe acute respiratory syndrome coronavirus (sars-cov- ) has posed great threat to human health. t cells play a critical role in antiviral immunity but their numbers and functional state in covid- patients remain largely unclear. methods: we retrospectively reviewed the counts of t cells and serum cytokine concentration from data of patients with laboratory-confirmed covid- and healthy controls. in addition, the expression of t cell exhaustion markers were measured in covid- cases. results: the number of total t cells, cd (+) and cd (+) t cells were dramatically reduced in covid- patients, especially in patients requiring intensive care unit (icu) care. counts of total t cells, cd (+) t cells or cd (+) t cells lower than , , or /μl, respectively, were negatively correlated with patient survival. t cell numbers were negatively correlated to serum il- , il- , and tnf-α concentration, with patients in the disease resolution period showing reduced il- , il- , and tnf-α concentrations and restored t cell counts. t cells from covid- patients had significantly higher levels of the exhausted marker pd- . increasing pd- and tim- expression on t cells was seen as patients progressed from prodromal to overtly symptomatic stages. conclusions: t cell counts are reduced significantly in covid- patients, and the surviving t cells appear functionally exhausted. non-icu patients with total t cells counts lower than /μl may still require urgent intervention, even in the immediate absence of more severe symptoms due to a high risk for further deterioration in condition. in december , a series of acute respiratory illnesses were reported in wuhan, hubei province, china ( , ) . a novel coronavirus, initially named severe acute respiratory syndrome coronavirus (sars-cov- ), was identified as the cause of this disease by the chinese center for disease control and prevention (cdc) ( ) . this disease, now designated as coronavirus disease (covid- ) by the who, rapidly spread to other cities of china, and has become a public health emergency of international concern (pheic) following its global spread. covid- clinically manifests as fever, cough, muscle pain, fatigue, diarrhea and pneumonia, and can cause death in severe cases ( ) ( ) ( ) . up through march , , china has reported cases of confirmed covid- and , fatalities ( ) . since an effective immune response against viral infections depends on the activation of cytotoxic t cells that can clear infection by killing virus-infected cells ( ) , boosting the numbers and function of t cells in covid- patients is critical for successful recovery. a recent study reported that the . % of covid- cases displayed low circulating lymphocyte counts ( ) ( ) ( ) . however, the factors which might cause the reduction in count, and the activation status of t cells in covid- patients, remain uninvestigated. we retrospectively analyze here the clinical data from cases of covid- who were admitted into the general hospital of central theater command and hanyang hospital in wuhan from december to january . we also compare the expression of exhaustion markers pd- and tim- on the surface of cd + and cd + t cells from covid- patients and healthy controls. our results thus provide a preliminary demonstration of t cell exhaustion during covid- infection and suggest that more urgent, early intervention may be required in patients with low t lymphocyte counts. medical records from patients (aged from days to years) with confirmed covid- and admitted into the general hospital of central theater command or hanyang hospital in wuhan from december to january , and healthy people (aged from to years), who came to the hospitals for routine physical examination, were collected and retrospectively analyzed. diagnosis of covid- was based on the new coronavirus pneumonia prevention and control program ( th edition) published by the national health commission of china ( ) . all the patients were laboratory-confirmed positive for sars-cov- by use of quantitative rt-pcr (qrt-pcr) of throat swab samples. this study was approved by the national health commission of china and ethics commission of general hospital of central theater command ([ ]- - ) and hanyang hospital ( ). written informed consent was waived by the ethics commission of the designated hospital for emerging infectious diseases. the classification of clinical types, which consist of mild/moderate/severe/critical, was based on the new coronavirus pneumonia prevention and control program ( th edition) published by the national health commission of china ( ) . within the analyzed cohort, patients were admitted to the intensive care unit (icu), because they required high-flow nasal cannula or higher-level oxygen support measures to correct hypoxaemia. hypoxaemia was defined as arterial oxygen tension (pao ) over inspiratory oxygen fraction (fio ) of < mm hg or arterial oxygen saturation of % or lower. according to the staging of infectious disease ( ), the prodromal period is a phase in which the host begins to experience general signs and symptoms. the illness period (overtly symptomatic period) is a phase in which the signs or symptoms of disease are most obvious and severe, with positive laboratory findings and chest/lung pathological manifestations. for icu patients, icu period is a phase in which the symptoms are most obvious and severe. the decline period is a phase in which the clinical symptoms begin to decline, laboratory findings and chest pathological signs improve, and arterial oxygen saturation normalizes. we reviewed clinical records, nursing records, laboratory findings, and chest x-rays or ct scans for all the patients and physical examination records of the healthy people. all information was obtained and curated with a customized data collection form. three investigators (c wang, z fen, and y chen) independently reviewed the data collection forms to verify data accuracy. peripheral blood samples from patients and healthy volunteers were simultaneously processed in the central lab of general hospital of central theater command to isolate peripheral blood mononuclear cells (pbmcs) for further testing. the peripheral blood was supplemented with anticoagulants (edta-k ) and pbmcs were harvested by density gradient centrifugation. isolated pbmcs were stained with a bd multitest imk kit (cat , bd biosciences) to analyze the frequency and cell number of total, cd + and cd + t cells, as well as b and nk cells in healthy controls and patients. the exhaustion of t cells was detected using human cd -percp (rpa-t , biolegend), cd -apc (sk , bd biosciences), cd -pe (sk , biolegend), pd- -pe (eh . h , biolegend), and tim- -fitc (f - e , biolegend) antibodies. after being stained, the cells were measured by flow cytometry on an lsr fortessa cell analyzer (bd biosciences) and data analyzed using the flowjo software (treestar). all experimental procedures were completed under biosafety level ii plus condition. statistical analyses were performed using graphpad prism version . (graphpad software, inc., san diego, ca, usa). the funding agencies did not participate in study design, data collection, data analysis, or manuscript writing. the corresponding authors were responsible for all aspects of the study to ensure that issues related to the accuracy or integrity of any part of the work were properly investigated and resolved. the final version was approved by all authors. from our retrospective analysis of patients, cases had lymphocyte count recorded. . % ( / ), . % ( / ), and . % ( / ) patients had remarkably low total t cell counts, cd + and cd + t cell counts, respectively. among milder disease patients in the non-icu group, the median value of total t cells, cd + and cd + t cell counts were , , and , respectively; the median value decreased to , , and . , respectively, in the icu group ( figure a) . the counts of total t cells, cd + , and cd + t cells were significantly lower in icu patients than non-icu cases ( figure b) . all these patients were further categorized into three groups based on age (< years old, - years and ≥ years), and an age-dependent reduction of t cell numbers was observed in covid- patients, with the lowest t cells numbers found in patients ≥ years old (figure c) , suggesting a potential cause for increased susceptibility in elderly patients. it is worth noting that the age range of the icu patients was - years [ . ( - . ), median (iqr)], suggesting that some young patients can become critically ill. we next retrospectively reviewed t cell numbers in cases from non-icu patients within one center (the general hospital of central theater command). the non-icu patients were further divided into four groups based on clinical outcomes. among these patients, , and cases had mild/moderate, severe and critical disease, respectively, while patient deaths occurred, in the perished group. statistical analysis showed that t cell numbers including total t cells, cd + and cd + t cells in the severe and critical disease groups as well as the perished group were significantly lower than in the mild/moderate disease group. most importantly, the numbers of total t cells, cd + t cells and cd + t cells in severe and perished groups were lower than , , or /µl, respectively ( figure d ). this result suggests that there is a profound t cell loss in covid- disease. the expression of angiotensin converting enzyme (ace ), the predicted receptor of sars-cov- viruses, is absent on t cells ( ) , suggesting that the depressed t counts in covid- patients mentioned above (figure ) were likely not caused by direct infection of t cells. we therefore examined the concentrations of serum cytokines, including tnf-α, ifn-γ, il- , il- , il- , and il- , from these covid- patients to explore the influence of cytokine signaling. we only found the levels of tnf-α, il- , and il- were significantly increased in infected patients, and statistical analysis illustrated that their levels in icu patients were significantly higher than in non-icu patients (figure a) . it is also worth noting that cytokine levels frontiers in immunology | www.frontiersin.org of some icu patients were relatively normal, suggesting that a small minority of icu patients were immunocompromised. the levels of ifn-γ, il- , and il- showed no significant difference among different groups (supplementary figure a) . we next investigated the relationships between il- , il- , tnf-α, and t cell count within non-icu patients. interestingly, the concentration of these three cytokines was negatively correlated with total t cell counts, cd + counts, and cd + counts, respectively ( figure b) . we subsequently summarized the follow-up data of cytokine concentrations and t cell numbers in ten patients that were followed over the course of inpatient care. interestingly, serum levels of ifn-γ, il- , il- , and tnf-α were significantly decreased in these patients in the decline (i.e., disease resolution) compared to the illness period, while counts of total t cells, cd + , and cd + t cell subsets recovered during the decline period ( figure c) . we also noted that serum levels of il- and il- had no difference between these two periods (supplementary figure b) . the phenomena suggests that the decrease of t cells seen in covid- patients may be the result of high serum concentration of tnf-α, il- , and il- negatively regulating t cell survival or proliferation. beyond changing in numbers during the course of infection, t cells may display limited function during prolonged infection as a result of exhaustion, which has been associated with the expression of some immune-inhibitory factors including pd- , tim- on cell surface ( ) . we therefore examined whether t cells in covid- patients have exhaustion phenotypes. facs analysis illustrated that, in comparison to healthy controls, especially icu patients with covid- disease showed markedly higher percentages of pd- + cd + and cd + t cells, with a trend to show higher pd- levels on pd- + cd + or cd + t cells (figures a,b) , indicating that sars-cov- can drive t cell exhaustion in covid- patients, particularly in those requiring icu care. three patients were followed during inpatient care, and the expression of the exhaustion markers including pd- and tim- on surface of t cells during disease progress was detected. facs analysis showed that these patients had very low percentages of pd- + and tim- + on cd + and cd + t cells in the prodromal stage of disease, however pd- and tim- expression tended to progressively increase in cd + t cells during overtly symptomatic and icu period disease stages (figures c,d) . similarly, higher percentages of tim- + cells were observed in cd + t cells from patients in the icu stage, although pd- expression in cd + t cells was not obviously affected during disease progression (figures c,d) . furthermore, pd- and tim- protein expression tended to increase throughout the stages discussed above in pd- + or tim- + cd + t cells (supplementary figure c) . these results demonstrated that t cells are exhausted in covid- patients during sars-cov- infection. t cells play a vital role in viral clearance, with cd + cytotoxic t cells (ctls) capable of secreting an array of molecules such as perforin, granzymes, and ifn-γ to eradicate viruses from the host ( ) . at the same time, cd + helper t cells (th) can assist cytotoxic t cells and b cells and enhance their ability to clear pathogen ( ) . however, persistent stimulation by the virus may induce t cell exhaustion, leading to loss of cytokine production and reduced function ( , ) . earlier studies have been unclear regarding the numbers and function of t cells in covid- patients, albeit with suggestions of depressed lymphocyte counts ( , ) . in this report, we retrospectively reviewed the numbers of total t cells, cd + , cd + t cell subsets in a total of covid- patients. in non-icu patients, we found that over . % cases had a decrease in total, cd + and cd + t cells. however, in the icu group, a total of % ( / ) patients showed a decrease in both total t cells and cd + t cells, and most importantly, all of the patients displayed decreases in cd + t cells. we also analyzed non-icu patients in greater detail, and found that urgent intervention may be necessary to preempt the development of severe symptoms in patients with low t cell counts. cytokine storm is a phenomenon of excessive inflammatory reaction in which cytokines are rapidly produced in large amount in response to microbial infection. this phenomenon has been considered an important contributor to acute respiratory distress syndrome (ards) and multiple organ dysfunction syndrome (mods) ( , ) . it has been also implicated in the setting of respiratory viral infections, such as sars in , avian h n influenza virus infection in and h n infection in ( ) ( ) ( ) ( ) . huang et al. showed that the levels of il- , il- , il- , tnf-α, g-csf, ip- , mcp- , and mip- a were significantly higher in covid- patients ( ). consistent with this report, here we found that the secretion of cytokines including tnf-α, il- , and il- was increased in covid- patients. interestingly, the numbers of total t cells, cd + t and cd + t cells are negatively correlated to levels of tnf-α, il- , and il- , respectively (figure b ), suggesting these cytokines may be involved in the decrease of t cells detected in covid- . tnf-α is a pro-inflammatory cytokine which can promote t cell apoptosis via interacting with its receptor, tnfr , which expression is increased in aged t cells ( , ) . our current analysis demonstrated that patients over years old have lower t cell numbers, indicating that tnf-α might be directly involved in inducing t cell loss in these patients. il- , when promptly and transiently produced in response to infections and tissue injuries, contributes to host defense through the stimulation of acute phase responses or immune reactions. dysregulated and continual synthesis of il- has been shown to play a pathological role in chronic inflammation and infection ( , ) . tocilizumab, a humanized anti-il- receptor antibody, has been developed and approved for the treatment of rheumatoid arthritis (ra) and juvenile idiopathic arthritis ( , ) . moreover, tocilizumab has been shown to be effective against cytokine release syndrome resulting from car-t cell infusion against b cell acute lymphoblastic leukemia ( ) . whether tocilizumab can restore t cell counts in covid- patients by suppressing il- signaling remains uninvestigated. the source of these cytokines during covid- disease remains an open interesting issue. while previous studies have validated that the secretion of cytokines, including il- , il- , and tnf-α, mostly derives from t cells, macrophages and monocytes etc. ( , ) , based on our (inverse correlation) results, we suggest that the secretion of these cytokines does not originate from t cells. however, the cytokine storm in turn may promote apoptosis or necrosis of t cells, and consequently leads to their reduction. our previous work demonstrated that monocytes and macrophages can produce pro-inflammatory cytokine during murine hepatitis virus strain- infection ( , ) , yet whether sars-cov- also triggers cytokine release from monocytes and macrophages in covid- patients needs further investigation and current work around this is in progress in our hospital. t cell exhaustion is a state of t cell dysfunction that arises during many chronic infections and cancer. it is defined by poor effector function, sustained expression of inhibitory receptors, and a transcriptional state distinct from that of functional effector or memory t cells ( ) . by facs analysis, we found that both cd + t cells and cd + t cells have higher levels of pd- in virus infected patients, particularly in icu patients (figure ) . il- , an inhibitory cytokine, not only prevents t cell proliferation, but also can induce t cell exhaustion. importantly, blocking il- function has been shown to successfully prevent t cell exhaustion in animal models of chronic infection ( , ) . we demonstrate here that covid- patients have very high levels of serum il- following sars-cov- infection, while also displaying high levels of the pd- and tim- exhaustion markers on their t cells, suggesting that il- might be mechanistically responsible. the application of potent antiviral treatments to prevent the progression to t cell exhaustion in susceptible patients may thus be critical to their recovery. we have read with great interest the successful application of remdesivir to cure a covid- patient in the us, and clinical trials indicate that this drug may have significant potential as an antiviral ( , ) . taken together, we conclude that t cells are decreased and exhausted in patients with covid- . cytokines such as il- , il- , and tnf-α might be involved in t cell reduction. thus, new therapeutic measures are needed for treatment of covid- icu patients, and perhaps these are necessary even early on to preempt disease progression in higher-risk patients with low t cell counts. the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. the studies involving human participants were reviewed and approved by national health commission of china and ethics commission of general hospital of central theater command ([ ]- - ) and hanyang hospital ( ). written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. written informed consent was obtained from the individual(s), and minor(s)' legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article. this manuscript has been released as a preprint at medrxiv ( ). the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material a novel coronavirus from patients with pneumonia in china early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding clinical features of patients infected with novel coronavirus in wuhan epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive 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coronavirus in the united states reduction and functional exhaustion of t cells in patients with coronavirus disease key: cord- -j no authors: de stefano, ludovico; bobbio-pallavicini, francesca; manzo, antonio; montecucco, carlomaurizio; bugatti, serena title: a “window of therapeutic opportunity” for anti-cytokine therapy in patients with coronavirus disease date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: j no the effects of cytokine inhibition in the different phases of the severe coronavirus disease (covid- ) are currently at the center of intense debate, and preliminary results from observational studies and case reports offer conflicting results thus far. the identification of the correct timing of administration of anti-cytokine therapies and other immunosuppressants in covid- should take into account the intricate relationship between the viral burden, the hyperactivation of the innate immune system and the adaptive immune dysfunction. the main challenge for effective administration of anti-cytokine therapy in covid- will be therefore to better define a precise “window of therapeutic opportunity.” only considering a more specific set of criteria able to integrate information on direct viral damage, the cytokine burden, and the patient’s immune vulnerability, it will be possible to decide, carefully balancing both benefits and risks, the appropriateness of using immunosuppressive drugs even in patients affected primarily by an infectious disease. the coronavirus disease , caused by severe acute respiratory syndrome coronavirus (sars-cov- ), has a wide spectrum of clinical expressiveness ranging from asymptomatic or paucisymptomatic infection to life-threating multiple organ failure. in most severe forms, sars-cov- infection leads to fulminant pneumonia and acute respiratory distress syndrome (ards) with a mortality rate approaching - % ( ) . clinical deterioration generally occurs several days after the onset of symptoms, in association with declining viral titers ( ) , suggesting that part of pathophysiology may be driven by dysregulated immune responses rather than by direct viral damage. uncontrolled hyperinflammation has indeed been recognized as a pivotal pathogenetic event in , with the release of inflammatory cytokines which are injurious to host cells similarly to what happens in other hyperinflammatory syndromes characterized by cytokine storms, such as the cytokine release syndrome (crs), secondary hemophagocytic lymphohistiocytosis (shlh) and macrophage activation syndrome (mas) ( ) . the known sensitivity of these syndromes to cytokine-directed therapies has fueled many expectations also for patients with covid- . however, the precise nature and role of hyperinflammation in severe covid- remain poorly defined, and the risk-benefit ratio of cytokine inhibition in the different phases of the disease is largely debated ( ) ( ) ( ) ( ) . as a matter of facts, preliminary clinical results on the efficacy of therapeutic blockade of interleukin (il)- , il- , tumor necrosis factor (tnf), and janus kinase signaling are thus far mixed ( - ) ( table ) , possibly also due to the different timing of drug administration in different reports. early immunosuppression during an infectious disease indeed incurs risks; on the other hand, however, suppression of pathogenic hyperinflammation may be more effective in the initial stages before clinical deterioration occurs. the identification of the correct timing of administration of immunosuppressive drugs remains therefore a research priority for the personalized management of covid- . the health analytic platform opensafely established in the united kingdom has recently confirmed that covid- -related deaths are strongly associated with demographic factors and comorbidities such as increasing age, male gender, obesity, cardiovascular and respiratory diseases ( ) . risk prediction and tailored treatment solely based on such generic parameters is however largely inaccurate, and covid- outcomes likely depend on a number of other variables influencing host-pathogen interactions, including sars-cov- genetic variants as well as host genetic susceptibility. research in this area is only in its infancy. also building on previous experience with other coronaviridae, several studies are investigating the link between sars and genetic variants in innate immune response ( ) , including mannosebinding lectin deficiency and polymorphisms, which are involved in the inactivation of a variety of respiratory pathogens through direct binding and complement activation ( ) . similarly, adaptive immune dysfunction is appearing as a key, and recent studies have confirmed the role of cd + t cell cytopenia in determining covid- progression and fatality ( ) . discovery of virus and host genomic factors will undoubtedly support risk stratification and targeted treatment; however, as genomic studies require long times before entering clinical practice, it is urgent to integrate easily accessible information on the dynamics and pathogenicity of the immune response during the different phases of sars-cov- infection. the relative contribution of viral damage and of innate and adaptive immune responses in course of covid- greatly varies over time ( ) ( ) ( ) . in the early phases, common to all patients, pathogenic mechanisms are mainly driven by the cytotoxic role directly exerted by sars-cov- , which usually affects only the upper respiratory tract. the early antiviral response mostly relying on type-i interferon (ifn) and natural killer (nk) cells, and the subsequent cd + t cell-mediated killing of virally infected cells as well as cd + t cell-dependent antibody production, allow the rapid reduction of the viral load, the paucisymptomatic character of the disease, and its recovery. however, in up to % of the patients, virus-induced immunosuppression occurs. this is highlighted by a marked reduction of cd + and cd + t lymphocytes in peripheral blood, defective cd + t cell and nk cell function, low ifn-g production, and low specific igg antibodies ( ) ( ) ( ) ( ) . the impairment of the initial antiviral defense mechanisms favors a sustained increase in the viral load capable of conditioning disease progression, especially in organs that have high angiotensin-converting enzyme expression, such as the lungs but also the endothelium, the heart, the kidney, and the intestine ( , ) . the clinical picture can evolve rapidly, with the development of interstitial pneumonia which can progress into ards ( ) . in these early stages, lung damage is mainly driven by sars-cov- cytotoxicity, while inflammatory responses aim at eliminating the pathogen ultimately leading to tissue repair. accordingly, longitudinal immune profiling of hospitalized covid- cases with different outcomes has recently shown that, despite similar levels of inflammatory cytokines in the first days from symptom onset, patients with less severe disease evolution also express mediators of wound healing and tissue repair ( ) . in this phase, the therapeutic strategy should thus include the use of antiviral drugs and of treatments aimed at cautiously enhance immune responses. conversely, the use of drugs that compromise the efficiency of the immune system could be counterproductive, as patients with high viral loads and long virus-shedding periods are at higher risk of severe covid- ( ) . in immunocompromised patients in whom the therapeutic strategies adopted and the presence of comorbidities do not allow adequate control of the viral load, extensive tissue damage, and subsequent uncontrolled inflammatory innate responses with exaggerated myeloid-derived cytokine production can occur ( ) ( ) ( ) ( ) . the clinical picture suddenly and unexpectedly changes with fever, respiratory failure, and ards associated with increased levels of acute phase reactants, neutrophilia, thrombocytosis, anemia, signs of coagulopathy, and cell lysis. these acute inflammatory mechanisms may precipitate tissue damage both locally (especially at the lung level) ( ) and systemically, thus playing an even greater pathogenetic role than that played by direct viral damage, and significantly affecting mortality. this hyperinflammatory syndrome shares pathophysiological similarities with cytokine storms in crs, shlh and mas, conditions that sometimes complicate viral infections, systemic autoimmune and autoinflammatory diseases, hematologic diseases, and medications such as engineered t cell therapy ( ) ( ) ( ) ( ) . experience from these conditions has encouraged the use of anti-cytokine therapy also for the management of covid- . however, the clinical picture of the cytokine storm in covid- , especially when it is not associated with multi-organ damage secondary to de stefano et al. frontiers in immunology | www.frontiersin.org october | volume | article coagulopathy, is different from that of the classic shlh/mas: it is mostly anatomically compartmentalized to the lungs, not associated with organomegaly and not accompanied by pancytopenia ( ) . accordingly, serum levels of il- are lower in covid- compared to other crs ( ) . furthermore, hypercytokinemia should be considered as a general marker of sars-cov- , even in the absence of a cytokine storm, and this makes covid- crs not easily distinguishable from ards pathogenetically correlated only with viral cytotoxic activity, also because levels of inflammatory markers and cytokines not always correlate with outcome ( ) . finally, compared to shlh/mas, the crs in covid- occurs in the context of immunodeficiency and immunoparalysis ( , , ) , thus imposing caution on further iatrogenic immunosuppression. the identification of the correct timing of administration of anti-cytokine therapies and other immunosuppressants in covid- should thus take into account the intricate relationship between the viral burden, the hyperactivation of the innate immune system, and the adaptive immune dysfunction. although the therapeutic window in crs is narrow, and timely control of the cytokine storm is crucial to reduce short-term mortality, premature use of immunosuppressants could indeed further compromise viral shedding with the risk of increasing viral replication and tissue damage directly induced by the virus ( ) . furthermore, iatrogenic immunosuppression could promote bacterial, fungus, or viral infectious complications, as shown with the targeting of il- with tocilizumab in observational studies on patients not requiring mechanical ventilation ( , ) . also at later stages, pharmacologically induced immunosuppression in already immunocompromised patients could adversely affect the course of the disease, as suggested by the finding of significantly longer periods of hospitalization and higher rates of mortality in ventilated patients treated with tocilizumab ( , ) or high-doses corticosteroids ( , ) . the main challenge for effective anti-cytokine therapy in covid- will be therefore to better define a precise "window of therapeutic opportunity," a phase of the disease during which the benefits of cytokine inhibition are prevalent on the inevitable consequences of immunosuppression. although > randomized clinical trials (rcts) on different cytokine inhibitors in covid- are underway, the identification of such a "window" remains elusive, and only few studies have tried to integrate further patients' characterization beyond the clinical status among the inclusion criteria ( table ) . most of the protocols essentially require, among the inclusion criteria, the presence of pneumonia not otherwise defined if not by the extent of functional impairment, with a tendency to focus mainly on the early stages of the disease, in the absence of ards or systemic involvement. some observational studies continue to suggest that immunosuppressants such as glucocorticoids and il- antagonists (alone or in combination) are advantageous if administered early at hospital admission ( , , ) (table ) . however, the recovery trial has clearly shown that benefits from dexamethasone are restricted to those patients with at least days of symptoms and those requiring invasive or non-invasive ventilation ( ) , suggesting that, based on the clinical criterion alone, only a late phase of covid- is dominated by pathogenic immunity. accordingly, the use of early short-term corticosteroid therapy, even at low-doses, has been associated with worse clinical outcomes in non-severe covid- pneumonia in observational studies ( ) ( table ). in line with these findings, following preliminary results, rcts on sarilumab (another il- receptor antagonist) have been amended to enroll only critical patients (https://investor.regeneron.com/newsreleases/news-release-details/regeneron-and-sanofi-provideupdate-us-phase- -adaptive), and the covacta trial on tocilizumab recently ended recruitment because neither primary nor secondary end-points were met (https://www. roche.com/media/releases/med-cor- - - .htm). better and still clinically feasible characterization of those patients with an hyperinflammatory syndrome potentially susceptible to immunosuppression could derive from the integration of markers of inflammation, tissue damage, cell lysis, and coagulopathy. this has indeed been the rationale for the development of the h-score for the diagnosis of shlh and mas, which includes, among the others, hyperferritinemia, cytopenias, and liver damage ( ) . however, the many clinical and pathogenetic differences between covid- hyperinflammation and slhl/mas limit the generalizability of the h-score to covid- associated cytokine storm, and only few patients with severe covid- achieve diagnostic cut-offs of > , mainly due to lower ferritin levels, absence of pancytopenia and hypofibrinogenemia ( ) . similarly, the prevalence of the hyperinflammatory phenotype in ards due to covid- is lower compared to that observed in non-covid- ards despite its higher mortality ( ) . however, laboratory indicators of hyperinflammation may still be of value in the identification of those covid- patients more at risk of escalation of respiratory support and death. using cut-off values of c-reactive protein (crp) concentrations greater than mg/l or ferritin concentrations greater than , mg/l, manson jj and colleagues ( ) recently described an hyperinflammatory phenotype of covid- characterized by poor clinical outcomes irrespective of other demographic and clinical variables. patients' stratification based on this approach could be of promise. accordingly, in an observational study on hospitalized covid- cases, early use of glucocorticoids was effective only in patients with high crp levels, being instead associated with increased mortality in case of low crp ( ) . elevation of inflammatory markers such as crp, ferritin, and others is however non-specific for cytokine storms. cytokine assays are ready available in the clinic and testing for serum levels of cytokines known to be involved in pathogenic inflammation could help guiding the rational use of targeted immunomodulatory therapeutic strategies. accordingly, il- and other proinflammatory cytokines have been shown to be predictive of patient outcomes in terms of both disease severity and survival, and integrated models taking into account demographics, comorbidities, markers of inflammation and tissue damage, and cytokines show good performance with areas under the receiver operating characteristic curve approaching . ( ) . several limitations however still exist even using similar approaches. apart from il- , the trend of other cytokines whose targeting is already available in the clinic does not appear uniform in covid- ( , , , ) , hampering the possibility of tailored treatments and contrasting with the apparent efficacy of some agents, such as il- antagonists, in preliminary observational studies ( , ) . even in the case of il- , no established cut-offs exist. furthermore, the release of inflammatory cytokines is also part of a well-conserved innate immune response necessary for efficient clearance of infectious agents. accordingly, levels of il- are similarly increased in the hyperinflammatory phenotype of non-covid- ards irrespective of its causative mechanism, but interventions targeting single cytokines in this setting have a long history of failure ( ) . collectively, current clinical, laboratory, and even biological parameters thus do not appear optimal at distinguishing an appropriate from a dysregulated inflammatory response in the context of covid- . ideally, in light of the very pulmonarycentric character of this condition, better knowledge could arise from the analysis of parameters deriving from bronchoalveolar lavage fluid and microscopic examination of transbronchial lung biopsies ( table ) . these include cytological analyses with prevalence of granulocytes, macrophages, and lymphocytes; pro-inflammatory cytokine and chemokine secretion including neutrophil recruiting mediators and other attractants of monocytes and immune cells; lung infiltration by monocytes, macrophages, and neutrophils; thrombosis ( ) ( ) ( ) ( ) . the clinical, radiological, and laboratory characteristics already identified in patients with covid- would thus be put in the context of more specific histological, cytological, and immuneinflammatory criteria in order to identify those parameters more accurately signaling the presence of a predominantly immuno-inflammatory pathogenesis which might benefit from immunosuppression ( table ). the evidence of ongoing sars-cov- replication late in disease would in any case support the associated use of antiviral therapy, even at a point when immunopathology is dominant ( ) . in conclusion, despite the emergency situation imposes speed in the identification of the therapeutic options for patients with covid- , better harmonization of inclusion criteria and patient stratifications for studies on immunomodulatory therapies should remain a priority. only considering a more specific set of clinical and pathological criteria, together with the extent of the viral load present in the alveolar bronchial lavage fluid and parameters useful to quantify the patient's immune vulnerability (lymphocyte count, cd + and cd + t cell levels, markers of lymphocyte exhaustion, development of specific antibodies), it will be possible to decide, carefully balancing both benefits and risks, the appropriateness of using immunosuppressive drugs even in patients affected primarily by an infectious disease. the original contributions presented in the study are included in the article/supplementary material; 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blander, j. magarian title: sensing microbial rna in the cytosol date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: atnrw ew the innate immune system faces the difficult task of keeping a fine balance between sensitive detection of microbial presence and avoidance of autoimmunity. to this aim, key mechanisms of innate responses rely on isolation of pathogens in specialized subcellular compartments, or sensing of specific microbial patterns absent from the host. efficient detection of foreign rna in the cytosol requires an additional layer of complexity from the immune system. in this particular case, innate sensors should be able to distinguish self and non-self molecules that share several similar properties. in this review, we discuss this interplay between cytosolic pattern recognition receptors and the microbial rna they detect. we describe how microbial rnas gain access to the cytosol, which receptors they activate and counter-strategies developed by microorganisms to avoid this response. when janeway formulated the theory of pattern recognition in , he proposed that host cells could sense microbial infection owing to receptors able to recognize invariant molecular structures defined as pathogen-associated molecular patterns (pamps). these patterns would be present in groups of pathogens, but absent in the host ( ) . years later, janeway and medzhitov described the activity of the first mammalian member of the toll-like receptor (tlr) family, toll-like receptor ( ) . tlrs comprise a family of transmembrane proteins able to recognize conserved microbial features and activate the immune response ( ) . once activated, tlrs and others pattern recognition receptors (prrs) initiate several intracellular pathways, including those mediated by nuclear factor-κb (nf-κb), mitogen-activated protein kinases (mapks), and interferon regulatory factors (irfs). another outcome of activation of distinct members of cytosolic prrs is their oligomerization into multimeric cytosolic structures called inflammasomes, which activate the cysteine protease caspase- , subsequently leading to the production of biologically active forms of pro-inflammatory cytokines ( ) . initially thought to detect exclusively microbial derived ligands, prrs were later shown to recognize host derived danger signals, which are released in response to stress conditions such as cellular damage or tissue injury ( ) . under normal physiological conditions, these ligands are not accessible to their respective prrs and do not activate the immune system. conversely, it was first suggested that self-dna artificially introduced into the cytoplasm by transfection could activate nf-κb and the mapk pathway ( ) . evidence that any dna, regardless of its origin, can engage innate immune receptors when localized outside of the nucleus was further confirmed by the identification of several endosomal and cytosolic dna sensors [reviewed in ref. ( ) ]. in contrast to cytosolic dna, rna sensing in the cytoplasm raises many questions on the mechanisms used by the innate system to specifically distinguish non-self-rna from self-rna. during infection, microbial rnas share the cytosolic cellular compartment with several host rna species, including messenger rna (mrna), transfer rna (trna), ribosomal rna (rrna), microrna, and other small regulatory rnas. as a consequence, cytosolic sensors must display a high affinity for specific microbial features to avoid activation by host molecules that would otherwise elicit autoimmune responses. despite this apparent challenge, efficient detection of foreign rna in the cytosol is essential for innate immunity. during certain viral infections, rna may be the only microbial pamp produced throughout most of the replication cycle. additionally, our laboratory previously showed that recognition of bacterial mrna in the cytosol was critical to elicit a robust innate response against bacterial infection ( ) . finally, cytosolic sensing of pathogen invasion by non-immune infected cells provides the very first steps of innate response against infection, before phagocytosis-competent immune cells are recruited to the site of infection. in this review, we summarize the current understanding of cytosolic rna sensing. we describe instances in which microbial rnas gain access to the cytosol, the prrs they activate, their corresponding ligands and strategies developed by microorganisms to conceal their rnas. rna entry into host cells generally takes place during the first steps of a microbial infection. we distinguish four processes leading to the presence of microbial rna in the cytosol of eukaryotic cells, where it can engage host prrs (figure ). figure | cytosolic recognition of microbial rna. genomic rna from rna viruses access the cytosol immediately after the cell entry step of the replication cycle, where it may be amplified by viral rna-dependent rna polymerase (rdrp). genomic dna from dna viruses is transcribed by viral or cellular rna polymerase, including the cytosolic rna polymerase iii. bacterial rna can access the cytosol through the activity of auxiliary secretion systems or during passive leakage of phagosomal products. once in the cytosol, microbial rna binds different families of prrs classified as rlrs, non-rlr helicases, and other receptors. downstream signaling pathways include activation of mavs, trif, and the nlrp inflammasome. black arrows, rna entry; red arrows, signaling pathways. step of the replication cycle. viruses can directly release their genome at the plasma membrane after binding to a receptor. alternatively, they can be first internalized through endocytosis or macropinocytosis. endocytosed virus particles will typically traffic through endosomal vesicles by actin-dependent and/or microtubule-dependent transport ( ) . specific environmental triggers like endosomal ph acidification induce either fusion of enveloped virus with the endosome, or membrane penetration of viral proteins, allowing viral genetic material to be released into the cytoplasm ( ) . alternatively, viruses can spread by direct cell-cell contact ( ) . cell-to-cell transmission of viral material can activate cytoplasmic innate pathways, as exemplified with hepatitis c virus ( ), lymphocytic choriomeningitis virus ( ) , or human immunodeficiency virus transmission ( ) . other viral rna pamps can be produced during viral replication. david baltimore has defined a classification of viruses based on the mechanism of mrna production ( ) . viruses are clustered in seven groups depending on their genomes (dna, rna), strandedness (single or double), sense or antisense, and method of replication. the type of rna ligands produced during viral replication will depend on the type of viral genome and the strategy used to generate mrna. rna ligands can be generated by dna viruses and retroviruses via genome transcription, or by synthesis of mrna and replication intermediates by rna-dependent rna polymerases (rdrps) of rna viruses ( ) . it has been shown that ligands generated in phagolysosomes after phagocytosis of bacteria by immune cells can engage cytosolic innate immune receptors ( ) . similarly, we showed that rna from escherichia coli could activate receptors in the cytosol after phagocytosis by macrophages ( ) . we demonstrated that phagosomes carrying e. coli exhibit intrinsic leakiness, suggesting a mechanism by which bacterial rna, irrespective of the activity of virulence factors, can gain access to the cytoplasm ( ) . alternatively, bacteria express secretion systems to translocate products outside of the bacterial cell wall. in the case of intracellular bacteria, auxiliary secretion systems like seca in listeria monocytogenes have been shown to actively translocate listeria rna into the cytoplasm, resulting in activation of cytosolic sensors ( , ) . similarly, another study proved that cytosolic rna sensors participate in the type interferon (ifn-i) response to legionella pneumophila. although the authors did not demonstrate the translocation of legionella rna into the cytosol of infected cells, they discuss their data through a model where it would be the case ( ) . future studies looking for additional secreted rna will likely provide additional insights on their interaction with the innate immune system. two independent groups have demonstrated that cytoplasmic dsdna triggers ifn-i production via rna polymerase iii, which transcribes dna into '-triphosphate ( -ppp) rna, subsequently recognized by cytosolic rna receptors ( , ) . this pathway has been involved in the sensing of dna viruses, like epstein-barr virus, or intracellular bacteria, like l. pneumophila ( , ) . although the rna intermediate produced is not sensu stricto microbial, its generation is due to the activity of a microbial invader. the best-studied cytosolic rna sensors are the three members of rig-i-like receptors (rlrs), a subfamily of the dexd/hbox family of helicases. they consist of retinoic acid-inducible gene i (rig-i), melanoma differentiation factor (mda ), and laboratory of genetics and physiology (lgp ). they share a similar organization with three distinct domains: (i) a c-terminal repressor domain (rd) embedded within the c-terminal domain (ctd); (ii) a central atpase containing dexd/h-box helicase domain able to bind rna; and (iii) a n-terminal tandem card domain that mediates downstream signaling, and which is present in rig-i and mda but absent in lgp . upon activation by rna ligands, rig-i and mda are subsequently recruited to the adaptor protein mitochondrial antiviral signaling (mavs) via a card-card interaction and lead to activation of nf-κb and irfs ( ) ( ) ( ) ( ) . in contrast to tlr expression that is predominantly expressed in specialized immune cells such as macrophages and dendritic cells (dcs), rlrs are found in the cytosol of most cell types and are strongly induced in response to ifn-i ( , ) . the rig-i ligand has been characterized as an rna molecule with two distinct features: (i) a -ppp moiety ( , ) and (ii) bluntend base pair at the '-end ( , ) . blunt-end base pairs can be found in double-stranded rna (dsrna) and secondary rna structures such as hairpin or panhandle conformations ( , ) . recent structural studies have contributed toward a better understanding of ligand binding and activation of rig-i. specificity of -ppp binding is conferred by the ctd, and the helicase domain binds the double-stranded part of the rna. rig-i is normally held in an auto-repressed conformation, and ligand binding results in a conformational change, releasing the card domain which can subsequently initiate signaling by association with mavs ( ) ( ) ( ) . despite the increasing amount of high-resolution crystal data, the consensus definition of rig-i ligand remains controversial. other rig-i ligands have been indeed described in the literature including long ( ) or short dsrna ( ) ( ) ( ) lacking the -ppp. however, thermodynamic analysis have shown that the full-length human rig-i protein binds '-ppp dsrna with -fold higher affinity than '-oh dsrna, and dsrna with a -fold higher affinity than short single stranded rna (ssrna) lacking secondary structure ( ) . many viral families display blunt-end base-paired rna with a -ppp, directly in their genomic rna or in replication intermediates. consistent with this notion, rig-i has been shown to be involved in the recognition of many viruses, either antisense (−)ssrna viruses ( , ) or sense (+)ssrna/dsrna viruses ( , ) . notably rig-i can detect panhandle structures found in lacrosse viral particles ( ) or in influenza genomic rna ( , ) . sendai virus (sev) and other mononegavirales produce defective interfering (di) viral genomes containing panhandle structures that activate rig-i in infected cells ( ) . retinoic acid-inducible gene i recognition has not been limited to rna virus since rig-i is involved in recognition of dna viruses, such as epstein-barr virus or adenovirus through the rna polymerase iii pathway ( , , ) . moreover, rig-i is also able to detect bacterial infections. bacterial mrna are not capped and it has been estimated that approximately % of rna oligonucleotides in e. coli have a -ppp ( ) . reports in the literature describe sensing of l. monocytogenes secreted rna ( , ) or purified legionella ( ) and helicobacter pylori rna ( ) by rig-i. finally, rig-i can also sense shigella flexneri infection in macrophages through the rna polymerase iii pathway ( ) . melanoma differentiation factor ligand is less characterized than rig-i. using poly(i:c) as a synthetic dsrna mimic, studies have shown that mda binds long, but not short dsrna ( , , ) . structural analyses have demonstrated that mda specifically recognizes the internal duplex structure of dsrna and uses it as a platform to stack along dsrna in a head-to-tail arrangement. this mechanism promotes stochastic assembly of the tandem card oligomers that activates the signaling adaptor mavs ( ) . melanoma differentiation factor detects infection by viral families known to produce long dsrna structures during their replication cycle, including (+)ssrna viruses like picornaviruses, dsrna viruses like reoviruses, or dna viruses like poxviruses ( , ( ) ( ) ( ) ( ) . in the case of (+)ssrna virus infection, fluorescent imaging studies have confirmed that mda recognizes preferentially the dsrna generated during the replication of these viruses, but not the genomic ssrna ( ) . prior to the structural study mentioned above, multiple observations raised the possibility that there may exist additional mda ligands, different from the consensus long dsrna. thus, a study has shown that mda cooperates with the ribonuclease rnase l to induce ifn-i in response to a viral mrna from parainfluenza virus ( ) . interestingly, rnase l converts rna into small rna products, with shorter length than the current mda www.frontiersin.org ligand definition ( ) . another work published the same year has shown that mrna lacking '-o-methylation at their ' cap structure induces production of ifn-i through mda activation ( ) . however, the data published, which focus on coronavirus infection, did not elucidate whether the absence of methylation was directly recognized by mda or via another intermediate ( ) . after binding to their specific ligands, both rig-i and mda activate mavs to trigger a common signaling pathway. the majority of mavs is localized on the mitochondrial membrane and its engagement by rlrs causes a conformational change that propagates to adjacent un-activated mavs in a prion-like behavior ( ) . the formation of these very large mavs aggregates results in a largescale amplification of the signaling cascade. this cascade involves the recruitment of cytosolic adaptor molecules, followed by the activation of the canonical ikks, ikk-α, ikk-β, and ikk-γ, the mapk and the non-canonical ikk-related kinase, tbk and ikki/ε. ultimately, specific transcription factors, such as irf , nf-κb, and depending on the cell type irf and irf , are translocated to the nucleus where they promote the expression of ifn-i genes and pro-inflammatory cytokines [reviewed in ref. ( ) ]. finally, mavs has been recently shown to interact with nodlike receptor family, pyrin domain containing (nlrp ) and promote its recruitment to the mitochondria. the authors emphasize the central role of mavs in innate immune signaling events by showing its importance in the functioning of nlrp inflammasome and the production of il- β ( ) . of note, mavs independent activation of the nlrp inflammasome by rig-i has also been reported ( , ) . the third member of rlrs, lgp , is able to bind dsrna ( , ) , however, its role in immune activation is poorly understood. lgp was proposed to be a modulator of rlr signaling. studies showed that lgp was required for rig-i and mda activity, in particular during picornaviral infection ( ) ( ) ( ) . another work proposed that lgp would inhibit rig-i through competition with its ligand ( ) . it is however unclear whether lgp binds microbial rna in an infectious context, and what specific features of the rna it would recognize. further studies will be required to clarify the precise role of lgp . apart from rlr, several recent studies have highlighted the importance of other dexd/h-box helicases in microbial rna sensing. rna helicases of the dead box family are involved in various different steps of rna metabolism [reviewed in ref. ( ) ]. they share eight conserved motifs that are involved in atp binding, atp hydrolysis, nucleic acid binding, and rna unwinding activity. additionally, most dexd/h-box helicases contain auxiliary nand c-terminal domains that confer on them functional specificities, such as an ability to induce downstream signaling or to bind specific rna targets ( ) . ddx (ddx x) can bind poly(i:c) or vesicular stomatitis virus (vsv) rna and was shown to enhance the ifn-i response to vsv infection by interaction with the rlr-mavs complex. overexpression assays suggest that ddx precipitates with rig-i and mda ( ) . since ddx is easily detected in resting cells, the authors propose a sentinel role for this helicase, the activity of which would be required during the initial steps of viral infection. another study showed that upon sev infection, ddx interacts with ikkε, an essential component of the irf signaling pathway, increasing the induction of the ifn-β promoter ( ) . moreover, ddx is targeted by vaccinia virus protein k ( ) , an inhibitor of ifn-β production, and by hcv core protein, which can disrupt its interaction with mavs ( ) . these observations highlight the importance of ddx in efficient viral sensing. using overexpression and knock-down experiments, dhx was shown to be required for the production of ifn-i and proinflammatory cytokines in response to poly(i:c), influenza virus, and reovirus by a murine splenic dc line and bone-marrow derived dcs. dhx can bind dsrna via its dsrna-binding motif and interact with mavs through both its helicase c-terminal domain and ha -duf ( ). myeloid dcs have also been shown to express a complex composed of ddx , ddx , and dhx that triggers an antiviral program in response to poly(i:c), in a pathway dependent of the adapter molecule tir-domain containing adapter-inducing interferon-β (trif). ddx binds to poly(i:c) via its helicase a domain, while dhx and ddx bind the tir domain of trif via their ha -duf and prk domains, respectively. this complex seems to be required for the innate response against influenza or reovirus infection ( ) . notably, a separate study also characterized dhx and dhx as a sensor for the dsdna species cpg-a and -b, respectively. in this case, both dhx and dhx activate the cytosolic adapter protein myeloid differentiation primary response gene (myd ) by binding to its tir domain ( ) . another recent study by yong-jun liu's group identified another helicase, dhx , as a cytosolic rna receptor able to activate the nlrp inflammasome ( ) . dhx is involved in inflammasome activation after sensing cytosolic rna such as poly(i:c) or reoviral rna when directly delivered by lipofection to the cytoplasm of a macrophage cell line or human monocyte-derived macrophages. additional experiments suggested that dhx could also possibly be involved in detection of cytosolic bacterial rna. the authors showed that dhx can bind to dsrna through its helicase c domain and to nlpr through its dead domain ( ) . a few months later, another study performed on myeloid dcs confirmed the role of dhx in the sensing of cytosolic poly(i:c) and reoviral rna. surprisingly, in this case, poly(i:c)-induced activation of mapk, nf-κb, and irf was mediated by mavs, which binds the helicase c domain of dhx ( ) . ddx has also been shown to enhance the ifn-i response to rna and dna stimulation through formation of complexes with frontiers in immunology | molecular innate immunity rig-i, mda , and lgp but not with mavs. this complex formation has been deciphered with overexpression assays in the case of mda and lgp , and with endogenous rig-i during vsv infection. ddx expression is induced by viral infection and its helicase domain can bind ds-or ss-vsv rna generated in vitro, independently of the -ppp ( ) . interestingly, ddx can also bind dsdna, and was shown to play role in ifn-i expression after infection with herpes simplex virus- , a dna virus. this ability to bind both dsrna and dna raises the question of the feature ddx recognizes. it should be finally noted that the role of ddx in the ifn-i pathway has been questioned ( ) . several other cytoplasmic receptors have been shown to play a role in microbial rna recognition. this is the case for the cytoplasmic protein kinase r (pkr), which is important for antiviral activity. pkr is activated by dsrna from viruses and is a component of mapk and nf-κb signaling pathways [reviewed in ref. ( ) ]. activation of pkr can also be mediated by short -ppp rnas containing limited secondary structures ( ) . proteins from the interferon-induced protein with tetratricopeptide repeats (ifits) family, such as ifit and , bind -ppp of viral rna ( ) . using short in vitro transcribed oligonucleotides, crystal structure studies have demonstrated that ifit proteins contain a positively charged cavity designed to engage, without any particular sequence specificity, ssrna with a -ppp end. contrary to rig-i, ifit proteins cannot bind blunt-ended -ppp dsrna, and owing to the limitations imposed by their rna-binding pockets, ifit and ifit require '-overhangs of at least or nt, respectively ( ) . using a -o-methyltransferase mutant of japanese encephalitis virus, another study showed that ifit preferentially binds to capped -o-unmethylated mrna ( ) , confirming previous findings showing that -o-methylation of viral mrna caps promotes ifit evasion ( , ) . the mechanism of ifit antiviral action is not completely understood, and it has been proposed that ifit might sequester viral rnas ( ) or inhibit viral mrna translation ( ) . the crystal structure of ifit (known as isg ) was also described. ifit specifically binds adenylate uridylate (au)rich rnas in vitro, independently of the presence of a -ppp ( ) . the authors showed that rna-binding capacity of this protein mediates its antiviral properties, using a model of hek t cells infected by newcastle disease virus or sev ( ) . nucleotide-binding oligomerization domain containing protein (nod ) is a member of the nod /apaf- family and encodes a protein with two card domains and six leucinerich repeats (lrrs). nod is primarily known for its ability to recognize bacterial peptidoglycan, but it also plays a role in the antiviral response. nod has been shown to activate mavs after stimulation with viral ssrna or human respiratory syncytial virus infection ( ) . nlrp is involved in cytosolic rna sensing. caspase- cleavage triggered by influenza virus, sev, or bacterial mrna is dependent on nlrp inflammasome activation ( , , ) . however, direct binding of nod or nlrp to microbial rna has not been established. leucine-rich repeat flightless-interacting protein (lrrfip ) contributes to the production of ifn-β induced by vsv and l. monocytogenes in macrophages ( ) . mostly located in the cytosol, lrrfip can also be found in rna-containing lysosomes ( ) . lrrfip can bind both dsrna and dsdna and subsequently induce ifn-i expression through β-catenin phosphorylation. activated β-catenin is translocated to the nucleus and increases ifn-β expression by binding to the c-terminal domain of the transcription factor irf and promoting the recruitment of the acetyltransferase p to the ifnb promoter. several other microbial rna features have been suspected or proposed to act as potential signals for cytosolic sensing, suggesting the existence of receptors detecting these characteristics. a computational analysis identified cpg motifs in an au-rich rna as an immunostimulatory feature. this sequence motif is underrepresented in both ssrna viruses and host innate immune gene mrna, and its frequency in influenza virus genomes has decreased throughout evolution ( ) . since this evolutionary pressure seems to also be applied on host mrna, the implication of a cytosolic receptor is possible, although experimental studies identified endosomal tlr as a potential prr ( ) . another study identified the nucleotide bias of a-rich hiv- genome as a strong inducer of ifn-i and potent mediator of lentiviral pathogenicity. the authors showed that the ability of rna sequences derived from the hiv- genome to induce an interferon response correlated with their nucleotide bias and that codon-optimized sequences lost their stimulatory activity ( ) . the experimental procedure used in this study consisted of direct delivery via lipofection of in vitro transcribed rna sequences into the cytosol of a reporter cell line, suggesting a potential role for a cytoplasmic rna sensor ( ) . recently, our group identified bacterial mrnas as an activator of the nlrp inflammasome. polyadenylation of these rnas abrogated their immunostimulatory activities, suggesting that features at the end of mrna, rather than the end, could engage cytoplasmic cellular sensors ( ) . philip bevilacqua's group has shown that different nucleoside modifications on rna, such as base or sugar internal modifications, suppress their intrinsic ability to activate immune sensors, notably pkr. the authors propose that self-rna editing could be a mechanism used by the innate immune system to discriminate self-transcripts from "unmodified" microbial rnas ( , ) . conversely, microbial rna editing by cellular deaminase enzymes such as dsrna-specific adenosine deaminase (adar) have been shown to enhance its recognition by cytosolic sensors ( ) . other host transcript specificities, like association to cellular components that prevent prr binding, or specific tertiary structure such as the eukaryotic mrna closed loop conformation ( ) , could be determinants for the differentiation of host mrnas from microbial rnas. identification of receptors able to recognize such features are lacking so far. infectious microorganisms have developed several strategies to evade cytosolic sensing. one of these strategies, which we only mention briefly here, is the direct targeting by microbial proteins www.frontiersin.org some (−)ssrna viruses edit the -ppp moieties in their genomes as well as replication intermediates into mono-phosphates to avoid recognition by rlrs ( ) . arenaviruses produce rna panhandle structures with a -ppp containing a gtp overhanging nucleotide. this viral structure is suggested to act as a rig-i ligand decoy, by trapping rig-i but not activating it ( ) . we are beginning to understand how eukaryotic cells use nucleoside modifications in order to protect self-rnas from innate sensing. for example, higher eukaryotes have acquired the ability to -omethylate their mrnas, allowing cellular receptors to distinguish self from unmethylated non-self mrna through specific types of antiviral sensors such as mda and ifits ( , ) . consistent with the red queen hypothesis ( ) , which postulates that parasites have to constantly evolve in order to adapt to their host species, the same immune escape strategy has been mimicked by several pathogens, like flaviviruses ( , ) . similarly, -o-methylation of g (gm ) on bacterial trna suppresses activation of the immune response in plasmacytoid dcs ( , ) . flaviviruses and other viruses are also known to induce cellular membrane reorganization that allows them to replicate in subcellular compartments, creating new replication-dependent organelles ( ) . thus, tick-borne encephalitis virus or japanese encephalitis virus have been shown to rearrange endoplasmic reticulum membranes to provide a compartment where viral dsrna is concealed from prr recognition. this hijacking of internal cell membrane induces a delayed cytosolic exposure of viral rna to innate receptors and accordingly, ifn-i responses are only measured late in the replication cycle ( ) ( ) ( ) . the ns protein from influenza virus can prevent rna sensing through the formation of a chain of ns molecules along the influenza dsrna backbone ( ) . picornaviruses mask their ppp with a viral encoded protein, vpg, which functions as a cap and as a primer during rna synthesis. interestingly, studies have shown that vpg could be used to evade rig-i recognition ( ) . similarly, ebola virus vp assembles into dimmers to cap the ends of viral dsrna and hide the specific rig-i recognition site ( ) . while one vp monomer binds the terminus and backbone of dsrna, the other vp monomer binds only the phosphate backbone of the dsrna, displaying a unique mode of dsrna concealing from prr ( ) . another hemorrhagic fever virus, lassa fever virus, uses the '- ' exonuclease activity of its nucleoprotein (np) to degrade stimulatory dsrna ( ) . this activity seems to be shared by other arenaviruses ( ) . finally, the protein c from human parainfluenza virus type (hpiv ), a paramyxoviridae, has been shown to limit the accumulation of dsrna. cell infection by a virus mutant defective for the c protein displays higher accumulation of several viral rnas, including viral genome, antigenome, and mrna, eventually leading to the accumulation of dsrna. thus, by limiting intracytosolic quantities of viral dsrna, the c protein of hpiv avoids dsrna triggering of mda and pkr in infected cells ( ) . the multiplicity of prr pathways is an essential determinant of the immune system's ability to sense with precision the level of frontiers in immunology | molecular innate immunity microbial threat and to respond accordingly ( ) . however, as far as cytosolic rna sensors are concerned, it is striking to observe the contrast between the high number of prrs that have been isolated and the similarities of the pamps they recognize ( table ) . while -ppp and dsrna are undoubtedly powerful triggers of the innate immunity, they cannot account for the diversity of responses that the organism is able to elicit against a wide range of pathogens. our understanding of how the immune system distinguishes between foreign and self-nucleic acids will continue to improve over time. this will help us better define the precise role played by cytosolic rna sensors in the global immune response against pathogens. approaching the asymptote? evolution and revolution in immunology a human homologue of the drosophila toll protein signals activation of adaptive immunity approaching the asymptote: years later beyond pattern recognition: five immune checkpoints for scaling the microbial threat activation of target-tissue immune-recognition molecules by double-stranded polynucleotides immune sensing of dna detection of prokaryotic mrna signifies microbial viability and promotes immunity principles of virology cell-to-cell transmission of viruses plasmacytoid dendritic cells sense hepatitis c virus-infected cells, produce interferon, and inhibit infection human pdcs sense lcmv infected cells in vitro innate sensing of hiv-infected cells expression of animal virus genomes bacterial ligands generated in a phagosome are targets of the cytosolic innate immune system rig-i detects infection with live listeria by sensing secreted bacterial nucleic acids rig-i detects triphosphorylated rna of listeria monocytogenes during infection in non-immune cells identification of host cytosolic sensors and bacterial factors regulating the type i interferon response to legionella pneumophila rig-i-dependent sensing of poly(da:dt) through the induction of an rna polymerase iii-transcribed rna intermediate rna polymerase iii detects cytosolic dna and induces type i interferons through the rig-i pathway ips- , an adaptor triggering rig-i-and mda -mediated type i interferon induction cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf visa is an adapter protein required for virus-triggered ifn-beta signaling expression analysis and genomic characterization of human melanoma differentiation associated gene- , mda- : a novel type i interferon-responsive apoptosis-inducing gene the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses '-triphosphate rna is the ligand for rig-i rig-i-mediated antiviral responses to single-stranded rna bearing '-phosphates recognition of ' triphosphate by rig-i helicase requires short blunt doublestranded rna as contained in panhandle of negative-strand virus '-triphosphate rna requires base-paired structures to activate antiviral signaling via rig-i structural basis of rna recognition and activation by innate immune receptor rig-i structural basis for the activation of innate immune pattern-recognition receptor rig-i by viral rna structural insights into rna recognition by rig-i molecular mechanism of signal perception and integration by the innate immune sensor retinoic acid-inducible gene-i (rig-i) a structural basis for discriminating between self and nonself double-stranded rnas in mammalian cells length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-i and melanoma differentiation-associated gene nonself rna-sensing mechanism of rig-i helicase and activation of antiviral immune responses the thermodynamic basis for viral rna detection by the rig-i innate immune sensor rig-i detects viral genomic rna during negative-strand rna virus infection incoming rna virus nucleocapsids containing a '-triphosphorylated genome activate rig-i and antiviral signaling differential roles of mda and rig-i helicases in the recognition of rna viruses innate immunity induced by composition-dependent rig-i recognition of hepatitis c virus rna preference of rig-i for short viral rna molecules in infected cells revealed by next-generation sequencing epstein-barr virus-encoded small rna induces il- through rig-i-mediated irf- signaling adenovirus virus-associated rnas induce type i interferon expression through a rig-i-mediated pathway the ' ends of rna oligonucleotides in escherichia coli and mrna degradation extracellular and intracellular pattern recognition receptors cooperate in the recognition of helicobacter pylori ifngamma inhibits the cytosolic replication of shigella flexneri via the cytoplasmic rna sensor rig-i essential role of mda- in type i ifn responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus structural basis for dsrna recognition, filament formation, and antiviral signal activation by mda double-stranded rna is produced by positive-strand rna viruses and dna viruses but not in detectable amounts by negative-strand rna viruses activation of mda requires higher-order rna structures generated during virus infection mda detects the double-stranded rna replicative form in picornavirus-infected cells innate immune response after adenoviral gene delivery into skin is mediated by aim , nalp , dai and mda visualisation of direct interaction of mda and the dsrna replicative intermediate form of positive strand rna viruses activation of ifn-&# ; expression by a viral mrna through rnase l and mda new insights into the role of rnase l in innate immunity ribose '-o-methylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda mavs forms functional prion-like aggregates to activate and propagate antiviral innate immune response intracellular pathogen detection by rig-i-like receptors the adaptor mavs promotes nlrp mitochondrial localization and inflammasome activation recognition of rna virus by rig-i results in activation of card and inflammasome signaling for interleukin beta production type i ifn triggers rig-i/tlr /nlrp -dependent inflammasome activation in influenza a virus infected cells the rig-i-like receptor lgp recognizes the termini of double-stranded rna the regulatory domain of the rig-i family atpase lgp senses doublestranded rna lgp is a positive regulator of rig-i-and mda -mediated antiviral responses atp hydrolysis enhances rna recognition and antiviral signal transduction by the innate immune sensor, laboratory of genetics and physiology (lgp ) lgp plays a critical role in sensitizing mda- to activation by double-stranded rna from unwinding to clamping -the dead box rna helicase family dexd/h-box rna helicases as mediators of anti-viral innate immunity and essential host factors for viral replication dead/h box (ddx ) helicase binds the rig-i adaptor ips- to up-regulate ifn-beta-inducing potential viral targeting of dead box protein reveals its role in tbk /ikkepsilon-mediated irf activation hepatitis c virus core protein abrogates the ddx function that enhances ips- -mediated ifn-beta induction dhx pairs with ips- to sense double-stranded rna in myeloid dendritic cells ddx , ddx , and dhx helicases form a complex with the adaptor molecule trif to sense dsrna in dendritic cells aspartateglutamate-alanine-histidine box motif (deah)/rna helicase a helicases sense microbial dna in human plasmacytoid dendritic cells the dhx rna helicase senses cytosolic rna and activates the nlrp inflammasome the interaction between the helicase dhx and ips- as a novel pathway to sense double-stranded rna and rna viruses in myeloid dendritic cells ddx , a dexd/h box helicase, is a novel antiviral factor promoting rig-i-like receptor-mediated signaling cytosolic sensing of viruses interferon-inducible antiviral effectors '-triphosphate-dependent activation of pkr by rnas with short stem-loops ifit is an antiviral protein that recognizes '-triphosphate rna structural basis for viral '-ppp-rna recognition by human ifit proteins ifit inhibits japanese encephalitis virus replication through binding to ' capped '-o unmethylated rna '-o methylation of the viral mrna cap evades host restriction by ifit family members '-o methylation of the viral mrna cap by west nile virus evades ifit -dependent and -independent mechanisms of host restriction in vivo crystal structure of isg reveals a novel rna binding structure and potential functional mechanisms activation of innate immune antiviral responses by nod critical role for cryopyrin/nalp in activation of caspase- in response to viral infection and double-stranded rna the nlrp inflammasome mediates in vivo innate immunity to influenza a virus through recognition of viral rna the cytosolic nucleic acid sensor lrrfip mediates the production of type i interferon via a betacatenin-dependent pathway characterization of lrrfip patterns of oligonucleotide sequences in viral and host cell rna identify mediators of the host innate immune system oligonucleotide motifs that disappear during the evolution of influenza virus in humans increase alpha interferon secretion by plasmacytoid dendritic cells the biased nucleotide composition of hiv- triggers type i interferon response and correlates with subtype d increased pathogenicity nucleoside modifications modulate activation of the protein kinase pkr in an rna structure-specific manner native tertiary structure and nucleoside modifications suppress trna's intrinsic ability to activate the innate immune sensor pkr inosine-containing rna is a novel innate immune recognition element and reduces rsv infection the mechanism of eukaryotic translation initiation and principles of its regulation targeting of immune signalling networks by bacterial pathogens processing of genome ' termini as a strategy of negative-strand rna viruses to avoid rig-i-dependent interferon induction short doublestranded rnas with an overhanging ' ppp-nucleotide, as found in arenavirus genomes, act as rig-i decoys a new evolutionary law identification of modifications in microbial, native trna that suppress immunostimulatory activity the '-o-methylation status of a single guanosine controls transfer rna-mediated toll-like receptor activation or inhibition viral reorganization of the secretory pathway generates distinct organelles for rna replication tick-borne encephalitis virus delays interferon induction and hides its double-stranded rna in intracellular membrane vesicles delayed cytosolic exposure of japanese encephalitis virus double-stranded rna impedes interferon activation and enhances viral dissemination in porcine cells formation of membrane-defined compartments by tick-borne encephalitis virus contributes to the early delay in interferon signaling x-ray structure of ns from a highly pathogenic h n influenza virus the genome-linked protein vpg of vertebrate viruses -a multifaceted protein ebolavirus vp uses a bimodal strategy to bind dsrna for innate immune suppression structure of the lassa virus nucleoprotein reveals a dsrna-specific ' to ' exonuclease activity essential for immune suppression structures of arenaviral nucleoproteins with triphosphate dsrna reveal a unique mechanism of immune suppression the c proteins of human parainfluenza virus type limit double-stranded rna accumulation that would otherwise trigger activation of mda and protein kinase r the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord- -yovp squ authors: duan, liangwei; zheng, qianqian; zhang, hongxia; niu, yuna; lou, yunwei; wang, hui title: the sars-cov- spike glycoprotein biosynthesis, structure, function, and antigenicity: implications for the design of spike-based vaccine immunogens date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: yovp squ the ongoing pandemic of coronavirus disease (covid- ), caused by severe acute respiratory syndrome coronavirus (sars-cov- ), poses a grave threat to global public health and imposes a severe burden on the entire human society. like other coronaviruses, the sars-cov- genome encodes spike (s) glycoproteins, which protrude from the surface of mature virions. the s glycoprotein plays essential roles in virus attachment, fusion and entry into the host cell. surface location of the s glycoprotein renders it a direct target for host immune responses, making it the main target of neutralizing antibodies. in the light of its crucial roles in viral infection and adaptive immunity, the s protein is the focus of most vaccine strategies as well as therapeutic interventions. in this review, we highlight and describe the recent progress that has been made in the biosynthesis, structure, function, and antigenicity of the sars-cov- s glycoprotein, aiming to provide valuable insights into the design and development of the s protein-based vaccines as well as therapeutics. the coronavirus disease (covid- ) global pandemic represents an unprecedented public health, social and economic challenge ( , ) . the etiological agent of covid- is a new member of the coronaviridae family that is closely related to severe acute respiratory syndrome coronavirus (sars-cov) and was recently referred to as sars-cov- by the coronavirus study group of the international committee on taxonomy of viruses ( ) . the virus has spread rapidly and sustainably around the global resulting in over twenty-one million cases and more than , deaths as of august , ( ) . coronaviruses (covs) are enveloped positive-sense rna viruses ( ) . enveloped covs entering host cells and initiating infection is achieved through the fusion of viral and cellular membranes ( , ) . membrane fusion is mediated by the large type i transmembrane s glycoprotein on the viral envelope and the cognate receptor on the surface of host cells ( ) ( ) ( ) . the surfaceexposed location of the s glycoprotein not only allows it to carry out membrane fusion but also renders it a direct target for host immune responses, making it the major target of neutralizing antibodies ( ) . because of its central roles in viral infection and eliciting protective humoral and cell-mediated immune responses in hosts during infection ( ) , the s protein is the primary target for vaccine design as well as antiviral therapeutics ( ) . here, we provide a comprehensive overview of the wealth of research related to the sars-cov- s glycoprotein biosynthesis, structure, function, and antigenicity, aiming to provide useful insights into the design and development of the s protein-based vaccines as well as therapeutics to prevent or treat the ongoing global spread of sars-cov- /covid- . the sars-cov- s glycoprotein is synthesized as a -amino acid polyprotein precursor on the rough endoplasmic reticulum (rer) (figure ) ( ) . the unprocessed precursor harbors an endoplasmic reticulum (er) signal sequence located at the n terminus, which targets the s glycoprotein to the rer membrane and is removed by cellular signal peptidases in the lumen of the er ( , ) . a single stop-transfer, membrane-spanning sequence located at the c terminus of the s protein prevents it from being fully released into the lumen of the er and subsequent secretion from the infected cell ( , ) . co-translationally, n-linked, highmannose oligosaccharide side chains are added during synthesis ( , ) . shortly after synthesis, the s glycoprotein monomers trimerize, which might be thought to facilitate the transport from the er to the golgi complex. once in the golgi complex, most of the high-mannose oligosaccharide side chains are modified to more complex forms ( , ) , and o-linked oligosaccharide side chains are also added ( , ) . in the trans-golgi network, the sars-cov- s glycoprotein is proteolytically cleaved by cellular furin or furin-like proteases at the s /s cleavage site, comprising multiple arginine residues that are not found in the closely related sars-cov ( , ) . cleavage at the s /s site yields a surface subunit s , which attaches the virus to the host cell surface receptor, and a transmembrane subunit s , which mediates the fusion of viral and host cell membranes ( ) . the s and s subunits remain associated through noncovalent interactions in a metastable prefusion state ( ) . furin-like cleavage is essential for the sprotein mediated cell-cell fusion and viral infectivity, and is required for efficient sars-cov- infection of human lung cells ( ) and airway epithelial cells ( ) . following cleavage, an er retrieval signal (errs) consisting of a conserved kxhxx motif ( ) located at the extreme c terminus ensures that the mature sars-cov- s protein accumulates near the er-golgi intermediate compartment (ergic) ( , ) , where driven by interactions with another structural protein, the membrane (m) protein, the s protein participates in virus particle assembly and is incorporated into virus envelope ( figure ) ( , ) . besides, a fraction of mature sars-cov- s proteins travel through the secretory pathway to the plasma membrane, where they can mediate fusion of infected with uninfected cells to form multinucleated giant cells (syncytia) ( , ) . this may allow direct spreading of the virus between cells and potentially alter the virulence of sars-cov- ( ) . notably, a deletion of~ amino acid containing the errs from the cytoplasmic tail of the sars-cov- s protein has been shown to increase the infectivity of single-cycle vesicular stomatitis virus (vsv)-s pseudotypes ( ) and replicationcompetent recombinant vsvs bearing the s glycoprotein ( , ) , which likely could be translated to single-cycle human immunodeficiency virus (hiv)-s or other retrovirus-s pseudotypes straightforward ( ) . presumably, this deletion may enhance the cell surface expression of the sars-cov- s glycoprotein ( ) , thereby facilitating the s protein incorporation into pseudovirions and replication-competent virions. as mentioned above, the sars-cov- s glycoprotein plays pivotal roles in viral infection and pathogenesis. mature s glycoprotein on the viral surface is a heavily glycosylated trimer, each protomer of which is composed of amino acids (residues - ) ( figure a) . the surface subunit s is composed of amino acids (residues - ) and organized into four domains: an n-terminal domain (ntd), a c-terminal domain (ctd, also known as the receptor-binding domain, rbd), and two subdomains (sd and sd ) ( figure a ) ( ) . the transmembrane s subunit is composed of amino acids (residues - ) and contains an n-terminal hydrophobic fusion peptide (fp), two heptad repeats (hr and hr ), a transmembrane domain (tm), and a cytoplasmic tail (ct), arranged as fp-hr -hr -tm-ct ( figure a ) ( ) . as a typical class i viral fusion protein ( ) , the sars-cov- s glycoprotein shares common structural, topological and mechanistic features with other class i fusion proteins, including hiv envelope (env) glycoprotein and influenza virus haemagglutinin (ha) ( ) ( ) ( ) . like other class i viral fusion proteins, the sars-cov- s glycoprotein is also a conformational machine that mediates viral entry by rearranging from a metastable unliganded state, through a prehairpin intermediate state, to a stable postfusion state ( , ) . since the first genome sequence of sars-cov- became publicly available ( ) , a number of structures have been determined for the sars-cov- s glycoprotein trimer fragments in both the prefusion and postfusion states ( figures b-d) ( , , ) . the overall architecture of the prefusion sars-cov- s ectodomain stabilized by two consecutive proline mutations in two conformations determined by single particle cryo-electron microscopy (cryo-em) is a~ Å long trimer with a triangular cross-section, with the s subunit adopting a "v" shape contributing to the overall triangular appearance and the s subunit forming the stalk ( figures b, c) ( , ) . the structural difference between these two conformations only lies in the position of one of the three s rbds ( figures b, c) ( ) . when all three rbds are in the "down" position, the resulting s ectodomain trimer assumes a closed conformation, in which the receptor-binding surface of the s rbd is buried at the interface between protomers and cannot be accessible by its receptor ( figure b ) ( ) . the s ectodomain trimer with one single rbd in the "up" position assumes a partially open conformation and represents the functional state, as the receptorbinding surface of the "up" rbd can be fully exposed ( figure c ) ( , ) . the structural information provides a blueprint for structure-based design of vaccine immunogens and entry inhibitors of sars-cov- . in the closed sars-cov- s ectodomain trimer, interprotomer interactions occur through the s ctd packed against the other two s ctds and one ntd from an adjacent protomer because of domain swapping and through s , primarily between helical interactions formed by the upstream and central helices from each subunit around the trimer axis ( figure b ) ( ) . the s subunits rest above the s trimer, the life cycle of sars-cov- begins with membrane fusion occurring at the plasma membrane or within acidified endosomes after endocytosis, which is mediated by conformational changes in the s glycoprotein triggered by angiotensin-converting enzyme (ace ) binding. following viral entry, sars-cov- releases its genomic rna into the host cell cytoplasm. genome rna is first translated into viral replicase polyproteins (pp a and ab), which are further cleaved by viral proteases into a total of nonstructural proteins. a replication-transcription complex (rtc) is formed based on many of these nonstructural proteins. in the process of genome replication and transcription mediated by rtc, the negative-sense (− sense) genomic rna is synthesized and used as a template to produce positive-sense (+ sense) genomic rna and subgenomic rnas. the nucleocapsid (n) structural protein and viral rna are replicated, transcribed, and synthesized in the cytoplasm, whereas other viral structural proteins, including the s protein, membrane (m) protein and envelope (e) protein, are transcribed and then translated in the rough endoplasmic reticulum (rer) and transported to the golgi complex. in the rer and golgi complex, the sars-cov- glycoprotein is subjected to co-translational and post-translational processing, including signal peptide removal, trimerization, extensive glycosylation and subunit cleavage. the n protein is subsequently associated with the positive sense genomic rna to become a nucleoprotein complex (nucleocapsid), which together with s, m, and e proteins as well as other viral proteins, is further assembled and followed by budding into the lumen of the er-golgi intermediate compartment (ergic) to form mature virions. finally, the mature virions are released from the host cell, waiting for a new life cycle to start. this figure is adapted from the template in biorender (https://biorender.com/). stabilizing the later in the prefusion conformation ( figure b ) ( ) . when the s ectodomain trimer adopts a partially open conformation, the rbd in the "up" position will abolish the contacts with the s subunit of an adjacent protomer, destabilizing the partially open conformation ( figure c ) ( , ) . this will be beneficial to the dissociation of the s subunit and facilitate conformational rearrangements that the s trimer undergoes to mediate viral entry. prefusion structures of human coronavirus hku (hcov-hku ) and mouse hepatitis virus s protein ectodomains without two consecutive proline mutations reveal only fully closed conformation ( , ) , similar to that observed for a full-length, wild-type prefusion form of the sars-cov- s glycoprotein ( ) . notably, it is well established that trimeric prefusion hiv- env primarily resides in a closed configuration that is conformationally masked to evade antibody-mediated neutralization ( , ) and can spontaneously sample a transient, functional configuration ( ) . it can thus be speculated that native cov s glycoproteins on mature and infectious virions share a similar conformational masking feature ( ) , concealing the receptor-binding surface (for those utilizing ctds as rbds) ( figure c ), which is further discussed below. several lines of research have established that angiotensinconverting enzyme (ace ) is an entry receptor for sars-cov- ( ) ( ) ( ) . detailed interactions between the sars-cov- rbd and its receptor ace have been revealed by several structures of ace in complex with rbd ( ) ( ) ( ) ( ) . structurally, rbd consists of two subdomains: a core and an external subdomain ( , ) . an extended loop (residues - ), which lies on one edge of the core subdomain, presents a gently concave surface to cradle the n-terminal helix (a ) of ace . analysis of the interface between the sars-cov- rbd and ace reveals that a total of residues in rbd are in contact with amino acids in ace , forming a network of hydrophilic interactions that are suggested to predominate the virus-receptor engagement ( ) . outside this extended loop, residue lys located in helix a of the core subdomain, was shown to form ionic interactions with asp of ace . as the extended loop contains almost all the amino acids of the sars-cov- rbd that contact ace , it is referred to as the receptor-binding motif (rbm) ( ) . it has been proposed that inhibiting the interaction between rbd and ace might be useful in treating sars-cov- infection. recombinant soluble ace ( ) and ace -fc ( , ) have been shown to have potential applications in the prevention and treatment of sars-cov- infection in vitro. as the interaction between the rbd and ace is extensive, small molecules probably cannot be used as entry inhibitors to effectively block the virus entry by targeting the interaction interface. however, peptides would be able to engage most of the residues belonging to rbm ( ) . a pioneering study demonstrated that a -amino acid peptide (residues - ), derived from the n-terminal helix (a ) of ace , specifically associates with the sars-cov- rbd with low nanomolar affinity and disables receptor interactions ( ), representing a promising strategy for preventing the virus from invading human cells. in another study, a -amino acid peptide (residues - ), derived from the n-terminal back-to-back helices (a and ) and composed of most of the residues of ace that mediate interactions with the s protein, shows a similar but probably more potent inhibitory effect ( ) . the formation of a trimer-of-hairpins structure (also known as six-helix bundle) comprising hr and hr in the postfusion conformation is a unifying feature of class i viral fusion proteins ( ) . the crystal structure of a protein construct in which sars-cov- hr and hr were connected by a six-residue hydrophilic flexible linker was determined to be a canonical six-helix bundle structure with a rod-like shape ∼ Å in length and ∼ Å in diameter ( ) . three hr helices form a parallel central coiled-coil with three hr helices packing in an oblique, antiparallel manner against deep hydrophobic grooves on the surface of the central coiled-coil ( ) . notably, when a full-length s protein construct bearing the native furin-like cleavage site was transiently expressed by expi f cells, the purified s proteins contained the dissociated s trimer in the postfusion conformation ( ) . the cryo-em structure of this trimeric postfusion s shows that the central helix (ch) extended regular helices from the central coiled-coil, oriented toward target cells ( figure d ) ( ) , which forms the longest central triple helical coiled-coil (~ Å) among all known class i transmembrane subunit structures. the sars-cov- s trimer in the pre-hairpin intermediate state is very unstable and is just transiently present in vivo after triggering by ace engagement, stymieing structural characterization of the s protein in this state ( ) . however, although this fusion-intermediate phase is very short, it is enough for inhibitory peptides to associate with the prehairpin intermediate and block the six-helix bundle formation ( ) . furthermore, it has already been shown that the hr regions in various human covs are highly conserved ( ) , and therefore could serve as an attractive target for the design and development of potent and broad-spectrum inhibitors of pan-covs, including sars-cov- . a highly potent pan-coronavirus fusion inhibitor, ek c , has been reported to have good prophylactic and therapeutic potential against sars-cov- infection ( ). as mentioned earlier, the sars-cov- s proteins are heavily decorated by heterogeneous n-linked glycans projecting from the s trimer surface. the sars-cov- s sequence encodes up to n-linked glycan sequons per protomer, which likely plays an important role in protein folding ( ) and host immune evasion as a glycan shield ( ) . of the potential n-linked glycosylation sites on the s protein, were identified to be predominantly occupied by processed, complex-type glycans ( ) . the remaining eight sites were found to be dominated by oligomannose-type glycans, which are divergent from those founded on host glycoproteins ( ) . although glycosylation sites (n , n , n ) proximal to the receptor-binding sites on the sars-cov- s protein can be observed, ace bound to the glycosylated and deglycosylated s ectodomains with nearly identical affinity ( . nm vs . nm) determined by a biolayer interferometry binding assay ( ) . this observation suggests that the high binding affinity between the sars-cov- s protein and ace does not depend on the s protein glycosylation. when the site-specific n-linked glycans are mapped onto the prefusion structure of the sars-cov- s ectodomain ( ), the resulting model exhibited substantially higher levels of glycanfree surface than that revealed by structures of fully glycosylated, trimeric hiv- env ectodomains ( , ) . this suggests that the sars-cov- s protein is covered by a less dense and less effective glycan shield compared to viral glycoproteins from hiv- ( , ) and lassa virus ( ) , which may be beneficial for the induction of humoral immunity and could be good news for a sars-cov- vaccine ( ) . notably, it has been shown that multiple major viral surface antigens have neutralizing epitopes that are partly or even exclusively composed of carbohydrate moieties ( , ) , exemplified by the hiv- env spike, which could be recognized by a large number of carbohydrate-binding antibodies, including g , pg , pg , ch , pgt , pgt , pgt , and pgt ( , ) . in the case of sars-cov- , more recently a potent neutralizing antibody against both sars-cov and sars-cov- , s , has been shown to recognize a highly conserved glycan-containing rbd epitope ( ) . these observations suggest that carbohydrate moieties could be immunogenic and highlight the need for immunogens to display the glycans important for the recognition of neutralizing antibodies ( ) ; in support of this, specific n-linked glycans on hemagglutinin has been shown to be essential for the elicitation of broadly neutralizing antibodies against influenza ( ) . accordingly, there has been mounting interest in exploring the potential of immunogenic glycan moieties as vaccine candidates against multiple viruses, including sars-cov- ( , ) . membrane fusion and viral entry of sars-cov- is initiated by binding of rbd in the viral s glycoprotein transiently sampling the functional conformation to ace on the surface of target cells (figure ) ( ). after receptor engagement at the plasma membrane or ensuing virus endocytosis by the host cell ( ), a second cleavage (s ′ cleavage site) is generated, which is mediated by a cellular serine protease tmprss ( ) or endosomal cysteine proteases cathepsins b and l ( ) (figure ) . protease cleavage at s ′ site frees the fusion peptide from the new s n-terminal region, further destabilizes the sars-cov- s glycoprotein and may initiate s -mediated membrane fusion cascade. following the second cleavage, the fusion peptide at the n terminus of the s trimer is inserted into the host membrane ( ) , forming the pre-hairpin intermediate state ( ) . since the pre-hairpin intermediate state is extremely unstable, the s fusion protein is refolded quickly and irreversibly into the stable postfusion state ( , ) . these large conformational rearrangements pull the viral and host cell membrane into close proximity, leading ultimately to the membrane fusion ( , ) . since sars-cov- was identified as the causative agent of covid- , and its first genome sequence was released immediately and freely by a chinese research group ( ), sars-cov- vaccine candidates based on various vaccine platforms, such as inactivated or live attenuated vaccines, dna and mrna vaccines, viral vector-based vaccines, and recombinant protein-based vaccines, have been developed ( , ) . most of these vaccine strategies are based on the full-length s glycoprotein, the major viral surface antigen ( ) . when a vaccine strategy requires that the sars-cov- s protein be recombinantly expressed in the human body, the errs should be omitted to enhance the cell surface expression level of the resulting protein. theoretically, the native hiv- env trimer present on the surface of intact virions is thought to be a most ideal immunogen ( ) , as most of the neutralizing antibodies thus far described could recognize and bind to the prefusion form of trimeric hiv- env, although it is with great difficulty that such neutralizing antibodies against this glycan-covered, sequence-variable native form are induced ( ) . for sars-cov- , different lines of research have shown that convalescent sera from sars-cov and sars-cov- patients showed no or limited crossneutralization activity against these two viruses by pseudotyped and authentic viral infection assays, despite significant crossreactivity in binding to the s glycoproteins of both viruses ( , ( ) ( ) ( ) . similar results were also observed in infected or immunized animals ( , , ) . together with the finding that although the sars-cov- s protein shares a high degree of amino acid sequence identity with that of sars-cov (~ % overall), the rbm is less conserved (~ % identity) than any other functional region or domain ( ) , it can thus been surmised that the rbm has the most immunodominant neutralizing epitope(s) of the whole s protein, capable of readily eliciting strong neutralizing antibody responses. however, the native trimeric sars-cov- s protein could conceal each of its immunodominant rbms by adopting the closed conformation ( , ) . therefore, sars-cov- evades immune surveillance also through conformational masking, which is well-documented for hiv- ( , ) ; while at the same time, the s protein could transiently sample the functional state to engage ace , consistent with the notion that the fusion glycoprotein of highly pathogenic viruses have evolved to perform its functions while evading host neutralizing antibody responses. another concern for vaccine candidates based on the fulllength s glycoprotein of sars-cov- is raised by the observation that the s subunit could spontaneously dissociate from the s glycoprotein probably as a trimer that still assumes the rbd closed conformation, leaving only the postfusion s trimer ( ) . the resulting s and s subunits might expose immunodominant, nonneutralizing epitopes that are utilized by sars-cov- to serve as decoys to distract the host immune system, inducing a large proportion of ineffective antibody responses, as documented for hiv- ( ) and respiratory syncytial virus (rsv) ( ) . it should be noted that although vaccine candidates based on the full-length s protein of the closely related sars-cov could elicit neutralizing antibody responses against infection of sars-cov, they may also induce harmful immune responses, including liver damage of the vaccinated animals, infection of human immune cells by sars-cov, and antibody-dependent enhancement of sars-cov infection ( ) ( ) ( ) ( ) ( ) . therefore, although the s proteins of both sars-cov and sars-cov- are thought to be promising vaccine immunogens for generating protective immunity, optimizing antigen design is critical to ensure an optimal immune response through exposing more neutralizing epitopes and displaying fewer potentially weakly or non-neutralizing epitopes ( ) . vaccines containing or expressing the full-length s protein or its soluble ectodomain form should thus be engineered to sample a rbd(s) "up" conformation while the rest is still kept in the prefusion state ( , ) . apart from recombinant, soluble, stabilized ectodomains that are engineered to expose the immunodominant rbd by adapting the rbd(s) "up" conformation, rbd proteins of sars-cov and sars-cov- have also been widely used as recombinant protein-based vaccines ( , ( ) ( ) ( ) . the rbd of sars-cov is highly immunogenic ( , ) and is targeted by most of the neutralizing monoclonal antibodies that have been characterized ( ) . based on the observation that a -amino acid fragment (residues - ) was previously identified to be the minimal rbd region of sars-cov ( ), a corresponding -amino acid fragment (residues - ) can be readily selected as the minimal rbd region of sars-cov- and has already been characterized ( ) . this minimal form of rbds of both viruses could serve as a vaccine candidate ( ) . however, a conserved cysteine residue is located immediately upstream of the minimal rbd fragments of both viruses and always forms a disulfide bond in nearly all published structures containing this residue ( , ) ; this is also the case for middle east respiratory syndrome coronavirus (mers-cov) ( , ) and hcov-hku ( ), consistent with the observation that all rbds of these viruses share a conserved structural core. the disulfide bond contributes to stabilization of the rbd structure and likely modulates the protein immunogenicity. this notion is consistent with the observation that mice immunized with a longer form of the sars-cov rbd (residues - ) produced a higher titer of neutralizing antibodies compared with mice immunized with the minimal rbd region (residues - ) ( ) . therefore, when each of the minimal rbd fragments of sars-cov and sars-cov- is used as vaccine candidates, the critical cysteine residue should not be ignored and thus should be included ( ) . besides the rbd, which has been shown to a major target for human neutralizing antibody responses ( ), the ntd was recently identified to be a new vulnerable site of the sars-cov- s protein for antibody neutralizing and therefore could also serve as a recombinant protein-based vaccine ( ) ( ) ( ) . as expected, ntd-specific neutralizing antibodies could target the s protein in both closed and open conformations ( ) . in addition, the apparent accessibility of the fusion peptide and hr region in published structures of the sars-cov- s ectodomain trimer as well as their high sequence conservation among covs suggests that they would be good immunogen candidates for epitope-focused vaccine design aimed at raising broadly cov neutralizing antibodies ( ) . the epitope-focused vaccine design has proven to be successful in generating neutralizing antibodies against rsv fusion glycoprotein ( ). however, neutralizing antibodies targeted against these two regions still need to be isolated in infected individuals to support this notion. unlike wild-type full-length s protein of sars-cov- , the above monomeric fragments do not induce any infection-enhancing antibodies or harmful immune or inflammatory responses ( , ) , all of which could be potentially avoided through structure-based immunogen design to improve immunogenicity ( , ) . however, wide-type full-length or soluble ectodomain form of the sars-cov- s protein could trigger stronger cellular immune responses ( ) , which have been demonstrated to play an important role in controlling diseases caused by covs ( , ) , including sars-cov- ( ) , and are probably also an important determinant of effective vaccines against sars-cov- ( , ) . additionally, when more than one rbd of the s protein trimer is engineered to be locked in the "up" conformation ( , ) , the antigenicity and immunogenicity of the resulting rbds would be significantly enhanced compared to monomeric rbd form ( , ) . moreover, improved protection is likely to be achieved when vaccinated with full-length or soluble ectodomain form of the sars-cov- s protein in that both forms can elicit neutralizing antibodies directed against non-rbd sites, as observed for mers-cov ( ) . genetic variation has been used by many viruses that have rna genomes ( ) , including hiv and influenza, as a mechanism to avoid antibody-mediated immunity, and is partially responsible for the great difficulty in developing effective and durable vaccines against these viruses ( ) . as an rna virus, however, sars-cov- has a very low mutation rate overall ( ) likely because covs have a genetic proofreading mechanism ( ) . all reported variations occurred in the sars-cov- s glycoprotein have a prevalence of no more than % ( ) , with an exception of d g, which has become the most prevalent genotype in the global covid- pandemic ( ) . fortunately, although the d g mutation of the sars-cov- s protein has been shown to enhance viral infectivity ( ) ( ) ( ) , until now there is no evidence that infection with sars-cov- carrying the g mutant will be associated with disease severity ( , ) . furthermore, assays using both monoclonal and polyclonal antibodies generated from individuals naturally infected with d -or g -carrying viruses demonstrated that the d g mutation retains or even increases viral susceptibility to neutralization ( , , , ) . this suggests that the d g mutant maintains or favors an open, functional conformational state ( ) . although at an extremely low frequency, natural variations, including l r a v, v a, and f l that render the s glycoprotein resistant to certain neutralizing antibodies targeting the rbd, emerged under no selection pressure exerted by approved vaccines or neutralizing antibodies or entry inhibitors ( , ) . however, it has been shown that sars-cov- escape mutants could be easily selected and quickly amplified under the selection pressure of single antibody treatment ( ) . these observations suggest that a combination of at least two neutralizing antibodies that recognize and bind to distinct and non-overlapping epitopes on the sars-cov- s glycoprotein (e.g., rbd and ntd, as well as hr and glycan) is required to restrict the possible occurrence of viral escape mutants and potential subsequent loss of single antibody-mediated neutralization ( ) ( ) ( ) ( ) . when these observations are taken into consideration for vaccine design and development, an ideal sars-cov- immunogen should contain as many exposed neutralizing epitopes as possible, although the rbd also possesses extra epitope(s) besides the epitope in the rbm region ( , ( ) ( ) ( ) . sars-cov- is a highly contagious pathogen that continues to spread quickly around the globe, causing covid- to be one of the worst pandemics in recorded history. a safe and efficacious vaccine represents one of the best ways to reduce or eliminate the covid- pandemic ( ) . unfortunately, no vaccines for any of the known human covs have been licensed ( , ) , although several potential sars-cov and mers-cov vaccines have advanced into human clinical trials for years ( , ) , suggesting the development of effective vaccines against human covs has always been challenging. however, it has been shown that both sars-cov and sars-cov- could readily induce neutralizing antibodies following natural infection or immunization ( ) ( ) ( ) ( ) . moreover, a growing number of neutralizing monoclonal antibodies targeting the sars-cov- s glycoprotein with high potency have been isolated from plenty of convalescent donors ( ) as well as humanized mice ( , ) , some of which have been shown to afford protection against sars-cov- challenge in animal models. it thus seems that vaccine candidates designed to elicit such neutralizing antibodies are feasible. it is widely accepted that the s protein of sars-cov- is a most promising immunogen for producing protective immunity ( ) . however, it is likely that the s protein has evolved to perform its functions while evading host neutralizing antibody responses and thus should be engineered to ensure an optimal immune response ( , ) . the immunogen design strategies described in this review based on the wealth of the sars-cov- s glycoprotein research related to its biosynthesis, structure, function, antigenicity as well as immunogenicity will likely contribute to the ultimate success of safe and efficacious vaccines against sars-cov- /covid- . covid- : emergence, spread, possible treatments, and global burden highlight of immune pathogenic response and hematopathologic effect in sars-cov, mers-cov, 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antibody signature induced by sars-cov- spike protein immunogens in rabbits progress and prospects on vaccine development against sars-cov- . vaccines (basel) ( ) : what are the most powerful immunogen design vaccine strategies? reverse vaccinology . shows great promise structure-based vaccine antigen design all authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication. we would like to thank prof. xinqi liu for critical reading of the manuscript; and drs. yanbin feng, mengyuan xu, jing ma and jianrong feng for helpful comments and discussions on the manuscript. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © duan, zheng, zhang, niu, lou and wang. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -mvqoyror authors: al-herz, waleed; essa, sahar title: spectrum of viral infections among primary immunodeficient children: report from a national registry date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: mvqoyror objective: to present the frequency and spectrum of viral infections in primary immunodeficient children. methods: the data was obtained from the kuwait national primary immunodeficiency disorders (pids) registry during the period of - . results: a total of pid children were registered in knpidr during the study period with predominance of immunodeficiencies affecting cellular and humoral immunity, followed by combined immunodeficiencies with associated syndromic features and diseases of immune dysregulation. overall infectious complications affected . % of the patients, and viral infections affected . % of the registered patients. forty-five patients ( . %) developed viral infections caused by at least organisms, among those patients were affected by three or more viral infections. there was a statistically significant association between viral infections and pid category. however, there was no statistically significant association between viral infections and gender or the patients' onset age. there was a total of viral infections during the study period and the causes of these infections were predominated by cmv ( . %), adenovirus ( . %), ebv ( . %), and enteroviruses ( . %). cmv and parainfluenza infections were more common in the group of immunodeficiencies affecting cellular and humoral immunity while ebv and human papilloma virus (hpv) were more common in the immune dysregulation group and combined immunodeficiencies with associated syndromic features, respectively. the most common presentation was viremia ( . %) followed by pneumonia ( . %) and skin infections ( . %). the most common causes of viremia were cmv followed by adenovirus and ebv, while the most common organisms causing pneumonia were cmv followed by rhinovirus and parainfluenza. there were deaths among the registered patients, % were caused by viral infections. conclusions: viral infections are common in pids and result into a wide-range of clinical manifestations causing significant morbidity and mortality. primary immunodeficiency disorders (pids) are monogenic defects affecting the innate and/or adaptive immune systems ( ) . patients are at increased risk of a wide range of manifestations including autoimmunity, immune dysregulation, and malignancies, but infectious complications are the commonest ( ) ( ) ( ) . historically, pid patients used to die before recognition because of infections due to the lack of effective measures to either prevent or treat them. advances in public health and the discovery of antimicrobial agents made the diagnosis of pids possible ( ) . although therapeutic interventions like intravenous immunoglobulins and hematopoietic stem cell transplants have helped to decrease morbidity and mortality, physicians caring for pid patients frequently struggle with treating infections which are usually recurrent or chronic, severe and are frequently caused by opportunistic organisms. among these microbes are viruses that in pid patients can be challenging with an increased risk of mortality and may predispose to malignancies ( ) ( ) ( ) . pids may also lead to reduced clearance and prolonged shedding of certain viruses like rhinovirus and poliovirus ( , ) . while most pids predispose to a wide spectrum of viral infections, certain diseases enhance vulnerability to specific viral infections ( ) . whereas, the risk of viral infections in pids is well-established and was recently reviewed ( , ) , we are not aware of any report that characterizes such infections in a large cohort of patients with different types of pids. in this report, we present the frequency and spectrum of viral infections in primary immunodeficient children from kuwait between january and december . the data was obtained from the kuwait national primary immunodeficiency disorders registry (knpidr) which was approved by the research and ethics committee of the ministry of health in kuwait and the kuwait university health sciences center ethical committee in accordance with the declaration of helsinki. the patients were followed prospectively and classified according to the international union of immunological societies, primary immunodeficiency diseases committee report on inborn errors of immunity ( ) ( ). secondary immunodeficiencies (drug induced, hiv induced, and immunodeficiency associated with metabolic disorders... etc.), were ruled out by obtaining a detailed history and by performing appropriate testing when these disorders were suspected. the clinical diagnosis was based on the standard of care depending on the patient's signs and symptoms supported by laboratory and/or radiologic findings. for example, patients who were diagnosed with pneumonia presented with respiratory signs and symptoms associated with radiologic findings. the diagnosis of herpes simplex virus (hsv) keratitis and stomatitis, warts caused by human papilloma virus (hpv), varicella-zoster virus (vzv) and molluscum contagiosum infections was based on clinical evaluation. in-house polymerase chain reaction (pcr) was used initially to test for cytomegalovirus (cmv), epstein-barr virus (ebv), herpesvirus (hhv- ), adenovirus, respiratory and gastrointestinal viruses. this was replaced later by commercial kits as indicated below. for patients with gastrointestinal manifestations stool or colonic samples were collected. deep nasopharyngeal aspirate or bronchoalveolar lavage samples were collected from patients with respiratory manifestations using sterile nylon flocked swab or by bronchoscopy and placed in viral transport medium. serum and cerebrospinal fluid samples were collected for viral infections as required. all samples were labeled and transported the earliest to virology laboratory, faculty of medicine, kuwait university for day-to-day routine screening for viral infections and storage. viral nucleic acid from samples was extracted using an automated magna pure lc . system (roche diagnostic systems, branchburg, nj). hsv ( ) keratitis ( ) hpv ( ) warts ( ) cmv ( ) viremia ( ), pneumonia ( ) adenovirus ( ) viremia ( ), pneumonia ( ) enterovirus ( ) hemophagocytosis ( ), viremia ( ), pneumonia ( ) norovirus ( ) immunodeficiency with centromeric instability and facial anomalies ( ) sapovirus ( ) enteritis ( ) adenovirus ( ) enteritis ( ), viremia ( ) predominantly antibody deficiencies (n = ) btk deficiency ( ) enterovirus ( ) meningo-encephalitis µ heavy chain deficiency ( ) enterovirus ( ) ( ) vzv ( ) chickenpox lyst deficiency ( ) ebv ( ) hemophagocytosis cmv ( ) colitis ( ), viremia ( ) ebv ( ) stool samples were homogenized. prior to extraction, a mg aliquot was suspended in ml of nuclease-free water or ml of stool transport and recovery buffer (roche diagnostics, meylan, france). then the suspension was immediately clarified by centrifugation at , g for min. according to the manufacturer's instructions rt-pcrs was performed on µl of nucleic acid by using lightcycler rt-pcr thermocycler (roche, meylan, france). cmv, ebv, enterovirus, hadv, and hhv- detection were performed on serum, stool/colonic, respiratory or csf samples by lightmix r kits (tib molbiol, berlin, germany). amplifications were performed according to the manufacturer's instructions on lightcycler rt-pcr thermocycler (roche, meylan, france). the presence of poliovirus rna in clinical samples was confirmed by one-step reverse transcription-pcr, followed by a direct sequencing of pcr products, as described previously ( ) . data were processed using ibm spss, version (ibm corporation, armonk, ny, usa ). pearson's chi-square test was used to assess the association between two categorical variables. the non-parametric mann-whitney u-test was applied to assess whether the patients' ages at onset of a symptom of pid have a significant effect on the risk of viral infection. the effect of age at onset was assessed both as quantitative and qualitative variables after dividing them into groups ( - , - , - , - , > months). the p ≤ . was used as the cut-off level for statistical significance. a total of pid children ( males and females) were registered in knpidr during the study period. the distribution of these patients according to pid categories is: immunodeficiencies affecting cellular and humoral immunity, patients ( . %); combined immunodeficiencies with associated syndromic features, patients ( . %); predominantly antibody deficiencies, patients ( . %); diseases of immune dysregulation, patients ( . %); congenital defects of phagocyte number or function, patients ( . %); autoinflammatory disorders, patient ( . %); and complement deficiencies, patients ( %). no patients with defects in innate immunity were registered. seventy-one patients were treated with hematopoietic stem cell transplant (hsct) and received intravenous immunoglobulins. it is important to mention that of the reported viral infections occurred prior to hsct in patients who received such treatment. overall infectious complications affected patients ( . %), and viral infections affected patients ( . % of the registered patients). forty-five patients ( . %) developed viral infections caused by at least organisms, mostly in the category of immunodeficiencies affecting cellular and humoral immunity ( patients). among those, patients were affected by three or more viral infections. there was a statistically significant association between viral infections and pid category after excluding patients who belong to congenital defects of phagocyte number or function, autoinflammatory disorders and complement deficiencies due to low numbers (p < . ) ( table ) . however, there was no statistically significant association between viral infections and gender (p = . ), or the patients' onset age when assessed both as quantitative and qualitative variables (p-values . and . , respectively). there was a total of viral infections during the study period, % were detected at the time of pid diagnosis while % were documented after establishing the diagnosis. the causes of these infections were: cmv ( . %); adenovirus ( . %); ebv ( . %); enteroviruses ( . %), hsv and hpv ( . % each); vzv and rhinovirus ( . % each); molluscum contagiosum ( . %) (figure ) ; norovirus and parainfluenza virus ( % each); h n virus ( . %); rotavirus, rsv, sapovirus, hhv- and corona virus ( . % each). a patient with severe combined immunodeficiency presented with myocarditis caused by poliovirus type . two patients ( with severe combined immunodeficiency and with mhc ii deficiency) had prolonged excretion of poliovirus type in the stool. the details of the viral infections are presented in table . figure shows the number of patients affected by different viruses according to pid categories. the most prominent findings are that cmv and parainfluenza infections are more common in the group of immunodeficiencies affecting cellular and humoral immunity while ebv and hpv are more common in the immune dysregulation group and combined immunodeficiencies with associated syndromic features, respectively. the most common presentation was viremia ( . %) followed by pneumonia ( . %) and skin infections ( . %) ( table ) . the most common causes of viremia were cmv followed by adenovirus and ebv, while the most common organisms causing pneumonia were cmv followed by rhinovirus and parainfluenza ( table ) . in the current study, we present the characteristics of viral infections in a large cohort of pid children who were followed prospectively over a period of years. viral infections affected more than / of the registered patients, many of whom were affected by more than virus. the patients were affected by a range of viral organisms but cmv, adenovirus and ebv were the culprits in almost half of the cases. the high frequency of cmv infections (> %) in our cohort can be explained by the fact that most of the cases are affected by combined immunodeficiencies. patients with such defects are extremely susceptible to progressive infection with cmv ( ) . our finding that cmv and parainfluenza infections are more common in the group of immunodeficiencies affecting cellular and humoral immunity has been documented previously ( , ) . the observation that ebv is more common in the immune dysregulation group specifically triggering hlh is also welldocumented ( , ) . we have found that patients with dock deficiency are particularly predisposed to mucocutaneous viral infections like molluscum contagiosum and hsv infections. this is probably since dock is an important regulator of the actin cytoskeleton that is critical for cell migration through collagendense tissue, hence playing an important antiviral immunity in the skin ( ) . the presented cohort of patients are characterized by the high frequency of combined immunodeficiencies which are more severe with a higher predisposition to viral infections compared to other pid categories. another prominent feature of our cohort is that none of the registered patients suffer from adenovirus ( ) ebv ( ) enteroviruses ( ) hhv- ( ) pneumonia . cmv ( ) rhinovirus ( ) parainfluenza ( ) adenovirus ( ) h n ( ) coronavirus ( ) rsv ( ) ebv ( ) enterovirus ( ) skin infections . hpv ( ) vzv ( ) molluscum contagiosum ( ) gastrointestinal infections . norovirus ( ) enteroviruses ( ) adenovirus ( ) cmv ( ) sapovirus ( ) rotavirus ( increased susceptibility to specific viral infections. examples of such diseases are tlr , trif, or unc b deficiencies which predispose to hsv- encephalitis and epidermodysplasia verruciformis or cxcr deficiencies which predispose to hpv. it is important to stress that physicians should be aware of pids and consider them in patients with severe or recurrent viral infections. importantly, they should be aware that many pids result in poor antibody response, hence serologic testing should be avoided while testing a patient for infectious complications and antigenic detection method should be used instead. health care providers should also be aware of the recommendations for live viral vaccines in immunodeficient patients and their close contacts ( ) . live vaccines, such as the chicken pox, measles, mumps, rubella (mmr), rotavirus, yellow fever, oral polio, and the influenza nasal spray should be avoided in certain types of pids. furthermore, any infants born into a family with a suspicious history of pid should avoid all live viral and bacterial vaccines until pids is ruled out. historically, oral polio vaccine (opv) was the only form used in the vaccination schedule in kuwait. since the first dose of opv given at the age of months was replaced with the inactivated formulation. fortunately, only patient from our cohort who was diagnosed with rag deficiency developed opv related complication (i.e., myocarditis). two more patients with cid had prolonged excretion of poliovirus type in the stool. unfortunately, stool surveillance program of pid patients for vaccine derived polio virus is not available in the country. the present study has some limitations since we did not determine the true burden of viral infections in pids. this could be established by documenting the number of admissions to the intensive care unit and the type of care provided like mechanical ventilation and the use of inotropes during these admissions. other important variables that can be considered are the number of viral infection reactivations, the number of admissions to the hospital, the length of stay, and duration of using antiviral treatments. however, an important strength of the study is that the patients were followed prospectively by the same clinical immunologist. another important strength of the study is that most patients were diagnosed at the molecular level. this may help in determining the genotype-phenotype correlation. yet, collaborative efforts will be needed to collect a bigger number of patients. viral infections in pids should be treated aggressively with appropriate antiviral medications and definitive treatments like hsct when possible since failure to eradicate viral pathogens creates an inflammatory environment that promotes cell survival and proliferation and may predispose to malignancy ( ) . innovative treatments like virus-specific t cells should be explored to improve clinical outcomes for this group of patients ( ) . all datasets generated for this study are included in the manuscript and/or the supplementary files. the data was obtained from the kuwait national primary immunodeficiency disorders registry (knpidr) which was approved by the research and ethics committee of the ministry of health in kuwait and the kuwait university health sciences center ethical committee in accordance with the declaration of helsinki. wa-h: development of the research concept and goals, design of methodology, data collection and analysis, writing the initial manuscript draft, approval of the submitted manuscript, and agreement to be accountable for the content of the work. se: contributed to the research idea, writing of the manuscript and approval of the submitted manuscript and agreement to be accountable for the content of the work. knpidr was funded by kuwait foundation for the advancement of sciences. international union of immunological societies: primary immunodeficiency diseases committee report on inborn errors of immunity primary immunodeficiency disorders in iran: update and new insights from the third report of 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viral infections and primary immunodeficiencies echovirus type is an important cause of viral encephalitis among infants and young children in kuwait respiratory virus infection in immunocompromised patients dock regulates lymphocyte shape integrity for skin antiviral immunity recommendations for live viral and bacterial vaccines in immunodeficient patients and their close contacts inflammation as a tumor promoter in cancer induction virus-specific t cells: current and future use in primary immunodeficiency disorders the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © al-herz and essa. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - lf no u authors: zayed, hatem title: vaccine development against covid- prior to pandemic outbreaks, using in vitro evolution and reverse genetics date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: lf no u nan the coronavirus disease (covid- ) is caused by severe acute respiratory syndrome coronavirus (sars-cov- ), which is an enveloped, non-segmented, positive-sense rna virus ( ) . the complete genome of sars-cov- is . kb ( , ). the virus genome contains four essential proteins that are believed to be important for the infectious ability of the virus, the glycoprotein spike (s), nucleocapsid (n), matrix (m), and small envelope (e) proteins ( ) . the s glycoprotein, which mediates entry of the virus into the target cells, is the main target for host defense antibodies ( ) . as of may, , the covid- pandemic has spread to countries and territories worldwide with nearly million confirmed cases and ∼ % mortality (who.int). as the outbreaks spread, scientists across the globe are racing to develop vaccines against covid- . since coronaviruses are increasing alarmingly, there is an urgent need for a safe and effective vaccine to prevent the spread of the virus during pandemic outbreaks, and stop deaths associated with the virulent covid- . however, developing vaccines that are safe and effective requires a lot of time and testing. it is estimated that months are needed to develop such a vaccine. although it is challenging to predict the severity, time, and location of future coronavirus pandemics, we can be prepared for the highly pathogenic strains that are likely to reemerge and cause future pandemics. this can be done using previous epidemiological studies on coronaviruses. for example, in , chinese scientists anticipated that there would be a potential bat coronavirus that would likely emerge and infect humans, and might cause an imminent outbreak in china ( ) . unfortunately, the efforts of these chinese scientists were met with no interest from the chinese government, evidenced by the lack of proper preparation for the current pandemic when it appeared in china a few months ago. we now know that sars-cov- shares % identity with two sars-like coronaviruses (bat-sl-covzxc and bat-sl-covzc ) that both originated in china, and use the same human angiotensin-converting enzyme receptor for cell entry during the process of infection ( ). if we had reacted to these predictions, then we would very likely have avoided the current crisis. in response to such forewarnings from scientists, a predictive vaccine could have been designed and developed for the potential virus pandemic. developing a vaccine during or after the pandemic outbreaks is too slow to provide timely responses against covid- , and risks many lives. producing an efficient and safe vaccine ready for human use can take up to months, according to the world health organization (who). therefore, anticipating the virus mutations responsible for the possible reemergence of highly pathogenic virulent strains may be a means by which to prepare for future, newly emerging, pandemic strains. the process of preparing a predictive vaccine can be summarized as follows: ( ) the sars-cov- genome would be used as a template for in vitro evolution through dna shuffling techniques ( , ) . random recombination of a viral genome in a test tube mimics the possible assortment and mutations that occur in the virus in nature, creating all possible random recombination. these changes could be incorporated into the four essential viral genes (s, n, m, and e). ( ) these genes would then be subcloned individually into integrating gene delivery vehicles, such as lentiviral vectors ( ) or transposons ( , ) . ( ) using reverse genetic strategies ( , ) , the recombinant constructs would be transfected into cell lines susceptible to coronaviruses ( ) , leading to secretion of viruslike particles (vlps) from the cells into the culture media. ( ) the recombinant vlps could then be harvested and purified from the supernatant of the culture media. ( ) vlps would then be tested for proper assembly and integrity using electron microscopy and different methods of protein quantification. ( ) all possible mutant vlps would be tested using different functional assays to check for possible antigenicity. ( ) the candidate vlps proven to be functional and highly immunogenic could then be used in challenge experiments using animal models and recombinant live virulent viruses believed to be highly pathogenic. ( ) the vlps that are highly protective against the highly pathogenic recombinant strains would be further selected and stable cell lines made from all candidate vlp vaccines. ( ) these cell lines could be expanded using bioreactors and stored for further use. lentiviral vectors could generate a stable cell line that is transgenic for the highly immunogenic antigenic determinants of covid- ( ) , and would be able to continuously secrete vlps into the culture media ( ) . thereafter, during the time of pandemic, suitable stored transgenic cell lines could be used, based on the abbreviations: covid- , coronavirus disease ; sars-cov- , severe acute respiratory syndrome coronavirus ; vlp, virus-like particle; who, world health organization. reemergent pandemic viral mutant strain, and could be easily shipped across the globe, thawed, and manufactured on a large scale in customized large-sized bioreactors (figure ) . vlp vaccines could be used as therapeutic vaccines and administered to infected individuals ( ) , or as vaccines into healthy noninfected individuals. the immunodominant epitopes ( ) of the viral mutants specific for the virus would elicit potent immune responses that could be life-saving ( ) . the genomeless hollow shells would mimic the actual live virus in terms of eliciting a strong immune response; however, these shells are neither replicative nor infectious by themselves ( ) . such a project should be done through international collaborations and under the supervision of the who. stocks of these vlp vaccines could be stored as vials of transgenic cell lines, able to be regularly expanded and checked for their quality and ability to generate vlp vaccines. stocks of these vials could be kept in different countries with satellite distributors managed and administered by the who. this project would require scientists with high degrees of skill that are trained in the field of vaccine design and development, and trained in several other fields such as molecular biology, virology, infectious diseases, and cell biology. the development of vlp vaccines against reemerging viral pandemics would be far affordable than the economic costs of the current covid- pandemic. such project requires concerted global efforts of multiple organizations, which is expected to save thousands of lives. i do believe that the time has come for all government officials and policymakers to listen very carefully to science and scientists' recommendations to ensure the health and well-being of people of our planet. hz conceptualized the study and wrote the manuscript. a novel coronavirus from patients with pneumonia in china a new coronavirus associated with human respiratory disease in china genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding origin and evolution of pathogenic coronaviruses structure, function, and antigenicity of the sars-cov- spike glycoprotein bat coronaviruses in china dna shuffling and family shuffling for in vitro gene evolution discovery of human-like lasparaginases with potential clinical use by directed evolution. sci rep membrane embedded hiv- envelope on the surface of a virus-like particle elicits broader immune responses than soluble envelopes a lentiviral vector expressing japanese encephalitis virus-like particles elicits broad neutralizing antibody response in pigs the sleeping beauty transposable element: evolution, regulation and genetic applications rna virus reverse genetics and vaccine design hemagglutinin amino acids related to receptor specificity could affect the protection efficacy of h n and h n avian influenza virus vaccines in mice sars-associated coronavirus replication in cell lines rapid generation of stable cell lines expressing high levels of erythropoietin, factor viii, and an antihuman cd antibody using lentiviral vectors production of virus-like particles for vaccination therapeutic vaccines against infectious diseases preliminary identification of potential vaccine targets for the covid- coronavirus (sars-cov- ) based on sars-cov immunological studies development of rabies virus-like particles for vaccine applications: production, characterization, and protection studies lentivirus-based virus-like particles as a new protein delivery tool the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © zayed. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -tx s ggu authors: restori, katherine h.; srinivasa, bharat t.; ward, brian j.; fixman, elizabeth d. title: neonatal immunity, respiratory virus infections, and the development of asthma date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: tx s ggu infants are exposed to a wide range of potential pathogens in the first months of life. although maternal antibodies acquired transplacentally protect full-term neonates from many systemic pathogens, infections at mucosal surfaces still occur with great frequency, causing significant morbidity and mortality. at least part of this elevated risk is attributable to the neonatal immune system that tends to favor t regulatory and th type responses when microbes are first encountered. early-life infection with respiratory viruses is of particular interest because such exposures can disrupt normal lung development and increase the risk of chronic respiratory conditions, such as asthma. the immunologic mechanisms that underlie neonatal host–virus interactions that contribute to the subsequent development of asthma have not yet been fully defined. the goals of this review are ( ) to outline the differences between the neonatal and adult immune systems and ( ) to present murine and human data that support the hypothesis that early-life interactions between the immune system and respiratory viruses can create a lung environment conducive to the development of asthma. are well known and include, among others, respiratory syncytial virus (rsv), influenza a/b viruses, rhinoviruses (rvs), human metapneumovirus, parainfluenzaviruses - (piv), bocaviruses, coronaviruses, and certain adenovirus strains. while infant-pattern immune responses may enhance susceptibility to some infections, mounting adult-type responses may not be optimal for overall development either. the neonatal immune system must first and foremost distinguish between self and nonself, and then rapidly proceed to distinguish between benign or even "helpful" non-self (e.g., the commensal microbiome) and potential pathogens. during this period of immune education, the developing immune system has to strike a delicate and pathogenspecific balance between the induction of potentially damaging, pro-inflammatory responses mediated by t helper (th) cell -type (th ) and some th -type cells, less damaging inflammatory responses (e.g., th -type) or even suppressive responses mediated by regulatory t cells (treg) . although the induction of suppressive responses may seem counterintuitive when dealing with potential pathogens, such responses appear to be essential to protect the fetus from reacting too vigorously to maternal antigens ( ) or to colonization of the neonatal gut and other body surfaces with the normal microbial flora ( ) . the "cost" to the infant of mounting an aggressive, pro-inflammatory response can be very high. early-life inflammation has been associated with a wide range of negative long-term consequences including overall development of the body and the brain, neuro-psychological reactions (e.g., to pain) and patterns of inflammatory response ( , ) [reviewed in ref. ( , ) ]. it is, therefore, entirely plausible that early-life experiences with respiratory pathogens can have long-lasting effects on the lung. a number of epidemiological studies have linked respiratory viral infection during infancy with the later development of asthma [reviewed in ref. ( ) ( ) ( ) ]. it is currently unknown whether this association is a direct effect of viral replication in respiratory tissues or results from a virus-induced exacerbation of an underlying predisposition to atopy. however, there is evidence from both human and animal studies to support both hypotheses. the purpose of this review is to examine the immunology of the host-virus interaction during the neonatal period in the context of asthma development. we will first briefly describe how the neonatal immune system differs from that of the adult. because rsv, influenza, and rvs cause a large proportion of respiratorytract infections in neonates, we will focus primarily on these three pathogens as models to better understand how early-life infection and antiviral immune responses might contribute to the subsequent development of asthma. adaptive immunity in the neonate prior to the s, it was commonly thought that the neonatal immune system was in a state of tolerance ( ) , as demonstrated by mouse experiments in the s showing the apparent lack of recall responses to antigens injected soon after birth ( ) . indeed, neonates were considered by some to be "immunodeficient" ( ) . upon discovery of different patterns of t cell response in the mid- s [e.g., th vs. th type: reviewed in ref. ( ) ], it subsequently became clear that what appeared to be a deficiency in responding to recall antigens was instead a reflection of an intrinsic th /treg bias in neonatal mice ( , ) . a quarter of a century later, th -and th -type responses are two of the most well characterized aspects of cellular immunity. the classical th response is characterized by production of the cytokines interferon (ifn)γ and interleukin (il)- , whereas th responses are typified by production of il- and il- . th -type responses tend to be pro-inflammatory in nature, with increases in cd + t cells, and are most often associated with challenges posed by intracellular organisms, including viruses. th responses typically play a central role in defending against invasive helminths, but are also important in modulating potentially damaging inflammatory responses as well as in driving allergic pathologies (e.g., atopy, asthma). other more recently recognized patterns of immune response have also been implicated in either promoting or preventing the development or evolution of asthma, including th -, th -, and treg. in particular, th -and th driven responses may promote exacerbations of severe asthma [reviewed in ref. ( ) ] and/or promote allergic airways disease through interactions with th cells ( , ) . tregs, on the other hand, play a central role in limiting excessive reactivity to self and innocuous allergens ( ) . while th skewing may be a natural or default response pattern to microbial insults in the neonate, the neonatal immune system can induce adult-like, th /th balanced or even th predominant responses under certain conditions ( , , ) . experimental situations in which such responses can be generated typically require potent th inducers such as cpg motifs found in dna vaccines ( ) and oligonucleotide adjuvants ( , ) , mycobacterial cell wall antigens either in the form of live bacille calmette-guerin (bcg) ( ) or complete freund's adjuvant ( ) , or lipid-based adjuvants, such as liposomes ( ) and oil-in-water emulsions ( ) . several of these powerful stimuli have recently been shown to act via ligation of pattern recognition receptors (prr) [e.g., toll-like receptor (tlr) in the case of cpg motifs] ( ) . even when an apparently "balanced" th /th response has been generated in neonates, recall responses may still reveal an underlying th bias ( ) . the mechanisms by which subsequent antigen recall results in a th -biased response are still under active investigation. neonatal mouse t cells produce significantly higher levels of il- within h of in vitro t cell receptor (tcr) stimulation ( ) compared to adult t cells, which typically take several days to produce high levels of th cytokines ( ) . this readiness to produce th cytokines has been attributed to hypomethylation of the th cytokine regulatory region on chromosome in neonatal murine cd + t cells. a similar state of epigenetic control over th cytokine production has also been observed in human neonatal cd + t cells ( ) . yoshimoto and colleagues have recently shown that epigenetic modifications of the th regulatory regions occur during fetal development in the mouse ( ) . the th regulatory regions in the -day fetal thymus are hypomethylated and adult-like methylation patterns appear only - days after birth along with a decreased propensity to produce th cytokines. interestingly, this hypomethylation is restricted to the th regulatory regions, and is not seen in the regulatory regions of the ifnγ or foxp loci. other factors that could potentially influence th -biased immune responses in neonates include relative antigen loads ( ) , lower total numbers of t cells in neonatal organs ( ) and the presence of fetal-origin t cells that are strongly th -skewed ( ) . cross-regulation between th subsets is well known and may be particularly effective in the very young. for example, there is evidence that th -type signaling is actively suppressed by th cytokines in the context of both antigen challenge and recall in neonates. using tcr transgenic mice, li et al. showed not only that recall responses are primarily th -like but that these responses are accompanied by apoptosis of antigen-specific th cells ( ) . this apoptosis of th cells is driven predominantly by il- , is enhanced by il- , and occurs only in neonatal t cells ( ) . neonatal (but not transplanted adult) th cells co-express il- rα and il- rα. upon exposure to il- after antigen challenge, th cells that carry this heteroreceptor undergo apoptosis ( ) , resulting in a th -biased antigen recall response. il- rα upregulation on these th cells is due to relatively low frequencies of il- -producing (cd α + cd − ) dendritic cells (dcs) in the neonate-a phenomenon that is reversed by day of life when cd α + cd − dcs mature and begin to secrete il- ( ) . by ~ days after birth, neonatal murine t cells respond to gradual increases in il- by upregulating il- rβ and suppressing il- rα , contributing to the sustained survival of antigen-specific th -type t cells ( ) . although the differences between neonatal and adult t cell responses are particularly striking, the capacity to mount competent humoral responses also varies considerably with age as different "waves" of b cell development occur through early-life ( ) . for example, antibody responses to t cell independent antigens such as polysaccharide antigens can be very weak during the first years of life. compared to the adult humoral response to any given antigen, the neonatal response is characterized by generally lower levels of antibodies produced with a delayed onset, less efficient antibody affinity maturation ( ) and higher levels of b cell apoptosis ( ) . despite these clinical observations, major differences between neonatal and adult b cells have not yet been identified at the molecular level. as a result, the relative "defects" in antibody production for t cell independent antigens are likely attributable to other factors such as delayed appearance of b cells fully competent to handle such antigens or deficiencies in the organization of secondary response sites, such as lymphoid and follicular centers, which develop only a few weeks after birth in mice [reviewed in ref. ( ) ]. innate immunity in the neonate striking differences in innate immunity between neonates and adults have also been characterized, particularly with regard to il- , a key innate th cytokine. the half-life of il- p mrna is decreased in cord blood mononuclear cells and these cells produce far less il- than adult peripheral blood mononuclear cells (pbmc) in response to lps ( ) . the addition of il- to cord blood cultures increases th -type responses to recall antigens in vitro ( ) , increases the activity of natural killer (nk) cells and enhances ifnγ production ( ) , and suppresses the induction of il- rα ( ) . however, the administration of supplemental il- to -week-old mice in vivo is not well tolerated, suppressing weight gain and increasing mortality compared to older mice ( ) . dcs are the main producers of il- ( ) and neonatal mice have fewer splenic dcs than adult animals ( ) , as well as weaker responses to antigen stimulation in vitro and in vivo ( ) . it is interesting that neonatal b cells can suppress both il- production and signaling in neonatal dcs in an il- -dependent manner ( , ). because dcs are positioned at the nexus of the innate and adaptive immune responses, the characteristics of neonatal dcs are likely central to the relatively weak th responses observed in neonates. monocyte-derived dcs from human cord blood have decreased markers of activation, as well as lower il- p mrna expression upon stimulation with ligands such as lps, cd ligation, or poly i:c, and are poor inducers of ifnγ from adult t cells ( ) . neonatal dcs also produce less ifnα and ifnβ than adult cells ( , ) . recently, it has been suggested that poor production of ifn by early-life dcs is due, at least in part, to posttranscriptional downregulation of tlr / signaling and increases in the regulatory mirnas, mir and mir ( ) . one important outcome of decreased type ifn signaling would be diminished ifnγ responses in neonates. both cell surface and cytoplasmic prrs, including tlrs, nod-like receptors, and retinoic acid induced gene i (rig-i)like receptors, play important roles in the recognition of common microbial products (e.g., lps, double stranded rna) and in triggering immune responses in key innate effector cells such as monocytes and dcs. responses of cord blood dcs to tlr ligation are both qualitatively and quantitatively different from adult dc responses, despite having similar levels of mrna ( , ) . using agonists specific to each tlr, kollmann et al. ( ) studied single cell responses of dcs and monocytes in human cord blood compared to neonatal or adult cells differentiated from pbmc. overall, the neonatal monocytes and dcs produced more il- , il- , and il- β, which can induce th differentiation/activity, as well as il- , a classic immunosuppressive cytokine, but less of the th -associated cytokines: ifnα, ifnγ, tumor necrosis factor (tnf)α, and il- p . in another study of early-life responses, the ratio of tlr-induced il- /tnfα production by neonatal monocytes was high compared to adult cells. newborn serum was found to have a similarly skewed ratio of these cytokines ( ) . serial measurements in human infants suggest that the ratio of tlr-driven inflammatory (e.g., ifnα, il- p , ifnγ) vs. suppressive/regulatory cytokines (e.g., il- ) shifts slowly toward "adult" values over the first years of life ( ) . several factors likely contribute to the sluggish activity of tlrs in neonatal immune cells, including lower myd levels ( ) , decreased dna and coactivator binding of ifn regulatory factor ( ), and decreased nuclear translocation of ifn regulatory factor ( ) . together, these data suggest that stimulation of innate immune cells in neonates/infants favors th /th and possibly treg differentiation rather than th development. this age-related bias in t cell education could plausibly play a pivotal role in the subsequent development of asthma. there are still other differences between neonatal and adult innate response capabilities. for example, fetal and the neonatal invariant natural killer t cells (inkt) cells produce equal amounts of ifnγ and il- upon receptor stimulation in sharp contrast to adult inkt cells that produce predominantly ifnγ ( ) . although nk cells are present in higher numbers in the peripheral blood of neonates compared to adults, they express more inhibitory receptors, have truncated maturation and have lower overall functionality ( ) . the nk maturation defect in neonates has also recently been linked to transforming growth factor-beta (tgfβ), since murine nk cells engineered to lack tgfβ receptor were capable of fully maturing by days after birth ( ) . neonatal macrophages, upon stimulation with lps and polysaccharide antigens, produce more il- than adult macrophages, and secrete less il- β, il- , tnfα, probably due to weak tlr signaling ( ) . neonatal macrophages are also less responsive to ifnγ due to a defect in stat phosphorylation ( ) despite having adult levels of phagocytic function ( ) . walk and colleagues studied the expression of a number of inhibitory receptors on neonatal cells, and found increased expression of lair- , cd , and cd on a wide range of innate immune cells including neutrophils, monocytes, and nk cells as well as cd + and cd + t cells ( ) . at least some of the apparent "defects" in neonatal immune cell function can be overcome using specific stimuli either alone or in combination. as mentioned above, tlr signaling can drive a strong pro-inflammatory response from neonatal monocytes but can also induce neonatal nk cells to produce adult levels of ifnγ in an il- -dependent fashion ( ). during the postnatal period, the lung continues maturation begun in utero and undergoes alveolarization (mice, pnd - ; humans, weeks preterm to - years) and microvascular maturation (mice, pnd - mice; humans, - years) ( , ) . a strong type- immune cell bias characterizes the lung mucosa during alveolarization in mice as populations of innate type lymphoid cells (ilc ), mast cells, eosinophils, and basophils increase ( ) . furthermore, a large proportion of tissue resident alveolar macrophages, unique populations that are self-maintained throughout life and which develop from fetal liver monocytes beginning at pnd in mice ( ) , express cd , indicative of a type- alternatively activate phenotype ( ) . conventional cd b + neonatal murine lung dcs, though few in number, process antigen more efficiently and more readily express ccr ( , , ) than adult dcs and, compared to cd + dcs, preferentially migrate to the draining (mediastinal) lymph node and promote th responses ( ) . in mice, lung delivery of house dust mite (hdm) extracts at this early stage of development promotes enhanced allergic airways disease [i.e., increased eosinophil recruitment to the lung, airway hyperresponsiveness (ahr)] ( , , ) , in a manner that is dependent upon il- (discussed in detail below) and influenced by preferential differentiation of cd + t cells to a th -type phenotype expressing il- , il- , and il- . in fact, additional studies in mice have investigated canonical t lymphocyte responses in the neonatal vs. adult lung ( ) . in response to anti-cd treatment in vitro, cd + , cd /cd double negative t cells, which are present in greater frequency during the neonatal period than in adulthood, more readily express gata- and produce more th cytokines, il- and il- ( ) . interestingly, priming of neonatal or adult lung dcs with bcg induced a th response when cocultured with neonatal lymph node t cells, a response that switched to th when cocultured with lymph node t cells from adult mice ( ) . altogether, these data highlight the importance the temporal window of the alveolarization stage of lung development to promote rapid, detrimental th -type inflammatory responses in the lung. compelling new data implicate changes to the lung microbiome in differential regulation of maladaptive type- allergic responses between neonates and adults. the predominance of firmicutes and gammaproteobacteria in the lung is associated with allergic airways disease in both murine models of asthma ( ) and human asthmatics ( ) and are the major families that colonize the lung during the alveolar stage of development. these commensals change throughout ontogeny as the lung milieu adapts to harbor bacteroidetes in adulthood ( ) . interestingly, helios + treg (cd + foxp + cd + ) cells are abundant in the neonatal lung during alveolarization, but are non-tolerogenic as repeated hdm exposure causes robust eosinophilic airway inflammation, ahr, and mucus production associated with allergic airways disease. later in life, when the lung microenvironment shifts to support bacteroidetes, helios − tregs are abundant and successfully promote regulatory, antiinflammatory responses thereby providing protection to adult mice from allergic airways disease upon exposure to hdm ( ) . the neonatal lung is comprised of type innate immune cells and dcs, which rapidly home to the mediastinal lns to educate naïve cd + t cells to develop th responses. furthermore, the postnatal lung supports a microbiome that promotes allergy and ineffectual treg responses. altogether, this type- biased neonatal mucosal lung environment has the potential to create a "perfect storm" for asthma development upon exposure to viruses or allergens. how this naturally biased state might synergize with early-life respiratory infections to create a lung microenvironment that favors the development of asthma is discussed in the next section. allergic asthma is defined as a chronic inflammatory response to inhaled allergens characterized by intermittent airway obstruction, increased th cytokine production, ahr and mucus production. at least million people worldwide have asthma ( , ) . despite intensive study, the etiology of asthma is still poorly understood, although a family history of atopy is consistently shown to be an important risk factor [reviewed in ref. ( , ) ]. genetics undoubtedly play a pivotal role in the development of asthma but genotype is not fully determinative. environmental factors influence not only the overall risk of asthma but also the onset and severity of disease. as outlined above, respiratory infections are major causes of short-term morbidity and mortality in the first years of life ( ) and viral infections are associated with up to % of all asthma exacerbations in the young ( ) . in addition to this apparent "direct" association with exacerbations ( ) ( ) ( ) , it is also possible that early-life exposure to specific viruses acts in young children (< years of age) with a high risk of atopy, both rsv and rv detection in nasal aspirates was associated with asthma development at years of age ( ) wheezing in young children (< years of age) at a high risk for developing asthma (one parent with asthma or respiratory allergies) that tested positive for rsv and rv, was strongly associated (or = ) with asthma at years of age ( ) a greater number of respiratory infections (viral or bacterial) in young children (< years of age) were associated with asthma development at years of age ( ) rsv in a prospective cohort study with matched controls, infants hospitalized with severe bronchiolitis and a family history of atopy/asthma had a greater prevalence of rsv-specific igg antibodies at the first year follow-up and asthma, atopy at the second year follow-up in comparison to the control group ( ) in young children (< years of age) hospitalized with lower respiratory-tract illness, rsv was an independent risk factor for the development of wheezing at , but not at years of age, though no association was found between rsv lower respiratory-tract illness and the development of atopy ( ) severe rsv bronchiolitis during infancy was associated with increased prevalence of allergic asthma at years of age (e.g., increased asthma, sensitization to perennial allergens, persistent/relapsing wheeze in association with early allergic sensitization, reduced spirometric function) wheezing in young children (< years of age) at a high risk for developing asthma (one parent with asthma or respiratory allergies) that tested positive for rsv was associated (or = . ) with asthma at years of age ( ) in twins, - years of age, severe rsv infection does not cause asthma, but rather indicates a genetic predisposition to asthma. hospital discharge registries and parent-completed questionnaires were fitted to genetic variance components models and direction of causation models in a prospective cohort study in twins, - years of age, hospitalization for rsv infection was associated with asthma shortly after discharge and hospitalization for asthma increased long-term susceptibility to severe rsv infection ( ) hospitalization during infancy was associated with the development of childhood asthma: % of asthma prevalence in children hospitalized with rsv vs. % non-hospitalized (overall comparison estimates-systematic review of articles) in young children (< years of age) hospitalized for wheezing respiratory illness, rv detection in nasopharyngeal aspirates was associated (or = . ) with asthma development years later in comparison to children that were rv negative ( ) wheezing in young children (< years of age) at a high risk for developing asthma (one parent has asthma or respiratory allergies) that tested positive for rv was strongly associated (or = . ) with asthma at years of age in a prospective population-based surveillance of children to prime some individuals for development of asthma later in life. indeed, a growing body of epidemiological evidence suggests that early-life respiratory virus infections predispose infants to the development of asthma (see below). murine models of both the induction of ahr and the exacerbation of existing airway disease in neonatal and infant animals are beginning to provide some mechanistic understanding of these phenomena. because rsv, influenza viruses, and rvs are ubiquitous causes of respiratory infection in young infants, these agents will be our primary focus. table provides an overview of studies linking viral respiratory infections and wheeze or asthma in children. background, prevention, and treatment there are two principal antigenic sub-types of rsv (a and b), each with multiple genotypes based on their surface glycoprotein g ( ) . although both sub-types can infect humans, most (> %) symptomatic disease is attributable to type a viruses. these viruses are the leading global cause of serious viral respiratory illness in infants ( , ) . based on seroepidemiology, nearly % of all infants are infected at least once by the age of two. there is no vaccine for rsv and treatment options are limited. although aerosolized ribavirin may provide marginal benefit in severely ill children, this approach is cumbersome and used infrequently ( ) . the monthly prophylactic use of a monoclonal antibody that targets the surface fusion (f) glycoprotein (e.g., palivuz-imab™ and others) can reduce hospitalization rates by about % ( ) as well as the total number of wheezing days in the first year of life ( ) . however, the cost of prophylaxis is so high that this approach is generally restricted to infants at greatest risk of severe disease (preterm infants, infants with immunodeficiency, etc.) [reviewed in ref. ( )]. a broad range of epidemiological data strongly supports the association between early-life rsv-related hospitalization, and the subsequent development of asthma in childhood ( ) with effects lasting into early adulthood ( ) . a recent meta-analysis of infants hospitalized with severe rsv during infancy found a % prevalence of asthma in children ≤ years of age that increased to % in children between the ages and . after years of age, asthma prevalence in these children falls to % but all of these numbers are striking compared to background asthma rates of - % in children with no history of early-life, rsv-associated hospitalization ( ) . the mechanisms that underlie this strong epidemiologic association are not yet fully understood and it has been argued that hospitalization with rsv is simply a "marker" for children genetically predisposed to asthma as opposed to an environmental risk factor for asthma development ( ) . in fact, it is likely that both are true to some degree. given the frequency of rsv infection in the first years of life, hospitalization due to severe infection is relatively rare regardless of genetic background, occurring only when rsv moves into the lower respiratory tract (lrt), causing pneumonia and bronchiolitis ( ) . in most healthy, full-term babies and infants, rsv infection is limited to the upper respiratory tract and causes only mildmoderate symptoms. in a -year study in the us, the rates of rsv-related hospitalization ranged from . per , in infants less than months of age to per , in infants older than year ( ). why some children progress to lrt complications is still unknown, but risk factors include a family history of atopy, preterm birth, congenital heart disease, and low levels of maternal anti-rsv antibodies [reviewed in ref. ( )]. the tissue tropism of rsv is narrow; restricted to cells of the airway epithelia both in vitro and in vivo. rsv entry on the apical side of these cells ( ) is mediated by the viral attachment (g) and fusion (f) glycoproteins ( ) that utilize surface glycosaminoglycans such as heparin and nucleolin ( ) , respectively, as receptors. as outlined above, a th -biased and/or immunosuppressive microenvironment in the lungs is associated with both the development and severity of asthma. there is now considerable evidence that the major rsv surface glycoproteins can directly impact the th / th balance in the lung ( ) . th -biased airway inflammatory responses, including th cytokine production and influx of eosinophils into the airways, are induced upon vaccination of balb/c mice with vaccinia virus expressing the rsv g-protein ( ) ( ) ( ) . compelling data implicate il- , a cytokine associated with the development of asthma ( ) in rsv g-protein-dependent induction of th -biased airway inflammation in these mice ( ) . the rsv f protein may also contribute to the development of a th microenvironment in the lung by signaling through tlr ( , ) and there is strong recent data linking tlr genotype to severe rsv disease in humans ( ) . rsv infection of airway epithelial cells (both immortalized cell lines and primary human cells) in vitro increases expression of tlr ( ) and epithelial cell-specific tlr signaling is required for th -type responses in the murine lung ( ) ( ) ( ) . for example, allergic airways disease induced by hdm depends upon airway epithelial cell-specific tlr expression and is associated with secretion of several innate cytokines associated with th -type responses [e.g., il- , il- , and thymic stromal lymphopoietin (tslp)], as well as the classic th cytokines il- and il- ( ) . rsv infection of bronchial epithelial cells also induces production of tslp via activation of retinoic acid induced gene i (rig-i) and downstream activation of nuclear factor-κb ( ) . importantly, rsv infection of mice sensitized to the model allergen ovalbumin (ova) leads to an increase in th cytokine production in the lung following ova challenge suggesting that the rsv effect on th /th balance in the respiratory tract is not restricted to viral antigens ( , ) . more recently, prior rsv infection of young mice has been shown to enhance allergic airways disease induced by hdm ( ) . recognition of the powerful effects of il- , il- , and tslp produced by respiratory epithelial cells is an important new element in understanding the potential for respiratory viruses to cause and/or exacerbate asthma. these epithelial-origin cytokines play a pivotal role in both the initiation of th type responses and the progression of allergic diseases ( ) . blockade of il- or the absence of its receptor, il- rb, reduces th cytokine production, mucus production, and/or ahr in murine rsv infection and rsv-induced asthma exacerbation models ( ) . moreover, in the absence of nk cells, il- secreted from airway epithelial cells upregulates the notch ligand, jagged on the surface of dcs, which induces the development of an rsv-specific th response ( ) . treatment of murine bone marrow-derived macrophages with exogenous il- or il- can induce production of il- and il- in vitro ( ) . intraperitoneal treatment of mice with il- results in the differentiation of macrophages from the small intestinal lamina propria into an alternatively activated (m or aam) phenotype in a stat -independent fashion. such peritoneal macrophages are major producers of il- in vivo. furthermore, il- links viral infection, macrophages, and ilc dependent production of il- to ahr ( - ) and induces ilc -and il- -dependent dc trafficking to the lymph nodes, promoting th adaptive immunity ( ) . in the case of rsv infection models, il- gene expression as well as the number of leukocytes expressing the il- receptor (st ) increase in adult balb/c mice infected with rsv and interestingly, st blockade decreases lung eosinophil recruitment, il- levels and mucus production, without affecting ifnγ levels ( ) . ilc s are an important source of il- secretion post-rsv infection and evidence that they play a role in enhanced disease is provided by data showing that adoptive transfer of lung ilc s purified from rsvinfected mice h prior to rsv infection dramatically increases production of il- and subsequent eosinophil infiltration ( ) . similarly, il- production in the lungs of rsv-infected neonatal mice contributes to ilc expansion as well as th -biased airway inflammatory responses following rsv re-infection in adults ( ) . evidence that il- may contribute to pathogenesis in human disease is provided by data showing that il- levels are increased in nasal aspirates of infants hospitalized with rsv infection ( ) . similarly, il- mrna levels are greater in nasal aspirates of infants hospitalized with rsv bronchiolitis with a family history of atopy compared to those with no such history ( ) . thymic stromal lymphopoietin likely also plays a role in the development of th responses in rsv infection. in rat primary airway epithelial cell cultures, infection with rsv triggers an immediate increase in tslp mrna and protein, which induces myeloid dcs to express markers associated with th polarization ( ) . ex vivo rsv infection of human airway epithelial cells of healthy children and children with asthma results in tslp induction and has been shown to contribute to th inflammation ( ) . infection of human primary bronchial airway epithelial cells also results in upregulation of functional tslp receptor (tslpr) on these cells, suggesting a feedback loop in which tslp binding to its receptor increases production of more tslp ( ) . tslp also acts directly on dcs. lung dcs, along with alveolar macrophages and ilc s are among the "first responders" to viral infections and allergen exposure in the lung. the relationship between lung epithelial cells and dcs and the development of asthma has been the subject of several recent reviews ( ) ( ) ( ) and provides a framework for understanding how rsv-infected epithelial cells may program lung dcs to promote th immunity. tslp secreted from epithelial cells infected with rsv ( , ) induces ox l expression on the surface of dcs ( ) that, in turn, drives t cells toward a th cell phenotype ( ) . a further link between tslp and il- in rsv infection is provided by evidence that il- enhances the memory th response induced by tslp-activated dcs ( ) . il- also upregulates ox l surface expression on dcs, effectively promoting th immunity in both allergy and rsv infection models ( , ) . tslp signaling in ilc s is also necessary for il- production by these cells as tslpr ko mice and blockade of tslp signaling with anti-tslp neutralizing antibody decrease il- lung protein levels, mucus, ahr and weight loss during rsv infection ( ) . in humans, when rsv infects primary myeloid and plasmacytoid-derived dcs ( ) from healthy volunteers, the viral g-protein decreases dc activation ( ) . suppression of dc maturation has also been observed by the viral non-structural (ns) proteins, ns and ns ( ) . moreover, ns -dependent activity in rsv-infected dcs suppresses the activation and proliferation of both migratory cd + t cells (cd + cd + ) and th cells while supporting the activation and proliferation of th cytokine-producing cd + t cells ( ) . together, these data suggest that rsv infection of epithelial cells and dcs may act synergistically to elicit a th -biased response in both mice and humans. this response pattern is likely accentuated by the already th -biased nature of the neonate. yet another piece of this puzzle may be the induction by rsv of long-lived, lung-resident macrophage populations. rsv infection in adult mice induces polarization of macrophages toward an aam (m ) phenotype ( , ) . similar polarization is observed upon rsv infection of peritoneal macrophages ex vivo ( ) . the classification of macrophages is largely based on cytokine production profiles upon activation and, as is the case with th and th lymphocytes, the cytokines ifnγ and il- appear to play central roles in the development of m and m macrophages, respectively. upon activation, m macrophages (also called classically activated macrophages or cam) typically secrete inflammatory cytokines, such as ifnγ, il- , and il- , whereas m macrophages secrete il- , il- , and il- ( ). m macrophages play an important role in tissue repair ( , ) [reviewed in ref. ( ) ] mediated, at least in part, by tgfβ and platelet-derived growth factor. studies in both human infants ( ) and neonatal mice ( ) have demonstrated that the production of tgfβ is increased upon rsv infection. on the one hand, this increase could reflect an adaptive m reparative response to the lung injury caused by rsv infection as shown by shirey and colleagues ( ) . on the other hand, over-enthusiastic repair of virally damaged airways could be maladaptive with longterm changes in macrophage phenotype that promote pathologic changes in airway structure and function ( , , ) . it is, therefore, interesting that rsv infection of neonatal mice is associated with long-term increases in collagen deposition and airway remodeling ( , ) . although the precise mechanisms by which rsv infection induces m polarization are still unknown, it seems likely that production of tslp, il- , and/or il- by rsv-infected or -exposed respiratory epithelial cells contributes to both m differentiation and long-term maintenance of a th biased macrophage population in the lung ( , , , ) . furthermore, as noted above, the ns and ns proteins of rsv inhibit the type-i ifn response in human macrophages ( ) , which could also contribute to the induction of m macrophages. much of the evidence outlined above in support of a th -biasing effect of rsv in the lung has been obtained using adult mice and human cells/cell lines. neither of these strategies is ideal. the limitations of cell lines and even primary cells are obvious since these reductionist models cannot reproduce the complexity of in vivo host-virus interactions. the adult mouse model of rsv infection also has important drawbacks since adult mice are not susceptible to rsv. this model requires the instillation of high titers of virus ( - % tissue culture infective dose/ml) directly into the lrt. furthermore, rsv replication in adult mice is relatively poor and occurs primarily in alveolar pneumocytes rather than the bronchiolar epithelial cells as occurs in humans ( , ) . perhaps most importantly, rsv infection of adult mice induces a potent th response that rapidly clears the virus. the role of rsv-specific th responses in this model may be restricted to the prevention of exaggerated th -mediated lung pathology ( ) . in the case of human clinical data, it is difficult to resolve the "chicken-and-egg" problem: do children with a genetic propensity to mount th responses and develop asthma suffer more severe early-life rsv or does early and severe rsv infection cause the subsequent development of asthma ( , )? in an attempt to address these limitations, several groups, including ours, have developed models of rsv infection in neonatal and young mice with re-infection or exposure to allergens later in life ( , , , , ) . these models demonstrate that early-life rsv infection (within days of birth) elicits little ifnγ production in contrast to adult animals ( ) and leads to a "proasthmatic" phenotype characterized by ahr, mucus production, airway remodeling, and severe disease upon subsequent allergen exposure or live rsv challenge ( , , , ) . consistent with these th -biased responses, il- rα is increased on pulmonary cd + t cells following rsv reinfection of adults ( ) . the exaggerated th -inflammatory response upon re-infection dendritic cells (dcs), macrophages, and innate type lymphoid cell (ilc s) are among the first responders in the lung. each of these cytokines increases activation of dcs, including increased expression of ox l. ox l expressing dcs drive th differentiation in the lung mediastinal lymph nodes. these innate type- cytokines also promote differentiation of m alternatively activated macrophages (aam), which secrete type- cytokines il- , il- , and il- and orchestrate type- inflammation and tissue repair in the lung. m macrophage cytokine/chemokine production contributes to enhanced type- biased inflammation and airway remodeling. il- and il- production from ilc s promotes eosinophil influx and responses in dcs and macrophages that further enhance type- inflammation, th differentiation, and airway hyperresponsiveness (ahr). each of these responses is enhanced in neonates upon exposure to respiratory viruses, setting the stage for enhanced type- inflammation upon exposure to allergens. is dependent upon the age at initial infection and production of both il- and il- ( , ) and is enhanced by the presence of anti-rsv ige antibodies ( ) . specifically targeting th responses in the neonate can reduce these exaggerated responses upon adult rsv reinfection. for example, delivery of antisense oligonucleotides targeting the il- rα ( ) or a cell penetrating peptide targeting the stat transcription factor (activated by both il- and il- ) ( ) to neonatal mice at the time of rsv infection reduces enhanced disease upon rsv re-infection of adults. interestingly, human cord blood cd + t cells also upregulate il- rα upon ex vivo rsv stimulation ( ) . consistent with these data, development of enhanced disease is t cell dependent in the neonatal challenge-adult rechallenge model. however, reduced inflammatory responses are seen only if cd + t cells are depleted at the time of adult re-infection but not during the neonatal exposure ( ) . on the other hand, depletion of cd + t cells during either the neonatal infection or adult re-infection significantly decreases enhanced disease in mice ( ) . how cd + t cells exposed to rsv during neonatal infection promote disease in adult re-infection is unclear, and somewhat paradoxical, given the protective role that cd + t cells are believed to play in recovery from viral infections. it is also possible that cd + t cells play slightly different roles in mouse vs. human disease ( ) . how cd + and cd + t cell populations influence the pathogenesis of rsv infection in reinfected adult mice is intriguing and deserves further evaluation. finally, repeated rsv infection of weanling mice interferes with treg-mediated tolerance and increases susceptibility to allergic asthma ( ) . as described in the first section of this review, the neonatal immune system is characterized by a pre-existing th bias. the addition of rsv infection of airway epithelial cells with production of the type- innate cytokines, il- ( ) , tslp ( ) , and il- ( ) , would, therefore, be predicted to create an even more exaggerated type- -biased microenvironment in the lung with activation of other immune cells (e.g., m macrophages, dcs, and/or ilc cells) and the development of ahr. when neonatal mice are treated with antibodies to neutralize tslp, il- or their target expressed on dcs, ox l, the ability of early rsv infection to prime pathological th responses upon re-infection of adults is reduced ( , ) . as discussed above, tslp and the other innate type- cytokines, il- and il- , all promote amplification, differentiation, and maintenance of m macrophages ( , , ) . together, these data suggest that many different cells and cytokines, as well as other yet to be determined factors have the potential to contribute to the rsvinduced, th microenvironment and the subsequent development of asthma (figure ). an alternative to the "too much th " explanation for th biased neonatal immune system would be "too little th ". ifnγ plays a key role in the induction and maintenance of th responses and infection of neonatal mice with recombinant rsv expressing ifnγ prevents enhanced disease upon re-infection ( ) . a large proportion of the immune cells present in the neonatal mouse lung are macrophages, and these cells are particularly sensitive to stimulation by ifnγ. indeed, the work of empey and colleagues shows that ifnγ plays a central role in the balance between the inflammatory m macrophage phenotype and the immunosuppressive, m phenotype ( ) . while lung macrophages from adult mice effectively induce m macrophage markers in response to rsv, neonatal macrophages require exogenous ifnγ to express m markers, produce inflammatory cytokines and drive efficient viral clearance ( ) . moreover, administration of intranasal clodronate liposomes that depletes macrophages significantly reduces the ability of ifnγ to promote viral clearance ( ) . furthermore, yamaguchi and colleagues ( ) have shown that treatment of neonatal mice with the powerful th adjuvant, cpg, prior to rsv infection prevents enhanced disease upon re-infection, possibly by promoting activation of antigen-presenting cells and production of ifnγ from nk cells. remarkably, cpg treatment even weeks after early-life infection provides protection ( ) . these observations raise the possibility that rsv vaccines incorporating cpg or other th -biasing adjuvants might be useful not only to prevent infection ( ) but also to block the later development of asthma. whether or not the effects of "more" ifnγ are antigenspecific or simply reflect an adjuvant-modified baseline "potential to respond" in the lung is also an important question. this last possibility is strongly suggested by the work of remot and colleagues who observed that mucosal vaccination of neonatal mice with nanostructures formed by the rsv nucleoprotein (n) and a combination of th -biasing adjuvants (e. coli enterotoxin lt and cpg) provides greater protection from immunopathology upon reinfection by rsv later in life compared to nanostructures containing the n protein + lt ( ) . together, these data suggest that preventing too much th activity (driven by rsv or other stimuli) or promoting th -type activity (or both) may establish a long-term more "balanced" lung microenvironment that resists subsequent development of asthma. as discussed above, rsv infection inhibits type i ifn production and plasmacytoid dendritic cell (pdc) responses ( , , ) .as a result, the maintenance of pdcs during rsv infection may promote beneficial th -type responses including the expansion of rsv-specific cd + t cells. in the challengerechallenge model, treatment of neonatal mice during the initial rsv infection with either ifnα or adoptively transferred adult pdcs leads to upregulation of ifnα expression and diminished th -biased lung inflammation when these mice are reinfected as adults ( ) . further support for the role of pdcs comes from experiments in which adult mice are exposed to fms-like tyrosine kinase ligand (flt -l) prior to rsv infection. in response to flt -l, both conventional dc (cd b + cd c + ) and pdc (cd b − cd c + b + ) populations expand in the lung and draining lymph nodes but only the pdcs protect against airway hyperreactivity, exaggerated th cytokine expression, airway inflammation and mucus production ( ) . these flt -l-induced pdcs have upregulated type i ifn (ifn-α/β) expression and support the expansion of cd + t cell populations that decrease viral load. depletion of these pdcs reduces the protective cd + th response with greater ahr, mucus, viral titers, and th cytokine expression ( , ) . similarly, flt -l administration prior to neonatal rsv infection reduces airway mucus secretion and airway eosinophilia but promotes th rsv-specific cd + t cells upon adult re-infection ( ) . although these observations suggest that flt -l protects against rsv infection by inducing pdcs to produce type i ifn, alveolar macrophages (cd c + siglecf + ) rather than pdcs (or epithelial cells), are thought to be the main producers of type i ifn in the adult mouse lung ( , ) . regardless of whether pdcs or alveolar macrophages are the predominant source of type i ifn, these studies strongly suggest that promoting type i ifn production in the lung during early-life rsv infection is likely to lead to adaptive th responses while suppression of type ifn production may favor maladaptive th responses. taken together, the data summarized above strongly suggest that rsv infection of the neonatal lung adds a further th influence to an already th -biased respiratory microenvironment, with likely contributions from respiratory epithelial cells as well as multiple innate cell populations (e.g., macrophages, dcs, nk cells, ilc s). the presence of multiple autocrine and paracrine amplification loops between these cells likely leads to the production of excessive il- and il- in the lung that increase both the short-and long-term risk that exposures to otherwise innocuous allergens will lead to immunopathologic responses including asthma (figure ) . influenza viruses are far more diverse than rsv due, in large part, to their genetic organization (eight independently segregating genes in the a and b viruses that most commonly infect humans) and the enormous genetic reservoir of influenza viruses in aquatic birds and a number of mammalian species (e.g., most notably pigs but also cats, whales, elephants, skunks, among others) ( ) . the influenza viruses remain a leading cause of childhood pneumonia globally ( ) and early-life infections are common despite the near universal availability of influenza vaccines in resource-rich settings ( ) . it has been estimated that without vaccination - % of young children are infected by an influenza virus every year ( ) . although a number of antivirals are available for influenza infections (e.g., m and neuraminidase inhibitors), these drugs have only modest efficacy and their use is further limited by resistance that is either preexisting or is rapidly induced during treatment ( ) . although influenza infections are clearly associated with exacerbations of asthma [reviewed in ref. ( ) ], the putative link between earlylife influenza and the subsequent development of asthma is much more tenuous. compared to the large body of work implicating rsv (above), far less work has been done to address this question for influenza viruses. at the current time, there is neither epidemiologic nor experimental evidence in humans suggesting a strong association between early-life influenza infection and asthma. although work with murine models tends to support an association, these data are not consistent from one model or laboratory to another. differences in the age of infection, both mouse and virus strain (i.e., human vs. mouse-adapted) and the infective dose may all contribute to these apparently contradictory results. for example, a study by dahl et al. ( ) showed that lrt infection of adult balb/c mice with influenza a (hkx strain) at - weeks of age could predispose to ahr upon subsequent allergen sensitization (keyhole limpet). surprisingly, ahr in this model was associated with both th -type (ifnγ, igg a) and th -type (igg ) antigenspecific responses and the allergic phenotype could be adoptively transferred with pulmonary dcs. using a similar protocol with ova as a model allergen in balb/c mice, barends et al. found that infection with a mouse-adapted influenza a strain (a/ pr/ / ) at the time of ova challenge decreased th cytokine production in the lungs but increased infiltration by eosinophils ( ) . chang cells (i.e., ilc s) to produce il- that promotes the development of ahr ( ) . recently, elevated pleural il- and ilc s were found to mediate the induction of asthma-like responses (e.g., ahr, production of th cytokines) following pdmh n infection in rag −/− mice (on a c bl/ background) that lack functional t and b lymphocytes ( ) . like the observations in adult mice, the reported outcomes after early-life influenza a infection are highly variable, sometimes protecting against, but sometimes promoting the development of ahr. when suckling balb/c mice ( weeks old) are infected with influenza h n (again mem ) and challenged with allergens as adults, ahr typically does not occur and the protective effect appears to be mediated by a subset of nkt cells that produce large amounts of ifnγ ( ) . at high inocula, many influenza viruses are lethal for young mice ( ) . using low titer h n virus (pr ), lines and colleagues found a delayed t cell response with a pronounced influx of eosinophils into the lungs of c bl/ j neonates compared to adult mice ( ) . ex vivo stimulation of lung cells with phorbol -myristate -acetate/ ionomycin in these experiments showed delayed and lower levels of ifnγ in the neonatal immune t cells ( ) . others have shown that infection of balb/c mouse pups with pr on day of life can result in long-term pulmonary dysfunction, ahr and an increase in inflammatory cytokines, neutrophils and alveolar macrophages in the lungs ( ) . in this model, ifnγ was not produced by the neonatal pulmonary cd + t cells, but adoptive transfer of adult cd + t cells prevented the long-term ahr ( ) . although this body of work is much smaller than that dealing with rsv, the available data raise the possibility that deficient ifnγ production in the neonate during influenza virus infection may play a role in the induction of an asthmatic phenotype upon subsequent exposure to viruses or allergens. further studies, including the development of a "standard" early-life mouse model of influenza infection are required to begin to understand the potential contribution of these viruses to asthma initiation. rhinoviruses are the most important etiological agents of the "common cold". similar to rsv, rv can cause both upper and lower respiratory-tract illness at all ages. there are - strains of rv, divided into three groups: a, b, and c. groups a and b were discovered in the s, while rv-c, a new group with over strains, was identified only in ( , ) . most rv infections are thought to be minimally symptomatic or completely asymptomatic. under years of age, at least one rv can be found by nasal swab in - % of asymptomatic children [reviewed in ref. ( ) ]. although both the scientific literature and the internet offer a wide variety of therapies to prevent or cure the common cold ( , ) , at the current time neither antivirals nor vaccines are available for rv infections. similar to rsv, the rvs, and particularly the rv-c group, have been implicated in both the development of asthma and in exacerbation of wheezing illness ( , , ( ) ( ) ( ) . interestingly, rv-associated wheezing tends to be more common in older infants (> year old) in contrast to rsv infection in which the most serious manifestations (i.e., bronchiolitis) occur primarily in those < year old ( , ) . however, both rsv and rv are frequently isolated in infants hospitalized with severe respiratory symptoms and wheezing ( ) . as noted above for rsv, it is very difficult to know if this observation means that children with a genetic propensity to wheeze are more likely to be hospitalized or if rv infections are a causal factor in the onset of wheezing illness ( , ) . until recently, animal models suffered from the absence of a receptor for human rv on mouse cells. most human rv strains bind to intracellular adhesion molecule- (icam- ) but not mouse icam- and only a minority (~ %) binds to low-density lipoprotein of both humans and mice ( ) . bartlett et al. ( ) have described two mouse models of rv infection, one in which balb/c mice are infected with a low-density lipoprotein-binding isolate and a second based upon transgenic expression of human icam- . infection of adult mice in the latter model results in a strong th response with abundant ifnγ production as well as exacerbation of allergic responses. to date, the transgenic model has not been used to study rv infection and asthma initiation in neonatal mice. a third model was recently described by schneider et al. ( ) in which -day-old balb/c mice are infected with rv- b, leading to the development of ahr and mucus production weeks later. surprisingly, this neonatal infection resulted in the production of ifnγ along with increases in inflammatory cytokines and chemokines, including tnfα, cxcl , and cxcl in the lungs. these observations are very different from the findings in rsv-infected neonates. however, il- was also strongly induced by rv- b infection in neonatal mice but not in adult mice ( ) . analysis of the lungs days after infection showed that a majority of cells producing il- were nkt cells, while cd t cells predominantly expressed ifnγ. there was also an increase in m macrophages in the lungs, cells that contribute to rv- b-induced ahr in allergen-sensitized mice infected as adults ( ) . neutralizing il- or il- r decreased ahr in mice infected as neonates, suggesting that mechanisms similar to those operating in influenza ( ) and rsv ( , , ) may also play a role in rv infection. in hdm (dermatophagoides farina)-sensitized balb/c mice, rv infection decreases il- and increases production of il- , rantes and tnfα as well as eosinophil infiltration into the lungs ( ) . th cytokines (il- , il- ) increase the expression of the rv receptor (icam- ) on human respiratory epithelial (h ) cells ( ) . antiviral responses (ifnβ/λ) appear to be decreased in primary bronchial epithelial cells isolated from children with atopy/asthma and are more permissive of rv replication ( ) . serum ige levels in these infants are positively correlated with viral rna levels in ex vivo-infected epithelial cells and negatively correlated with rv-induced ifnβ/λ. other investigators have found a negative correlation between ifn-induction by rv and airway th responses, including eosinophilia and detectable il- , in children with a history of asthma both with and without atopy ( ) . in a human challenge study with rv , both atopic and non-atopic subjects with low ifnγ/il- ratios had more symptoms and prolonged viral shedding ( ) . the dearth of studies on rv infection in neonatal mice makes it hard to draw conclusions about potential mechanisms of rv-mediated asthma initiation. further work on neonatal models, particularly using the group c strains associated with clinical asthma in infants ( , , ( ) ( ) ( ) , would be useful. like rsv, rv primarily targets respiratory epithelial cells, although rv infections are typically "patchy" and thus may not cause the same degree of damage as rsv. recent investigations of rv infection in both animal models and human studies have focused on the innate type cytokines il- , tslp, and il- . tslp is secreted at a low concentration from primary human bronchial epithelial cells infected ex vivo with rv, but rise dramatically with the addition of il- ( ). tlsp and il- are also upregulated in bronchial epithelial cells in response to in vitro rv infection but even greater increases in il- are seen in cells from asthmatic patients post-rsv infection ( , ) . rv-induced production of il- and/or tslp blocks ova-induced inhalational tolerance, with resultant lung eosinophilia and neutrophilia, as well as increased th and decreased treg cells. delivery of neutralizing antibodies that target st (the il- receptor) or rv infection of tslpr knock-out mice both disrupt the ability of rv to block tolerance in this model ( ) . these data suggest that rv may increase susceptibility to allergic airways disease, by increasing tslp and/or il- , generating a lung environment conducive to immune recognition of normally harmless antigens/allergens. in a murine model of rv-induced asthma exacerbation using ova, lung levels of il- , expressed by epithelial cells and infiltrating immune cells are greatest in mice exposed to both ova and rv ( ) . ova-rv exposed mice have increased levels of il- + basophils, il- + cd + t and icos + st + non-t (ilc ?) cells in the bronchoalveolar lavage (bal) fluid, as well as enhanced airway eosinophilia and neutrophilia, and exacerbated th cytokine production, all of which are reduced upon delivery of neutralizing il- receptor antibody (α-il- rb) prior to rv infection. interestingly, α-il- rb treatment also decreases lung tissue il- and tslp protein levels. these data suggest that targeting il- receptor may hold therapeutic promise through mitigation of viral-and allergen-induced inflammation promoted by innate type cytokines. interleukin- is produced by cultured bronchial epithelial and smooth muscle cells when infected with rv ( , ) . in response to in vivo infection with rv , asthmatics have greater viral loads, increased bal eosinophilia, and greater respiratory symptom scores compared to non-asthmatic controls. these parameters are also associated with reductions in both peak expiratory flow and fev . moreover, elevated levels of the th cytokines (il- , il- , il- ) as well as il- are present in the bal fluid of rv -infected asthmatics. ex vivo incubation of activated (undifferentiated) cd + t cells (th ) or ilc cells with supernatants from rv-infected bronchial epithelial cells leads to production of il- and il- by both cell types in an il- -dependent manner ( ) . significantly, production of il- and il- is -to fold greater in ilc cells compared to cd + t cells suggesting that il- -dependent activation of ilc cells during rv infection could play a major role in the development of asthma. taken together, these data suggest that pronounced il- secretion by smooth muscle cells and/or il- , tslp, and il- secretion by epithelial cells in response to rv infection may skew the lung microenvironment toward th -biased allergic airways disease through activation of both innate (e.g., macrophage, dcs, and ilc cells) and adaptive immune cells. at the current time, a large body of rsv data in both humans and murine models strongly suggest that there is a maladaptive and "asthma-genic" interaction between the th -biased nature of the infant immune system and the th -promoting effects of the virus itself. it is also very likely that host genetics play an important role in determining both the short-term (e.g., hospitalization) and long-term (e.g., asthma) consequences of this interaction ( , ) . to date, the only factors known to increase susceptibility to rsv-induced asthma are family history of atopy, premature birth, and certain host genetic polymorphisms that are linked with severe disease ( ) . whether or not any child, regardless of his/her genetic background can be "made" asthmatic by early-life rsv infection is an important question that cannot be answered at the current time. in favor of the hypothesis that asthmatics are "born" and not "made", gern and colleagues have reported bi-directional cytokine responses in cord blood mononuclear cells stimulated with mitogen or viral antigens (e.g., rsv, rv) from children who were later classified as either wheezing vs. non-wheezing at one year of age ( ) . whether or not rsv is unique among early childhood respiratory virus exposures with regard to asthma development is also of great interest. the more limited data available suggest that the rv but not influenza virus infection may have effects similar to rsv. bonnelykke et al. ( ) reported that the number of viral or bacterial respiratory infection episodes within the first year of life is a greater predictor of asthma development than the particular viral or bacterial type(s) ( ) . in contrast to the contribution of respiratory viruses to asthma development, the role of several viruses, including rsv, rv, and influenza viruses, in asthma exacerbations is very clear ( , , , , ) . of course, neonates and infants are not just exposed to respiratory viruses in the first weeks-months of life. rather, they experience a wide range of immunologic challenges with both commensal and potentially pathogenic organisms at multiple epithelial sites throughout childhood. for example, ege and colleagues have shown that children exposed to wide range of microbes early in life, including children that live on farms, have a reduced risk of developing asthma (the hygiene hypothesis) ( ) and germ-free mice can more easily be made airway hyperresponsive than their non germ-free littermates ( ) . a great deal of work remains to be done to fully understand how the ubiquitous respiratory viruses discussed in this review, as well as other early-life microbial exposures, contribute to the development and perpetuation of asthma. such an improved understanding is essential to develop strategies to both prevent asthma development and to mitigate the symptoms of asthma once developed. this review has focused on how a small number of viruses may also contribute to the induction of asthma. in particular, we have described the growing evidence that innate immune effector cells may orchestrate earlylife events in the lung to "set the stage" for the later development of asthma. until very recently, the potential asthma-inducing role of respiratory epithelial cells, macrophages, dcs, ilc s, and nk/ nkt cells has been under-appreciated. although vaccination to protect the very young from early-life respiratory viruses (including maternal immunization) is one possible strategy ( ), these new data suggest that modulation of innate responses during early-life viral infections may also be successful. of course, any strategy that seeks to alter either the antigen-specific or the "overall" immune response pattern of neonates/infants would have to be approached with great caution (e.g., risk of exaggerated th immunopathology, less effective responses to other pathogens or vaccines). as outlined above, the th -biased nature of the neonatal immune system is a strategy that has been "proven" by evolution. nonetheless, given that asthma has now reached essentially "pandemic" proportions, very few questions in clinical medicine are of greater importance. the concepts described in this review raise the possibility of completely novel strategies for dealing with this pandemic. kr, bs, bw, and ef contributed equally to the writing of the manuscript and production of the accompanying figure. global, regional, and national causes of child mortality: an updated 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stromal lymphopoietin, and il- il- -dependent type inflammation during rhinovirus-induced asthma exacerbations in vivo rhinoviral stimuli, epithelial factors and atp signalling contribute to bronchial smooth muscle production of il- bidirectional interactions between viral respiratory illnesses and cytokine responses in the first year of life viral respiratory tract infection and exacerbations of asthma in adult patients asthma exacerbations. : pathogenesis exposure to environmental microorganisms and childhood asthma microbial exposure during early life has persistent effects on natural killer t cell function vaccination against rsv: is maternal vaccination a good alternative to other approaches? the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord- -r lih j authors: okamoto, masaaki; kouwaki, takahisa; fukushima, yoshimi; oshiumi, hiroyuki title: regulation of rig-i activation by k -linked polyubiquitination date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: r lih j rig-i is a pattern recognition receptor and recognizes cytoplasmic viral double-stranded rna (dsrna). influenza a virus, hepatitis c virus, and several other pathogenic viruses are mainly recognized by rig-i, resulting in the activation of the innate immune responses. the protein comprises n-terminal two caspase activation and recruitment domains ( cards), an rna helicase domain, and the c-terminal domain (ctd). the ctd recognizes ′-triphosphate viral dsrna. after recognition of viral dsrna, the protein harbors k -linked polyubiquitination essential for rig-i activation. first, it was reported that trim ubiquitin ligase delivered k -linked polyubiquitin moiety to the cards. the polyubiquitin chain stabilizes a structure called the card tetramer, in which four cards assemble and make a core that promotes the aggregation of the mitochondrial antiviral-signaling (mavs) protein on mitochondria. mavs aggregation then triggers the signal to induce the innate immune responses. however, subsequent studies have reported that riplet, mex c, and trim ubiquitin ligases are also involved in k -linked polyubiquitination and the activation of rig-i. mex c and trim mediate polyubiquitination of the cards. by contrast, riplet ubiquitinates the ctd. the physiological significance of each ubiquitin ligases has been shown by knockout and knockdown studies, but there appears to be contradictory to evidence reported in the literature. in this review, we summarize recent findings related to k -linked polyubiquitination and propose a model that could reconcile current contradictory theories. we also discuss the physiological significance of the ubiquitin ligases in the immune system against viral infection. rig-i is a pattern recognition receptor and recognizes cytoplasmic viral double-stranded rna (dsrna). influenza a virus, hepatitis c virus, and several other pathogenic viruses are mainly recognized by rig-i, resulting in the activation of the innate immune responses. the protein comprises n-terminal two caspase activation and recruitment domains ( cards), an rna helicase domain, and the c-terminal domain (ctd). the ctd recognizes ′-triphosphate viral dsrna. after recognition of viral dsrna, the protein harbors k -linked polyubiquitination essential for rig-i activation. first, it was reported that trim ubiquitin ligase delivered k -linked polyubiquitin moiety to the cards. the polyubiquitin chain stabilizes a structure called the card tetramer, in which four cards assemble and make a core that promotes the aggregation of the mitochondrial antiviral-signaling (mavs) protein on mitochondria. mavs aggregation then triggers the signal to induce the innate immune responses. however, subsequent studies have reported that riplet, mex c, and trim ubiquitin ligases are also involved in k -linked polyubiquitination and the activation of rig-i. mex c and trim mediate polyubiquitination of the cards. by contrast, riplet ubiquitinates the ctd. the physiological significance of each ubiquitin ligases has been shown by knockout and knockdown studies, but there appears to be contradictory to evidence reported in the literature. in this review, we summarize recent findings related to k -linked polyubiquitination and propose a model that could reconcile current contradictory theories. we also discuss the physiological significance of the ubiquitin ligases in the immune system against viral infection. keywords: rig-i, ubiquitin, innate immunity, virus, signaling pathway introduction pattern recognition receptors (prrs) recognize viral nucleic acids and trigger a signal to induce the innate immune responses during viral infection ( , ) . rig-i is a cytoplasmic rna helicase and a prr that recognizes cytoplasmic ′ tri-or diphosphate double-stranded rna (dsrna) ( ) ( ) ( ) . rig-i binds relatively short dsrna (< kbp) and is involved in the recognition of various viral infections, such as influenza a and b viruses, japanese encephalitis virus, hepatitis c virus (hcv), dengue virus, and west nile virus ( ) ( ) ( ) . after recognition of viral rna, rig-i associates with an adaptor protein, mitochondrial antiviral-signaling (mavs) protein, also called ips- , cardif, and visa ( ) ( ) ( ) ( ) , resulting in the aggregation of mavs on the outer membrane of mitochondria. this ubiquitination of rig-i frontiers in immunology | www.frontiersin.org january | volume | article aggregation triggers a signal to induce the expression of type i interferon (ifn) and other inflammatory cytokines ( ) . the rig-i protein comprises two caspase-activation and recruitment domains ( cards) at the n-terminal region, an rna helicase domain, and a c-terminal domain (ctd) ( ) ( ) ( ) . viral dsrna binds to the rna helicase domain and the ctd, and ′ tri-and diphosphate are recognized by the ctd ( , ) . the n-terminal cards are responsible for the association with mavs and, therefore, are required for triggering downstream signaling ( ) . in resting cells, the c-terminal region, which includes the ctd and the linker region between the ctd and the helicase domain, suppresses the n-terminal cards ( , ) . binding of the ctd to dsrna induces the conformational change of the rig-i protein, resulting in the release of the cards ( ) . subsequently, the proteins assemble along viral dsrna and form a nucleoprotein filament ( ) . the released cards also assemble and form a card tetramer structure ( ) . the structure functions as a core for mavs aggregation on mitochondria ( ) . the rig-i protein harbors lys -linked (k -linked) polyubiquitination required for its activation ( ) . trim is a ubiquitin ligase and delivers k -linked polyubiquitin moiety to the rig-i cards ( , ) . the polyubiquitin chains stabilize the card tetramer structure ( ) . the physiological significance of trim in rig-i activation has been shown by several studies ( ) ( ) ( ) ( ) ( ) ( ) . however, recent studies have reported three other ubiquitin ligases, ring finger protein leading to rig-i activation (riplet), mex- rna-binding family member c (mex c), and trim , which are required for the polyubiquitination and activation of rig-i ( - ). ubiquitin ligases add a ubiquitin chain at k but not r residues of the target protein. there are k residues in the rig-i cards, and mass spectrometry analysis revealed that the cards fragment carried k -linked polyubiquitin chains at k , k , k , k , k , and k ( ) . knockdown of trim abrogated polyubiquitination of the cards fragment, suggesting that trim mediates k -linked polyubiquitination at the k residues of the cards ( ) . the cards fragment has an ability to bind to mavs, and overexpression of the cards fragment leads to auto-activation of signaling ( , ( ) ( ) ( ) ( ) . an amino acid substitution assay revealed that the substitution of k , but not of other k residues, with r abrogated the signaling induced by the cards fragment ( ) . knockout of trim severely reduced rig-i-mediated type i ifn production during viral infection. these observations indicate the importance of trim -mediated k ubiquitination ( ) . evidence also suggests that trim produces unanchored k -linked polyubiquitin chains in response to viral infection and delivered them to rig-i ( ) . the same study also showed that the k residue of rig-i was important for non-covalent binding of rig-i with unanchored polyubiquitin chains ( ) . considering that mass spectrometry analysis revealed the covalent binding of rig-i with k -linked polyubiquitin chains, these observations indicate that either covalent or non-covalent binding with polyubiquitin chains is sufficient for rig-i cards activation ( , ) . a structural study of rig-i cards tetramer provided evidence that both covalent and non-covalent binding of polyubiquitin chains promotes the formation of the card tetramer structure ( , ) . the trim activity itself is regulated by the physical interaction between the trim spry domain and rig-i cards ( ) . the cooperative assembly of trim and rig-i facilitates the dimerization of the trim ring domain, which is required for trim to make polyubiquitin chain ( ) . an accumulating body of evidence has shown that trim delivers k -linked polyubiquitin moiety to rig-i cards for rig-i activation and that the k residue is important for the binding of rig-i to polyubiquitin chains ( , , ) . however, subsequent studies revealed that not only k but also other k residues are also important for the binding of rig-i to k linked polyubiquitin chains. first, shigemoto et al. reported that the expression of the rig-i k r full-length protein could compensate for a defect in rig-i knockout mouse embryonic fibroblasts (mefs) after sendai virus infection ( ) . second, two other ubiquitin ligases, mex c and trim , were reported to mediate polyubiquitination of the rig-i cards at other k residues ( , ) . kuniyoshi ( ) . mass spectrometry analysis has also revealed the ubiquitination at k , k , k , as well as k and k of the cards ( ) . they reported that simultaneous amino acids substitutions at k , k , and k substantially reduced the polyubiquitination of rig-i ( ). these observations suggest that k -linked polyubiquitination at these k residues can compensate for the loss of k binding to the ubiquitin chain under certain conditions (figure ). riplet, another ubiquitin ligase, is also involved in k -linked polyubiquitination and activation of rig-i. riplet was first isolated by our yeast two-hybrid screening using the rig-i ctd fragment as a bait ( ) . an immunoprecipitation assay then confirmed that the protein bound to the ctd fragment, and our studies also indicated that riplet mediates k -linked polyubiquitination of the rig-i ctd ( ) . our mutation analysis indicated that k and k of the ctd were important for riplet-mediated polyubiquitination, and riplet also targeted other k residues, including k , k , and k of the ctd and k in the linker region between the ctd and the helicase domain ( , ) (figure ) . to assess the physiological significance of the protein, we generated riplet knockout mice. knockout of riplet severely impaired the type i ifn and il- production in mef, macrophages, and conventional dendritic cells following influenza a virus and an rig-i c-terminal fragment ( - aa region), which includes the linker region and the ctd, suppresses the card activation ( ) , and kageyama et al. reported that the linker region ( - aa) was responsible for the auto-suppression ( ) . riplet targets the k in the linker region. therefore, it is possible that the ubiquitination of the linker region disrupts the auto-suppression. the structure analysis revealed that k , k , k , and k of the ctd bind to ′ triphosphate of dsrna ends ( ) . the k , k , and k of the ctd are targeted by riplet. binding of the ctd to ′ triphosphate of dsrna was reported to induce conformational change of the rig-i protein ( ) . although several rig-i molecules assemble along dsrna, only one rig-i molecule binds the ′ triphosphate at the dsrna end ( ) (figure a) . therefore, riplet could access the k , k , and k of the ctds of the rig-i molecules associating with dsrna (but not the end of dsrna) to induce conformational change of rig-i. these k residues are located at the edge of the ctd basic cleft, which is an rna-binding site ( ) . further studies are required to reveal underlying mechanism of riplet-mediated rig-i activation. despite the identification of four ubiquitin ligases, we found that knockout of riplet alone could abolish the polyubiquitination of the endogenous rig-i protein ( , ) . recently, shi et al. also reported that knockout of riplet is sufficient to abolish the polyubiquitination of rig-i and, therefore, claimed that riplet is a primary ubiquitin ligase and mediates k -linked polyubiquitination of the cards ( ) . however, their model appears to be contradict to previous papers showing that trim plays a crucial role in rig-i activation. previously, we have postulated a sequential ubiquitination model that riplet-mediated polyubiquitination of rig-i c-terminal region is a prerequisite for the polyubiquitination of the cards (figure ) ( ) . this model could explain the apparent discrepancy in the literature, because due to the initial failure to polyubiquitinate the c-terminal region, this would obstruct the subsequent polyubiquitination of the cards by other ubiquitin ligases. this indicates that knockout of riplet alone is sufficient to abolish the polyubiquitination of the ctd, the linker region, and the cards. in a previous study, we have shown that riplet promotes the binding of trim to rig-i ( ). this observation supports the sequential model. it is expected that riplet-mediated polyubiquitination leads to the release of auto-suppression and/or conformational change of rig-i, which would allow the access of trim to the cards and/or promote rig-i assembly along dsrna (figure b) . considering that higher-order oligomerization of trim with the cards is required to induce trim -mediated polyubiquitination ( ), it is not surprising that riplet-mediated c-terminal ubiquitination is a prerequisite for the second ubiquitination by trim (figure b) . mex c or trim might compensate for the defect of trim under certain experimental conditions, because these two ubiquitin ligases target the cards in a similar way (figure ) . although we failed to detect an interaction between riplet and the cards fragment, other groups have reported that riplet bound to the cards fragment and was involved in the k -linked polyubiquitination of the cards ( , ) . these observations do not conflict with the sequential ubiquitination model because several ubiquitin ligases can compensate for the loss of trim in some conditions. we do not exclude the possibility that riplet is not only involved in the primary ubiquitination of the ctd and the linker region but also the secondary ubiquitination of the cards (figure ). type i ifn exhibits a strong antiviral effect, and hence several viruses have evolved to suppress the type i ifn production. hcv is a major cause of hepatocellular carcinoma and persistently infects hepatocytes over several decades without exclusion by the host immune system. a viral ns - a protease is required to cleave viral polypeptides and produce mature viral proteins; however, it is also important to suppress the host innate immune response. ns - a of hcv cleaves mavs, which results in the release of mavs from mitochondria ( ) . several reports have shown that released mavs protein fails to trigger signaling to induce type i ifn production ( ) . accordingly, ns - a-mediated cleavage of mavs abrogates rig-i-mediated type i ifn production. ns - a protein also targets the riplet protein. the ring finger domain is a catalytic domain of the riplet protein, and viral ns - a protease cleaves the domain and destabilizes the protein ( ) . ns protein of influenza a virus also has the ability to suppress type i ifn production ( ) . although several mechanisms have been postulated, gack et al. reported that viral ns bound to trim and inhibited trim -mediated polyubiquitination of rig-i ( ) . in further study, they reported that ns protein also targeted the riplet protein and inhibited rig-i polyubiquitination ( ) . severe acute respiratory syndrome coronavirus (sars-cov) also interferes trim function ( ) . the nucleocapsid protein of sars-cov physically interacted with trim and inhibited the binding of trim to rig-i, resulting in the attenuation of rig-i-signaling ( ) . these data imply that viruses obtained the ability to suppress the ubiquitin ligases to escape innate immune responses. conversely, these data indicate the importance of the two ubiquitin ligases, trim and riplet, for the antiviral innate immune response. trim is also called efp, and it has been shown that trim / efp mediates the polyubiquitination of - - σ and promotes its proteolysis to suppress the growth of breast tumor cells ( ) . although other targets of riplet have not been reported, it was shown that mutations on human riplet genes (also called rnf ) are linked to learning disabilities and several neuropsychiatric disorders ( , ) . thus, it is expected that riplet targets the proteins involved in these conditions. there are several reports that riplet and trim are involved in tumorigenesis ( , , ) . as several viruses have the ability to abrogate the described ubiquitin ligases, it is expected that viral protein-mediated inhibition of the ubiquitin ligases affects both innate immunity and other phenomena, such as virus-induced tumorigenesis and neuropsychiatric disorders. there are two protein families related to rig-i called rig-ilike receptors (rlrs). lgp is an rlr, and the ctd structure of the protein is similar to that of rig-i ( ) . initial studies reported that lgp is a negative regulator for rig-i signaling ( , ) . however, knockout and biochemical studies have revealed that lgp functions as a positive regulator of the rig-i pathway ( , ) . lgp is also expressed in cd + t cells and is required for cd + t cell proliferation ( ) . it remains unclear whether lgp carries a ubiquitin chain. considering the conservation of the ctd between rig-i and lgp , it is possible that riplet also targets the ctd of lgp and affects lgp -mediated rig-i activation and cd + t cell proliferation. further studies are required to fully elucidate the role of the ubiquitin ligases in the antiviral immune response. ho and mo wrote the manuscript. tk and yf helped the discussion. immune signaling by rig-i-like receptors toll-like receptors and their crosstalk with other innate receptors in infection and immunity antiviral immunity via rig-i-mediated recognition of rna bearing '-diphosphates '-triphosphate rna is the ligand for rig-i the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-i and melanoma differentiation-associated gene distinct rig-i and mda signaling by rna viruses in innate immunity differential roles of mda and rig-i helicases in the recognition of rna viruses identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf ips- , an adaptor triggering rig-i-and mda -mediated type i interferon induction visa is an adapter protein required for virus-triggered ifn-beta signaling cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus mavs forms functional prion-like aggregates to activate and propagate antiviral innate immune response regulation of innate antiviral defenses through a shared repressor domain in rig-i and lgp shared and unique functions of the dexd/h-box helicases rig-i mda , and lgp in antiviral innate immunity nonself rna-sensing mechanism of rig-i helicase and activation of antiviral immune responses the c-terminal regulatory domain is the rna '-triphosphate sensor of rig-i amino acid linker between helicase and carboxyl terminal domains of rig-i functions as a critical repression domain and determines inter-domain conformation rig-i forms signaling-competent filaments in an atp-dependent, ubiquitin-independent manner structural basis for dsrna recognition, filament formation, and antiviral signal activation by mda structural basis for ubiquitin-mediated antiviral signal activation by rig-i trim ringfinger e ubiquitin ligase is essential for rig-i-mediated antiviral activity reconstitution of the rig-i pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity a distinct role of ripletmediated k -linked polyubiquitination of the rig-i repressor domain in human antiviral innate immune responses hepatitis c virus reveals a novel early control in acute immune response post-translational control of intracellular pathogen sensing pathways viral evasion of intracellular dna and rna sensing riplet/rnf , a ring finger protein, ubiquitinates rig-i to promote interferon-beta induction during the early phase of viral infection pivotal role of rna-binding e ubiquitin ligase mex c in rig-i-mediated antiviral innate immunity trim modulates type i interferon induction and cellular antiviral response by targeting rig-i for k -linked ubiquitination mechanism of trim catalytic activation in the antiviral rig-i pathway ubiquitin-mediated modulation of the cytoplasmic viral rna sensor rig-i identification of loss of function mutations in human genes encoding rig-i and mda : implications for resistance to type i diabetes ube d and ube n are essential for rig-i-mediated mavs aggregation in antiviral innate immunity the ubiquitin ligase riplet is essential for rig-i-dependent innate immune responses to rna virus infection visualizing the determinants of viral rna recognition by innate immune sensor rig-i reul is a novel e ubiquitin ligase and stimulator of retinoic-acid-inducible gene-i viral infection switches non-plasmacytoid dendritic cells into high interferon producers influenza a virus ns targets the ubiquitin ligase trim to evade recognition by the host viral rna sensor rig-i species-specific inhibition of rig-i ubiquitination and ifn induction by the influenza a virus ns protein the severe acute respiratory syndrome coronavirus nucleocapsid inhibits type i interferon production by interfering with trim -mediated rig-i ubiquitination efp targets - - sigma for proteolysis and promotes breast tumour growth mutations in rnf , a gene within the nf microdeletion region, cause phenotypic abnormalities including overgrowth mutation screening of the ubiquitin ligase gene rnf in french patients with autism ring finger protein, promotes the proliferation of human glioblastoma cells in vivo and in vitro via the erk pathway the e ubiquitin ligase rnf regulates the tumorigenesis activity of tongue cancer scc cells loss of dexd/h box rna helicase lgp manifests disparate antiviral responses lgp is a positive regulator of rig-i-and mda -mediated antiviral responses the innate immune sensor lgp activates antiviral signaling by regulating mda -rna interaction and filament assembly the rig-i-like receptor lgp controls cd (+) t cell survival and fitness the authors thank dr. t. seya and dr. m. matsumoto for their helpful discussion. this work was supported in part by grantsin-aid from ministry of education, science, and culture, and presto jst. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -h j iqbm authors: sinha, sushmita; boyden, alexander w.; itani, farah r.; crawford, michael p.; karandikar, nitin j. title: cd (+) t-cells as immune regulators of multiple sclerosis date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: h j iqbm the vast majority of studies regarding the immune basis of ms (and its animal model, eae) have largely focused on cd (+) t-cells as mediators and regulators of disease. interestingly, cd (+) t-cells represent the predominant t-cell population in human ms lesions and are oligoclonally expanded at the site of pathology. however, their role in the autoimmune pathologic process has been both understudied and controversial. several animal models and ms patient studies support a pathogenic role for cns-specific cd (+) t-cells, whereas we and others have demonstrated a regulatory role for these cells in disease. in this review, we describe studies that have investigated the role of cd (+) t-cells in ms and eae, presenting evidence for both pathogenic and regulatory functions. in our studies, we have shown that cytotoxic/suppressor cd (+) t-cells are cns antigen-specific, mhc class i-restricted, ifnγ- and perforin-dependent, and are able to inhibit disease. the clinical relevance for cd (+) t-cell suppressive function is best described by a lack of their function during ms relapse, and importantly, restoration of their suppressive function during quiescence. furthermore, cd (+) t-cells with immunosuppressive functions can be therapeutically induced in ms patients by glatiramer acetate (ga) treatment. unlike cns-specific cd (+) t-cells, these immunosuppressive ga-induced cd (+) t-cells appear to be hla-e restricted. these studies have provided greater fundamental insight into the role of autoreactive as well as therapeutically induced cd (+) t-cells in disease amelioration. the clinical implications for these findings are immense and we propose that this natural process can be harnessed toward the development of an effective immunotherapeutic strategy. introduction studies addressing the immunobiology of multiple sclerosis (ms) and its animal model experimental autoimmune encephalomyelitis (eae) have focused on cd + t-cells as the main orchestrators of pathogenesis and regulation. cd + t-cells are the most abundant t-cells in cns lesions of ms patients ( ) and exhibit oligoclonal expansion ( ) ( ) ( ) . this indicates an important role for these cells in the target organ. however, the functional nature of these cells during disease and its treatment is unclear and somewhat controversial. there are abundant cns-specific ( , ) and therapeutically induced cd + t-cell responses in ms patients ( ) ( ) ( ) ( ) . recent studies suggest that certain mhc class i alleles can be associated with genetic risk or protection in ms ( ) ( ) ( ) . functional roles for some of these mhc class i molecules have been tested in the eae models. d -tcr humanized transgenic mice, expressing ms risk variant hla-a together with tcr that recognizes myelin proteolipid protein (plp), develope spontaneous eae in only % of mice and mild eae early on when immunized with plp peptide. a quarter of these mice went on to develop a severe disease course with d + -tcr + -cd + t-cells present in the cns of these mice, suggesting a pathogenic role for hla-a -restricted myelin-specific cd + t-cells ( ) . however, introduction of hla-a alleles in the same model completely abrogates spontaneous and induced eae, providing evidence for the protective role for hla-a -restricted cd + t-cells ( ) . we are only beginning to understand these responses and here attempt to provide an overview of such studies. we will summarize the evidence for both pathogenic and regulatory functions of cd + t-cells in ms and eae. we will provide an overview of the various cellular and molecular interactions that mediate the role of these cells and develop a model for such functions during disease. much of the focus regarding the pathogenesis of eae has revolved primarily around myelin-specific cd + t-cells. adoptive transfer of cd + t-cells isolated from myelin antigen-primed animals is sufficient to induce disease. this observation partly facilitated the overall ignorance surrounding cd + t-cells and their potential contribution to disease. a pathogenic role first became evident when a cd + t-cell-mediated model of eae was developed using the self-protein myelin basic protein (mbp) ( ) . in attempts to prime an mhc class i-restricted t-cell response, c h.fej, and c h mbp-deficient shiverer mice were infected with mbpexpressing vaccinia. cd + t-cell lines specific for mbp - drove pathogenesis and demyelination when transferred into wildtype (wt) c h recipients. mice developed neurological symptoms including ataxia, spasticity, and lost weight when compared to control animals that received vaccinia-specific cd + t-cells. histologically, perivascular cuffs composed primarily of lymphocytes and macrophages were detected in the brain but not in the spinal cord. ifnγ was found to play an important role in mediating mbp-specific cd + t-cell-driven disease, as its neutralization reduced severity. the break of peripheral tolerance following viral infection was also shown to induce cd + t-cell-mediated cns autoimmunity ( ) . in this report, dual tcr-expressing cd + t-cells recognizing both viral antigen and mbp triggered disease. following viral infection, cd + t-cells, macrophages, and activated microglia infiltrated both the brain and spinal cord. clinically, mice lost weight and exhibited symptoms of ataxia, impaired mobility, and tail weakness. cd + t-cell-mediated eae has also been induced in c bl/ (b ) mice through transfer of myelin oligodendrocyte glycoprotein (mog)-specific cd + t-cells ( ) . mog-specific cd + t-cells isolated from mice immunized with mog - peptide were encephalitogenic, and transferred severe paralytic disease to b mice. one caveat to this study is that cells were nylon woolenriched, calling purity into question. disease was transferred using < e mog - cd + t-cells and resulted in more severe eae compared to active immunization. transferred cells could be re-isolated - months later, possibly due to additional il- stimulus. how these cells induced pathology was not investigated. a separate group identified mog - -specific cd + t-cells as autoaggressive effectors ( ) . in this system, mog-specific cd + t-cells were generated following immunization. restimulation with antigen and il- readily yielded ifnγ from these cells, but not tgfβ or il- . these cells, which were found to be h- d brestricted, could induce eae when transferred into scid or naïve wt b recipients. mog - elicited the best ifnγ response from mog - -primed lymph node cells, although bound mhc poorly. when used to induce active eae in b mice, mog - led to similar disease as mog - -immunized mice. using mog - /h- d b tetramers, mog-specific cd + t-cells were found to persist within the cns. while these studies utilized myelin-components to examine the potential pathogenic role of cd + t-cells in eae, nonmyelin antigen-driven systems have been used as well. one report describes cd + tcr transgenic mice recognizing glial fibrillary acidic protein (gfap), an intermediate filament protein expressed in the cns by astrocytes and in various peripheral tissues ( ) . bg transgenic mice are reactive to the gfap - peptide presented on h- k b , and develop spontaneous inflammatory cns disease by - months of age. interestingly, gfap-expressing vaccinia induced distinct disease pathology compared to spontaneous disease. lesion localization and clinical manifestations of disease was dependent upon how cns-reactive cd + t-cells were activated. cd + t-cells isolated from brains of wt bg mice were poor secretors of ifnγ, il- a, and granzyme b, suggesting alternative effector mechanisms. efforts to study the role of src homology domain-containing protein tyrosine phosphatase (shp- ) in eae demonstrated that disease could be ameliorated through phosphatase inhibition ( ) . the competitive inhibitor, nsc- led to reduced demyelination and blocked cd + but not cd + t-cell migration into the cns, suggesting a pathogenic role for cd + t-cells in this model. a study of engineered transgenic nod mice expressing a mog - -reactive tcr ( c ) lends further support for pathogenic cd + t-cells in eae ( ) . c mice spontaneously generated mog-specific cd + and cd + t-cells that secrete pro-inflammatory cytokines. c cd + t-cells could recognize mog - in the context of mhc class i and ii, and when adoptively transferred into nod. scid recipients, induced optic neuritis and mild eae, while c cd + t-cells induced severe eae. cd + t-cells' ability to target cns components has also been evaluated in several viral models ( ) ( ) ( ) . lcmv gp peptidespecific cd + t-cells can induce lesions in cultured murine neurons presenting gp in mhc class i. while this report relies on peptide pulsing and artificial upregulation of mhc class i, viral infection-induced upregulation of class i has been demonstrated in borna disease virus-infected rat neuronal cultures, which could be targeted by antiviral cd + t-cells, eventually leading to apoptosis of neurons ( ) . although electrical signals were not initially disrupted in this model and longer incubation times were needed for neuronal apoptosis, another study has demonstrated impaired murine neuronal signaling following neuron/cd + t-cell interactions along with eventual apoptosis which interestingly occurred independent of perforin/granzymes ( ) . to this end, ifnγ-production from cns cd + t-cells and subsequent ifnγ signaling in neurons has been shown to be significant for intracranial lcmv disease in mice ( ) . in another study, ot-i cd + t-cells formed immune synapses with mhc class i (h- k b )-expressing axons presenting siinfekl peptide, and loss of axon integrity was observed. additionally, axonal injury was dependent upon antigen-specific tcr recognition and granzyme b ( ) . another report also described mice expressing neo-self antigen in oligodendrocytes (odcs) targeted by transgenic cd + t-cells ( ) . in this model, ovalbumin was expressed exclusively in the cytosol of odcs and therefore ignored by cd + t-cells and b-cells. following immunization, mild eae was observed in some odc-ova mice. studies using double transgenic odc-ova/ot-i mice demonstrate treatment with d mab (specific for h- k b /ova) prevented the lethal eae normally observed in these animals ( ) . double transgenic mice were also given d prophylactically, which in certain instances led to spontaneous disease remission. cd + t-cells have also been shown to indirectly influence cns autoimmunity. tc cells, coined for their ability to produce il- a, were detected in the lymph nodes and cns of mog - eae mice ( ) . tc s differ from conventional cd + t-cells regarding granzyme b and ifnγ expression, and thus are impaired in their cytotoxic capacity. in a separate study implementing cd + and cd + t-cell co-transfer, tc cells were found to help cd + th cells accumulate in the cns and induce eae ( ) . furthermore, their ability to produce il- a was required to render cd + t-cells encephalitogenic. while evidence exists to suggest a pathogenic role for cd + t-cells in ms and eae (reviewed in ref. ( ) and discussed above), there is a growing body of evidence supporting the opposite conclusion -cd + t-cells play an important regulatory role in the pathogenesis of ms and ms-like disease. ultimately, cd + t-cell subsets likely perform varying effector functions in the context of ms/eae. however, the seeming discrepancy is in part due to a lack of concrete in vivo evidence demonstrating a cytotoxic effect of cd + t-cells in ms lesions. furthermore, it has been demonstrated that depletion of cd + t-cells prior to eae induction results in exacerbated disease ( ) . similar results are seen in mice lacking mhc class i (although a role for nk cells can be argued) ( ) and in cd -deficient mice ( , , ) . this is in addition to work from our lab, which clearly demonstrated -in marked contrast to their cd + counterparts -neuroantigenspecific cd + t-cells failed to adoptively transfer eae disease to naïve recipient mice ( ) . we have seen this protective cd + t-cells phenotype very robustly in several models of eae ( ) . the notion of a regulatory cd + t-cell subset (cd + tregs) in ms is not a new idea. studies spanning several decades point to the suppressive potential of cd + t-cells in ms patients ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . in lieu of these examples, t-cell-mediated tolerance studies have largely focused on cd + cd + foxp + t-cells. although full appreciation of cd + treg function and significance in ms and eae is lacking, the last years have seen a steady growth toward this understanding. cd + t-cells' suppressive ability has been described in many mouse models, including cancer ( ) , diabetes ( ) , colitis ( ) , sle-like disease ( ), grave's disease ( ) , and transplant tolerance ( ) . inhibitory cd + t-cell subsets involved in autoimmunity in both mice and humans have been exhaustively reviewed in ref. ( ) . these regulatory cd + t-cells have been extensively studied in t d where it has been shown that lowavidity autoreactive cd + t-cells convert into memory-like autoregulatory cells and blunt diabetes progression ( , ) . however, cd + treg participation in eae is less-widely studied. moreover, unlike murine cd + foxp + tregs, a universal cd + treg phenotype has yet to be described. for example, in eae, cd + cd − t-cells have been shown to play an inhibitory role ( ) while others show cd + cd + t-cells to be protective ( ) ( ) ( ) . little is known concerning the induction of these cells in ms-like disease, though the involvement of one subtype versus another surely is influenced by disease setting and may depend on the cell's antigen specificity/mhc-restriction. studies of anterior chamber-associated immune deviation (acaid) represent some of the best efforts to understand antigen-specific cd + tregs, which appear to be qa- -restricted ( ) ( ) ( ) . several acaid studies further complicate the cd + treg phenotyping picture (e.g., foxp + , cd + , cd + , tgfβ-producing, etc.) ( ) ( ) ( ) ( ) ( ) . interestingly, immune deviation can be elicited against myelin antigens ( , ) , pointing to the potential role for qa- -restricted cd + t-cells in eae disease. qa- -restricted cd + t-cells have been described as being important for protection in mbp-driven eae ( ) . we have demonstrated that qa- -restricted cd + t-cells suppress eae. we have also demonstrated that ga treatment induces cd + treg in mice, and that these cd + t-cells are required for ga to be therapeutically effective in ameliorating eae disease ( ) . while little is still known about qa- -restricted cd + tregs, even less was understood about cns-specific cd + t-cells until very recently. we observed the surprising result that neuroantigen-specific cd + t-cells could suppress eae induction and even ameliorate established eae disease ( ) . to our knowledge, this was the first documentation of neuroantigenspecific cd + tregs in mice. in our recently published and unpublished results, adoptive transfer of both mog - -and plp - -specific cd + t-cells can suppress eae ( , ) . due to mechanistic studies, we will elaborate upon later that these cells are quite distinct from previously described qa- restricted cd + tregs ( ) . recent work has suggested a role of il- -producing cd + t-cells in diminishing disease pathology in virus-induced encephalitis models. these il- -producing cd + t-cells display a more functional profile including increased expression of pro-inflammatory cytokines and chemokines, are immunosuppressive, and their presence in the cns following coronavirus infection reduces tissue destruction and morbidity in these mice ( ) . interactions between cd + tregs and other cell types in eae/ms advancement in therapy for ms patients, particularly cellular immunotherapy, necessitates the full understanding of regulatory immune cell interplay. studies concerning the functional interactions between cd + tregs and other cells in the context of ms and ms-like disease are therefore of paramount interest. the next several sections will provide mechanistic insights into cd + t-cell-mediated modulation of other immune cells including cd + t-cells and antigen presenting cell (apc) populations. qa- -restricted cd + t-cells have been shown to modulate eae disease through action on cd + t-cells. it has been demonstrated in a model of mbp-driven eae that cd + t-cell vaccination protocol-mediated protection against eae disease is dependent on the presence of qa- -restricted cd + t-cells that recognize specific tcrvβ molecules on mbp-reactive cd + t-cells ( ) . in this particular example, cd + t-cells mediated their control by preferentially suppressing th cd + t-cells during eae. while this report did not directly test cytotoxic killing as a means of suppression, the group had previously established this capability in t-cell vaccination scenarios. data from another group later confirmed a cytotoxic effect by demonstrating that cd αα + tcrαβ + t-cells from lines that recognize tcrvβ . + (mbp-reactive) cd + t-cells could protect against eae disease in recipient mice by the targeted killing of these pathogenic cells via qa- -recognition ( ) . we have showed that the disease-ameliorating effect of ga-therapy in eae is dependent upon qa- -restricted cd + tregs ( ) . in this report, we demonstrated that the protective ability of cd + t-cells was completely lost or diminished when unable to produce ifnγ or perforin, respectively. these cd + t-cells could kill ga-loaded target t-cells and even limited the proliferation of ex vivo neuroantigen-specific cd + t-cells ( ) . furthermore, the ga-induced qa- -restricted cd + t-cells in this study were important for generation of cd + tregs ( ). these ga-specific cd + t-cells have the potential to kill ga-expressing cd + t-cells and limit proliferation of neuroantigen-specific and anti-cd -stimulated cd + t-cells ( , ) . we have also demonstrated that ga therapy, whose effects require cd + t-cells in mice ( ) , was able to increase the induction of cd + cd + tregs from the cd + cd − t-cell population in ms patient blood ( ) . distinct from the non-classical hla-e-like qa- -restricted murine cd + tregs, we have also demonstrated the existence of neuroantigen-specific cd + tregs in ms and eae. neuroantigen-specific, mhc class ia-restricted cd + t-cells can kill mog-loaded cd + t-cells in mice ( , ) and mediate their disease-ameliorating effects via the targeting of encephalitogenic cd + t-cells during eae disease ( ) . we have also demonstrated an ability of neuroantigen-specific cd + tregs to induce anti-inflammatory profiles in cd + t-cells during eae ( ) . importantly, we have also shown that neuroantigen-specific cd + t-cells are detectable in ms patient blood, and possess capacity to suppress cd + t-cell proliferation ( , ) . the potential for cd + t-cells to alter cd + t-cell priming through direct effects on dcs is worth investigation. cd + cd − t-cells have been implicated as regulators of eae disease. it has been demonstrated that dcs have reduced costimulatory molecule (cd , cd , and cd ) expression after culture with cd + cd − t regulatory cells, rendering these dcs as substandard apcs ( ) . it has been similarly demonstrated that dcs cultured with cd + cd + t-cells had a reduction in cd / and mhc molecules and showed inferior antigen-presentation ability compared to dcs cultured with cd + cd − t-cells ( ) . while it remains unclear whether qa- -restricted cd + tregs have a direct effect on dcs, we have shown that neuroantigen-specific cd + tregs can both kill and suppress antigen presentation of mog-loaded bulk apcs (contains dcs) ( ) . interestingly, we have demonstrated that neuroantigen-specific cd + tregs have little effect on dc surface expression of mhc or costimulatory molecules, but rather shift the inflammatory profiles of cd c + dcs from il- to il- ( ). early human ms work from our lab points to the potential of ga therapy-induced cd + tregmediated killing of apcs, as cd + t-cells were only a part of the larger target pool ( ) . another potential mechanism of suppression is cd + t-cellmediated regulation of monocytes or macrophages, which are present in ms lesions and important for pathology in the cns of eae mice. interestingly, ga treatment has been demonstrated to affect monocyte populations in eae. for example, anti-inflammatory type ii monocytes are induced in ga-treated mice, which can shift inflammatory cytokine profiles toward immunosuppressive il- , expand th cells, and induce cd + tregs capable of ameliorating eae ( ) . we have observed similar results and have further demonstrated that the action of ga on monocytes elicits cd + tregs and actually requires cd + t-cells for its ameliorative effects in eae ( ) . this ga-induced monocyte-cd + t-cell interaction is largely unknown in ms, as is the effect of ga-induced cd + t-cell targeting of other macrophage populations. while a direct link to cd + t-cells has yet to be confirmed, studies from us and others have shown modulation of monocytes following ga therapy in humans ( , , ) . as mentioned in the section above, ga-induced cd + t-cell-mediated killing of apc populations like dendritic cells and monocytes/macrophages while unconfirmed, cannot be ruled out, as cd + t cells were only a portion of a larger affected target pool ( ) . refining these assays for direct detection of killed targets is needed going forward. beyond ga-induced cd + tregs, neuroantigen-specific cd + tregs could conceivably modulate monocytes/macrophages in eae. we have demonstrated that these cells can kill mog-loaded bulk apcs, which may contain monocytes/macrophages, and can suppress their antigen presentation ( ) . however, we did not observe a substantial neuroantigen-specific cd + treg effect on monocytes during eae ( ) . furthermore, neuroantigen-specific cd + tregs from ms patients do not appear to specifically target monocytes. more work is needed to understand the potential functional interactions between cd + t-cells and monocytes/ macrophages during ms and ms-like disease, and may ultimately be a ga treatment-specific phenomenon. potential cd + t-cell: b-cell interactions in ms/eae? in light of depletion therapy success, more focus is now being given to b-cells and their role in ms. the literature supports both a pathogenic ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) and regulatory ( , ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) role for b-cells in ms/eae, and it is intriguing to speculate about the potential immune cell interplay between cd + t-cells, b-cells, and cd + t-cells therein. there is evidence in the literature to support a b-cell effect on cd + t-cells ( , , , ( ) ( ) ( ) ( ) ( ) ( ) . many of these reports point to b-cell antigen presentation to cd + t-cells and even a b-cell requirement for cd + treg function in some models. there is also literature supporting a role for bregs in controlling cd + t-cell responses ( , ( ) ( ) ( ) ( ) ( ) . additionally, cd + t-cells can be detected in follicles and modulate b-cell biology, such as germinal centers and antibody production ( , ( ) ( ) ( ) ( ) ( ) ( ) . the significance of these cd + t cell and b-cell subset interactions in the context of ms/eae remains to be seen. due to the inherent complexity of studying cd + t-cell function in the human brain, only circumstantial evidence exists regarding a pathogenic role for cd + t-cells in ms. cd + t-cells are the most abundant t-cells found in the cns lesions of ms patients, far outnumbering cd + t-cells ( ). in patients with active disease, cd + t-cells were detected in increasing amounts from the center to the edge of the lesions studied ( ) . the cd /cd ratio is shown to have been as high as / in the lymphocytic perivascular cuffs at the edge of active plaques ( ) . cd + t-cells displaying activated and memory phenotypes (suggesting previous interaction with local antigens) have also been detected in the cns and csf of ms patients ( , ) . cd + t-cell clones have also been shown to move throughout the affected cns and into normal appearing white matter (nawm) ( ) . one study demonstrated that there is diffuse infiltration by cd + t-cells combined with microglial activation and meningeal inflammation in the nawm of ms patients ( ) . unfortunately, assigning function to these cd + t-cells remains a challenging task, although speculations have been made that cd + t-cells present in the cns lesions of ms patients may be cytotoxic toward cns cells including glia and axons. cd + mhc class i-restricted myelin peptide-specific t-cells have been shown to cause injury to human odcs in vitro ( ) . similarly, an mbp-specific memory phenotype cd + t-cell line generated from the peripheral blood of ms patients, in addition to secreting ifnγ and tnfα, was able to lyse cos-mbp/hla-a -transfected cells that were presenting endogenous mbp ( ) . cd + t-cells have also been detected near or attached to odcs and demyelinated axons in ms patients ( ) ( ) ( ) . importantly, mhc class i molecules are present on astrocytes, odcs, neurons, and endothelial cells ( , ) . furthermore, mhc class i molecules are upregulated -depending on disease severity -and can be induced by ifnγ ( ) . cns blood vessel endothelium as well as several apcs also express mhc class i molecules, which can cross-present exogenous peptides ( ) . thus, it is not surprising that cd + t-cells have been demonstrated to interact with apcs at cns plaque margins ( ) . the potentially detrimental nature of this interaction is supported by a study that showed that the amount of cd + t-cells and macrophages present in an ms lesion is proportional to the amount of acute axonal damage present ( ) . effector cytokines from cd + t-cells can also enhance their cytotoxic function and activate other immune cells to amplify inflammatory cascades in the cns. for example, neuroantigenspecific cd + t-cells present in the peripheral blood express ifnγ and tnfα in response to their cognate antigen ex vivo ( , , ) . ifnγ-and il- -producing cd + t-cells can be recruited into the cns when responding to apoptotic t-cellassociated self-epitopes ( ) . one report demonstrated that cd + but not cd + t-cells from patients with acute rrms had increased ability to be recruited in inflamed cns venules ( ) . additionally, cd + il- -secreting t-cell numbers have been shown to be significantly elevated in acute cns lesions of ms patients ( ) . ifnγ-and il- -secreting cd + cd + t-cells were also found to be elevated in the peripheral blood of ms patients ( ) . higher frequency of cd + t-cells expressing cytotoxic molecules like perforin has been shown to be present in ms patients, particularly during a relapse ( ) . in light of the present literature, it can be appreciated that cd + t-cells in ms and other autoimmune diseases are phenotypically and functionally diverse, and can potentially regulate the pathogenic immune processes. besides cytolytic molecules like perforin and granzyme, cd + t-cells are armed with immunosuppressive cytokines, such as il- , that can dampen the inflammatory response. the evidence for cd + t-cell regulatory function in ms has existed for a long time and has been largely ignored by the field. cd + t-cells from the peripheral blood of ms patients displaying reduced levels of suppressor function was the first report that suggested a regulatory function for cd + t-cells in ms ( ). this was followed by another study that demonstrated a similar defect in cd + t-cell-mediated suppression in patients with chronic progressive ms ( ) . since then, mounting evidence has accumulated in the field of ms disease and others that collectively points toward a regulatory role for cd + t-cells in autoimmune diseases ( , , ) . more recently, our lab has provided direct evidence for cd + t-cell regulatory function in ms and has established clinical correlations with the disease activity ( ) . as in the mouse, phenotypic identification of human cd + tregs has been challenging. human cd + cd − t-cells have been shown to possess suppressor activity and are the most extensively studied population of cd + tregs. in ms, they were found to be present at significantly reduced frequency in the blood of rrms patients as compared to healthy donors ( ) . although it is not a marker for cd + tregs, foxp -expressing cd + t-cells are present in human blood. they possess regulatory activity ( ) , which is foxp -dependent ( ) , and are associated with autoimmune diseases such as ibd and ms ( , ) . cd + foxp + cells are present at reduced levels in the csf of ms patients during acute exacerbation ( ) . cd + cxcr + t-cells are human counterparts of the well-known regulatory cd + cd + t-cells found in mouse. human cd + cxcr + t-cells are suppressive in nature and their function is il- dependent ( ) . although all of these cd + treg subsets have potent immunosuppressive functions, so far their antigen specificity remains unknown. our lab showed for the first time that cns-specific cd + t-cells have potent suppressor activity toward myelin antigenspecific cd + t-cells ( ) . these cns-specific cd + t-cells were reactive to several myelin antigens including mog, plp, mbp, mag and others and are present in the peripheral blood of healthy donors and ms patients ( ) . mechanistically, these cns-specific cd + t-cells are mhc class i-restricted, and their suppressive function is ifnγ-and perforin-dependent ( , ) . our findings lend credence to the hypothesis that cns-specific cd + t-cells in the cns would function to dampen the inflammatory response by targeting pathogenic cd + t-cells and apcs, rather than causing damage themselves. phenotypically, these cells are cd + cd − cd − cd ro − cd l − cd + or a terminally differentiated subset of cd + t-cells ( ) . similar to qa -restricted cd + t in murine models, hla-erestricted cd + t-cells in humans perform a regulatory function and are involved in the maintenance of self-tolerance ( ) . the nature of nkg receptors present on cd + t-cells determines the functional outcome of their interaction with qa -expressing t-cell targets. for example, nkg c-expressing cd + t-cells suppress qa -expressing target t-cells while nkg a-expressing cd + t-cells get suppressed by these targets, and therefore cannot perform regulatory functions. a recent study showed reduced expression of foxp and cd in nkg c-expressing cd + t-cells from ms patients compared to healthy controls, suggesting a reduced regulatory potential of these cells in ms patients ( ) . although, there are only a handful of studies that report the phenotypic and functional significance of cd + t-cells in ms patients, one prominent feature that emerges from these studies is an underlying defect in the cd + treg component. of note, this defect is found specifically during ms relapses. since a relapse represents the active phase of the disease, any significant differences in the phenotype and functions of immune cells between relapse and remission may be directly correlated with the immunopathogenesis of ms. interestingly, frequency of circulating cd + foxp + t-cells was found to be significantly lower in the peripheral blood of ms patients during relapse as compared to remission ( ) . another study showed that cd + cd + cd − t-cells harbored potent suppressive activity and were lower in ms patients during relapse when compared to healthy controls ( ) . importantly, treatment with glucocorticoids leads to a significant increase in the frequency of these cd + tregs in the blood of ms patients. this was an interesting observation, suggesting that recovery from relapse under glucocorticoid treatment might be mediated by the regulatory function of cd + t-cells. furthermore, deficiency in cd + treg function is not limited to the blood, as evidenced by the significantly reduced cd + t-cell cloning frequency in the csf during ms relapse as compared to remission, suggesting loss of cd + tregs in the csf during relapse ( ) . our own studies show that the terminally differentiated cd + t-cell pool, which harbors the cns-specific cd + tregs, is significantly reduced during ms relapse as compared to remission ( ) . furthermore, relapses in ms are associated with significantly lower cns-specific cd + t-cell suppressor ability, while this potential in ms patients during quiescence is similar to healthy donors, suggesting a role with disease activity ( ) . of clinical significance, we showed that the cns-specific cd + treg suppressive function is restored in ms patients during remission and this recovery in cd + treg-mediated suppression correlated with the distance in time from an acute clinical episode. this suggests that the correction of the neuroantigen-specific cd + suppressor deficit would correlate with recovery from an acute relapse ( ) . one caveat to the study is that the quiescence samples could still potentially have pseudo relapses in the cns in the absence of any clinical signs. nonetheless, these findings raise the possibility that reduction in cns-specific cd + t-cell suppression might be used as a marker to predict relapses in ms patients. although etiology of ms remains unknown, epidemiological studies suggest an association between epstein-barr virus (ebv) and ms ( ) . ebv-reactive cd + t-cells are present in the peripheral blood of ms patients ( ) . by using high throughput sequencing, a recent study demonstrated intrathecal enrichment of ebv-reactive cd + t-cells in ms patients ( ) . however, the function of these cd + t-cells in the cns remains speculative. interestingly, adoptive immunotherapy with in vitro-expanded autologous ebv-specific cd + t-cells in secondary progressive ms had no adverse effects and was associated with clinical improvement and reduced disease activity on mri ( ) . this study suggests that the ebv-specific cd + t-cells in the cns of ms patients might be playing a regulatory role by limiting ebvinfected b-cells and antibody production. the pathogenic function of cd + t-cells in ms is believed to be largely derived from its cytotoxic potential toward cns tissues including glial cells and axons. however, there is a clear lack of evidence in this area in human ms. interestingly, a recent study demonstrated that cd + but not cd + t-cells from peripheral blood of ms patients expressed nkg c and had elevated levels of cytotoxic molecules fasl, granzyme b, and perforin. intriguingly, these cd + t-cells were cytotoxic toward hla-e-positive human odcs in vitro ( ) . this study suggested a novel mechanism for cns damage in ms which is, in contrast to the widely held view, potentially mediated by cd + t-cells. although the pathogenic role of cd + t-cells in ms remains largely speculative, the studies discussed above strongly suggest that there is now ample evidence for the regulatory role for cd + t-cell subsets in the disease process. lack of their regulatory function specifically during relapses should be probed further, as this could be a major underlying factor leading to relapse in ms. the majority of drugs used for the long-term management of ms are immunomodulatory in nature. the precise mechanisms by which these drugs act are under constant investigation. we have convincingly demonstrated that cd + tregs not only exist physiologically but can also be induced therapeutically by ga treatment. both, cd + and cd + t-cells reactive to ga are present in the peripheral blood of healthy donors and ms patients ( ). although cd + t-cell responses are comparable between the two groups, untreated ms patients have reduced ga-induced cd + t-cell responses and this deficiency is corrected after ga therapy ( ) . functionally, these ga-reactive cd + t-cells are hla-erestricted and have a strong suppressive potential against cd + t-cells ( ) . interestingly, ga-reactive cd + t-cells obtained from untreated ms patients have reduced suppressor ability and ga therapy restores the cd + t-cell suppressive potential in ms patients ( ) . these were the pioneering findings that linked the regulatory function of cd + t-cells with the therapeutic action of the drug. the proof of principle came from our eae studies discussed above where we showed that ga does not work in the absence of cd + t-cells in mice ( ) , suggesting that cd + t-cells are absolutely required for ga action and all the other reported immunomodulatory effects of ga might lie downstream to the induction of cd + tregs by the drug. the idea is also supported by our surprising observation that ga reverses the cd /cd t-cell ratio and increases cd + t-cell-mediated suppression as early as h after ga therapy initiation in humans ( ) . similar to our findings, a -year follow-up study after ifnβ treatment showed expansion of regulatory cd + t-cell subsets (cd + cd + and cd + cd + cd − ) in the responder cohort ( ) . another study found a higher frequency of regulatory cxcr + cd + t-cells months after ifnβ therapy ( ) . collectively, these studies suggest that therapeutic induction of cd + tregs might be the underlying factor in other ms therapies as well. natalizumab treatment results in a decreased cd + /cd + ratio in the csf and peripheral blood of ms patients ( ) . fingolimod therapy is associated with altering the cytokine status of cd + t-cells in peripheral blood ( ) . however, detailed dissection of the role of cd + t-cells has not been performed in the setting of these treatments. the potential roles of cd + t-cells in ms is summarized in the model shown in figure , where the various pieces of evidence supporting the potential of cd + t-cells for both pathogenic and regulatory roles in ms/eae disease are depicted. on the pathogenic side of the model, cd + t-cells, whose antigenic specificity has yet to be fully elucidated, have been shown to be involved in several disease-driving mechanisms, ranging from cytotoxicity and demyelination to pro-inflammatory cytokine production. this is in addition to hallmark activation behavior in disease lesions, such as oligoclonal expansion and ifnγ production. interestingly, this fails to rule out the activation of a regulatory population, as indicated in the bottom portion of the model. as illustrated on the regulatory side -to which our lab has made several novel contributions -several lines of evidence exist demonstrating the regulatory mechanisms performed by cd + t-cells in the context of ms/eae, which can either be neuroantigen specific (mhc class a-restricted) or ga/copaxone ® specific (hla-e/qa- restricted). their protective functions, which seem to depend on ifnγ and perforin production, range from direct cytotoxicity to pathogenic cd + t-cells to modulation of pro-inflammatory cytokine profiles to inhibition of apc function. it is still unclear to what extent cd + t-cells affect other cell populations such as b-cells, but some evidence demonstrates a suppressive effect on monocytes and macrophages. these all serve to suppress cns auto-inflammation and protect myelinated axons -effectively limiting eae disease pathogenesis. the potential role for these cd + tregs in ultimately modulating ms disease is of high interest. this work was supported, in part, by grant awards (to njk) from the nih and national ms society. immunohistological analysis of t lymphocyte subsets in the central nervous system in chronic progressive multiple sclerosis clonal expansions of cd (+) t cells dominate the t cell infiltrate in active multiple sclerosis lesions as shown by micromanipulation and single cell polymerase chain reaction oligoclonal expansion of memory cd + t cells in cerebrospinal fluid from multiple sclerosis patients multiple sclerosis: t-cell receptor expression in distinct brain regions neuroantigen-specific cd + regulatory t-cell function is deficient during acute exacerbation of multiple sclerosis high prevalence of autoreactive neuroantigen-specific cd + t cells in multiple sclerosis revealed by novel flow cytometric assay glatiramer acetate (copaxone) therapy induces cd (+) t cell responses in patients with multiple sclerosis therapeutic induction of regulatory, cytotoxic cd + t cells in multiple sclerosis hla-a confers an hla-drb independent influence on the risk of multiple sclerosis multiple sclerosis: a modifying influence of hla class i genes in an hla class ii associated autoimmune disease genes in the hla class i region may contribute to the hla class ii-associated genetic susceptibility to multiple sclerosis opposing effects of hla class i molecules in tuning autoreactive cd + t cells in multiple sclerosis a pathogenic role for myelin-specific cd (+) t cells in a model for multiple sclerosis viral infection triggers central nervous system autoimmunity via activation of cd + t cells expressing dual tcrs myelin antigen-specific cd + t cells are encephalitogenic and produce severe disease in c bl/ mice specificity, magnitude, and kinetics of mog-specific cd + t cell responses during experimental autoimmune encephalomyelitis relapsingremitting central nervous system autoimmunity mediated by gfap-specific cd t cells blocking initial infiltration of pioneer cd (+) t-cells into the cns via inhibition of shp- ameliorates experimental autoimmune encephalomyelitis in mice a transgenic model of central nervous system autoimmunity mediated by cd + and cd + t and b cells a critical role for virus-specific cd (+) ctls in protection from theiler's virus-induced demyelination in disease-susceptible sjl mice bystander cd t cell-mediated demyelination after viral infection of the central nervous system adoptively transferred cd + t lymphocytes provide protection against tmev-induced demyelinating disease in balb/c mice transection of major histocompatibility complex class i-induced neurites by cytotoxic t lymphocytes cytotoxic cd + t cell-neuron interactions: perforin-dependent electrical silencing precedes but is not causally linked to neuronal cell death neuroprotective intervention by interferon-gamma blockade prevents cd + t cell-mediated dendrite and synapse loss axons are injured by antigen-specific cd (+) t cells through a mhc class i-and granzyme b-dependent mechanism induction of experimental autoimmune encephalomyelitis in transgenic mice expressing ovalbumin in oligodendrocytes antigenspecific blockade of lethal cd t-cell mediated autoimmunity in a mouse model of multiple sclerosis a th -like developmental process leads to cd (+) tc cells with reduced cytotoxic activity il- a secretion by cd + t cells supports th -mediated autoimmune encephalomyelitis immune regulation of multiple sclerosis by cd + t cells regulatory functions of cd +cd -t cells in an autoimmune disease model eae in beta- microglobulin-deficient mice: axonal damage is not dependent on mhc-i restricted immune responses the disease-ameliorating function of autoregulatory cd t cells is mediated by targeting of encephalitogenic cd t cells in experimental autoimmune encephalomyelitis cd + t cells in inflammatory demyelinating disease immune regulatory cns-reactive cd +t cells in experimental autoimmune encephalomyelitis autoregulatory cd t cells depend on cognate antigen recognition and cd /cd* myelin determinants defective suppressor cell function mediated by t + cell lines from patients with progressive multiple sclerosis suppressor and cytolytic cell function in multiple sclerosis. effects of cyclosporine a and interleukin modulation of immune function occurs within hours of therapy initiation for multiple sclerosis hla-e restricted cd + t cell subsets are phenotypically altered in multiple sclerosis patients lag- regulates cd + t cell accumulation and effector function in murine self-and tumor-tolerance systems cd + regulatory t cells are responsible for gad-igg gene-transferred tolerance induction in nod mice cd +cd + regulatory t cells (tregs) and cd + tregs cooperatively prevent and cure cd + cell-induced colitis inhibition of follicular t-helper cells by cd (+) regulatory t cells is essential for self tolerance cd +cd + t cells, a newly identified regulatory t subset, negatively regulate graves' hyperthyroidism in a murine model mechanism and localization of cd regulatory t cells in a heart transplant model of tolerance inhibitory cd + t cells in autoimmune disease development of memory-like autoregulatory cd + t cells is cd + t cell dependent reversal of autoimmunity by boosting memory-like autoregulatory t cells essential role of cd +cd + regulatory t cells in the recovery from experimental autoimmune encephalomyelitis two discreet subsets of cd t cells modulate plp( - ) induced experimental autoimmune encephalomyelitis in hla-dr transgenic mice il- -dependent cd + cd + t cells ameliorate experimental autoimmune encephalomyelitis by modulating il- production by cd + t cells ocular immune privilege promoted by the presentation of peptide on tolerogenic b cells in the spleen. ii. evidence for presentation by qa- splenic b cells act as antigen presenting cells for the induction of anterior chamber-associated immune deviation implication for the cd / nkg a-qa- system in the generation and function of ocular-induced splenic cd + regulatory t cells cd + t regulatory cells use a novel genetic program that includes cd to suppress th immunity in eye-derived tolerance increased expression of foxp in splenic cd + t cells from mice with anterior chamber-associated immune deviation upregulation of cd on cd + t cells in anterior chamber-associated immune deviation splenic cd + t cells secrete tgf-beta to exert suppression in mice with anterior chamber-associated immune deviation in vitro-induced cell-mediated immune deviation to encephalitogenic antigens the in vivo and in vitro induction of anterior chamber associated immune deviation to myelin antigens in c bl/ mice cd + t cells control the th phenotype of mbp-reactive cd + t cells in eae mice cd t cells are required for glatiramer acetate therapy in autoimmune demyelinating disease neuroantigen-specific autoregulatory cd + t cells inhibit autoimmune demyelination through modulation of dendritic cell function highly activated cytotoxic cd t cells express protective il- at the peak of coronavirus-induced encephalitis regulation of immunity by a novel population of qa- -restricted cd alphaalpha+tcralphabeta+ t cells disease exacerbation of multiple sclerosis is characterized by loss of terminally differentiated autoregulatory cd + t cells type ii monocytes modulate t cell-mediated central nervous system autoimmune disease type monocyte and microglia differentiation mediated by glatiramer acetate therapy in patients with multiple sclerosis multiple sclerosis: glatiramer acetate inhibits monocyte reactivity in vitro and in vivo a monoclonal antibody against a myelin oligodendrocyte glycoprotein induces relapses and demyelination in central nervous system autoimmune disease critical role of antigen-specific antibody in experimental autoimmune encephalomyelitis induced by recombinant myelin oligodendrocyte glycoprotein a comparative analysis of b cell-mediated myelin oligodendrocyte glycoprotein-experimental autoimmune encephalomyelitis pathogenesis in b cell-deficient mice reveals an effect on demyelination pathogenic myelin oligodendrocyte glycoprotein antibodies recognize glycosylated epitopes and perturb oligodendrocyte physiology regulatory b cells inhibit eae initiation in mice while other b cells promote disease progression b-cell activation influences t-cell polarization and outcome of anti-cd b-cell depletion in central nervous system autoimmunity the role of antibodies in multiple sclerosis the immunopathophysiology of multiple sclerosis b cell depletion therapy ameliorates autoimmune disease through ablation of il- -producing b cells b cells and antibodies in multiple sclerosis pathogenesis and therapy b cells promote induction of experimental autoimmune encephalomyelitis by facilitating reactivation of t cells in the central nervous system experimental autoimmune encephalomyelitis induction in genetically b cell-deficient mice b cells are critical to induction of experimental allergic encephalomyelitis by protein but not by a short encephalitogenic peptide b cells regulate autoimmunity by provision of il- not always the bad guys: b cells as regulators of autoimmune pathology regulatory b cells (b cells) and regulatory t cells have independent roles in controlling experimental autoimmune encephalomyelitis initiation and late-phase immunopathogenesis glatiramer acetate for treatment of ms: regulatory b cells join the cast of players a case for regulatory b cells in controlling the severity of autoimmune-mediated inflammation in experimental autoimmune encephalomyelitis and multiple sclerosis immune regulatory function of b cells regulatory b cells control t-cell autoimmunity through il- -dependent cognate interactions il- -producing regulatory b cells (b cells) in autoimmune disease cytokine-producing b cells as regulators of pathogenic and protective immune responses il- -producing b cells are critical regulators of immunity during autoimmune and infectious diseases suppression of immune responses by cd cells. ii. qa- on activated b cells stimulates cd cell suppression of t helper responses cpg-dna aided cross-priming by cross-presenting b cells b-cell cross-presentation of autologous antigen precipitates diabetes role of splenic b cells in the immune privilege of the anterior chamber of the eye splenic b cells are required for tolerogenic antigen presentation in the induction of anterior chamber-associated immune deviation (acaid) peripheral tolerance via the anterior chamber of the eye: role of b cells in mhc class i and ii antigen presentation role of il- -producing regulatory b cells in control of cerebral malaria in plasmodium berghei infected mice the expanding family of regulatory b cells infiltrating regulatory b cells control neuroinflammation following viral brain infection regulatory b cells inhibit cytotoxic t lymphocyte (ctl) activity and elimination of infected cd t cells after in vitro reactivation of hiv latent reservoirs regulatory b cell frequency correlates with markers of hiv disease progression and attenuates anti-hiv cd (+) t cell function in vitro cd t cells are required for the formation of ectopic germinal centers in rheumatoid synovitis a specific role for b cells in the generation of cd t cell memory by recombinant listeria monocytogenes mesenteric b cells centrally inhibit cd + t cell colitis through interaction with regulatory t cell subsets cxcr + ccr -cd t cells are early effector memory cells that infiltrate tonsil b cell follicles helper b cells promote cytotoxic t cell survival and proliferation independently of antigen presentation through cd /cd interactions integration of b cells and cd + t in the protective regulation of systemic epithelial inflammation distribution of t cells, t cell subsets and ia-positive macrophages in lesions of different ages immunohistochemical analysis of the cellular infiltrate in multiple sclerosis lesions increased cd + cytotoxic t cell responses to myelin basic protein in multiple sclerosis cortical demyelination and diffuse white matter injury in multiple sclerosis mhc class i-restricted lysis of human oligodendrocytes by myelin basic protein peptide-specific cd t lymphocytes the immunopathology of multiple sclerosis: an overview cytotoxic t lymphocytes in autoimmune and degenerative cns diseases dendritic cells in multiple sclerosis lesions: maturation stage, myelin uptake, and interaction with proliferating t cells expression of major histocompatibility complex class i molecules on the different cell types in multiple sclerosis lesions cell type-specific regulation of major histocompatibility complex (mhc) class i gene expression in astrocytes, oligodendrocytes, and neurons an antigen-specific pathway for cd t cells across the blood-brain barrier acute axonal injury in multiple sclerosis. correlation with demyelination and inflammation autoantigen recognition by human cd t cell clones: enhanced agonist response induced by altered peptide ligands autoreactive cd + t-cell responses to human myelin protein-derived peptides increased cd + t cell responses to apoptotic t cell-associated antigens in multiple sclerosis cd + t cells from patients with acute multiple sclerosis display selective increase of adhesiveness in brain venules: a critical role for p-selectin glycoprotein ligand- interleukin- production in central nervous system-infiltrating t cells and glial cells is associated with active disease in multiple sclerosis cd (high)cd + t cells bear pathogenetic potential in multiple sclerosis circulating cd +cd -perforin+ t cells are increased in multiple sclerosis patients comparison of t + cell-mediated suppressor and cytotoxic functions in multiple sclerosis inhibition of t cell responses by activated human cd + t cells is mediated by interferon-gamma and is defective in chronic progressive multiple sclerosis defects in cd + regulatory t cells in the lamina propria of patients with inflammatory bowel disease characterization of effector memory cd + t cells in the synovial fluid of rheumatoid arthritis numerical defects in cd +cd -t-suppressor lymphocyte population in patients with type diabetes mellitus and multiple sclerosis tlr agonists enhance cd +foxp + regulatory t cells and suppress th immune responses during allergen immunotherapy role of cd + cd + foxp + regulatory t cells in multiple sclerosis cd (+)foxp (+) t cells in peripheral blood of relapsing-remitting multiple sclerosis patients human cd +cxcr + t cells have the same function as murine cd +cd + treg hla-e-restricted regulatory cd (+) t cells are involved in development and control of human autoimmune type diabetes expansion of regulatory cd + t-lymphocytes and fall of activated cd + t-lymphocytes after i.v. methyl-prednisolone for multiple sclerosis relapse isolation and characterization of cd + regulatory t cells in multiple sclerosis th dahlem conference on infection, inflammation and chronic inflammatory disorders: epstein-barr virus and multiple sclerosis: epidemiological evidence strong ebv-specific cd + t-cell response in patients with early multiple sclerosis high-throughput sequencing of tcr repertoires in multiple sclerosis reveals intrathecal enrichment of ebv-reactive cd + t cells epstein-barr virus-specific adoptive immunotherapy: a new horizon for multiple sclerosis treatment? cytotoxic nkg c+ cd t cells target oligodendrocytes in multiple sclerosis ifnbeta- a therapy for multiple sclerosis expands regulatory cd + t cells and decreases memory cd + subset: a longitudinal -year study immune response during interferon beta- b treatment in patients with multiple sclerosis who experienced relapses and those who were relapse-free in the start study fingolimod modulates peripheral effector and regulatory t cells in ms patients altered cd +/cd + t-cell ratios in cerebrospinal fluid of natalizumab-treated patients with multiple sclerosis key: cord- -tyimwctm authors: farr, laura; ghosh, swagata; moonah, shannon title: role of mif cytokine/cd receptor pathway in protecting against injury and promoting repair date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: tyimwctm wound healing after an injury is essential for life. an in-depth understanding of the healing process is necessary to ultimately improve the currently limited treatment options for patients suffering as a result of damage to various organs and tissues. injuries, even the most minor, trigger an inflammatory response that protects the host and activates repair pathways. in recent years, substantial progress has been made in delineating the mechanisms by which inflammatory cytokines and their receptors facilitate tissue repair and regeneration. this mini review focuses on emerging literature on the role of the cytokine macrophage migration inhibitory factor (mif) and its cell membrane receptor cd , in protecting against injury and promoting healing in different parts of the body. whenever an injury occurs, the body needs to repair it efficiently in order to protect from further damage and restore function. from minor scratches to myocardial infarction, we continually experience traumatic events throughout life. therefore, the healing process is essential for survival. further understanding of the mechanisms that promote healing could lead to new therapeutic opportunities to improve the lives of individuals with illnesses that resulted from organ and tissue injury ( , ) . in addition to protecting against invading pathogens, an appropriate inflammatory response activates repair pathways that are essential for healing, without causing unwanted damage to the host tissue. cytokines play a crucial role in inflammation-driven repair. cytokines act by binding to specific receptors on certain cell types triggering downstream signaling events that ultimately promote the healing process ( , ) . this review focuses on the recent advances that have greatly contributed to our current understanding of the link between the signaling pathways activated upon binding of macrophage migration inhibitory factor cytokine to its membrane receptor cd and wound healing in different body parts (figure ). macrophage migration inhibitory factor (mif) is one of the first described cytokines, identified as a soluble immune cell-derived factor over years ago in . similar to cytokines such as tumor necrosis factor (tnf), mif's range of functions has exceeded what is implied by the historical name ( , ) . the mif gene was cloned in , and subsequent studies have demonstrated a wide range of roles for mif. mif is a truly pleiotropic inflammatory cytokine that is expressed by a variety of cells, and is a critical upstream mediator of innate immunity. given its important role in immunity, it is not surprising that excess mif expression has been linked to exaggerated inflammation and immunopathology. in addition, mif demonstrates well-documented proliferative properties. mif is secreted by many different types of cells and interacts with several receptors, which helps to explain the variety of biological functions. receptors that interact that bind mif include cd , and chemokine receptors cxcr and cxcr ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . cd is a type ii transmembrane protein consisting of an nterminal cytosolic tail, a short transmembrane region, and a long c-terminus luminal region. human cd is encoded on chromosome and consists of four isoforms. isoforms p and p are generated by alternative splicing, that is, the p isoform is created by excluding exon b from p cd transcript. isoforms p and p originate from an alternative start site ( ) ( ) ( ) ( ) ( ) ( ) . while cd was first discovered in through coimmunoprecipitation of the major histocompatibility class ii antigen (mhcii), it wasn't until the antigen presentation function of cd was recognized. cd is expressed on classical antigen presenting cells (apcs), such as dendritic cells and macrophages, acts as a chaperone that binds mhcii, and is commonly referred to as the class ii invariant chain (ii) ( , , , ) . subsequently, a growing body of evidence supported the concept that cd could have additional functions as a receptor. surface expression of cd occurred independently of concomitant mhcii expression. additionally, cd expression was found on the surface of non-apcs such as endothelial cells, and epithelial cells in the kidney, lung, gut, and skin ( , ) . the receptor that mediated mif activity remained elusive until a study in , which utilized a cdna library and fluorescently conjugated mif to screen for a receptor and identified cd as the mif receptor. the authors described that mif bound to the extracellular domain of cd , resulting in extracellular signal-regulated kinase (erk) pathway activation ( ) . mif-induced erk activation through cd appears to depend on cd forming a complex with co-receptor cd (cd /cd ) ( , ) . in addition to erk, stimulation of cd has been shown to trigger activation of the pi k-akt signal transduction cascade, nf-κb, and the amp-activated protein kinase (ampk) pathways. these pathways play important roles in cell proliferation and survival ( ) . d-dopachrome tautomerase (d-dt, mif- ) was recently described as a member of the mif protein superfamily, demonstrating overlapping inflammatory and proliferative properties with mif. d-dt and mif genes are located in close proximity on chromosome , ∼ kb apart. the amino acid sequence of human mif and d-dt shows % identity, however, the structure of the two proteins is highly conserved. d-dt binds cd and initiates similar signaling pathways ( , ) . mif homologs are also expressed by parasites. these mif homologs are structurally and functionally similar to human mif and interact with cd . while it may seem counter-intuitive for protozoans to secrete mif, parasite mif appears to contribute to immune evasion and invasion ( ) ( ) ( ) . regulation of mif-cd interactions occurs at several levels. mif is constitutively expressed with increased mif secretion occurring early in the inflammatory response. triggers of increased mif release include lipopolysaccharide (lps) and cell injury. secreted mif then interacts with cd to carry out some if its functions ( , ) . cd activity is regulated by changes in expression, proteolytic processing, and mifinteracting proteins that prevent binding to cd . similar to mif, cd is expressed on multiple cells: immune cells (e.g., b lymphocytes, macrophages, dendritic cells) and non-immune cells including epithelial cells. information on the regulation of cd expression in these different cells remains limited. increased cd expression is observed in injury, inflammation, and cancer. ifn-γ, a cytokine crucial to both innate and adaptive immunity, increases cd expression in a variety of cells ( ) ( ) ( ) . intracellular binding partners released in the extracellular space can regulate cytokine activity. both ribosomal protein s (rps ) and c-jun activation domain binding protein (jab ) were shown to have regulatory effects by binding to mif, inhibiting its interaction with cd ( , ) . cd also exists in a soluble cd ectodomain form which results from proteolytic shedding of the ectodomain region. however, the molecular mechanism including the protease responsible for releasing cd ectodomain remains poorly understood. ectodomain shedding decreases the amount of cd surface receptors available to interact with mif. also, cd ectodomain regulates mif activity by acting as a decoy receptor, sequestering free mif to negatively regulate mif signaling ( ) ( ) ( ) . another proteolytic step involves signal peptide peptidase-like a (sppl a), which is an aspartic intramembrane protease. sppl a has shown to play an important role in cd proteolysis ( , ) . yet, the exact role of sppl a-mediated cd proteolysis in mif signaling and whether modulating sppl a enzyme activity affects mif proinflammatory and proliferative functions remain to be fully investigated ( ) . in the following sections, we summarize the recent data supporting the reparative role of mif-cd signaling in different organs and tissues during injury. the role of cd in other disease processes, antigen presentation, and cancer has been well-reviewed elsewhere ( , - , , - ) . inflammatory bowel disease (ibd), exemplified by crohn's disease (cd) and ulcerative colitis (uc), is a growing public health challenge and socio-economic problem that affect millions with rapidly increasing incidence worldwide ( , ) . mucosal healing has been established as an important treatment predictor of sustained clinical remission and resection-free survival in ibd ( ) . unfortunately, a significant number of ibd patients do not respond to current treatment (including corticosteroids or biologics), and as many as % of cd and % of uc patients require surgical resection of affected regions of their intestine ( ) . current therapeutic strategies focus on limiting inflammation, thus, there is an urgent need to develop new approaches that also facilitate tissue repair and mucosal healing. our understanding of the genetic contributions to ibd has seen significant advances over the past few decades. genome-wide association studies (gwas) have identified new single nucleotide polymorphisms (snps) associated with ibd predisposition and treatment failure ( , ) . a recent study aimed at determining genetic factors associated with poor response to anti-tnf therapy, found that a strong association between a cd polymorphism and anti-tnf failure in patients with ulcerative colitis. the rs snp is located in the cd promoter region. the odds ratio for non-response to anti-tnf therapy with this snp was relatively high at ( ) . cd gene expression is increased in patients with ibd ( , ) , which occurs in the inflamed areas compared with noninflamed and healthy intestine (figure ). cd overexpression was most noticeable in proliferating crypt epithelial cells of patients with ibd and amebic colitis, a condition often misdiagnosed as ibd ( , ) . cd is almost undetectable in the epithelium of non-inflamed human and mice intestine when analyzed by immunohistochemistry ( , , ) . therefore, it was not too surprising to find that cd deficient mice had normal colon, histology, and barrier integrity, and lacked spontaneous colitis in the absence of pathologic insults ( ) . on the other hand, mif is expressed by epithelial cells that line the intestine and mif-deficient mice have impaired intestinal barrier integrity ( ) . using a combination of genetic knock-out, bone marrow chimera mice, chemical, non-chemically-induced, acute, and chronic mouse models of colitis, cd was found to be essential for mucosal healing in colitis-associated injury. at the cellular level, mif stimulation of cd on intestinal epithelial cells increased cell proliferation and wound closure, an effect that was lost in cd -deficient cells. mechanistically, mif, which also is increased in colitis, stimulated the cd receptor, activating proproliferative akt and erk pathways ( ) . so while dispensable in steady state conditions, cd appears to be necessary for reparative inflammation. based on these findings, enhancing the cd pathway might represent a unique treatment approach for promoting healing in ibd. though, finding the right ligand to stimulate cd may present a challenge. that is, stimulation of cd with exogenous mif might lead to an excessive inflammatory state, as mif is capable of stimulating cxcr and cxcr receptors in addition to cd . cxcr and cxcr receptors when activated promote influx of neutrophils and lymphocytes, respectively ( , ) . lung injury arises from a wide variety of insults, which include pulmonary infections, such as bacterial and viral pneumonia caused by influenza and coronavirus, vaping-associated pulmonary illness (vapi), ischemia-reperfusion-induced lung injury, and ventilator-induced lung injury ( ) ( ) ( ) . in the - season, influenza caused around , hospitalizations and , deaths ( ) . the emerging covid- has an increasing impact through infections and deaths as well as the economic impacts of quarantines and event cancellations to reduce infection spread ( , ) . lung injury causes damage to the epithelium. the alveolar epithelial barrier consists of two main cell types: alveolar epithelial type i and type ii cells. type i cells are flat cells through which gas exchange takes place and occupies most of the alveolar surface area. type ii cells serve as progenitor cells for the alveolar epithelium. type i cells are more sensitive to injury and are predominantly destroyed during lung damage. type ii cells proliferate and differentiate into type i cells, thus actively reforming the alveolar epithelium after damage and promoting alveolar repair ( ) . type ii cells express cd on their surface. during acute injury such as viral infection, type i cells release mif. extracellular mif binds to cd on adjacent type ii epithelial cells, activating akt and erk pathways, resulting in cell proliferation and differentiation to restore the alveolar barrier ( ) . lung endothelial cells display almost undetectable amounts of cd at baseline. a recent study found that chronic hyperoxia led to cd upregulation in endothelial cells ( ) . hyperoxia is common in patients with adult respiratory distress syndrome (ards), which is due to the requirement for high levels of supplemental oxygen. endothelial injury is a key feature of hyperoxic acute lung injury ( ) . mif-cd activation was found to protect from oxidative stress in an animal model. mif and cd genetic knock-outs, and pharmacological inhibition of cd resulted in loss of the protective effects of cd . this led to increases in inflammatory cytokines, apoptosis, and mortality. at the molecular level, cd activation during hyperoxia induced proliferative and pro-survival effects through erk and akt activation ( ) . neutrophils appear to play a significant role in tissue damage and the development of acute lung injury ( ) . it is important to mention that excess mif was shown to correlate with neutrophil accumulation into the lung ( ) . however, it remains unclear how much mif-cxcr interaction is contributing to leukocyte recruitment. acute kidney injury (aki) remains a significant medical problem and is associated with increased hospital mortality, length of stay, and costs. individuals who survive an aki hospitalization are likely to fail renal function recovery and go on to develop chronic kidney disease and hypertension ( ) . most cases of aki are due to ischemia, but our kidneys are also vulnerable to damage by toxins, infection, and immune-mediated insults. ischemic aki, for example, results in significant renal tubular cell damage. free radicals formed during ischemia and reperfusion (i/r) also contribute to renal damage. surviving cells undergo epithelium regeneration to restore healthy renal function ( , ) . a better understanding of the repair processes underlying kidney repair will facilitate therapies that will prevent injury, promote recovery, and minimize the progression to chronic kidney disease. cd is expressed on the surface of renal tubular epithelial cells. also, these cells express low levels of mif which is increased following aki to ensure adequate supplies at the site of damage ( , ) . a spontaneous pathological renal phenotype is absent mif knock-out mice, suggesting little to no effect on healthy organs ( ) . however, high mif levels can be found in the serum of patients following cardiac surgery and correlates with protection from aki ( ) . in a murine model of experimental ischemia-reperfusion injury, mif, mif- , and cd knock-out mice had worse tubular injury compared to wild type control mice. mif- improved the recovery of injured epithelial cells by enhancing cell regeneration through secretory leukocyte proteinase inhibitor (slpi) and activating transcription factor (atf) -dependent mechanisms ( ) . slpi has proliferative, antioxidant and cytoprotective properties, and is being evaluated as a biomarker for aki after surgery ( ) ( ) ( ) . while mif/mif- are likely protective in ir, this might not be the case for all renal diseases depending on the underlying pathology. for example, mif has been linked to injury and inflammation in models of glomerular diseases ( ) ( ) ( ) . therefore, additional studies are required to determine which patient conditions would benefit from blockade vs. stimulation strategies. cardiovascular disease is the leading cause of death in the united states. risk factors for cardiovascular disease include smoking, obesity, and hypertension. myocardial infarction, or heart attack, occurs in one american every s ( ) . treatment for mi is composed of anti-coagulant medication, thrombolytics, and surgical intervention to restore normal blood flow. however, damage to cardiomyocytes caused by ischemia is not addressed in the standard treatment regimen and can lead to heart failure. targeting repair of heart tissue during mi may improve patient outcomes and prevent chronic disease. cd signaling was shown to have protective effects in cardiomyocytes in cardiac i/r injury animal model. mif is secreted from the cardiomyocytes during i/r and acts in an autocrine-paracrine manner, stimulating cell surface cd receptor. activation of cd with exogenous mif- improved cell survival and infarct size both in wild-type control and conditional mif- knockout mice, while cd deletion led to worse injury. mechanistically, mif- binding to cd quickly activates the amp-activated protein kinase (ampk) cascade via a calcium dependent kinase, cammk ( ) . activation of the ampk pathway in cardiomyocytes decreases apoptosis, necrosis, and contractile dysfunction following ischemia ( , ) . mif- in contrast to mif appears to lack the necessary cxcr-interacting motifs necessary for activation, and it is believed to exert a more selective action in activating the tissue-protective cd signaling pathway. that said, mif triggers the cd /cd /ampk receptor signaling pathway, which promotes glucose uptake in cardiomyocytes and protects the heart during ischemia-reperfusion injury ( , ) . further studies are required to determine the potential of mif/mif- as a treatment strategy to protect the heart against ischemic injury. impaired wound healing in the setting of non-healing surgical or traumatic wounds, pressure ulcers, diabetic foot ulcers, venous, and ischemic ulcers, presents a substantial healthcare burden. chronic non-healing wounds contribute to significant healthcare costs, poor quality of life, and serious outcomes such as amputations ( , ) . following injury, several cytokines play important roles during tissue repair and promote cutaneous wound healing by the classic stages of wound repair: inflammation, new tissue formation, and remodeling ( , ) . therefore, cytokine pathways have been targeted when designing regenerative strategies to promote chronic wound repair ( ) . gene expression studies have been valuable for identifying cytokines expressed during the inflammatory process in a wound setting ( ) . a study analyzing gene expression profiles in patients with punch biopsies found mif gene expression increased during cutaneous wound healing ( ) . the role of mif in promoting wound healing was investigated using an animal model of skin injury. mif levels were elevated early after injury and facilitated proliferation and migration of keratinocytes from the edge of the wound ( ) . these results support a reparative response of mif to cutaneous injury. in addition, transcriptomic analysis revealed cd upregulated in pressure ulcers in a neuropathic ulcer mouse model ( ) . it is plausible that the mif-cd pathway promotes cutaneous wound repair, however, further studies will be required to characterize the role of cd signaling in cutaneous wound healing. cd signaling has also been found to play a potential role in healing in other tissues such as the nervous system and liver. sciatica is a chronically painful disease caused by injury to the sciatic nerve. schwann cells express cd , and mif is upregulated following sciatic nerve injury. mif-stimulated cd activation of the erk pathway led to schwann cell proliferation and subsequent nerve regeneration. also, in vitro studies show that mif facilitates schwann cell migration. both schwann cell proliferation and migration promote nerve regeneration ( ) . a separate in vitro study demonstrated that cd activation by mif promoted cell survival and proliferation of neural progenitor cells ( ) . further studies will be required to determine if mif-induced proliferation of neural progenitor cells can be a therapeutic option in brain disorders. in the liver, cd -mif signaling plays a protective role in nonalcoholic fatty liver disease (nafld) by enhancing ampk ( ) . while this review focuses on the protective role of mif-cd signaling, it should be noted that this is not the case for all diseases ( , , ) . the complex pathological processes that result in disease combined with cd 's expression on a variety of cell types, and its multiple co-receptors with diverse downstream signaling pathways contribute to these varied outcomes. for example, lupus nephritis is inflammation of the kidney that is caused by the autoimmune disease systemic lupus erythematosus (sle) ( ) . b cells participate in sle immunopathogenesis ( ) . b lymphocytes express elevated levels of cd in mouse models of sle and lupus-prone mouse strains have elevated mif. both mif and cd elevated expression positively correlated with worsening inflammation. mif inhibition and cd deficiency protected against glomerulonephritis in lupusprone mice ( , ) . despite these results that suggest mif-cd pathway plays a role in lupus pathology, a phase clinical trial of an anti-mif monoclonal antibody in lupus nephritis was terminated early for unclear reasons ( ) . these findings suggest that mif-cd functions with differential outcomes occur in a context-and cell type-dependent manner. given this complexity, additional research is needed to determine when and how to inhibit or stimulate the mif-cd pathway to achieve benefit. also, whether disease associations are a result of different coreceptor involvement on different cell types should be a focus of future research. mif's proinflammatory effects involve enhancing the expression of various cytokines such as tnf-α, il- , il- ( ) . cytokines like il- are now recognized for their roles triggering tissue repair and regeneration ( , ) . while these downstream proinflammatory mif effects have been linked to immune disorders, it remains possible that they play a role in the healing effects of mif-cd signaling. this would be an interesting area for future investigation as balancing the positive and negative effects of mif appears to be key. discussed above is the recurrent observations of the protective effects of mif-cd signaling in wound-healing. recent studies have furthered our understanding of the mechanisms by which cd stimulation leads to tissue repair in multiple parts of the body involving some of the most important diseases. despite these advances, key questions remain unanswered. for example, although there is mechanistic overlap, the downstream pathways that are important for cd -mediated repair appear to vary with the tissue or cell type. in epithelial cells, such as those that line the gut and alveoli of the lungs, mif-cd interaction triggers the activation of pro-survival and proliferative akt and erk pathways. in contrast, activation of the pro-survival kinase ampk seems to play a more significant role in cardiomyocytes and hepatocytes. the molecular reason for the different downstream signaling pathways beyond differences in cell types is not fully understood and present worthy unknowns to be solved by future studies. furthermore, a selective agonist that will stimulate cd -mediated repair with little or no unwanted side effects remains poorly defined. the answers to such questions may allow us to translate these recent scientific discoveries into clinical interventions, and ultimately benefit those suffering as a result of injury to various organs and tissues. lf, sg, and sm wrote, edited and reviewed the manuscript. all authors contributed to the article and approved the submitted version. this work was supported by national institutes of health (nih) r ai -s , k ai , uva seed grant, and the robert wood johnson foundation-harold amos medical faculty development program award. tissue repair: the hidden drama wound repair and 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macrophage migration inhibitory factor (mif) promotes cell survival and proliferation of neural stem/progenitor cells protective role of macrophage migration inhibitory factor in nonalcoholic steatohepatitis contribution of the macrophage migration inhibitory factor superfamily of cytokines in the pathogenesis of preclinical and human multiple sclerosis: in silico and in vivo evidences update on lupus nephritis mechanisms of b cell autoimmunity in sle cd deficiency mitigates systemic lupus erythematosus-like autoimmunity and pathological findings in mice a role for the b-cell cd /macrophage migration inhibitory factor pathway in the immunomodulation of systemic lupus erythematosus by a therapeutic tolerogenic peptide anti-macrophage migration inhibitory factor (anti-mif) antibody in lupus nephritis a gp -src-yap module links inflammation to epithelial regeneration we thank richard bucala md, ph.d. (yale university) for the support. we apologize to those colleagues whose work could not be included due to space limitations. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © farr, ghosh and moonah. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - vtv xuh authors: schramm, markus a.; venhoff, nils; wagner, dirk; thiel, jens; huzly, daniela; craig-mueller, nils; panning, marcus; hengel, hartmut; kern, winfried v.; voll, reinhard e. title: covid- in a severely immunosuppressed patient with life-threatening eosinophilic granulomatosis with polyangiitis date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: vtv xuh immunosuppressive therapies increase the susceptibility of patients to infections. the current pandemic with severe acute respiratory syndrome coronavirus (sars-cov- ) compels clinicians to develop recommendations for successful clinical management and surveillance of immunocompromised patients at high risk for severe disease progression. with only few case studies published on sars-cov- infection in patients with rheumatic diseases, we report a -year-old male who developed moderate coronavirus disease (covid- ) with fever, mild dyspnea, and no major complications despite having received high-dose prednisolone, cyclophosphamide, and rituximab for the treatment of highly active, life-threatening eosinophilic granulomatosis with polyangiitis (egpa). with a wide range of clinical outcomes in coronavirus disease (covid- ), from being asymptomatic to fatal acute respiratory distress syndrome, questions have been raised about the safety of immunosuppressive therapies ( ) . individuals with anti-neutrophil cytoplasm autoantibody (anca)-associated vasculitides require particular care, especially considering the life-threatening course of disease with multi-organ manifestations. pulmonary disease manifestations and immunosuppression with glucocorticoids combined with cyclophosphamide and/or rituximab are associated with infectious complications. thus, inadequate immune response to severe acute respiratory syndrome coronavirus (sars-cov- ) in such patients may predispose to severe covid- . we report the case of a -year-old male with nosocomial covid- while receiving immunosuppressive treatment for eosinophilic granulomatosis with polyangiitis (egpa). egpa was newly diagnosed in early january when the patient presented at the emergency room with sinusitis, asthma, and a life-threatening myocardial infarction, resulting in a decreased ejection fraction of %. blood eosinophils and serum concentrations of immunoglobulin e (ige) and c-reactive protein (crp) were increased, anca-testing was negative, and a pulmonary ct scan unremarkable ( figure a) . immediately initiated immunosuppression with intravenous high-dose prednisolone and cyclophosphamide showed adequate therapeutic response. with conversion to oral glucocorticoid treatment at the end of january , the patient unexpectedly developed a serious relapse of disease with peripheral neuropathy, pulmonary hemorrhage ( figure b) and a second myocardial infarction. thus, due to severity and refractory disease the previously healthy patient was continuously hospitalized from january to march , receiving intravenous cyclophosphamide (cyclops-protocol, cumulative dose . g), rituximab ( × mg/m ), and a long-term, slowly tapered high-dose prednisolone treatment (up to g/day). on presumed day of covid- (ongoing oral treatment with mg prednisolone only, days after last of five cyclophosphamide infusions and days after the last of four rituximab infusions), he reported catarrh and a mild cough. a sars-cov- real-time reverse transcription pcr (rt-pcr) from oropharyngeal swab was positive (figure a) . on day , treatment with hydroxychloroquine (for days) and lopinavir/ritonavir (for days) was initiated while daily prednisolone was reduced from to mg. he developed a sore throat, hyposmia, headaches, myalgias, and diarrhea. despite rhonchi/crackles on auscultation and a ct scan consistent with bilateral viral pneumonia (figure c) , the patient only reported mild dyspnea. short-term decrease of oxygen saturation (minimal sao %) required oxygen supplementation for days (low flow l/min). with spiking serum concentrations of crp ( . mg/l, reference range < mg/l), procalcitonin ( . ng/ml, reference range < . ng/ml), and interleukin- (il- , pg/ml, reference range < pg/ml), concomitant with decreasing cd + and cd + t-cell counts, the patient developed fever (max. . • c) on day ( figure b) . anti-il -receptor treatment was considered, however the patient steadily recovered and was free of covid- symptoms weeks after onset. nevertheless, subsequent oropharyngeal swabs confirmed active sars-cov- infection with gradual decrease of viral rna. relapsing neurological symptoms of egpa urged us to re-administer high-dose glucocorticoids and cyclophosphamide on days and , respectively, without causing recurrence of covid- -related symptoms. despite severe immunosuppression and complete peripheral b-cell depletion, sars-cov- rna copy numbers in oropharyngeal swabs were below the threshold for reliable detection on days , , and . by day , there were no antibodies to sars-cov- spike protein detectable by elisa (euroimmun) . remarkably, interferon-gamma release upon polyclonal t-cell stimulation was normal on day . given the high-risk profile with sustained cardiac dysfunction, previous pulmonary hemorrhage, continued high-dose glucocorticoids, b-cell depletion, decreased t-cell counts, and secondary hypogammaglobulinemia (minimal igg . g/l, reference range > g/l), it is remarkable that our patient overcame covid- in a rather timely manner without complications. the effects of potential anti-viral agents hydroxychloroquine and lopinavir/ritonavir on the disease course remain unclear. despite initially higher than average copies per oropharyngeal swab, which could be explained by the effect of immunosuppression during virus contraction, our patient showed a temporal pattern of viral load peaking within the first week after onset of symptoms and gradually declining over the following three weeks as previously described in covid- patient cohorts ( , ) . our observations might therefore suggest that a functional adaptive immune system with an effective b-cell response is not required to survive covid- . moreover, our data points to an important role of innate immune mechanisms and perhaps t cells for sars-cov- control based on the coinciding increase of cd + and cd + t-cell numbers with declining viral rna load. notably, antibodies may often be insufficient for viral clearance ( ). there is even evidence that antibodies against the sars-cov- spike protein can exacerbate pulmonary inflammation due to immunocomplex-mediated complement and fcγ receptor activation with consecutive immune cell infiltration ( , ) . while our knowledge of covid- pathogenesis continues to evolve, strategies to avoid unfavorable outcomes of sars-cov- infection should continue to be mindful of potentially greater adverse outcome caused by tempering existing immunosuppressive or immunomodulatory treatment in autoimmune diseases. the original data generated and analyzed for this study are included in the published article. further inquiries can be directed to the corresponding author. written informed consent was obtained from the individual for the publication of any potentially identifiable images or data included in this article. american college of rheumatology guidance for the management of adult patients with rheumatic disease during the covid- pandemic temporal dynamics in viral shedding and transmissibility of covid- virological assessment of hospitalized patients with covid- long-term coexistence of sars-cov- with antibody response in covid- patients the potential danger of suboptimal antibody responses in covid- anti-spike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection this work was supported by the german research foundation (dfg): trr , project to rv. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © schramm, venhoff, wagner, thiel, huzly, craig-mueller, panning, hengel, kern and voll. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - fz authors: kleen, thomas-oliver; galdon, alicia a.; macdonald, andrew s.; dalgleish, angus g. title: mitigating coronavirus induced dysfunctional immunity for at-risk populations in covid- : trained immunity, bcg and “new old friends” date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: fz the novel, highly contagious coronavirus sars-cov- spreads rapidly throughout the world, leading to a deadly pandemic of a predominantly respiratory illness called covid- . safe and effective anti-sars-cov- vaccines are urgently needed. however, emerging immunological observations show hallmarks of significant immunopathological characteristics and dysfunctional immune responses in patients with covid- . combined with existing knowledge about immune responses to other closely related and highly pathogenic coronaviruses, this could forebode significant challenges for vaccine development, including the risk of vaccine failure. animal data from earlier coronavirus vaccine efforts indicate that elderly people, most at risk from severe covid- disease, could be especially at risk from immunopathologic responses to novel coronavirus vaccines. bacterial “new old friends” such as bacille calmette-guérin (bcg) or mycobacterium obuense have the ability to elevate basal systemic levels of type cytokines and immune cells, correlating with increased protection against diverse and unrelated infectious agents, called “trained immunity.” here we describe dysfunctional immune responses induced by coronaviruses, representing potentially difficult to overcome obstacles to safe, effective vaccine development for covid- , and outline how trained immunity could help protect high risk populations through immunomodulation with bcg and other “new old friends.” in recent months, a novel severe acute respiratory syndrome (sars) coronavirus (cov), sars-cov- , which causes covid- , has spread rapidly throughout the world ( ) . as of july , , more than million infections and over , covid- related deaths have been confirmed worldwide. based on a chronic lack of adequate testing capabilities in many countries worldwide, including large industrialized nations like the united states, a large amount of undiagnosed infection and mortality from covid- must be assumed. the unprecedented pandemic seriously challenges the world's health care systems and available hospital capacities to treat seriously ill patients. these challenges are amplified by frequent sars-cov- infection of healthcare workers (hcw), leading to hospital-acquired infection of hcw and patients, and significant mortality within that group ( ) . other high-risk groups of infection include the elderly, with age-related immunosenescence and "inflammaging" having been suggested as a mechanism responsible for lowered immunological competence and the high mortality of the elderly in the current covid- pandemic ( ) . age-related risks are a particular issue in assisted care facilities and individuals with serious, non-covid underlying health conditions like cardiovascular disease, chronic kidney disease, diabetes, chronic respiratory disease, immunosuppression, and cancer ( , ) . in the case of cancer, many malignancies require active treatment, making isolation -even social distancing -impossible, based on the need to commute to the hospital regularly to receive treatments. therefore, there is an urgent need to protect individuals aged years and older with co-morbidities. throughout the public discourse, there has been little attention given to the observations that these populations are historically the same populations that are most unlikely to develop efficient and protective immune responses to standard respiratory viruses. consequently, this is likely to be the same case for sars-cov- . indeed, for these populations, other more potent vaccines, compared to the general population, are required, e.g., "high dose" influenza shots for the elderly. nevertheless, those more potent vaccines often still result in less than ideal outcomes in these vulnerable populations ( ) . in order to avoid the need for achieving herd immunity by infection or mass vaccinations before safely reopening societies and economies, a priority would be immunizing the most at-risk populations first. there is a justified concern that suboptimal vaccine efficacy for at-risk populations and the elderly could place the goal of not having to achieve herd immunity first in jeopardy. at the same time, a non-efficacious vaccine for at-risk populations could increase the probability of second and subsequent waves of infection in these populations ( ) . worldwide availability of safe, effective, prophylactic vaccines is likely the only approach that will ultimately control this deadly pandemic. however, such vaccines may not be available until early next year, even in the most optimistic scenarios ( ) . despite numerous efforts, no vaccine, proven safe and effective in humans, has ever been developed against any coronavirus ( , ) . questions about the potential lack of sufficient vaccine efficacy in elderly populations have not yet been widely discussed. therefore, strategies to prevent covid- morbidity and mortality in high risk groups are desperately needed in order to safeguard the most vulnerable individuals, as well as maintaining continuous patient care and functioning hospital systems. both humans and animals are susceptible to disease caused by covs. three highly pathogenic covs are known, sars-cov, middle east respiratory syndrome (mers)-cov and sars-cov- . all three are now known to efficiently infect and replicate in the lower respiratory tract, frequently causing substantial immunopathology, acute lung injury (ali), acute respiratory distress syndrome (ards), and fatal pneumonia, resulting in high morbidity and mortality ( ) . sars-cov and sars-cov- are both members of the betacoronavirus genus and share more than % of their genetic code ( ) . however, it is noteworthy that sars-cov- is closest related to the bat coronavirus ratg , with % genetic similarity compared to all known genetic coronavirus sequences ( ) . four additional, circulating but low pathogenic human coronaviruses (hcov) are known and will not be reviewed here, but preexposure to them could impact the immune response to sars-cov- in patients ( ) . all four, hcov- e, hcov-oc , hcov-nl , and hcov-hku , display a winter seasonality, causing comparatively mild to moderate upper respiratory illnesses and only occasionally, bronchiolitis and pneumonia symptoms ( , ) . all hcovs share a minimum of four, genome encoded, major structural proteins: the spike (s) glycoprotein, nucleocapsid (n) protein, membrane protein (m), and the envelope protein (e), all of which are required to produce a structurally complete viral particle ( ) . the pandemic, which originally emerged from wuhan, china, has been characterized by a rapidly increasing morbidity and mortality rate associated with older age, beginning around age years ( ) . multiple aspects of immunity can be influenced by ageing, prompting scrutiny of which components of the immune response might be responsible for higher mortality in older people ( ) . in general, an early and robust innate immune response to viral infections permits more rapid and effective viral clearance and may even prevent symptomatic infection or diminish the severity of the infection ( ) . no correlates of protection have yet been formally established for the recently emerged sars-cov- . however, mouse model data from studies with the first sars-cov that emerged in , suggested a delayed innate immune response during infection is linked to a more severe course, with immunopathology in the lungs and high mortality ( ) . initial observational studies suggest that a failure of antiviral immunity, including depleted natural killer (nk) cells, at an early stage in covid- , may lead to severe clinical course and an inability to recover from infection ( ) . in addition, it has previously been shown that the sars-cov macrodomain suppresses the innate immune response during infection, whereas an early strong innate immune response can protect mice from lethal disease and prevent detrimental downstream effects on the immune system ( ) . on the other hand, in later stages of infection, it appears that a dysregulated immune system, including excessive inflammatory responses by innate cells in the lungs, and selective immunosuppression of the adaptive immune system, can be detrimental for the host ( , ) . acute lung injury caused by viruses like respiratory syncytial virus, influenza a virus and sars-cov have been described previously ( , , ) . aberrant expression of the antiviral cytokine type i interferon (ifn), interferon stimulated genes, and other inflammatory cytokines, were observed in patients with severe sars-cov disease compared to healthy individuals, providing evidence that sars-cov is partly an innate dysregulated immune disease ( , ) . the innate immune system recognizes pathogen-associated molecular patterns (pamps) of viral or bacterial intruders via pattern recognition receptors (prrs). toll-like receptors (tlrs), a family of type i transmembrane prrs that consists of related, transmembrane proteins, play a central role in the initiation of inflammatory responses against pathogens, including the secretion of cytokines and chemokines. tlr is known to sense lipopolysaccharide (lps) from gram-negative bacteria, but, based on its additional function as sensor for damage-associated molecular patterns (damps), tlr has been suggested to play a central role in the induction of damaging inflammatory responses during several acute viral infections ( ) . in addition, oxidized phospholipids (oxpls), damps which lead to ali in patients infected with sars-cov, also accumulate in lungs of patients infected with sars-cov- and activate monocyte-derived macrophages through tlr ( , ) . interfering with innate cell activation by tlr in response to ligands such as oxpls may therefore help prevent thrombotic complications, recently identified as a major factor in mortality of covid- patients ( ) ( ) ( ) . endothelial cell activation, infection and dysfunction has been implicated in severe covid- by altering vessel barrier integrity, promoting a pro-coagulative state, inducing vascular inflammation, endotheliitis, and mediating inflammatory cell infiltration. the proposed mechanism is disruption of vascular integrity and endothelial cell death, which leads to exposure of the thrombogenic basement membrane and results in the activation of the clotting cascade ( ) . altered platelet gene expression and functional responses in patients infected with sars-cov- may additionally contribute to observed hemostatic abnormalities like disseminated intravascular coagulopathy ( ) . neutrophils are an important component of the general response to infection in the respiratory system and capable of recognizing viruses via viral pamps ( ) . in the context of potentially excessive neutrophil activation in late stage covid- disease, neutrophil extracellular traps (nets) in the lungs can drive severe pathologies by accumulation of mucus in the airways of patients, contributing to ards ( ) . more importantly, nets have been proposed to contribute to organ damage and mortality, since excess net formation can trigger a cascade of inflammatory reactions that destroys surrounding tissues and facilitates atherosclerosis, aortic aneurysms, as well as thrombosis, including microthrombosis, in the vascular system, with devastating effects on organ function ( ) . macrophages are key innate immune cells in any infection setting ( , ) . they are highly flexible innate cells that can, simplistically, be functionally and phenotypically divided into pro-inflammatory "m " macrophages (capable producers of inflammatory cytokines and mediators, that kill infectious organisms, virus-infected cells, or tumor cells) and more regulatory "m " macrophages (that are important for wound healing and parasite infections) ( , ) . both activation states are needed for a "balanced" immune response, although the m /m paradigm of macrophage activation is an over-simplistic definition of these complex and diverse innate cells ( , ) . during ageing and chronic inflammatory diseases, macrophages may switch to a more m -like phenotype ( , ) . importantly, nearly all identified high-risk factors for severe covid- disease, like cardiovascular disease, diabetes, age, chronic obstructive pulmonary disease, and smoking, generally share a shift from more m to more m phenotype and function ( ) . classical activation of m macrophages is induced by lps/ifnγ exposure, while alternately activated m macrophages are stimulated by il- , il- , il- and glucocorticoids ( ) . the activation of innate immune cells such as macrophages can be heavily influenced by the character of the t cell response and, in particular, the cytokines produced by t cells during infection ( ) . sars-cov replication has previously been shown in human peripheral monocytes and macrophages, with varying efficacy. importantly, the infection efficiency was shown to be donor dependent, with % infection in some and less than % in others ( ) . in adults, vγ vδ cells are the dominant γδ t cell population, however, in elderly individuals the variability increases ( , ). an analysis of t cell repertoires in hcw who survived sars-cov infection during the outbreak revealed that an innate-like subpopulation of effector memory t cells, γδ-t cells, specifically vγ vδ t cells, were selectively expanded approximately months after the onset of disease ( ) . importantly, no such expansion of non-innate αβ t cells was detected at the same time point. furthermore, expansion of the vγ vδ t cell population was associated with higher anti-cov igg titers, and in vitro experiments demonstrated that vγ vδ t cells display an ifn-γ -dependent ability to directly kill cov infected target cells. therefore, innate-like vγ vδ t cells may play a protective role during sars-cov and other cov infections. a recent study analyzed the number and activation status of vγ vδ t cells in hospitalized patients with covid- . they found significantly lower levels of vγ vδ t cells than that of matched healthy control and concluded that this could indicate that elderly with lower frequencies of vγ vδ t cells constitute a sars-cov- vulnerable population or that the vγ vδ t cells in these patients have migrated to the lungs to kill sars-cov- infected cells ( ) . sars-cov infection leads to lymphopenia and strongly reduced peripheral t cell levels, with low cd + and cd + t cell counts associated with adverse outcome, and a rapid and dramatic restoration of peripheral t cell subsets in the periphery of recovering patients ( ) ( ) ( ) . in addition, sars-cov can infect and replicate within pbmcs of sars-cov patients, with viral replication appearing to be self-limiting but leading to leukopenia or lymphopenia ( ) ( ) ( ) . patients with clinical symptoms of severe covid- also commonly present with lymphopenia, including dramatically reduced numbers of nk cells, cd + t cells, cd + t cells and b cells, which has not been observed in mild cases ( ) ( ) ( ) ( ) . further studies have shown exhaustion markers like nkg a on cytotoxic lymphocytes, including nk cells and cd + t cells, are upregulated in patients with covid- , and that for recovered patients, numbers of nk cells, cd + t cells, cd + t cells, and b cells normalize, along with markers of exhaustion on cytotoxic lymphocytes ( , ) . reduced functional diversity and increased t cell exhaustion in peripheral blood could predict severe progression in covid- patients, supporting the role of functional t cells in controlling covid- ( ) . importantly, it was recently shown that a patient with mild to moderate covid- symptoms had a broad-based robust immune response across different immune cell types, which was associated with rapid recovery ( ) . this observational study identified the presence of activated cd + t cells, cd + t cells, and follicular helper t cells in the blood, along with increased antibody-secreting cells and igm and igg antibodies. the study did not investigate the neutralization capabilities of the observed antibodies. cell-mediated type immune responses are therefore theorized to be a major component necessary to overcome covid- infection ( ). this is further supported by a study that screened for the presence of sars-specific t cells in a cohort of three sars-cov-recovered individuals, where cd + t cell responses targeting the sars-cov membrane and nucleocapsid proteins were found to persist up to years post-infection ( ) . characterization of sars-cov-specific memory t cells from recovered individuals years after infection indicated that the majority of memory cd + t cells produced ifn-γ, whereas memory cd + t cells produced ifn-γ, il- , or tnf-α ( ). multiple other independent studies established that sars-cov specific memory cd + and cd + t cells persisted for up to years after infection ( ) ( ) ( ) . s protein-derived epitopes of sars-cov elicited recall cd + t cell secretion of ifn-γ as well as intracellular production of ifn-γ, tnf-α, perforin, and granzyme a from recovered patients over -year post infection, indicating that sars-cov infection can induce strong and longlasting cytotoxic t lymphocyte (ctl)-mediated immunity in patients ( , ) . high frequencies of cd + tc -type t cells, reactive against mers-cov, were observed in a large proportion of patients with severe and moderate mers at acute stage before detection of humoral and cd + t cell responses. another report emphasizing the importance of t cells demonstrated that years after the sars outbreak, sars-cov-recovered patients still maintained long-lasting memory t cells reactive to the n protein of sars-cov, which notably exhibited robust cross-reactivity to the n protein of sars-cov- ( ) . a recent study showed predominant th responses in convalescing covid- cases, with little to no th responses. it demonstrated sars-cov- specific cd + t cells in % of covid- convalescent patients, with the majority of responses against s protein, correlating with the magnitude of anti-sars-cov- igg and iga titers, but as well responses against m and n proteins in all patients, accounting for - % of the total cd + responses. the same study found sars-cov- specific cd + cells against s and m proteins in about % of patients, and interestingly, t cell reactivity to sars-cov- epitopes was also detected in nonexposed individuals, likely cross-reactive from previous, seasonal hcov infections ( ) . however, at the convalescent phase, the magnitude of the cd + t cell response was not increased further. although it seems clear that robust inflammatory and ctl responses are required to clear the invading virus, when excessive, they can also lead to lung tissue destruction and pneumonia ( ) . early pathological findings of covid- patients with ards, showed not only reduced counts of peripheral cd + and cd + t cells, but that remaining t cells were found in a hyperactivated state, with high proportions of hla-dr and cd double-positive fractions ( ) . it is noteworthy here that, in patients hospitalized with avian h n , survival reflected an early, but transient, prevalence of highly activated cd +cd +hla-dr+pd- + t cells, but prolonged cd +hla-dr+pd + coexpression predicted fatal outcomes ( ) . cd + t cells in patients that died of h n were non-functional, as reflected by a lack of ifnγ production, but displayed high and continued expression of the cd +hla-dr+ activation markers, together with the inhibitory pd- immune checkpoint receptor. similar studies in ebola, dengue, and pandemic h n have also mentioned the presence of these "non-survival" peripheral lymphocyte populations, with high and prolonged frequency of activated cd +cd +hla-dr+ cells ( ) ( ) ( ) . we hypothesize that, as suggested for h n disease ( ), in covid- patients this could also be associated with defective t cell activation and a lack of relevant t cell receptor (tcr) specificities. it is known that infection with human immunodeficiency virus (hiv) induces broad lymphocyte activation, with an increase in t cell activation markers such as cd ( ) . several studies have shown that such increased cd + expression on cd + t cells is a strong predictive marker for disease progression in hiv- infection ( ) . not only does the cd +cd + t cell count predict progression of hiv disease to aids and death, but it is also independently predictive for evaluation of high plasma virus load and low cd + t cell counts ( ) . in early hiv infection, during onset of viremia, cd +cd +hla-dr+ t cells correlate inversely with viral set point. however, hyperacute hiv infection leads these cells to be short-lived effector cells that do not persist, characterized by marked apoptosis, upregulation of cd and failure to upregulate the il- receptor cd ( ) . strikingly, in a recent study in covid- patients, considerable proportions of peripheral cd + and cd + t cells co-expressed cd and hla-dr, but those cells could not be re-activated with peptide pools of the s protein in vitro, supporting the notion of sars-cov- specific refractory t cells and/or different specificities ( ) . no data about the pd- status of t cells was provided. the same remarkable study showed that, while the majority of s-reactive cd + t cells from covid- patients co-expressed cd and hla-dr, s-reactive cd + t cells from healthy donors, proposed to be cross reactive to other hcovs, only expressed cd and hla-dr at very low frequencies and coexpression was not observed. in cancer therapy models, depleting "dysfunctional" cd +cd hipd- + cells enhanced therapeutic outcomes, and patients who did not respond to immunotherapy showed more cd +cd hipd- + in tumor and blood compared to responders ( ) . the potential significance of levels and timing of prolonged expression of cd , hla-dr, and pd- on dysregulated t cells and the utility of cd +cd +hla-dr+pd- + t cells as a prognostic marker could be important and should be investigated in more detail. these could serve as indicators of sars-cov- immunosuppression, exhaustion and immune evasion, predicting divergent disease outcomes. the suggestion that a dysfunctional immune response is at the heart of covid- pathology is further supported by the recent finding that, compared to patients with moderate disease, significantly reduced frequencies of cd + t cells, as well as diminished frequencies of cd + and cd + t cell subsets with activated differentiated memory/effector phenotype and migratory capacity, are found in peripheral circulation of patients with severe covid- ( ) . antibody responses elicited by coronaviruses, including sars, have been described as comparatively short lived and inconsistent ( , ) . studies with human volunteers that were infected with a seasonal coronavirus hcov- e showed that individuals could get infected and display symptoms, including lymphocytopenia, regardless of preexisting antibodies ( ) . one study showed that six years post infection, sars-cov specific igg was undetectable in of former patients, and no sars-cov specific memory b cell responses could be detected in any of the patients ( ) . another study revealed that sars-cov antibodies could be seen up to months after infection ( ) . interestingly, longevity of mers-cov antibody response correlated with disease severity. in one study, patients with severe mers-associated pneumonia had a persistent antibody response detected for about months after infection, while patients with infection limited to the upper respiratory tract or who had no clinical signs had no detectable mers-cov antibody response ( ) . in another report, the more severe the illness, the greater the antibody response, including igm, igg, and neutralizing ab (nabs). patients in the convalescent phase, with mild or asymptomatic disease, rarely developed antibody responses ( ) . a strong antibody response developed in most mers patients only after - weeks of illness, but the antibody responses were not correlated with the elimination of the virus from the body ( , ) . this was confirmed in two more studies that showed mers infections are frequently characterized by low nabs, despite patient recovery ( , ( ) ( ) ( ) . it is noteworthy that this was also recently shown for covid- patients, where seroconversion has been observed in mild to moderate cases after - days, but, despite covid- antibodies arising at that time, no rapid decline of viral loads was observed, as would be expected with highly effective and neutralizing antibodies ( ) . since anti-sars-cov antibody responses are short-lived in patients who have recovered from sars, there are early indications that antibodies, and especially nabs, may not be the predominant mechanism necessary for effective viral clearance and for infected individuals to overcome a covid- infection ( , ( ) ( ) ( ) . this is further reinforced by the first longitudinal study in covid- patients, which showed that some individuals who have recovered and displayed a strong nab response shortly after infection, had titers fall as much as -fold, and in some cases back to baseline within months ( ) . the authors speculated that the observed transient nab response could be a feature shared by both a sars-cov- infection that causes low disease severity, and the circulating seasonal coronaviruses. other recent data supports the notion of an unclear role of abs, by reporting short duration of ab and nab titers after sars-cov- infection. compared with responses of patients with symptoms, asymptomatic individuals (arguably with the more effective immune response), had weaker ab responses to infection, with a reduction of igg levels already occurring in the early convalescent phase ( ); viral load and duration of infection are likely to be factors. remarkably, in this study, % of asymptomatic patients had undetectable levels of protective antibodies two to three months after infection, compared to % of the symptomatic patients with covid- . an even more notable finding, further indicating a limited role for abs in overcoming sars-cov- infection, is that intrafamilial exposure to sars-cov- induces a cellular immune response without seroconversion ( ) . of the different proteins that characterize coronaviruses, the s protein is an important determinant of virulence, tissue tropism and host range ( ) . trimers of s form the characteristic large spikes on the coronavirus envelope and both sars-cov and sars-cov- use the protein angiotensin converting enzyme (ace- ) as primary receptor for docking and infecting human host cells. priming of the virus s protein by host cell proteases is essential for entry. when sars-cov- docks to the cell via the ace- receptor, the host transmembrane serine protease (tmprss ) is responsible for cell entry ( ) ( ) ( ) . tmprss also aids the mers-cov to penetrate the cell ( ), but its primary receptor for entry is dipeptidyl peptidase (dpp ) ( ) . virus s glycoproteins are postulated to elicit an immune response in humans that could protect against future infection ( , ) . many vaccine approaches against covid- that are currently in development are focusing primarily on the generation of antibody responses against the sars-cov- s protein ( ) . however, despite the great urgency for making an effective vaccine against covid- available, this approach must be undertaken with great caution. several sars-cov vaccines that initially induced antibodies and short-term protection in mouse models of sars-cov led to dysfunctional or type helper t cell (th )-type immunopathology on challenge, with prominent eosinophil infiltration in the lungs, suggesting hypersensitivity to sars-cov components was induced ( ) . several other independent studies with animal models used to develop vaccine candidates against sars-cov exposed signs of lethal vaccine failure based on induction of cell-mediated type enhanced immunopathology, with associated eosinophilic infiltrates causing severe pneumonia, especially in aged mice. a vaccine based on sars-cov s protein protected against viral challenge when young mice were vaccinated, but it failed to efficiently protect older mice ( ) . another study indicated poor vaccine performance as well as th -based eosinophilic immune pathology in the lungs that was shown to be caused by alum adjuvanted and unadjuvanted sars-cov vaccines in aged animals ( ) . all this requires that particular attention be given to the strongly increased mortality rate already evident in older sars-cov- patients and patients with comorbidities. sars-cov has been shown to dysregulate the immune response in sars patients by biased activation of a th response, which can counter-regulate the type response that normally attacks bacteria and viruses ( ) . there was a significant increase in th cytokines il- , il- and il- during acute infection in fatal sars cases, once again indicating that the character of cellular immune response induced by any covid- vaccine will be critical in determining whether it will succeed ( , ) . four earlier vaccines against mers-cov- have been tested in rhesus macaques (rm), but no reports of efficacy of a single-dose mers-cov vaccine in non-human primates (nhps) had been made until a recent study reported that rm seroconverted after a single intramuscular vaccination with the experimental chadox mers vaccine ( ) . the study showed that vaccinated animals developed a neutralizing antibody response, were protected against respiratory injury and pneumonia, and showed reduced viral load in lung tissue and reduced disease severity. in addition, a phase trial in healthy individuals aged - years has been conducted, with no adverse safety signal reported ( ) . neither study has provided data in either aged animals or elderly humans. most relevant in this context are early sars-cov- vaccine trial data. a phase / study in adults aged to years of a covid- rna vaccine candidate (bnt b ), utilizing mrna that encodes trimerized sars-cov- spike glycoprotein, showed the generation of nab titers days after the first injection and one week after the second dose ( ). it is not yet known what kind of immune response the vaccine will elicit in older people or long-term. an additional mrna nano-particle based vaccine candidate (mrna- ) has been reported to induce both potent nabs and cd + t cell responses and to protect against sars-cov- infection in the lungs and noses of a mouse model, without evidence of immunopathology ( ) . importantly, it showed spike peptide-reactive cd + and cd + t cells producing ifn-γ, il- , and tnf, which would be encouraging if corroborated in ongoing phase clinical trials and phase efficacy evaluation of the same vaccine candidate. another advanced sars-cov- vaccine candidate in phase clinical studies is adenovirus-vectored vaccine chadox ncov- , which has been reported to prevent sars-cov- pneumonia in rm and not to be th dominated, determined by igg subclass and cytokine expression profiling ( ) . notably, no evidence of immune-enhanced disease following viral challenge twenty-eight days after vaccination was observed in the respective animals. the levels of abs produced by the vaccine in these rm were lower than many ab responses in humans infected with sars-cov- . while the vaccine protected rm from severe infection, they became infected with evident active virus replication, which does not rule out the potential of maintained ability to transmit virus. despite the inherent challenges of adopting new routes of routine vaccine administration during an ongoing pandemic, recent evidence would encourage consideration of intranasal administration, inhalation or other vaccine strategies that directly target the mucosal surfaces of the airways, because of distinct functional responses by respective tissue-resident memory t cells ( ) . it was shown, for example, in a mouse model, that conserved epitopes shared by sars-cov and mers-cov could induce airway memory cd + t cells producing ifn-γ which were phenotypically and functionally different from lung-derived cells and crucial for protection against both covs. it is particularly noteworthy in this study that intranasal (but not subcutaneous) vaccination protected mice from pathogenic human covs, and that protection required ifn-γ and was depended on early induction of robust innate and virus-specific cd + t cells ( ) . sars-cov- has shown replication, not only in human peripheral monocytes and macrophages, but also to directly infect t lymphocytes during primary infection through s proteinmediated membrane fusion, likely contributing to the severe lymphocytopenia that is a diagnostic indicator common in covid- patients ( , , ) . sars-cov has also been shown to infect dendritic cells (dc), the central coordinators of the immune response, leading to impaired dc maturation and their high expression of the pro-apoptotic protein trail ( ) . instead of facilitating lymphocyte activation and expansion in numbers, this likely induces lymphocyte death and represents another mechanism of immune escape and intensification of the immunocompromised state of sars-cov patients ( ) . similar mechanisms could contribute to lymphopenia and dysfunctional immune responses observed in severe covid- patients. in the elderly, immune evasion by sars-cov- is probably made worse due to the reduced number and function of antigen presenting cells (apcs) ( ) . multiple studies have been performed in mouse models describing the importance of type cd + and cd + t cells in sars-cov ( , ) , with one study establishing that virus-specific memory cd + t cells provided substantial protection from lethal closely related sars-cov infection in a mouse model, emphasizing the importance of a cell-based type immune response for survival of sars infections ( ) . the majority of the many current vaccine strategies against sars-cov- rely on unadjuvanted or selfadjuvanted vaccines (e.g., rna and dna vaccines), or type immune response promoting vaccines (e.g., alum adjuvanted, or unadjuvanted peptide or protein based vaccines) ( , , ) . rather than promoting type immunity, such approaches are likely to mostly lead to induction of type responses which, as previously discussed, are unlikely to be effective against sars-cov- ( ). existing cov antibodies have, in the case of host challenge with the same virus, enhanced viral load and disease severity in feline coronavirus or feline infectious peritonitis virus (fipv) infections. this phenomenon is known as antibody-dependent enhancement (ade) of viral infection ( , ) . in fipv infection ade can be induced by the presence of sub-neutralizing levels of anti-fipv spike antibodies ( ) . unlike in dengue virus infections, ade in feline coronavirus infection is caused by re-infection with the identical serotype virus ( ) . it should be noted that mice, often used for preclinical safety evaluation of vaccines, lack fcγriia, the main fcγr on human cells linked to ade induction ( , ) . increasing viral entry into permissive cells and/or triggering excessive production of pro-inflammatory cytokines has made ade a significant concern with several viruses, including the closely related sars-cov ( , ) . concerns have also been raised that anti-sars-cov- non-neutralizing antibodies, or even declining nab titers over time, could lead to ade and enhanced disease after such vaccinations, antibody-based drug therapies, or treatment with convalescent plasma from recovered patients ( , ) . however, none of the early clinical trial results of the most advanced vaccine candidates described above have reported signs of ade ( ) . demonstration of a lack of ade induction of different experimental vaccines against sars-cov- in nhps and humans will remain critical for other vaccines advancing through the pipeline. one recent example of the need for continued vigilance is a study using chinese macaques indicating cause for concern by showing that vaccine-induced, s-specific immunity in the form of anti-spike igg resulted in severe ali by skewing macrophage responses during subsequent, acute infection with closely related sars-cov ( ) . given all of the above, it is likely that successful vaccines against covid- will require appropriate dc activation, leading to induction of a multifaceted and long-lived type immune response that includes memory cd + th cells, cd + ctls, and nabs. most importantly, they will need to be effectively induced and sustained in older individuals without generating type responses or ade. it may remain a challenge to achieve this formidable goal and more creative approaches to vaccination may be required, but early data from pre-clinical and clinical trials of sars-cov- vaccines seem encouraging that they will provide some protection. direct comparisons in the literature of clinical observations in covid- patients with il- induced "cytokine storm" or cytokine release syndrome (crs) should be made with caution ( , , ) . for example, cytokine levels during hyperinflammation in covid- are multiple orders of magnitude lower than has been observed during cancer treatments by adoptive cell transfer of autologous t cells modified with chimeric antigen receptors (car-t cell therapy), a classical example for crs ( , ) . although, crs is normally treated with extensive use of steroids, the clinical evidence does not support corticosteroid treatment for covid- induced lung injury and interfered with clearance ( ) . in sars and mers, corticosteroid use did not improve patient mortality and also resulted in delayed viral clearance ( ) . it should be noted that a recent preprint of a randomizedcontrolled trial observed that therapy with dexamethasone lead to a significant reduction of death in ventilated patients, as well as for patients on supplemental oxygen, while no benefit was shown in mild cases ( ) . a recent review of corticosteroid use in the management of covid- revealed a mixed picture from five available studies. in four retrospective studies and one quasi-prospective study, three studies indicated a benefit, while the other two studies showed no benefit, and one sub-study even suggested significant harm in critical cases ( ) . based on success in hematological and oncology settings, several il- antagonists (tocilizumab, sarilumab as well as siltuximab) have been utilized as emergency interventions in covid- patients with ards and hypotension, although so far with mixed results ( ) . il- is an indispensable cytokine that initiates innate defence after pathogen invasion or tissue damage by stimulating acute phase reactions, immune responses, hematopoiesis, and activation of numerous internal organs to prepare for host defence ( ) . therefore, il- and other cytokines like tumor necrosis factor (tnf)-α are indispensable during functional activation of monocytes, macrophages and dcs before or early during covid- disease, as they are in diseases caused by other respiratory viruses ( ) . however, in later disease stages increasing immune dysregulation and t cell apoptosis, macrophages and il- may accelerate immune imbalance ( ) . preventing and treating coronavirus infections will likely need a multiphasic approach to prophylaxis and therapy, especially in vulnerable populations. it will be important to use the right tools at the right time to avoid unintended and potentially counterproductive consequences. the right set of immunomodulators would likely be able to prepare and boost innate immune defences to either ensure appropriate, effective responses to infection and/or guide the development of suitable, protective immunity in response to potentially suboptimal adjuvanted first generation vaccines. antiviral treatments or combinations of them will be most useful during early infection, while a different set of immunomodulators may be needed in late stage and severe disease, where a dysregulated antiviral response can cause deadly collateral damage. some microbes have existed throughout human history, with evidence of their presence in hunter-gatherer societies, shaping the evolution of the human immune system ( ) . some of these microbes, branded as "old friends" or "old infections, " are thought to be so intricately involved in this process that they are required for human immunity to develop and function properly ( , ) . examples of such microbes are harmless mycobacteria that are present in the environment and used to be prevalent in water and food, where they were postulated to have a "training" impact on the human immune system ( ) . in addition, "paleolithic" strains of mycobacterium tuberculosis (mtb) that were less pathogenic than modern strains could have contributed to this process ( ) . environmental mycobacteria can provoke type responses, as has been shown in mouse models and human cell-based in vitro studies for heat killed mycobacterium obuense, nctc (imm- ) and mycobacterium vaccae, nctc (imm- ) ( ) ( ) ( ) ( ) . this is also the case for the attenuated strain of mycobacterium bovis, bcg ( ) . however, modern, urban societies are often missing frequent exposure to environmental bacteria such as m. obuense and m. vaccae -they literally have lost touch with their "old friends" and may need "new old friends, " to support type immune responses. remarkably, several observational studies have recently proposed that countries with active bcg vaccination in place had fewer confirmed covid- cases and related deaths ( ) ( ) ( ) . these observational studies should be appraised with caution, since there are many confounding factors in interpreting such correlative data in the context during the covid- pandemic ( ) . there is no peerreviewed data yet, or a clear scientific hypothesis about the proposed mechanism of action, to explain how decades later a single bcg vaccination could provide long lasting, heterologous protection against a viral disease. in contrast, there are evidence-based arguments, acutely relevant to the covd- pandemic, regarding how bcg or type immune inducing environmental mycobacteria could provide protection against severe covid- in the form of the trained immunity hypothesis. contact with specific microbial stimuli can induce long-lasting epigenetic changes in innate immune cells, which not only results in an enhanced response to a second challenge by the same microbe, but also to unrelated microbial insults ( ) . referred to as "trained" immunity or innate immune memory, this process was originally shown for the bcg vaccine ( , ) . this concept may help explain previous observations that, after infection or vaccination, prototypical innate immune cells like monocytes, macrophages and nk cells undergo longterm changes in their functional programs, promoting host resistance against a wide spectrum of pathogens, including fungi, bacteria and viruses ( ) . trained immunity is thought to be responsible for the observation in clinical studies that childhood vaccination with bcg correlates with protection against - % of infections with any known pathogen, including viruses ( , ) . additionally, a reduction in childhood mortality, unrelated to the prevention of tuberculosis (tb), has been observed ( ) . similar positive effects have been shown for bcg vaccinations in adults, including improving responses to influenza vaccination ( ) . a study in guinea-bissau showed that bcg reduced the incidence of respiratory syncytial virus infection ( ) . importantly for the at-risk populations for severe covid- , it was shown that bcg had a similar protective effect on respiratory tract infections in older individuals in indonesia ( ) . in addition, a clinical trial performed in older individuals in japan established protection against pneumonia after pneumococcal, influenza and bcg vaccinations ( ) . further confirmation of this effect has been demonstrated in a randomized controlled trial in which bcg vaccination protected against experimental infection of a yellow fever virus ( ) . in summary, bcg vaccination has been shown to protect against a range of viral infections ( ) . related to this, when vaccination against smallpox was introduced around years ago, positive side-effects such as protection against measles, scarlet fever and whooping cough, among others, were noticed ( ) . monocytes from healthy human volunteers were stimulated ex vivo with unrelated pathogens and displayed enhanced proinflammatory cytokine production of il- β, tnf and il- after bcg vaccination ( ) . experimental studies in mice have delineated that some of the mechanisms by which bcg induces these protective effects. for example, in mice, reduced viral titers of influenza a virus rely on macrophages ( ) . subcutaneous administration in mice of muramyl dipeptide (mdp), part of the mycobacterial cell wall, protected against vaccinia virus and herpes simplex virus type (hsv ) infections ( ) . newborn mice could be protected with bcg from infection by hsv ( ) . more recently, other inducers of trained immunity have also been identified, including β-glucan, which has been shown to induce protective trained immunity in human monocytes and against mtb infection in mice ( ) . the combination of these observations and others led to the proposal of the development of trained immunity-based vaccines (tibv). tibvs aim to induce a pre-activated or "poised" activation state in innate immune cells. in this way they are, unlike conventional vaccines, theoretically able to stimulate much broader immune responses that are not focused on just one specific pathogen ( ) . this capacity of tibvs to promote responses beyond their nominal antigens may be particularly useful when conventional vaccines are not available, or when multiple co-infections and/or recurrent infections arise in susceptible individuals at the same time, as is the case in the current pandemic covid- health emergency. at least six different countries, including the netherlands and australia, have initiated clinical trials with the intent of investigating bcg vaccination as tibv to protect hcw from symptomatic or serious covid- infections ( , , ) . in general, bcg is regarded a safe vaccine in young and healthy individuals. however, as is the case with any vaccines containing live attenuated organisms, there is a possibility of adverse events, such as disseminated bcg disease, in the elderly and immunocompromised. for this reason, in cancer patients, who represent a high-risk group for severe covid- infection, bcg is contraindicated in several countries highly impacted by the pandemic, including the united states and canada ( , ) . as a result, populations likely to benefit most from the potential of tibvs and at the highest risk of a severe covid- disease (e.g., cancer patients, frail elderly, or other people with impaired immune systems), cannot be included in bcg vaccination strategies. despite the potential promise for mitigation of the covid- pandemic, a major obstacle to its quick, rational deployment is the fact that the bcg vaccine comprises of a number of genetically distinct substrains ( ) . these have subsequently been shown to have different immunological properties, such as variable virulence and efficacy as a tuberculosis vaccine in mice ( ) . this substrain diversity may also help explain some inconsistencies following bcg use, such as variable th or th induction and side-effects ( ) . in clinical use, no evidence was found that vaccination efficacy against tb was associated with a specific bcg strain; however, a th or th bias was not investigated in that study ( ) . it has also been shown that the immune response can be directed from th to mixed th /th , depending on the dose of bcg used ( ) . bacille calmette-guérin is not routinely injected more than once, but an earlier study showed that, of six patients who were given a second inoculation of the bcg vaccine, three showed persistent cutaneous granulomas ( ) . a recent clinical study also observed evidence of a protective effect against persistent mtb infection after bcg revaccination ( ) ; although repeat treatment with bcg has been used in the past in oncology as an adjuvant to boost cell-based cancer vaccines ( ) . imm- is a preparation of heat killed, whole cell, m. obuense national collection of type cultures (nctc) , one of over named species within the genus mycobacterium, and an "old friend." m. obuense is a rapidly dividing mycobacterium that normally grows as an environmental saprophyte ( ) . since imm- is a heat killed preparation, treatment is not associated with the potential side-effects of delivering live or attenuated organisms ( ) . moreover, one can speculate that imm- , by virtue of its potent type inducing ability, will counter-regulate type responses, helping to explain the encouraging clinical results to date in melanoma and pancreatic cancer ( , ) . an open label, phase study of imm- in combination with checkpoint inhibitor therapy nivolumab is currently underway in patients with advanced melanoma in the united kingdom ( ) . the total number of patients exposed to imm- across clinical trials and compassionate programs without any unexpected adverse events has been over . the mode of action of imm- is in the process of being elucidated, but it has been shown to be a multifaceted modulator of both innate and adaptive arms of the immune system ( ) . experiments with mouse and human immune cells have shown that imm- is very effective in inducing cytokine expression by innate immune cells, including m polarization and enhanced antigen presentation by dcs, leading to a typical type -biased immune response (figures , ) ( , ) . systemic activation of, and ifnγ production by, multiple immune cell types ( ) , including innate immune cells like nk cells, t cells expressing gamma/delta receptors (γδ-t cells) and natural killer t (nkt) cells ( , ) (figure ) , is based in part on the promotion and activation of cd + th , and cd + ctls, with increased production of the cytokine ifn-γ in in vitro and in vivo ( ) ( ) ( ) ( ) ( ) . it is also possible that, in this setting, imm- may act to train monocytes for enhanced m function (figure ) . nk, γδ-t, nkt, th cells, and ctls, are well-known to play crucial roles in anti-viral and anti-tumor responses that can kill infected or tumor cells. this diverse mechanism of action of imm- , the safe promotion of a broad, systemic innate and adaptive type immune response, may provide a rationale for considering its use against sars-cov- . interestingly, bcg has been shown to promote activation of vγ vδ t cells, the major subset of γδ t cell pool in human peripheral blood with a previously proposed protective role against sars-cov (see above) ( ) . vδ t cells are exactly the cell-subtype that has been shown to also be activated by imm- stimulation, in some experiments showing a stronger ability to do so than bcg ( ) . γδ t cells normally only represent a minor subset in peripheral blood, but can rapidly proliferate following infection with certain pathogens, expanding from % to over % of circulating t cells within a week ( , ) . , , [ ] [ ] [ ] [ ] [ ] . imm- activated dendritic cells (dc) directly promote the proliferation of cd + cytotoxic t-lymphocytes (ctl) and type- polarised cd + t cells, whereas innate-like cells including natural killer (nk), nkt and γδ t cells can be activated either by direct interaction with imm- or indirectly via recognition of dc secreted cytokines ( , ) . this local dc activation eventually leads to a systemic increase in immune cells secreting anti-viral interferon (ifn)-γ, perforin and granzyme b ( , ) . th, helper t cell. tnf, tumour necrosis factor. frontiers in immunology | www.frontiersin.org figure | bcg and environmental mycobacteria promote m macrophages and are likely to induce trained immunity. (a) treatment with mycobacterial immunomodulators induce polarization of m macrophages along with "trained" inflammatory monocytes with enhanced m function, which can result in enhanced viral clearance ( ) ( ) ( ) ) . (b) during innate immune training, innate cells undergo long-term cellular reprogramming. unlike classical antigen-specific responses seen with adaptive immunity, this reprogramming results in increased capacity to respond to secondary challenges from a variety of pathogens and forms the basis of trained-immunity based vaccines ( ) ( ) ( ) ) . it is noteworthy that a large majority of vδ t cells co-express vγ in humans, and were shown to be important to overcome sars-cov infection ( , ) . in addition to th cells, ctls and γδ t cells, nk and nkt cells also play key protective roles during viral infection ( , ) , and the potential importance of improving the nk cell and ctl response at the early stage of sars-cov- infection has already been highlighted ( ) . under the umbrella of trained immunity, broad protection could be achieved by systemically increasing the non-specific effector response of innate immune cells (e.g., macrophages, nk, nkt, and γδ t cells) while also enhancing dc activation and ability to promote adaptive t cell (e.g. th and ctl) and b cell responses to both specific and nonrelated (bystander) antigens, all of which have been shown for imm- (figure ) ( , ) . several studies have shown that the effects of imm- are in part mediated by tlr / , and to a lesser extent, tlr / ( , ) . tlr has been shown to directly trigger th effector functions in mice ( ) . subsequently, it was shown that imm- activates human mincle reporter cell lines ( , ) . it is noteworthy that mincle can suppress tlr activation ( ) and tlr has been proposed to have a central role in the initiation of damaging inflammatory responses during different acute viral infections ( ) . in contrast to bcg, imm- does not activate tlr ( , , ) . in a similar manner, mincle suppresses th immune responses, which as well have been suggested in coronavirus immunopathology and vaccine-induced immune enhancement ( , ) . it was only recently discovered that activation of the mincle receptor is a key activation pathway for complete freund's adjuvant (cfa), the "gold standard" adjuvant for eliciting cell-mediated immunity (cmi) in research models ( ) ( ) ( ) . effective and enhanced viral and tumor antigen crosspresentation requires tlr or tlr activation of human dcs ( ) . mouse cd α+ dcs express tlr and tlr , in addition to the tlr family and tlr , whereas the only relevant corresponding cross-presenting human cd + dcs in lymph nodes exclusively express the tlr family and tlr ( , ) . importantly, analysis of the susceptibility of primary human dc subsets to viral infections has shown that figure | bcg and "new old friends" have potential utility for prevention of severe covid- in a number of ways. bacille calmette-guérin (bcg) and other mycobacterial immunomodulators initiate robust type immune responses and innate immune training, leading to tissue type immune cell infiltration and elevated basal systemic type inflammation ( - , - , - ) . this allows for potential alteration of disease trajectory through prevention of viral establishment, enhanced viral killing or as a vaccine adjuvant to enhance immunity. cd + dcs have an innate resistance to infection by a broad range of enveloped viruses, including hiv and influenza virus. in contrast, cd c+ dcs are susceptible to infection, which enables viral antigen production, but impairs their immune function and survival. this has led to the conclusion that inclusion of tlr or tlr agonists would be the most direct mechanism to enable enhanced viral and tumor antigen crosspresentation, likely necessary for effective cancer immunotherapy ( ) and viral clearance ( ) . interestingly, previous work has suggested that vaccine-induced eosinophil immunopathology in the lungs after sars-cov infection could be avoided with the use of tlr agonists as adjuvants ( ) . however, use of tlr agonists may have to be viewed with caution in the context of covid- , based on observations of harmful contributions of tlr to influenza a virus-induced acute pneumonia in mice. in that scenario, tlr -influenza a virus interaction critically contributed to the debilitating effects of a detrimental host inflammatory response ( ) . further, it has been shown that tlr signaling induces tlr up-regulation in alveolar macrophages during ali, and that tlr and tlr in macrophages are an important determinant in ali ( ) , and that there is an association between respiratory syncytial virus tlr -mediated immune responses and chronic obstructive pulmonary disease exacerbation frequency ( ) . tlr activation of macrophages leads to m polarization, and a shift from m into m macrophages ( ) . in addition, it has been shown that tlr activation of macrophages can impair activity of m -like macrophages ( ) . imm- activates tlr and not tlr and leads to m macrophage polarization (figure ) ( , ) . the combined characteristics of imm- have led to the approval by health canada of a randomized, phase trial of immunization with imm- , versus observation, for the prevention of severe respiratory and covid- related infections in cancer patients at increased risk of exposure ( ) . in this review, we have presented an overview of current knowledge of the innate, adaptive and dysfunctional immune responses to sars-cov- , in relation to other closely related coronaviruses. we have outlined the responses that may be required for successful vaccine development against covid- , while highlighting potential risks during this development, especially for the elderly. early clinical data look promising, but continued studies of human and nhp immune response to different sars-cov- vaccines in the pipeline are required to mitigate potential dangers of well-intended, but potentially flawed, vaccines that are being expedited to large parts of highrisk populations around the globe. in addition, the potential utility of "new old friends" as tibvs like bcg or heat killed environmental bacteria such as imm- , that act as multitargeted, systemic immunomodulators of the innate and adaptive immune system have been described. studies to show bcg's and imm- 's potential utility for the prevention of severe covid- are underway or planned, with the potential to change immune status and alter disease trajectory in multiple ways (figure ) : (i) as prophylaxis, with enhanced innate memory and increased basal systemic type immunity preventing viral establishment; (ii) as a treatment for patients in early stages of disease, with increased local and systemic type inflammation enhancing killing of virally infected host cells; (iii) as an adjuvant for future covid- vaccines. thus, bcg and imm- have the potential to be rapidly deployed to address the covid- emergency and the challenge posed by the current lack of effective treatments and vaccines, leading to a high unmet medical need. with other routes of vaccine and therapy development likely to take many months or years to develop, or even reformulate, the help of "new old 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detrimental contribution of the toll-like receptor (tlr) to influenza a virus-induced acute pneumonia tlr signaling induces tlr up-regulation in alveolar macrophages during acute lung injury association of respiratory syncytial virus toll-like receptor -mediated immune response with copd exacerbation frequency tlr /tlr signaling blocks the suppression of monocytic myeloid-derived suppressor cell by promoting its differentiation into m -type macrophage tlr stimulation impairs anti-inflammatory activity of m -like macrophages, generating a chimeric m /m phenotype novel immunomodulators and their combinations require a broad, adaptive clinical biomarker strategy the authors thank b. trease, mediscribe consulting ltd., for technical editing. t-ok is an employee of immodulon therapeutics ltd. am is a member of the scientific advisory board for immodulon therapeutics ltd. ag is a recipient of an mrc (uk) npif phd studentship, which is part funded by immodulon therapeutics ltd. ad is a member of the scientific advisory board for immodulon therapeutics ltd.copyright © kleen, galdon, macdonald and dalgleish. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - xorq ce authors: naz, anam; shahid, fatima; butt, tariq tahir; awan, faryal mehwish; ali, amjad; malik, arif title: designing multi-epitope vaccines to combat emerging coronavirus disease (covid- ) by employing immuno-informatics approach date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: xorq ce a recent pandemic caused by a single-stranded rna virus, covid- , initially discovered in china, is now spreading globally. this poses a serious threat that needs to be addressed immediately. genome analysis of sars-cov- has revealed its close relation to sars-coronavirus along with few changes in its spike protein. the spike protein aids in receptor binding and viral entry within the host and therefore represents a potential target for vaccine and therapeutic development. in the current study, the spike protein of sars-cov- was explored for potential immunogenic epitopes to design multi-epitope vaccine constructs. the s and s domains of spike proteins were analyzed, and two vaccine constructs were prioritized with t-cell and b-cell epitopes. we adapted a comprehensive predictive framework to provide novel insights into immunogenic epitopes of spike proteins, which can further be evaluated as potential vaccine candidates against covid- . prioritized epitopes were then modeled using linkers and adjuvants, and respective d models were constructed to evaluate their physiochemical properties and their possible interactions with ace , hla superfamily alleles, tlr , and tlr . a rapid increase in the human population and its mobility has led to urbanization and subsequent climate and ecological changes, catering to emerging infectious diseases that galvanize an implacable threat to human health around the world ( ) . the human race has encountered multiple bacterial and viral pathogens, some being inconsequential while others causing global chaos. interestingly, before the twenty-first century, human coronaviruses were thought to be trivially harmful, causing only common cold in healthy individuals ( ) . coronaviruses have an enveloped positive-sense rna genome comprising about - kilobases. they have been identified in multiple mammalian hosts, including dogs, cats, bats, camels, pigs, and civets ( ) . according to centers for disease control and prevention (cdc), common human infecting coronaviruses include e coronavirus, nl coronavirus, oc beta coronavirus, hku coronavirus, mers-cov, sars-cov, and the recently emerged deadly coronavirus disease . the first four account for - % of upper respiratory tract infections in human adults. while the latter three have emerged as perpetual challenge for the scientific community. in november , an outbreak of severe acute respiratory syndrome coronavirus (sars-cov) in guangdong, china, led to the deaths of around out of ∼ , infected individuals from different countries ( ) . common symptoms in sarsinfected individuals were documented as cough, fever, dyspnea, and occasional diarrhea. although sequence analysis of the virus depicted that bats were its hosts, human-to-human transmission was also observed ( , ) . likewise, in , the emergence of middle east respiratory syndrome coronavirus (mers-cov) was reported in saudi arabia ( ) . the symptoms included atypical pneumonia along with gastrointestinal problems and kidney failure. as a result, out of , reported cases, patients have died to date as of november (world health organization report). in december , covid- was initially encountered in wuhan, china, and has now rapidly spread to multiple countries. the affected individuals exhibit mild symptoms that turn into pneumonia as the illness progresses ( ) . according to nature news, as of february th, this virus is responsible for infecting about , humans in china, leading to the death of patients. the majority of the cases tend to have some connection to the seafood and animal market, which indicates the virus is zoologically transmitted. this situation has gained the attention of authorities at both a local and state level and has highlighted an urgent need to devise a method for rapid treatment of the deadly pathogen ( , ) . recent research has established that the rna genome of recently discovered sars-cov comprises of , amino acids. it features two untranslated regions at both flanking ends while only a single polyprotein encoding open reading frame is present between them. the genome is organized in a sequential manner starting from ' replicase, and it is followed by structural proteins: the spike, envelope, and nucleocapsid at the n terminal ( ) . reportedly, the spike protein acts as multifunctional molecular machinery to mediate viral entry into host cells and is involved in viral transmission. initially, it binds the host cell-surface receptor via the s subunit domain and afterwards carries out the fusion of host and viral cell membranes with the help of the s domain. a wide variety of host receptors can be recognized by two subsequent domains in s region of sars-cov- , leading to viral attachment. the n-terminal peptide domain (ranges from amino acid - in the sequence) as well as the c-terminal peptide domain (the receptor binding domain ranging from amino acid number to ) of the s zone have the ability to bind host cell receptors. it has been suggested that sars-cov- exploits angiotensin-converting enzyme (ace ) as a cell receptor ( , , ) . outbreaks of infectious disease like covid- poses a serious challenge to the scientific community since they usually arise from unrecognized zoonotic sources or due to scarcity data. viruses can emerge by evolving from their animal-restricted form to another form that can infect humans by attainment of their receptors and biosynthetic machinery. a majority of the recently emerging pathogens are difficult to treat due to the lack of specific therapeutic options ( ) . so far, no therapeutic vaccine for either sars-cov, mers-cov, or sars-cov- currently exists in the market, although some clinical trials are in progress ( ) . innovative computational biology approaches have enabled us to obtain immunogenic and highly conserved epitopes from bacterial and viral antigens ( ) ( ) ( ) ( ) . both cd + and cd + epitopes can be used separately or in combination to construct broad spectrum vaccine candidates. the proposed vaccines can combat a wide variety of pathogens and possess the ability to elicit cellular and humoral responses in human hosts. once administered, the mock epitopes from the vaccine are presented by mhc. the presented epitopes are recognized by their corresponding t-cell receptors that proliferates and generates suitable immune responses. considering this, tcell epitopes from deadly pathogens can facilitate t-cell-based vaccine development (cd + and cd +) . more precisely, a cd + -based subunit vaccine usually deals with exogenous antigens that are phagocytosed by apcs and subsequently bind to mhc-ii, which presents them to cd + t cells. accordingly, a cd + -based t-cell vaccine encompasses endogenous antigens that are degraded by apcs and later presented via mhc-i to cd + t cells ( , , ) . epitope-based chimeric/subunit vaccines have many advantages when compared to vaccines produced via conventional vaccinology. for instance, they are cheaper to develop, do not require microbial culturing, and can surpass many wet lab experiments, saving time. they are a safer option, as they do not contain the entire pathogen and are highly specific and stable ( ) . nevertheless, due to the presence of mutable hla variants, epitope-based vaccines targeting limited hla alleles usually do not produce the required/equal effect among the human population. hence highly promiscuous epitopes can bind multiple alleles at a time and can ensure the desired immune response among a heterogeneous human population ( ) . the current study focuses on finding promiscuous cd + and cd t + cell epitopes for chimeric covid- vaccine development using a variety of web-based tools. the proposed potential vaccine is then checked for its binding affinity with suitable receptors. the surface glycoprotein sequence of the pneumonia virus discovered at the wuhan seafood market (qhd . from mn . reference genome) was retrieved from ncbi ( ) . to scrutinize required hla binding epitopes, a tepitool from iedb was used ( ) . a set of mhc class i super-types (a * : , a * : ,a * : , a * : ,a * : , b * : , b * : , b * : , b * : , b * : , b * : , and b * : ) were used, and the two highest-scoring epitopes (based on percentile rank and ic values) for each allele were selected. a percentile rank is calculated by the comparison of the peptide's predicted bindingaffinity against a panel of a variety of peptides randomly selected from the swiss-prot. hence, a lower percentile rank numerical value depicts better binders. additionally, all the predicted peptides were checked for their ic value, and those with ic ≤ nm were taken into account. specific immune responses are based on cd + and cd + t cells, and protective vaccines should thus induce specific t-cell responses based on peptides represented by mhc-i and mhc-ii alleles. the rationale behind prioritizing hla binding epitopes is to ensure the specific immune response in infected macrophages. for mhc-ii-binding peptide epitopes, the sevenallele method was used. this selection is based on the median of consensus percentile ranks among the seven commonly encountered dr alleles, namely, hla-drb * : , hla-drb * : , hla-drb * : , hla-drb * : , hla-drb * : , hla-drb * : , and hla-drb * : . epitopes with a median consensus percentile rank ≤ . were designated as good binders. the scrutiny of promiscuous peptide epitopes was established based on the median of the consensus percentile rank of the seven preselected alleles. for b-cell epitope prediction, bepipred . from immune epitope database analysis resource (iedb-ar) was used ( ) . iedb-ar is linked to iedb and offers computational analysis regarding both b and t cell epitope prediction and their subsequent analysis. bepipred . works on the basis of a randomly chosen forest algorithm that has been trained on epitopes acquired from antibody-antigen models obtained from interactive protein structures ( ) . owing to the significance of spike protein, the selected epitopes were manually screened for their presence in this zone. the epitopes were further examined for antigenic potential via vaxijen version . ( ) . a threshold value of . was taken into account. non-antigenic peptides (having vaxijen score < . ) were discarded, while antigenic epitopes (with threshold value > . ) were further prioritized for their immunogenicity. the immune epitope database (iedb) tool for immunogenicity score calculation was used to predict immunogenicity scores for all mhc-i predicted epitopes ( ) . this tool is designed to predict immunogenicity of the peptide based on amino-acid position and properties. immunogenic epitopes were then verified for their presence in iedb database. shortlisted top-scoring epitopes were checked for their binding affinity with each other for determining the final sequence of the chimeric vaccine. the epitopes were analyzed using a haddock web server (guru interface) ( ) . clusters representing two epitopes, which possessed the highest interaction scores, depicting their maximum interaction, were refined by removing the water molecules, which may hinder their interaction, and then having them dock to the third epitope. likewise, evaluation of clusters with three epitopes was done. the refined and the highest-scoring cluster was docked to the fourth epitope to obtain the final sequence. to facilitate the process of vaccine development, a flexible linker ggggs was added between each epitope. this helps to restore protein folding by allowing interaction between different domains ( , ) . additionally, another linker eaaak was added at the n terminal to separate bi-functional domains. designed vaccines were then tested with different epitopes, including truncated ov-asp- protein (residues - ) and beta defensin ( residues long), and constructs having higher antigenicity and that are predicted to produce high antibody titers were added with the multi epitope vaccine construct to the enhance immune response ( ) . three different constructs were designed in this study, one comprising the top-scoring cd and cd epitopes lying in the s domain, while another is formed by taking two epitopes from the s domain and two from the s domain, representing mhc-i and mhc-ii binders. finally, the third one is formed by adding a b-cell epitope to the second one but with a different adjuvant. the final sequences of the chimeric vaccine constructs were screened for its antigenic potential and solubility using antigenpro and solpro ( ) . allertop version . was used to check the probability of the construct to cause an allergic reaction ( ) . sequence of the finalized vaccine candidate in fasta format was given as an input to expasy server, in order to calculate various parameters like molecular weight, theoretical pi, half-life of the protein, instability index, amino acid composition, aliphatic index, and gravy ( ). secondary structures of the vaccine constructs were predicted using pdbsum ( ) . this step was executed to better understand the structures of predicted vaccines. pdbsum is a database that is exclusively designed to show the molecules that build dna or proteins, ligands, and metal ions along with the illustration of graphical representation of their interactions with each other. to generate d structures of the vaccine candidates, dpro was used ( ) . the predicted models were then refined using galaxy refine server ( ) . this server is responsible for subjecting the predicted d model to structural perturbations and subsequent structural relaxations. it generates five different models. all five models for each vaccine construct were screened for gdt-ha, rmsd, and poor rotamers, and the finest predicted models were taken to the next step. the finalized models were further evaluated using errat scores and ramachandran plot analysis for verification. in order to obtain stabilized vaccine constructs, energy minimization was carried out using online yasara server. yasara deals with molecular-dynamics simulations of the given models in solvent, using an exclusive forcefield that has been derived from amber, whose constraints have been improved to minimalize the impairment done to protein structure during the process of energy minimization ( ) . in order to study the binding affinity of the putative vaccine candidates with immune receptors, molecular docking technique was adopted. prioritized vaccine constructs were docked to ace receptor (pdb id: sci), tlr (pdb id: z x), and tlr (pdb id: g a). vaccine , having a b-cell epitope, was also checked for its interaction with a b-cell receptor (bcr) cd (pdb id: kg ). for this protein-protein docking validation process, the haadock server (guru interface and refinement interface) was used ( ) . additionally, to obtain a graphical illustration of the interactions between vaccine and receptor, pdbsum was used ( ) . moreover, in order to verify the binding affinity of our multiepitope peptide vaccines with hla alleles, all our vaccine constructs were docked with class i and class ii superfamily alleles to reveal the interaction of epitopes with mhc alleles when combined as well. hence, for this purpose, class i [hla a * (pdb id u y), hla b * (pdb id mji)] and class ii [hla-drb * (pdb id atf)] were used; they represent broad-spectrum peptide-binding repertoires. population coverage of epitopes was determined using iedb for prioritized epitopes, as it helps to determine the percentage population that can respond to the particular epitope and can elicit an immune response against it. initially, , hla class i epitopes have been predicted within spike glycoprotein of covid- . scrutiny on the basis of percentile rank filtered peptide epitopes. each of them had a considerable binding affinity for the superfamily alleles. all of these epitopes, along with their features and respective binding alleles, are reported in table s . further analysis revealed that predicted epitopes lie within the s domain of the spike protein, eight epitopes lie in the n terminal domain ( - aa), and three epitopes are in the receptor-binding domain ( - aa). vaxijen antigenic score prediction at a threshold of . was used to detect the antigenicity of peptide epitopes. antigenic epitopes tend to trigger a large number of antibody titers to fight the infection. among predicted epitopes of covid- virus, six epitopes showed considerable antigenic potential, including five from the n-terminal domain and one from the receptor binding domain. an immunogenicity analysis was then carried out for further filtration, and, consequently, five epitopes were screened out; one of the epitopes lying within n-terminal domain showed relatively less immunogenicity value. out of these five mhc-i epitopes, two epitopes from s domain with high antigenic and immunogenicity score were further selected for multiepitope vaccine construction. these were gvyfastek and stqdlflpf . gvyfastek is a part of the n-terminal binding domain with antigenicity and immunogenicity scores of . and . , respectively. epitope stqdlflpf also lies within the n-terminal domain and has an antigenicity and immunogenicity score of . and . , respectively. moreover, another hla class i epitope ktsvdctmy from the s domain of the spike proteins was also screened to be potential candidates for multi-epitope vaccine construction. a total of , unique epitopes against seven drb alleles were identified. twenty ( -mer epitopes) epitopes were screened out via filtration on the basis of median percentile rank < ( table s ). the major portion of binding energy between a peptide epitope and mhc class ii receptor molecule is delivered through the basic peptide core, comprising ∼ amino acids in length. nevertheless, the existence of extra amino acids around the basic binding core seems to play a significant role in stable binding even if they do not precisely bind the peptide-bindinggroove of the mhc receptor. the -mer epitopes for binding with mhc-ii are thus usually recommended ( ) . ten of mhc class ii epitopes (three in the receptorbinding area and seven in the n-terminal domain) were found to be a part of the s domain. while considering a total of s epitopes, four were found to be highly antigenic (threshold > . ). among these, three belonged to the nterminal domain of s while was a part of the receptorbinding domain. two epitopes efvfknidgyfkiys and qpyrvvvlsfellha were selected for vaccine construction on the basis of their high antigenic potential. the former belonged to the n-terminal domain with an antigenicity score of . , while the later was a part of the receptorbinding domain and had an antigenicity score of . . an epitope mtktsvdctmyicgd from the s domain was also prioritized and was used along with the s epitope qpyrvvvlsfellha for vaccine construction. to the best of our knowledge, none of the epitopes reported in this study have been previously added to the iedb database. table shows the final epitopes picked for vaccine development. an iedb server was used to identify b cell epitopes. out of these, were found to be antigenic in nature (threshold > . ). they were further checked for their allergenicity, and the highly antigenic epitope, found to be non-allergenic in nature ( ynsasfstfkcygvsptklndlcft ), was picked. this epitope was conjugated with the s and s epitopes along with a beta defensin adjuvant to design the vaccine construct. envelope-affixed spike protein of coronaviruses plays an important role in receptor recognition. several virology studies have been carried out to discover the exact mechanism of receptor binding and subsequent entry into the host cells. the sars-cov- spike protein has been found to be % identical to the sars-cov urbani stains' spike protein and % identical to the bat sarsr-cov zxc and zc spike protein ( ) . the shortlisted epitopes have also been subjected to conservation analysis, hence manifesting cross protection against other species. conservation analysis revealed the high similarity between the prioritized epitopes of the sars-cov- spike protein with mers and sars spike protein epitopes ( table ). all our seven epitopes were found to be a part of at least eight viral sequences present on ncbi, while one of the prioritized epitopes, ktsvdctmy, was found to be % identical in available coronavirus sequences (table s ) . the finalized epitopes in table were examined for their interactive ability with one another using haddock. all possible combinations of epitopes along with a flexible linker ggggs between them were explored. for vaccine , the e s -e s combination had the highest haddock refinement score. the binding affinity of the e s -e s combination with the other two epitopes was determined to find the best combination of three epitopes. e s -e s -e s was thus formed. finally, the vaccine construct obtained after combination analysis was e s -e s -e s -e s (table ) . similarly, for vaccine , e -s -e s was the first combination, and it was followed by e s -e s -e s and e s -e s -e s -e s . each best combinations haddock refinement score vaccine e s -e s − . +/- . e s -e s -e s − . +/- . e s -e s -e s -e s − . +/- . vaccine e s -e s − . +/- . e s -e s -e s − . +/- . e s -e s -e s -e s − . +/- . vaccine e s -e s − . +/- . e s -e s -e s − . +/- . e s -e s -e s -e s − . +/- . e s -e s -e s -e s -e s − . +/- . probable combination lined up for putative vaccine design along with their corresponding haddock scores is present in table . moreover, truncated ov-asp (ivvavtgyncpgg kltalerkkivgqnnkyrsdlingklknrngtymprgk nmleltwdcklessaqrwanqcifghsprqqregvgen vyaywssvsveglkktagtdagkswwsklpklyennpsn nmtwkvagqgvlhftq) was attached to the n terminal of both the putative vaccines using another linker, eaaak. the finalized vaccines together with the linkers and adjuvant were amino acids long. ov-asp- reportedly has ability to activate antigen-processing cells (apcs) which define its good adjuvanticity for a number of vaccines and antigens ( ) . they are thus added in vaccine constructs to improve the efficacy of these new generation subunit vaccines. in order to ensure both cell and humoral mediated responses, a potent b-cell epitope was added to vaccine based on the best docking scores predicting the combination pattern of epitopes. vaccine was created with an order; e s -e s -e s -e s -e s and the corresponding docking score are enlisted in table . for comparison purposes, another adjuvant betadefensin (giintlqkyycrvrggrcavlsclpkeeqigkcstr grkccrrkk) was added to this combination. beta defensin has previously been reported as a potent adjuvant when conjugated with mers-cov antigens ( ) . vaccines containing defensins as adjuvants have been shown, both in vivo and in vitro, to activate the primary innate antiviral immune response and mediate other immunomodulatory activities against a number of viruses, including coronaviruses ( , ) . vaccine , after addition of this adjuvant at the n terminal along with eaaaak and ggggs linkers, consisted of residues. the final combination of epitopes of all three vaccine constructs have been shown in figure . various physiochemical properties were examined for both the constructs. the molecular weight of vaccine is . g/mol while the theoretical pi is . , depicting the basic nature of the peptide construct. the instability index ii showed that the construct is stable with a score of . . the gravy (grand average of hydropathy) index was calculated to be − . , validating the hydrophilic nature of the construct that can form interactions with surrounding water molecules. the aliphatic index . illustrated that the construct is thermostable in nature. vaccine has a molecular weight of . g/mol, and its theoretical pi is . . hence, this construct was also found to be basic in nature. likewise, instability analysis showed that the protein is stable with a score of . . the gravy index testified the hydrophilic nature of this construct as well (− . ). the thermostable nature of the construct was established by the value of aliphatic index, . . the predicted values of antigenicity for both the vaccines were found to be . and . , respectively. this ensured highly antigenic nature of the constructs. similarly, the solubility upon overexpression was predicted to be . and . . furthermore, both vaccine constructs designed in this study were designated as non-allergenic by allergenpro. vaccine has a molecular weight of . g/mol and its theoretical pi is . . therefore, this vaccine construct was also found to be basic in nature. the gravy index testified the hydrophilic nature of this construct as well (− . ). the thermostable nature of the construct was established by the value of aliphatic index, . . the predicted values of antigenicity for this particular the vaccine was . . this ensured highly antigenic nature of the construct. similarly, the solubility upon overexpression was predicted to be . . furthermore, like both the previous vaccine constructs designed in this study, this vaccine was also found to be non-allergenic by allergenpro. the secondary structure of vaccine includes six helices, beta turns, seven gamma turns, and nine helix-helix interactions. the secondary structure of vaccine has eight helices, beta turns, gamma turns, and nine helix-helix interactions. for vaccine , secondary structure consisted of two beta strand, one hairpin, one sheet, four helices, beta turns, gamma turns, and one helix-helix interaction. helix-helix interaction presents facts about different pairs of helices, interacting with each other with the vicinity of the protein structure, whereas beta turns depict frontiers in immunology | www.frontiersin.org four consecutive residues. these four residues are represented by i, i + , i + , and i + . this is possible when the measured distance between the alpha carbon atom of the first residue (i) and alpha carbon atom of the fourth residue (i + ) is < Å plus the two residues between them are not helical. a gamma turn comprises of three residues i, i + , and i + . this is possible when a hydrogen bond is present between the two residues (i.e., i and i + ). moreover, the phi angle and the psi angle of the second residue i.e., i + lies within a range of degrees in one of the next two cases: ( ) classic [phi i + ( ), psi i + (− )] or ( ) inverse [phi i + (− ), psi i + (− )]. the dpro tool, which works on the basis of ab initio method for predicting tertiary structure, was used to predict three dimensional structures of proposed vaccine constructs. this strategy was adopted due to the lack of fine homolog proteins that could be exploited for homology modeling. the obtained models were then refined via several structure perturbations and subsequent structure relaxations using glaxyrefine server. the obtained best models are shown in figure . the errat score for d models of three vaccines were calculated as . , . , and . , respectively. while ramachandran plot analysis showed . % residues in favored region for vaccine , . % residues in the favored region for vaccine and . % for vaccine (figure ) . these analyses authenticated the reliability and stability of the predicted structures. energy minimization by a yasara server was performed. for vaccine , the yasara force field was applied to , atoms. a total of , water molecules were found. the initial energy was − . kj/mol (z score − . ), which was minimized to − . kj/mol (− . ). for vaccine , the yasara force field was applied to , atoms while the water molecules were , . initial energy was − . kj/mol (z score − . ); however, the final energy was . kj/mol (z score − . ). for vaccine , the yasara force field was applied to , atoms while the water molecules were , . initial energy was − . kj/mol (z score − . ); however, the final energy was − . kj/mol (z score − . ). sars-cov spike protein has been studied previously for its exceptional binding affinity with human ace- . it should be noted that, structurally, sars-cov- and sars-cov spike proteins are highly homologous in nature, sharing . % identical amino acids. atomic level studies between sars-cov and ace- show promising interactions between the two, and therefore, owing to the structural and sequence similarity, it is anticipated that an ace- blocker might be handy in curbing sar-cov- ( ) . for vaccine and the ace- receptor, therefore, docking was carried out. a haddock server clustered probable structures into seven different clusters, which represented a total of . % of the water-refined models. the top-scoring cluster had a score of . +/- . and a z score of − . . similarly, for vaccine and the ace- receptor, haddock clustered structures in three clusters, which represented . % of the water-refined models. the topscoring cluster had a value of . +/- . and a z score of − . . likewise, for vaccine , haddock clustered structures into five clusters, which depicted % of the water refined models generated by haddock. here, the best cluster had a score of . +/- . and a z score of − . (figure ) . tlr and tlr are well-studied toll-like receptors that identify both structural and non-structural proteins of the virus and subsequent cytokine production and inflammation. they are present on the surface of cells and are triggered by viral glycoproteins. tlr agonists have the potential to initiate an immune response and actively participate in viral clearance ( ) . the prioritized vaccine constructs were therefore also explored for their interaction with toll-like receptors tlr and tlr . vaccine and tlr interaction revealed structures in a total of six clusters that represented . % of the water-refined models. the model with the highest score, − . +/- . had a z value of − . . likewise, for vaccine and tlr , haddock clustered structures in clusters, which represented . % of the water-refined models. here, the highest-scoring model had a score of − . +/- . with a z-value of − . . for vaccine and tlr , haddock clustered structures in clusters, which represented . % of the water-refined models. the highest scoring model had a score of − . +/- . with a z-value of − . . moreover, haddock clustered structures in clusters to determine vaccine and tlr interaction, which represented . % of the water-refined models. the top-scoring model had a score of . +/- . and a z-value of − . , whereas the interaction of vaccine and tlr is determined by structures in nine cluster(s), which represents . % of the water-refined models. the top-scoring model had a score of − . +/- . (z-value − . ). similarly, haddock clustered structures in eight clusters to determine vaccine and tlr interaction, which represented . % of the water-refined models. the top-scoring model had a score of . +/- . and a z-value of − . . models from top clusters were refined using haddock refinement interface. this server was used to cluster structures, obtained via haddock, into one cluster. this final cluster symbolized % of water-refined models that were generated by haddock. the statistics observed in interactions of vaccine , vaccine , and vaccine from their refined clusters can be seen in table , and complexes are shown in figure . the pdbsum analysis of vaccine with ace showed hydrogen bonds and one salt bridge. additionally, interface residues of vaccine , representing an interface area of , (a ), were found while the corresponding ace had interface residues covering an area of , (a ). for vaccine and ace , there were two salt bridges and seven hydrogen bonds predicted by pdbsum. additionally, and residues from vaccine and ace interacted with each other covering an area of , and , , respectively. likewise, for vaccine there was one salt bridge and hydrogen bonds predicted by pdbsum. additionally, and residues from vaccine and ace interacted with each other, covering an area of , and , , respectively. an interaction analysis of vaccine with the tlr interacting complex via pdbsum exhibited hydrogen bonds and one salt bridge. furthermore, interface residues of vaccine , representing an interface area of , (a ), were found while a corresponding tlr had interface residues encompassing an area of , (a ). for vaccine and tlr , there were two salt bridges and hydrogen bonds predicted by pdbsum. additionally, and residues from vaccine and tlr interacted with each other, covering an area of , and , , respectively. lastly, for vaccine and tlr pdbsum, hydrogen bonds and five salt bridges were found. furthermore, interface residues of vaccine , representing an interface area of , (a ), were found while corresponding a tlr had interface residues, encompassing an area of , (a ). similarly, the interaction of vaccine with tlr exhibited eight hydrogen bonds and interface residues of vaccine , representing an interface area of , (a ) while a corresponding tlr had interface residues, encompassing an area of , (a ). for vaccine and tlr , there were three salt bridges and hydrogen bonds predicted by pdbsum. additionally, and residues from vaccine and tlr here, a lower haddock score indicates the higher strength of interaction between the proteins. z-score of all docking complexes came out to be . interacted with each other, covering an area of , and , , respectively. in case of vaccine and tlr , hydrogen bonds and three salt bridges were found, while interface residues of vaccine represented an interface area of , (a ) and a corresponding tlr had interface residues, encompassing an area of , (a ). for the interaction analysis of vaccine and bcr (cd ), the haddock server clustered probable structures into different clusters, which represented a total of % of the water-refined models. the top scoring cluster had a score of − . +/- . and a z score of − . . models from top clusters were refined using haddock refinement interface. this server was used to cluster structures, obtained via haddock, into one cluster. this final cluster symbolized % of waterrefined models that were generated by haddock. the statistics observed in interactions of vaccine and bcr from its particular refined clusters can be seen in table . pdbsum analysis showed that and residues from vaccine and bcr interacted with each other covering an interface area (a ) of , and , , respectively. they formed one salt bridge and hydrogen bonds. for interaction analysis of vaccine and hla a allele, the haddock server clustered probable structures into different clusters, which represented a total of . % of the waterrefined models. the top scoring cluster had a score of − . +/- . and a z score of − . . similarly, for vaccine and the hla a allele, haddock clustered structures in clusters, which represented . % of the water-refined models. the top scoring cluster had a value − . +/- . and a z score of − . . likewise, for vaccine haddock clustered structures into three clusters, which depicted . % of the water refined models generated by haddock. here the best cluster had a score of − . +/- . and a z score of − . . for vaccine and hla b allele, probable structures were clustered by haddock into different clusters, which represented a total of . % of the water-refined models. the top scoring cluster had a score of − . +/- . and a z score of − . . similarly, for vaccine and the hla b allele, haddock clustered structures into nine clusters, which represented % of the water-refined models. the top scoring cluster had score of − . +/- . and z score of − . . likewise, for vaccine haddock clustered structures into clusters, which depicted % of the water refined models were generated. here, the best cluster had a score of − . +/- . and a z score of − . . furthermore, for vaccine and the hla drb allele docking, the haddock server clustered probable structures into different clusters, which represented a total of . % of the waterrefined models. the top-scoring cluster had a score of − . +/- . and a z score of − . . similarly, for vaccine and hla drb allele, haddock clustered structures in clusters, which represented % of the water-refined models. the topscoring cluster had score of − . +/- . and z score of − . . likewise, for vaccine , haddock clustered structures into clusters, which depicted . % of the water refined models generated by haddock. here the best cluster had a score of − . +/- . and a z score of − . . models from top clusters were refined using haddock refinement interface. this server was used to cluster structures, obtained via haddock, into one cluster. this final cluster symbolized % of waterrefined models that were generated by haddock. the statistics observed in interactions of vaccine , vaccine , and vaccine from their particular refined clusters can be seen in table s . epitope population coverage was checked by iedb population coverage tool. resultantly, all epitopes had a combined class i and class two average coverage score of %. this step was performed by using the entire world population datasets and the mhc restricted alleles used in this case were (a * : , coronavirus can reportedly spread from person to person via droplet transmission. however, there is currently no available fda-approved vaccine against covid- ( , ) . a vaccination regime, if successfully developed against covid- , has the ability to improve global human health statistics. the advent of immuno-informatics approaches has revolutionized the area of vaccine development. antibody response as well as cell mediated immunity can be established by using proper protein antigens ( ) . notably, the natural infections elicit a minimal immune response that can be enhanced by developing epitope-based vaccines. therefore, rational selections are done to separate the constituents required for the desired immune response. efforts to identify suitable tcell epitopes as well as the design of effective strategies in order to deliver those epitopes are under consideration. the benefits of epitope-based vaccine construction includes improved safety levels, time saving, and, additionally it can provide the opportunity to specifically attach/engineer combinations of epitopes for augmented potency. this also facilitates to emphasize the required immune responses on antigenic/ conserved epitopes ( ) . spike proteins of coronaviruses are responsible for selection and entry into the target cells. any therapeutic approach to target the spike protein can prove to be fruitful to curb the deadly pathogen. moreover, it has been reported that like sars-cov, sars-cov- uses the ace human receptor to bind and enter the cells ( ) . peptides that potentially interact with the functional domain of the coronavirus spike protein, can be designated as viral entry inhibitors. in our study, the chosen cd + and cd + t cell epitopes are predicted to be antigenic and immunogenic, and they can thus play a vital role in viral clearance mechanisms. to further validate the authenticity of our proposed vaccines, more detailed docking analysis and experimentation has to be performed. nevertheless, it might take months to years to actually derive a vaccine against covid- , we believe that our contribution in this case might be a useful to initiate the process. for vaccine , four epitopes from the s domain were picked. the s domain, which comprises of amino acids from to , is further divided into the n-terminal domain and receptor-binding domain. analysis showed that three of our chosen epitopes lied in the n-terminal domain of the s protein while one " qpyrvvvlsfellha " was a part of receptorbinding domain ( - ). viral infections are prompted by the interaction of the spike-protein with the receptor, present on the surface of the target cell. this process is mediated by the receptor binding portion of the s domain. hence, it plays a significant role in the attachment, and subsequent fusion and entry of the virus into the host cell. hence this particular portion can be targeted for designing antiviral agents ( ) . vaccine is comprised of a combination of strong and weak epitopes. it had two epitopes (mhc-i and mhc-ii) that were found to be the best epitopes for s domain. regardless of the fact that efvfknidgyfkiys had a higher antigenicity score compared to qpyrvvvlsfellha ( . and . ), the latter was used in vaccine construction due to its presence in receptor binding domain. additionally, another experimental strategy was applied; comparatively weak epitopes from s were selected and their binding affinity was checked. docking with tlrs and ace showed that they bind effectively; from mtktsvdctmyicgd , thr , val , lys , thr , and ser bound to tlr ; lys had affinity for ace receptor. the other s epitope ktsvdctmy completely overlapped with mtktsvdctmyicgd . vaccine is a modified form of vaccine with an additional mer b-cell epitope ynsasfstfkcygvsptklndlcft integrated. the whole idea to include b-cell epitopes along with was to ensure both cellular and humoral defense responses ( ) . b-cell epitopes are precisely amino acids clusters present at the cell surfaces that are identified by certain antibodies or bcell receptors, that in turn elicit cellular or hormonal immune response ( ) . antibodies released by b-cells can neutralize toxins and thus label them for destruction ( , ) . in this case, in addition to considerable interactions with tlrs and hla superfamily alleles, notable interactions were observed between cys and phe from b cell epitope and arg and glu from bcr, respectively. designed vaccines have been tested against different receptors to identify their potential to induce immune response within the host. results revealed that proposed vaccines are likely to be presented by mhc-i and mhc-ii, as that was the prime objective of this study. also, they may interact with human tlr and tlr to induce innate immune response, as these receptors have been revealed to play a key role in the induction of immune responses ( ) . moreover, the spike protein of sars-cov has been reported to play a significant role in the induction of neutralizing-antibodies and t-cell responses as well as protective immunity during the infection ( ) . therefore, keeping in view the importance of spike proteins in immunity, we applied this predictive framework to identify potential vaccine candidates in spike protein of sars-cov- against its potential host receptor ace as well as against tlr and tlr . recent studies have strongly suggested that covid- uses angiotensin-converting enzyme (ace ) as its potential receptor. several critical residues in covid- receptor binding motif (rbm) of s domain particularly gln provide favorable interactions with human ace ( ) . thus, it has been proposed in several studies that spike-protein-based vaccines can be potential therapeutic targets against sars-cov- , as they may block the viral interaction with ace and may thus prevent the downregulation of ace and ultimately the pulmonary vascular permeability ( ) . vaccines designed in this study may also interact with ace resulting interrupted interaction of the receptor with the viral spike protein and thus can be a potential therapeutic target against covid- . the overall effect of all these interactions within the host is still unknown and requires further experimental studies for their clear role in the immune regulation and virus clearance. concisely, we have combined several immuno-informatics tools to propose a set of potentially antigenic and immunogenic peptide epitopes that can facilitate vaccine design. the predicted vaccine constructs consist of distant epitopes. the authenticity of these constructs must be validated via further experimentation. however, further experimental authentication is required to verify this study. we anticipate promising outcomes from the predicted peptide epitopes to curb the deadly covid- pandemic. all datasets presented in this study are included in the article/supplementary material. an, fs, and fa conceptualized the study, validated the study, and wrote the original draft. fs performed the data curation and developed the software. an and fs performed the formal analysis, methodology, and visualized the study. tb, aa, and am performed the funding acquisition. an performed the investigation and supervised the study. an and am performed the project administration. an, tb, fa, aa, and am wrote, reviewed, and edited the manuscript. all authors contributed to the article and approved the submitted version. does urbanization make emergence of zoonosis more likely? evidence, myths and gaps coronavirus infections-more than just the common cold 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the application of human defensins as antivirals angiotensin-converting enzyme (ace ) as a sars-cov- receptor: molecular mechanisms and potential therapeutic target toll-like receptors in antiviral innate immunity clinical features of patients infected with novel coronavirus in wuhan early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia genome-based approaches to develop vaccines against bacterial pathogens epitope-based vaccines: an update on epitope identification, vaccine design and delivery receptor-binding domains of spike proteins of emerging or re-emerging viruses as targets for development of antiviral vaccines in silico screening of antigenic bcell derived t-cell epitopes and designing of a multi-epitope peptide vaccine for acinetobacter nosocomialis current progress of immunoinformatics approach harnessed for cellular-and antibody-dependent vaccine design fundamentals and methods for t-and b-cell epitope prediction toll-like receptors: the swiss army knife of immunity and vaccine development the spike protein of sars-cov-a target for vaccine and therapeutic development receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars a review of sars-cov- and the ongoing clinical trials we want to acknowledge institute of molecular biology and biotechnology (imbb) at the university of lahore for providing the facilities and confidence to publish this article. we also want to acknowledge atta-ur-rehman school of applied biosciences (asab) at the national university of sciences and technology for collaboration and support. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © naz, shahid, butt, awan, ali and malik. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -mqq fmsp authors: waumans, yannick; baerts, lesley; kehoe, kaat; lambeir, anne-marie; de meester, ingrid title: the dipeptidyl peptidase family, prolyl oligopeptidase, and prolyl carboxypeptidase in the immune system and inflammatory disease, including atherosclerosis date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: mqq fmsp research from over the past years has implicated dipeptidyl peptidase (dpp) iv and its family members in many processes and different pathologies of the immune system. most research has been focused on either dppiv or just a few of its family members. it is, however, essential to consider the entire dpp family when discussing any one of its members. there is a substantial overlap between family members in their substrate specificity, inhibitors, and functions. in this review, we provide a comprehensive discussion on the role of prolyl-specific peptidases dppiv, fap, dpp , dpp , dipeptidyl peptidase ii, prolyl carboxypeptidase, and prolyl oligopeptidase in the immune system and its diseases. we highlight possible therapeutic targets for the prevention and treatment of atherosclerosis, a condition that lies at the frontier between inflammation and cardiovascular disease. research from over the past years has implicated the dipeptidyl peptidase (dpp) family in various physiological processes and pathologies of the immune system. usually only four prolyl-specific peptidases are considered: dppiv (ec . . . ), fibroblast activation protein α (fap; ec . . .b ), and the more recently discovered dpp and dpp (ec . . ). however, due to similarities in substrate specificity and structural homology, it is more relevant to consider a broader family that also includes prolyl oligopeptidase (prep; ec . . . ), dipeptidyl peptidase ii (dppii) (ec . . . ), and prolyl carboxypeptidase (prcp; ec . . . ). first, dppii and prcp share the α/β hydrolase fold with the other dpps and the catalytic triad is completely conserved in both enzymes ( ) . moreover, dppii can cleave several dppiv substrates in vitro ( ) . conversely, due to its substrate preference for tripeptides ( ) , dppii could actually be considered as a prolyl carboxytripeptidase, emphasizing its similarities to prcp. another argument for considering a broader family stems from the fact that functional studies on the role of peptidases rely heavily on the use of enzyme inhibitors and many of the inhibitors used in earlier studies are now known to inhibit more than one family member. for example, early studies on dppiv used inhibitors which we now know also inhibit dppii, dpp , dpp , fap, and/or prep due to their sequential and/or structural similarity [e.g., ref. ( ) ( ) ( ) ( ) ( ) ]. prcp is known to be inhibited by kyp- and z-pro-prolinal at higher concentrations, which have often been used for the functional study of prep [e.g., ref. ( ) ( ) ( ) ]. table summarizes the most commonly used dpp inhibitors and their selectivity compared to dppiv. in view of the aforementioned reasons and for the sake of simplicity, we will use "dpp family" as a blanket term, which includes dppii, prcp, and prep even though strictly speaking they are not dpps. figure provides a general overview of this broadly defined dpp family. the roles of various family members in certain aspects of the immune system or immune dysfunction have been reviewed in the past [e.g., ref. ( ) ( ) ( ) ]. in this review, we provide a comprehensive discussion and update on the roles of dppiv, dppii, dpp , dpp , fap, prep, and prcp in the immune system and inflammatory disease. we highlight the role of these enzymes in atherosclerosis, a condition that lies at the frontier between inflammation and cardiovascular disease, as the dpp family encompasses possible therapeutic targets for the prevention and treatment of this disease. the prototypical dpp, dppiv (often dpp in medical jargon) cleaves off an n-terminal dipeptide from peptides with pro or ala on the penultimate position. its localization as a soluble enzyme in body fluids, or anchored in the plasma membrane of cells provides it with the necessary access to cleave a wide range of bioactive peptides. as such, it can modify their biological activity. glucagon-like peptide (glp)- and - , and glucosedependent insulinotropic peptide (gip) ( , ), substance p ( ), neuropeptide y (npy) ( ), stromal cell-derived factor- α/β (sdf- α/β or cxcl ) ( ), granulocyte macrophage colony-stimulating factor (gm-csf) ( ), cxcl ( - ), and high-mobility group box (hmgb ) ( ) have been identified as physiological substrates, while others, such as rantes, have been proposed based on in vitro experiments [e.g., ref. ( )]. dppiv also performs many of its physiological functions through interactions with other proteins, such as collagen, fibronectin, adenosine deaminase (ada), caveolin- , and the mannose- -phosphate/insulin-like growth factor ii receptor (m p/igfiir) ( - ). some of those will be discussed in more detail below. dipeptidyl peptidase iv is well known for its role in glucose homeostasis. it has become a validated therapeutic target for the treatment of type diabetes (t d) ( ). dppiv inhibitors reduce the rate of glp- inactivation (boxes and ). it has also been shown to be involved in cancer biology. the role of the dpp family in cancer has been addressed in several other reviews ( , - ). finally, dppiv has recently come back into the center of attention as the receptor for the mers coronavirus ( ). the incretins are a group of glucose-lowering molecules produced by the intestines. the best known incretin is glucagon-like peptide- (glp- ). this incretin is derived from proglucagon and secreted after a meal from l-cells in the distal ileum and colon. in the pancreas, it induces insulin secretion and biosynthesis while lowering glucagon secretion. in addition, glp- increases the β-cell mass, thereby restoring insulin production. it is clear that glp- also has functions outside glucose metabolism. its receptor, glp- -r, is not only found in the pancreas but also expressed in brain, lung, kidney, stomach, and heart ( , ). recently, it was shown that stimulation after myocardial infarction reduces the infarct size ( , ). currently, glp- agonists are approved for the treatment of type diabetes. these incretin mimetics seem to have a slightly better efficacy as dppiv inhibitors and lead more frequently to weight loss. unfortunately, an important drawback for their therapeutic use is that they can only be administered by subcutaneous injection ( ). fibroblast activation protein α, also known as seprase can present itself as a type ii transmembrane protein or as a shedded plasma protease ( ). in the latter case, it is also known as antiplasmincleaving enzyme, which converts α -antiplasmin into a more active form, suppressing fibrinolysis ( ). some of the known dppiv substrates were later found to be cleaved in vitro by fap as well ( ), though any physiological relevance remains unclear. unlike dppiv, fap also possesses a gelatinase activity. this enables fap to degrade proteins of the extracellular matrix ( ). this is of particular interest with regard to its involvement in a number of pathological processes ( ). fap is highly induced during inflammation, activation of hepatic stellate cells in liver cirrhosis and strongly expressed by mesenchymal cells of remodeling tissue ( , ). fap is also a key regulator during tumor growth and metastasis ( ). as all these processes require degradation of the extracellular matrix, fap's involvement in these pathologies is most likely associated with its gelatinase activity ( ). its role in cancer biology has been reviewed before ( , ). it is interesting to note that, so far, in clinical trials talabostat has shown minimal or no clinical benefit for the treatment of metastatic colorectal cancer, advanced non-small cell lung cancer, or stage iv melanoma ( - ). it should be mentioned, however, that talabostat is a broad-range inhibitor also targeting dppiv, dpp , and dpp . dipeptidyl peptidases and dpp show dppiv-like activity and share a very high-sequence similarity to each other ( % aa similarity, % aa identity) ( ). these cytoplasmic enzymes have several isoforms. it has been a matter of debate whether all are expressed as protein in cells and, if so, whether they are active ( - ). interestingly, the n-terminal extension of the longer dpp variant contains a nuclear localization signal and, indeed, this form localizes to the nucleus ( ). dpp has been shown to cleave a number of dppiv chemokine substrates in vitro ( ). another dppiv substrate, npy, has indirectly been shown to be box dppiv inhibitors. dipeptidyl peptidase iv inhibitors prolong the biological half-life of the incretins and are therefore used for the treatment of type diabetes. sitagliptin, vildagliptin, saxagliptin, linagliptin, and alogliptin are dppiv inhibitors currently available on the market for treatment of type diabetes. sitagliptin and alogliptin are highly selective toward dppiv in vitro, whereas vildagliptin and saxagliptin are less selective with regard to dpp and , and linagliptin with regard to fap ( ). their clinical efficacy and safety in the use of type diabetes seem comparable as far as can be judged from the data available. there is a growing interest toward a use outside type diabetes as it has become clear that dppiv inhibitors have pleiotropic effects. while negative effects have been found in heart failure ( ), some studies suggest them as a possible therapeutic strategy in cardiovascular pathologies ( , ). the sitagrami trial and follow-up studies revealed that the combination of a dppiv inhibitor with granulocyte-colony-stimulating factor or in monotherapy presents a therapeutic option after myocardial infarction ( , ). as stated above, the mechanism is not yet clear but may be explained by a longer biological half-life of dppiv substrates, glucagon-like peptide- , b-type natriuretic peptide, and stromal cell-derived factor- α/β. all three peptides have a cardioprotective effect that is abolished by dppiv-mediated cleavage. a dpp and dpp substrate as well ( ). efforts have been made to find intracellular dpp and substrates using a peptidomic approach ( ), but so far it has been hard to attribute physiological relevance to the possible substrates beyond the role of dpp and in intracellular peptide turnover ( ). the physiological functions of dpp and dpp are still not properly understood. mainly, a lack of available knockout animals, specific inhibitors, and substrates has hampered progress ( ). a mouse model has been established with a targeted inactivation of dpp enzymatic activity ( ), but homozygous dpp inactive neonates die within - h after birth. despite these limitations, some indications toward their role are surfacing. using immunohistochemistry, dpp and were found associated with spermatozoids and spermatids and the short mrna of dpp is predominantly expressed in testes ( , ), suggesting a role in spermatogenesis and male fertility. recent work has found sumo to be an allosteric activator of dpp ( ), whereas a small peptide corresponding to the interaction surface of sumo is a non-competitive inhibitor of dpp and dpp ( ). a genomewide association study has linked dpp to idiopathic pulmonary fibrosis ( ). finally, a number of studies have shown a role for dpp and dpp in apoptosis ( , - ). two studies showed that overexpression enhanced induced apoptosis and impaired cell adhesion and migration ( , ). conversely, dpp / inhibition in tumor cells decreased the number of viable cells because of a decreased cleavage of pro-apoptotic npy ( ). in macrophages, inhibition caused a marginal, yet significant increase in apoptosis, independent of npy cleavage ( ). interestingly, vildagliptin, a dppiv inhibitor already on the market to treat type diabetes, but with poorer selectivity toward dpp and , was shown to enhance parthenolide's anti-leukemic activity through its inhibition of dpp and , and not dppiv ( ). prolyl carboxypeptidase, also called angiotensinase c or lysosomal pro-x carboxypeptidase, is a lysosomal carboxypeptidase sharing strong sequence homology with the likewise lysosomal dppii ( , ) . prcp preferentially cleaves off the c-terminal amino acid when ala or pro is in the penultimate position, while dppii targets n-terminal x-pro or x-ala dipeptides ( , ). in addition to a structural similarity, prcp and dppii have partially overlapping substrate specificities due to dppii's preference for tripeptide substrates ( ) . perhaps surprisingly, gly-pro-pna and ala-pro-pna, two typical synthetic dpp substrates, have actually been used to perform prcp activity measurements ( ). prolyl carboxypeptidase is particularly known as one of the key enzymes of the renin-angiotensin system (ras). it inactivates the vasoactive peptides angiotensin ii and angiotensin iii by cleaving off the c-terminal phe ( ). α-melanocyt-stimulating hormone - , an anorexigenic neuromodulator, is inactivated by prcp, implying a role in body weight control ( ). based on the involvement of prcp in the conversion of these peptide hormones, the enzyme has also been associated with diseases, such as hypertension, diabetes mellitus, obesity, inflammation, and cardiovascular dysfunction ( , ). dipeptidyl peptidase ii has no known natural substrates. the dppiv substrate substance p has been shown to be cleaved by dppii in vitro ( ), but much less efficiently, casting doubt over any physiological relevance. it has been shown that inhibition or silencing of dppii causes apoptosis of quiescent g lymphocytes ( - ). on the other hand, a highly specific dppii inhibitor, uamc , did not induce apoptosis, autophagy, or necrosis in human leukocytes ( , ), but this study did not specifically look at quiescent cells or lymphocytes. finally, changes in dppii activity levels have been observed in a number of pathologies, such as neurodegenerative disorders, myopathies, cancer, and gastro-intestinal disorders ( ). prolyl oligopeptidase is an oligopeptidase with endopeptidase activity. it has been shown to be localized in the cytoplasm ( ) ( ) ( ) ( ) , but given its ability to inactivate several neuropeptides in vitro by limited proteolysis ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , its involvement in the in vivo generation of immunoactive peptides n-acetyl-prolyl-glycylproline and n-acetyl-seryl-aspartyl-lysyl-proline ( , ) , and its presence in plasma ( , ) , it most likely also has an extracellular role. initial interest for prep derived from the positive effects of prep inhibitors on scopolamine-induced amnesia in rats ( ) ( ) ( ) ( ) . prep inhibition was also found to promote neuronal survival and neurite outgrowth of cerebellar granule cells ( ) . however, a recent study in mice shows that the lack of prep in vivo causes a reduction of synaptic spine density in the hippocampal region along with reduced long-term potentiation and memory functions ( ) . many of prep's functions are mediated through its interactions with other proteins. prep is known to interact with gap- ( , ) , α-tubulin ( ) , and gadph ( ) . its most studied interaction is with α-synuclein ( ), reviewed in ref. ( ) . prep and α-synuclein have been shown to co-localize in cell models of stress and in the substantia nigra of post-mortem parkinson's disease brain ( , ) . in vitro, the aggregation rate of α-synuclein increases in the presence of high concentrations of prep, which is abolished through active site inhibitors of prep and absent with a catalytically impaired prep mutant ( ) . in vivo, prep inhibition reduces α-synuclein aggregates in a cellular and animal model for parkinson's disease ( ) . the role of dppiv in monocytes and macrophages has been somewhat contested. whereas dppiv's presence on monocytes and macrophages has been shown repeatedly in mice and rats ( ) ( ) ( ) , its expression in human monocytes and macrophages is less obvious. figure shows an overview of the expression of dppiv throughout the immune system. in visceral obesity, dppiv expression is low on peripheral blood monocytes, macrophages, and dendritic cells, but it is upregulated in vitro frontiers in immunology | www.frontiersin.org august | volume | article after differentiation and activation of isolated monocytes into macrophages or dendritic cells, and in vivo locally in adipose tissue ( ) . interestingly, the authors showed that macrophage-or dendritic cell-associated dppiv most likely binds ada, promoting local degradation of adenosine, a t-cell proliferation suppressor, thereby inducing t-cell proliferation ( ) . three other studies also found no to low dppiv expression or activity associated with human monocytes and/or macrophages ( , [ ] [ ] [ ] . others have investigated dppiv in monocyte-or macrophage-like cell lines ( , , ( ) ( ) ( ) ( ) ( ) ( ) . in hl- cells, its expression has been found to be regulated by differentiation into macrophage-like cells ( ) . dppiv inhibitor alogliptin can affect erk activation, mmp and il- secretion in u cells ( , ) . however, these studies employed alogliptin at concentrations lower than its ic for dppiv. it is therefore questionable whether the observed effects were mediated by dppiv at all. on the other hand, proliferation is reduced in the presence of a dpp inhibitor in u cells expressing high levels of dppiv, but not in the same cell type expressing low levels of dppiv ( ) . moreover, the same inhibitor causes the former cells to secrete lower amounts of il- β, but higher amounts of tnfα ( ) . it could be that inhibition merely increases tnfα's half-life, as dppiv has been implicated in its degradation in u cells ( ) . in thp- cells, dppiv inhibitors alogliptin and sitagliptin both reduced these cells' chemotactic potential ( ) . dppiv inhibitors sitagliptin and nvpdpp also reduced nlrp , tlr , and il- β expression and increased glp- r expression in thp- cells and this effect was blocked through pma differentiation ( ) . importantly, such cell lines have been derived from different types of myeloid leukemia, and as it is known that dppiv expression is often dysregulated in cancer ( - ), the physiological relevance of these findings remains uncertain. fap has been shown on tumor-associated macrophages in human breast cancer ( ) . dipeptidyl peptidase / activity has been found in human monocytes and u cells ( ) . dpp was found associated with activated microglia/macrophages in a rat model of cerebral ischemia ( ) . dpp and are abundantly present in macrophage-rich regions of atherosclerotic plaques ( ). interestingly, dpp is upregulated after in vitro monocyte-to-macrophage differentiation. moreover, inhibition or rna silencing of dpp attenuates pro-inflammatory m , but not m , macrophage activation ( ). in rats, dppii is expressed in tissue-resident macrophages ( , ) . humans show dppii activity in monocytes as well as u cells ( , ) . human blood derived alveolar macrophages show high-prcp activity ( , ) . interestingly, in a mouse in vivo angiogenesis assay, macrophage infiltration into the wound was increased in mice with a prcp deletion ( ) . prolyl oligopeptidase activity has been shown in mouse and rat peritoneal macrophages and in rat pulmonary macrophages ( , , ) . its activity in mouse peritoneal macrophages is increased after thioglycollate ellicitation ( ) . in addition, prep has been identified as a neurotoxic component in the supernatant of activated thp- cells, which are monocyte-like cells ( ) . apparently, these cells secrete prep upon activation with ifnγ and lps and partly because of this, their supernatant is toxic to neuroblastoma sh-sy y cells, as shown through the use of prepspecific inhibitors ( ) . prep's mode of action in this remains unclear. recently, a study showed that dppiv acts as a chemorepellent for human and murine neutrophils ( ) . adding recombinant dppiv to purified human neutrophils in an insall chamber causes the neutrophils to migrate away from the higher concentration of dppiv. this effect is blocked by dppiv inhibitors, meaning that the effect is mediated through dppiv's enzymatic activity, although a candidate substrate is not obvious. moreover, in a mouse model of acute respiratory distress syndrome, oropharyngeal aspiration of dppiv prevented accumulation of neutrophils in the lung ( ) . by contrast, prep is involved in the generation of prolyl-glycyl-proline, a collagen fragment that is an efficient neutrophil chemoattractant ( ) . human peripheral blood neutrophils contain prep activity and are themselves capable of generating prolyl-glycyl-proline after lps-activation, alluding to a self-sustaining pathway of neutrophil inflammation ( ) . prcp is also abundantly expressed in human neutrophils ( ). the recruitment of eosinophils is affected by dppiv activity. ccl , also known as eotaxin, is a dppiv substrate and cleavage by dppiv prevents the activation of its receptor ccr ( ) . in rats, it was shown that administration of ccl results in eosinophil recruitment and this recruitment is significantly more effective in dppiv-deficient f mutants ( ) . finally, dppii activity has been reported in the granules of mast cells in several publications ( , , ) . it is released from peritoneal mast cells upon degranulation and is apparently inhibited by histamine and zn + at concentrations present in the granules of mast cells ( ) . dipeptidyl peptidase is present in low amounts on freshly isolated human nk cells and its expression is only upregulated in a small subpopulation after il- stimulation ( ) . in that study, it was also shown that dppiv inhibition suppresses dna synthesis and cell cycle progression of nk cells, but these effects may be dpp / mediated as the inhibitors used in that study are now known to also inhibit dpp / activity ( ) . another study shows that dppiv is actually only expressed by a small subpopulation of peripheral nk cells ( ) . the natural cytotoxicity of nk cells is not influenced by the presence or absence of dppiv on their cell surface ( , ) . however, dppiv-negative nk cells show significantly less cd -dependent lysis than dppiv-positive nk cells ( ) . interestingly, nk cytolytic function against tumor cells was diminished in dppiv-deficient rats in a model for lung metastasis ( ) . figure shows an overview of published data on the dpp family in the innate immune system. only about % of freshly isolated cd -positive b cells express dppiv, but this fraction grows significantly upon pokeweed mitogen (pwm) or s. aureus protein stimulation ( ) . similar to nk cells, dppiv inhibitors significantly suppress dna synthesis in b-lymphocytes ( ) , but again these inhibitors are now known to also inhibit dpp and ( ) . mouse spleen-derived b-lymphocytes only express low amounts of dppiv mrna ( ) . dpp and mrna, on the other hand, are expressed at much greater levels in these cells, and they are upregulated in raji cells, a b-lymphocyte-like cell line, after pwm, lps stimulation or mitomycin c treatment, and downregulated after dtt treatment ( ) . dpp and have also been shown immunohistochemically in human lymph follicular lymphocytes ( ) . dppii activity has also been shown in human b-lymphocytes ( ). dipeptidyl peptidase iv was originally described as a surface marker for t-lymphocytes, in which case it is better known as cd , and later more specifically for a subset of cd -positive memory cells, cd + cd ro + cd + cells, which respond maximally to recall antigen tetanus toxoid and induce b-cell igg synthesis ( , ) . indeed, cd surface expression is augmented along with the antigen sensitivity of a particular cd + t-cell clone ( ) . cd high cd + t-cells belong to the early effector memory t-cell subset ( ) . cd is also a marker for t-cell activation ( , ( ) ( ) ( ) . cd expression on cd + tcells correlates with t h responses. stimuli that typically induce a t h phenotype tend to induce cd expression ( ) . additionally, the cd + t cells capable of transendothelial migration in vitro are characterized by a bright expression of cd ( , ) , but cd does not seem to be actually involved in t-cell adhesion to endothelial cells or fibroblasts ( ) . recently, it was shown that up to % of all t h cells show very high cd expression, with mean fluorescent intensity on these cells almost twice as high as on t h or t h cells. therefore, the authors of this study suggest cd as a marker for t h cells ( ) . conversely, cd has been proposed as a negative marker for the selection treg cells due to its very low-surface expression on these cells ( ) ( ) ( ) . cd is also a costimulatory molecule for t-cell activation. crosslinking of cd , along with cd , stimulates t-cell activation and proliferation ( , , ) . cd can also directly activate t-cells in an alternative activation pathway, but this requires the presence of the tcr/cd complex ( ) ( ) ( ) . during costimulation, cd is mannose- phosphorylated and internalized, the latter of which is mediated in part by its interaction with m p/igfiir ( ) . it then localizes to lipid rafts where it might interact with cd , required for tcr signaling, facilitating colocalization of this molecule with tcr signaling molecules ( , ) . a number of candidate binding partners for costimulation have been proposed. ada and cd are known binding partners ( ) . even though ada binding to cd does not seem to be essential for immune functions in humans ( ) , the nanomolar affinity of this interaction probably reflects its importance ( ) . indeed, association with free ada or ada presented by ada-anchoring proteins on dendritic cells seems to costimulate t-cells through cd binding ( , ) . on the other hand, it has been shown that soluble dppiv enhances t-cell proliferation independent of its enzyme activity or ada-binding capability ( ) . interestingly, the ada-cd interaction can be inhibited by hiv- external envelope protein gp and this requires interaction of gp with cxcr ( ) . in fact, evidence suggests a physical association between cxcr and cd on peripheral blood lymphocytes ( ) . fibronectin is another known binding partner of cd involved in t-cell costimulation ( ) ( ) ( ) . finally, cd interacts with caveolin- on monocytes. this interaction causes an upregulation of cd on these cells, which potentiates antigen-specific t-cell activation ( ) . most studies seem to find no need for dppiv's enzymatic activity for succesful costimulation, as evidenced through the use of inhibitors and catalytically impaired dppiv mutants ( ) ( ) ( ) ( ) . dipeptidyl peptidase and are present in baboon spleen interfollicular t-lymphocytes and jurkat t cells ( ) . they are upregulated in the latter after pwm and lps, but not pha, stimulation ( , , ) . activation of pwm-stimulated t-cells is suppressed after dppiv/ / inhibition. moreover, dna synthesis and t-cell proliferation are reduced, as well as production of il- , - , - , and ifn-γ. this is due to an induction of tgf-β secretion ( , ( ) ( ) ( ) ( ) . inhibition also upregulates ctla- and downregulates dppiv expression ( , ) . these observations might be physiologically relevant as endogenous inhibitors of dpps are known which have similar effects in cell-based experiments as the synthetic inhibitors ( , ) . dipeptidyl peptidase ii activity is higher in t-lymphocytes than in b-lymphocytes ( ) and absence of dppii steers t-lymphocytes toward a t h phenotype. t-lymphocytes of dppii ko mice hyperproliferate and secrete il- after cd crosslinking or after in vivo priming and in vitro antigen-specific restimulation ( ) . prep activity has also been shown in mouse t-lymphocytes ( ) . its activity is significantly higher in immature, double-positive thymocytes compared to mature, single-positive thymocytes, or peripheral t-cells. t-cells stimulated with con a followed by il- show a time-dependent increase in prep activity and pretreatment of cells with a prep inhibitor renders them resistant to activation-induced cell death ( ) . figure shows an overview of in vitro data on dpp involvement in primary human t cell activation. the dpp family has been reported to be dysregulated or even involved in a number of inflammatory disorders. expression levels of a number of family members are modulated in rheumatoid arthritis. whereas the density of cd on peripheral t cells is increased in patients, it is low on synovial fluid t cells ( ) ( ) ( ) . dppiv activity in plasma, serum, or synovial fluid of patients has also been found to be decreased, similar to results in several rat models of arthritis ( ) ( ) ( ) ( ) ( ) ( ) ( ) . interestingly, rats resistant to induction of arthritis show higher plasma dppiv levels ( ) . by contrast, dppii and prep activity are increased in serum or synovial fluid of arthritis patients ( ) ( ) ( ) . likewise, fap immunoreactivity is much higher in fibroblast-like synoviocytes of rheumatoid arthritis patients compared to osteoarthritis controls ( ) . dppiv's involvement in rheumatoid arthritis has been studied, but remains unclear. on the one hand, inhibition can suppress development of arthritis in rats ( ) . note, however, that effects mediated through other dpps are hard to exclude as these inhibitors were developed before dpp and dpp were discovered. on the other hand, induced arthritis is more severe in dppiv-deficient mice ( ) . this may be due to increased levels of circulating cxcl ( ), a dppiv substrate shown to be involved in rheumatoid arthritis. several case reports in patients seem to suggest a link between the development of rheumatoid arthritis and the use of dppiv inhibitors ( ) ( ) ( ) . prcp has also been associated with rheumatoid arthritis as its activity was shown in synovial fluid isolated from arthritic joints ( ) . inflammatory bowel disease shows a distinct expression pattern of the dpp family. dppiv serum or plasma activity seems to be lower in patients, whereas there is an increase of circulating cd + cd + cells with a higher cd surface expression ( , ) . fap is heavily expressed by myofibroblasts in the submucosa strictures in crohn's disease, and is upregulated after stimulation with tnfα or tgfβ ( ) . in a mouse model, colonic dppii and dpp mrna and dppii activity are increased, while colonic dpp / activity only increases significantly in mice that are also dppiv knockouts ( ) . in mouse models, inhibition or abrogation of dppiv seems to at least partially ameliorate symptoms, possibly by increasing circulating glp- , impairing neutrophil recruitment, and maintaining treg populations ( ) ( ) ( ) ( ) ( ) ( ) . some of those beneficial effects may be mediated in part by the other dpps, as additive effects were found for dppiv ko and the dpp inhibitors ( , , ) . a recent study suggests that the ameliorative effects of dpp inhibitors are most likely not mediated through glp- protection ( ) . the dpp family has also been studied in neuroinflammation. ischemia-induced neuroinflammation in rats prompts a distinct expression and activity pattern of the dpps. in the days following ischemia, the brain of these rats undergoes a complex reorganization of dpp expression with changes in mrna, protein, and activity levels of dppii, , , and in cortical neurons, microglia, and macrophages ( ) . similarly, prep seems to be associated with astrocytes and microglia in lesioned inflamed brains of rats ( ) . dppiv and prep also may be involved in multiple sclerosis. cd + t cells were found to correlate with disease scores ( ) . soluble dppiv levels are elevated in cerebrospinal fluid of patients ( ) . plasma prep activity, on the other hand, is lower in patients with relapsing-remitting or primary progressive multiple sclerosis and in clinically isolated syndrome ( , ) . interestingly, prep inhibition seems to aggravate symptoms in a mouse model of multiple sclerosis ( ) . in systemic lupus erythematosus, dpps also seem to be dysregulated. in mouse models, dppii and prep activities are increased in plasma, spleen, kidney, and liver, whereas dppiv activity is decreased ( , ) . human patients also show elevated dppii and reduced dppiv activities in serum, along with reduced numbers of cd + t cells ( , ) . interestingly, serum dppiv levels are inversely correlated with disease score ( ) . fap immunoreactivity is decreased in the synovium of lupus patients ( ) . finally, dppiv has been studied in psoriasis, an immunemediated chronic inflammatory disorder with primary involvement of skin and joints. its mrna, protein levels and activity are higher in psoriatic skin samples ( , ) . by contrast, serum dppiv levels and activity seem to be lower in patients ( , ) , accompanied by a reduction of peripheral cd + cd + t cells ( , ) . two case reports suggest a link between the use of dppiv inhibitor sitagliptin and psoriasis. while one woman developed a psoriaform eruption days after starting sitagliptin treatment ( ), another patient's psoriatic lesions gradually diminished and were effectively gone months after the start of sitagliptin treatment ( ) . dipeptidyl peptidase iv has recently received much attention for its potential as a therapeutic target for the treatment of atherosclerosis (box ) ( ). this is not surprising considering the current use of dppiv inhibitors in the treatment of t d and the fact that t d is associated with a higher risk for atherosclerosis ( , ). in the apoe −/− mouse model of atherosclerosis, dppiv inhibition generally reduces plaque area and monocyte and macrophage plaque infiltration ( ) ( ) ( ) . a reduction in the number of plaque lesions or in smooth muscle cell content have also been observed ( , ) , as well as lower plaque mmp and higher plaque collagen levels, suggesting increased plaque stability ( ) . one study reported effects of dppiv inhibition on atherosclerotic plaques of only diabetic apoee −/− mice ( ), but more recently, terasaki et al. found similar effects in non-diabetic and diabetic apoe −/− mice ( ) . likely, such differences can be explained by the fact that different dppiv inhibitors were employed. effects of dppiv on atherogenesis similar to those observed in apoe −/− mice have been reproduced in ldlr −/− mice ( , ) . in human atherosclerotic plaques, atherosclerosis is the most common underlying cause of cardiovascular diseases and should be regarded as an inflammatory disease. it starts with dysfunction of the endothelium leading to the expression of leukocyte adhesion molecules, such as selectins and integrins. locally produced proinflammatory cytokines attract the immune cells into the inner layer of the endothelium. however, not only leukocytes are found in the plaque but also low-density lipoprotein particles (ldl) and their oxidized counterparts (oxldl). in the plaque, monocytes differentiate into macrophages, phagocytose the oxldl and turn into so-called pro-atherogenic foam cells. this process leads to a self-sustaining, local inflammation leading to plaque growth, and migration of smooth muscle cells into the core. a plaque is defined as stable as long as it is contained by a thick fibrous cap. however, the latter is slowly degraded by the proteolytic enzymes from the leukocytes. this eventually leads to rupture and the formation of arterial thrombi ( , dppiv immunoreactivity could only be found on endothelium of neovessels ( ). it was recently found that dppiv activity may be a predictor for the onset of atherosclerosis in otherwise healthy chinese individuals ( ) . another prospective study investigated the influence of vildagliptin or sitagliptin treatment on intima-media thickness, a surrogate marker for atherosclerosis. this study found that treatment with vildagliptin or sitagliptin reduced intima-media thickness, suggesting that dppiv inhibition might be beneficial in atherosclerosis in humans as well ( ) . moreover, treatment naïve t d patients treated with alogliptin for months saw a significant decrease in their circulating atherogenic lipids ( ) . it has been suggested that dppiv inhibitors' anti-atherogenic effects are mainly mediated through decreased monocyte infiltration, as dppiv inhibitors suppress monocyte activation and chemotaxis in vitro ( , ) . dppiv inhibition also reduces in vitro foam cell formation in exudate peritoneal macrophages from apoe −/− mice ( ) . moreover, soluble dppiv stimulates in vitro proliferation of smooth muscle cells and this can be reduced through the addition of a dppiv inhibitor ( , ) . finally, active circulating glp- levels are augmented and this improves endothelial dysfunction ( , ) . probably, dppiv inhibition improves atherosclerosis through a combination of all these mechanisms. indeed, incretin antagonists only partially attenuate the anti-atherogenic effects of dppiv inhibition, suggesting that other mechanisms beyond incretin preservation are in play ( ) . interestingly, monocyte-endothelial cell adhesion is abrogated by an anti-sdf- α antibody in vitro ( ) . ldl seems to induce sdf- α expression and leads to smooth muscle cell proliferation and inhibition of cell apoptosis ( , ) . sdf- α is a dppiv substrate, which loses its biological activity after cleavage ( ) . as dppiv inhibition seems to improve atherosclerosis, whereas intact sdf- α appears to be deletorious, it could be argued that sdf- α cleavage by dppiv does not play a major role in atherosclerosis. dipeptidyl peptidase and have been found to be abundantly present in the macrophage-rich regions of human atherosclerotic plaques and considering dpp 's role in macrophage activation, it might potentially be involved in atherogenesis ( ). fap expression is enhanced in some, but not all types of human atheromata. it is found on smooth muscle cells, and its expression correlates with macrophage burden, probably due to the fact that tnfα upregulates fap in smooth muscle cells in vitro. as it is mainly associated with collagen-poor regions and can digest type i collagen and gelatin in vitro, fap probably contributes to plaque instability ( ) . interestingly, many of the studies reviewed above show the potential of targeting dpp family members for the treatment of atherosclerosis (see figure ). fap inhibition might reduce plaque instability by decreasing collagen breakdown; dpp inhibition is likely to attenuate m macrophage activation, reducing the local inflammatory cascade; dppiv inhibition may decrease monocyte infiltration, foam cell formation, improve endothelial dysfunction, and reduce smooth muscle cell proliferation; and finally, prep inhibition might reduce neutrophil infiltration, preventing endothelial dysfunction, and monocyte infiltration. all of this shows the possibilities of repositioning dppiv inhibitors, currently being used to treat type diabetes, as well as the potential of targeting other members of the dpp family. caution should be taken when interpreting results from literature data based on dpp inhibitors, especially from older studies. it is now known that, under the experimental conditions used, many of these inhibitors are not specific for one particular family member. the reported findings, however, remain interesting. this review has shown extensive involvement of members of the dpp family in the immune system. it is clear that these enzymes hold great potential as targets for the treatment of certain inflammatory disorders. particularly, the possibility of targeting dpp family members for the prevention and treatment of atherosclerosis warrants further investigation. in vivo effects of a potent, selective dppii inhibitor: uamc is a possible tool for the elucidation of the physiological function of dppii. adv exp med dipeptidylpeptidase negatively regulates colony-stimulating factor activity and stress hematopoiesis structures of human dpp reveal the molecular basis of specific inhibition and the architectural diversity of proline-specific peptidases purification of two dipeptidyl aminopeptidases ii from rat brain and their action on proline-containing neuropeptides dipeptidyl peptidase ii (dppii), a review thioxo amino acid pyrrolidides and thiazolidides: new inhibitors of proline specific peptidases structure-activity relationships of boronic acid inhibitors of dipeptidyl peptidase iv. . variation of the p position of xaa-boropro dipeptides structure-activity relationship of diaryl phosphonate esters as potent irreversible dipeptidyl peptidase iv inhibitors rapid parallel synthesis of dipeptide diphenyl phosphonate esters as inhibitors of dipeptidyl peptidases fluoro-olefins as peptidomimetic inhibitors of dipeptidyl peptidases prolyl oligopeptidase induces angiogenesis both in vitro and in vivo in a novel regulatory manner a prolyl oligopeptidase inhibitor, kyp- , reduces α-synuclein protein levels and aggregates in cellular and animal models of parkinson's disease subcellular localization suggests novel functions for prolyl endopeptidase in protein secretion subcellular distribution of prolyl endopeptidase and cation-sensitive neutral endopeptidase in rabbit brain expression and traffic of cellular prolyl oligopeptidase are regulated during cerebellar granule cell differentiation, maturation, and aging distribution of prolyl oligopeptidase in the mouse whole-body sections and peripheral tissues partial purification and characterization of post-proline cleaving enzyme: enzymatic inactivation of neurohypophyseal hormones by kidney preparations of various species changes in prolyl endopeptidase during maturation of rat brain and hydrolysis of substance p by the purified enzyme porcine muscle prolyl endopeptidase and its endogenous substrates purification and characterization of prolyl endopeptidase from pig brain purification and characterization of prolyl endopeptidase from rat skin effect of s , a novel prolyl endopeptidase inhibitor, on substance p and alpha-melanocyte-stimulating hormone breakdown in the rat brain an evaluation of the role of a pyroglutamyl peptidase, a post-proline cleaving enzyme and a post-proline dipeptidyl amino peptidase, each purified from the soluble fraction of guinea-pig brain, in the degradation of thyroliberin in vitro prolyl oligopeptidase: a potential target for the treatment of cognitive disorders on the role of prolyl oligopeptidase in health and disease degradation of neurotensin by rabbit brain endo-oligopeptidase a and endo-oligopeptidase b (prolineendopeptidase) proline specific peptidases brain endo-oligopeptidase b: a post-proline cleaving enzyme that inactivates angiotensin i and ii inactivation of thyrotropin-releasing hormone (trh) and ( me-his) trh by brain peptidases studied by highperformance liquid chromatography evaluation of the role of prolyl endopeptidase and pyroglutamyl peptidase i in the metabolism of lhrh and trh in brain proline-specific proteases in cultivated neuronal and glial cells effect of a novel prolyl endopeptidase inhibitor, jtp- , on prolyl endopeptidase activity and substance pand arginine-vasopressin-like immunoreactivity in the brains of aged rats neutrophils contain prolyl endopeptidase and generate the chemotactic peptide, pgp, from collagen prolyl oligopeptidase is involved in release of the antifibrotic peptide ac-sdkp alteration of prolyl oligopeptidase and activated α- -macroglobulin in multiple sclerosis subtypes and in the clinically isolated syndrome prolyl oligopeptidase is inhibited in relapsing-remitting multiple sclerosis specific inhibitors for prolyl endopeptidase and their anti-amnesic effect jtp- : a novel prolyl endopeptidase inhibitor with potential as a cognitive enhancer pharmacological studies of a novel prolyl endopeptidase inhibitor, jtp- , in rats with middle cerebral artery occlusion effects of prolyl endopeptidase inhibitors and neuropeptides on delayed neuronal death in rats ono- , a potential antidementia drug, delays age-induced apoptosis and suppresses overexpression of glyceraldehyde- -phosphate dehydrogenase in cultured central nervous system neurons prolyl endopeptidase-deficient mice have reduced synaptic spine density in the ca region of the hippocampus, impaired ltp, and spatial learning and memory prolyl oligopeptidase binds to gap- and functions without its peptidase activity gap shows partial co-localisation but no strong physical interaction with prolyl oligopeptidase prolyl oligopeptidase is a glyceraldehyde- -phosphate dehydrogenase-binding protein that regulates genotoxic stress-induced cell death interaction of prolyl oligopeptidase with α-synuclein prolyl oligopeptidase colocalizes with α-synuclein, β-amyloid, tau protein and astroglia in the post-mortem brain samples with parkinson's and alzheimer's diseases prolyl oligopeptidase stimulates the aggregation of alpha-synuclein the anti-inflammatory effect of neuropeptide y (npy) in rats is dependent on dipeptidyl peptidase (dp ) activity and age soluble dpp originates in part from bone marrow cells and not from the kidney representative aminopeptidases and prolyl endopeptidase from murine macrophages: comparative activity levels in resident and elicited cells a potential role for dendritic cell/macrophage-expressing dpp in obesity-induced visceral inflammation dipeptidyl peptidase / -like activity in human leukocytes human u cell surface peptidase activities: characterization and degradative effect on tumor necrosis factor-alpha plasma membrane-bound and lysosomal peptidases in human alveolar macrophages divergent regulation of cell surface protease expression in hl- cells differentiated into macrophages with granulocyte macrophage colony stimulating factor or neutrophils with retinoic acid dpp- (cd ) inhibitor alogliptin inhibits tlr -mediated erk activation and erkdependent mmp- expression by u histiocytes dpp- (cd ) inhibitor alogliptin inhibits atherosclerosis in diabetic apolipoprotein edeficient mice longterm dipeptidyl-peptidase inhibition reduces atherosclerosis and inflammation via effects on monocyte recruitment and chemotaxis dpp- inhibitors repress nlrp inflammasome and interleukin- beta via glp- receptor in macrophages through protein kinase c pathway inhibitors of dipeptidyl peptidase iv (dp iv, cd ) specifically suppress proliferation and modulate cytokine production of strongly cd expressing u cells fibroblast activation protein expression by stromal cells and tumor-associated macrophages in human breast cancer dipeptidyl peptidase iv, aminopeptidase n and dpiv/apn-like proteases in cerebral ischemia cytochemical localization and biochemical characterization of dipeptidyl aminopeptidase ii in macrophages and mast cells cytochemical localization and biochemical evaluation of a lysosomal serine protease in lung: dipeptidyl peptidase ii in the normal rat prolylcarboxypeptidase (angiotensinase c) in human lung and cultured cells prolylcarboxypeptidase promotes angiogenesis and vascular repair a prolyl endopeptidase from murine macrophages, its assay and specific inactivation cathepsin b and prolyl endopeptidase activity in rat peritoneal and alveolar macrophages. stimulation of peritoneal macrophages by saline lavage prolyl endopeptidase is revealed following silac analysis to be a novel mediator of human microglial and thp- cell neurotoxicity dipeptidyl peptidase iv is a human and murine neutrophil chemorepellent a novel proteolytic cascade generates an extracellular matrix-derived chemoattractant in chronic neutrophilic inflammation inhibition of cd /dipeptidyl peptidase iv enhances ccl /eotaxinmediated recruitment of eosinophils in vivo rat peritoneal mast cells release dipeptidyl peptidase ii expression and functional role of dipeptidyl peptidase iv (cd ) on human natural killer cells dipeptidyl peptidase iv inhibition for the treatment of type diabetes, potential importance of selectivity over dipeptidyl peptidases and the cd antigen is coupled to protein tyrosine phosphorylation and implicated in cd -mediated lysis in natural killer cells cd expression determines lung metastasis in mutant f rats: involvement of nk cell function and soluble cd functional role of cd on human b lymphocytes regulation of dipeptidyl peptidase and expression in activated lymphocytes and injured liver the in vivo expression of dipeptidyl peptidases and f , a novel cell surface molecule, involved in helper function of cd cells dipeptidyl peptidase iv of human lymphocytes -evidence for specific hydrolysis of glycylproline p-nitroanilide in t-lymphocytes influence of cd and integrins on the antigen sensitivity of human memory t cells cd -mediated co-stimulation in human cd (+) t cells provokes effector function via proinflammatory cytokine production dipeptidyl peptidase iv in human t lymphocytes. an approach to the role of a membrane peptidase in the immune system dipeptidyl peptidase iv in the immune system a novel pathway of human t cell activation via a kd t cell activation antigen cell surface characterization of t lymphocytes and allergen-specific t cell clones: correlation of cd expression with t(h ) subsets phenotypic characterization of cd + t cells that exhibit a transendothelial migratory capacity characterization of the c antigen involved in transendothelial migration of cd hi t cells after tight adhesion to human umbilical vein endothelial cell monolayers cd (dipeptidyl peptidase iv) on human t lymphocytes does not mediate adhesion of these cells to endothelial cells or fibroblasts human th cells express high levels of enzymatically active dipeptidylpeptidase iv (cd ) cd : a negative selection marker for human treg cells human treg cells are characterized by low/negative cd expression cloning and functional expression of the t cell activation antigen cd costimulation of cd + and cd + t cells through cd : the ada-binding epitope is not essential for complete signaling triggering of cytotoxic t lymphocytes and nk cells via the tp pathway is dependent on the expression of the t cell receptor/cd complex function of dipeptidyl peptidase iv (cd , tp ) in transfected human t cells fcr-mediated crosslinking of ta (cdw ) induces human t lymphocyte activation internalization of cd by mannose -phosphate/insulin-like growth factor ii receptor contributes to t cell activation cd -mediated signaling for t cell activation occurs in lipid rafts through its association with cd ro coassociation of cd (dipeptidyl peptidase iv) with cd on the surface of human t lymphocytes direct association of adenosine deaminase with a t cell activation antigen the binding site of human adenosine deaminase for cd /dipeptidyl peptidase iv: the arg gln mutation impairs binding to cd but does not cause immune deficiency the hiv- gp inhibits the binding of adenosine deaminase to cd by a mechanism modulated by cd and cxcr expression cd , adenosine deaminase, and adenosine receptors mediate costimulatory signals in the immunological synapse expression of ecto-adenosine deaminase and cd in human t cells triggered by the tcr-cd complex. possible role of adenosine deaminase as costimulatory molecule soluble cd /dipeptidyl peptidase iv enhances human lymphocyte proliferation in vitro independent of dipeptidyl peptidase enzyme activity and adenosine deaminase binding comodulation of cxcr and cd in human lymphocytes a novel consensus motif in fibronectin mediates dipeptidyl peptidase iv adhesion and metastasis fibronectin promotes proliferation of naive and memory t cells by signaling through both the vla- and vla- integrin molecules vla- mediates cd -dependent cd + t cell activation via the cs alternatively spliced domain of fibronectin cd up-regulates expression of cd on antigen-presenting cells by means of caveolin- the costimulatory activity of the cd antigen requires dipeptidyl peptidase iv enzymatic activity enzymatic activity of cd (dipeptidylpeptidase iv) is not required for its signalling function in t cells molecular analysis of cd -mediated signal transduction in t cells unchanged signaling capacity of mutant cd /dipeptidylpeptidase iv molecules devoid of enzymatic activity biochemical properties and expression profile of human prolyl dipeptidase dpp cloning, expression and chromosomal localization of a novel human dipeptidyl peptidase (dpp) iv homolog, dpp role of dipeptidyl peptidase iv (dp iv)-like enzymes in t lymphocyte activation: investigations in dp iv/cd -knockout mice inhibitors of dipeptidyl peptidase iv induce secretion of transforming growth factor-ß in pwm-stimulated pbmc and t cells dipeptidyl peptidase iv (dp iv/cd ) mrna expression in pwm-stimulated tcells is suppressed by specific dp iv inhibition, an effect mediated by tgfbeta( ) dipeptidyl peptidase iv on activated t cells as a target molecule for therapy of rheumatoid arthritis the expression of t-cell surface antigens ctla- , cd , and cd is modulated by inhibition of dipeptidylpeptidase iv (dpp iv, cd ) activity in murine stress-induced abortions downregulation of t cell activation following inhibition of dipeptidyl peptidase iv/cd by the n-terminal part of the thromboxane a receptor non-substrate peptides influencing dipeptidyl peptidase iv/cd activity and immune cell function th differentiation is the default program for dpp -deficient t cell differentiation murine t cells expressing high activity of prolyl endopeptidase are susceptible to activationinduced cell death expression and functional role of f (cd ) antigen on peripheral blood and synovial fluid t cells in rheumatoid arthritis patients cd surface molecule involvement in t cell activation and lymphokine synthesis in rheumatoid and other inflammatory synovitis in active chronic rheumatoid arthritis, dipeptidyl peptidase iv density is increased on monocytes and cd (+) t lymphocytes circulating cd is negatively associated with inflammation in human and experimental arthritis serum levels of soluble cd and cd and their clinical significance in patients with rheumatoid arthritis levels of dipeptidyl peptidase iv/cd substrates neuropeptide y and vasoactive intestinal peptide in rheumatoid arthritis patients activities of dipeptidyl peptidase ii and dipeptidyl peptidase iv in synovial fluid from patients with rheumatoid arthritis and osteoarthritis activities of dipeptidyl peptidase ii, dipeptidyl peptidase iv, prolyl endopeptidase, and collagenaselike peptidase in synovial membrane from patients with rheumatoid arthritis and osteoarthritis activities of dipeptidyl peptidase ii and dipeptidyl peptidase iv in mice with lupus erythematosus-like syndrome and in patients with lupus erythematosus and rheumatoid arthritis neutral aminopeptidase and dipeptidyl peptidase iv in the development of collagen ii-induced arthritis fibroblast activation protein is expressed by rheumatoid myofibroblast-like synoviocytes antiarthritic effects of the novel dipeptidyl peptidase iv inhibitors tmc- a and tsl- polyarthropathy in type diabetes patients treated with dpp inhibitors sitagliptin (dpp- inhibitor)-induced rheumatoid arthritis in type diabetes mellitus: a case report acute onset of rheumatoid arthritis associated with administration of a dipeptidyl peptidase- (dpp- ) inhibitor to patients with diabetes mellitus dipeptidyl peptidase iv (dp iv, cd ) in patients with inflammatory bowel disease dipeptidyl peptidase- expression is reduced in crohn's disease fibroblast activation protein expression in crohn's disease strictures dipeptidyl peptidase expression during experimental colitis in mice the dpp-iv inhibitor er- has a proliferative effect on the colonic epithelium and a minimal effect in the amelioration of colitis dipeptidyl peptidase iv (dp iv, cd ) and aminopeptidase n (apn, cd ) as regulators of t cell function and targets of immunotherapy in cns inflammation inhibiting dipeptidyl peptidase activity partially ameliorates colitis in mice contribution of dipeptidyl peptidase iv to the severity of dextran sulfate sodium-induced colitis in the early phase dipeptidyl peptidase- inhibitor anagliptin facilitates restoration of dextran sulfate sodium-induced colitis biochemical and histological changes in the small intestine of mice with dextran sulfate sodium colitis the effects of a tgr agonist and a dipeptidyl peptidase iv inhibitor on dextran sulfate 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patients treated with cyclosporine, etanercept, and psoralen plus ultraviolet a phototherapy serum soluble cd levels: diagnostic efficiency for atopic dermatitis, cutaneous t-cell lymphoma and psoriasis in combination with serum thymus and activation-regulated chemokine levels expression of dipeptidyl-peptidase iv (cd ) on cd + t cells is significantly decreased in patients with psoriasis vulgaris and atopic dermatitis reduced cd bright expression of peripheral blood cd + tcell subsets in psoriatic patients psoriasiform eruption triggered by a dipeptidyl peptidase iv inhibitor sitagliptin, a dipeptidyl peptidase-iv inhibitor, improves psoriasis dpp- inhibitors and atherosclerosis: the promise primary prevention of cardiovascular diseases in people with diabetes mellitus: a scientific statement from the american heart association and the american diabetes association effects of pkf - , a dipeptidyl peptidase- inhibitor, on the development of atherosclerotic lesions in apolipoprotein e-null mice dpp- inhibitor, suppresses proliferation of vascular smooth muscles and monocyte inflammatory reaction and attenuates atherosclerosis in male apo e-deficient mice dipeptidyl peptidase- inhibitor, sitagliptin, improves endothelial dysfunction in association with its anti-inflammatory effects in patients with coronary artery disease and uncontrolled diabetes sitagliptin reduces plaque macrophage content and stabilises arteriosclerotic lesions in apoe (-/-) mice preventive effect of dipeptidyl peptidase- inhibitor on atherosclerosis is mainly attributable to incretin's actions in nondiabetic and diabetic apolipoprotein e-null mice dipeptidyl-peptidase- inhibitor, alogliptin, attenuates arterial inflammation and neointimal formation after injury in low-density lipoprotein (ldl) receptor-deficient mice increased plasma dpp activities predict new-onset atherosclerosis in association with its proinflammatory effects in chinese over a four year period: a prospective study decreased carotid atherosclerotic process by control of daily acute glucose fluctuations in diabetic patients treated by dpp-iv inhibitors alogliptin: a new dipeptidyl peptidase- inhibitor with potential anti-atherogenic properties inflammation and atherosclerosis the immune response in atherosclerosis: a doubleedged sword a dipeptidyl peptidase- inhibitor, des-fluoro-sitagliptin, improves endothelial function and reduces atherosclerotic lesion formation in apolipoprotein e-deficient mice upregulation of sdf- is associated with atherosclerosis lesions induced by ldl concentration polarization sdf- promotes ox-ldl induced vascular smooth muscle cell proliferation fibroblast activation protein is induced by inflammation and degrades type i collagen in thin-cap fibroatheromata key: cord- - s wtj authors: ruscitti, piero; berardicurti, onorina; di benedetto, paola; cipriani, paola; iagnocco, annamaria; shoenfeld, yehuda; giacomelli, roberto title: severe covid- , another piece in the puzzle of the hyperferritinemic syndrome. an immunomodulatory perspective to alleviate the storm date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: s wtj the coronavirus disease (covid- ), an acute respiratory disease caused by severe acute respiratory syndrome-coronavirus- (sars-cov- ), has been declared as a worldwide public health emergency. interestingly, severe covid- is characterized by fever, hyperferritinemia, and a hyper-inflammatory process with a massive release of pro-inflammatory cytokines, which may be responsible for the high rate of mortality. these findings may advocate for a similarity between severe covid- and some challenging rheumatic diseases, such as adult onset still's disease, secondary hemophagocytic lymphohistiocytosis, and catastrophic anti-phospholipid syndrome, which have been included in the “hyperferritinemic syndrome” category. furthermore, as performed in these hyper-inflammatory states, severe covid- may benefit from immunomodulatory therapies. the coronavirus disease (covid- ) is an acute respiratory disease caused by a novel coronavirus (severe acute respiratory syndrome-coronavirus- , sars-cov- ), identified in wuhan, china in december ( ) . since then, the covid- outbreak has spread worldwide, becoming a pandemic, causing a public health emergency, according to the world health organization (who), and resulting in thousands of deaths ( ) . sars-cov- is a β-coronavirus, an enveloped non-segmented positive-sense rna virus, which could be transmitted from bats via unknown intermediate hosts to infect humans, using the angiotensin-converting enzyme (ace ) receptor ( ) . the latter, is more expressed in adults than children, and thus possibly explains why the disease is more aggressive in older patients ( ) . covid- shows a heterogeneous course, from patients affected by mild flu-like symptoms to patients with unremitting fever and severe respiratory involvement. on this basis, markers of poor prognosis have recently been investigated to effectively prioritize resources to patients with more severe symptoms ( ) . interestingly, this study identified hyperferritinemia and interleukin (il)- , as predictors of poor outcome, thus suggesting a hyper-inflammatory process as the major cause of death ( , ) . in severe covid- , a specific cytokine profile resembling the pattern of a secondary hemophagocytic lymphohistiocytosis (hlh) has been shown, due to significant increases of il- , il- , granulocyte colony stimulating factor (gcsf), interferon-γ inducible protein (ip- ), monocyte chemoattractant protein (mcp- ), macrophage inflammatory protein -α, and tumor necrosis factor (tnf) ( ) . contextualizing unremitting fever, hyperferritinemia, and the hyper-inflammatory process, severe covid- shows similarity to disorders comprised in the so-called hyperferritinemic syndrome ( ) . this syndrome includes adult onset still's disease (aosd), systemic juvenile idiopathic arthritis (sjia), secondary hlh, catastrophic anti-phospholipid syndrome (caps), and septic shock ( ) . hyperferritinemia is a common trait of all these forms, which could be an active pathogenic mediator and not only a consequence of the inflammation ( ). on these bases, we aimed to review the similarities between severe covid- and diseases included in hyperferritinemic syndrome, from a pathogenic, clinical, and therapeutic point of view, thus proposing new insights to improve the management of those patients. coronavirus rnas may act as pathogen-associated molecular patterns, which are detected by the pattern recognition receptors and activate downstream cascades pro-inflammatory pathways ( , ) . in the endosome, toll-like receptor (tlr) , tlr , tlr , and tlr may sense viral rna and dna ( ) , whereas, in the cytoplasm, the viral rna receptor retinoic-acid inducible gene i ( ), cytosolic receptor melanoma differentiation-associated gene , and nucleotidyltransferase cyclic guanosine monophosphateadenosine monophosphate (gmp-amp) synthase may recognize viral rna and dna ( ) . consequently, downstream cascades molecules are triggered, involving adaptor molecule myeloid differentiation primary response (myd ), transcription factor nuclear factor-κb (nf-κb), and interferon regulatory factor , leading to the production of pro-inflammatory molecules ( , ) . in fact, plasma cytokines and chemokines were increased in covid- patients, including il- β, il- , il- , il- , il- , il- , il- , il- , gcsf, ip- , interferonγ (ifn-γ), and tnf ( ) . during severe covid- , these mechanisms could be exaggerated, probably because of a specific genetic susceptibility ( ) , and these patients are characterized by very high blood levels of pro-inflammatory mediators and ferritin ( , ) . the latter is an iron-binding molecule, which is produced after pro-inflammatory stimuli, in addition to iron availability ( ) . furthermore, ferritin comprises subunits, codified according to their molecular weight into heavy (feh) and light (fel) subunits. remarkably, increased expression of feh and of cd +/feh+ macrophages may be observed in inflammatory infiltrate of aosd and secondary hlh ( , ) . additionally, a stimulatory effect of feh on nf-kb has been described, acting as a pro-inflammatory cytokine on hepatic stellate cells ( ) . in this work, ferritin was shown to regulate an iron-independent signaling pathway that resulted ultimately in nf-kb activation ( ) , thus converging on the same pathway elicited by sars-cov- rnas ( , ) . on the contrary, the deletion of feh reduced the inflammatory burden in the model of sepsis by lipopolysaccharide-induced endotoxemia, cecal ligation, and puncture ( ) . such protection was predominantly mediated by the compensatory increase in fel, associated with an inhibitory action on nf-kb ( ) . the proinflammatory cytokines, which are elevated in hyperferritinemic syndrome, have also been described in severe covid- ( ) , and may preferentially induce the expression of feh, via fer , a regulatory element acting as a binding site to nf-kb. the latter, in turn, stimulates the synthesis of further feh and pro-inflammatory cytokines, thus perpetuating a vicious inflammatory loop ( ) . in addition, feh+/il- + macrophages have been shown in the infiltrate of aosd and secondary hlh ( , ) , which may further release feh, following inflammatory stimuli ( ) , and thus contributing to the inflammatory loop ( ) . on these bases, we hypothesize that severe covid- shares common pathogenic mechanisms with other diseases of hyperferritinemic syndrome ( ), with ferritin enhancing the inflammatory burden and triggering a vicious pathogenic loop. lung involvement and hyper-inflammation are at the crossroad between severe covid- and the hyperferritinemic syndrome. as observed in other β-coronaviruses diseases, covid- is characterized by fever, dry cough, increasing dyspnoea with hypoxemia, and bilateral ground-glass opacities and patchy shadowing with a peripheral or posterior distribution, mainly in the lower lobes, on chest ct scans ( ) . in fact, an anatomy report of a covid- pneumonia cadaver showed that sars-cov- invades the respiratory mucosa and infects other cells, thus provoking an inflammatory response in the lower airway and causes lung injury ( ) . considering that coronavirus binds to the host cells using the ace receptor, which is highly represented in the lower respiratory tract, a persistent and repeated stimulation of tlrs in the lung may occur, hence triggering an aberrant immune response and the production of a cytokine storm ( ) . the latter is the result of overwhelming systemic inflammation with a massive release of pro-inflammatory cytokines, quickly progressing to multiple organ dysfunction syndrome and eventually to death ( ) . in spite of various inflammatory etiologies, cytokine release syndrome is supported by an essential underlying hypothesis: the massive release of cytokines as a consequence of: (i) excessive and repeated inflammatory stimuli, and (ii) an inadequate regulation of inflammation, (iii) an uncontrolled release of cytoplasmic cytokines from destroyed lymphocytes after anti-cancer therapies ( , ) . in addition, it has been shown that increased amounts of pro-inflammatory cytokines, including il- β, il- , il- , ifn-γ, ip- , and mcp , were associated with pulmonary inflammation and extensive lung damage in sars patients ( ) , thus suggesting a further pathogenic loop in inducing the cytokine storm. although the mechanisms of how covid- and, in other more general viral infections, would prompt the cytokine storm syndrome are not fully elucidated, it has been suggested that the ifn-γ, which is largely released by a variety of hematopoietic cells in response to viral infection, may facilitate the occurrence of hyperinflammation ( ) . in patients with sjia, lung involvement may trigger systemic inflammation and the development of secondary hlh and ifn-γ plays a central pathogenic role ( , ) . in fact, in lung biopsies in patients with sija, the analysis of expressed genes revealed that many of the up-regulated targets were in gene pathways related to an ifn-γ signature, including human leukocyte antigen (hla)-d family members and other ifnrelated genes ( , ) . two of the most highly up-regulated non-hla genes were chemokine (c-x-c motif) cxcl , and cxcl ( ) , which are ifn-induced chemokines strongly correlated with the occurrence of secondary hlh ( ) . in addition, the lung is one of the major physiological producers of il- β and il- ( ), which are also involved in pathogenic steps, leading to the occurrence of secondary hlh ( , ) . considering all of these findings, it is possible to postulate that during the acute respiratory distress syndrome of covid- , the sars-cov- may trigger a hyper-inflammatory reaction strongly resembling that observed in the lung involvement of sjia, in which the lung acts as a trigger to amplify the immune response. the final result is the uncontrolled proliferation of activated immune cells, the massive production of pro-inflammatory mediators, and the development of cytokine storm syndrome, either in severe covid- or sjia. from a clinical point of view, severe covid- and the diseases included in hyperferritinemic syndrome share a fever as the main clinical symptom. in these conditions, the analysis of fever pattern would also suggest a useful clue to assess the severity of the disease and the occurrence of complications. in sjia and aosd, a typical change from the high-spiking intermittent typical quotidian pattern, to a continuous unremitting pattern suggests the occurrence of secondary hlh, and the worsening of the clinical situation toward a life-threatening hyperinflammatory complication ( ) . during covid- , on the basis of observations from clinicians on the frontlines, the occurrence of unremitting fever would similarly identify a more aggressive subset of patients, at higher risk of a poor prognosis. in addition, in severe covid- , hyperferritinemia is observed, suggesting a marker of severity ( , ) . although it has poor specificity, a -fould increase of ferritin is strongly suggestive of the diseases included in hyperferritinemic syndrome, and is a useful marker to assess disease activity and to predict a poor prognosis ( ) . in fact, hyperferritinemia is associated with increased mortality in sepsis, multiple organ dysfunction syndrome, and critical illness ( ) ( ) ( ) . thus, the clinical phenotype, characterized by unremitting fever and hyperferritinemia, identifies the most severe subset of covid- as observed in the diseases included in hyperferritinemic syndrome. considering the lack of efficacy of antiviral therapy for severe coronavirus infection, it is reasonable to postulate the clinical usefulness of specific immunomodulatory therapies (figure ) , as observed for other diseases included in hyperferritinemic syndrome such as intravenous immunoglobulins (ivigs) and tocilizumab, the humanized monoclonal antibody against il- receptor ( ). ex juvantibus, one of the best criteria for identifying a common pathogenic mechanism, among different diseases, is that the clinical manifestations were reversed upon initiation of the same therapy. it has been shown that, after ivigs therapy, a significant reduction of hyperferritinemia, both in sepsis and secondary hlh was observed, correlating with an improvement in patients ( ). considering their proposed anti-viral activity, possibly comprising many cross-reacting antiviral antibodies and per se immunomodulatory activities ( ), ivigs has also been proposed to treat severe covid- ( ) . another therapeutic immunomodulatory possibility in severe covid- is the administration of hydroxychloroquine (hcq). this drug, has been a licensed treatment for rheumatoid arthritis for many years, and was shown to reduce the viral load, favoring the disappearance of sars-cov- ( ) . however, although it seems promising, a recent meta-analysis, including , patients, suggested that more data are required for a definitive conclusion on the use of hcq in this setting, since no difference was observed in virologic cure, death, or clinical worsening of disease between hcq-treated patients and control groups ( ) . as far as tocilizumab is concerned, the rationale for its use in severe covid- derived from evidence of its beneficial effect on cytokine release syndrome. this is a clinically significant, on-target, off-tumor side effect of the chimeric antigen receptor t-cell therapies administered for treatment of malignancies ( ) . characteristics of cytokinerelease syndrome include fever, encephalopathy, hypotension, and coagulopathy, leading to multiorgan failure, associated with very pronounced levels of hyperferritinemia and il- ( ). the latter provided an effective therapeutic target in cytokine release syndrome ( ) . mirroring this finding, tocilizumab has been used to treat severe covid- with promising results, as observed in other diseases of hyperferritinemic syndrome ( ) . furthermore, a reduction of ferritin, obtained by combing immunomodulatory drugs, was associated with a lower mortality rate in caps and hlh ( ), thus possibly suggesting the use of, in a more aggressive subset of covid- , a combination therapy with both antiviral and antiinflammatory drugs, at the same time ( ) . in addition, the repurposing of these drugs in severe covid- could benefit from the findings of previous reports, and thus, on this basis, many clinical trials are ongoing in different countries (chictr , nct , nct , and nct ). as far as other immunomodulatory strategies in covid- are concerned, il- inhibition showed benefits in sepsis, in which both hyperferritinemia and hyper-inflammation, may be observed, contributing to the dysregulation of the host immune system ( ) . a post-hoc analysis of data from a phase randomized controlled trial showed some improvement of patients with sepsis, following anakinra, a recombinant non-glycosylated form of human il- receptor antagonist, thus suggesting its possible use in those patients ( ) . as a consequence, it is possible to hypothesize that anakinra may also relieve severe covid- . reported data suggest the possible efficacy of emapalumab, a monoclonal antibody neutralizing ifn-y, approved in the treatment of hlh and its massive production of pro-inflammatory cytokines ( ) . due to the important role of ifn-y in driving hyper-inflammation during viral infections, emapalumab may be an additional immunomodulatory therapy that could be employed in the treatment of severe covid- . in addition, available literature suggests that janus kinase (jak) inhibition might affect covid- twice as much, by targeting both inflammation and cellular viral entry ( ) . it has been proposed that baricitinib, a jak /jak inhibitor, may control the hyper-inflammatory steps in those diseases, characterized by a cytokine storm, since a plethora of cytokine receptors indiscriminately use these jaks as mediators of ligands binding and consequent activation of the inflammatory cascade ( ) . furthermore, the disruption of p -associated protein kinase , a known regulator of viral endocytosis into the cell, by baricitinib, could possibly be an additional positive effect in covid- , decreasing the viral entry ( ) . finally, considering ferritin as a pathogenic mediator, this could also be proposed as a therapeutic target in these conditions. high-volume hemofiltration and plasma exchange, extracorporeal blood purification techniques, have been employed to treat secondary hlh to sepsis ( ) ( ) ( ) . interestingly, in parallel with the clinical efficacy, these procedures induce a ferritin reduction ( ) ( ) ( ) , suggesting that the mechanical removal of ferritin could have a possible therapeutic role. in this work, we discuss the similarities, from a pathogenic, clinical, and therapeutic point of view, between severe covid- and four conditions; secondary hlh, aosd, caps and septic shock, which are included in hyperferritinemic syndrome. all these diseases are characterized by very high levels of ferritin, which could not only be the product of the inflammation but rather may play a pathogenic role. possibly, in an inflammatory environment, as observed in these diseases, hyperferritinemia may be involved in a vicious pathogenic loop prompting its pro-inflammatory properties. in severe covid- , ferritin could be a further possible enhancer of the cytokine storm. clinically, unremitting fever is a common feature of severe covid- , suggesting that a change from the intermittent quotidian pattern to a continuous unremitting form would indicate a worsening toward the cytokine storm, as in aosd and sjia. the hyperferritinemia seems to be a marker of poor prognosis and response to treatment, in both severe covid- and hyperferritinemic syndrome. finally, the good response to immunomodulatory therapies, observed during severe covid- , strongly supports the link between this form and other diseases included in hyperferritinemic syndrome. in addition, targeting the hyperinflammatory process, through immunomodulatory therapies, decreases the high mortality rate of all these diseases ( , ( ) ( ) ( ) , thus proposing additional therapeutic options to improve the survival of severe covid- patients, the latter characterized by an over-exuberant pro-inflammatory response, in which the viral load is not correlated with the worsening of symptoms ( ) . in conclusion, we hypothesize that severe covid- shares pathogenic mechanisms, a clinical picture, outcomes, and therapeutic strategies with disorders included in hyperferritinemic syndrome. the hyperferritinemia, characterizing all these diseases may be a pathogenic mediator, enhancing the inflammatory burden, and, as observed in aosd, caps, and secondary hlh, its reduction is associated with a lower mortality. thus, at present, severe covid- , seems to be a new entity in hyperferritinemic syndrome. in addition, since accumulating evidence suggests that severe covid- is associated with a cytokine storm syndrome, therapeutic strategies combining immunomodulatory therapies, may improve the management of those patients. furthermore, in this setting, high levels of ferritin, identifying a more aggressive subset of covid- , may drive clinicians to apply more aggressive therapies and resources in those patients, thus balancing appropriate escalation of therapy and minimizing the exposure to iatrogenic harm. sars-cov- and consequent covid- are a new and great challenge for health systems worldwide, requiring a multidisciplinary approach and a large body of knowledge. all the authors meet all criteria for authorship in the icmje recommendations, since all authors made substantial contributions to the conception or design of the work, the acquisition and interpretation of data. all authors contributed to the critical review and revision of the manuscript and 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fortes, puri title: type i interferon regulates the expression of long non-coding rnas date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: h q m ed interferons (ifns) are key players in the antiviral response. ifn sensing by the cell activates transcription of ifn-stimulated genes (isgs) able to induce an antiviral state by affecting viral replication and release. ifn also induces the expression of isgs that function as negative regulators to limit the strength and duration of ifn response. the isgs identified so far belong to coding genes. however, only a small proportion of the transcriptome corresponds to coding transcripts and it has been estimated that there could be as many coding as long non-coding rnas (lncrnas). to address whether ifn can also regulate the expression of lncrnas, we analyzed the transcriptome of huh cells treated or not with ifnα by expression arrays. analysis of the arrays showed increased levels of several well-characterized coding genes that respond to ifn both at early or late times. furthermore, we identified several ifn-stimulated or -downregulated lncrnas (isrs and idrs). further validation showed that isr , , and expression mimics that of their neighboring genes gbp , irf , and il , respectively, all related to the ifn response. these genes are induced in response to different doses of ifnα in different cell lines at early (isr or ) or later (isr ) time points. ifnβ also induced the expression of these lncrnas. isr and were also induced by an influenza virus unable to block the ifn response but not by other wild-type lytic viruses tested. surprisingly, both isr and were significantly upregulated in cultured cells and livers from patients infected with hcv. increased levels of isr were also detected in patients chronically infected with hiv. this is relevant as genome-wide guilt-by-association studies predict that isr , , and may function in viral processes, in the ifn pathway and the antiviral response. therefore, we propose that these lncrnas could be induced by ifn to function as positive or negative regulators of the antiviral response. interferons (ifns) are key players in the antiviral response. ifn sensing by the cell activates transcription of ifn-stimulated genes (isgs) able to induce an antiviral state by affecting viral replication and release. ifn also induces the expression of isgs that function as negative regulators to limit the strength and duration of ifn response. the isgs identified so far belong to coding genes. however, only a small proportion of the transcriptome corresponds to coding transcripts and it has been estimated that there could be as many coding as long non-coding rnas (lncrnas). to address whether ifn can also regulate the expression of lncrnas, we analyzed the transcriptome of huh cells treated or not with ifnα by expression arrays. analysis of the arrays showed increased levels of several wellcharacterized coding genes that respond to ifn both at early or late times. furthermore, we identified several ifn-stimulated or -downregulated lncrnas (isrs and idrs). further validation showed that isr , , and expression mimics that of their neighboring genes gbp , irf , and il , respectively, all related to the ifn response.these genes are induced in response to different doses of ifnα in different cell lines at early (isr or ) or later (isr ) time points. ifnβ also induced the expression of these lncrnas. isr and were also induced by an influenza virus unable to block the ifn response but not by other wildtype lytic viruses tested. surprisingly, both isr and were significantly upregulated in cultured cells and livers from patients infected with hcv. increased levels of isr were also detected in patients chronically infected with hiv. this is relevant as genome-wide guiltby-association studies predict that isr , , and may function in viral processes, in the ifn pathway and the antiviral response. therefore, we propose that these lncrnas could be induced by ifn to function as positive or negative regulators of the antiviral response. transcriptome analysis by tiling arrays and rna sequencing has led to the conclusion that while - % of the genome is transcribed, only % is dedicated to the transcription of proteincoding sequences ( , ) . among the non-coding transcriptome, there is a group of poorly studied transcripts longer than nt and with low coding potential that have been collectively called long non-coding rnas (lncrnas) ( , ) . it has been estimated that there could be as many lncrna genes as coding genes, but the number of lncrnas is still growing and some authors consider that it could increase to up to~ ( , ) . therefore, there is a great need to identify novel lncrnas and to understand their function and regulation. long non-coding rnas genes are very similar to coding genes at the chromatin, dna, and rna level ( ) . compared to mrnas, most lncrnas are more cell-type specific, less expressed, and less conserved at the nucleotide sequence level ( ) . many lncr-nas have been shown to be functional. some lncrnas function to regulate the expression of neighboring or antisense genes by transcriptional interference, by recruitment of chromatin modifiers and remodelers, or by regulation of imprinting, editing, splicing or translation, and stability ( ) ( ) ( ) ( ) ( ) . enhancer rnas (ernas) and lncrna-activating rnas (lncrna-a) are transcripts that control the expression of neighboring genes in "cis" ( ) ( ) ( ) . however, lncrnas can also function in "trans," away from their site of synthesis. for instance, some pseudogenes regulate the expression of their parental gene, located in a distant genomic location ( ) ( ) ( ) ( ) . lncrnas have especially emerged as regulators of development, pluripotency, and proliferation as some function as oncogenes or tumor suppressors ( , , ( ) ( ) ( ) ( ) . therefore, several lncrnas have been implicated in cancer and in other human diseases ( ) ( ) ( ) . proliferation, differentiation, and pluripotency factors regulate the expression of some lncrnas ( ) . besides, several signaling molecules, including those involved in the immune response, have been shown to induce the expression of specific lncrnas ( ) ( ) ( ) ( ) . induction of tlr , tlr , or tlr leads to the activation of lncr-nas, including lncrna-cox , which regulates the expression of several immune genes or neat , which functions to increase the expression of some antiviral genes such as il ( ) ( ) ( ) . downregulation of il β-erna and il β-rbt lncrnas decreases il β and the accumulation of lps-induced rnas ( ) . similarly, downregulation of lnc-il r decreases the lps-induced inflammatory response ( ) . treatment of thp macrophages with an innate immunity activator also induces the expression of several lncrnas. one of them, linc (or thril) activates the expression of tnfα and other genes involved in the immune response ( ) . in turn, tnfα also induces many lncrnas in fibroblasts, including lethe, a pseudogene that responds to nfκb and inhibits nfκb dna-binding activity leading to reduced inflammation ( ) . besides, dendritic cells (dcs), cd +, and cd + t-cells express a specific set of lncrnas that may regulate cell activation and differentiation ( , , ) . nest lncrna controls the ifnγ locus in cd + t-cells causing decreased salmonella enterica pathogenesis ( , ) . downregulation of lnc-dc, expressed in conventional dcs, impairs dc differentiation from monocytes, and reduces the capacity of dcs to activate t-cells ( ) . lncr-nas also respond to viral infections. infection with enterovirus, influenza virus, hiv, hepatitis b, and c (hcv) viruses as well as the sars coronavirus leads to altered levels of lncrnas ( , ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) (carnero et al., in prep) . from the collection of infectionaltered lncrnas, it is difficult to distinguish those that respond to the virus from those that respond to the cellular antiviral pathways activated by the infection. recently, some lncrnas regulated by infection have also been found to be regulated by ifnα in mice ( ) . interferon is a key molecule in the cellular antiviral response ( ) . detection of pathogens by the cell triggers transcription of the interferon (ifn) genes. when type i/iii ifn is released, it is sensed by the ifn receptors, which induce the jak/stat pathway. stat and coupled to irf form a complex that binds ifnstimulated response elements (isre) in the promoters of ifnstimulated genes (isgs) and activates their transcription. isgs induce an antiviral state by several means, including inhibition of viral replication, transcription, and translation. well-characterized isgs are mx , oas, gbp , but also stat and irf , which amplify the ifn response. further, stat induces the expression of proinflammatory genes such as irf , a transcription factor that also activates isgs, and whose induction is dependent on de novo protein synthesis ( , ) . besides, ifn also induces the expression of negative regulators that limit the strength and duration of the ifn response ( ) ( ) ( ) . finally, ifn activates expression of several mirnas that contribute to the antiviral state or to the control of the ifn response ( ) . here, we have postulated that ifn could also regulate the expression of lncrnas that may have key roles in the antiviral response. therefore, we have performed a high-throughput analysis of lncrnas whose expression is deregulated in response to ifnα. the conditions we used aimed to identify genes controlled by the ifn pathway directly or by other isgs. the results show that several lncrnas are controlled in response to type i ifn in several cell lines tested. the best candidates are lncrna genes upregulated in response to ifn that are found in the genome adjacent to ifn-related coding genes. they have been called isr , , and . interestingly, guilt-by-association genome-wide studies predict that the function of these lncrnas is related to the cellular antiviral response and to viral infections. in fact, isr , , and their neighboring genes are also increased after infection of cultured cells with hcv. a similar increase is detected in the livers of patients infected with this virus or, in the case of isr , in blood cells of patients infected with hiv. huh cells, derived from a human hepatocarcinoma, were provided by dr. chisari's lab (scripps research institute, la jolla, ca, usa). a and thp cells were kindly provided by estanislao nistal (cima, university of navarra, spain), and hela and cells were obtained from atcc. liver samples from patients with or without hcv infection were obtained from the biobank of the university of navarra under approval from the ethics and scientific committees. liver tissue sections were snap frozen and stored at − °c. the clinical data from hcv and hiv-infected subjects are shown in table s and s in supplementary material. cells were grown in dulbecco's modified eagle medium (dmem) enriched with % fetal bovine serum (fbs) and % penicillinstreptavidin in a % co atmosphere. twenty-four hours before treatment with ifn, huh , a , thp , , or hela cells were seeded in six-well plates. then, , , , , , or u/ml of ifnα (sicor biotech) or ifnβ (pbl pestka biomedical laboratories) were used in a final volume of ml. huh cells were also treated with ng/ml il b/ifn-λ (r&d systems) in a final volume of ml. cells were harvested for rna extraction , , , , and/or h after treatment. hcv jfh- was obtained from an initial viral stock from the genotype a jfh- plasmid (pjfh- ) previously described by wakita et al. ( ) . to amplify the virus, huh cells were infected at low multiplicity of infection (moi) with the initial viral stock and supernatants from the cells were harvested at different days postinfection. the presence of virus was evaluated by infecting fresh cells with the supernatant and checking infected cells by immunofluorescence against the hcv core protein. the supernatants with higher titers were selected to perform the experiments. influenza virus strain a/pr / wt (pr ) and the mutant lacking ns (∆ns ) were kindly provided by estanislao nistal (cima, university of navarra, spain) ( ), semliki forest virus (sfv) was a gift from cristian smerdou (cima, university of navarra, spain), and adenovirus serotype (ad ) was amplified as described ( ) . twenty-four hours before infection, cells were seeded in six-well plates in a final volume of ml. cells were infected with hcv at a moi of . , and with a moi of of influenza a, ∆ns , ad , and sfv. in the case of the lytic viruses, we used a moi of as this led to cytopathic effects at h (for influenza and sfv) or h (for ad ) in huh cells. infection with hcv was performed for h, versus h in the case of ad and h in the case of the other viruses. a final volume of ml was used for infection. after infection, the virus was removed and fresh medium was added to the cells. cells were harvested for rna extraction at the indicated times post-infection. two million huh cells were incubated in µl of cytoplasmic buffer ( mm tris hcl ph . , mm edta, and % np ) for min at °c. then, cells were centrifuged for min at g and the supernatant was used to isolate cytoplasmic rna. the pellet was washed with cytoplasmic buffer and centrifuged as before. the supernatant was discarded and the pellet was used to isolate the nuclear rna. rna from nuclear and cytoplasmic fractions was isolated with maxwell research system (promega). total rna from tissue samples was extracted in ml trizol (sigma-aldrich) using the ultra-turrax homogenizer (t basic ika-werke) ( ) . then, µl chloroform was added and the samples were mixed vigorously and then centrifuged at g for min at °c. the aqueous phase was mixed with µl isopropanol and centrifuged at g for min at °c. the pellet of total rna obtained from the centrifugation was washed with % ethanol. finally, the pellet was resuspended in µl dnase/rnase-free pcr water (bioline). dnase i (fermentas) treatment was performed to eliminate dna from the samples before the reverse transcription (rt)-pcr reactions. to isolate rnas from total blood, . ml blood were collected into paxgene blood rna tubes with rna stabilization solution. total rna was extracted by using the paxgene blood rna kit (qiagen gmbh) according to the manufacturer's instructions. briefly, prior to the actual rna isolation, the frozen samples were first incubated at rt for at least h to achieve complete lysis of blood cells. then, the paxgene blood rna tubes were centrifuged for min at g, the supernatant was removed and the pellet was washed with ml rnase-free water. the pellet was then dissolved in µl of the provided lysis buffer bm and transferred into a . ml microcentrifuge tube. to this mixture, µl buffer bm and µl proteinase k were added and incubated for min at °c in a shaking incubator at rpm. the samples were next transferred to a paxgene shredder spin column and centrifuged for min at full speed. the flow-through was transferred into a new tube without disturbing the pellet and mixed with µl isopropanol ( %, purity grade p.a.). this mixture was then passed through a paxgene rna spin column by centrifugation for min at g. following a wash step ( µl bm ), an on-column dnase digest (rnase-free dnase set, qiagen) was performed by addition of dnase i and incubation on the benchtop ( - °c) for min. the column was washed three times, before rna was eluted with µl buffer br . the final eluate was incubated for min at °c, and several aliquots of the rna were stored at − °c. rna extraction from cells or cellular fractions was performed using the maxwell research system from promega according to the manufacturer's recommendations. for each condition, a minimum of a confluent well of a m -plate was used in order to obtain enough rna. in all cases, the rna concentration was measured using a nanodrop spectrophotometer. the quality of the rna was determined in a bioanalyzer (agilent technologies). for microarray hybridization, the samples were processed using manufacturer protocols and hybridized to the agilent sureprint g human gene expression × k microarray. transcriptome data are available at the ncbi gene expression omnibus (geo) data repository . reverse transcription was performed using . µl m-mlv-rt and µl m-mlv-rt × buffer (promega), µl mm dntps, µl random primers at ng/µl, µl dtt . m, and µg rna in a final volume of µl. the reaction was run in the c touch thermal cycler from bio-rad. the samples were incubated at °c for min, then at °c for s, and next immediately placed at °c. quantitative polymerase chain reaction was performed in the cfx real-time system from bio-rad. for the reaction, µl iq syber green mix from bio-rad, . µl of each primer at µm, and µl of the dna sample at . µg/µl were mixed in a final volume of µl. the mixture was first incubated at °c for min, and then at °c for s, °c for s, and °c for s for cycles. the pcr ended after min at °c and min at °c. the results were analyzed with bio-rad cfx-manager software. gapdh levels were evaluated in all cases as a reference. only the samples with similar gapdh amplification were analyzed further. the primers used are listed in table s in supplementary material and were designed using the primer program . initial setups included gc percentage between and %, product size from to bp and primer length between and nt. microarray data normalization was performed using the quantile algorithm. after quality assessment, a filtering process was carried out to eliminate low expression probe sets. applying the criterion of an expression value > in the three samples of at least one of the experimental conditions, probe sets were selected for statistical analysis. limma (linear models for microarray data) ( ) was used to identify the probe sets with significant differential expression between experimental conditions. genes were selected as significant using a b statistic cut-off of b > . . data processing and statistical analyses were performed with r and bioconductor ( ) . functional enrichment analysis of gene ontology (go) categories was carried out using standard hypergeometric test ( ) . the biological knowledge extraction was complemented through the use of ingenuity pathway analysis (ingenuity systems) , whose database includes manually curated and fully traceable data derived from literature sources. all the differentially expressed sequences obtained by the analysis were compared to the ensembl and encode databases and searched for in the genome browser from ucsc for more information ( , ) . orf finder (ncbi) was used to evaluate the length of all probable orfs in isr , , and . coding potential was assayed with the coding potential assessment tool (cpat) ( , ) and by searching the lncipedia database ( ) for the presence of our candidates in the pride archive ( ) or in lists of transcripts associated with ribosomes ( , ) . phylogenetic codon substitution frequencies (phylocsf) was also used to predict the coding potential of isr , , and ( ) . a guilt-by-association approach was used to predict the go categories ( ) in which the differentially expressed lncrnas could be implicated. first, we collected data from samples hybridized to sureprint g microarrays. these samples include the rnas isolated from huh cells treated or not with ifn, and rnas obtained from human samples of different origin, including healthy tissues and several leukemias and other tumors. then, a pearson correlation analysis was performed between isr , , and and all the genes represented in the sureprint g human microarray. coding genes related to cellular antiviral pathways were randomly selected and included in the analysis as positive controls. the obtained correlation matrix was used as input for gitools ( ) where an enrichment analysis of go categories was performed using z -score ( ) and fdr ( ) . statistical analysis of the expression levels obtained by quantitative rt-pcr (qrt-pcr) was performed using graphpath. statistical significance of treated or infected versus nontreated or non-infected samples was calculated using a two-tailed non-parametric mann-whitney t -test. in correlation studies, a two-tailed non-parametric spearman analysis was used. similar results were obtained by pearson correlation. p values lower than . were deemed as significant. we wanted to identify lncrnas that respond to ifn. ifn induces the expression of isgs very fast. some isgs are transcription factors able to regulate the expression of genes with antiviral potential in a secondary wave of ifn response. other isgs are inhibitory factors that function to decrease the response. therefore, to identify lncrnas regulated by ifn or by isgs, one should analyze the transcriptome of cells treated by ifn at different time points. however, to simplify the analysis, we decided to check first whether we could find conditions showing a wide ifn response at a single time point. to this aim, we tested whether high doses of ifn could lead to increased expression of well-known isgs even at late times post-ifn treatment. huh cells were treated for , , , or h with increasing doses of ifnα up to , units/ml, and the expression levels of gbp , irf , bst , oas, il , and isg were evaluated by qrt-pcr (figure ) . the results show that gbp and irf are induced to highest levels at h post-treatment, while bst and oas are induced to similar levels at all times tested. in contrast, il is only significantly induced at days post-treatment (figure and data not shown). however, compared to untreated cells, a significant upregulation of all the genes, including gbp and irf , can be detected at days post-treatment with , units/ml of ifnα ( figure b) . in fact, this dose induced the highest levels of these transcripts at most of the time points. before analyzing the transcriptome of cells treated with , units/ml of ifnα for days, we confirmed that these conditions induced antiviral effects. when huh cells infected with hcv were treated with these conditions, we indeed detected a drastic decrease in viral protein expression by immunofluorescence and a decrease in the levels of hcv viral genomes by qrt-pcr (data not shown). an agilent array that evaluates expression of entrez genes and lncrnas was used to hybridize rna isolated in three independent experiments from control cells or huh cultures treated with , units/ml of ifnα for days. analysis of the array showed that genes upregulated with a high statistical significance (b > ) includes well-known ifn-related genes from the gbp, ifi, oas, isg, mx, or irf families (figure a) . analysis using less stringent criteria (b > . ) showed that % of the genes were upregulated in response to ifn treatment ( figure b) . ingenuity analysis of this set indicated that ifn signaling is the pathway with the highest enrichment followed by other antiviral responses ( figure c) . similarly, the ifn-induced stat pathway and the tlr/irf network are well represented in the set of upregulated genes ( figure s in supplementary material). we selected the probes described as long intergenic non-coding rnas (lincrnas) that showed a significantly altered expression by the ifn treatment (b > . ). first, we determined the position in the genome of the sequences from these probes using the blat searches available at ucsc ( , ) . many sequences corresponded to coding genes or seemed to be utr extensions of coding genes and were discarded for further analysis. the remaining frontiers in immunology | molecular innate immunity www.frontiersin.org sequences corresponded to genes, including genes annotated as lncrnas and located in non-annotated areas of the genome ( figure d , table s in supplementary material) . surprisingly, while % of coding genes were upregulated by ifn, only % of the lncrnas were upregulated. most of the downregulated lncr-nas corresponded to the non-annotated category. this suggests that there could be a relevant ifn-mediated repression of genes that more strongly affects lncrnas. we named these genes idrs, for ifn-downregulated rnas, and accordingly named the ifnstimulated rnas isrs. the fact that almost % of the isrs and idrs correspond to non-annotated areas of the genome suggests that the percentage of the genome able to react to different stimuli could be even larger than expected. as many lncrnas have been described to regulate the expression of neighboring genes, we looked for the closest coding gene for each isr or idr (table s in supplementary material). we considered candidates to have no neighbor when the closest coding gene was not within a distance of kb from the start or the end of the candidate or when the closest gene was non-coding. forty candidates had neighboring coding genes according to these criteria. half of the coding-non-coding pairs were in tandem, convergent, or divergent, were antisense to each other, were overlapping, and pairs seem to share the same promoter according to the dnase i hypersensitivity and the histone marks described by encode for the respective area. therefore, these couples of coding-non-coding genes could be coregulated. three upregulated candidates, isr , isr , and isr were neighbors of the ifn-related genes gbp , irf , and il , respectively. we next wanted to validate the ifn effect on these candidates in independent samples using a different technique. furthermore, we wanted to determine whether the levels of isrs and idrs were altered early after ifn treatment. therefore, the expression levels of isrs and idrs were evaluated by qrt-pcr in huh cells treated with or , units/ml of ifnα for , , , , or h. the fold-change observed for each candidate at each time point is shown in figure and table s in supplementary material. at h post-treatment, isrs and idrs showed a fold-change higher or lower, respectively, than . . only isr and were not significantly upregulated, or idr , , , , and were not significantly downregulated, at any time tested. however, the foldchange was relatively low in most of the cases, indicating a weak response to ifn. moreover, % of the candidates showed low overall expression levels ( table s in supplementary material and data not shown). interestingly, isr and isr were induced more than -fold at h post-ifn treatment, and isr was induced more than -fold at later times. therefore, we decided to study these candidates further. we also opted to focus on isr and , as they were upregulated at later time points. idr and idr , which were downregulated at most times studied, were also analyzed further. as many lncrnas are cell-specific, we decided to study the response to ifn of these selected isrs/idrs in different cell lines. hela, , a , or thp cells were treated with or , units/ml of ifnα for , , , , or h and rna was isolated and used to evaluate the expression of isr , , , , and as well as idr and . the results showed that in the new cell lines tested, expression of isr and was not detected and idr and showed only a mild downregulation in response to ifn (data not shown). importantly, isr , , and were well expressed in all cell lines tested and their expression was strongly induced by ifn at early (isr and ) or late times (isr ) (figure a and data not shown). interestingly, isr , , and have neighboring genes related to ifn response (figure b ). isr is located in tandem with gbp , at the end of the cluster of gbp genes formed by gbp , , , , , , and . in fact, isr is gbp p , a pseudogene of gbp . this may be interesting as some pseudogenes have been described to regulate the expression of their parental genes ( ) ( ) ( ) ( ) . isr is located in tandem with il and isr is convergent with irf . therefore, we decided to evaluate the expression of gbp , irf , and il in response to ifn in the same cell lines. the results showed a similar induction pattern of each isr and of the corresponding neighboring coding gene in response to ifn (figures a,c) . note that in the case of isr , we evaluated the expression of its parental neighboring gene gbp instead of the closest neighbor gbp , as we speculated that there could be a co-regulation of the parental gene and the pseudogene. furthermore, we could not detect expression of gbp in control or ifnα-treated huh cells (data not shown). all the experiments performed so far with these lncrnas have studied the response to high doses of ifn. to determine whether isr , , and also respond to lower doses, their expression level was evaluated in huh cells treated for , , , or h with , , , , or , units/ml of ifnα (figure ) . the results show that induction of these lncrnas is similar to that observed for their corresponding neighboring coding genes (compare figure with figure ). similar results were observed when ifnβ was used instead of ifnα (data not shown). further, we evaluated whether expression of these transcripts was induced after treatment with tnfα. tnfα, similarly to some pathogenassociated molecular patterns, induces nfκβ signaling and expression of pro-inflammatory genes. however, the nfκβ pathway is a poor inducer of isgs. a treatment of huh cells with ng/ml of tnfα for h, induced the expression of cxcl , used as a positive control, more than -fold. a significant increase in expression of only - -fold was observed after tnfα treatment for gbp , irf , il , and isr , but not for isr . similar results were obtained in huh cells treated with lps or polyi:c (data not shown). to obtain more information about these isrs, we looked in detail at the data from encode. even though we did not observe a strong activation after tnfα treatment, transcription factor chip seq from encode showed that isr and isr promoters have nfκb binding sites. interestingly, the isr promoter has sites for stat and as well as irf and , suggesting that isr could be a bona fide isg. besides the non-coding transcript that we name isr , six other transcripts could be expressed from the isr area according to ucsc and ensembl databases ( figure s a in supplementary material). these transcripts have certain coding capacities and could be translated to proteins of up to amino acids named c orf . the syntenic region in the mouse also comprises non-coding transcripts together with transcripts with certain coding potential that lead to peptides no longer than amino acids (data not shown). only five amino acids in the n-terminal part of the putative protein predicted in mouse are conserved in human. given the poor conservation and the short size of the predicted peptides all the transcripts from this mouse region could be classified as non-coding ( , ) . to analyze the expression of isr and all the putative coding transcripts annotated in the human isr area, we used qrt-pcr. the results show that most of the transcripts are poorly detected in huh or hela cells treated or not with ifn. the highest expression is observed for the isr lncrna ( figure s b in supplementary material). finally, ucsc database also shows that isr is convergent to irf and antisense to a longer irf transcript with poor coding capacity that we named lncirf (figure b , figure s a in supplementary material). if expressed, lncirf could regulate the level of isr via antisense mechanisms. therefore, we have evaluated the expression of lncirf in response to ifn. the results show that lncirf is expressed and its levels are induced at short times after ifn treatment ( figure s c in supplementary material). unlike isr , isr , or isr transcripts did not overlap with annotated transcripts from gbp or il ( figure s in supplementary material). we evaluated the coding capacity of isr , , and bioinformatically. orf finder (ncbi) was used to determine all possible open reading frames in these isrs ( figure s a in supplementary material). the analysis shows that all putative orfs are shorter than aa. then, we evaluated their coding potential with the cpat ( , ) ( figure s b in supplementary material). cpat uses a model built with open reading frame size and coverage together with codon (ficket score) and hexamer (hexamer score) usage bias. according to this program, isr , , and are non-coding as they have a coding probability much lower than . , used as a threshold with the highest sensitivity and specificity to differentiate between coding and non-coding transcripts in humans ( ) . isr , , and were also described as non-coding in lncipedia ( ) . this lncrna database shows that isr , , or are not found in the pride archive, a database for proteomic data, or in lists of transcripts associated to ribosomes in ribosome profiling experiments ( ) ( ) ( ) . isr and isr were also described as non-coding by the analysis of phylocsf, which uses multiple alignments to calculate www.frontiersin.org the phylogenetic conservation score and determines whether a multi-species nucleotide sequence alignment is likely to represent a protein-coding region ( ) . finally, we evaluated the subcellular localization of isr , , and in huh cells mock-treated or treated with , units/ml of ifnα. rna was isolated from nuclear or cytoplasmic fractions and quantified by qrt-pcr. the results show that the coding gapdh or isg mrnas accumulate preferentially in the cytoplasm while the nuclear lncrna malat is preferentially nuclear (figure ) . similarly, isr , , and accumulate preferentially in the nucleus. this result, together with the bioinformatic analyses, strongly suggests that isr , , and are non-coding rnas. as indicated above, each isr and its neighboring coding gene have similar induction patterns in response to ifn (compare figure and figure or figures a,c) . this suggests that they could be co-regulated and therefore, that they could share similar functions. to analyze in more detail whether the expression level of each isr correlates significantly with the expression level of its neighboring coding gene, we performed correlation studies. we compared the levels of each coding/non-coding pair in all the samples evaluated in figures , , and . the results show a highly significant positive correlation between isr and gbp or isr and irf . in contrast, isr had a non-significant correlation with il ( figure a) . expression of neither isr nor isr significantly huh cells were mock-treated or treated with , units/ml of ifnα and divided into nuclear and cytoplasmic fractions. rna was isolated from each fraction and used to evaluate the expression levels of isr , , and by qrt-pcr. malat , gapdh, and isg mrna was also quantified and used as a reference to calculate the relative levels of each transcript and as a control to evaluate the subcellular fractionation. the ratio of cytoplasmic to nuclear levels is shown. the experiment was performed three times and each value shows the average of three replicas from a representative experiment. error bars indicate standard deviations. correlated with the expression of other isgs such as oas or bst (data not shown). arguably, this correlation analysis has been done with few isgs and using homogeneous samples. therefore, we decided to perform a more stringent high-throughput analysis of correlation. accordingly, we carried out a guilt-by-association genome-wide frontiers in immunology | molecular innate immunity analysis ( ) , which also predicts the function of unknown genes with high statistical confidence ( figure b) . we compared the expression levels of isr , , and and coding genes related to cellular antiviral pathways, used as positive controls, with the expression levels of all the genes represented in a sureprint g microarray. we used data obtained from microarray experiments performed with human samples of different origin. the results show a significant positive correlation between isr and irf (corr = . and p < e− . ), indicating that these genes are coregulated. significant correlations were not observed for gbp and isr or il and isr . furthermore, the correlation analysis of each candidate organized all the microarray genes from the ones with the highest positive correlation to the ones with the highest negative correlation. this matrix was used to search for go categories with highly significant enrichment in genes that correlate positively (positive z-score) o negatively (negative z-score) with isr , , or . this analysis revealed that isr , , and clustered very closely but away from other genes related with the ifn pathway and the antiviral response. isr , , and showed a negative correlation with genes that significantly enriched go categories related to viral processes including viral life cycle and viral transcription. isr and also shared a negative correlation with response to viral infection and ifn pathway genes. however, isr , similar to irf and tlr , showed a positive correlation with ifn signaling and immune response genes. the results obtained in the guilt-by-association analysis led us to hypothesize that isr , , and could respond strongly to viral infections or to the ifn response induced by viral infections. to study this hypothesis, we evaluated the expression of these lncr-nas in cells infected with ad , a dna virus, or rna viruses such as influenza virus, sfv, or hcv. all of them have developed mechanisms to block the cellular antiviral response. influenza virus control of ifn is exerted primarily by the influenza ns protein ( ) . therefore, we also infected cells with an influenza virus mutant that lacks ns . all the viruses used, with the exception of hcv, lead to a fast lytic infection that initiates cell death at h post-infection in the case of influenza virus and sfv, or at h post-infection in the case of ad . therefore, several time points post-infection were evaluated in each case. the results show that isr expression was not altered by infection (data not shown). isr and isr www.frontiersin.org expression was only induced in cells infected with the influenza virus unable to control ifn at later times post-infection, when the ifn response is strongest (figure ) . in general, the induction pattern was similar for gbp and irf . we were surprised to see that the strongest increase in isr and was observed in cells infected with hcv, an ifn-sensitive virus that employs several viral proteins to block the ifn pathway. increased expression was also observed for gbp and irf but not for other isgs such as oas (figure and data not shown) . to determine whether a similar upregulation could be observed in hcv patients, levels of isr , , and were evaluated in livers from hcv-negative (n = ) to hcv-positive (n = ) patients. the results show that both isr and are significantly upregulated in hcv patients (figure a) . no differences were observed in the levels of isr in the same samples. finally, we wanted to determine whether these lncrnas also respond to other chronic viral infections relevant for human health. therefore, we evaluated the expression of isr , , and in blood cells isolated from healthy patients or from patients chronically infected with hiv. we could not detect expression of isr or in these samples. however, both isr and gbp were significantly upregulated in hiv-infected patient cells ( figure b ). in this work, we show that ifn treatment alters the expression of several lncrnas in human cells. these lncrnas were identified in a high-throughput analysis using conditions that detect increased levels of coding genes that respond to ifn both at early or late times (figures and and figure s in supplementary material). in the cells treated with ifn for days, we found, with a very high statistical significance (b > ), an upregulation of well-characterized isgs such as mx , stat , irf , isg , bst and several members of the gbp, oas, and ifi families (figure ) . besides, the ifninduced stat pathway shows the highest enrichment by ingenuity analysis ( figure s in supplementary material). this suggests that high levels of ifn could maintain an active jak/stat pathway in huh cells even at late times post-ifn treatment. therefore, we feel that among the lncrnas identified in this work, there may be some candidates whose expression is controlled directly by the jak/stat pathway, while other candidates could be controlled by other isgs or by later downstream effectors of the ifn response. comparison of the results obtained in the array between coding and lncrna genes gave an unexpected result: % of the altered coding genes but only % of the lncrnas were upregulated by ifn. this suggests that there could be an ifn-mediated repression of genes that affects more strongly lncrnas. however, even if idrs showed a generally decreased expression in the presence of ifn (figure b) , the downregulation was mild. validation of downregulated genes showed that only idr was strongly affected by ifn at late times post-treatment (table s in supplementary material). further experiments will be required to determine whether there is a relevant downregulation of lncrna genes in response to ifn. notably, the majority of the idrs match with genomic regions that have not been associated with active transcription in public databases. this is surprising, as some of them such as idr , , , or are expressed at high levels in huh cells according to huh cells were mock-treated or infected with wild-type influenza virus (pr ) or a mutant that lacks ns (∆ns ), sfv, ad , or hcv for the indicated times. rna was isolated and the expression levels of isr , gbp , isr , and irf were evaluated by qrt-pcr. gapdh expression was also evaluated and used as a reference to calculate the relative levels of each transcript. the experiment was performed three times. the fold-change of infected versus non-infected cells is indicated. each value shows the average of three replicas from a representative experiment. error bars indicate standard deviations. the fold-change of treated versus non-treated cells is indicated at the top of each bar when higher than . . the qrt-pcr data (table s in supplementary material). therefore, it would be interesting to analyze ifn regulation of lncrnas using rnaseq, as this may yield a more comprehensive picture frontiers in immunology | molecular innate immunity of the lncrna transcriptome and its manipulation by ifn. in fact, during the revision of this paper, another manuscript was accepted showing that transcriptome analysis by rnaseq allowed the identification of several lncrnas whose expression is altered in response to ifn ( ) . similar results have been obtained using rnaseq in our lab (barriocanal et al., submitted). several isrs were clearly validated by qrt-pcr. specifically, isr , , , , and were upregulated more than fivefold, and isr and isr even more than -fold. isr was expressed at very low levels. in contrast, isr , , and were well expressed in all cell lines tested and their expression was strongly induced by ifn at early (isr and ) or late times (isr ) (figure a) . furthermore, low doses of type i ifnα or ifnβ also induced the expression of these isrs (figure and data not shown). we did not detect significant induction of these isrs, gbp , irf , or il with type iii ifnλ using doses able to induce other isgs such as oas, isg , or bst (data not shown). further experiments are required to determine whether these genes could show some specificity for type i ifn, as the repertoire of genes that are induced by type iii ifns is essentially the same as those induced by type i ifns ( ) . finally, isr was also induced after the activation of the nfκβ pathway by tnfα, lps, or polyi:c (data not shown). this result is in line with the identification of nfκβ binding sites in isr promoter by chip seq. our molecular and bioinformatic analyses strongly suggest that isr , , and are indeed long non-coding rnas. the reasons are: (i) they accumulate preferentially in the nucleus of ifn-treated or untreated cells ( figure ) ; (ii) they are marked as lncrnas with high sensitivity and specificity after analysis of their orf size and coverage, analysis of their codon and hexamer usage bias, or analysis of phylocsf; (iii) they have not been identified as associated with ribosomes in ribosome profiling experiments; and (iv) if they are translated to small peptides, such peptides have not been identified by proteomic analyses ( figure s in supplementary material). given that some lncrnas regulate the expression of neighboring genes, we searched for the closest coding gene for each isr or idr. interestingly, isr , , and have neighboring genes related to the ifn response (table s in supplementary material, figure b , figure s and s in supplementary material). isr is in tandem and downstream of gbp . it is very unlikely that isr results from run-off transcription from gbp , as gbp expression could not be detected in huh cells (data not shown). isr is in tandem and upstream of il while isr is convergent with irf . none of the transcripts annotated for isr or isr overlaps with the transcripts annotated for gbp or il , respectively ( figure s in supplementary material). however, the region of isr and irf is more complex. while isr does not overlap with its coding neighbor irf , the irf gene also transcribes a longer transcript of poor coding capacity called lncirf ( figure b , figure s a in supplementary material). lncirf expression is induced by ifnα to similar levels than isr ( figure s c in supplementary material). as isr is antisense to lncirf , they could potentially regulate each other by transcriptional interference or by antisense mechanisms, although only nt of the mature form of isr are antisense to the mature form of lncirf ( figure b and figure s a in supplementary material). none of the coding/non-coding pairs share the promoter, making it unlikely that they are co-regulated at the transcriptional level, which would otherwise be a way to explain that both are induced in response to ifn. instead, the promoters of these isrs seem to be independent. isr is located within the gbp locus, which could be indicative of a general co-regulation in response to ifn. the isr and isr promoters have nfκb binding sites, although only isr is reproducibly induced in response to tnfα under the conditions tested. furthermore, isr seems a bona fide isg as the promoter has sites for stat and as well as irf and . in spite of this, the possibility exists that ifn activation of a coding isg could result in an unintended recruitment of transcription factors to the promoter of lncrnas located nearby. we do not think that this is a general phenomenon, as in the microarray or rnaseq analysis, we do not observe that many lncrnas located close to isgs are induced after ifn treatment. besides, if such unintended transcription occurs, we would expect that the expression of isr , , and should always correlate with the expression of their neighboring coding genes. this, however, is not the case. upon further analysis of the results in figures , , and , we observed a highly significant positive correlation between the expression levels of isr and gbp , or of isr and irf ( figure a) . these correlations may reflect the fact that isr and isr are genes induced by ifn at early time points. in fact, the expression of isr and isr also correlated significantly with the expression of irf and gbp , respectively (data not shown). therefore, to analyze correlation in a more stringent manner, we compared the expression of isr , , and with the expression of all the genes represented in the sureprint g microarray using data from human samples. in this case, we only observed www.frontiersin.org a significant correlation for isr and irf (corr = . and p < e− . ), suggesting that these two genes are co-expressed. in fact, the isr promoter has a conserved irf binding site. guilt-by-association genome-wide analysis predicts that isr , , and could function in the ifn pathway and the antiviral response ( figure b) . furthermore, they could be involved in viral processes including viral life cycle and viral transcription. in fact, isr and are upregulated at later times post-infection with an influenza virus mutant that lacks ns , unable to block the ifn response (figure ) . this suggests that isr and are increased in response to the physiological amounts of ifn secreted by the cells as a consequence of infection. we did not observe significant responses of isr , , and to other lytic viruses able to block the ifn response. however, both isr and were significantly upregulated in cells infected with hcv compared to controls. this was observed in hcv-infected cells in culture but also in the livers of hcv-infected patients ( figure a) . intriguingly, increased levels of isr and gbp were also detected in patients chronically infected with hiv ( figure b) . we did not observe significant correlations between the levels of isr and isr and clinical symptoms, although patients with higher hiv load tend to have higher levels of isr and gbp (data not shown). hcv is a chronic virus that employs several viral proteins to block the ifn pathway ( ) . however, the isg expression profile of some patients with chronic hcv infections indicates that ifn is being produced by the infected cells as well as by neighboring cells ( ) . this may in turn explain upregulation of isr and isr . upregulation was also observed for the neighboring genes gbp and irf but not for other isgs such as oas (figure and data not shown). this raises the question why the infection persist in spite of increased levels of antiviral factors? different viruses are targeted by unique sets of isgs ( ) . in the case of hcv both gbp and irf have been shown to have antiviral potential ( ) ( ) ( ) . irf overexpression on its own can activate a similar set of genes as ifn and can lead to a control of the replication of hcv and other viruses ( ) . therefore, for infection to persist, the effects of irf and probably other isgs should be inhibited in infected cells ( ) . one intriguing hypothesis for future work is that this inhibition is in fact exerted by isg lncrna neighbors. further experiments will be required to determine whether these isrs have a proviral or an antiviral role by affecting the function of isgs. the only preliminary evidence of an anti-ifn role is the highly significant anti-correlation between isr /isr and key factors of the ifn pathway found by genome-wide guiltby-association studies. similarly, inhibition of a lncrna located close to viperin, an isg that also inhibits hcv replication, has been shown to increase the levels of many ifn-inducible genes ( , ) . therefore, several lncrnas could act as negative regulators of the ifn pathway. the guilt-by-association study also shows a positive correlation of isr /irf and the ifn response, suggesting that isr could have a positive role in the ifn pathway. one possible approach to further dissect the function of these isrs in the future will be their inhibition via rnai. however, resistance to rnai is a common feature of many lncrnas that locate in the nucleus away from the rnai machinery or contain poorly accessible structured sequences. an alternative could be the use of gene editing technologies, provided that the function of the targeted lncrnas is not essential for the cell, which would prevent their complete deletion. moreover, it may also be feasible to overexpress selected lncrnas from plasmids or viral vectors, in order to study gain-offunction phenotypes. in the long run, we are optimistic that these and other approaches will help to further delineate the potential role of lncrnas in the ifn pathway and in the antiviral response. elena carnero and marina barriocanal designed and performed the experiments, analyzed and interpreted the data, and contributed to the writing of the manuscript; celia prior performed some of the experiments; victor segura and elizabeth guruceaga were in charge of all the bioinformatics analyses; kathleen börner and dirk grimm prepared and provided the rna samples from hiv-infected patients; and puri fortes conceived the project and the required experiments, provided the budget, interpreted the data, and wrote the manuscript. we thank nerea razquin for excellent technical assistance, estanis nistal, ruben hernandez, cristian smerdou, and rafael aldabe for influenza, adenovirus, sfv, and hcv, respectively. we also thank pablo gastaminza for the huh cells sensitive to hcv infection used in all experiments and esther larrea for ifnα and primers for isgs. we would like to thank patients for the generous donation of samples and virginia villar and the biobank of the university of navarra for their mediation. we extend our thanks to the nurses monika arnold and linda strauch, the hiv-infected blood donors, as well as hans-georg kräusslich, martin hartmann, and paul schnitzler (all heidelberg university hospital). this work was supported by grants from ministerio de ciencia e innovacion bio / , and saf - , feder funding, funds from the "ute project cima" and by the project rnareg (csd - ), funded by the ministry of science and innovation under the program consolider ingenio . an integrated encyclopedia of dna elements in the human genome non-coding rnas: the architects of eukaryotic complexity the rise of regulatory rna human cancer long non-coding rna transcriptomes specific expression of long noncoding rnas in the mouse brain chromatin signature reveals over a thousand highly conserved large non-coding rnas in mammals landscape of transcription in human cells control of alternative splicing through sirna-mediated transcriptional gene silencing 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replication by interferon regulatory factor the antiviral protein viperin inhibits hepatitis c virus replication via interaction with nonstructural protein a conflict of interest statement: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest the supplementary material for this article can be found online at http://www.frontiersin.org/journal/ . /fimmu. . / abstract key: cord- -adlp rjy authors: de rivero vaccari, juan carlos; dietrich, w. dalton; keane, robert w.; de rivero vaccari, juan pablo title: the inflammasome in times of covid- date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: adlp rjy coronaviruses (covs) are members of the genus betacoronavirus and the coronaviridiae family responsible for infections such as severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), and more recently, coronavirus disease- (covid- ). cov infections present mainly as respiratory infections that lead to acute respiratory distress syndrome (ards). however, covs, such as covid- , also present as a hyperactivation of the inflammatory response that results in increased production of inflammatory cytokines such as interleukin (il)- β and its downstream molecule il- . the inflammasome is a multiprotein complex involved in the activation of caspase- that leads to the activation of il- β in a variety of diseases and infections such as cov infection and in different tissues such as lungs, brain, intestines and kidneys, all of which have been shown to be affected in covid- patients. here we review the literature regarding the mechanism of inflammasome activation by cov infection, the role of the inflammasome in ards, ventilator-induced lung injury (vili), and disseminated intravascular coagulation (dic) as well as the potential mechanism by which the inflammasome may contribute to the damaging effects of inflammation in the cardiac, renal, digestive, and nervous systems in covid- patients. symptoms and systemic complications associated with these infections in order to develop better therapies against covid- . here we review the literature on the role of the inflammasome in cov infections, which includes how covs activate inflammasomes upon infection, the role of the inflammasome in acute respiratory distress syndrome (ards), how ventilator-induced lung injury (vili) activates the inflammasome, how the inflammasome plays a role in the systemic complications associated with covid- , and how the inflammasome is involved in the process of disseminated intravascular coagulation (dic). the inflammasome is a multiprotein complex of the innate immune response initially described as a regulator of caspase- activation and processing of the pro-inflammatory cytokines interleukin (il)- β and il- ( ) . these multiprotein complexes are comprised of three basic components: ( ) a sensor such as a nod-like receptor (nlr) or an aim- like receptor (alr) ( ) the adaptor protein apoptosis-associated speck-like protein containing a caspase-recruitment domain (asc) and ( ) the inflammatory cysteine aspartase caspase- . inflammasomes are named after their sensor proteins which include nlrp , nlrp , nlrp , nlrc , and aim , with nlrp being the most extensively studied inflammasome to date ( ) . in addition to its role in cytokine production, the inflammasome is also involved in the cleavage of gasdermin-d (gsdm-d) in the cell death process of pyroptosis ( ) . gsdm-d is cleaved at the linker region between the amino (n) and carboxy (c) terminus by either caspase- and/or caspase- or - in humans (caspase- in rodents), resulting in freeing of the n-terminus from autoinhibiting the c-terminus, which allows formation of a pore by the n-terminus (gsdm-d-n) in the cell membrane (figure ) ( ) . inflammasomes were initially described for their role in mounting an innate immune response against bacterial ( ) , viral ( ) , and fungal ( ) infections as well as in autoimmune diseases ( ) . however, lately, most attention has been paid to the role of inflammasomes in diseases such as rheumatoid arthritis ( ) , gout ( ) , diabetes ( ) , heart disease ( ) , renal diseases ( ) , hepatic diseases ( ) , psoriasis ( ) , vitiligo ( ) , multiple sclerosis ( , ) , alzheimer's disease ( ), parkinson's disease ( ), as well as central nervous system (cns) injury ( - ), among others. each inflammasome is activated by different ligands which can either be endogenous or exogenous. endogenous ligands are referred to as damage/danger-associated molecular patterns (damps), and exogenous ligands are referred to as pathogenassociated molecular patterns (pamps). examples of damps include adenosine tri-phosphate or mitochondrial dna ( ). however, in the context of covid- , sars-cov- represents a pamp capable of activating the inflammasome. during viral infections, inflammasomes play a role in the response to influenza virus ( ), encephalomyocarditis virus ( ) the process of inflammasome activation involves a twostep process (figure ). the first step is referred to as signal and represents the priming step of inflammasome activation in which a pamp or damp binds to a pattern recognition receptor (prr) such as toll-like receptor (tlr)- to stimulate the synthesis of pro-il- β and pro-il- in a nuclear factor (nf)-κb-dependent manner ( ). once these pro-inflammatory cytokines are formed, then a second signal is needed to induce inflammasome formation and subsequent processing of pro-il- β and pro-il- into its active forms which are then secreted by different mechanisms. one of these mechanisms includes the gsdm-d pore, which is formed by the n-terminus of gsdm-d that is inserted in the membrane (gsdmd-n) following gsdm-d cleavage ( ). inflammasome formation involves a process in which the sensor molecule such as nlrp oligomerizes and then the adaptor protein asc is recruited into the complex, followed by incorporation of caspase- , which is then autoproteolytically cleaved into its active form. this cleaved or active form of caspase- then exerts its catalytic activity on the pro-inflammatory cytokines that after their release perpetuate the inflammatory response ( ). although there is no unifying consensus regarding the mechanism of inflammasome activation, various processes have been proposed to contribute to the second signal of inflammasome activation such as high extracellular k + concentration, k + efflux, mitochondrial dysfunction, formation of reactive oxygen species (ros), oxidized mitochondrial dna, lysosomal degradation, and ca + imbalance (figure ) ( , ). viroporins are hydrophobic proteins that facilitate release of viral proteins from infected cells by modifying the cell membrane, figure | mechanisms of inflammasome activation. inflammasome activation in general relies on two signals for its activation. first, a pamp binds to a prr resulting in synthesis of nlrp and pro-il- β. then a second signal leads to inflammasome assembly, leading to the activation of caspase- , processing of pro-il- β into il- β and pyroptosis. the second signal may come from a variety of pathways including k + efflux. lysosomal rupture or mitochondrial dysfunction. mitochondrial dysfunction results in release of ros, ca + and mitochondrial dna (mtdna), all of which have been shown to activate the inflammasome. pyroptosis occurs as a result of caspase- -mediated cleavage of gsdm-d at the linker region of gsdm-d. following gsdm-d cleavage, the amino terminus of gsdm-d (gsdm-d-n) forms a non-selective pore at the cell membrane through which il- β is then released. and they are involved in the activation of inflammasomes. viroporins are associated in viral pathogenesis, and their low ionic selectivity makes them ideal candidates for ionic exchange, which is critical for viral infection and inflammasome activation (figure ) . when viroporins are blocked or deleted, the severity of infections tends to decrease ( ), making viroporins attractive for the development of therapies to prevent exacerbation of the inflammatory response associated with viral infections ( table ) . the e glycoprotein of cov forms a membrane pore that allows the passage of ions ( ). mice infected with e protein viruses developed pulmonary edema ( ), which is characteristic of ards, and a main cause of death associated with cov infections ( , ). in addition to edema following pore activity mediated by the e protein, high levels of the inflammasome-mediated proinflammatory cytokine il- β have been detected in the lung parenchyma ( ). this finding suggests the involvement of the inflammasome in the mechanism of cov infection. lack of ion exchange through the e protein following sars-cov infection results in lower levels of il- β and lower immunerelated pathology. the events associated with over-activation of the immune response tend to be more damaging to the host than the response associated with cell death in the host induced by the virus ( ). thus, a modulation of the immune inflammatory response is as critical, if not more, than preventing cell death induced by viruses. this observation makes the inflammasome, the master regulator of il- β, a key target for cov infections. in addition to the role of viroporin e in ca +mediated inflammasome activation, the accessory protein a, which potentially acts as a k + channel, has also been shown to activate the nlrp inflammasome (figure ) ( ). like viroporin e, sars-cov open reading frame a (orf a) acts as an ion channel (na + , k + , ca + ) ( ). however, in regards to inflammasome activation, it seems that orf a promotes nlrp inflammasome activation by modulating the ubiquitination of the inflammasome adaptor protein asc and the production of pro-il- β by activation of nf-κb, which is independent of the ion-channel role that orf a plays. furthermore, release of active il- β following activation of the nlrp inflammasome is dependent on tumor necrosis factor (tnf) receptor-associated factor (traf ) ( ). however, a similar effect was not detected for the aim inflammasome. the effects of orf a on the priming (signal ) and the processing (signal ) of pro-il- β on inflammasome activation suggests a mechanism by which one protein is responsible for both signaling events needed for inflammasome activation, unlike other infectious mechanisms that rely on lipopolysaccharide (lps) for the priming step (step ) and adenosine tri-phosphate (atp) for the activation step (step ) (figure ) . interestingly, orf a is also able to process pro-il- β in an asc-dependent but nlrp -independent manner ( ). shi et al. showed that orf b triggers nlrp inflammasome activation and il- β release by binding to the leucine rich repeat (lrr) domain of nlrp , resulting in macrophage pyroptosis ( ). in this study, the authors demonstrated that orf b formed insoluble protein aggregates. moreover, orf b aggregates seemed to interact with nlrp and asc forming a single speck (figure ) . considering that asc specks have prionoid properties that interact with other pathogenic protein aggregates such a amyloid-β, resulting in a more exacerbated inflammatory response, a deeper understanding regarding the role of orf b aggregates following cov infections would be beneficial to understanding the innate immune response mounted by these infections ( ). in the lungs when the s protein of sars-cov- binds to the ace receptor, the virus is internalized by endocytosis, leading to translation and rna replication of genomic and sub-genomic rna including orf a, orf b, and the viral structural proteins (n, s, m, and e proteins). the e protein is involved in ca + release from the golgi apparatus. this ca + has the potential to activate the inflammasome. in addition, orf a interacts with traf to ubiquitinate asc, and orf b interacts with nlrp , resulting in inflammasome activation and pyroptosis. il- β is released through the gsdm-d-n pore during pyroptosis, while na + and h o molecules enter the cell, resulting in cell swelling, which then manifests as pulmonary edema. furthermore, orf a also acts in the cell membrane as a k + channel, which causes an ionic imbalance also capable of promoting inflammasome activation, and mitochondrial dysfunction produces ros that also contribute to inflammasome activation. consistent with previous studies regarding the mechanisms of inflammasome activation, it has been shown that ca + imbalance is a common denominator in a variety of viral infections that result in inflammasome activation such as influenza ( ) , and encephalomyocarditis virus ( ) . ionic imbalance has been associated with inflammasome activation in the lung following infections ( ) , and consistent with this finding is a recent study by nieto-torres et al. showing that e protein from sars-cov makes a ca + permeable channel in the endoplasmic reticulum (er)/golgi intermediate compartment (ergic)/golgi membrane that results in nlrp inflammasome activation and increased levels of il- β (figure ) ( ). the macrodomain of sars unique domain (sud) known as sud-mc is involved in the activation of the chemokine cxcl and il- β in lung epithelial cells as determined by the levels of il- β in bronchioalveolar lavage fluid (balf) ( ) . this process is mediated by the nlrp inflammasome in a c-jun-dependent pathway. however, a similar effect was not detected in nlrp knockout mice, indicating that nlrp is the main inflammasome responsible for this sud-mc-mediated effect. in contrast, the inflammasome also plays a protective role following murine cov infection ( ) . the mouse hepatitis virus (mhv) strain-a (mhv-a ) is a positive-strand rna virus, like sars-cov- , that is used to study cov infections with effects in the cns, liver, spleen, and lungs. using this model, zalinger et al. illustrated that inflammasome activation contributes to control of viral replication through il- . il- knockout mice presented poor survival and increased viral replication ( ) . furthermore, il- was involved in production of interferon (ifn)-γ in activated t-cells. the authors showed that caspase- and caspase- knockout mice were more susceptible to mhv infection. nonetheless, survival increased in il- knockout mice when compared to wildtype, even when the viral load was higher in the il- knockouts ( ) . therefore, as a result of the different effects of each inflammasome signaling protein on viral load and survival, care must be taken when considering therapies that aim to block inflammasome activation for in certain infections, the lack of inflammasome activation may result in death, which is probably due to the negative consequences associated with a suppressed immune response ( ) . accordingly, asc and caspase- have been shown to be necessary for protective adaptive immunity against influenza. however, in that study, a similar role was not found for nlrp ( ), yet other reports have shown an important involvement of nrlp following influenza infection ( , ) , which adds further complexity to the understanding of inflammasome signaling following viral infections. mhv strain- (mhv- ) causes a viral fulminant hepatitis that results in production of fibrinogen-like protein- (fgl ), a monocyte/macrophage-specific procoagulant ( ) . this procoagulant effect may contribute to pathomechanisms that trigger dic. guo et al. showed that mhv- infection increased the levels of il- β in the serum and liver of mice. they also showed that the levels of fgl from macrophages was diminished in il- r knockout mice, and this finding was consistent with decreased infiltration of cd + gr- high neutrophils. in addition, ros derived from the nadph oxidase complex (nox) resulted in nlrp inflammasome activation; thus, nlrp and caspase- knockouts showed lower levels of il- β ( ) . the inflammatory response following cov infection has an anti-viral and a pro-viral role. in regard to the anti-viral response, inflammation restricts viral replication and infection. however, inflammation plays a pro-viral role when it acts to release virions. current understanding of sars-cov- infection indicates that the virus infects the cell through angiotensinconverting enzyme- (ace ) receptors in host cells by binding to the s glycoprotein. ace is part of the renin-angiotensin system (ras) and is involved in the regulation of blood pressure and fluid homeostasis ( ) . recently, shao et al. demonstrated that ace receptor stimulation results in nlrp inflammasome activation in podocytes; thus, leading to cell death. interestingly, this effect did not affect blood pressure ( ) . however, whether sars-cov- binding through the s glycoprotein to ace results in inflammasome activation has yet to be determined. in addition, binding of angiotensin ii receptor (at ) also results in nlrp inflammasome activation ( ) . interestingly, bats that are known to be infected by several covs such as ebola, mers, sars-co-v, and potentially sars-cov- remain asymptomatic following infection even in the presence of high viral loads in blood and tissues ( ) . it has been suggested that covid- was passed to humans by an intermediate host between bats and humans, similar to previous cov infections that were transmitted to humans through camels (mers) or civets (sars-cov). nevertheless, the intermediate host for covid- remains unknown. recently, ahn et al. showed that following infection with mers-cov, bats are able to fight cov infections with lower levels of nlrp inflammasome activation when compared to humans, without affecting viral load ( ) . therefore, the molecular mechanism employed by bats to limit the damaging effects of cov infections should be explored to develop better interventions for the care of covid- -positive patients. traditionally, acute lung injury (ali) is defined by pulmonary infiltrates and edema present in the chest as determined by radiography procedures in the absence of left atrial hypertension, or a pulmonary wedge pressure lower than mmhg and an arterial oxygen to inspired oxygen fraction (pao /fio ) lower than mmhg ( ) . on the other hand, when the pao /fio is below mmhg the ali is referred to as ards ( ) . hence, according to this definition ards is a more severe form of ali. however, the modern definition of ards, eliminates the use of ali for humans, and limits its use to animal studies. moreover, this modern definition of ards divides ards into mild ( - mmhg), moderate ( - mmhg) and severe (below mmhg) based on the berlin definition ( ) . more recently, ards has been stratified based on different phenotypes such as those that are physiologically derived, which separates patients according to the pao /fio ratio, the pulmonary dead space, the ventilator ratio, and the driving pressure ( , ) . the clinically derived phenotype considers whether the etiology is direct (pulmonary origin) or indirect (extrapulmonary origin) ( , ) . the biologic phenotype relies on biomarkers associated with ards and considers whether there is a hypo or hyperinflammatory response, which can be used as a guide for potential therapies ( , ) . examples of these inflammatory markers that are associated with the cytokine storm are il- , il- , il- , and tnf. in addition, other markers associated with ards are proteins of endothelial injury such as surfactant protein-d or coagulation-associated proteins such as plasminogen activator inhibitor- and protein c. moreover, recently it has been shown that increased levels of il- were consistent with increased mortality in sepsis-induced ards ( ) . therefore, the hyperinflammatory phenotype is characterized by increased inflammation, less ventilator-free days, and increased mortality when compared to the hypoinflammatory phenotype ( , ) . thus, supporting a strong role for the inflammatory response in ards that is capable of determining favorable or unfavorable outcomes depending on whether there is hyperinflammation or hypoinflammation. finally, another phenotype that has been described is the omics derived phenotype which stratifies patients based on genomewide association and microrna transcriptomic analysis ( , ) . of the well-known covs (hcov-oc , hcov-nl , hcov-hku , mers-cov, sars-cov, and sars-cov- ), sars-cov- has garnered especial attention due to the level of infectivity as well as lethality in vulnerable populations. the acute stage of cov infections is characterized by infiltration of immune cells into lung tissue, whereas the post-acute stage is characterized by pulmonary fibrosis ( ) . covid- , like other cov infections, causes ali with high viral titers, high levels of the inflammatory cytokines il- β and il- as well as infiltration of macrophages and neutrophils into the lungs ( ) . high mobility group box protein (hmgb ), which activates the inflammasome in the lungs leading to ards/ali ( ) , is upstream of il- release ( ), and has been suggested to play a key role in the inflammatory response occurring in the lungs of covid- patients ( ) . covid- infections are associated with bacterial and viral pneumonia. pneumonia following cov infection can be either viral that may result in secondary bacterial pneumonia, or due to a combination of viral and bacterial pneumonia, however, the combined type has a lower incidence. following sars-cov infection, secondary bacterial (methicillin-resistant staphylococcus aureus) pneumonia has been described with ventilator-associated pneumonia (vap) ( ) . although the role of the inflammasome in viral pneumonias has not been thoroughly examined, several studies point to inflammasome activation after bacterial infections by different organisms. for instance, nlrp , asc, and caspase- are upregulated following streptococcus pneumoniae (s. pneumoniae) infection, resulting in production of il- β ( ) . the authors showed that nlrp knockout cells were able to produce il- β. however, asc knockouts significantly decreased the levels of il- β ( ), pointing to the possibility that even if the nlrp inflammasome is blocked, other inflammasomes that require asc may compensate for the role of nlrp such as the aim inflammasome or other nlr-dependent inflammasomes, yet nlrp knockout mice are more susceptible to the effects of pneumococcal pneumonia infection than wild types. moreover, levels of infiltrated leukocytes into the lungs was not affected by knockdown of nlrp , but pulmonary edema did increase in the nlrp knockout mice as determined by decreased dynamic lung compliance, which probably explains why nlrp knockouts were more likely to die. however, a better measure of edema would have been the determination of the wet to dry lung ratio or balf total protein. similarly, mice deficient in nlrp are also susceptible to the effects of α-hemolysin-expressing staphylococcus aureus (s. aureus) in murine pneumonia and are able to produce il- β, suggesting that other inflammasomes besides nlrp may be involved in the innate immune response to s. aureus pneumonia ( ) . on the other hand, knocking out the nlrc inflammasome has been shown to be protective following pseudomonas aeruginosa (p. aeruginosa) pneumonia as determined by improved bacterial clearance, decreased mortality and decreased lung damage ( ) . in this study, the authors suggested that the inflammasome may not be needed to fight the infection. however, the inflammasome seemed to play a role in increasing levels of il- β and il- , resulting in decreased bacterial clearance and increased lung toxicity. in contrast, during influenza a infection, the aim inflammasome is activated, leading to lung injury and mortality ( ) . moreover, in this study the authors showed that aim knockout mice presented less ali and increased survival without affecting viral load in the lungs ( ) . mechanical ventilation is used as a treatment for ards in order to expand collapsed alveoli. the high pressure generated by mechanical ventilation leads to vili ( ) . vili has been described in sars ( ) and covid- ( ) . wu et al. showed that this process was mediated in part by the nlrp inflammasome by sensing lung alveolar stretch ( ) , suggesting that stretch-injury in vili activates an innate immune response that was partially mediated by the inflammasome. in addition, activation of the nlrp inflammasome by stretched injury seems to be regulated by an interaction between nek- and nlrp , which can be treated with glibenclamide (glyburide) in mice ( ) . furthermore, a study by dolinay and colleagues showed upregulation of il b in a rodent model of vili ( ) . the mrna transcript levels of caspase- , il- β, and il were higher in patients with sepsis/ards when compared to patients with systemic inflammatory response syndrome and controls ( ) . moreover, deletion of il- and caspase- were shown to be protective following vili, and delivery of a neutralizing antibody against il- resulted in decreased neutrophil counts in balf ( ) . moreover, another complication associated with mechanical ventilation in covid- patients is vap ( , ) . in general, vap takes place in ∼ % of patients who undergo mechanical ventilation for over h ( ) , resulting in ∼ % mortality rate ( ) . escherichia coli (e. coli), klebsiella pneumoniae, p. aeruginosa, acinetobacter baumannii, and s. aureus are the main causative organisms of vap ( ) . mortality in the intensive care unit associated with vap is usually related to multi-drug resistant pathogens, and early diagnosis of vap by proper identification of the causative agent is paramount to increase patient survival ( ) . in a study analyzing balf and serum from patients suspected to have vap and age-matched volunteer controls, it was found that il- β and il- in balf were higher in the vap suspected cases when compared to controls ( ) . it has been suggested that a major contributor to poor outcomes in patients with covid- is an exacerbated immune response ( ) . this heightened immune response is characterized by unusually high levels of inflammatory cytokines such as il- β (the main cytokine activated by the inflammasome together with il- ), il- , monocyte chemoattractant protein- (mcp- ), macrophage inflammatory protein- α (mip a), il- , il- , il- , tnf, granulocyte-macrophage colony-stimulating factor (gm-csf), cc-chemokine ligand (ccl ), ccl , cxc-chemokine ligand (cxcl ), and the soluble form of the α-chain of the il- receptor, among others ( , ) . the exacerbated immune response is referred to as cytokine storm syndrome or cytokine release syndrome (crs). however, despite the increased levels of a variety of cytokines in covid- patients, those protein levels seem to be to times lower in covid- patients than in patients with ards ( ) . thus, crs may not fully explain the poor outcomes experienced by covid- patients and further investigation into the inflammatory response in these patients is granted. the heightened inflammatory response in covid- patients presents with decreased cd- + t cells in blood (lymphopenia) probably due to infiltration of these cells into tissues or due to a response to the steroid treatment given to these patients ( ) . in post-mortem studies, lymphocytic cell death has been detected in the lymph nodes and spleen, which could also explain the lymphopenia. crs may result in hemophagocytic lymphohistiocytosis (hlh) or macrophage activation syndrome (mas), leading to high fever, high levels of ferritin, and hypertriglyceridemia ( ) . symptoms of crs range from mild to high fever, fatigue, headache, rash, arthralgia, myalgia, hypotension, circulatory shock, vascular leakage, dic, and multi-organ dysfunction syndrome (mods) ( ) . patients with crs also present with cytopenia, and elevated c-reactive protein (crp), creatinine levels, liver enzymes, and d-dimer values. additionally, von willebrand factor (vwf), a marker of endothelial activation is also increased in crs and has been described in covid- patients ( ) . this suggests that endothelial cells may be an attractive therapeutic target for the treatment of covid- -related hyperinflammation, especially in cases presenting capillary leakage, hypotension and coagulopathy. il- is induced by il- β, the main cytokine activated by the inflammasome ( ) . therefore, inhibition of the inflammasome could be expected to help treat patients with crs. il- , the other cytokine controlled by inflammasome activation is also elevated in patients with crs ( ) . since there is no fda-approved drug that directly inhibits the inflammasome, to this extent, anti-il- and anti-il- β signaling therapies are being tested in patients with covid- ( , ) . increased il- levels lead to vascular leakage, dic and myocardial dysfunction ( ) . in addition, type i ifn signaling has been reported to be decreased in patients with covid- ( ) . it is possible that decreased type i ifn signaling in the presence of an exacerbated inflammatory response may be related to increased inflammasome signaling ( ) . however, viral infections are capable of generating high levels of type i ifn, and deletion of ifnar , a receptor involved in type i ifn signaling, or downstream type i ifn signaling pathways increases the replication, dissemination and lethality associated with viral infections ( ) , indicating that type i ifn are also involved in viral clearance. in a mouse model of s. suis infection, lin et al. studied the systemic effects of streptococcal toxic-shock-like syndrome (stsls) on cytokine production. stsls is characterized by fever, blood spots (purpura), hypotension, shock and mods, similar to what is seen in patients with crs. in that study, the nlrp inflammasome was activated by s. suis leading to production of il- β, resulting in crs ( ) , further highlighting that the inflammasome is a contributor to the effects of crs following infections. thus, the cytokine storm results in mods, which can severely affect patients by inducing an inflammatory response that spreads to other organs beyond the lungs. as a result, a concern in covid- patients is not only what happens due to the pulmonary infection but also the non-respiratory manifestations associated with the inflammatory response caused by sars-cov- infections. in addition to the respiratory effects associated with covid- , other manifestations affecting a variety of organ-systems have been recognized ( table ) . patients with pre-existent cardiovascular conditions tend to have worse outcomes due to covid- than patients who do not present a cardiovascular comorbidity, including hypertension. due to the manifestation of a cardiovascular involvement in more severe cases, it is likely that the effects on the heart are due to sequelae associated with the crs. thus, a reduction and control of the cytokine storm may alleviate the deleterious effects in these patients. covid- exacerbates underlying cardiovascular conditions such as ischemic heart disease and chronic heart failure ( ) . in addition, it may cause myocardial injury, myocarditis, arrhythmia, acute coronary syndrome, cardiogenic shock, stroke, venous thromboembolism, and pulmonary embolism ( ) . non-ischemic events in the heart, such as pressure overload, activate the inflammasome in the heart probably as a result of ros production following stimulation of β-adrenergic receptors, resulting in higher levels of nlrp and asc as well as production of il- from myocardial cells ( ) . similarly the inflammasome was shown to be involved in cardiac arrhythmias ( ) , and higher levels of il- β and il- have been associated with hypertension ( ) . in a mouse model of hypertension, the inflammasome is activated in the kidneys, resulting in production of il- β but not il- ( ); whereas inhibition of the inflammasome resulted in lower blood pressure ( ) . taken together, considering the exacerbated inflammatory response that covid- patients present, the effects of the inflammasome on the cardiovascular system could explain, in part, some of the adverse cardiovascular events seen with covid- ; however, a direct role between inflammasomes and cardiac complications has not been tested in an animal model of cov infections. problems with the gastrointestinal system such as diarrhea, abdominal pain, vomiting and lack of appetite have been described in covid- patients. in some cases, these symptoms occur even in the absence of any respiratory symptoms, and sometimes correspond to a longer time between disease onset and hospitalization when compared to patients who do not present any digestive symptoms ( ) . lack of appetite may be associated with the anosmia that characterizes some of the symptoms experienced by some patients. however, the cause of digestive symptoms in some covid- cases is not well-understood, yet it is possible that the effects are due to an alteration of the intestinal microbiome by the infection, or the result of the effects of the virus binding to the liver, which expresses ace receptors. accordingly, pan et al. suggest that an alteration in the gut-lung axis ( ) may be responsible for the digestive symptoms in some covid- patients, which could also explain how a problem affecting the lungs also affects the gastrointestinal system ( ) . similar to covs, enteroviruses are also positive-sense single stranded rna viruses, and several enteroviruses have been shown to activate the inflammasome by acting on nlrp , caspase- , asc, il- β, and gsdm-d ( ) , indicating that the inflammasome can be activated by infections that affect the gastrointestinal system, and that the machinery responsible for the inflammatory response mediated by the inflammasome is present in the gastrointestinal system. however, whether covs activate the inflammasome directly in the gastrointestinal tract is yet to be determined. it is possible that the ionic imbalances associated with sars-cov- infections also results in inflammasome activation in the gut ( ) , moreover, there is ample evidence on inflammasome regulation of the inflammatory response in intestines in chronic diseases like chron's disease and colitis ( ) , further highlighting the relevance of this innate immune complex in inflammatory events in the gastrointestinal tract. in the liver, covid- increases the levels of alanine aminotransferase (alt) and aspartate aminotransferase (ast), particularly in severe cases ( ) . although no-link between inflammasome and liver problems have been described following cov infections, previous studies have shown that inhibition of the nlrp inflammasome lowers the levels of alt and ast, improving outcomes in liver fibrosis and nonalcoholic steatohepatitis (nash) in mice ( ) , suggesting that inflammasome inhibition may be beneficial to control the effects caused by covs infections in the liver. the pancreas also expresses ace receptors, making the pancreas a target for cov infections. previously, sars-cov was shown to damage pancreatic islet cells leading to diabetes ( ) . furthermore, following covid- , there is an association between poorer outcomes and diabetes. however, poorer outcomes in diabetics seemed to be less frequent in older individuals and those with hypertension ( ) . however, the mechanism for this finding is presently unknown. it is possible that medications used to treat these patients also might serve to treat some of the symptoms associated with covid- , including hyperactivation of the immune response. in addition, nlrp inflammasome proteins are elevated in monocyte-derived macrophages and peripheral blood mononuclear cells from patients with diabetes ( ) . acute pancreatitis, which has been described in covd- patients and is worse in diabetics, also activates the nlrp inflammasome ( ) . thus, a heightened inflammatory response in patients with diabetes could be a risk factor in patients with diabetes and covid- . cerebrovascular complications, convulsions, encephalitis, change in mental status, confusion, headaches and febrile seizures as well as taste (hypogeusia/ageusia) and smell (anosmia/hyposmia) dysfunction have been described in patients with covid- ( , ) . although, the mechanism of taste and smell dysfunction is not known, it is possible that the virus binds to ace in the oral mucosa ( ) , affecting this sensing function. although smell and taste dysfunction are common in several upper respiratory infections, it seems that in covid- , these are even more prevalent ( ) . moreover, covs have been detected in the cerebrospinal fluid (csf) of patients with sars-cov ( ), suggesting the possibility of a similar neurological involvement in patients with covid- . in addition, sars-cov- has been shown to affect human neural progenitor cells and brain organoids ( ), indicating the possibility of direct infection by cov in brain tissue. inflammasomes have been shown to be activated in a variety of diseases and injuries affecting the cns ( , - ). thus, the cns is capable of mounting an immune response through the inflammasome. recently a mechanism was described by which inflammasome proteins are carried in extracellular vesicles (ev) to the lungs from the brain following brain injury, thus inducing ali ( ) . therefore, it is also possible that the lung secretes inflammasome proteins in extracellular vesicles that are carried to the cns following infection, causing neurological symptoms. although ev are capable of carrying viral components ( ) , whether ev are secreted during covid- as part of the neuro-respiratory lung axis is yet to be determined. studies indicate that patients with severe covid- may present conjunctivitis ( ) , and covs have been previously detected in tears ( ) . covs gain direct access to the conjunctival mucosa as the viral particles from an infected patient travel in droplets that reach the eye. however, how cov reach the tear film in the absence of direct access from the virus to the conjunctival mucosa is yet to be fully elucidated. evidence from feline cov suggests that infected macrophages and monocytes extravasate immune tissues and cause endothelial cell dysfunction that leads to vasculitis ( ) . it is the vasculitis that is believed to be an underlying contributor to the ocular manifestations seen following feline cov infections, which include conjunctivitis, retinal vasculitis, pyogranulomatous anterior uveitis and choroiditis with retinal detachment ( ) . in addition, in mouse cov, inflammation in the eye results in optic neuritis and retinal degeneration that affects photoreceptors and ganglion cells ( ) . although conjunctivitis remains the only ocular manifestation widely reported in regards to covid- , it is possible that figure | role of the inflammasome on clot formation. thrombin binds to a gpcr in platelets, resulting in ros-dependent activation of the inflammasome and release of il- β into the cell. il- β stimulates production of il- . il- stimulates tissue factor (tf) to convert prothrombin into thrombin. tf-containing microvesicles are released by pyroptosis following inflammasome activation. thrombin then converts fibrinogen into fibrin, leading to fibrin cross-linking, clot formation and dic. other conditions may arise upon closer examination of covid- patients. moreover, in goblet cells of the conjunctiva s. aureus activates the nlrp inflammasome as well as the purinergic receptors p x and p x which have been shown to be involved in inflammasome signaling ( , , ) . in addition, s. pneumoniae and p. aeruginosa activate the nlrc inflammasome in corneal ulcers ( ) . thus, the ocular surface has the inflammasome machinery necessary to mount an innate immune response against conjunctivitis in the presence of covid- . however, further studies are needed to understand the type of conjunctivitis present in covid- patients. acute kidney injury (aki) is a significant problem in patients with covid- ( ) . patients develop aki during hospital admission and when disease is severe, such as in patients with ards or on mechanical ventilation, as well as in patients with hypertension or diabetes ( ) . it is possible that in covid- , the exacerbated inflammatory response or vascular thrombosis can damage the kidneys. in addition, ace receptors are present in the kidney, making this organ a potential direct target of sars-cov- infection. a potential contributor to the exacerbated inflammatory response is the inflammasome that besides being involved in crs, it is present in the kidneys where it contributes to inflammation in several renal diseases and complications, inhibits tlr and tlr , which have been described in inflammasome activation ( ) ( ) ( ) anakinra il- receptor blocker ( ) tocilizumab therapeutic monoclonal antibody that blocks il- signaling ( ) including aki ( ) . previously, the zika virus has been shown to induce aki through nlrp inflammasome activation ( ); however, whether sars-cov- is responsible for inducing aki through the inflammasome in covid- has yet to be tested. severe cases of covid- may present with coagulation complications that manifest as thrombi, high levels of d-dimers (a sign of fibrin degradation), prolonged prothrombin time, and low platelet count (thrombocytopenia) that may lead to dic. thrombi in covid- patients have been described in the lungs, heart, brain, liver, kidneys and lower limbs ( ) . dic tends to present in cases of sepsis where it blocks microvessels and leads to organ dysfunction. in addition to dic, some patients with covid- may present pulmonary embolism ( ) and deep vein thrombosis ( ) . the nlrp inflammasome has been described as a signaling intermediate between inflammation and thrombosis by modulating clot retraction and platelet spreading ( ) . nlrp knockout mice present abnormal hemostasis and arterial thrombosis, probably as part of a mechanism that involves binding of thrombin to g protein-coupled receptors (gpcr), which stimulates ros production in platelets. ros activates the inflammasome, resulting in il- β signaling that is followed by platelet spreading and clot retraction ( ) . il- stimulates tissue factor (tf) to transform prothrombin into thrombin. thrombin then converts fibrinogen into the fibrin that is characteristic of thrombi. thus, there is a clear association between the inflammasome and clot formation. furthermore, inflammasome activation causes release of microvesicles containing tf by pyroptosis, resulting in systemic coagulation and death ( ) , which provides a mechanism by which dic may contribute to the poor outcomes experienced by covid- patients (figure ) . to date, therapies intended to treat covid- include remdesivir or favipiravir to control translational replication of viral rna, tocilizumab to block the il- receptor, bevacizumab to block vascular endothelial growth factor (vegf), anakinra to block il- receptor activity, lopinavir or ritonavir to target proteolysis, losartan to target ace receptors, corticosteroids such as dexamethasone to target the exacerbated inflammatory response, heparin to treat dic and intravenous immunoglobulins to target crs, or convalescent plasma, among others ( ). thus, great efforts are being undertaken to develop therapeutics against the pulmonary and systemic manifestations of covid- . this review highlights the inflammasome as a target to interfere with different aspects associated with this pandemiccausing virus. however, care must be taken since inflammasome signaling may be necessary to fight the actual viral infection, while at the same time inflammasome activation may be responsible for the hyperactivated inflammatory response that leads to sepsis, dic, aki and death. mechanisms employed by bats to dampen cov infections indicate that inflammation signaling pathways are probably better targets than reduction of viral load in controlling covid- . thus, a better understanding of the role of inflammasomes and inflammatory processes in covs and those regulating viral load are critical for development of therapeutics to treat these diseases, and although to date there are no fda-approved drugs that directly target the inflammasome, in regards to inflammasome signaling and therapeutics that can be considered for covid- treatment, potential therapies that are currently being manufactured for the treatment of inflammasome-related diseases include mcc that interferes with nlrp inflammasome activation by binding to the nacht domain of nrlp , hence preventing its oligomerization ( ) , as well as ic , a monoclonal antibody with intracellular and extracellular action that interferes with asc speck formation ( ) ( table ) . on the other hand, there are some therapies that are already fda-approved and have been shown to interfere with inflammasome signaling activation which are already being considered for the treatment of covid- such as enoxaparin ( , ) , anakinra ( ) , tocilizumab ( ) , and dexamethasone ( , ) . moreover, another drug that is already approved by the fda that can also be used to inhibit the inflammasome is ifn-β ( ) , which is already used to treat multiple sclerosis ( ) , and is currently being tested for its effects on covid- patients ( ) . moreover, tlr and tlr , which have been implicated in inflammasome signaling ( ) have been suggested to play an underlying role in covid- severity ( ) . tlr / are activated by single stranded rna viruses like sars-cov- , and in addition to their role on inflammasome activation ( ) , these prr are better known for their involvement in type i ifn synthesis and a variety of ifn stimulated genes (isg), which when deregulated are capable of contributing to an exacerbated inflammatory immune response ( ) . as a result, m , a tlr / inhibitor, is currently being tested in clinical trials for the treatment of severe symptoms of covid- as a potential treatment for crs ( ) ( table ) . given the number of people that have been affected with covid- worldwide, a better understanding of the systemic effects associated with covid- 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(covid- ) affect the eyes? a review of coronaviruses and ocular implications in humans and animals p x receptors influence inflammasome activation after spinal cord injury acute kidney injury in critically ill patients with covid- role of the nucleotide-binding domain-like receptor protein inflammasome in acute kidney injury coexistent covid- pneumonia and pulmonary embolism: challenges in identifying dual pathology incidence of asymptomatic deep vein thrombosis in patients with covid- pneumonia and elevated d-dimer levels nlrp regulates platelet integrin alphaiibbeta outside-in signaling, hemostasis and arterial thrombosis inflammasome activation triggers blood clotting and host death through pyroptosis emerging therapies for covid- pneumonia mcc directly targets the nlrp atp-hydrolysis motif for inflammasome inhibition after years of regulating immunity, dexamethasone meets covid- dexamethasone alleviate allergic airway inflammation in mice by inhibiting the activation of nlrp inflammasome enoxaparin attenuates acute lung injury and inflammasome activation after traumatic brain injury attention should be paid to venous thromboembolism prophylaxis in the management of covid- the role of interferons in inflammation and inflammasome activation interferon beta- a for covid- : critical importance of the administration route a tolllike receptor , , and antagonist inhibits th and th responses and inflammasome activation in a model of il- -induced psoriasis presence of genetic variants among young men with severe covid- targeting human tlrs to combat covid- : a solution early identification of covid- cytokine storm and treatment with anakinra or tocilizumab effect of genetic polymorphisms on therapeutic response in multiple sclerosis relapsing-remitting patients treated with interferon-beta figures in this manuscript were created using biorender.com. key: cord- - xcbtn q authors: borghi, maria orietta; beltagy, asmaa; garrafa, emirena; curreli, daniele; cecchini, germana; bodio, caterina; grossi, claudia; blengino, simonetta; tincani, angela; franceschini, franco; andreoli, laura; lazzaroni, maria grazia; piantoni, silvia; masneri, stefania; crisafulli, francesca; brugnoni, duilio; muiesan, maria lorenza; salvetti, massimo; parati, gianfranco; torresani, erminio; mahler, michael; heilbron, francesca; pregnolato, francesca; pengo, martino; tedesco, francesco; pozzi, nicola; meroni, pier luigi title: anti-phospholipid antibodies in covid- are different from those detectable in the anti-phospholipid syndrome date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: xcbtn q background: critically ill patients with coronavirus disease (covid- ) have a profound hypercoagulable state and often develop coagulopathy which leads to organ failure and death. because of a prolonged activated partial-thromboplastin time (aptt), a relationship with anti-phospholipid antibodies (apls) has been proposed, but results are controversial. functional assays for apl (i.e., lupus anticoagulant) can be influenced by concomitant anticoagulation and/or high levels of c reactive protein. the presence of anti-cardiolipin (acl), anti-beta -glycoprotein i (anti-β( )gpi), and anti-phosphatidylserine/prothrombin (aps/pt) antibodies was not investigated systematically. epitope specificity of anti-β( )gpi antibodies was not reported. objective: to evaluate the prevalence and the clinical association of apl in a large cohort of covid- patients, and to characterize the epitope specificity of anti-β( )gpi antibodies. methods: elisa and chemiluminescence assays were used to test sera of patients suffering from severe covid- . of them, displayed major thrombotic events. results: anti-β( )gpi igg/iga/igm was the most frequent in . / . / . % of patients, while acl igg/igm was detected in . / . % by elisa. comparable values were found by chemiluminescence. aps/pt igg/igm were detectable in . and . % by elisa. no association between thrombosis and apl was found. reactivity against domain and - of β( )gpi was limited to / ( . %) tested sera for each domain and did not correlate with acl/anti-β( )gpi nor with thrombosis. conclusions: apl show a low prevalence in covid- patients and are not associated with major thrombotic events. apl in covid- patients are mainly directed against β( )gpi but display an epitope specificity different from antibodies in antiphospholipid syndrome. critically ill patients with coronavirus disease (covid- ) have a profound hypercoagulable state and often develop thrombosis in veins, arteries and in the microcirculation ( , ) . a recent analysis showed several coagulation abnormalities in these patients, including prominent elevation of fibrin/fibrinogen degradation products (i.e., d-dimer) and a prolonged activated partial-thromboplastin time (aptt). while high levels of d-dimer are consistent with sustained activation of the clotting and fibrinolytic cascades, the combination of prolonged aptt and both arterial and venous thrombosis was, however, surprising, and it is reminiscent of a clinical scenario known as antiphospholipid syndrome (aps) ( ) . looking at the causes of aptt prolongation, recent studies have shown that lupus anticoagulant (la) can be detected in a significant percentage of covid- samples ( ) ( ) ( ) . since la is often caused by anti-phospholipid antibody (apl), these findings support the idea that apl may play a role in covid- ( ) . however, it is important to point out that la is a very sensitive assay and its outcome can be influenced by several factors, most notably heparin administration ( ) and a profound inflammatory state characterized by high levels of c reactive protein (crp) ( , ) . both of them are present in covid- patients ( ) . another method to detect apl that is in principle insensitive to anticoagulation and other confounding agents relies on the detection and quantification of autoantibodies using solid-phase assays ( ) . using this method, the presence of apl was recently reported in a handful of case reports and small cohorts of patients ( , , , , ) . while encouraging, this data is limited and its interpretation remains controversial, with some investigators proposing an important role of apl in covid- patients ( ) while others suggesting a very poor correlation between apl and thrombotic events ( ) . there is no information on the antigen specificity of covid- apl in comparison with aps antibodies. such information and a larger study, possibly multicenter, may be instrumental to clarify the real clinical value of these autoantibodies. a total of patients were enrolled from two covid- referral centers in lombardia. all patients tested positive to sars-cov- , and classified as severe or critical covid- ( ) . the mean age was . (± sd . ) years; were men and women. no diagnosis of previous autoimmune diseases was made; six patients had a thrombotic event (three arterial and three venous) in the past clinical history. the presence of antinuclear antibodies (anas) was investigated in patients at istituto auxologico italiano by hep -iif and solid phase ctd screening following the guidelines described in agmon-levin et al. ( ) . of the samples, none was positive for ana. eighty-seven patients suffering from aps were also tested for anti-cardiolipin (acl) and anti-b gpi igg/igm ( ) .the study was approved by the ethics committees (istituto auxologico italiano - - -milan and asst spedali civili np -brescia). acl and anti-b gpi igg/iga/igm were detected by chemiluminescence immunoassay (cia; quanta flash, inova, san diego, ca, us) and a home-made elisa as described ( , ) . anti-b gpi domain igg (anti-d ) were detected by cia ( , ) , igg anti-d - by a home-made elisa, as described ( , ) . detailed methods are reported in the supplementary material. anti-phosphatidylserine/prothrombin (aps/pt) igg/igm were detected by a commercial elisa as reported ( ) . blood samples were collected in the first week after hospital admission. data were analyzed using r v . . . descriptive statistics was used to summarize data. associations and differences between categorical or continuous variables were tested by fisher's exact test and non-parametric mann-whitney test, respectively. a p-value < . was considered statistically significant. table reports the median with minimum and maximum values for different coagulation and inflammation parameters in severe or critical covid- patients. in particular, prolonged aptt (> s) was found in . % while pt inr values were above the cut-off in . % of the cases. most of the patients ( / ) were on anticoagulation with low molecular weight heparin ( % on therapeutic and the remaining on prophylactic dosage). despite anticoagulation, we observed sixteen thrombotic events ( . %, in veins and in arteries). these statistics are in agreement with previous reports ( , ( ) ( ) ( ) ( ) ( ) ( ) and document a systemic inflammation and a coagulopathy in our patients. in the aps field, testing for la is not recommended when patients are on heparin, since the presence of heparin, even if neutralized, may lead to false-positive results ( ) . likewise, high levels crp, such as those found in our cohort of patients, have been shown to prolong aptt independently from the presence of apl ( , ). on these bases, the presence of apl was researched using solid-phase assays, and not la. first, we investigated the presence of acl and anti-b gpi, two aps classification criteria ( ) . testing was independently performed in milan and brescia, using harmonized methodologies ( ) . the prevalence of covid- patients positive for acl and anti-b gpi igg/iga/ igm detected by elisa and cia is summarized in table . the elisa raw data are shown in figure . we found igg/igm acl in . / . % of patients, whereas anti-b gpi igg/iga/igm were found in . / . / . % of patients. similar values were obtained for acl antibodies using cia ( table ) , whereas a slightly lower sensitivity was obtained for anti-b gpi antibodies ( ) . the positivity for acl and anti-b gpi antibodies was at medium/ low titer in contrast with the medium/high titers found in the control group of primary aps ( figure ). there is no association between apl positivity and thrombotic events. fifty-eight sera were also tested with d and d - -coated plates in order to characterize their epitope specificity. figure b shows that three out of samples reacted with d , while in figure c , three samples tested positive for d - . none of the sera was positive for both domains and all displayed a weak reactivity with no association with thrombosis. prolonged aptt (> s) was found in . % of the patients. although aps/pt are not included in the aps classification laboratory tests, they can be associated with a prolonged aptt and with the presence of la ( ) . consequently, we looked at the presence of aps/pt antibodies in our cohort and we found fifteen out of sera positive for aps/pt ( . %), mostly of the igm isotype ( out ) and at a low titer (figure ). there was no association between prolonged aptt and the presence of aps/pt antibodies nor with thrombotic events in our covid- cohort. taken together, our data show a low prevalence of classification criteria apl in covid- patients. in this regard, our study confirms recent studies obtained with smaller cohorts of patients ( , , ) . importantly, our data also shows that apl are slightly more reactive towards b gpi-coated plates as compared to clcoated ones and that, regardless of the nature of apl, there is no association between apl positivity and thrombotic events (p = ). a striking difference between the autoantibody profile in covid- patients as compared to the one in aps concerned the titers of apl. medium/low apl titers were consistently found in patients with covid- . by contrast, medium/high titers are usually found in aps patients (figure ) . this difference suggests that apl found in covid- may be different from apl found in aps and led us to further investigate the epitope specificity of antib gpi antibodies. we focused on autoantibodies directed against the n-terminal domain (anti-d ) or the c-terminal domains - (anti-d - ) of the molecule ( ) (figure a) . this is because anti-d antibodies are associated with an increased risk of thrombosis and pregnancy complications in aps ( , , ) . by contrast, anti d - antibodies are associated neither with vascular nor obstetric aps manifestations ( , ) . furthermore, anti d - antibodies are also reported at high levels in the so called asymptomatic apl carriers and are frequently found in non-aps (e.g., patients with leprosy, atopic dermatitis, atherosclerosis, and in children born to mothers with systemic autoimmune diseases) ( ) . we found that three out of samples reacted with d , and three samples tested positive for d - . none of the sera was positive for both domains and all displayed a weak reactivity. although the number of the investigated sera is relatively small, this finding is quite different from the results found in aps in which almost all the sera positive for the whole b gpi molecule also reacted with domain d at high titer ( , ) . furthermore, at variance with aps patients, none of the anti-d positive patients displayed thrombotic events ( ) . approximately, % of covid- patients have prolonged aptt. yet, only a small proportion of covid- patients carry acl and anti-b gpi antibodies. this suggests that other factors must be responsible for the prolonged aptt phenomenon and likely for the la activity. la may be affected by the concomitant heparin treatment and the high crp levels. although more sensitive and specific diagnostic algorithms have been suggested ( ), we followed the isth guidelines available at the beginning of the study ( ) . since aps/pt can be associated with a prolonged aptt and with the presence of la ( ), we tested our cohort for aps/pt antibodies. we found a small percentage ( . %) of positive sera, mostly of the igm isotype ( out ) and at a low titer. again, there was no association between prolonged aptt and the presence of aps/pt antibodies nor with thrombotic events in our covid- cohort. this indicates that aps/pt are not responsible for the prolongation of aptt nor are predictors of adverse clinical outcomes. furthermore, in contrast to what we would have expected in aps ( ), we found no associations between the presence of aps/ pt, acl, and anti-b gpi antibodies. this data is in line with the unusual epitope specificity of anti-b gpi antibodies documented in figure , supporting the hypothesis that apl found in covid- patients are different from apl found in aps patients. whether covid- apl are similar to the ones found in other infectious diseases such as hcv, hbv and hiv ( ) remains to be determined. despite heparin treatment, . % of our patients displayed thrombotic events. although we cannot exclude that treatment could be protective, the prevalence of vascular events was in line with that reported by other studies as recently reviewed ( ) . in conclusion, while the medium/high apl titers with d specificity are associated with vascular events in aps, low antibody titers with reactivity against b gpi epitope(s) different from d or d , can be found in covid- . this may explain the lack of association with thrombotic events in covid- . in addition, our data do not support the hypothesis that apl can be the main cause of prolonged aptt in these patients. although low titer apl are not predictive of vascular events in the aps, it is important to keep in mind that covid- patients suffer from an acute form of systemic inflammation with complement activation ( ) , which may be responsible for endothelial perturbation. in this context, since b gpi can accumulate on the activated endothelium at high density, even low titers of apl may become pathogenic thus potentiating or even triggering thrombus formation, especially when anticoagulation is suspended. a comparable condition in which low titers of apl can cause substantial damage is seen in obstetric aps, where high levels of b gpi can be found in the placenta ( ) . hence, while transitory apl are likely to be clinically irrelevant in covid- patients as in other infections ( ) , detection of apl may be useful for identifying patients potentially at risk of thrombosis after the hospital discharge. accordingly, anticoagulant prophylaxis or therapies affecting cell signaling involved in inflammatory and coagulation responses could be justified before a confirmatory assay ( , ) . the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. the studies involving human participants were reviewed and approved by istituto auxologico italiano - - -milan and asst spedali civili np -brescia. the ethics committee waived the requirement of written informed consent for participation. this article has been released as a pre-print at [medrxiv pulmonary post-mortem findings in a series of covid- cases from northern italy: a two-centre descriptive study abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus 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to detect antiphospholipid antibody syndrome in patients with or without sle subcommittee on lupus anticoagulant/antiphospholipid antibodies. laboratory criteria for antiphospholipid syndrome: communication from the ssc of the isth tetra positive thrombotic antiphospholipid syndrome: major contribution of antiphosphatidyl-serine/prothrombin antibodies to lupus anticoagulant activity infections and the antiphospholipid syndrome coagulation abnormalities and thrombosis in patients infected with sars-cov- and other pandemic viruses complement activation in patients with covid- : a novel therapeutic target eureka algorithm predicts obstetric risk and response to treatment in women with different subsets of anti-phospholipid antibodies metabolic pathways mediate pathogenesis and offer targets for treatment in rheumatic diseases antiphospholipid antibodies in covid- are different from those detectable in the anti-phospholipid syndrome. version medrxiv the authors would like to thank n. carabellese and g. martini (department of laboratory diagnostics; asst spedali civili, brescia, italy) for their valuable collaboration; all the physicians of the covid- units of the irccs istituto auxologico the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material conflict of interest: mm was employed by inova diagnostics, inc.the remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © borghi, beltagy, garrafa, curreli, cecchini, bodio, grossi, blengino, tincani, franceschini, andreoli, lazzaroni, piantoni, masneri, crisafulli, brugnoni, muiesan, salvetti, parati, torresani, mahler, heilbron, pregnolato, pengo, tedesco, pozzi and meroni. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - nnobx g authors: herrmann, marissa; schulte, sophia; wildner, nils h.; wittner, melanie; brehm, thomas theo; ramharter, michael; woost, robin; lohse, ansgar w.; jacobs, thomas; schulze zur wiesch, julian title: analysis of co-inhibitory receptor expression in covid- infection compared to acute plasmodium falciparum malaria: lag- and tim- correlate with t cell activation and course of disease date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: nnobx g coronavirus disease (covid- ) which is caused by the novel sars-cov- virus is a severe flu-like illness which is associated with hyperinflammation and immune dysfunction. the virus induces a strong t and b cell response but little is known about the immune pathology of this viral infection. acute plasmodium falciparum malaria also causes acute clinical illness and is characterized by hyperinflammation due to the strong production of pro-inflammatory cytokines and a massive activation of t cells. in malaria, t cells express a variety of co-inhibitory receptors which might be a consequence of their activation but also might limit their overwhelming function. thus, t cells are implicated in protection as well as in pathology. the outcome of malaria is thought to be a consequence of the balance between co-activation and co-inhibition of t cells. following the hypothesis that t cells in covid- might have a similar, dual function, we comprehensively characterized the differentiation (ccr , cd ro) and activation status (hla-dr, cd , cd , cd ), the co-expression of co-inhibitory molecules (pd , tim- , lag- , btla, tigit), as well as the expression pattern of the transcription factors t-bet and eomes of cd (+) and cd (+) t cells of pbmc of n = sars-cov- patients compared to n = p. falciparum infected patients and n = healthy controls. overall, acute covid- and malaria infection resulted in a comparably elevated activation and altered differentiation status of the cd (+) and cd (+) t cell populations. t effector cells of covid- and malaria patients showed higher frequencies of the inhibitory receptors t-cell immunoglobulin mucin- (tim- ) and lymphocyte-activation gene- (lag- ) which was linked to increased activation levels and an upregulation of the transcription factors t-bet and eomes. covid- patients with a more severe disease course showed higher levels of lag- and tim- than patients with a mild disease course. during recovery, a rapid normalization of these inhibitory receptors could be observed. in summary, comparing the expression of different co-inhibitory molecules in cd (+) and cd (+) t cells in covid- vs. malaria, there is a transient increase of the expression of certain inhibitory receptors like lag- and tim- in covid- in the overall context of acute immune activation. coronavirus disease which is caused by the novel sars-cov- virus is a severe flu-like illness which is associated with hyperinflammation and immune dysfunction. the virus induces a strong t and b cell response but little is known about the immune pathology of this viral infection. acute plasmodium falciparum malaria also causes acute clinical illness and is characterized by hyperinflammation due to the strong production of pro-inflammatory cytokines and a massive activation of t cells. in malaria, t cells express a variety of co-inhibitory receptors which might be a consequence of their activation but also might limit their overwhelming function. thus, t cells are implicated in protection as well as in pathology. the outcome of malaria is thought to be a consequence of the balance between co-activation and co-inhibition of t cells. following the hypothesis that t cells in covid- might have a similar, dual function, we comprehensively characterized the differentiation (ccr , cd ro) and activation status (hla-dr, cd , cd , cd ), the co-expression of co-inhibitory molecules (pd , tim- , lag- , btla, tigit), as well as the expression pattern of the transcription factors t-bet and eomes of cd + and cd + t cells of pbmc of n = sars-cov- patients compared to n = p. falciparum infected patients and n = healthy controls. overall, acute covid- and malaria infection resulted in a comparably elevated activation and altered differentiation status of the cd + and cd + t cell populations. t effector cells of covid- and malaria patients showed higher frequencies of the inhibitory receptors t-cell immunoglobulin mucin- (tim- ) and lymphocyte-activation gene- (lag- ) which was linked to increased activation levels and an upregulation of the transcription factors t-bet and eomes. covid- patients with a more severe disease course showed higher levels of lag- and tim- than patients with a mild disease course. during recovery, a rapid normalization of these inhibitory receptors could be observed. in summary, comparing the expression of different co-inhibitory molecules in cd + and cd + t since december , when the emerging coronavirus sars-cov- was first described in wuhan, china, a rapid increase in cases and deaths worldwide could be recorded. the virus, responsible for coronavirus disease (covid- ) , belongs to the coronavirus family and causes respiratory tract infections. sars-cov- is a . nucleotide large, single-strand rna virus coding for four structural proteins: the spike protein, the membrane protein, the envelope protein, and the nucleocapsid protein ( , ) . previous studies have shown that recovered covid- patients developed anti-spike-protein immunity ( ) . while most patients are asymptomatic or display only mild symptoms such as fatigue, fever, and dry coughs, some individuals develop pneumonia, severe acute respiratory distress syndrome, sepsis, and septic shock with an overall case fatality rate of % ( ) ( ) ( ) . in a subset of patients, covid- is associated with a so-called cytokine storm, lymphopenia, and dysregulation of the immune system, yet the underlying mechanisms and cellular sources of these immunological complications remain to be fully understood ( , ) . first studies have shown that reduced t cell counts correlated with disease severity in covid- patients ( ) . broadly directed, antigen-specific t cell responses could be detected in the effector and central memory subsets of cd + and cd + t cells of covid- patients ( , ) . cd + t cells predominantly produced ifn-γ, while cd + t cells produced th and th cytokines ( , ) . the exact role and function of t cells in covid- patients with regards to immunological complications like cytokine storm, however, needs to be elucidated in further studies ( ) . from other viral infections, we know that cytotoxic t cells play an important role in killing virus-infected cells and thus contribute to the clearance of the infection ( , ) . detailed studies of the phenotype and function of virus-specific t cells are important to understand their potential role in the pathophysiology of the disease and to develop future treatment strategies ( ) ( ) ( ) . in order to induce effector t cells with high proliferative and cytotoxic activity and to prevent excessive host immune responses, a balanced t cell response is shaped by the simultaneous upregulation of co-stimulatory and coinhibitory receptors ( , ) . inhibitory receptors and their role in t cell exhaustion have been extensively studied in cancer as well as chronic infections such as hcv and hiv ( , ( ) ( ) ( ) ( ) ( ) . however, in the early stages of an acute infection, the kinetics and role of co-inhibitory molecules are not well-understood. it is not clear how early t cell exhaustion and loss of effector function set in and whether exhaustion plays a role in the pathophysiology of acute infections ( , , , ) . previous studies of t cells and their role in covid- patients have shown an upregulation of inhibitory receptors like pd . therefore, it has been suggested that t cell exhaustion might play a role in the pathophysiology of covid- infection ( ) . however, an upregulation of inhibitory receptors in acute infections does not necessarily correlate with terminal exhaustion of these cells but can be regarded rather as a characteristic of the overall immune activation to counterbalance excessive immune responses ( ) . a similar, strong up-regulation of a combination of several coinhibitory receptors in the context of massive t cell activation and secretion of pro-inflammatory cytokines has been observed in acute malaria ( ) ( ) ( ) . it has been shown that the expression of these co-inhibitory receptors has a double-edged role with potentially detrimental, but also beneficial effects ( , ( ) ( ) ( ) ( ) : on the one hand, over-expression of inhibitory receptors can hinder the hosts ability to clear the infection. on the other hand, this inhibition of immune cells can also dampen hyperinflammation by down-regulating t cell effector functions ( , ) . these studies highlight the importance of intricate modulation of the t cell response in malaria rather than supporting the concept of development of terminal t cell exhaustion at early stages of infection. we hypothesize that t cells are modulated in a similar way in covid- and thus compared the pattern of coinhibitory receptor expression in malaria vs. covid- . in the case of immunological analysis of patients with covid- , it will be important to assess the level of co-expression and the ranges of the different co-inhibitory receptors to understand their adverse effects and interpret their role in the overall context of acute infection ( , ) . comparing the t cell response in covid- patients not only with samples of healthy individuals but also with samples of patients with other acute infections will help to identify the specific immune signature of covid- infection. in this current study we comprehensively examined the t cell expression profile of inhibitory and stimulatory receptors as well as the expression of the transcription factors t-bet and eomes in a cohort of n = covid- and n = malaria patients in order to obtain a more detailed understanding of the nature of t cells in the context of acute infection and disease severity. both, acute infection with sars-cov- and plasmodium falciparum, can cause excessive host immune activation that can in terms cause severe damage and even limit the hosts ability to clear the disease ( , ) . pbmc (peripheral blood mononuclear cells) of sars-cov- infected patients (n = ) and p. falciparum infected patients data are expressed as absolute numbers n (n/n) or n (range) or mean with standard deviation. (n = ) as well as uninfected healthy individuals (n = ) were collected at the university medical center hamburg eppendorf. blood samples of p. falciparum infected patients and healthy controls were collected prior to the covid- outbreak. the study was approved by the local ethics board of the Ärztekammer hamburg (pv , pv , pv ) and written consent was obtained by all study participants. sars-cov- infection was verified by rt-pcr of nasopharyngeal swabs as previously described ( ) . plasmodium falciparum infection was confirmed microscopically at the bernhard-nocht-institute for tropical medicine. thick and thin blood smears were stained with % giemsa and examined under oil immersion (original magnification × ). clinical and laboratory data were obtained by structured chart review. intracellular as well as surface staining was performed as previously described ( ) . cryopreserved pbmc were thawed and stained with the live/dead tm fixable near-ir dye (thermo fisher, schwerte, germany). cells were then stained with the following surface antibodies flow cytometric data was analyzed with flowjo version (treestar, ashland, or, usa). statistical analysis was performed using the graphpad prism software (graphpad software, san diego, ca). unpaired groups were analyzed using the mann-whitney test while paired groups were analyzed with the wilcoxon test. for bivariate correlation analysis the spearman correlation was applied. data are expressed as mean with standard deviation. p < . were considered to be significant. the clinical cohorts consisted of sars-cov- and p. falciparum infected patients and healthy individuals. all patients were admitted to the university medical center hamburg eppendorf due to the severity of their symptoms. none of the patients needed to be admitted to the intensive care unit at the time of blood collection. during the course of the hospital stay, a total of three covid- patients were transferred to the intensive care unit, one of whom unfortunately later died due to a pulmonary embolism. on average, the covid- patients spent . days and the malaria patients spent . days in the hospital. the mean age of the covid- patients was years compared to years of patients with malaria. of note, one covid- patient had received treatment with rituximab months prior to blood sample collection and another patient had received treatment with a pd- antibody several weeks prior to the infection with sars-cov- . we enrolled pbmc of healthy volunteers into our study, eight female and five male with a mean age of ± . the analysis of lymphocyte subsets in the clinical charts showed a significant generalized decrease of the t lymphocyte count affecting both cd + and cd + t cells with a resulting normal cd /cd ratio in covid- patients ( table ). in accordance with these results we found a significantly lower t lymphocyte frequency as measured by cd + frequency in our facs analysis in samples of covid- patients but not in malaria patients ( figure a) . a more detailed description of the cohort is given in table . for a description of the activation status, we assessed the frequencies of cd and hla-dr/cd on cd + and cd + t cells in covid- and malaria patients as well as in healthy controls. there were increased levels of cd and hla-dr/cd co-expression on cd + and cd + t cells in both covid- and malaria patients compared to healthy controls which is characteristic for acute infections ( figure b ) ( , , ) . the gating strategy for cd + and cd + t cells is illustrated in supplementary figure . based on the established differentiation markers ccr , cd ro, and cd ( ), we defined naïve (ccr + cd ro − ), central memory cm (ccr + cd ro + ), transitional memory tm (ccr − cd ro + cd + ), effector memory em (ccr − cd ro + cd − ) and terminal effector memory emra (ccr − cd ro − ) cd + and cd + t cells and assessed the frequency of the activation markers on each subset ( ) . overall, we saw an increase of cd + em t cells and cd + and cd + cm t cells in both covid- and malaria compared to healthy individuals ( figure c) . representative dot plots for the gating of naive and memory subsets of cd + and cd + t cells can be seen in figure d . cd was more frequently expressed on cm, tm, and emra cd + and cd + t cells in covid- and malaria patients compared to the respective subsets of t cells in healthy individuals. the same statistical trend toward higher cd frequencies was evident in the em subset of cd + and cd + t cells in covid- and malaria patients compared to healthy controls (figure a) . looking at the activation as measured by hla-dr and cd expression, we observed an increase of hla-dr + cd + cells in the tm and em subset of cd + t cells and in the cm and tm subset of cd + t cells in both covid- and malaria patients compared to healthy controls. of note, cd + cm, tm, and em t cells showed particularly high frequencies of hla-dr + cd + cells in covid- and patients infected with p. falciparum (figure a) . together with the overall increase of cm and em cd + t cells in covid- , this could imply an important role for these cells during the acute infection. in addition to the analysis of the traditional activation markers, we also looked at the expression of the co-stimulatory receptor cd on cd + and cd + t cells in covid- and malaria patients compared to healthy individuals. the cd molecule enhances cytotoxic effector functions when upregulated on cd + t cells ( , , ) . indeed, a significantly higher frequency of cd was detectable on bulk cd + t cells in both covid- and malaria compared to healthy individuals. in covid- patients, the tm, em, and emra cd + t cell subsets showed a higher frequency of cd compared to healthy controls, while in malaria patients the upregulation of cd was significant across all differentiation subsets compared to healthy individuals which implies a highly activated and functional phenotype of these cells ( figure b and supplementary figure a) . representative dot plots for the gating of cd , hla-dr/cd and cd on cd + and cd + t cells are illustrated in figure c . overall, we observed a strong activation of t cells in covid- and malaria with an increased expression of both early (cd + ) and late (hla-dr + cd + ) activation markers as well as the co-stimulatory receptor cd . accompanied by elevated frequencies of lag- and tim- on cd + and cd + t cells next, we examined the expression of the inhibitory receptors pd , tigit, lag- , tim- , and btla of t cells in covid- and malaria compared to healthy controls. previous research has established inhibitory receptors to play a critical role in t cell exhaustion in chronic infections after prolonged antigen exposure ( , , , ) . however, inhibitory receptors also have important functions and are transiently expressed during acute infections ( , , ( ) ( ) ( ) . many studies have described an increased expression of inhibitory receptors on both cd + and cd + t cells during malaria infection ( , , , ) . whether this increase has a beneficial or damaging effect, however, is not yet fully understood. to see whether similar effects can be observed in covid- , we assessed the frequencies of the coinhibitory receptors pd , tigit, btla, lag- , and tim- on cd + and cd + t cells in covid- and malaria patients and compared them with healthy individuals (figure a) . we also assessed the expression of these inhibitory receptors on the different memory subsets to detect potential influences on the expression due to a change in subset distribution ( figure b and supplementary figure ) . a trend toward higher pd expression on cd + and cd + t cells in covid- could be observed although with no statistical significance ( figure a) . however, malaria patients showed a strong upregulation of pd on both cd + and cd + t cells ( figure a ). this increase of pd on t cells in malaria was significant across all memory subsets of cd + t cells and on naïve and emra cd + t cells (supplementary figure b) . the mean fluorescence intensity (mfi) of pd showed an increased expression of pd on bulk cd + t cells in covid- and malaria patients compared to healthy individuals. in contrast, while we could not see any difference in the pd mfi on cd + t cells between covid- patients and healthy donors, malaria patients showed a significantly increased pd mfi on bulk, naïve, and all cd + memory t cell subsets compared to healthy donors. interestingly, the em subset of cd + t cells, which presumably includes a high proportion of sars-cov- -specific t cells ( ), showed a comparable pd mfi in healthy donors and covid- and malaria patients (supplementary figure c) . we did not detect increased tigit expression on bulk t cells in covid- patients compared to healthy controls ( figure a) . the em cd + t cell subset in covid- even showed a significant decrease of tigit expression. the same trend was evident on cd + em t cells although this trend did not reach statistical significance. tigit expression in malaria patients was significantly increased on bulk cd + and cd + t cell populations. however, when looking at the expression pattern of tigit on different memory subsets, only tm cd + t cells showed a significantly higher tigit frequency (supplementary figure d) . overall it seems that in acute covid- infection there is a trend toward a decreased tigit frequency on t cells compared to healthy donors while t cells in acute malaria patients generally showed a trend toward higher tigit frequencies compared to healthy individuals. btla inhibits lymphocyte effector functions during immune response, similar to pd ( ). it is well-established that btla expression is downregulated during cd + and cd + t cell differentiation, with the highest expression on naïve t cells ( ) . indeed, a lower btla frequency in memory compared to naive subsets of cd + and cd + t cells could be detected in covid- and malaria patients as well as healthy donors ( figure a) . however, the decrease of btla on cd + t cell subsets detected in both, covid- and malaria, was not as strong as in healthy subjects. we observed a significantly higher expression of btla on tm and em cd + t cells in covid- and malaria patients compared to healthy donors ( figure a) . lastly, we looked at the expression of lag- and tim- . both inhibitory receptors have previously been described to be upregulated during acute malaria infection with complex effects on t cell function ( ) . accordingly, we observed a significant increase of lag- and tim- on cd + and cd + t cells in malaria patients when compared to healthy donors. in covid- patients we saw a similar upregulation of lag- and tim- on t cells of even higher frequencies on cd + t cells (figure a) . a significant increase of lag- and tim- on both cd + and cd + t cells across all subpopulations in covid- patients could be detected (figure b) . to further characterize the expression pattern of lag- and tim- we examined the co-expression of pd /lag- and pd /tim- on cd + and cd + t cells of covid- and malaria patients compared to healthy controls ( figure c) . interestingly, we found differences in the distribution of these receptors, not only when comparing t cells of covid- and malaria patients with t cells of healthy donors, but also when comparing covid- with malaria. cd + and cd + t cells of covid- patients showed high frequencies of single-positive pd − lag- + and pd − tim- + cd + and cd + t cells with a significant increase compared to healthy controls. however, in pbmc of malaria patients we only observed an increase of pd − lag- + cd + t cells when compared to the frequency of pd − lag- + cd + and cd + t cells in healthy controls. double-positive pd + lag- + and pd + tim- + cells were increased in both covid- and malaria when compared with healthy donors. looking at the single-positive pd + lag- − and pd + tim- − cells we found no difference between covid- patients and healthy donors in the cd + t cell compartment. only cd + t cells in covid- showed a higher frequency of pd + lag- − and pd + tim- − t cells compared to healthy donors although the frequency of these cells was highest in malaria with a significant increase of both pd + lag- − and pd + tim- − on cd + and cd + t cells compared to healthy controls. overall, we saw a shift toward double-positive pd + lag- + and pd + tim- + and singlepositive pd − lag- + and pd − tim- + cells in the cd + t cell subset of covid- patients whereas in malaria, the distribution changed more toward double-positive pd + lag- + and pd + tim- + and single-positive pd + lag- − and pd + tim- − t cells. lastly, we looked for pd + lag- + tim- + t cells. however, the frequency of these pd + lag- + tim- + t cells was comparably low ranging from only . - . % of cd + t cells in covid- (data not shown). overall, we saw a strong increase of lag- and tim- expression on t cells in covid- and malaria. pd was only slightly increased on t cells in covid- patients while the frequency of tigit on t cells did not differ between covid- patients and healthy controls. in malaria we observed an upregulation of both pd and tigit on cd + and cd + t cells compared to healthy donors. t cells of patients with covid- and acute malaria showed high frequencies of the activation markers cd and hla-dr/cd as well as high frequencies of the inhibitory receptors lag- and tim- . to characterize whether stimulatory and inhibitory receptors are co-expressed on, or limited to distinct t cells we analyzed the expression of cd and the co-expression of hla-dr/cd on pd − lag- − and pd − tim- − t cells and compared it to the expression on double-positive pd + lag- + and pd + tim- + t cells in covid- and malaria patients. double-negative t cells did not co-express hla-dr and cd while double-positive t cells showed high frequencies of hla-dr + cd + cd + and cd + t cells (figure a ). this upregulation of hla-dr/cd on t cells co-expressing pd and lag- and pd and tim- could be observed on both cd + and cd + t cells in covid- and malaria patients. similarly, the frequency of cd was lower on pd − lag- − and pd − tim- − cd + and cd + t cells compared to the expression on double-positive t cells in covid- and malaria patients ( figure b ). in summary, the expression of activation markers (cd , hla-dr, cd ) on t cells was closely linked to the co-expression of inhibitory receptors (pd , lag- , tim- ) on t cells in covid- and malaria patients. in a next step, we analyzed the transcription factors t-bet and eomes in covid- and malaria patients. for this, we stained pbmc with the transcription factors t-bet and eomes and analyzed their expression in t cells in relation to the co-inhibitory receptors pd and lag- . the transcription factors t-bet and eomes have important functions in the immune response to acute infections and are upregulated to preserve cytotoxic effector functions in t cells ( ) ( ) ( ) ( ) ( ) . in chronic settings it has been shown that exhaustion and functional impairment of t cells is accompanied by an upregulation of eomes and downregulation of t-bet in exhausted t cells ( , , ) . looking at the expression of t-bet and eomes in cd + and cd + t cells in covid- and malaria patients we saw an upregulation of t-bet in covid- and an upregulation of both t-bet and eomes in malaria ( figure c ). next, we aimed to look at t-bet and eomes in the context of the co-inhibitory receptors pd and lag- . for this, we compared the expression of t-bet and eomes in pd + lag- + t cells to that in pd − lag- − t cells in covid- and malaria ( figure d) . similar to the expression of activation markers on these cells, t-bet and eomes were both more frequently expressed in pd + lag- + t cells than in their double-negative counterparts. altogether, the simultaneous upregulation of t-bet and eomes in t cells co-expressing pd and lag- further supports the notion that an upregulation of coinhibitory receptors can be seen as a result of the overall immune activation during covid- and acute malaria infection. additionally, we examined the frequency of cd expression on cd + t cells in covid- and malaria patients in relation to the expression of the co-inhibitory receptors pd and lag- . cd as part of the il- receptor is hugely important for the survival and differentiation of t cells and is involved in memory development ( , ) . cd low t cells are considered short-lived effector cells that undergo apoptosis after antigen clearance ( ) . in some viral infections such as hiv and hcv the downregulation of cd on bulk cd + t cells has been demonstrated to have damaging effects on viral control and promote t cell exhaustion ( , ) . bulk cd + t cells of covid- and malaria patients, however, showed no difference in cd expression compared with healthy individuals (supplementary figure a) . cd on pd + lag- + cd + tells in covid- and malaria was downregulated compared to their double-negative pd − lag- − counterparts (supplementary figure b) . to further evaluate whether the expression of lag- and tim- on t cells was related to the severity and disease course in covid- , we sub-stratified patients into a mild and a severe table ) . patients with mild disease course were defined to have a maximum hospital stay of five days and absence of any complications. severe disease course was defined as hospital stay longer than days, oxygen saturation lower than %, transfer to the icu or death. according to this definition, six patients fitted into the mild group and five patients fitted into the severe group. next, we compared the activation and inhibitory marker expression on t cells between these two groups. interestingly, we saw a higher expression of lag- and tim- on cd + and cd + t cells in patients with a severe disease course compared to patients with a mild disease course ( figure a) . however, there was no significantly higher expression of other co-inhibitory receptors on t cells in the severe group compared to the mild group (supplementary figure a) . additionally, we also compared the peak levels of the clinical inflammation parameters c-reactive protein (crp), il- , or ferritin with the expression of activation and inhibitory receptors on cd + and cd + t cells in covid- . cd on cd + t cells and tim- on cd + t cells correlated with crp levels (figure b) . a similar trend of correlation between crp levels and the frequency of tim- on cd + t cells could be observed. il- and ferritin levels did not correlate with the expression of co-inhibitory molecules (data not shown). finally, we investigated the expression kinetics of inhibitory receptors during the course and recovery of covid- infection. for this, pbmc of three covid- patients were collected at a second time point on day , , or after the first sample date. all three patients were discharged in good health from the hospital shortly after the second sample collection. frequencies of inhibitory receptor expression on cd + and cd + t cells were analyzed and compared to those measured at the first time point. a rapid decrease and normalization in all three patients for lag , tim , and pd expression on cd + and cd + t cells was observed, independently of the individual expression level measured at the first time point (figure c ). one of the main results of this cross-sectional side-by-side comparison of co-inhibitory and co-stimulatory receptors on t cells isolated from pbmc of patients with acute covid- and malaria infection was the comparable t cell activation and phenotype observed, following sars-cov- and p. falciparum infection. of interest, a strong upregulation of lag- and tim- on t cells in both covid- and malaria patients was linked to the co-expression of activation markers and correlated with disease course (figures - ) ( ) . lag- and tim- are coinhibitory receptors with multiple functions in modulating proinflammatory t cell responses ( ) . elevation and sustained co-expression of inhibitory receptors such as pd , lag- , tim- , ctla , and btla is the hallmark of exhausted t cells ( , , ) . this unique t cell population is the result of persisting antigen stimulation found in cancer and chronic infections. previous studies of t cells in malaria have shown that the upregulation of co-inhibitory molecules such as lag- and tim- in response to acute infection can have both beneficial and detrimental effects ( ) ( ) ( ) ( ) . it is still highly debated whether t cell exhaustion can occur during early malaria infection or other acute viral infections since the expression of inhibitory receptors is also induced through t cell activation and differentiation ( , , , ) . legat et al. showed that stimulating human cd + t cells induced a strong and rapid upregulation of inhibitory receptors after h while cytokine production was not necessarily affected ( ) . additionally, in a murine lcmv model it was demonstrated that irreversible t cell exhaustion occurred only after about days of infection ( ) . our data demonstrate a strong upregulation of lag- and tim- on t cells in covid- and malaria patients across all differentiation stages. in covid- patients we found high frequencies of single-positive pd − lag- + and pd − tim- + cd + and cd + t cells and fewer doublepositive pd + lag- + and pd + tim- + t cells. however, in malaria patients we found high levels of single-positive pd + lag- − and pd + tim- − as well as high levels of double-positive pd + lag- + and pd + tim- + cd + and cd + t cells. triple-positive pd + lag- + tim- + t cells were nearly absent in both covid- and malaria patients. double-positive cells were highly activated compared to their double-negative counterparts and showed increased levels of the transcription factors t-bet and eomes in covid- and malaria. a previous study by niu et al. demonstrated that the expression of pd and lag- on t cells in patients with acute sepsis had a regulatory impact on cytokine production ( ) . the degree of inhibition was dependent on the expression pattern of pd and lag- on t cells. in comparison, single-positive pd − lag- + t cells showed the highest cytokine production followed by single-positive pd + lag- − t cells while doublepositive pd + lag- + t cells produced the least amount of cytokines ( ) . overall, when comparing the activation and expression signatures of different co-inhibitory receptors in covid- and malaria patients we found relatively subtle differences between t cells of patients with either infection. the activation markers cd and hla-dr/cd were elevated on t cells in covid- and malaria patients. the upregulation of the co-inhibitory receptors lag- and tim- was more pronounced on cd + t cells in covid- patients while malaria patients showed especially high expression of lag- and tim- on cd + t cells. in line with this, acute malaria infection has been shown to result in a strong polarization of cd + t cells ( ) while it can be speculated that there seems to be a polarization toward cd + t cells in covid- . furthermore, t cells of malaria patients showed even higher frequencies of pd and tigit than covid- patients and healthy donors ( figure a) . overall, our data show a rather similar t cell signature of co-inhibitory molecules in covid- and malaria, an infection in which the upregulation of co-inhibitory receptors is a result of their strong activation ( , ) . however, acute malaria generally seemed to result in an upregulation and co-expression of a broader range of co-inhibitory receptors on t cells ( figure a) . importantly, we also observed a strong upregulation of the transcription factors t-bet and eomes in t cells co-expressing inhibitory receptors. t-bet and eomes are important mediators during acute infections and promote effector functions and cytotoxicity in cd + and cd + t cells ( ) ( ) ( ) ( ) . t-bet drives the expansion of cytokine producing th cells and inhibits the formation of th and th cells ( , ) . in this context t-bet has also been shown to repress the expression of pd and counterregulate the th response by inducing tim- on cd + t cells. in cd + t cells t-bet promotes cytotoxicity by inducing the production of granzyme b and perforin ( , ) . eomes is also found in both cd + and cd + t cells during acute infection. in cd + t cells eomes can either induce the production of ifn-γ by th cells or promote tr cells by driving il- production although the exact mechanisms underlying this are not yet fully understood ( , ) . in cd + t cells eomes is needed for a t-bet independent induction of ifn-γ and later during infection drives the formation of memory cells by aiding the responsiveness to il- ( ) . regarding t cell exhaustion, t-bet and eomes have distinct roles compared to those in effector and memory t cells. exhausted t cells express high levels of eomes and decreased expression of t-bet ( , , ) . we observed an increase of t-bet in bulk cd + and cd + t cells and high frequencies of both t-bet and eomes in cd + and cd + t cells coexpressing pd and lag- indicating strong effector functions in these cells. further and more comprehensive studies of the transcription factor profile of t cells in covid- and malaria are needed to understand and interpret the expression pattern of inhibitory receptors. recently, the cd /tigit axis has been identified as an important regulator in anti-tumor and anti-viral t cell responses ( ) . tigit expression on cd + and cd + t cells in covid- patients did not differ greatly from healthy individuals. we even observed a downregulation of tigit on some t cell subsets (supplementary figure b) . considering that tigit and cd compete for the same ligand cd on apcs and binding of cd to this ligand promotes cytotoxicity in cd + t cells the simultaneous increase of cd and decrease of tigit expression could benefit cd + t cells to maintain cytotoxic effector functions in covid- ( , ) . in malaria patients we observed an increase of tigit on bulk cd + and cd + t cells. however, looking at the different t cell subsets no significant changes could be seen which indicates that the increase in tigit expression might be linked to the differentiation status and change in distribution away from naïve toward effector cells. however, the overall frequency of tigit + t cells in malaria and covid- patients was lower than the frequency observed in chronic settings such as hcv ( ) . expression of lag- and tim- on t cells in covid- patients was closely related to disease course with higher levels detectable in severe patients compared to the mild patient group. toward recovery of covid- infection, the expression of inhibitory receptors on t cells decreased rapidly. taken together, our results do not hint toward the development of terminal t cell exhaustion during acute covid- infection. however, whether the upregulation of inhibitory receptors on t cells in covid- has detrimental or beneficial effects needs to be further investigated. moreover, we only analyzed the bulk expression levels of co-inhibitory molecules of t cells and pathogen-specific t cells might show a different pattern. on the same note, blockade of the inhibitory receptors lag- and tim- as a therapeutic strategy in covid- or malaria must be critically evaluated ( ) . on the one hand, it could enhance the effector t cell response to sars-cov- infection, on the other hand, such an intervention could also increase hyperinflammation. interestingly, robilotti et al. describe that therapy with immune checkpoint inhibitors correlated with a more severe outcome in covid- patients ( ) . interestingly, two patients of our covid- cohort were previously treated with biologicals (rituximab and pembrolizumab) but neither patient showed a severe disease course, increased hyperinflammation or differing t cell pattern in terms of activation, differentiation, or expression of coinhibitory molecules (data not shown). nonetheless, study of the immune response following sars-cov- infection in patients receiving immunomodulatory therapy can give us further insight into the role and function of different immune cells. disease progression and adverse outcome in covid- seemed to be associated with the degree of hyperactivation leading to cytokine storm and lymphopenia ( ) . the question remains, whether and in how far t effector cells are involved in the pathophysiology and hyperinflammation of covid- . the so-called cytokine storm is known from other infections such as severe acute respiratory syndrome (sars) and ebola ( , ) . high levels of cytokines like il- , or interferon-y could be measured in the serum of covid- patients, hinting toward a dysbalanced, overreacting immune system ( ). the underlying mechanisms and driving forces of this cytokine storm in covid- remain to be fully understood. however, from our results and findings in initial covid- studies, it appears that t cells do not play a direct role in the development of hyperinflammation ( , ) . the main source of il- does not appear to be t cells but rather monocytes and macrophages ( , ) . it can even be speculated whether the upregulation of lag- and tim- is a reaction of the immune system to counteract systemic hyperinflammation. interestingly, a study on ebola has demonstrated that fatal ebola infections were associated with higher expression of ctla and pd on cd + and cd + t cells and was accompanied by strong inflammation while ebola patients who survived showed less inflammation markers and lower ctla and pd expression on t cells ( ) . in accordance, our data show a correlation between disease severity of covid- and expression of inhibitory receptors like lag- and tim- on t cells. our study was limited by the relatively small and heterogeneous cohort and we assessed only few patients longitudinally. in order to further investigate the exhaustive state of the sars-cov- -specific t cell response in covid- , intracellular cytokine assays (ics) after antigen-specific short term stimulation, mhc class i and class ii multimer-staining and further functional assays in larger prospective cohorts with broader variations of the clinical course and worse outcome are needed to understand the role and function of antigenspecific cells. additionally, functional t cell subsets such as follicular helper cells, th , and regulatory t cells need to be characterized in parallel. of note, the frequency of peripheral tregs as measured by clinical immunology did not differ between patients with covid- and healthy individuals ( table ) . in our study, we examined the phenotype of peripheral cd + and cd + t cells. it has been shown that the expression pattern of co-inhibitory receptors on t cells depends on their localization ( ) . t cells in the peripheral blood can greatly differ in their expression pattern to those in the tissue ( ) . considering this, further studies that examine t cells in the lung, liver, and other organs in covid- and malaria are needed. the direct comparison of the t cell response between an acute viral and an acute parasitic infection does not seem selfevident. however, there are only few acute infections which are accompanied by hyperinflammation with high levels of proinflammatory cytokines and in which the t cell response is regulated by a strong upregulation of a broad spectrum of coinhibitory receptors, such as ebola, sars, mers, and malaria ( , ) . interestingly, we found relatively minor differences of the t cell signature and co-expression of co-inhibitory molecules of t cells in the direct comparison of patients with covid- and acute malaria supporting the notion that co-inhibitory molecules have a dual function, with potentially beneficial but also detrimental effects on t cell function ( , , , ) . our data suggest that there is a common pattern of t cell regulation in acute infections and that the up-regulation and co-expression of co-inhibitory molecules is as a consequence of their strong activation. nonetheless, comparisons of the t cell phenotype and functional immune response with other acute viral respiratory infections like influenza are needed in future studies. in summary, comparing the expression of different coinhibitory molecules of cd + and cd + t cells in covid- vs. malaria there is a transient increase of the expression of certain inhibitory receptors like lag- and tim- in covid- in the overall context of acute immune activation. during recovery, a rapid normalization of these inhibitory receptors could be observed. the results of this study will be the basis for further analysis regarding t cells and their potential role for immune pathogenesis during covid- infection. the datasets generated for this study are available on request to the corresponding author. the studies involving human participants were reviewed and approved by Ärztekammer hamburg (pv , pv , and pv ). the patients/participants provided their written informed consent to participate in this study. mh, ss, tj, and js designed the study. mh, ss, nw, mw, tj, and js designed the panels. mh, ss, nw, rw, and mw conducted experiments. mh and ss analyzed the data. js, tb, and mr recruited the patients. js supervised the study at all stages. mh, ss, tj, and js wrote the first draft. js and al gave institutional support. all authors reviewed the manuscript and gave important input. this project has been funded by the deutsche forschungsgemeinschaft sfb (ss, js, tj, nw, and al) and sfb (js) and deutsches zentrum für infektionsforschung dzif (mh, js, tj, tb, and mw). the funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript. a sequence homology and bioinformatic approach can predict candidate targets for immune responses to sars-cov- a new coronavirus associated with human respiratory disease in china severe acute respiratory syndrome coronavirus -specific antibody responses in coronavirus disease patients clinical features of patients infected with novel coronavirus in wuhan clinical and 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unique human immune signature of ebola virus disease in guinea extended co-expression of inhibitory receptors by human cd t-cells depending on differentiation, antigen-specificity and anatomical localization liver environment and hcv replication affect human t-cell phenotype and expression of inhibitory receptors we thank all patients who participated in this study. the authors also thank benno kreuels and matin kohsar for help with patient recruitment, sophie pflüger and marc van der meirschen for data collection, silke kummer for technical assistance, and christin ackermann for advice on the interpretation of the data. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material august | volume | article the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © herrmann, schulte, wildner, wittner, brehm, ramharter, woost, lohse, jacobs and schulze zur wiesch. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -gmc qrci authors: de miranda santos, isabel kinney ferreira; costa, carlos henrique nery title: impact of hydroxychloroquine on antibody responses to the sars-cov- coronavirus date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: gmc qrci nan recent large observational studies indicate that hydroxychloroquine (hy) does not affect outcomes of patients hospitalized with covid- ( , ) and may even be harmful ( ) . results of double-blind, randomized studies to assess efficacy of hy more rigorously are still not available. in spite of these facts, officials are currently advocating use of hydroxychloroquine (hy) for treatment and even prevention of covid- . in view of this situation and of the importance of correct interpretation of antibody profiles for planning preventive measures for covid- , we would like to bring the attention of readers to studies that raise concerns about the possible impact of hy upon antibody responses to sars-cov- . hy and chloroquine are lysosomotropic drugs that increase the ph of the lysosome, thus affecting functions of proteins involved in antigen presenting pathways and in b-cell activation ( ) . to the best of our knowledge, there are no new facts in the scientific and medical literature that indicate that the same mechanism could not operate in hy-treated patients suffering from covid- and negatively impact their sars-cov- -specific antibody responses. indeed, recent findings indicate that some individuals, including hospitalized patients, who have recovered from covid- have not made vigorous igg antibody responses. however, the most comprehensive publications addressing antibody responses, wherein study subjects presented viability in levels of igg antibody responses, have not detailed the treatment regimens delivered to the subjects ( ) ( ) ( ) ( ) . plans for employing immunity profiles against sars-cov- to relax social distancing and other epidemic mitigation measures and to create "immunity passports" to control spread of covid- have recently been questioned by the world health organization because of uncertainty regarding antibody responses ( ) . as more needs to be learned about the role of antibodies in recovery from and protection against infection with sars-cov- , the impact of hy and other treatment regimens on antibody responses requires systematic evaluation. observational study of hydroxychloroquine in hospitalized patients with covid- association of treatment with hydroxychloroquine or azithromycin with in-hospital mortality in patients with covid- in new york state hydroxychloroquine or chloroquine with or without a macrolide for treatment of covid- : a multinational registry analysis antibody response to preexposure human diploid-cell rabies vaccine given concurrently with chloroquine humoral immune response to tetanus-diphtheria vaccine given during extended use of chloroquine or primaquine malaria chemoprophylaxis effect of antimalarial drugs on the immune response to intramuscular rabies vaccination using a postexposure prophylaxis regimen mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infections by sars-cov- : an observational cohort study detection of sars-cov- -specific humoral and cellular immunity in covid- convalescent individuals evaluation of the euroimmun anti-sars-cov- elisa assay for detection of iga and igg antibodies a systematic review of antibody mediated immunity to coronaviruses: antibody kinetics, correlates of protection, and association of antibody responses with severity of disease available online at: www.who.int/newsroom/commentaries/detail/immunity-passports-in-the-context-of-covid- all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. key: cord- -e p lrlf authors: li, yunchuan; zhang, hao; zhu, bibo; ashraf, usama; chen, zheng; xu, qiuping; zhou, dengyuan; zheng, bohan; song, yunfeng; chen, huanchun; ye, jing; cao, shengbo title: microarray analysis identifies the potential role of long non-coding rna in regulating neuroinflammation during japanese encephalitis virus infection date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: e p lrlf japanese encephalitis virus (jev) is the leading cause of epidemic encephalitis worldwide. jev-induced neuroinflammation is characterized by profound neuronal cells damage accompanied by activation of glial cells. albeit long non-coding rnas (lncrnas) have been emerged as important regulatory rnas with profound effects on various biological processes, it is unknown how lncrnas regulate jev-induced inflammation. here, using microarray approach, we identified lncrnas and , mrnas differentially expressed in jev-infected mice brain. the functional annotation analysis revealed that differentially regulated transcripts were predominantly involved in various signaling pathways related to host immune and inflammatory responses. the lncrnas with their potential to regulate jev-induced inflammatory response were identified by constructing the lncrna-mrna coexpression network. furthermore, silencing of the two selected lncrnas (e and n ) resulted in reducing the phosphorylation of jnk and mkk , which are known to be involved during inflammatory response. collectively, we first demonstrated the transcriptomic landscape of lncrnas in mice brain infected with jev and analyzed the coexpression network of differentially regulated lncrnas and mrnas during jev infection. our results provide a better understanding of the host response to jev infection and suggest that the identified lncrnas may be used as potential therapeutic targets for the management of japanese encephalitis. approximately , - , cases of japanese encephalitis are reported annually in the region with , deaths, and nearly half of the survivors suffer from perpetual neuropsychiatric sequelae ( , ) . during infection, jev invades the central nervous system, wherein virus replication triggers a massive inflammatory response, and subsequently causes neuronal cell death ( ) . the crucial factor in jev-induced neuroinflammation is the uncontrolled activation of microglia, which release proinflammatory cytokines and chemokines such as interleukin (il)- β, il- , monocyte chemotactic protein , tumor necrosis factor alpha (tnf-α), and chemokine (c-c motif) ligand ( , ) . the higher levels of proinflammatory mediators appear to commence an irreversible inflammatory response leading to neuronal death ( ) . many studies have shown that microglia can be directly infected with jev, and serves as long-lasting reservoir for jev ( ) . therefore, the activation of microglia is a key event during jev-caused neuroinflammation. previously, we have investigated various molecular mechanisms of jev pathogenesis ( ) ( ) ( ) ( ) ; however, further studies are still required to advance the understanding of jev-induced neuroinflammation. recent advances have revealed that both mouse and human genomes contain a large number of long non-coding rna (lncrna) genes ( ) . lncrnas have been implicated as regulators of diverse cellular processes such as regulation of cell cycle, chromatin structure, and rna stability ( ) ( ) ( ) . however, the precise molecular mechanisms by which lncrnas act are largely unknown. recently, the transcriptomic analysis has revealed that a large number of lncrnas were differentially expressed upon infection with severe acute respiratory syndrome coronavirus and enterovirus ( , ) . furthermore, several lncrnas have been reported to be involved in virus replication. for instance, an lncrna, nrav, can regulate the replication of influenza a virus through inhibition of interferon-stimulating genes such as ifitm and mxa ( ) . many lncrnas have also been shown to modulate inflammatory response ( ) ( ) ( ) ( ) . of these, lethe, a pseudogene lncrna, interacts with nuclear factor-kappa b to inhibit the binding of rela with dna and to suppress the activation of downstream signaling cascades ( ) . these findings suggest that lncrnas play important roles in virus infection and inflammatory response. thus, further efforts toward accurate annotation and functional significance of lncrnas will be critical for our understanding of disease processes. among non-coding rnas, micrornas (mirnas) have been extensively studied in posttranscriptional regulation of gene expressions in various biological processes ( ) . accumulating evidence also suggests a key role for mirnas in various neuroinflammatory diseases ( , ) , including japanese encephalitis ( ) . however, the role of lncrnas in jev-induced neuroinflammation and virus replication is still unknown. in the present study, transcriptomic profiling of lncrnas and mrnas from jev-infected mice brain was performed using a microarray platform. our results identified jev-induced changes in expression patterns of lncrnas and mrnas in jev-infected and non-infected mice brain samples. this study may provide new insights into the pathogenic mechanisms associated with jev infection. postnatal - -day-old suckling balb/c mice were infected with p strain of jev. upon onset of clinical manifestation of jev such as poor brain response, limb paralysis, and whole body tremor, mice were killed and their brains were excised. brain homogenate prepared in dulbecco's modified eagle's medium (dmem) was centrifuged at , g to remove cellular debris. the resultant suspension was filtered through . µm sterile filters to obtain viral suspension. immediately, aliquots of filtered virus suspension were stored at − °c until further use. the virus titer was determined by plaque formation assay on baby hamster syrian kidney (bhk- ) cells as described previously ( ) . adult balb/c mice ( weeks old) were purchased from hubei provincial center for disease control and prevention, wuhan, china. mice were randomly assigned to two groups (n = for each group): the jev-infected group and the control group. for the infection group, mice were inoculated intracranially with plaque-forming units (pfu) jev (p strain) in µl dmem, whereas mice belonging to the control group were injected intracranially with equal volume of dmem. on day postinfection, mice infected with jev showed signs of acute encephalitis. mice from both groups were euthanized, and brain samples were collected for further studies. all animal experiments were performed following the national institute of health guide for the care and use of laboratory animals, and the experimental protocols were approved by the research ethics committee of college of veterinary medicine, huazhong agricultural university, hubei, wuhan, china (no. s m). all of our virus experiments are under biosafety level containment. we performed double-blind procedure in our in vivo experiments, blinding investigators, participants, and outcome assessors. the raw . cells were purchased from the american type culture collection, and the bv cells were kindly provided by professor yuanan lu from university of hawaii, manoa, usa ( ) . the cells passage numbers were below . bv and raw . cells were cultured and maintained in dmem that was supplemented with % (v/v) heat-inactivated fetal bovine serum, u/ml penicillin, and mg/ml streptomycin sulfate at °c in % co atmosphere. cells were seeded in multiwell plates, and grown to % confluency. non-adherent cells were removed by washing with non-supplemented dmem prior to further treatment. all of our virus experiments are under biosafety level containment. the cells were mock-infected or infected with jev p strain at multiplicity of infection (moi) of for h. at , , and h postinfection, cells were fixed and blocked with % bovine serum albumin in phosphate-buffered saline (pbs, ph . ) for min. then, cells were incubated with monoclonal antibody recognizing jev ns ( ng/ml for immunofluorescence assay, prepared by our laboratory) for h. after washing three times with pbs, cells were incubated with alexa fluor -conjugated secondary antibody (invitrogen) for min. cells nuclei were stained with ′, -diamidino- -phenylindole dihydrochloride (invitrogen). the staining was observed using a fluorescence microscope (zeiss) with × magnification. small-interfering rnas (sirnas) corresponding to the sequence of lncrna-e and lncrna-n , which were used to inhibit endogenous expression of lncrna-e and lncrna-n , and the negative control sirnas, which exhibited no downregulation of any mouse genes, were synthesized by gene pharma. transfection was performed with lipofectamine (invitrogen). cells were transfected with nm of each sirna. the sequence of all sirnas used in this study is listed in table s in supplementary material. total rna was extracted from mouse brain or treated bv or raw . cells using trizol reagent (invitrogen), and subsequently, was reverse transcribed into cdna using first strand cdna synthesis kit (toyobo) following the manufacturer's instructions. quantitative real-time pcr was performed using a viia™ real-time pcr system (applied biosystems) and sybr green real-time pcr master mix (toyobo). amplification was performed for min at °c and min at °c, followed by cycles of °c for s, °c for s, and °c for s. the relative expression levels of lncrnas, tnf-α, il- , il- β, and ifn-β were normalized to that of β-actin within each sample using the −ΔΔct method. the specific primers for lncrnas, tnf-α, il- , il- β, ifn-β, and β-actin are listed in table s in supplementary material. total rna was extracted and purified using mirneasy mini kit and rnase-free dnase set. biotinylated cdna was prepared according to the standard affymetrix protocol from ng total rna by using genechip ® wt plus reagent kit. the resultant cdna was then transcribed to generate crna, which is reverse transcripted to yield second-cycle cdna, and then fragmented. following labeling, . μg of cdna was hybridized for h at °c on genechip mouse eg . st array in genechip ® hybridization oven . genechips were washed and stained in the affymetrix fluidics station . the array was scanned by genechip scanner g. the probe array was scanned by affymetrix ® genechip command console. the gene expression levels were normalized by expression console. expression data were generated by affymetrix expression console software and normalized by the robust multi-chip average (rma) method. rvm t-test performed by brb-array tools (v . . ) was applied to filter the differentially expressed genes for the control and experimental group. after significant analysis and false discovery rate (fdr) analysis, we selected the differentially expressed genes according to the p-value and fold change threshold (absolute ratio). p-value < . and fold change > for mrna or fold change > for lncrna, were considered as significant difference. the lncrnas and mrnas were annotated through the affymetrix power tools from the database of refseq, ensembl, and non-code. the microarray data are available on ncbi geo database, and geo accession number is gse . heat map and functional analysis of differentially expressed genes the statistical heat map of the two groups was constructed using software cluster (version . ) to show the difference of gene fold changes between both groups. to gain insight into the functions of differentially expressed genes, associated gene ontology (go) terms were identified using david bioinformatics resources (version . , http://david. abcc.ncifcrf.gov/). it can organize genes into hierarchical categories and uncovers the gene regulatory networks on the basis of biological process and molecular function ( , ) . specifically, we used two-side fisher's exact test and χ test to classify the go category, and the fdr ( ) was calculated to correct the p-value. smaller the value of the fdr, smaller will be the error in judging the p-value. we computed p-values for the gos of all differentially regulated genes. enrichment provides a measure of significance of the function: as the enrichment increases, the corresponding function is more specific, which helps us to find those gos with more concrete function description in the experiment. we also used david software to analyze the signaling pathways of differentially expressed genes according to kegg, biocarta, and reatome. the significance was determined using the fisher's exact test and χ test, and the threshold of significance was defined by p-value and fdr ( ) ( ) ( ) . the coexpression networks were built according to normalized signal intensity of specific gene expressions. for each pair of genes, the pearson correlation was calculated, and the significant correlation pairs were chosen to construct the network ( ) . the gene coexpression network was constructed in experimental group and control group, respectively. within the network analysis, a degree is the simplest and most important measure of centrality of a gene within a network and determines the relative importance. a degree is defined as the number of directly linked neighbors. degree in experimental group was recorded as exp_degree, whereas in control group was recorded as con_degree. a clustering coefficient is a measure of the degree to which nodes in a graph tend to cluster together. it was calculated by the local measure ( ) . to exclude other genes' impact in each coexpression network, we further performed normalization of the degree, i.e., divided by maximum value of the gene degree in each network [normalized degree(i) = degree(i)/degree(max)]. then, the difference value of a gene's normalized degree (delta normalized degree, represented as |diffk|) was calculated between the two coexpression networks. while considering different networks, core regulatory factors were determined based on degree differences between the two class samples ( ) . considering our pathway analyses, we chose some genes of our interest, and functions of most of the selected genes were related to inflammation. the cytoscape software (version . ) was used to build the interaction map. the lncrna e and n were amplified by polymerase chain reaction. the lncrnas were transcribed (t megascript™, ambion) and biotinylated (pierce™ rna ′ end desthiobiotinylation, thermo scientific) according to the manufacturer's protocol. the jev-infected bv cells were lysed in radioimmunoprecipitation assay buffer (sigma) containing protease and phosphatase inhibitors (roche). for pull-down experiments, biotin-labeled rna or antisense-rna were incubated with streptavidin magnetic beads on a rotator at room temperature for min in the cellular extract solutions, then washed with pbs for five times, and the resultant pellet was extracted rna for qrt-pcr ( ). total mice brain tissues lysates were generated using radioimmunoprecipitation assay buffer (sigma) containing protease and phosphatase inhibitors (roche). protein concentrations were measured with a bca protein assay kit (thermo scientific). equal protein quantities were separated by sds-page and transferred to a polyvinylidene fluoride membrane (millipore) using a mini trans-blot cell (bio-rad). blots were probed with the relevant antibodies, and proteins were detected using the ecl reagent (thermo scientific). mouse monoclonal antibodies against jev ns were generated in our laboratory. commercially obtained antibodies used were: mouse monoclonal antibody against gapdh, rabbit polyclonal antibodies against β-tubulin, mkk , iκbα, phosphor-mkk -thr (abclonal technology), phosphor-iκbα-ser , phosphor-sapk/jnk-thr /tyr (cst technology), and horseradish peroxidase-conjugated antimouse secondary antibodies (boster). bv and raw . cells were transfected with sie , sin , or their controls (final concentration, nm) for h, and subsequently infected with jev at moi of . at , , and h postinfection, cell supernatants were harvested, serially diluted, and then used to inoculate monolayers of bhk- cells. after removal of unbound jev virus particles, bhk- cells were further incubated for - days, and plaques identified. the visible plaques were counted and viral titers calculated. all data are expressed as the mean of triplicate samples. all of our virus experiments are under biosafety level containment. the culture supernatants were collected from treated cells at the indicated time points and stored at − °c. the protein levels of tnf-α, il- β, and il- in cell cultures or mouse brain tissue lysates were measured by elisa kits (ebioscience) following the manufacturer's instructions. hematoxylin-eosin (h&e) staining, immunohistochemistry (ihc), and the terminal deoxynucleotidyl transferased utp nick end labeling (tunel) assay mice were anesthetized with ketamine-xylazine ( . ml/ g of body weight) and perfused with pbs, followed by % paraformaldehyde. brain tissues were removed and embedded in paraffin for coronal sections. the sections were used for h&e staining, ihc, and the tunel assays as described previously ( ) . for ihc experiment, tissue sections were incubated overnight at °c with primary antibodies against ionized calcium-binding adapter molecule (iba- ) (wako), glial fibrillary acidic protein (gfap) (dako), and neuron-specific nuclear protein neun (chemi-con) at concentrations indicated in the manufacturer's guidelines. after washing, slides were incubated with antimouse horseradish peroxidase-conjugated secondary antibodies, washed, and , ′-diaminobenzidine (vector laboratories) was used for color development. for tunel assay, an in situ cell death detection kit (roche) was used according to the manufacturer's instructions. all results are expressed as mean ± sem. statistical analysis was performed by graphpad prism (graphpad software, san diego, ca, usa). differences were analyzed for statistical significance using two-sided unpaired t test for two groups or multiple comparison one-way of variance (anova) for more than two groups (bonferroni's multiple comparison test, p < . ). to start our analysis, we first established a mouse model for jev infection and then collected samples for the microarray analysis ( figure s in supplementary material). to confirm whether the mice have successfully acquired the jev infection, we examined the virus replication in mice brain samples using plaque assay and western blot analysis. the results showed that the infected mice had a high virus titer in the brain tissues ( figure a) , and these tissues exhibited an increased expression of jev ns ( figure b) . to visualize the effect of jev infection in mice brain, we examined the brain tissue sections processed for h&e staining and ihc analysis. the data demonstrated that jev-infected mice presented the emblematic histopathological features of encephalitis, whereas no pathological changes were noticed in the mice belonging to control group ( figure c) . furthermore, an aberrant increase in number of astrocytes and microglia was detected by ihc using anti-gfap and -iba- antibody, respectively ( figure d) . taken together, these findings indicate the establishment of a productive jev infection in the mice brain. to identify changes in the expression levels of lncrnas and mrnas in mice brain infected with jev, total rna was extracted from jev-infected or mock-infected mice brain. the array which covers , lncrnas and , proteincoding transcripts from data sources such as ensembl, frnadb, lncrnadb, nocode . , and ucsc known gene, was used to detect lncrnas and mrnas in the mice brain samples (table s in supplementary material). scatter plot was used to show variation in lncrnas and mrnas expressions between jev-infected and mock-infected mice brain samples (figures a,b) . rvm t-test revealed that lncrnas and , mrnas were differentially expressed in a significant manner between the two groups. among the differentially expressed lncrnas and mrnas, lncrnas and mrnas were found to be upregulated, whereas lncrnas and mrnas exhibited a downregulated pattern (p < . ; fold change > for mrna or fold change > for lncrna, table ; table s in supplementary material). the hierarchical clustering analysis was also employed to show the differential expressions of lncrnas and mrnas between both groups of samples (figures c,d) . to better understand the functions of differentially regulated mrnas and lncrnas in jev-induced inflammation, go term analysis of these mrnas and lncrnas was performed. we found that highly enriched go categories for mrnas included, but were not confined to, phosphoproteins, nuclear functions, signal transduction, acetylation, and nucleotide binding ( figure s a in supplementary material). the most significant gos associated with upregulated lncrnas were related to innate immune response, apoptotic process, and inflammatory response ( figure s c in to investigate the correlation of lncrnas and mrnas, we constructed the lncrna-mrna coexpression network in each of the two separate groups (table s in supplementary material). we used biostatistics to calculate the possibility of interaction between these genes. the solid line between the two nodes strands for positive correlation, whereas the dotted line indicates negative correlation ( figure s in supplementary material). the clustering coefficient represents the density of the gene in the network. higher value of the clustering coefficient indicates more dependent interaction with other genes in the network or vice versa. the genes with higher degree and higher clustering coefficient in both groups are listed in table . in order to interrogate interactions between the genes related to inflammation, we selected some of the lncrna genes that may participate in the inflammatory pathways, and then built the lncrna-mrna and lncrna-lncrna coexpression networks (figures a,b) . to confirm our coexpression network analysis, we chose lncrna e and n and performed rna pull down experiments. our results showed that nod , tap , and col a were pulled down by indicated bio-lncrnas compared with bio-anti-lncrnas (figures c,d) . in order to determine that which pathway plays an important role in our lncrna network, we used pathway studio (version ) and kegg pathway to predict the possibility of pathway enrichment. the findings showed that mkk / is the most enriched pathway. an altered gene status in the coexpression network between jev-infected and mock-infected groups suggest that the gene may perform a very important function during the jev infection. therefore, we compared the status of genes in both groups, and the genes exhibiting the most significant difference are enlisted in table . the differential expression levels of lncrnas found in our microarray analysis were validated by quantitative real-time pcr. from the list of differentially regulated lncrnas, we randomly selected five upregulated and five downregulated lncrnas for their expression validation in mice brain tissues. our results obtained from quantitative real-time pcr were analogous to those observed in microarray data (figures a,b) . we also detected the expression levels of lncrnas in jev-infected bv cells. most of the lncrnas results showed the same tendency as were observed in mice brain samples after jev infection (data not shown). however, some of the lncrnas were not detected in bv cells, which may happen due to tissue or cell specificity ( ) . to discern a possible role of differentially regulated lncrnas in jev-induced inflammation, we selected two lncrnas (lncrna e , lncrna n ) form the data validated using mice brain samples. since jev infection is associated with marked activation of glial cells and exorbitant release of inflammatory cytokines ( ), we chose mouse microglial cells (bv cells) for further analysis. to determine whether bv cells were permissive to jev, the successful infection of bv cells was verified by plaque assay (figure a ) and immunofluorescence analysis ( figure b) . next, we examined the expression levels of selected lncrnas in jev-infected bv cells by quantitative real-time pcr. our results were concordant to those as observed in jev-infected mice brain samples (figure c ). to examine whether lncrna e and n are involved in jev-mediated inflammatory process, the effect of these lncrnas on the regulation of inflammatory cytokine production was determined. we analyzed the outcomes of silencing of these two lncrnas using lncrna-specific sirnas in bv cells. first, we confirmed that the sirnas significantly inhibited the expressions of lncrnas in bv cells (figure a) . to determine the role of lncrnas in inflammatory cytokine production, the cells were transfected with sie , sin or non-specific control sirna, and then infected with jev. the results revealed that knockdown of these lncrnas significantly decreased the production of inflammatory cytokines at mrna and protein levels (figures b,c) . furthermore, we found that treatment of cells with sie or sin did not exhibit any effect on virus replication in jev-infected bv cells, as viral titers were similar to those in control cells ( figure d) . furthermore, to examine whether these two lncrnas are microglia specific, we selected another immune cell line (raw . ) as a control. the expression pattern of lncrnas and the effects of lncrnasspecific sirnas on inflammatory cytokine production and virus replication were analogous to those observed in bv cells ( figures s a-d in supplementary material). thus, these data indicate that lncrnas participates in regulating jev-mediated inflammation. our previous study demonstrated that jnk plays an important role in jev-mediated inflammation ( ) , and we found that mkk / , the upstream kinase of jnk, is the most enriched pathway in this study. therefore, we wondered whether the selected lncrnas (e and n ) can regulate the mkk/jnk pathway. to validate it, we first examined the phosphorylation of mkk at thr and jnk at thr and tyr in jev-infected mouse brain samples ( figure b) . each of these phosphorylation events is associated with increased kinase activity ( , ) . to examine the role of lncrna e and n in regulating the kinase activity of mkk /jnk pathway, bv cells were transfected with sie , sin or non-specific control sirna, and then infected with jev. our findings showed that reduction of these lncrnas significantly decreased the kinase activity of mkk and jnk after jev infection at and h (figures a,b) . on the other hand, nfκb is known as one of the key transcriptional factors of inflammatory cytokines in addition to ap- which can be activated by jnk. we also measured whether the lncrna e and n can modify the nfκb pathway. we found that the phosphorylation of iκbα, a direct inhibitor of nfκb, was increased and iκbα protein was degraded upon jev infection, but blocking the lncrnas did not alter the phosphorylation level and protein amount of iκbα (figures a,b) . thus, we consider that the selected lncrnas may regulate jev-induced inflammation through activation of mkk /jnk pathway but not nfκb pathway. the struggles between viruses and hosts have been existed for very long times. people usually gave attention to the interaction between protein-coding genes and viruses. interestingly, only % of the mammalian genome is translated into proteins, and the remaining genome contains the non-coding rna genes ( ) . several transcriptome profiling studies have provided compelling evidence highlighting the importance of non-coding rnas, such as mirnas, in modulating the immune response against various viral infections, including jev infection ( ) . however, the roles of lncrnas in viral infections and associated inflammatory conditions are largely unknown. long non-coding rnas have emerged as important regulators of gene expression, with an accumulating body of evidence linking lncrnas to pathologies, including inflammatory diseases ( ) . albeit, the precise functions of lncrnas in virusmediated pathogenesis remain poorly understood, evidence from recent studies indicates that lncrnas may play a key role in virus-mediated inflammatory responses ( , ) . herein, a transcriptomic profiling of lncrnas and protein-coding genes from jev-infected mice brain was performed using a microarray platform. the results of our study reveal the first experimental evidence demonstrating the complex regulation of lncrnas by jev infection in mice brain and microglial cells. the present study is unique from different angles. first, we used mice brain to understand the regulation of host lncrnas upon jev infection. this may provide us a way to elucidate the relationship between lncrnas and protein-coding genes in the brain, having the more complex biological system compare to cell culture system. second, we validated our findings using microglial cells, which play important role in eliciting innate immune response during jev infection ( ) . however, some of the lncrnas were not detected in bv cells, which may associate with cell type specificity. third, the integration of microarray platform, quantitative real-time pcr, go analysis, pathways analysis, and lncrna-mrna coexpression network analysis has allowed us to conduct an active comparative genomics and bioinformatics study to reveal host lncrnas expression patterns associated with jev infection. we have identified a unique series of host molecular responses involving different combinatorial contributions of multiple cellular lncrnas. this provides an opportunity to understand the unique cellular lncrnas-mrna interactome network, dynamically regulated by jev infection. since we confirmed our findings using microglia cell line, further validations using primary microglia remain to be done in future. several lncrnas have been investigated to play important roles in modulating the host immune response during viral infections ( ) . for instance, lncrna-cmpk and -nrav have been found to negatively regulate interferon response ( , ) , which is an important component of innate immune system against viral infections ( ) . jev non-structural protein ns can block interferon-α signaling as an immune evasion strategy ( ) . for this reason, we believe that lncrnas may contribute to regulation of jev-induced pathogenesis. to the best of our knowledge, this is the first report where complete lncrnas and mrnas are profiled in jev-infected mice brain using microarray approach, and differentially regulated lncrnas and mrna transcripts are reported. in order to understand the impact of global lncrnas modulation during jev infection, we further analyzed go terms and cellular pathways that may have significance in jev pathogenesis. we found that the most significant gos associated with upregulated transcripts were related to innate immune response, inflammatory response, apoptotic process, acetylation, nucleotide binding, and defense response to virus infections; whereas the gos terms associated with downregulated transcripts were mainly limited to ions transportation, cell adhesion, signal transduction, and synaptic transmission. similarly, the most enriched upregulated pathways included jak-stat signaling pathway, toll-like receptor signaling pathway, mapk signaling pathways, and pathways related to herpes simplex and influenza a viruses infection. in contrast, the most enriched downregulated pathways were related to calcium signaling, neuroactive ligand-receptor signaling, and retrograde endocannabinoid signaling. to find the potential key lncrnas involved during jev infection, we built the lncrna-mrna and lncrna-lncrnas coexpression networks. to verify the coexpression network, we performed rna pull-down experiments and found that three mrnas nod , tap , and col a can interact with indicated lncrnas. nod are known to play a remarkable role in host immune responses which can positively regulate jnk cascade, nfκb activity ( ) . this may explain the fact of reduced production of inflammatory cytokines upon silencing of target genes. our analysis revealed some important genes that may have their roles in regulating jev-induced neuroinflammation. considering the networks analyses and previous study, we speculated that the selected genes may trigger the mkk /jnk and nfκb pathway, and our experimental found the selected lncrnas may activate mkk / jnk pathway but not nfκb pathway. the lncrna e and n may also regulate other pathways to alter the jevmediated inflammation. however, we can not exclude all the other possibilities by experiments. we also compared our mrna transcriptomic profile with a recent study revealing the dynamic changes in mirnaome and transcriptome in jev-infected microglia ( ) . of the enlisted differentially expressed genes, genes are also found to be differentially expressed in our study. moreover, nine of pathways predicted in referenced study are also analogous to our prediction analysis, thus, supporting our predictions. in conclusion, we first generated the expression profile of lncrnas and related mrnas in jev-infected mice brain based on a microarray approach. using the bioinformatics tools, we found some important lncrnas that may involve during jev infection. furthermore, our experimental data revealed the role of selected lncrnas in regulating the kinase activity of mkk and jnk, which are considered to be involved in inflammatory pathway, and thus, confirmed our predictions. this study may provide insights into the molecular mechanisms involved in jev pathogenesis. further studies are still required to understand the biological functions of these identified lncrnas during jev infection. as the roles of lncrnas in viral infections have not yet been fully identified and understood, this study may also provide valuable resource for further studies. adult balb/c mice ( weeks old) were purchased from hubei provincial center for disease control and prevention, wuhan, china. mice were randomly assigned to two groups (n = for each group): the jev-infected group and the control group. for the infection group, mice were inoculated intracranially with pfu jev (p strain) in μl dmem, whereas mice belonging to the control group were injected intracranially with equal volume of dmem. on day postinfection, mice infected with jev showed signs of acute encephalitis. mice from both groups were euthanized, and brain samples were collected for further studies. figure s | selected lncrnas regulate jev-induced production of inflammatory cytokines. (a) raw cells were transfected with sie , sin , or their non-specific control sirna (final concentration, nm) for h, and then expression levels of lncrna nonmmut and ensmust were detected by quantitative real-time pcr. (b,c) raw cells were transfected with sie , sin , or their non-specific control sirna (final concentration, nm) for h, and then infected with jev at moi of for h. the mrna (b) and protein (c) levels of tnf-α, il- , and il- β were analyzed by quantitative real-time pcr and elisa, respectively. ifn-β mrna level was determined by quantitative real-time pcr. *p < . ; **p < . . (d) raw cells were transfected with sie , sin , or their non-specific control sirna (final concentration, nm) for h, and then infected with jev at moi of for the indicated times. the titers of infectious virus in the culture supernatants were determined by plaque assay. one-way anova with subsequent bonferroni's multiple comparison. all data are representative of three independent experiments. table s | the annotation of lncrnas and mrnas in the database of refseq, ensembl, and non-code. table s | differentially expressed lncrnas and mrnas between jev-infected and mock-infected groups. tables s and s | go terms and pathways analyses of differentially expressed lncrnas and mrnas. murray valley encephalitis: a review of clinical features, diagnosis and treatment review of climate, landscape, and viral genetics as drivers of the japanese encephalitis virus ecology past, present, and future of japanese encephalitis proinflammatory mediators released by activated microglia induces neuronal death in japanese encephalitis upregulation of rantes gene expression in neuroglia by japanese encephalitis virus infection regulation of microglia effector functions by tumor necrosis factor signaling role of pro-inflammatory cytokines released from microglia in neurodegenerative diseases highly permissive infection of microglial cells by japanese encephalitis virus: a possible role as a viral reservoir microrna- b modulates japanese encephalitis virus-mediated inflammation via targeting rnf microrna- a- p modulates japanese encephalitis virus replication by targeting eukaryotic translation elongation factor a microrna- b- p modulates japanese encephalitis virus-mediated inflammation via targeting rnf quantitative phosphoproteomic analysis identifies the critical role of jnk in neuroinflammation induced by japanese encephalitis virus lincrnas: genomics, evolution, and mechanisms long non-coding rna uca regulated cell cycle distribution via creb through pi -k dependent pathway in bladder carcinoma cells regulation of chromatin structure by long noncoding rnas: focus on natural antisense transcripts evidence for natural antisense transcript-mediated inhibition of microrna function lncrna expression signatures in response to enterovirus infection unique signatures of long noncoding rna expression in response to virus infection and altered innate immune signaling nrav, a long noncoding rna, modulates antiviral responses through suppression of interferonstimulated gene transcription the human long noncoding rna lnc-il r regulates the inflammatory response expression and regulation of long noncoding rnas in tlr signaling in mouse macrophages a long noncoding rna mediates both activation and repression of immune response genes a mammalian pseudogene lncrna at the interface of inflammation and antiinflammatory therapeutics micrornas: genomics, biogenesis, mechanism, and function neuroinflammation and depression: microglia activation, extracellular microvesicles and microrna dysregulation role of micrornas in the regulation of innate immune cells under neuroinflammatory conditions enhanced detection and study of murine norovirus- using a more efficient microglial cell line the gene ontology (go) project in gene ontology: tool for the unification of biology. the gene ontology consortium genomescale analysis of in vivo spatiotemporal promoter activity in caenorhabditis elegans the kegg resource for deciphering the genome wholepathwayscope: a comprehensive pathway-based analysis tool for high-throughput data a systems biology approach for pathway level analysis human gene coexpression landscape: confident network derived from tissue transcriptomic profiles collective dynamics of 'small-world' networks gene connectivity, function, and sequence conservation: predictions from modular yeast co-expression networks long non-coding rna neat promotes non-small cell lung cancer progression through regulation of mir- - p-e f pathway integrative annotation of human large intergenic noncoding rnas reveals global properties and specific subclasses p alpha (mapk ) critically regulates the immunological response and the production of specific cytokines and chemokines in astrocytes synergistic activation of stress-activated protein kinase /c-jun n-terminal kinase (sapk /jnk) isoforms by mitogen-activated protein kinase kinase (mkk ) and mkk diverse functions of jnk signaling and c-jun in stress response and apoptosis gencode: the reference human genome annotation for the encode project differentially expressed mirna in inflammatory mucosa of chronic rhinosinusitis transcriptomic landscape of lncrnas in inflammatory bowel disease lncrnas regulate the innate immune response to viral infection negative regulation of the interferon response by an interferon-induced long non-coding rna regulation of type i interferon responses blocking of interferon-induced jak-stat signaling by japanese encephalitis virus ns through a protein tyrosine phosphatase-mediated mechanism nod and nod signaling in infection and inflammation dynamic changes in global micrornaome and transcriptome reveal complex mirna-mrna regulated host response to japanese encephalitis virus in microglial cells key: cord- -hkjhqgie authors: jewett, anahid title: the potential effect of novel coronavirus sars-cov- on nk cells; a perspective on potential therapeutic interventions date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: hkjhqgie coronavirus-induced disease- (covid- ) continues to cause significant morbidity and mortality worldwide. while studies on sars-cov- effects on immune cell function continue to progress, we know very little about the significance of depletion of key immune effectors by the virus in the mortality and morbidity of the disease. this commentary outlines what is the reported literature thus far on the effect of virus on nk cells known to kill virally infected cells. it also underscores the necessity for the future comprehensive studies of nk cells in sars-cov- infected individuals and animal models to better understand the role and significance of reported nk cell depletion and functional inactivation in disease morbidity and mortality, in hope to design effective therapeutic interventions for the disease. coronavirus-induced disease- (covid- ) poses a great public health threat, and presents a complex challenge for epidemiologists and public health professionals around the planet, as the disease has shifted from a regional epidemic to a worldwide pandemic in a short period of time. the toll that the disease has had on the global level continues to increase as the virus reaches all continents, except antarctica, afflicting more than countries. initial reports of covid- disease came from wuhan, china in late december , as patients began complaining about unexplained respiratory infections, which later was coined as "pneumonia of unknown etiology" ( ) . shortly after surfacing of the virus several independent laboratories identified the causative agent of covid- disease, ultimately naming it as severe acute respiratory syndrome coronavirus (sars-cov- ) ( , ) . while the search is continuing to uncover the infectious path of sars-cov- , several key findings have led the infectious disease experts to partly uncover the mechanisms of the original spread to humans. by phylogenetically comparing sars-cov- to other coronaviruses, it was noted that the new virus was highly identical to other coronaviruses that had originated from bats ( , ) . however, to date the complete transmission route remains elusive. despite the novelty of this particular strain of coronavirus, the sars-cov- is not without precedent. outbreaks in the past decades, such as severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers), identified viruses that fall into the same category of coronaviruses, which are single-stranded rna viruses (+ssrna) that morphologically have been determined to express crown-like spikes on their surfaces. however, the difference seen between prior species of coronaviruses and sars-cov- partly lies in their respective symptom presentations in patients. compared to sars and mers, the symptoms of covid- disease are not presented earlier in the infectious cycle, which may be a reason for the greater ability of viral transmission in patients ( ) . the incubation period of the sars-cov- is relatively longer than those of sars and mers ( - days vs. . - . and . - . , respectively) ( ). in addition to its longer incubation period, the mean reproductive number (r ) of sars-cov- has also been estimated to range from . to . , indicating that each infected patient can on average transmit the disease to two to three other individuals ( , ) . according to the available covid- clinical data, most patients fall into the range of - years of age, although several cases have been identified in younger individuals and in children recently ( ) . for infected patients, severity of symptoms has been classified as mild, severe, and critical. this spectrum of disease widely varies, as clinical presentation in infected individuals have ranged from asymptomatic infection to severe respiratory failure ( ) . asymptomatic transmission of sars-cov- poses a great public health challenge in containment efforts, as previous reports have noted as much as . % of case reports to be presymptomatic transmission ( ) . however, the main characteristic symptoms of covid- disease have included fevers, fatigue, dry cough and respiratory distress. the number of sars-cov- infected cases will certainly continue to rise worldwide especially now that many countries have chosen to relax the rules of social distancing and isolation due to the reopening of the economy and the work force. one of the most troubling factors about this disease is the lack of adequate understanding of the virus and the mechanisms by which it mediates the underlying pathology in humans. the problem has been compounded by the limited ability of the research laboratories to conduct studies due to the implementation of social distancing since many academic university laboratories have either been shut down or been operating at a minimum capacity. although the existing novel therapeutic strategies and research on potential vaccines are important directions, they will not be sufficient to provide adequate progress to fully understand the potential of the virus to infect individuals and the underlying mechanisms by which the virus causes pathology. containment efforts, through quarantines and social distancing, hand washing and wearing mask are important directions to mitigate the spread of sars-cov- infections. however, at the moment, we do not have the capability of large scale testing which would be necessary for the identification and isolation of asymptomatic and symptomatic patients to halt the chain of viral transmission. therefore, until the existing public health measures are able to curtail the transmission and bring the disease somewhat under control, the research laboratories will not be able to fully engage in the studies of covid- disease worldwide, thereby slowing the discoveries of more effective treatments and vaccines. progression models of covid- disease paint a dismal forecast for the duration of the outbreak, and therefore, warrant the discovery of novel treatments to alleviate disease symptoms that will supplement containment efforts. researchers continue to search for viable treatments to alleviate symptoms of the disease and to eradicate the spread of the disease. the search for effective treatments for covid- disease has featured studies advocating for the potential use of the anti-malarial drugs, hydroxychloroquine. the placebo controlled studies of hydroxychloroquine have not shown any efficacy, and indeed, in certain cases they have exhibited dangerous side effects in patients. others have advocated the immunologic path in support of monoclonal antibody therapy ( , ) . on the other hand remdesivir, an antiviral drug, was shown to be superior to placebo in shortening the time to recovery in adults hospitalized with covid- disease with the evidence of lower respiratory tract infection ( ) . the development of a sars-cov- vaccine would utilize the adaptive immune system to combat symptoms in patients, along with providing a preventative measure for healthy individuals. however, current timelines estimate that vaccine development could take up to anywhere from to months. while studies on sars-cov- 's effects on immune functions continue to progress, published studies concerning other coronaviruses may shed some light on how the immune system may be employed to mitigate covid- disease. based on experiences with coronaviruses in sars and mers, it has been suggested that the sars-cov- infection may also trigger major immunological changes, such as delayed or suppressed type ifn response and the influx of activated neutrophils and inflammatory monocytes/macrophages ( ) . in addition, the effect of virus on host following infection has demonstrated severe changes in the proportion of different immune effectors ( ) . in particular, in the peripheral blood of patients that were infected with sars, it was noted that there were significantly lower numbers of natural killer (nk) cells compared to healthy subjects ( ) . such a profile has also been extended to the immune responses of covid- individuals. a study of covid- individuals demonstrated that the numbers of nk, b and t cells were significantly decreased, with more severe cases being associated with greater decreases in the numbers ( ) , please see the references ( ) ( ) ( ) ( ) for reviews on the potential mechanisms of inactivation and loss of nk cells. upon admission, the neutrophil counts were remarkably higher in patients with severe covid- disease than in the mild cases, whereas the total lymphocyte counts were significantly lower in severe cases when compared to the mild cases ( ) . the numbers of nk cells and total t cells as well as cd + t cells were significantly lower in patients exhibiting severe symptoms when compared to those with the mild symptoms and healthy controls ( ) . thus, a correlation could be seen with severe decrease in nk and t cell numbers and the extent of severity of the disease. furthermore, the function of nk and cd + t cells was found to be suppressed along with the increased expression of nkg a in covid- individuals ( ) . more importantly, in patients convalescing after therapy, the numbers of nk and cd + t cells were restored with reduced expression of nkg a. in addition, these results suggested that the functional exhaustion of cytotoxic lymphocytes was directly associated with severity of sars-cov- infection. hence, sars-cov- infection is likely to paralyze the antiviral immunity at an early stage and contribute to progression and severity of the disease ( ) (figure ) . in patients infected with sars-cov- , nkg a expression was increased significantly on nk and cd + t cells compared to those in healthy controls ( ) . lower percentages of cd a+ nk, ifn-γ+ nk, il- + nk, and tnf-α+ nk cells and decreased mean fluorescence intensities (mfi) of granzyme b+ nk cells were also reported in covid- individuals when compared to healthy controls ( ) . although these preliminary results are of significance, they still would need to be reproduced by other laboratories, however, they clearly point to the potential contribution of functional exhaustion of cytotoxic lymphocytes in covid- disease ( ) . thus, future studies should be focused to fully understand the contribution of dysfunctional nk cells in disease pathogenesis. in addition, the underlying mechanisms of depletion of nk cells such as viral infection of nk cells, mobilization and homing of nk cells to the tissues from the peripheral blood, and activation induced cell death would need to be investigated (figure ) . neutrophils are the most abundant white blood cells in the lung, and they are critical effectors against infections, in particular against bacterial infections, however they are also capable of inducing life-threatening morbidities. moreover, natural killer cells are the most abundant lymphocytes in the lung, and therefore, play an important role not only in curtailment of infection but also in exertion of significant regulatory effect ( ) . however, little is known regarding the specific mechanisms by which nk cells maintain local homeostasis. by using lung-intravital microscopy to directly visualize and quantify neutrophil and natural killer cell interaction within the lung of live mice, the authors reported in a preliminary study that nk cells were greatly responsible for the slower pace of scanning of endothelium by neutrophils over a large area, and they were able to reduce the number of neutrophils that accumulated in an lps-triggered inflammatory challenge ( ) . indeed, depletion of nk cells in mice exhibited severe respiratory distress associated with protein-rich, high-permeability alveolar edema accompanied by neutrophil infiltration in myocardial infarction model ( ) . thus, nk cells are important effectors in not only combating the infection directly but also indirectly by activating the local inflammatory processes to curtail infection. in addition, they are also able to limit local immune activation in the lung to a manageable level without causing or allowing significant pathologies to be induced by other immune effectors (figure ) . thus, nk cells are important in keeping the balance of immune activation in such a way that sufficient levels of activation will ensue to remove the infection in the presence of finely tuned inflammatory processes to avoid local tissue damage. as mentioned above the infectious agent of covid- disease depletes nk cells in the peripheral blood, and potentially even in the lung tissues of patients, thereby, disabling and depleting the core immune effectors necessary to remove the virus and regulate uncontrolled immune activation. indeed, nk cells are the army generals of the immune effectors which bring order and discipline to the infected tissue microenvironment. without the nk cells it is likely that immune anarchy may ensue and result in the uncontrolled expansion and activation of other immune effectors ( ) ( ) ( ) ( ) . we have previously shown that nk cells also curtail the numbers of cd + t cells and expand cd + t cells ( , ) and (manuscript submitted). therefore, lack of nk cells may also result in the decrease expansion of cd + t cells as seen in covid- individuals ( ) ( ) ( ) ( ) (figure ) . thus, it is no surprise that patients suffering from covid- disease have greater cd /cd ratios as reported previously. the covid- individuals suffer from increased viral replication as well as uncontrolled inflammation resulting in cytokine storm and widespread tissue and organ damage ( ) ( ) ( ) . nk cells are known to mediate cytotoxicity, and regulate both the innate and adaptive immune functions through the release of many pro-and anti-inflammatory growth factors, cytokines and chemokines ( - , , ) . they constitute - % of the peripheral blood mononuclear cells (pbmcs), and are cytotoxic effectors in the blood of healthy individuals with the ability to recognize and lyse virally infected cells, including sars-cov- infected cells and a number of different cancer stem cells (cscs) and undifferentiated or poorly differentiated tumors which constitute the most aggressive subpopulations of the tumors. morphologically, nk cells are large granular lymphocytes that develop in the bone marrow. after development, the majority of nk cells are found in peripheral blood as the third largest lymphocyte population, next to b and t cells ( ) . moreover, nk cells are also found in the tissues such as in healthy skin, gut, lung, liver, lymphoid organs and uterus during pregnancy ( ) . nk cells have two different effector functions: cytotoxicity and cytokine release. in contrast to cd + cytotoxic t lymphocytes, nk cells do not need priming with antigen in order to kill their target cells. their function is regulated by the sum of interactions between activating and inhibitory receptors on their surface and the ligands on the target cells ( ) . ligands for nk activating receptors are expressed on fast proliferating cells that are virally infected or malignantly transformed. cytotoxic activity of nk cells is executed by two distinct mechanisms. one is regulated by the cytotoxic granules containing perforin and granzymes. after the formation of the immune synapse between nk and target cells, cytotoxic granules are released. perforin alters the permeability of the target cell membrane, allowing the entry of granzymes. the second pathway is interaction of ligands on nk cells with their respective cell death receptors on target cells. target cell death can also be induced through antibody dependent cellular cytotoxicity (adcc) ( ) ( ) ( ) ( ) . indeed, nk cell mediated adcc is likely one of the key mechanisms by which antibodies induced by the virus in recovered patients known as convalescent plasma or serum are able to alleviate the symptoms and improve the disease outcomes in infected and recovering patients ( ) (figure ) . the second key effector function of nk cells is the release of cytokines and chemokines. two major cytokines released by nk cells are ifn-γ and tnf-α ( ) ( ) ( ) ( ) ) . released cytokines not only affect the function of innate and adaptive immune cells, but it can also impact the differentiation of both healthy and cancer cells (figure ) . similar to sars-cov- infection in patients, decreased nk cell function in the tumor microenvironment, and peripheral blood of cancer patients as well as down-modulation of cd receptors on the surface of nk cells have been reported previously ( , ( ) ( ) ( ) ( ) ( ) ( ) . decreased function of nk cells is associated with increased viral infection and cancer risk, whereas higher function was correlated with prevention of establishment and progression of infection and cancer ( ) ( ) ( ) ( ) . indeed, older patients and those with immunosuppression are more susceptible to severe form of sars-cov- infection, and are likely to die from it. thus, it is no surprise that the same subsets of population of individuals are found to have lower expansion and functions of nk cells as reported previously ( , ) . decreased nk and t cell numbers and function can be due to the activation induced cell death and/or direct infection of the immune cells by the virus (figure ) . indeed, recent studies indicated that similar to mers-cov infection, sars-cov- also infects t cells through receptor-dependent, s-protein mediated membrane fusion and that the infection can be inhibited by ek peptide ( ) . furthermore, the infection is abortive since sars-cov- does not have the capability to replicate in the 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their proliferation at ex vivo culture condition retracted article: sars-cov- infects t lymphocytes through its spike protein-mediated membrane fusion the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © jewett. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -xypg evo authors: market, marisa; angka, leonard; martel, andre b.; bastin, donald; olanubi, oladunni; tennakoon, gayashan; boucher, dominique m.; ng, juliana; ardolino, michele; auer, rebecca c. title: flattening the covid- curve with natural killer cell based immunotherapies date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: xypg evo natural killer (nk) cells are innate immune responders critical for viral clearance and immunomodulation. despite their vital role in viral infection, the contribution of nk cells in fighting sars-cov- has not yet been directly investigated. insights into pathophysiology and therapeutic opportunities can therefore be inferred from studies assessing nk cell phenotype and function during sars, mers, and covid- . these studies suggest a reduction in circulating nk cell numbers and/or an exhausted phenotype following infection and hint toward the dampening of nk cell responses by coronaviruses. reduced circulating nk cell levels and exhaustion may be directly responsible for the progression and severity of covid- . conversely, in light of data linking inflammation with coronavirus disease severity, it is necessary to examine nk cell potential in mediating immunopathology. a common feature of coronavirus infections is that significant morbidity and mortality is associated with lung injury and acute respiratory distress syndrome resulting from an exaggerated immune response, of which nk cells are an important component. in this review, we summarize the current understanding of how nk cells respond in both early and late coronavirus infections, and the implication for ongoing covid- clinical trials. using this immunological lens, we outline recommendations for therapeutic strategies against covid- in clearing the virus while preventing the harm of immunopathological responses. natural killer (nk) cells are a key component of the innate immune system and are critical in the response to many viral infections in humans and animal models ( ) ( ) ( ) . in addition to their beneficial antiviral role, nk cells have also been associated with immunopathology in infections such as respiratory syncytial virus (rsv) ( ), influenza a virus ( ) ( ) ( ) ( ) , and hepatitis b ( ) . additionally, in the context of non-respiratory viral infections by hiv and hcv, nk cells appear to act as a rheostat by eliminating activated cd + and cd + t cells, thus preventing t cell-mediated autoimmunity ( ) . the etiologic agent of the outbreak of pneumonia in wuhan, china, was identified as belonging to the coronaviridae family and named severe acute respiratory syndrome coronavirus (sars-cov- ). this virus causes the coronavirus disease (covid- ) which was declared a pandemic by the world health organization (who) on march th, ( , ) . with the paucity of information currently available, there is a lack of consensus on the role played by nk cells in the response to coronavirus (cov) infection. in this review, we will explore evidence for both the protective and pathological role that nk cells may play in cov infection. based on this knowledge we will comment on immune modulating treatment options that are being developed for the current covid- crisis. first discovered in the s, covs are part of the coronaviridae family of enveloped positive single-strand rna viruses ( , ) . the subfamily orthocoronaviridae includes four genera: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus ( ) . alpha-and betacoronaviruses circulate in mammals, including bats, gammacoronaviruses infect mostly avian species, and deltacoronaviruses infect birds and mammals ( ) . low pathogenic human covs (hcovs), such as hcov- e ( ) , infect upper airways and etiological studies suggest they account for - % of common colds ( , ) . on the other hand, highly pathogenic covs infect the lower respiratory tract and can cause severe pneumonia ( ) . these highly pathogenic covs include sars-cov- , the virus responsible for the - severe acute respiratory syndrome (sars) epidemic, and mers-cov, the virus responsible for the outbreak of middle eastern respiratory syndrome (mers) in ( ) ( ) ( ) . while highly pathogenic covs have become a relatively recent issue for humans; feline, canine, and bovine covs have long been recognized as significant pathogens with implications in veterinary medicine and agriculture ( , ) . all covs have a roughly kb genome packed into an enveloped helical capsid ranging from to nm ( ) . at minimum, coronaviridae members encode structural and non-structural proteins ( ) with the family owing its name to the crown-like appearance produced by their spike (s) proteins ( ) . mutations in the s protein have allowed sars-cov / to co-opt ace or mers-cov to co-opt dipeptidyl peptidase (dpp ) receptor/cd as viral entry receptors, thus facilitating the zoonosis of nonhuman covs ( , ( ) ( ) ( ) . in addition, another mechanism that may have allowed these viruses to adapt to human hosts is through s protein cleavage by host cell proteases to expose the s domain fusion peptide, which induces viral and cellular membrane fusion and results in the release of viral genome into the cytoplasm ( ) . genetic sequencing revealed sars-cov- to be a betacoronavirus that shares . % nucleotide identity with sars-cov- and . % identity to mers-cov ( ) . the epidemic of sars in - caused by sars-cov- illustrated the devastating potential of coronaviruses to cause serious disease in humans ( ) . sars ultimately reached countries and continents causing over , infections and over deaths. the basic reproductive rate (r ) or the number of expected cases arising from one infected individual, ranges from to ( , , ) . with its reservoir in bats, sars-cov- is a zoonosis that was transmitted to humans by palm civets ( , , ) . sars-cov- infects lung pneumocytes ( ) and enterocytes in the digestive tract ( ) most often producing flulike symptoms ( , ) . more severe presentations including pneumonia, pronounced lymphopenia, liver abnormalities, and acute respiratory distress syndrome (ards) were also reported, with most fatalities due to respiratory failure ( , ( ) ( ) ( ) ( ) . the subsequent mers-cov outbreak in also originated in bats, with dromedary camels being the intermediary host ( , , ) . the r for mers-cov is estimated to be under ( ) . the extent of mers-cov transmission was more limited than sars-cov- , but its case fatality rate was greater with , cases over countries and deaths being reported at the end of ( ) . common presentations for mers-cov include fever, dyspnea, muscle pain, and digestive tract symptoms and disease progression is more likely in those with comorbidities ( ) . like sars-cov- and mers-cov, sars-cov- is thought to have originated in bats through an unknown intermediary host ( ) . at the time of writing, the number of global infections is estimated to be over , , with over , deaths ( ) and the r is roughly . ( ) . like other diseases caused by infectious covs, most patients present with flu-like symptoms including fever, cough, and lethargy, with the development of pneumonia and ards often proving fatal ( ) . furthermore, patients with underlying conditions are at risk for further complications if infected with covid- , such as those with cardiovascular disease ( ) . sars-cov- has been posthumously detected in not only the lungs, but the pharynx, heart, liver, brain, and kidneys ( ) . transmission of sars-cov- is thought to mainly occur through direct contact/inhalation of respiratory droplets and aerosols from infected carriers, but indirect transmission by fomites has also been reported, although less efficient ( , ) . sars-cov- viral entrance is thought to be mediated by binding of the s protein to the ace receptor ( , ) , although this is still under debate ( ) . while direct cytopathic effects are thought to play a major role in cov pathology, studies have suggested that a dysregulated immune response resulting in pathological inflammation is also partly responsible ( ) . with the current pandemic already surpassing the previous cov outbreaks ( ) , rapid deployment of novel approaches to understanding and treating coronavirus infections are needed. innate immunity is essential in disease prevention and viral clearance. among the first responders to viral infections, tissueresident macrophages and dendritic cells (dcs) ( ) recognize evolutionarily conserved microbial structures termed pathogenassociated molecular patterns (pamps) via germline-encoded pattern recognition receptors (prrs) ( ). in the context of respiratory rna viruses, airway epithelial cells, that also express some prrs ( ) , are often infected and have a major role in the first line of defense. tlr , tlr , tlr , mda- , and rig-i are prr expressed by immune and non-immune cells that are especially relevant in fighting respiratory rna viruses, such as coronaviruses ( ) . sensing through prrs results in the transcription of genes involved in the inflammatory response, with type i interferons (ifns) (ifn-α/β) production being a critical part of the antiviral response ( ) . type i ifns are produced by many immune and non-immune cells ( , , ) and in addition to eliciting intrinsic antiviral responses ( ), they are also essential to prime innate and adaptive lymphocytes, including nk cells ( ) . nk cells are cytotoxic lymphocytes that directly target infected, stressed, or transformed cells and play a critical role in bridging the innate and the adaptive immune responses ( ) . in humans, mature nk cells comprise - % of total peripheral blood leukocytes and are described phenotypically as cd − cd − cd − cd + cd +/− ( ). nk cells do not undergo clonal selection but instead express several germline-encoded receptors that regulate their activity ( , , ) . upon viral infection, host cells become more susceptible to nk cell killing through: (i) upregulation of self-encoded molecules induced by infection/cellular stress ( , ) that bind activating nk cell receptors such as natural cytotoxicity receptors (ncrs) (nkp , nkp , and nkp ) ( ), c-type lectin-like receptors nkg d and nkp ( ) , and co-activating receptors such as dnam- ( ); (ii) downregulation of ligands for inhibitory receptors such as killer immunoglobulin-like receptors (kirs) ( ) ( ) ( ) and the c-type lectin-like receptor cd -nkg a ( , ) which suppress nk cell activation, and; (iii) direct recognition of viral moieties, via engagement of pamps ( ) or transmembrane activating receptors such as mouse ly h ( ) or human nkg c ( ) . moreover, nk cells can eliminate virus-infected cells via cd -mediated antibody-dependent cellmediated cytotoxicity (adcc), which has been shown to be particularly important for herpesvirus clearance ( ) . finally, nk cell activity is modulated by cytokines, including, but not limited to, the activating cytokines interleukin (il)- / / / ( ) and type i ifn, which can be produced by virally infected cells or activated antigen presenting cells ( , ) . il- / / / , alone or in combination, promotes nk cell survival, proliferation, cytotoxicity, and cytokine production, including ifn-γ ( ) . therefore, nk cells are uniquely equipped to sense and quickly respond to viral infections. nk cells are found in circulation and in peripheral tissues ( ) and can be quickly recruited to sites of infection where they facilitate and accelerate viral clearance. in fact, nk cells are not thought to have permanent tissue residency but instead move dynamically between the blood and tissues, such as the lungs ( ) . nk recruitment is regulated by chemokine gradients that are sensed via chemokine receptors ( , ) . activated nk cells induce the apoptosis of target cells through the engagement of death receptors, such as trail and fas ( ) or via direct cytotoxicity through ca + -dependent exocytosis of cytolytic granules (perforin and granzymes) ( ) . moreover, nk cells secrete cytokines, including ifn-γ, which have key anti-viral properties ( ) . in addition to being essential first responders to viral infection, nk cells can elicit a stronger secondary response resembling the memory features of adaptive lymphocytes ( , ) . nk cell memory has been initially described in mice infected by mcmv, where ly h + nk cells quickly expand and have stronger responses after a secondary encounter with the virus ( ) . interestingly, a similar nk cell subset has been identified in humans, where nk cells expressing nkg c are expanded and persist in cmv infected patients ( ) . both ly h and nkg c bind viral determinants, highlighting how nk cell memory is linked with the ability of nk cells to directly recognize viruses ( , ) . in addition to direct recognition of viral molecules, longlasting changes in nk cells are induced by the cytokine milieu ( , ) , which can be elicited by viral infection. the relevance of nk cells in fighting viral infections has been highlighted by several studies where nk cells, in mice and humans, were not present or had compromised functions ( ) . for example, individuals with nk cell deficiencies (nkd), a subset of primary immunodeficiency diseases, are highly susceptible to viral infection, particularly by herpesvirus and papillomavirus families ( ) . the seminal case of nkd in an adolescent female with severe herpesvirus infections (varicella pneumonia, disseminated cmv, and disseminated hsv) revealed how functional nk cell deficiencies have clinical consequences in terms of viral infections ( ) . cancer patients are also at risk of viral infections ( ) , which may be explained, at least in part, by an impairment of nk cell responses often observed in humans and in murine tumor models ( ) ( ) ( ) ( ) ( ) ( ) . unsurprisingly, cancer patients are at a significantly increased risk of severe covid- ( , ) . elderly patients are also more susceptible to viral infections ( ) . mouse studies highlighted how a decreased number of circulating mature nk cells in aged animals paralleled with increased susceptibility to viral infections ( ) . studies in humans suggest that although nk cell numbers can actually increase with aging, nk cell activity declines significantly ( , ) . przemska-kosicka et al. investigated nk cell function in response to seasonal influenza vaccination in young and old populations and observed quantitative and qualitative changes associated with impaired responses in the nk cell population and this was associated with poor seroconversion in the older population ( ) . additionally, obesity, which has been shown to cause systemic nk cell dysfunction ( , ) , has also been linked to increased covid- severity and could be the reason behind the high prevalence of severe covid- in younger people ( ) . in short, nkd and individuals with reduced nk cell numbers or function are more susceptible to viral infections. unsurprisingly, the cdc has already highlighted a higher risk of infection and severity of covid- in older individuals and individuals with comorbidities such as obesity and cancer ( ) . however, this point is still controversial as a systematic review showed that primary immunodeficiencies are not linked with increased covid- severity ( ) , but these data have to be interpreted keeping in mind that a large part of covid- pathology is caused by excessive immune activation, which is arguably harder to reach in immunocompromised individuals. given the paradoxical role of the immune response in covid- patients, it would be extremely useful to be able to rely on immunological functional biomarkers that could predict the outcome of disease severity. such assays are readily available for determining nk cell activity, e.g., nkvue tm , and there is therefore an opportunity to conduct studies that would link nk cell functions to disease severity. evasion of host immune responses is necessary for the successful propagation of a virus. mechanisms employed by covs to evade the immune response could provide insights into how the immune system, and nk cells in particular, responds to sars-cov- . covs have been shown to target components of the innate ifn response, employing non-structural proteins (nsps), structural proteins, and accessory proteins to achieve this goal. nsp methylates viral rna therefore preventing recognition by mda and dampening type i ifn production ( ) . nsps also suppress type i ifn responses via the inhibition of the transcription factor stat mrna transcription (nsp ) and deubiquitination of transcription factors like interferon regulatory transcription factor (irf) (nsp ) ( ) . moreover, viral-encoded accessory proteins from sars-cov- open reading frame (orf) b and mers-cov orf a/ b also block ifn production and signaling ( ) . in addition, the mers-cov orf -encoded protein blocks p-stat import, thus blocking ifn signaling ( ) . finally, the structural m protein of mers-cov ( ) physically sequesters kinase proteins rig-i, tbk , ikke, and traf and the sars-cov- n protein inhibits activator protein (ap)- signaling, protein kinase r function, and nfκb activation, all of which act to impede ifn responses ( ) . in vivo murine studies report young mice rapidly clear sars-cov- infection, while old mice do not and that this discrepancy is due to a delay in type i ifn. furthermore, early administration of ifn-β induces a stronger immune response and reduces mortality in old mice ( ) . since type i ifns are critical for nk cell activation and effector functions, it is possible that nk cell-mediated clearance of sars-cov- is being subverted by these mechanisms. further research into the role of nk cells in cov clearance and potential immune evasion mechanisms are necessary to inform therapeutic development and use. there is currently a paucity of studies into the role of nk cells not only in covid- pathophysiology, but also in other coronavirus infections. an in vivo study reported that beige mice on a b background cleared sars-cov- normally, indicating that functional lymphocytes, including nk cells, may not be required to eliminate sars-cov- in murine models ( ) . however, in a more recent study characterizing the cellular immune response to sars-cov- in - -month old balb/c mice, t cell depletion did not prevent control of sars-cov- replication ( ), suggesting a role for the innate immune system, and nk cells, in viral clearance. importantly, in this study cd -depletion resulted in enhanced lung immunopathology and delayed viral clearance, while cd -depletion did not affect viral replication or clearance, thus highlighting an important role for cd + t cells in coronavirus infection. these conflicting results may be due to the inherent limitations of cov murine models. in - week-old mice, sars-cov- is associated only with mild pneumonitis and cytokines are not detectable in the lungs ( , , ) . a sars-cov- isolate (ma- ) replicates to a high titer and is associated with viremia and mortality, however the model lacks significant inflammatory cell infiltration into the lungs ( ) . thus, mouse models developed for the study of sars fell short in terms of reproducing the clinical and histopathological signs of disease ( , ( ) ( ) ( ) . it is therefore necessary to develop a usable animal model that is capable of reproducing the clinical and histopathological signs on covid- . israelow et al. recently described a sars-cov- murine model based on adeno associated virus (aav) -mediated expression of human (h)ace , which replicated the pathologic findings found in covid- patients ( ) . this model, which overcame the inability of murine (m)ace to support sars-cov- infection, was used to show the inability of type i ifn to control sars-cov- replication ( ) . in a similar attempt to overcome the lack of infectability through mace , dinnon et al. recently described a recombinant virus (sars-cov- ma) with a remodeled s protein mace interface, which replicated in upper and lower airways in young and aged mice with disease being more severe in aged mice. the authors used this model to screen therapeutics from vaccine challenge studies and assessed pegylated ifn-λ- as a promising therapeutic. the authors suggested that this model has greater ease of use, cost, and utility over transgenic hace models ( ) to evaluate vaccine and therapeutic efficacy in mice ( ) . a preliminary analysis of nk cell function and phenotype has been performed by zheng et al. using peripheral blood from covid- patients ( ). on admission, nk cell levels in the peripheral blood inversely correlated with disease severity. furthermore, covid- patients with severe disease had significantly lower numbers of circulating nk cells, as compared to mild disease (p < . ) ( ) . additionally, circulating nk cells in severe disease displayed increased expression of the inhibitory receptor nkg a and had an hyporesponsive phenotype with lower levels of ifn-γ, tumor necrosis factor (tnf)-α, il- , and granzyme b, although degranulation was maintained ( ) . finally, as compared to patients with active disease, patients recovering from covid- had higher numbers of nk cells and lower nkg a expression ( ) . liao et al. performed singlecell rnaseq on the cells obtained from bronchoalveolar lavage fluid of severe and mild covid- patients and found that covid- patients had significantly more nk cell infiltrates into the lungs, however patients with severe disease had reduced proportions of nk cells ( ) . in addition, klrc (nkg a) and klrd (cd ) were highly expressed by nk cells ( ) . carvelli et al. analyzed myeloid and lymphoid populations by immunophenotyping from blood and bronchoalveolar lavage fluid (balf) in healthy controls, paucisymptomatic covid- patients, pneumonia patients, and patients with ards due to sars-cov- and found that absolute numbers of peripheral blood lymphocytes, including nk cells, were significantly reduced in the pneumonia and ards groups compared to healthy controls. furthermore, the proportion of mature nk cells was reduced in patients with ards and nk cells showed increased nkg a, pd- , and cd ( ) . finally, wilk et al. performed single-cell rna-sequencing on covid- patients and healthy controls and found that the cd bright population was depleted in all covid- patients but the cd dim population was depleted only in patients with severe covid- . furthermore, nk cells had increased expression of the exhaustion markers lag and havcr ( ) . nk cell cytopenia seems to be a consistent characteristic among sars-cov- infected patients ( ). altogether, these data indicate alterations in the nk cell phenotype and functional profile that are consistent with the hypothesis that to establish a productive and lasting infection, sars-cov- needs to dampen the nk cell response. nk cell dysfunctions were also observed in patients from the previous cov outbreaks. wang et al. assessed nk cell number and phenotype using peripheral blood from sars patients admitted to hospitals in beijing, china ( ) . nk cell proportion and absolute number were significantly reduced in sars patients as compared to healthy donors and patients infected with the bacterium mycoplasma pneumoniae ( ). nk cell number correlated inversely with disease severity and patients with anti-sars cov-specific igg or igm antibodies had significantly fewer nk cells ( ) . the patients assessed had varied disease duration from to days (mean . days) and this allowed for patient stratification by disease duration. within the first days of sars-cov- infection, nk cell numbers remained high but this period was followed by the development of lymphopenia with levels recovering only around day ( ) . dong et al. also observed a reduction of nk cell numbers in sars patients, and these levels were lower in patients with severe, as compared to mild, sars ( ) . in addition, mers infection is strongly associated with leuko-and lymphopenia ( , ( ) ( ) ( ) . the mechanisms underlying the reduction of circulating nk cells in patients infected with covs are still unclear. as most studies have focused on peripheral blood nk cells, it is possible that the reduced number of circulating nk cells is due to redistribution of blood nk cells into the infected tissues ( ) . while it is hard to assess nk cell migration to infected tissues in covid- patients, this hypothesis was corroborated by mouse studies, where nk cells have been shown to migrate to the lungs in cov infected animals ( ) . an abundance of inhibitory factors, such as tgf-β, may be partially responsible for the nk cell hyporesponsiveness observed in covid- patients. in support of this hypothesis, huang et al. found significantly higher tgf-β levels in sars patients compared to healthy controls and this positively correlated with length of stay ( ) . given the importance of tgf-β in suppressing nk cell functions, it is possible that the higher levels of tgf-β (as well as other inhibitory cytokines) in cov patients leads to suppression of nk cell antiviral activity ( ) . early studies of covid- patients report secondary (super-) infections, including nosocomial pneumonia or bacteremia as a complication of sars-cov- infection ( ) . since nk cells are critical first responders that play a role in preventing and clearing infections ( ) , a poor nk cell count or exhausted phenotype, in addition to negatively influencing covid- patient outcomes, could facilitate the development of secondary infections and have a significant negative impact on patient outcomes. one of the main barriers in studying the role of nk cell activation in the early clearance of cov infection in asymptomatic or mildly symptomatic patients is the fact that these individuals are rarely diagnosed in the clinic and therefore an opportunity to collect samples for research does not exist. thus, while there is currently no direct evidence to support a role for nk cells in the clearance of sars-cov- , evidence showing that viral infection has a negative effect on the nk cell compartment is accumulating. given the importance of nk cell activity in early viral clearance and late immunopathology, having a rapid and reliable test to predict nk cell function, such as nkvue tm (atgen canada/nkmax), whereby whole blood is stimulated by an nk cell-specific activating cytokine mix and activity is measured via ifn-γ production, might allow researchers to predict who will mount an adequate response with asymptomatic or minimally symptomatic viral clearance and who will need icu admission, as has been shown with cancer patients ( ) . further research will be required into the innate immune response to cov infection to more fully understand nk cell contributions to viral clearance. in the context of covs, the significant morbidity and mortality associated with severe disease is due to acute lung injury (ali) and the development of ards ( , ) . pathological analysis of tissues obtained from sars and mers patients showed edematous lungs with areas of consolidation, bronchial epithelial denudation, loss of cilia, squamous metaplasia, pneumocyte hyperplasia, and bronchial submucosal gland necrosis ( , ) . histological features include diffuse alveolar damage and acute fibrinous and organizing pneumonia ( ) . a heightened inflammatory response in the lungs resulting in tissue damage has been hypothesized to explain the development of ali. there are several key factors that may be responsible for the induction of this dangerous inflammation ( ) . both sars-cov- and mers-cov replicate to high titers early in infection, which could lead to enhanced cytopathic effects and increased production of pro-inflammatory cytokines/chemokines by infected cells. chen et al. developed a pneumonia model where pulmonary replication of sars-cov- was associated with histopathological evidence of disease, including bronchiolitis, interstitial pneumonitis, diffuse alveolar damage, and fibrotic scarring ( ) . they identified a biphasic cellular immune response in which cytokines (tnf-α and il- ) and chemokines [interferon gamma-induced protein (ip)- , monocyte chemoattractant protein (mcp)- , macrophage inflammatory protein (mip)- a, rantes] were produced early, likely by infected airway epithelial cells, alveolar macrophages, and recruited inflammatory monocyte-macrophages and neutrophils, which have been shown to replace resident alveolar macrophages ( , ) . sars-cov- and mers-cov encode structural and non-structural proteins that antagonize the interferon response, which may initially delay the innate immune response but eventually potentiate inflammatory monocyte-macrophage responses ( ) . in covid- patients, liao et al. reported increased lung infiltration by macrophages identified via rnaseq analysis of bronchoalveolar lavage fluid. patients with mild cases exhibited infiltration by alveolar macrophages [fatty acid binding protein (fabp) + ] while patients with severe ards exhibited infiltration by highly inflammatory [ficolin (fcn ) + ] monocyte-derived macrophages ( ) . in the sars-cov- pneumonia model, the first wave of cytokines and chemokines induced an accumulation of nk cells, as well as plasmacytoid (p)dcs, macrophages, cd + t cells and nkt cells in the lungs. a second wave of inflammatory mediators was detected later on day post-infection [cytokines tnf-α, il- , ifn-γ, il- , il- , and chemokines mcp- , mip- a, rantes, monokine induced by gamma interferon (mig), ip- ] and correlated with lung infiltration of t cells and neutrophils ( ) . these findings are consistent with studies that have shown increased levels of activating and inhibitory cytokines and chemokines in the blood and lungs of sars patients, as well as histological studies of sars and mersinfected lungs which show extensive cell infiltrates ( , , ( ) ( ) ( ) . when huang et al. investigated the cytokine/chemokine profile in the acute phase of sars infection in a cohort of taiwanese patients, they observed an ifn-γ-led cytokine storm ( ) . they assessed sera from hospitalized patients prior to the administration of immunomodulators and found significantly increased levels of ifn-γ, il- , ip- , mcp- , mig, and il- ( ) , which returned to basal levels in convalescent sera. ip- , mig, mcp- , and il- levels were all significantly increased in death vs. survival groups. interestingly, they found an inverse relationship between ifn-γ levels and lymphocyte numbers and suggested this could either be due to ifn-γ-induced lymphocyte apoptosis or sequestration of chemokine-recruited lymphocytes in the lungs ( ) . indeed, this hyper-cytokinemia has been consistently observed in sars-infected patients ( ) . however, a recent study found that levels of six pro-inflammatory cytokines (il- b, il- ra, il- , il- , il- , and tnf-α) implicated in the cytokine storm in covid- patients did not differ significantly from levels in cytokine storms caused by other conditions. they suggest that it is therefore possible that increased levels of proinflammatory cytokines in the context of severe covid- may simply reflect an increased viral burden rather than an exuberant immune response and suggest that immunotherapies should therefore be used with caution ( ) . altogether these studies show that during acute cov infection, inflammatory monocyte-macrophages and neutrophils accumulate in the lungs and produce cytokines and chemokines that induce the activation and migration of lymphocytes, including nk cells, to the lungs, where they could be one of the main producers of ifn-γ ( ). under normal conditions, human lung nk cells are typically hyporesponsive but dynamically migrate in and out of pulmonary tissues ( ) . this supports the hypothesis that during infectious respiratory diseases, an increased recruitment of hyperresponsive nk cells would worsen the festering immunopathology ( ) . in fact, through viral-track scanning of unmapped single-cell rnasequencing data, bost et al. showed that patients with severe covid- exhibited a hyperinflammatory response with an enriched and highly proliferative nk cell compartment ( ) . high levels of ifn-γ leads to epithelial and endothelial cell apoptosis and vascular leakage, suboptimal t cell response, accumulation of alternatively activated macrophages and altered tissue homeostasis, and ards ( ) , all of which may contribute to covid- disease severity. in summary, the evidence is consistent with the hypothesis that nk cells are involved in the cytokine storm associated with cov infection and that this hyper-cytokinemia contributes significantly to disease severity via inflammation-mediated lung damage (figure ) . interestingly, this duality of nk cell roles mirrors what is seen in critically ill patients with sepsis. studies suggest that while early nk cell stimulation and ifn-γ production is beneficial to combat infections, excessive and prolonged stimulation of nk cells leads to reduced nk cell numbers and an exhausted phenotype and was associated with increased systemic inflammation in systemic inflammatory response syndrome (sirs)/sepsis and increased mortality ( ) ( ) ( ) ( ) . this review of the literature suggests that nk cells may play an important role in both cov clearance and immunopathology. the continued probing of nk cell involvement is essential for a more complete understanding of cov pathophysiology and for the deployment of immunotherapeutics. depending on the patient, the stage of disease, and other still poorly understood factors, it may be necessary to either boost nk cell activity to ensure viral clearance, e.g., at exposure or during early infection, or to finely tune nk cell effector functions in late stage infections to prevent hyper-cytokinemia and inflammatory lung damage. indeed, all covs that infect humans are zoonoses and there is an extensive reservoir of covs that could serve as a source for future pandemics ( , ) . therefore, a broader understanding of the immune response to coronaviruses and insights into therapeutic implications will be of significant value not only for the current covid- pandemic, but also for potential future pandemics. the race to vaccinate and find a cure for covid- has resulted in a spectacular effort from researchers and medical practitioners around the world. early attempts at creating targeted therapeutics have mostly relied on historical evidence from related, but not identical, coronaviruses and on the paucity of studies investigating sars-cov- . these strategies have attempted to combat the virus by targeting various stages of its life cycle starting with neutralizing sars-cov- virions using monoclonal antibodies or plasma from convalescent patients ( ) . the entry mechanism of covs has been shown to rely on binding the ace receptor and using proteases such as tmprss for s protein priming ( ) . thus, preventing ace receptor binding through blocking antibodies or competitive binding with soluble ace and tmprss protease inhibitors (camostat mesylate) are being tested ( ) . upon viral entry, the viral proteolysis or replication cycle can be targeted with protease inhibitors (lopinovir and ritonavir) ( ) or rna-dependent rna polymerase inhibitors (remdesivir and ribavirin) ( ) . at the time of writing this review, the results of these trials have not been released or are still preliminary and will require further evaluation to assess their clinical efficacy in larger cohort studies. as nk cell activity is critical for viral clearance and may be involved in disease immunopathology, a rapid and reliable predictor of nk cell function may allow for the prediction of clinical progression and the stratification of patients to receive therapeutic intervention. the remainder of this review will discuss the various ways immunotherapies are being deployed to tackle covid- , with a focus on therapies that use nk cells (table ) . lastly, while nk cells play an important role in combating viral infections, we also need to be fully cognizant of the potential damage immunotherapies could have in severe cases of covid- , and how these adverse effects may need to be attenuated ( table ) . in the absence of a clinically approved vaccine against sars-cov- , scientists have begun developing therapeutics to halt the spread of covid- by alternative strategies. studies have reported that patients infected with sars-cov- have lower levels of circulating nk cells and these express a greater level of inhibitory receptors (e.g., nkg a) while producing less ifn-γ ( , , ) . these findings provide a rationale for pursuing nk cell-based therapies as a tool to fight covid- . although nk cell-based therapies have mostly been developed for use against cancer, similar concepts and mechanisms could provide guidance in the fight against viruses. therapeutic nk cell products can be thought of as "living drugs" as they generally use either primary nk cells isolated from peripheral blood mononuclear cells (pbmcs) or are generated from stem cell precursors or genetically engineered immortalized human nk cell lines ( ) . primary nk cell products are often pre-treated and expanded in vitro with cytokines or via co-culture with target cells before being infused into patients. patients can also receive immune stimulants [e.g. recombinant il- ( ) or il- ( ) ] with the goal of improving the in vivo activity and persistence of the nk cell products ( ) as is being tested in this covid- trial (nct ). the first cell-based investigational drug to be approved by the fda for clinical testing in covid- patients is an allogeneic, off-the-shelf, cryopreserved nk cell therapy made by celularity (cynk- ), originally developed for cancer immunotherapy ( ) . the trial (nct ) is split into two phases. phase i will assess the frequency and severity of adverse events in mild, non-icu covid- patients (n = ) following infusion of nk cells derived from placental cd + cells. the subsequent phase ii trial will recruit up to patients and include a standard of care comparator at a : allocation. genetically modified nk cells are also being investigated for efficacy against covid- . chimeric antigen receptor nk cells (car-nk cells) are engineered to express virtually any receptor(s) of interest and were originally designed to enhance the ability of nk cells to eliminate cancer cells via receptors targeting egfr ( ) or cd ( ) , which are present on many cancer types and b cell hematological malignancies, respectively ( ) . although the efficacy of car-nk cells to control viral infections has yet to be rigorously tested in large scale clinical trials, the promising safety profile of car-nk cells in cancer patients, who are often immunocompromised, suggests that car-nk therapy can be well-tolerated in early phase/mild covid- patients. notably, car-nk cells are considered "safe" largely because they are less likely to lead to cytokine release syndrome (crs), a severe adverse event of car-t cell therapy ( ) . but as these are unchartered waters, it is critical that car-nk cells are used cautiously and not given to late/severe covid- patients. a phase i/ii study in early stage covid- patients (within days of illness) employing car-nk cell therapy is currently being tested using off-the-shelf nk cells derived from human umbilical cord blood expressing nkg d and ace cars (nct ). this complex five-arm study will compare the efficacy of different car-nk constructs: (i) nk cells, (ii) nk cells secreting il- , (iii) nkg d car-nk cells, (iv) ace car-nk cells, and (v) nkg d-ace car-nk cells. nkg d car-nk cells have shown promising preclinical results in cancer studies ( ) , and although not proven for sars-cov- , the rationale for expressing nkg d derives from work showing that nkg d-ligands (nkg dl) are upregulated on virally infected cells ( ) . similarly, the investigators hypothesize that expressing ace on nk cells will facilitate the elimination of sars-cov- virions and infected cells by binding the viral spike proteins-but it is unknown whether or not car-nk cells can eliminate virions or if infected cells display sufficient levels of spike protein to be recognized by ace -nk cells upon viral infection. the investigators also suggest that expressing ace on nk cells may also have a secondary benefit as a decoy cell that will be infected by the virus thereby indirectly protecting lung epithelial cells. as described previously, it is unclear whether this strategy will work to stop viral spread to healthy epithelial cells or if it will serve to perpetuate viral spread if the virus can replicate in nk cells. in arms ii-v of this trial, the car-nk cells have been engineered to secrete il- based on studies showing improved in vivo persistence of car-nk cells in cancer patients ( ) . however, the addition of the proinflammatory cytokine il- to this treatment strategy should be monitored closely for life-threatening toxicities, as elevated il- has been previously reported to accompany chronic pulmonary inflammatory diseases ( ) and mers-cov infection ( ) even if no correlation has been reported for sars-cov- . interestingly, a study compared il- levels from lung tissue homogenates following sars-cov infection in aged vs. juvenile monkeys and showed that il- concentrations were only elevated in juvenile monkeys days post-infection ( ) . this study would suggest that il- therapy may be tolerated and effective in older covid- patients that may not be able to produce il- , however this has not been confirmed. lastly, all the car-nk cells in this trial secrete gm-csf neutralizing scfv antibodies, since this cytokine has a known role in crs in cancer patients treated with car-t cells ( ) , and has been shown to be correlated with covid- disease severity in association with pathogenic cd + th cells ( ) . although nk cell based therapies are versatile, have shown safety and efficacy in cancer patients, and can be utilized in immunocompromised individuals, their potential has yet to be fully realized as an antiviral therapy. furthermore, the logistics of manufacturing nk cell products (cost and time) may pose limitations and barriers to access. for this reason, therapies focused on stimulating a patient's own nk cells offer many advantages over adoptive transfer of nk cells. the importance of the interferon pathway is underscored by the fact that many viruses actively interfere with host interferon responses, for which coronaviruses are a prime example. as described above, covs utilize numerous tactics to avoid elimination by disrupting the host type i ifn response ( ) . therefore, since the majority of covs fail to induce any detectable type i ifn response, eliciting a type i ifn response is a very attractive therapeutic strategy ( , ) . given the robust immunomodulatory nature of type i ifns, uninfected or early symptomatic patients would benefit the most from this therapy to prevent exacerbating immunopathology at later stages of disease. numerous clinical trials have been initiated investigating type i ifns ( table ) . a large study (nct ) of ∼ , medical staff allocated participants to two trial arms: (i) low-risk (non-isolated wards or laboratories) or (ii) highrisk (isolated wards in direct contact with covid- patients). in addition to the ifn-α- b nasal drops, high-risk medical staff will also receive the immune-modulating tlr activator, thymosin α , which indirectly activates nk cells through pdcs ( , ) . interestingly, reports in sars-cov- studies showed that ifn-β therapy had a -fold greater anti-viral activity in vero cells than ifn-α treatment ( ) . promising results have been published from a phase ii study (nct ) ( ) , showing that complementing lopinavir-ritonavir and ribavirin with subcutaneous ifn-β- b in mild-to-moderate covid- patients is safe with no serious adverse events reported in the triple combination therapy group, and highly effective, with significant and clinically meaningful reductions in time to complete alleviation of symptoms, hospital length of stay, and time to negative viral load ( ) . despite our best efforts in timing type i ifn therapy to mitigate immunopathology, these treatments still increase the risk of excessive activation of proinflammatory signals, which could damage host tissues and perpetuate immunopathology ( , ) . for this reason, alternative therapeutic avenues to direct type i ifn administration are being explored. type iii ifns can be a valid alternative to type i ifns, because they maintain antiviral functions yet are less toxic and less prone to mediate immunopathology ( ) . the type iii ifn, ifn-λ, activates nk cells indirectly (compared to type i ifns which directly act on nk cells), resulting in a less potent and slower immune response ( , ) . ifn-λ activates nk cells by stimulating macrophages to produce il- which in turn induce nk cells to produce ifn-γ ( ) . pegylated ifnλ is being tested in covid- positive patients with mild symptoms in the absence of respiratory distress (nct ). while ifn-λ can lead to the eventual activation of nk cells, its primary utility is in preventing the tissue damaging potential of neutrophils at mucosal surfaces, such as the lungs. however, ifn-λ also has been shown to reduce the rate of tissue repair, which in the context of covid- which has a long disease course, could mean greater risk of secondary infections. since exogenous administration of any ifn therapy poses the risk of tipping the balance toward severe covid- immunopathology, broggi et al. assessed the levels of ifns in upper and lower respiratory samples from healthy and covid- patients. in this preprint, they report that while the upper airway swabs showed similar mrna expression levels of type i and iii ifn compared to healthy controls, the balf samples of severe covid- patients had significantly elevated type i and iii ifn levels ( ) . therefore, as with all of the therapies discussed in this review, careful consideration about safe and effective timing should guide our design of clinical trials. in addition to ifn cytokine therapy, interleukin cytokine therapy can enhance the effector functions of nk cells ( ) . the use of whole, unmodified recombinant cytokines as a monotherapy has resulted in minimal success in humans in cancer immunotherapy. the earliest cytokine therapies to gain fda-approval were ifn-α and recombinant il- , approved for renal cell carcinoma and metastatic melanoma ( ) . although approved, they were limited by their in vivo half-life, marginal anti-tumor activity, and associated toxicities. the next generation of cytokine therapies were created to address these issues by first improving their biological stability through pegylation and fusion to chaperone molecules and secondly improving their specificity by fusing cytokines with antibodies or intratumoral administration. these advances in the field have allowed for the reassessment of the therapeutic potential of specific cytokines ( ) . given the importance of il- signaling and nk cell function, researchers have developed il- "superagonists" which are il- :il- r heterodimers that have better in vivo stability and bioactivity compared to monomeric il- ( ) . although at the time of writing il- superagonists are not being studied for their efficacy in covid- patients, il- superagonists, such as alt- , are safe in humans ( ) and have been used in conjunction with many of the therapies being discussed in this review including: car-nk cell therapy, adoptive nk cell transfers, checkpoint inhibitors, and the bcg vaccine in cancer ( ). it should be noted that although the therapeutic potential of cytokine therapy to specifically stimulate nk cells is enticing, exogenous cytokine therapy has a high risk for exacerbating crs if given at the incorrect time. some viruses are known to induce a state of functional hyporesponsiveness in t cells that is essential for the productive establishment of chronic viral infections ( ) . a vast body of literature has identified inhibitory checkpoint receptors, including ctla and pd- , as key regulators of this process ( ) . interestingly, cancer exploits similar mechanisms to escape the immune response, which provided the rationale for the introduction of antibodies targeting checkpoint receptors for cancer immunotherapy ( ) . ctla and pd- /pd-l blockade have revolutionized cancer immunotherapy, and their success provides a strong rationale for the use of these drugs in covid- patients, where emerging evidence suggests that the immune response is also subverted. a clinical trial (nct ) is currently assessing the efficacy of pd- blocking antibodies in severe covid- patients within h of reported respiratory distress. pd- has also been shown to play a role in regulating nk cell responses, in addition to modulating t cell functions ( ) ( ) ( ) ( ) ( ) , and has been reportedly increased in covid- patients ( ) . inhibitory receptors on the surface of nk cells regulate nk cell activation and can be targeted by antibody therapy. one of the most promising is certainly the inhibitory receptor nkg a, which binds to hla-e ( , , ) . nkg a expression is increased in circulating ( ) and balf nk cells from covid- patients, in contrast to nkg c, an activating receptor closely related to nkg a, which remains unchanged ( ) . however, it is unclear whether the observed increase in nkg a + nk cells is due selective proliferation of nkg a + cells or if it is the result of nkg a negative cells migrating out of circulation to infected tissues. circulating nk cells from patients with active hepatitis b disease had higher levels of nkg a compared to patients without active disease, however antiviral administration was associated with a reduction in nkg a expression. additionally, blocking nkg a in vitro with nkg a monoclonal antibodies led to improved nk cytotoxicity ( ) . given the association between nkg a expression in patients with severe covid- ( , ) , a promising avenue of investigation would be anti-nkg a therapy, even in light of results showing that nkg a + nk cells are tuned to present a higher level of responsiveness to stimulation ( ) . while nk cells can be stimulated directly by cytokines such as interferons and interleukins, their activity can also be enhanced through a by-stander effect following stimulation of other innate immune cells, such as macrophages and pdcs ( table ). this type of coordinated innate immune response may be more effective at cov viral clearance and mitigation of severe covid- . trained immunity has been recently described as an epigenetic re-wiring occurring in myeloid cells and progenitors upon stimulation that primes for a stronger response to subsequent stimuli, even of a different nature ( , , ) . whereas, the consensus is that myeloid cells are primarily responsible for trained immunity ( ) , it is likely that the resulting alteration in the cytokine milieu also has an effect on nk cells ( , ) . this is the case for the bcg vaccine, which has been shown to provide non-specific protection against yellow fever viral infection ( , , ) . the bcg vaccine is composed of a live attenuated strain of mycobacterium bovis originally given to young children to protect against tuberculosis (m. tuberculosis) ( ) . this vaccine provides an initial boost to innate immunity, but more importantly, results in the secretion of il- β from monocytes/macrophages, which feeds back to further stimulate the innate response ( ) . the use of a heterologous vaccine to provide enhanced protection against non-specific/new pathogens makes this a compelling strategy against covid- that warrants thorough investigation in randomized controlled trials ( , ) . the bcg vaccine is undergoing clinical trials in healthcare workers in the netherlands (nct ), australia (nct ), egypt (nct ), and the usa (nct ) to enhance overall innate immunity and provide heterologous protection against sars-cov- . interestingly, an association was found that linked lower covid- -attributable mortality rates in countries using bcg in their national immunization schedules ( ) . on the contrary, a study that assessed the association of childhood bcg vaccination in adults living in israel did not show a beneficial difference in covid- infection rates. the discrepancy between these two reports likely stem from the fact that the latter study only included adults who were previously vaccinated during childhood, supporting the fact that heterologous vaccination may not result in long-term protection ( ) . childhood bcg immunization has a limited window of opportunity to protect younger individuals from infection ( ) , but it is hypothesized that reducing the number of infected children can have a meaningful impact on curbing the spread of covid- to the rest of the population ( , ) . another heterologous vaccine in the process of clinical trial development for covid- studies is imm- (cctg id# ic ). created by immodulon therapeutics ltd, imm- is composed of heatkilled mycobacterium obuense and may have an improved safety profile over the bcg vaccine ( ) . imm- has been studied in multiple clinical trials for its non-specific immune stimulating properties as a cancer immunotherapy in pancreatic ( ) and melanoma patients ( , ) . agonists of toll-like receptors (tlrs) have been shown to broadly activate different immune populations and have had both preclinical and clinical success as adjuvants in vaccination and in the treatment of a variety of viral pathogens ( ) . for example, cpg oligodeoxynucleotides (cpg odns) are short dna sequences that contain unmethylated cpg dinucleotides which activate tlr particularly on dcs and b cells ( ). bao et al. showed that their cpg odn construct, bw , had protective effects against sars-cov- in a mechanism that relied on nk cell activation likely through a dc intermediate ( ) . amidst the ongoing sars-cov- pandemic, two clinical trials (nct , nct ) have opened using the tlr / / agonist, pul- , in order to prevent infection. ascorbic acid, more commonly known as vitamin c, has been shown to exhibit potent immunomodulatory, antioxidant, and antimicrobial effects ( ) . vitamin c has been shown to restore nk cell cytotoxicity in individuals exposed to toxic chemicals through protein kinase c expression, a critical component in lymphocyte metabolism ( ) . additional reports have shown that vitamin c also enhances the expression of nkp , cd , cd and ifn-γ production by nk cells ( ) and can increase the expression of irf in lung tissues of influenza infected, pneumonia-induced mice ( ) . vitamin c also harbors potent antioxidant attributes which can scavenge reactive oxygen species (ros) and prevent lung injury ( , ) . although ros production is an important component in the host defense response to viruses, they can be harmful to cells and lead to the pathogenesis of viral-induced host injury ( ) . the underlying rationale to investigate the therapeutic potential of vitamin c has been based on two key observations: (i) critically ill patients have lower levels of vitamin c ( ) ( ) ( ) and (ii) vitamin c has pleiotropic immunomodulatory, antioxidant, and antiviral effects ( ) . it is important to underscore that reports on the clinical outcomes of vitamin c treatment in humans are mixed and context dependent. a thorough metaanalysis on vitamin c supplementation for the common cold has been reported by hemilä and chalker ( ) . briefly, they concluded that while the incidence of colds was not reduced, the duration and severity of colds was reduced when assessing studies of regular vitamin c intake ( ). interestingly, a separate metaanalysis on vitamin c and cardiac surgery showed a reduction in the length of icu stay and shortened the need for mechanical ventilation ( ) . this is an important correlation as clinical trials are currently investigating the efficacy of vitamin c to reduce mortality and hospital burden in covid- patients ( table ) . a phase ii clinical trial (nct ) was initiated in wuhan where covid- patients will be given a high dose intravenous infusion of vitamin c. lastly, whether oral dosing of vitamin c can achieve therapeutically relevant concentrations, as described in the above studies, is currently unknown, thus caution should be taken as exceeding the recommended dietary allowance of - mg/day may lead to mild toxicities including abdominal discomfort and diarrhea ( , ). the main cause of death for covid- patients has been pulmonary complications and respiratory failure often as a result of an unregulated cytokine storm ( ) . it is unclear whether the hyperinflammation seen in severe cases of covid- is the result of the viral replication within pulmonary epithelial cells or an overactive/avalanching immune response. however, studies in sars-cov- reported hyperinflammation in later stages of disease progression, despite reduced viral titers, suggesting that the damage was immune-mediated ( ) . the most appropriate course of therapy can only be determined by elucidating the pathophysiology of disease progression. scientists and physicians, however, have had to respond quickly to the growing number of severe covid- cases and this has resulted in therapy mainly through a combination of anti-inflammatory and anti-viral interventions ( table ) . as described above, there is a potential for nk cells to contribute to the cytokine storm and therefore the development of ali. a possible explanation for the observed lymphopenia in covid- patients is that nk cells and other lymphocytes migrate out of the circulation and into pulmonary tissues to aid in the elimination of infected epithelial cells ( ) . this could be the premise for the large, unintended, amount of tissue damage that worsen the respiratory distress ( ). for this reason, therapeutics that dampen the immune response have been effective in mitigating immunopathology in severe covid- patients. the following review papers have thoroughly discussed many of these immunotherapies already ( ) ( ) ( ) ( ) ( ) ( ) , therefore, this section will focus on immunotherapies and their potential implications on nk cells. the main cytokines responsible for the life threatening respiratory distress seen in reported cases of severe covid- are il- , il- , il- , il- , g-csf, ip- , mcp- , mip a, and tnf-α ( ) . many clinical trials have focused on targeting il- signaling with anti-il- r monoclonal antibodies (e.g., tocilizumab, sarilumab, siltuximab) because of the important role il- has in propagating crs ( ) . tocilizumab, in particular, is being used as the primary therapy in the majority of these trials, likely owing to its fda approved status as a therapeutic for crs in car-t cell therapy ( ) . a case report demonstrated the potential for tocilizumab therapy in treating severe covid- illness, where a single dose on day of symptoms led to progressive reduction in il- levels and resolution of symptoms ( ) . a phase iii study (nct ) led by hoffman-la roche is recruiting patients to study the safety and efficacy of tocilizumab therapy in a randomized, double-blind, placebocontrolled, multicenter study in over patients with severe covid- pneumonia ( table ) . targeting the il- axis in severe covid- patients may also serve to improve nk cell functions as cifaldi et al. showed that increased il- negatively impacts nk cell function ( ) . they also showed that tocilizumab treatment improved nk cell function in vitro ( ) . mazzoni et al. recently reported that serum il- levels were inversely correlated (p = . ) with nk cell function in covid- icu patients. additionally, in a small subset of covid- icu patients (n = ), nk cells displayed improved markers of activation (granzyme a and perforin) after tocilizumab treatment ( ) . similar therapies have emerged in the fight against covid- including an il- r antagonist (anakinra; nct ) ( ) and cytosorb (nct ) ( ) . high dose anakinra therapy has shown promising safety and efficacy in a small retrospective study, as part of the covid- biobank study (nct ) ( ) . cytosorb therapy is used in conjunction with conventional dialysis through a whole blood cartridgebased filtration system designed to remove middle molecular weight molecules (which include inflammatory cytokines < kda) through extracorporeal cytokine adsorption ( ) . it is reported to be effective at removing ferritin and il- in a case study of a -year-old with severe crs following car-t cell therapy ( ) . jak / inhibitors (jaki) are also undergoing clinical trials in moderate-severe covid- patients, such as baricitinib (nct ). in addition to their ability to impede the production of il- , thus curb the excessive inflammation, they may also block clathrin mediated endocytosis-indicating a dual role for jaki ( ) . however, jaki can also lead to the transient increase in nk cells as shown in baricitinib treated rheumatoid arthritis patients ( ) , which could be detrimental for severe covid- patients. corticosteroids have played a key role in the treatment of auto-immune diseases over the past years ( , ) . whether endogenous or exogenous, corticosteroids decrease the number of circulating monocytes and lymphocytes and decrease synthesis of pro-inflammatory cytokines (il- , il- , tnf-α) ( ) . their strong anti-inflammatory and immunosuppressive effects make them good candidates for rapidly suppressing inflammation during early auto-immune disease or viral infections. corticosteroids have been shown to inhibit nk cells in ex vivo experiments ( , ) . while corticosteroids may delay clearance of infections, their major benefit lies in suppressing excessive innate immune responses, thus preventing lung damage and ards commonly present in severe viral infections ( ) ( ) ( ) . in fact, this was the main rationale for the widespread use of corticosteroids during mers and sars infections ( , ) . specific to covid- , some groups have advocated for the use of low-dose corticosteroids in a specific subset of critically-ill patients with refractory ards, sepsis, or septic shock ( table ) ( ) . there is one known ongoing randomized clinical trial examining the effect of the corticosteroid ciclesonide in adults with mild covid- infections (nct ). this trial is based on preclinical studies showing in vitro antiviral activity of ciclesonide against sars-cov- . while there may be a benefit to using corticosteroids in a subset of critically-ill patients with refractory ards or sepsis ( ) , their routine use in covid- is not recommended outside of clinical trials, based on expert opinion and who recommendations ( ) ( ) ( ) . corticosteroids also cause a multitude of side effects, most notably diabetes mellitus, osteoporosis, and increased risk of infections ( ) . controversially, a systematic review of over , influenza patients showed that corticosteroids actually led to increased mortality, length of icu stay, and secondary infections ( ) . additionally, one retrospective observational study examined the use of corticosteroids in covid- patients, and reported no significant association between corticosteroids and viral clearance time, hospital length of stay, or duration of symptoms ( ) . these studies highlight the need to be vigilant in our attempts to fight covid- . healthy, uninfected individuals, who are at a high risk of becoming infected (through situational circumstances such as healthcare workers) would be most fit and suitable to receive investigational prophylactic therapies such as exogenous ifns and heterologous vaccines. (b) individuals who have tested positive for covid- that are asymptomatic or have mild to moderate disease progression may benefit from receiving investigational immune stimulating therapies, including nk cell-based therapies. it is critical that investigators must be vigilant to assess the safety profile and potential immunopathologies associated with these immunotherapies. (c) in severe covid- patients, the most appropriate therapies to investigate would be those that mitigate immunopathologies, such as anti-inflammatory and immunosuppressive therapies. given the relatively low chance of toxicity and the wide range of beneficial immune effects, natural health products such as vitamin c and vitamin d can be suitable for investigation at all categories of covid- patients. non-steroidal anti-inflammatory drugs, or nsaids, are one of the most commonly prescribed drugs for treating fever, pain, and inflammation. nsaids include over-the-counter household names such as ibuprofen, naproxen, and aspirin. given the widespread use of these medications it is appropriate that researchers have investigated the potential benefits and harms of nsaids in patients diagnosed with covid- . thus far, the evidence for using nsaids in the context of covs are mixed and might not be generalizable to all nsaids as reports tended to focus on specific nsaids. these studies also focused on the potential for nsaids to act as an antiviral, with a potential added benefit of being able to treat inflammatory symptoms. one report showed that the nsaid indomethacin could directly inhibit sars-cov replication in vero cell monolayers in a dosedependent manner ( ) . the antiviral properties of naproxen have been described in the context of influenza virus ( , ) and has prompted the initiation of a clinical trial investigating the efficacy of naproxen as a treatment for critically ill covid- infected patients (nct ). nsaid therapy should be used with caution as they have been shown to interfere with immune responses and ability to produce antibodies, with ibuprofen having the greatest suppressive effect ( ) . furthermore, ibuprofen has been reported to increase the expression of the ace receptor ( ) which could facilitate sars-cov- viral entry. this finding should be considered for any current (nct ) and potential covid- clinical trial assessing ibuprofen therapy. nsaids also have been shown to have a direct suppressive effect on nk cell ifn-γ and tnf-α production ( ) which may be beneficial for late stage covid- patients. the relevance of nk cells as antiviral first responders is highlighted in patients with nkd and immunocompromised individuals who show increased susceptibility to viral infections. while there is currently little direct evidence to support a role for nk cells in the clearance of sars-cov- there is a paucity of research in this field. however, studies in admitted covid- patients with mild and severe disease reported a reduction in circulating nk cell levels and function as compared to healthy individuals. furthermore, reduced nk cell levels and function were inversely correlated with disease severity, suggesting that nk cells may be involved in some capacity. one of the potential mechanisms by which nk cells may become hyporesponsive is via sars-cov- interference with type i ifn pathways. in investigating the pathogenesis of other cov infections, namely sars and mers, studies suggest that during acute cov infection, inflammatory monocyte-macrophages and neutrophils accumulate in the lungs and produce chemokines and cytokines that induce nk cell migration and activation. as nk cells are one of the main producers of ifn-γ, they may be involved in the ifn-γ-led cytokine storm that is responsible for the induction of inflammation-mediated ali, ards, and subsequent mortality associated with covid- . inarguably, more research into the role of nk cells in covid- is required. despite the knowledge gaps in covid- pathophysiology, there has been a surge of clinical trials as the fda continues to fast-track the approval of investigational therapeutics ( ). here we have outlined potential therapeutics with a focus on mediating nk cell activity, including prophylactic treatments that could boost innate immunity in addition to therapeutics that could mitigate the immunopathological consequences of covid- , thereby relieving the 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administration the authors would like to thank dr. doug gray for his thorough editing, proofreading, and thoughtful suggestions. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © market, angka, martel, bastin, olanubi, tennakoon, boucher, ng, ardolino and auer. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - n wwn authors: pal, anandita; gowdy, kymberly m.; oestreich, kenneth j.; beck, melinda; shaikh, saame raza title: obesity-driven deficiencies of specialized pro-resolving mediators may drive adverse outcomes during sars-cov- infection date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: n wwn obesity is a major independent risk factor for increased morbidity and mortality upon infection with severe acute respiratory syndrome coronavirus (sars-cov- ), which is responsible for the current coronavirus disease pandemic (covid- ). therefore, there is a critical need to identify underlying metabolic factors associated with obesity that could be contributing toward increased susceptibility to sars-cov- in this vulnerable population. here, we focus on the critical role of potent endogenous lipid metabolites known as specialized pro-resolving mediators (spms) that are synthesized from polyunsaturated fatty acids. spms are generated during the transition of inflammation to resolution and have a vital role in directing damaged tissues to homeostasis; furthermore, spms display anti-viral activity in the context of influenza infection without being immunosuppressive. we cover evidence from rodent and human studies to show that obesity, and its co-morbidities, induce a signature of spm deficiency across immunometabolic tissues. we further discuss how the effects of obesity upon sars-cov- infection are likely exacerbated with environmental exposures that promote chronic pulmonary inflammation and augment spm deficits. finally, we highlight potential approaches to overcome the loss of spms using dietary and pharmacological interventions. collectively, this mini-review underscores the need for mechanistic studies on how spm deficiencies driven by obesity and environmental exposures may exacerbate the response to sars-cov- . obesity is an independent risk factor for increased morbidity and mortality upon infection with the severe acute respiratory syndrome coronavirus (sars-cov- ) responsible for the current covid- pandemic. several studies underscore the notion that obesity, in addition to a range of other co-morbidities and dietary factors, may increase the risk for sars-cov- ( - ). as an example, in a study from mexico, the odds of having covid- among obese patients with a bmi > kg/m was % higher than that of control non-obese patients ( ) . generally, amongst patients with symptoms, those with severe or critical conditions had much higher bmi and prevalence of obesity than the normal population or covid- negative patients ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . one study used the uk biobank data (n = , ) to show that obesity almost doubled the risk of infection, adjusted for age, sex, ethnicity and socioeconomic status ( ) . thus, it is clear that obesity results in a higher risk of increased severity of infection with sars-cov- . these findings mirror influenza infection, as obesity also independently increases risk for influenza severity and death ( ) . the high rate of obesity worldwide (e.g., in the u.s. over % of the adult population is obese) combined with the enhanced morbidity and mortality in obese individuals from infection with sars-cov- represents a public health emergency. therefore, there is a critical need to identify the underlying factors by which obese patients are at high risk of infection and complications with sars-cov- . in this mini-review, we focus on a unique aspect of fatty acid metabolism that may provide a link between obesity and immune dysregulation to sars-cov- infection. these significant insights could evoke new areas of investigation at a mechanistic level and ultimately therapeutic strategies for this vulnerable population. a wide range of metabolic factors contribute toward impaired innate and adaptive immunity in obesity. here, we discuss the role of fatty acid-derived metabolites belonging to the specialized pro-resolving mediator (spm) family. these potent lipid autacoids known as resolvins, protectins, maresins, and lipoxins are synthesized during the transition of inflammation to resolution and are critical for turning damaged tissue to homeostasis ( ) . spms are predominately synthesized from the n- polyunsaturated fatty acids (pufa) known as eicosapentaenoic (epa) and docosahexaenoic (dha) acids ( figure a) . some spms are also synthesized from arachidonic acid, an n- pufa ( figure b) . for further details on these metabolites and their immunoresolvants properties, we refer the reader to elegant reviews from serhan et al. ( , ) . there is strong literature to support a role for spms in improving outcomes upon bacterial, parasitic, and viral infections ( , ) . to exemplify, the dha-derived spm known as protectin dx (pdx), an isomer of protectin d (pd ), enhanced mouse survival upon lethal h n infection including under conditions where antiviral drugs failed to confer protection ( , ) . mechanistically, pdx inhibited viral replication by targeting the nuclear export machinery for viral transcripts. pdx specifically blocked viral transcripts from being transported to nxf , an mrna transporter. furthermore, pulmonary pdx levels were lowered upon influenza infection and were dependent on / -lipoxygenase activity. these effects were unique to pdx as other pufa-derived metabolites did not confer any improvement in survival. another study suggested that metabolites of the dha-derived spm family have utility as adjuvants for influenza vaccination. the spm precursor -hydroxydocosahexaenoic acid ( -hdha) increased antibody levels and improved survival upon ph n influenza vaccination and infection in lean mice by promoting b cell differentiation toward the formation of cd + long-lived antibody secreting cells ( ) . at a molecular level, this was driven by -hdha upregulating the expression of key transcription factors including blimp- , the master regulator of b cell differentiation toward antibody secreting plasma cells. similarly, administration of dietary dha ethyl esters, the parent compound of dha-derived spms, also boost antibody levels of obese mice ( , ) . dha improved antibody levels upon influenza infection by increasing the concentration of hydroxydocosahexaenoic acid ( -hdha), which in turn drove the formation of long-lived cd + antibody secreting cells ( ) . therefore, these studies suggest that spms have a role in controlling influenza infection through differing mechanisms including improving aspects of humoral immunity. furthermore, there is also in vitro evidence that the n- pufa-derived spm known as lipoxin b can stimulate antigen-specific igg production from memory b cells in subjects that were vaccinated for influenza ( ) . in this case, lipoxin b upregulated the expression of blimp- and xbp to increase the abundance of memory b cells. the effects of spms are not just limited to influenza virus. for instance, aspirin-triggered resolvin d is reported to have anti-inflammatory effects on murine ocular inflammation driven by infection with herpes simplex virus ( ) . in addition, aspirin triggered resolvin d can clear mouse bacterial infections such as pulmonary pneumonia, which can lower the need for antibiotics ( , ) . the cellular targets of spms in the context of viral infection and obesity are emerging. there is strong evidence for the role of spms in controlling chronic inflammation in obesity by targeting monocyte and macrophage polarization ( ) . this is particularly relevant for covid- as adipose tissue presumably expresses high levels of the human angiotensin converting enzyme (ace ), the receptor for sars-cov- . ace expression levels are likely higher in adipose tissue of the obese compared to the lungs, suggesting that adipose tissue may be a major target for sars-cov- ( ). as described above, there is strong evidence on how spms drive b cell differentiation toward long-lived antibody secreting cells. however, it is unclear how spms influence other aspects of humoral immunity to promote antibody production. for instance, the abundance of t follicular helper cells, which are required to promote b cell activation and germinal center formation, is lowered in obesity ( ) . it remains unclear if spms could be targeting the abundance of these cells to improve germinal center formation and function. in addition, obesity impairs pulmonary outcomes upon influenza infection, including lung inflammation characterized by dysregulated memory cd + t cell metabolism ( ) . given evidence to show that spms can control t cell differentiation and function, there is a need to figure | metabolic pathways by which specialized pro-resolving mediators (spms) are synthesized from polyunsaturated fatty acids (pufa). (a) epa and dha are long-chain n- pufas that serve as precursors for the biosynthesis of spms of the resolvin, protectin, and maresin families through the use of differing enzymes. epa and dha can be synthesized from the essential short-chain n- pufa known as alpha-linolenic acid. (b) the biosynthesis of lipoxins from the n- pufa arachidonic acid. arachidonic acid can be synthesized from the essential n- pufa linoleic acid. key enzymes for fatty acid elongation and desaturation in addition to spm biosynthesis are indicated for the n- and n- pufa pathways. for simplicity, the biosynthesis of all spm intermediates is not shown for the n- and n- pathways. understand the mechanisms by which spms may control the abundance and function of pulmonary t cell populations ( ) . there is evidence that obesity generally drives a unique signature of spm deficiency ( , ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . table summarizes the results of these studies. to exemplify, obese mice compared to lean controls display a rapid reduction in dha-derived spm precursors and spms in white adipose tissue within days of consuming a high fat diet ( ) . others have also reported a reduction of not only dha-derived spms but also metabolites from the epa pathway upon long term consumption of obesogenic diets in white adipose tissue and liver, which are central in driving complications of obesity ( , , , ) . as described below, these deficiencies can be overcome through dietary administration of epa-or dha-enriched marine oils. on the contrary, one study demonstrated that in a model of liver steatosis, select spms were elevated, which may be due to an attempt to lower chronic inflammation ( ) . however, in this study, the liver content of epa and dha, the parent fatty acids of spms, were lower in obese mice relative to lean controls. spm deficiencies are not just limited to adipose tissue and liver. when mice were fed a western diet, there was a significant loss of pdx in the spleen, which was reversed upon administration of dha ethyl esters in the diet ( ) . a significant reduction of -hdha, -hdha, and pdx was also reported in mice consuming a high fat diet with a modest effect on -hdha in the bone marrow ( ) . the effects were evident in male but not female obese c bl/ j mice, suggesting sex differences c bl/ j mice spleen and bone marrow -hdha, -hdha and pdx were lower in obese male but not female mice. -hdha was lowered in the bone marrow of obese male but not female mice humans with the metabolic syndrome and weight loss neutrophils metabolic syndrome patients who lost weight in a weight loss program had a -fold increase in rve compared to those participants who were in the weight maintenance group and did not lose weight ( ) in spm deficiencies. in support of this notion, it is known that synthesis of dha is higher in women than men ( ) . the notion of sex-differences in spm metabolism is also consistent with a human study that showed females were protected from endothelial impairments driven by inflammation due to elevated levels of spms compared to males ( ) . the sex-differences are intriguing, as data on covid- prevalence shows that males are disproportionally at higher risk for becoming infected than females across all ages ( ) . studies with human samples have validated murine studies by demonstrating that obese humans compared to lean controls display deficiencies of key spm precursors in circulation. a major finding was that leukocytes isolated from obese patients had reduced levels of -hdha and an unbalanced formation of dha-derived resolvins along with an increased production of the potent chemokine leukotriene b ( ) . this study found impaired activity of -lipoxygenase, a key enzyme required for spm biosynthesis to be the cause of the deficiency. interestingly, the impairment was not due to reduced cellular uptake of dha, consistent with rodent studies that show no impairment in dha levels ( ) . furthermore, when leukocytes were treated in vitro with -hdha, the biosynthesis of downstream metabolites was rescued, demonstrating -lipoxygenase to be a potential therapeutic target for improving circulating levels of spms ( ) . the observations on spm deficiencies with obesity are generally consistent with models of type diabetes, a major comorbidity of obesity ( table ) . for instance, in wounds of db/db mice, select spms were lowered relative to littermate controls ( ) . in another study, -hdha and pd were decreased in white adipose tissue of db/db mice, consistent with studies using diet-induced obese mice, although -hepe levels were elevated compared to controls ( ) . in type diabetic subjects, circulating maresin (mar ) levels were decreased compared to controls; furthermore, mar was further decreased in those type diabetics with foot ulcers ( ) . mar is of significance given its role in regulating murine insulin sensitivity and adipose tissue inflammation in models of genetic and diet-induced obesity ( ). finally, a recent study showed weight loss elevated rve levels in human subjects with metabolic syndrome ( ) , suggesting that the effects of obesity on spms could be potentially reversed through weight loss ( table ) . recent studies have noted that individuals living in areas with higher levels of ambient air pollution are at a higher mortality risk from covid- ( , ) . this was also noted with previous sars pandemics ( ) . obese individuals are uniquely susceptible to environmental exposures and it is currently unknown whether there is a higher rate of mortality from covid- in obese patients that live in areas with increased air pollution. epidemiological studies have indicated an association between obesity and air pollution ( , ) . studies of obese humans and animal models have demonstrated a greater decrement in pulmonary function after exposure to the criteria air pollutant ozone (o ), enhanced production of proinflammatory cytokines, and markers of oxidative stress ( , ) . it is currently unclear why obese individuals are more susceptible to the health effects of environmental exposures. however, experimental data have noted that obese mice and humans exposed to air pollutants have increased pulmonary and systemic tnfα, il- , markers of lung injury, and airspace neutrophilia ( ) . in addition to increased inflammation, acute exposure to o significantly reduces pulmonary and systemic dha-derived spm precursors and spms ( ) . treatment of mice with -hdha, -hdha, and pdx significantly decreased o induced pulmonary inflammation ( ) . this suppression of spm production was also noted in a murine model of nanotoxicity wherein obese mice exposed to nanoparticles had a significant suppression in pulmonary expression of -lipoxygenase and / -lipoxygenase and the production of epa-and dhaderived spms ( ) . taken together, these data suggest that the susceptibility of obese individuals to environmental lung diseases may drive an altered pulmonary immune response and a state of spm deficiency that increases the morbidity and mortality to respiratory infections, including covid- . given that spm deficiencies in obesity are potentially contributing toward poor outcomes upon sars-cov- infection, administration of spms may be beneficial ( ) . this hypothesis assumes that spms would target key mechanisms by which sars-cov- drives an uncontrolled and dysregulated pulmonary response. sars-cov- can drive a cytokine storm, which may be a potential target for intervention as spms are known to have dual anti-inflammatory and pro-resolving properties including restricting excessive immune cell infiltration ( , ) . for instance, tnf-α, il- , il- β, il- , il- , monocyte chemoattractant protein (mcp ), interferon-gamma inducible protein (ip ) and macrophage inflammatory protein a (mip a) have been implicated in driving complications associated with sars-cov- ( ) . furthermore, uncontrolled infiltration of immune cells into the lungs, due to excessive reactive oxygen species and secretion of proteases promote pulmonary destruction and thereby lower blood oxygen upon sars-cov- infection ( ). thus, spms or their parent compounds may have utility in improving pulmonary cytokine production and recruitment of pulmonary immune cells upon infection. in support of this notion, in a mouse model of infection with non-typeable haemophilus influenzae, the aspirin triggered rvd decreased the concentration of pulmonary tnfα and il- in addition to driving the clearance of macrophages ( ) . there are several approaches that could increase levels of spms. one is through dietary intervention in which the parent compounds of spms, notably epa and dha, can be delivered as either over-the-counter supplements or as prescription supplements such as lovaza, vascepa, and epanova. it is important to note that over-the-counter formulations of these fatty acids are not the same as prescriptions due to differences in dose, purity, and composition of the fatty acids. nevertheless, a recent study showed that an spm precursor containing marine oil strongly upregulated spms of the epa and dha series within hours of administration accompanied by enhanced neutrophil and monocyte phagocytosis of bacteria ( ) . however, a major limitation of this approach is that dietary epa and dha may not be as potent as direct intervention with spms ( ) . a more directed approach is to deliver spms rather than the parent compounds although the mode of delivery remains to be established. one recent study showed that spms were delivered using nanoparticles in a model of intestinal wound healing, which led to activation of pro-repair pathways in the colonic mucosa ( ) . furthermore, changes in dietary patterns may be another viable option. the western diet is associated with impaired pulmonary outcomes and a shift toward a mediterranean diet may prevent a deficiency of spms ( ) . an additional consideration is the potential role of n- pufas on outcomes related to sars-cov- infection. n- pufas are highly abundant in the western diet and there is some suggestion that select n- pufas such as linoleic acid could be driving spm deficiencies due to competition between the n- and n- fatty acids for specific enzymes that control spm biosynthesis ( , ) . this is particularly important to consider given that parenteral nutrition in a hospital setting is enriched in n- pufa-enriched oils ( ) . thus, increasing n- pufa levels alone may not be enough to increase downstream spms in the obese but could require changes in the intake of n- pufas. of course, n- pufas themselves are also critical for synthesis of spms such as lipoxins ( ) . thus, additional studies on the complex relationship between dietary n- and n- pufas with downstream spm biosynthesis, particularly in the context of viral infection are essential. overall, there is no current evidence to support changes in dietary pufa intake for improving outcomes upon sars-cov- infection, but is an important area of investigation at the pre-clinical and clinical level. finally, our understanding of the mechanisms by which sars-cov- exerts its effects are just emerging ( ), although how the virus impairs outcomes in obese individuals currently remains unknown. there is no evidence for a role for spms in controlling the host's response upon sars-cov- infection. therefore, there is a critical need to evaluate and understand the kinetics of spm biosynthesis in human and animal models of obesity during sars-cov- infection using mass spectrometrybased lipidomics. supporting experiments with gain and loss of function approaches in animal models are also required to establish that spm deficiencies in obesity exacerbate the response to the infection. it is also important to consider the host genetic profile ( ) , which could be a major consideration in developing dietary or pharmacological approaches to overcoming spm deficiencies and improving outcomes to sars-cov- for the obese. in summary, spms are key players in inflammation resolution and the infectious response. deficiencies in spms, driven by obesity, its co-morbidities, and chronic pulmonary environmental exposures, could exacerbate the sars-cov- induced morbidities and mortalities. thus, there is an urgency for mechanistic studies on spms in the context of obesity and its co-morbidities upon sars-cov- infection. ultimately, targeting spm deficiencies through dietary and pharmacological interventions may be a therapeutic approach worth investigating in order to decrease the morbidity and mortality in response to sars-cov- infection in a highly vulnerable and metabolically impaired population. ap and kg wrote the manuscript. ko, mb, and ss wrote parts of the manuscript. ss assumes responsibility for the work. all authors contributed to the article and approved the submitted version. this work was supported by nih r at (ss), nih r es (kg and ss), and nih r ai (ko). predicting mortality due to sars-cov- : a mechanistic score relating obesity and diabetes to covid- outcomes in mexico clinical characteristics of patients with corona virus disease (covid- covid- in critically ill patients in the seattle regioncase series prevalence of malnutrition and analysis of related factors in elderly patients with covid- 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thoracic society workshop report: obesity and metabolism. an emerging frontier in lung health and disease interactive effects of maternal and weaning high linoleic acid intake on hepatic lipid metabolism, oxylipins profile and hepatic steatosis in offspring linoleic acid: a nutritional quandary parenteral nutrition and lipids the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © pal, gowdy, oestreich, beck and shaikh. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -faptufak authors: meini, simone; zanichelli, andrea; sbrojavacca, rodolfo; iuri, federico; roberts, anna teresa; suffritti, chiara; tascini, carlo title: understanding the pathophysiology of covid- : could the contact system be the key? date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: faptufak to date the pathophysiology of covid- remains unclear: this represents a factor determining the current lack of effective treatments. in this paper, we hypothesized a complex host response to sars-cov- , with the contact system (cs) playing a pivotal role in innate immune response. cs is linked with different proteolytic defense systems operating in human vasculature: the kallikrein–kinin (kks), the coagulation/fibrinolysis and the renin–angiotensin (ras) systems. we investigated the role of the mediators involved. cs consists of factor xii (fxii) and plasma prekallikrein (complexed to high-molecular-weight kininogen-hk). autoactivation of fxii by contact with sars-cov- could lead to activation of intrinsic coagulation, with fibrin formation (microthrombosis), and fibrinolysis, resulting in increased d-dimer levels. activation of kallikrein by activated fxii leads to production of bradykinin (bk) from hk. bk binds to b -receptors, mediating vascular permeability, vasodilation and edema. b -receptors, binding the metabolite [des-arg( )]-bk (dabk), are up-regulated during infections and mediate lung inflammatory responses. bk could play a relevant role in covid- as already described for other viral models. angiotensin-converting-enzyme (ace) displays lung protective effects: it inactivates dabk and converts angiotensin ii (ang ii) into angiotensin-( - ) and angiotensin i into angiotensin-( - ). sars-cov- binds to ace for cell entry, downregulating it: an impaired dabk inactivation could lead to an enhanced activity of b -receptors, and the accumulation of ang ii, through a negative feedback loop, may result in decreased ace activity, with consequent increase of bk. therapies targeting the cs, the kks and action of bk could be effective for the treatment of covid- . starting in december in wuhan (hubei province, china), a novel coronavirus, designated sars-cov- , has caused an international outbreak of a respiratory illness (covid- ) , rapidly evolving into a pandemic. the clinical spectrum of sars-cov- infection varies from asymptomatic or self-limiting mild forms, occurring in most cases, to severe progressive pneumonia with acute respiratory distress syndrome (ards), and death. in a yet to be defined percentage of cases, after about one week, there is a sudden and unpredictable worsening of clinical conditions ( ) . at present, there is no vaccine or pharmacological treatments of proven efficacy for covid- ( ) and further investigation on effective drugs is required to face the current pandemic. one factor determining the lack of effective treatments is that the pathophysiology of covid- remains largely unclear. in this review, we try to address the complex link between the pathophysiology of covid- and the different proteolytic defense systems operating in human vasculature, investigating the role of the mediators involved and speculating on the possibility of pharmacological modulation. covid- is mainly a respiratory illness, but a wide variety of clinical manifestations have been described, including the central nervous ( ) and the digestive ( ) systems. most symptomatic covid- patients display manifestations such as fever ( . %), dry cough ( . %), dyspnea ( . %), myalgias ( . %), sore throat ( . %), diarrhea ( . %), and other ( ) . olfactory and gustatory dysfunctions are common symptoms, occurring in about % of patients and often presenting early in the clinical course ( , ) . low blood pressure values are frequently observed in hospitalized patients: wang et al. ( ) in their cohort reported a median of mean arterial pressure values of mmhg despite % of patients having a history of hypertension. the predominant findings of lung computed tomography are images of bilateral, peripheral and basal ground-glass opacities, crazy-paving pattern, consolidations, often in association ( ) , and ultrasonography precociously demonstrates a lung interstitial syndrome ( ) . these findings are consistent with a lung injury characterized by increased permeability, leaky blood vessels and edema, and have been confirmed by the histopathological data obtained from the lungs of patients who died from covid- , showing diffuse alveolar damage with necrosis of alveolar lining cells, pneumocyte type hyperplasia, linear intra-alveolar fibrin deposition and increased lung weight due to edema; in addition, thrombi in pulmonary arteries with a diameter of - mm, without complete luminal obstruction, and massive alveolar capillary microthrombi were observed ( ) . concerning laboratory findings, an increase of lactate dehydrogenase levels and lymphocytopenia are common. elevated levels of serum ferritin, as commonly found in viral infections, are detected in most patients ( ) . levels of interleukin- (il- ) are typically in the upper limit of the reference range and appear to correlate with disease severity ( , ) . il- -induced high levels of c-reactive protein (crp) are typically more related to bacterial rather than to viral infections ( ): in covid- patients crp values are very variable. even in non-critical patients, high d-dimer levels are found in most patients. prothrombin time is often slightly increased. levels of inflammatory and coagulation biomarkers vary considerably among patients with covid- , suggesting the existence of different biochemical/clinical phenotypes, in which the predominant systems involved and the inflammatory and coagulopathy response patterns differ. from a pathogenetic point of view, it is clear that a link (to date not yet fully clarified) exists between the clinical manifestations and alterations of the inflammatory and coagulation systems, and that these different systems are only apparently unrelated. the contact system (cs) is part of the innate immune system and of inflammatory response mechanism against artificial material, misfolded and foreign proteins and microorganisms (including viruses), found in the intravascular compartment. it remains to be clarified whether contact factors bind and activate directly on the viral surface or on infected cells ( ) . the main proteins of the cs are the factor xii (fxii), the prekallikrein (pk) and the high-molecular-weight kininogen (hk). these proteins are produced by the liver and circulate as zymogens into the bloodstream. virtually all plasma pk circulates in complex with hk. auto-activation of fxii to fxiia by contact with a variety of artificial and biological negatively charged surfaces, including microorganisms, gives rise to cs cascade. biological substances with the potential to support its activation include: dna, rna, polyphosphates retained on activated platelet surface, aggregated proteins, neutrophil extracellular traps (nets) and ferritin ( ) ( ) ( ) ( ) ( ) . kannemeier et al. ( ) presented evidence that different forms of eukaryotic and prokaryotic rna serve as promoters of blood coagulation, enhancing auto-activation of proteases of the cs, such as fxii and fxi. as the extracellular rna derived from damaged or necrotic cells represented a "foreign surface" able to activate the cs, it could be speculated that the same process may be initiated by viral rna. in addition, at times of cellular stress (i.e., hypoxia, hyperthermia, oxygen radical production) such as that observed during covid- , endogenous "alarmins" named "danger-associated molecular patterns" (damps) are released from necrotic cells. these molecules are able to initiate appropriate defense reactions associated with "sterile" inflammation and tissue repair, engaging the "pattern-recognition receptors" (prrs), such as the cell membrane and endosomal toll-like receptors (tlrs) ( ) . moreover, during viral infections, tlrs represent a host primary line of defense for pathogen sensing, due to their properties to bind diverse exogenous ligands (the "pathogen-associated molecular patterns, " pamps), including viral rna ( ); damps and pamps are able to activate the fxii and the cs. hk, complexed with pk, binds to these "surfaces": the domain is the artificial surface-binding region of hk, while the domain binds pk and fxi in order to initiate the intrinsic coagulation ( ) . after hk binds to a surface, pk is exposed to conversion to plasma kallikrein (kal) by fxiia: the binding induces a conformational change in pk so that it acquires enzymatic activity and can stoichiometrically cleave hk ( ) . in turn, kal cleaves and activates more fxii, in a powerful positive feedback loop ( ) . in addition, a vessel wall-associated serine protease, prolylcarboxypeptidase (prcp), is able to activate pk to kal independent of fxiia ( ) . the cs is involved in inflammation and in coagulation: when sufficient amounts of fxii are activated, fxiia also activates fxi (to fxia), and the intrinsic (or contact) coagulation pathway can start, leading to subsequent thrombin activation and fibrin formation. kal can influence the fibrinolytic pathway by activating plasminogen into plasmin, thus leading also to fibrin degradation ( ) . d-dimer is a soluble fibrin degradation product deriving from the plasmin-mediated degradation of cross-linked fibrin: it can therefore be considered a biomarker of concomitant activation of both coagulation and fibrinolysis ( ) . it should be remembered that plasmin can also activate fxii ( ) , and that fxiia can act as a plasminogen activator too ( ) : it has been speculated that in the very early stages of in vivo contact activation, when pk has yet to become activated, plasmin could have an initiating role ( ) , however it should be noted that plasmin is hardly present in plasma as an active protease due to the very effective action of its specific inhibitor, the α -antiplasmin: plasmin is protected from inactivation by this inhibitor only when bound to fibrin. the coagulation cascade can be modernly considered as a component and one of the intravascular effectors of innate immunity (immunothrombosis) ( ) . it is debatable whether the main physiological function of fxii is the activation of the intrinsic coagulation pathway, or if to be a component of the cs should be considered its main physiological function. for physiological hemostasis to occur, fxii auto-activation is dispensable ( ) . the fxii-induced intrinsic coagulation pathway is involved in pathological thrombus formation but is not associated with abnormal hemostasis: fxii-deficient subjects present in fact a normal hemostatic capacity ( , ) . challenging the concept of the coagulation balance, targeting fxii or its activator polyphosphate can provide protection from thromboembolic diseases (and modulate immunothrombosis) without interfering with hemostasis and increasing the risk of bleeding ( , , , , ) . covid- is a condition clearly characterized by coagulopathy, as testified by the extensive microthrombosis reported in lung autopsies ( ) , and the high levels of d-dimer displayed by most patients indirectly testify the hyperactivation of both coagulation and fibrinolysis, and overwhelming immunothrombosis. it should be remembered that lowmolecular weight heparins (lmwhs) have been extensively used in hospitalized covid- patients for preventing venous thromboembolism and thrombotic complications, and are currently investigated in randomized controlled trials (i.e., clinicaltrials.gov identifier: nct ). it is interesting to note that in the physiological state fxii acts as a growth factor promoting angiogenesis and wound repair ( ) , but pathologically it can promote lung fibroblast proliferation leading to pulmonary fibrosis ( ): covid- may also evolve into pulmonary fibrosis. the archetypal contact activation disease state is sepsis from any etiology. there is no specific data on the model of sars-cov- , but data may be gathered from other viral models. it is known that herpes simplex virus type- (hsv ) can trigger and amplify coagulation through the contact phase and intrinsic coagulation pathway: both an inhibitor of fxiia (corn trypsin inhibitor), and anti-fxii, anti-kal and anti-fxi antibodies were able to inhibit hsv -initiated clotting ( ) . moreover, pk and fxii levels are significantly lower in patients with dengue hemorrhagic fever (dhf), probably due to activation and consumption ( ) . it has been mentioned that cs is part of the innate immune system: it is known that non-structural protein (nsp ) of coronaviruses results able to block the host innate immune response ( ) , and other nsp play a role in evading host recognition ( ) . the kallikrein-kinin system (kks) is mainly a host inflammatory response mechanism, and although kks and cs overlap and interact in the intravascular compartment (plasma kal is part of both systems), the use of the two terms has different implications. activation of kks finally leads to the liberation of bradykinin (bk), and plays an essential role in inflammation, but not in blood coagulation ( ) . upon activation by fxiia, kal cleaves hk, releasing from its domain the nonapeptide bradykinin (bk- - or bk) ( ); bk is converted by a carboxypeptidase to [des-arg ]-bk (bk- - or dabk), an active metabolite ( ) . during inflammation, plasmin potentiates the cleavage of hk by kal, thus enhancing bk production ( ) . bk and dabk bind to two pharmacologically distinct g protein-coupled receptors: the bradykinin b receptor (b r), whose ligand is bk, and the b receptor (b r), whose main agonist is dabk ( ) . the b r is widely and constitutively expressed in mammalian cells (e.g., endothelial and smooth muscle cells), whereas the b r is mostly inducible under the effect of cytokines during infections and immunopathology ( ) . after binding through its b r, bk activates signaling pathways resulting in increased vascular permeability, vasodilation, edema formation, hypotension, pain, fever ( ) : all typical clinical features of covid- . bk is one of the most potent vasodilatory substances in humans: it is known that the bk-mediated angioedema is responsible for a very high percentage of serious morbidity and mortality ( ) . bk is also one of the most potent inflammatory mediators, able to stimulate the production of superoxide radicals and nitric oxide and to modulate the mobilization and release of histamine, arachidonic acid, prostaglandin e , prostacyclin, pro-inflammatory interleukin- , and tumor necrosis factor (tnf)-alpha ( ) . thereafter, bk has shown to increase il- production via b r in colorectal cancer cell ( ) , and the b r-antagonist icatibant was able to inhibit the bk-induced il- release ( ) . this effect is interesting: also chloroquine, that has been extensively used and investigated for covid- treatment, was able to reduce il- production by monocytes/macrophages ( ) . bk also stimulates tissue plasminogen activator (t-pa) release from human endothelium through a b r-dependent mechanism: this effect was significantly reduced in smokers ( ) . a strong link between kks and the renin-angiotensin system (ras) is testified by the fact that b r forms homoand heterodimers with several receptors of the ras, that are important for some physiologic functions, including thrombosis risk regulation. the b r also complexes with endothelial cell nitric oxide synthase, while the b r couples with inducible nitric oxide synthase ( ) . b r mediates several responses including vasodilation, hypotension, and increased vascular permeability ( ) : all typical features of covid- . human kallikreins have been detected in many tissues ( ), including the epithelia of the upper and lower respiratory tract: there are in fact two classical pathways for the generation of kinins, the plasma and the tissue kks. as the substrate of plasma kal is hk (leading to bk), the substrate of tissue kallikreins is the low-molecular-weight kininogen, leading to formation of the decapeptide lys-bradykinin or kallidin (kd). a carboxypeptidase leads to the formation of the active metabolite [des-arg ]-kd (dakd) from kd. kd mainly binds to b r, while b r has a high affinity for dakd ( ) . it is not known if sars-cov- infection is specifically associated with kinins dysregulation, but this happens in several viral models. low levels of hk have been observed in dhf patients, perhaps due to proteolysis and generation of bk ( ). taylor et al. ( ) previously described a novel mechanism of hantavirus-induced vascular leakage involving activation of the kks, showing that incubation of fxii, pk, and hk with hantavirus-infected endothelial cells results in increased cleavage of hk, higher enzymatic activities of fxii/kal and increased liberation of bk, that dramatically increased cell permeability. furthermore, the alterations in permeability could be prevented using inhibitors directly blocking bk binding, the activity of fxii, or the activity of kal ( ) . infection of guinea pigs by nasal instillation of parainfluenza- virus induced airway hyperreactivity and influx of inflammatory cells into lung tissues, and these responses were attenuated by b rantagonists ( ). tissue kallikrein was shown to intervene early during influenza infection, enhancing the antiviral defense, and the decreased expression observed in patients with chronic obstructive pulmonary disease could contribute to the less favorable evolution of influenza in this group ( ) . therefore, the kks appears to be involved in vascular leakage and inflammatory response observed during different viral infections ( ) . we can speculate that modulation of the cs and the kks may limit the evolution towards a frankly dysregulated host response also in sars-cov- infection. moreover, a role of bk in covid- pathogenesis is suggested by several clinical features and symptoms observed in patients: given the close interconnection with the ras, these aspects will be further discussed in the next chapter. the renin-angiotensin system (ras) is classically known for its effects on the cardiovascular system and fluid homeostasis, but it has become clear that the ras is present in many tissues, where evidently has a role to play ( ) . starting from angiotensinogen, whose primary source is the liver, the ras leads to the production of the multi-functional peptide hormone angiotensin ii (ang ii). renin first catalyzes the cleavage of the peptide angiotensin i (ang i) from the n-terminus of the angiotensinogen molecule, then, sequentially, the dicarboxyl-peptidase angiotensin converting enzyme (ace) removes two amino-acids from the c-terminus of ang i to form ang ii ( ) . ang ii exerts its main functions binding to two specific g-protein coupled receptors: the atii type receptor (at r) and atii type receptor (at r) ( ) . ace is present in many tissues and is particularly abundant on the endothelium of the lungs: it is mainly anchored to the plasma membrane through a single trans-membrane domain, but a soluble form has also been described ( ) . apart from its well-known role as a peptidyl-dipeptidase forming ang ii, ace is also described as a kininase ii, able to inactivate bk, as well as kd ( ) . the affinity of ace appears to be higher for bk than for ang i, suggesting that ace-inhibition may really involve the bk degradation more than the ang ii production ( ) . bk-evoked sensitization of airway sensory nerves is believed to be the main mechanism for ace-inhibitor-induced dry cough ( ) : considering that dry cough is very frequently observed in covid- patients, this pathway could in part explain the pathogenesis of this symptom. additionally, a role of the bk has been hypothesized also for gustatory and olfactory dysfunctions ( ); again, ace-inhibitors can cause olfactory dysfunction ( ) . in addition, over the last years, knowledge of the biology and physiology of another enzyme besides ace, the angiotensin converting enzyme (ace ), has accumulated ( ): ace is widely expressed, including type alveolar epithelial cells, endothelial cells and enterocytes ( , ) . both ace and ace act as zinc metallopeptidases (ace only acts as a carboxypeptidase), but differ for substrate specificities, displaying counterbalancing roles in the ras. ace converts ang i into angiotensin ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , and ang ii into angiotensin ( - ); unlike ace, ace does not cleave bk, and is insensitive to conventional ace-inhibitors ( ) . ang ii can be converted to angiotensin ( - ) also by prcp in the low-ph areas of the kidney ( ) . angiotensin - , acting on mas receptor, exerts vasodilatory effects, thus diminishing and opposing the vasoconstrictor effect, mainly at r-mediated, of ang ii; moreover, it displays anti-fibrotic, anti-oxidant and anti-hypertrophic protective properties ( ) . therefore, ace expression seems to protect from lung injury. sodhi et al. ( ) observed that a reduction in pulmonary ace activity contributes to the pathogenesis of lung inflammation, resulting in prompt onset of neutrophil infiltration and more severe inflammation. imai et al. ( ) showed that the loss of ace expression in acute lung injury leads to leaky pulmonary blood vessels through at r stimulation, while the at r protects against lung injury during sepsis. angiotensin - has shown beneficial biological effects via the at r, resulting in protective effects on cardiac and vascular remodeling ( ) and against pulmonary arterial hypertension, inflammation and fibrosis ( ) . sars-cov- binds ace for host cell entry, through the binding of its major spike glycoprotein (s ) to the n-terminal region of the receptor ( ); chloroquine seems to interfere with ace glycosylation, thus possibly preventing sars-cov- binding to target cells ( ) . following binding with sars-cov- , a loss of ace function occurs, driven by endocytosis and activation of proteolytic cleavage and processing ( , ) . it can be assumed that this downregulation may be involved in the pathophysiology of covid- and its manifestations. dabk is a substrate of ace , and the attenuation of ace activity leads to impaired dabk inactivation and thus to enhanced b r signaling. in a mouse model, the lack of ace function with consequent accumulation of ang ii, through a negative feedback loop, frontiers in immunology | www.frontiersin.org resulted in a secondary reduction of ace activity (at the molecular level, ang ii downregulates renal ace gene and enzymatic activity levels, as well as renin gene expression): these crosstalk effects between ace and ace appeared to be sexdependent and more evident in males ( ) . it is known that covid- affects male patients in a larger percentage ( ) and with worse outcomes ( ) . the reduced activity of ace is also expected to result in further bk accumulation. moreover, it has been recognized in vitro that ang ii, through the stimulation of at r, is associated with increased expression of prcp, leading to a kal-mediated increased formation of bk ( ) . since sars-cov- binds to ace receptors to enter host cells, and intravenous infusion of ace-inhibitors and angiotensin receptor blockers (arbs) in experimental animal models increased the amount of ace receptors in the cardiopulmonary circulation, it has been speculated that patients chronically taking these drugs may be at increased risk of worse outcomes from covid- ( ) . however, to date, there are no conclusive data demonstrating beneficial or adverse outcomes with background use of ace-inhibitors, arbs or other ras antagonists among covid- patients with a history of cardiovascular disease treated with these drugs ( ) ( ) ( ) . for the pathophysiological considerations previously made, however, in our opinion, it remains debatable if ace-inhibitors, for their action on bk, should be temporarily suspended during the acute phase of illness, especially in the case of low blood pressure values. finally, it is interesting to observe that in an experimental malaria model (plasmodium parasites during blood stages release kinins), exposure to captopril (an ace-inhibitor that leads to the reduction of bk degradation) resulted in death in mice, while the concomitant administration of chloroquine protected them. b r-knockout mice presented a significant reduction of survival when compared with wild-type mice, unlike the b r-knockout ones ( ) . in this inflammation/infection model, chloroquineinduced upregulation of b r expression proved protective: the full meaning of this result is unclear but might indicate that the selective inhibition of b r could represent a rational modulation of dysregulated bk pathway during infection. could the same considerations apply to covid- ? hereditary angioedema (hae) represents the archetypal kks disorder and can be due to a deficiency of c -inh (type ), an abnormal c -inh molecule (type ), or a gain-in-function of fxii with consequent plasma c -inh consumption (type ) ( ). thrombin formation is not considered a feature of this disorder: even if patients with acute attacks have elevated d-dimer levels, they do not display an increased thrombotic risk ( ) . clinical pictures of activation of cs and kks without (such as hae) and with thrombin formation (such as sepsis) can be in fact distinguished ( ) ( ) . moreover, c -inh inhibits selectin-mediated leukocyte adhesion, regardless of its protease inhibitory activity ( ) . wygrecka et al. ( ) showed that c -inh is able to inhibit the cytotoxic activity of extracellular histones (that play a determining role in pulmonary injury leading to ards) and the release of several cytokines, such as tnf-alpha, il- b, and il- . it is interesting to note that accumulation of extracellular histones has been detected during infection due to influenza virus, and anti-histone antibodies have led to a marked decrease in the lung damage consisting of widespread pulmonary microvascular thrombosis, endothelial necrosis, hemorrhagic effusions and edema ( ) . these histopathological findings are observed also in covid- , although there are several differences compared to the influenza model ( ) whose discussion goes beyond the scope of this review. although there is actually no specific evidence regarding sars-cov- infection, it can be assumed that c -inh might have beneficial effects also in this case, both through the inhibition of the cs and kks, especially regarding the bkinduced vascular leakage and edema formation, and its antiinflammatory activity mediated by inhibition of complement activation and histone toxicity. figure shows the interconnection between the different human proteolytic systems operating in the vasculature, proposing a picture of an integrated host response to sars-cov- infection. table lists some available drugs potentially representing effective therapeutic approaches in covid- , by modulation of the pathways and systems whose involvement has been hypothesized in its pathogenesis. the hypothesis of the involvement of different human proteolytic defense systems operating in the vasculature in the pathogenesis of covid- has recently been proposed also by other authors. van de veerdonk et al. ( ) hypothesized that a kinin-dependent local lung angioedema via b r and eventually b r is an important feature of covid- and proposed that blocking the b r and inhibiting plasma kal activity might be beneficial in early disease, preventing ards. roche and roche ( ) emphasized the pivotal role of bk and dabk, suggesting that the b r-antagonist icatibant might be able to interrupt the dysregulated pathway, thereby improving clinical outcomes. colarusso et al. ( ) proposed instead to block pharmacologically the kks upstream of the bk, by means of lanadelumab. regarding b r-antagonists, several companies have in past developed orally available molecules, and some of these entered phase ii clinical trials, but none have been developed further; possible reasons for this failure may be inefficacy in humans due to species differences, or human specific adverse effects ( ) . in our opinion, the rational for modulating these pathways is strong but to date few data for covid- are available. however, the exceptional nature of this pandemic and the lack of effective interventions of proven efficacy makes it necessary to explore further therapeutic possibilities. understanding the pathogenetic mechanisms underlying covid- is crucial for the development of new effective therapeutic approaches modulating the cs, the kks, the ras and the coagulation/fibrinolysis system. the kks inhibitors lanadelumab and ecallantide, licensed for the treatment of hae, and several oral kks inhibitors in clinical development, should be assessed for their efficacy in the treatment of patients with covid- . the same holds for icatibant, a selective b r antagonist used for on demand treatment in hae. other promising cs-linked targets or mediators that should be explored in covid- include anti-fxiia antibodies and c -inh. this pathophysiological therapeutic approach could be of great value also for other viral infections. publicly available datasets were analyzed in this study. this data can be found at the appropriate doi link of every cited article. the csl behring funded the publishing support and journal styling services, but had no role in the conduct of the research, preparation of the article, in study design, in the collection, analysis and interpretation of data, in the writing of the report, and in the decision to submit the article for publication. evaluation and treatment coronavirus (covid- ) drug treatment options for the -new coronavirus ( 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protect against lung injury the role of extracellular histones in influenza virus pathogenesis kallikrein-kinin blockade in patients with covid- to prevent acute respiratory distress syndrome a hypothesized role for dysregulated bradykinin signaling in covid- respiratory complications we acknowledge seed medical publishers, that provided publishing support and journal styling services. key: cord- -bqh jkds authors: raony, Ícaro; de figueiredo, camila saggioro; pandolfo, pablo; giestal-de-araujo, elizabeth; oliveira-silva bomfim, priscilla; savino, wilson title: psycho-neuroendocrine-immune interactions in covid- : potential impacts on mental health date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: bqh jkds coronavirus disease (covid- ) is caused by the severe acute respiratory syndrome coronavirus (sars-cov- ). the impacts of the disease may be beyond the respiratory system, also affecting mental health. several factors may be involved in the association between covid- and psychiatric outcomes, such as fear inherent in the pandemic, adverse effects of treatments, as well as financial stress, and social isolation. herein we discuss the growing evidence suggesting that the relationship between sars-cov- and host may also trigger changes in brain and behavior. based on the similarity of sars-cov- with other coronaviruses, it is conceivable that changes in endocrine and immune response in the periphery or in the central nervous system may be involved in the association between sars-cov- infection and impaired mental health. this is likely to be further enhanced, since millions of people worldwide are isolated in quarantine to minimize the transmission of sars-cov- and social isolation can also lead to neuroendocrine-immune changes. accordingly, we highlight here the hypothesis that neuroendocrine-immune interactions may be involved in negative impacts of sars-cov- infection and social isolation on psychiatric issues. in december , a new outbreak of severe acute respiratory syndrome (sars) emerged in wuhan, china. caused by severe acute respiratory syndrome coronavirus (sars-cov- ), the coronavirus disease (covid- ) caused a national outbreak of severe pneumonia in china and quickly spread worldwide. according to the world health organization (who) official website, on may th, , , , people have been tested positive for sars-cov- infection and , deaths have resulted from sars-cov- worldwide ( ). the disease, initially restricted to china, is now a pandemic, comprising all continents so far except for antarctica, thus having become a major planetary health issue ( ) . the most common symptoms of covid- are fever, cough, dyspnea, sputum production, myalgia, headache, diarrhea, rhinorrhea, anosmia, and ageusia ( , ) . nevertheless, symptoms of post-traumatic stress disorder (ptsd), anxiety and depression have also been prevalent in patients infected with covid- ( , ) . besides, sars-cov- rna was detected in the cerebrospinal fluid of a patient ( ) and increasing evidence points out that coronaviruses (covs) may invade the central nervous system (cns) ( ) . thus, we describe here the likely routes by which sars-cov- can invade the brain. since covid- is associated with increased levels of pro-inflammatory cytokines ( ) , an immune signature shared with several psychiatric disorders, we propose how the relationship between sars-cov- /host can possibly impair interactions between the immune, nervous and endocrine systems, leading to psychiatric symptoms. furthermore, once millions of people worldwide are isolated in quarantine to minimize the transmission of sars-cov- ( ), we also discuss herein evidence on the negative impacts of social isolation measures upon mental health, gathering evidence that explains how social isolation can also lead to neuroendocrine-immune changes, impairing mental health. accordingly, it is likely that both sars-cov- infection and social isolation epidemiological measures to contain the pandemic can lead to changes in psychoneuroendocrine-immune circuits with impact on the appearance and/or evolution of mental health impairments in infected subjects, as well as in those individuals that, even though not being infected, are subjected to social isolation due to one or more risk factors. finally, we provide some suggestions for how future research could confirm the hypotheses outlined here, as well as intervention strategies that mitigate the impact of covid- pandemic on mental health. coronaviruses (covs) comprise a large enveloped nonsegmented positive-sense rna virus, which belong to the family coronaviridae, within the order nidovirales ( ). they are classified in four genera, namely alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus, based on their phylogenetic relationships and genomic structures ( ) . the α-cov and β-cov are able to infect mammals, whereas the γ-cov and δ-cov tend to infect birds ( ) . previously, six covs have been identified as capable of infecting humans (human coronaviruses-hcovs): α-cov hcov-nl and hcov- e, and β-cov hcov-oc , hcovhku , middle east respiratory syndrome coronavirus (mers-cov), and severe acute respiratory syndrome coronavirus (sars-cov). the last two hcovs are considered the most lethal among them. however, the novel sars-cov- has shown a mortality rate that is presently also expressive ( ) . sars-cov- infection leads to a clinical picture characterized by highly lethal pneumonia with symptoms similar to those reported for sars-cov and mers-cov ( ) . genomic analysis show that sars-cov- shares highly homological sequence with sars-cov ( ) . although the existence of more than one receptor for this virus cannot be excluded by now, evidence so far reveals that sars-cov- enters human host cells using the same receptor of sars-cov, the human angiotensin-converting enzyme (hace ) ( ) . consequently, most of the infection mechanisms detailed for sars-cov could be applied to this novel virus. hcovs may enter the cns through distinct routes: hematogenous and/or neuronal retrograde dissemination ( ) . the neuronal route can occur through at least two different pathways: (a) via olfactory nerves and/or (b) via enteric nervous system ( , ). an experimental study using k -hace transgenic mice for the expression of hace (i.e., human sars-cov receptor) showed that sars-cov, when given nasally, could invade the brain, likely via the olfactory nerves ( ) . however, the non-expression of ace in neurons in the olfactory system ( , ) leads to question whether this is really a possible route for sars-cov- entry into cns, although it is not yet possible to rule out the possibility that other ace -independent mechanisms are involved in the entry of sars-cov- into host cells. by contrast, ace expression is abundant in small intestine endothelial cells ( ) , which connect with neurons in the enteric nervous system. in addition, gastrointestinal symptoms are commonly seen in a part of patients with covid- ( , , ) and sars-cov- was isolated from oral and anal swabs of these patients ( ) . in this way, the enteric nervous system, via the vagus nerve, can also be a possible pathway for sars-cov- to enter the cns. similarly, the hematogenous route can occur by at least two mechanisms: (a) through infected leukocytes that cross the blood-brain barrier carrying the virus to the brain and/or (b) through direct infection of brain microvascular endothelial cells, which express ace ( ) . nonetheless, the hematogenous route does not seem to be involved in the cns invasion by sars-cov, since virtually no viral particles were detected in non-neuronal cells of the infected brain areas in the early stage of infection ( ) ( ) ( ) . yet, the precise route(s) by which sars-cov enters the cns remain(s) to be determined. the recent sars-cov- rna detection in the cerebrospinal fluid of a patient with covid- ( ), as well as its similarities with the sars-cov, emphasizes the need to conduct studies aiming at evaluating the neuroinvasive potential of sars-cov- in animal models and humans. sars-cov genomic sequences in human brain tissues were found mainly in neurons of the cerebral cortex and hypothalamus, but not in the cerebellum ( , ) . however, pre-clinical studies with k -hace mice infected by sars-cov revealed viral particles during acute phase in other brain regions besides the cortex and hypothalamus, such as cerebellum, midbrain (e.g., dorsal raphe and substantia nigra), thalamus, amygdala, hippocampus, basal ganglia (e.g., caudate-putamen and nucleus accumbens), cortex (e.g., frontal, infralimbic, and cingulate), and olfactory bulb ( , ) . in these animals, a rapid spread throughout the brain was accompanied by significant neuronal loss in the cingulate and infralimbic cortices and the anterior olfactory nucleus ( ) . interestingly, high levels of cytokines and chemokines, most notably interleukin- (il- ) and interferon gamma (inf-γ) were found in brain of k -hace transgenic mice infected by sars-cov ( , ) . rather surprisingly, minimal signals of local inflammation were observed, and apoptotic or necrotic cells were not detected ( ) . considering the high-expression of inflammatory mediators along with a lack of other inflammatory signals, how sars-cov can be leading to neuronal death remains unknown. cell death non-inflammatory processes, such as autophagy, may be an explanation ( ) . since autophagy is related to several neurodegenerative and psychiatric diseases ( ) , evaluating whether infection by sars-cov- can lead to neuronal death by autophagy may also be important for future relationships between sars-cov- infection and mental health outcomes. several studies have demonstrated psychiatric manifestations in patients with mers or sars during the acute phase, such as increased stress levels, impaired memory, symptoms of depression, anxiety, ptsd, psychoses, and suicidal behavior ( ) ( ) ( ) ( ) ( ) ( ) . long-term damage has also been seen in these patients. survivors of sars, months or years after the acute phase of the infection, may also exhibit impaired memory, sleep disturbances, increased levels of stress, depression, anxiety, and ptsd symptoms ( , ( ) ( ) ( ) ( ) ( ) . to date, few studies have evaluated the possible mental health outcomes of sars-cov- infection. however, corroborating the data observed in patients with sars, a study recently demonstrated a prevalence of . % of ptsd symptoms in patients with covid- during acute phase ( ) . another study reported a prevalence of . and . % of anxiety and depression symptoms, respectively, in patients with covid- ( ). taken together, these data indicate that infection with these hcov, especially sars-cov- , can yield a negative impact on mental health, both in the short-and longterm time windows. supplementary table summarizes studies that reported mental health outcomes in patients with mers, sars, or covid- . many factors can influence the results of studies that have reported symptoms or development of psychiatric disorders in patients with mers, sars, or covid- . among them (a) the work directly with health care, (b) the presence of family history of psychiatric illnesses, (c) less social support, (d) older age, (e) the isolation, and (f) the use of high doses of steroids during the acute phase (see supplementary table ) . however, some patients who survived sars displayed psychiatric manifestations that appear to be disproportionate to the extent of lung infection or expected side effects of corticosteroid therapy ( , , ) . furthermore, it has been reported that one patient developed progressive neurological symptoms starting at day after the onset of the disease. this patient eventually died due to the sars-cov infection, and an autopsy revealed the presence of the virus in the brain, together with neuronal necrosis, glial hyperplasia, and edema ( ) . although the studies cited above have been conducted with small samples of patients, they suggest that the psychiatric manifestations seen in at least some patients might be a direct effect of the infection of sars-cov. also, studies with humans are important to evaluate and highlight the possible psychiatric outcomes in patients with sars-cov- infection. a "cytokine storm" has been proposed as a key mechanism in the sars-cov- pathophysiology and related to lung damage and lethality observed in patients bearing covid- ( ) . accordingly, increased circulating levels of several cytokines have been found in patients with mers, sars, or covid- (see table ). interestingly, high levels of pro-inflammatory cytokines (e.g., il- and inf-γ) were also found in the cns of k -hace transgenic mice infected by sars-cov ( , ) . this evidence supports the existence of an immune signature characterized by increased levels of pro-inflammatory cytokines involved in the pathophysiology of different pathogenic sars-cov in humans. furthermore, higher serum levels of pro-inflammatory cytokines (e.g., il- and ifn-γ) and chemokines were found in sars patients with severe disease, as compared to individuals with uncomplicated sars ( ) ( ) ( ) . recently, dysregulation of the immune response similar to sars-cov infection has been observed in patients with sars-cov- in wuhan (china). particularly, a significant increase in the serum levels of several pro-inflammatory cytokines, or corresponding cytokine receptors, in severe patients (n = ) than the non-severe ones (n = ), including il- , tumor necrosis factor alpha (tnfα) and interleukin- receptor (il- r) ( ) . similarly, intensive care unit (icu) patients (n = ) with severe sars-cov- infection displayed higher plasma levels of cytokines, such as il- and tnf-α, when compared with non-icu patients (n = ) ( ) . a previous study identified psychiatric manifestations (e.g., psychosis, cognitive impairments, depression, and anxiety symptoms) in patients during the acute phase of sars-cov infection ( ) . the authors also found an association between the severity of symptoms and some psychiatric outcomes. if the increase in cytokine levels and the manifestation of psychiatric symptoms are related to the severity of the symptoms of sars-cov infection, the "cytokine storm" might also be related to the "mental health thunderstorms" seen in patients with covid- ? accordingly, a possible mechanism concerning the relationship between sars-cov- infection and mental health outcomes is the involvement of neuroimmune networks. table shows that increased levels of various cytokines can be seen in several psychiatric disorders, an immune signature shared with the sars-cov- infection. soluble cytokines that reach the brain, or corresponding local altered levels can influence synthesis, release and reuptake of several neurotransmitters, including monoamines, such as dopamine, norepinephrine, and serotonin ( ) . changes in the metabolism of neurotransmitters are involved in the pathophysiology of various psychiatric disorders, such as depression, anxiety, ptsd, and obsessive-compulsive disorder ( , ) . since changes in cytokine levels can lead to a disruption in the metabolism of neurotransmitters, triggering behavioral deficits, we hypothesize than the immune system can be placed as a link between sars-cov or sars-cov- infection and mental health impairments. evidence shows that cytokines also play a key role in learning and memory processes. in healthy conditions, an increase in gene il- n = icu vs. n = healthy n = severe vs. n = moderate n = critical > n = severe > n = mild n = / icu n = severe vs. n = mild n = severe vs. n = non-severe n = severe vs. n = moderate n = spo < % vs. n = spo ≥ % il- β n = infected vs. n = healthy n = severe vs. n = moderate tnf-α n = infected vs. n = healthy n = icu vs. n = non-icu n = severe vs. n = moderate n = critical vs. n = severe vs. n = mild n = severe vs. n = non-severe n = severe vs. n = moderate il- n = infected vs. n = healthy n = icu vs. n = non-icu n = severe vs. n = moderate n = critical vs. n = severe vs. n = mild n = / icu n = severe vs. n = non-severe n = severe vs. n = moderate n = spo < % vs. n = spo ≥ % il- n = icu vs. n = healthy n = icu vs. n = non-icu n = severe vs. n = non-severe il- r n = severe vs. n = moderate n = critical > n = severe > n = mild n = severe vs. n = moderate expression of il- β, il- receptor antagonist, il- , and il- occurs in hippocampus during long term potentiation (ltp), a process considered to underlie certain forms of learning and memory ( ) ( ) ( ) . while il- β is related to ltp maintenance, acquisition of learning and memory consolidation, il- has opposite effects. however, during peripheral and central diseases in which the brain levels of il- β and il- are increased, both cytokines tend to inhibit the synaptic plasticity, learning, and memory ( ) . importantly, high levels of il- were found in blood of sars-cov and sars-cov- infected patients (see table ), as well as in cns of k -hace transgenic mice infected by sars-cov ( , ) . impaired memory has also been observed in both acute and convalescent phases of sars infection in humans (see supplementary table ). therefore, it is possible that the increased levels of il- are related to the cognitive impairments observed in sars patients. such issue should be evaluated in future studies. interleukin- is a well-known pleiotropic cytokine expressed in low levels in healthy individuals, in the presence of homeostasis alterations it becomes higher and rapidly detected, and even after stress agent removal, its levels can be maintained elevated and cause diseases ( , ) . accordingly, a dysregulation of this cytokine expression counts for the development of psychiatric disorders ( ) , as seen in table . recently, gao et al. ( ) showed increased levels of cytokines in patients with sars-cov- , especially il- , which seems to be directly related to the severity of the disease. evaluating the blood parameters of adult patients positive to sars-cov- and subdivided in groups (mild and severe) they found a significant increase in the combined detection of il- and d-dimer specially in the severe cases, pointing out the il- and d-dimer combination as a potential biomarker to identify early stages or the prognosis of the covid- disease ( ). in another study, patients were subdivided in three groups (mild, severe, and critical) and had hematological parameters followed up during disease evolution. it was shown that the more severe the case was, the higher was the il- level ( ). liu et al. demonstrated that not only increased levels of il- related to the severity of covid- , but also that decreased levels of il- were positively correlated with the treatment effectiveness and remission of the disease ( ) . in this sense, the humanized anti-interleukin- -receptor (il- r) monoclonal antibody (tocilizumab), a drug used against rheumatoid arthritis ( ) that inhibits il- signaling, has been administered experimentally in treatment of covid- ( ) . the retrospective evaluation of patients demonstrated that tocilizumab was able to improve the respiratory function and restored the levels of lymphocytes in the blood, which can be promising ( ) . in a second vein, a meta-analysis study pointed out that treatment with anti-cytokine drugs, including tocilizumab, may have an antidepressant effect ( ) . accordingly, we can conceive that this type of treatment may represent a promising therapeutic alternative to be attempted in humans, not only has beneficial effects for respiratory symptoms associated with covid- , but also for possible depressive symptoms related to the disease. thus, it would be interesting for future clinical studies to evaluate the effects of tocilizumab and other pharmacological treatments not only on symptoms and tests related to respiratory and immune functions, but also on the psychiatric symptoms. it is important to notice that some individual biological characteristics associated with impaired immunity may influence not only the natural history of covid- , but also the associated psychiatric outcomes. in this context, obesity, which is linked with systemic inflammation and impaired immunity, can increase vulnerability for covid- ( , ) , contributes to neuroinflammation and constitutes an important risk factor for the development or worsening of psychiatric disorders [for review, see ( ) ]. another important factor is aging, which is related to an imbalance in the levels of proinflammatory (high levels) and anti-inflammatory (low levels) cytokines and decrease in t-cell-mediated function ( ) . these immunosenescence-dependent changes in the elderly may be associated with higher susceptibility to viral diseases, including covid- ( ) , as well as neuropsychiatric disturbances, such as cognitive impairments ( ) . it has been demonstrated the relationship between aging and symptoms of anxiety and depression in patients infected with sars-cov- during the acute phase ( ). therefore, both obesity and older age may increase the risk of psychiatric symptoms in patients with covid- ; and one hypothesis is that neuroimmune circuits may be involved in this association. in addition, since poor nutrition and sedentary lifestyle are frequent in the elderly population and in overfat individuals, actions that promote the practice of physical activity and adequate nutrition are crucial, as they can potentially be associated with a lower risk for covid- and mental health impairments. pregnancy is another important potential factor that can affect the neuropsychiatric outcomes of covid- . maternal immune activation (e.g., in response to infection) is a risk factor for neurodevelopmental disorders such as autism spectrum disorder (asd) ( ) . autism has a complex etiology, involving environmental, and genetic factors. one of the proposed etiologies for asd is viral infection in early stages of development ( ). although the mechanisms by which viral infection can lead to autism are not yet known, it is believed that they may occur through (a) direct infection of the infant cns, or (b) due to the inflammatory response of the mother and/or the fetus, which can lead to neuroinflammation, triggering changes in brain development ( ). in fact, clinical evidence supports the participation of the neuro-immune mechanisms in the pathophysiology of asd [for review, see ( ) ]. while increasing evidence supports the neuroinvasive potential of sars-cov- , there is still no consistent demonstration of vertical transmission of this virus. in this sense, a recent study reviewing the effects of sars, mers, and covid- on gestational outcomes, including vertical transmission, and demonstrated that fortunately this transmission mechanism does not appear to occur in these betacoronaviruses ( ) . however, the controversial data on this aspect and the high expression of ace detected in the human placenta ( ) revealed that the possibility of vertical transmission needs to be further explored in clinical settings. accordingly, it is important to point out that there is still insufficient evidence to support the association between sars-cov- infection during pregnancy and the development of asd. nonetheless, we cannot rule out that changes in the maternal immune response triggered by the sars-cov- infection may affect neurodevelopment, another aspect that also deserves the attention of the medical and scientific communities. in any case, since increased levels of cytokines have been observed in covid- and in psychiatric disorders, it is likely that changes in neuroimmune axes may be involved in the mental health outcomes occurring in covid- patients. although this hypothesis is based mainly on studies with other betacoronaviruses, it will be interesting if future clinical studies, for example, include the search for correlations between the levels of inflammatory markers and psychiatric symptoms in covid- patients and survivors. studies in animal models infected with sars-cov- may also assist in the investigation of possible pathological mechanisms involved in neurobehavioral disorders related to the viral infection. the activation of the hypothalamic-pituitary-adrenocortical (hpa) axis has been observed during pathologies involving an immune/inflammatory process, including viral infections ( ) . the activation of this neuroendocrine axis by pro-inflammatory cytokines causes increased glucocorticoid production, a physiological response that contributes to avoid the deleterious effects of excessive production of inflammatory mediators and a non-specific recruitment of cells with no or low affinity for triggering antigens ( ) . in this respect, it seems reasonable to imagine a state hyperactivity of the hpa axis in infected patients, due to the "cytokine storm" observed in these individuals ( figure a) . a second aspect deserving discussion is the fact that ace overexpression in corticotropin-releasing-hormone (crh)producing neurons in the hypothalamic paraventricular nucleus alters the processing of psychogenic stress in mice, decreasing the crh content in the hypothalamus and corticosterone plasma levels (i.e., less hpa axis activation), as well as anxiety-like behaviors ( ) . sars-cov infection decreases the expression of ace in the lungs and myocardium of infected mice ( , ) . also, patients who died from sars and had sars-cov detected in the hearts exhibited reduced ace levels, when compared to patients who died from a non-sars related sepsis ( ) . although sars-cov genomic sequences have been found in the hypothalamus of humans ( ) , it remains to be determined whether the virus also decreases ace contents in this brain region. in any case, a downregulation of hypothalamic ace levels may be considered as another potential mechanism by which sars-cov/sars-cov- induces hyperactivity of the hpa axis with consequent psychiatric disturbances that are observed in these patients, such as the anxiety for example ( figure b) . however, the role of ace in the sars-cov- pathogenesis is still unknown and more studies are needed to test this mechanism. by contrast, in a study that prospectively assessed the presence of hormonal changes in sars survivors (without pre-existing endocrine disorders) months following recovery, patients ( . %) displayed late hpa axis hypoactivity, with hypocortisolism ( ). this alteration appeared to be a pathological effect of sars-cov, since nearly two-third of the patients did not use steroids and the majority were young (mean age: . years) and previously healthy ( ) . retrospective data from sars survivors do not support changes in hpa axis activity during the acute phase, suggesting that sars-associated hypocortisolism is a late onset phenomenon ( ) . since the "cytokine storm" is seen in the acute phase of sars (see table ), increased cytokine levels are unlikely to be secondary to hpa axis hypofunction. although proinflammatory cytokines classically increase the activity of the hpa axis (i.e., a downregulation mechanism of the inflammatory figure | hypothetical mechanisms by which sars-cov- may lead to changes in the activity of the hypothalamus-pituitary-adrenal (hpa). (a) during a viral infection (e.g., sars-cov- ), pro-inflammatory cytokines are released by immune cells present in the periphery (e.g., macrophages, t and nk cells) and/or in the brain (microglia). these cytokines can act at three levels of the hpa axis: increasing (i) the secretion of the corticotrophin-releasing hormone (crh) in the hypothalamus, (ii) the secretion of adrenocorticotropic hormone (acth) in the pituitary, and (iii) release of glucocorticoids (e.g., cortisol) through the adrenal cortex. by any of these actions, the result is an increased release of glucocorticoids, which bind to their receptors present in immune cells, suppressing the synthesis and release of pro-inflammatory cytokines. therefore, it is possible that increased pro-inflammatory cytokine levels in covid- may lead to hyperactivity of the hpa axis. however, due to a dysfunction in the negative feedback between the hpa axis and the immune system, this neuroendocrine axis is not able to reduce the production of inflammatory mediators, a possible explanation for why sars-cov- infection leads to cytokine storm. (b) hypothalamic ace overexpression decreases the activity of the hpa axis in mice, reducing the crh content in the hypothalamus and corticosterone plasma levels. since sars-cov infection is able to reduce the expression of ace in other tissues, one hypothesis (based on molecular similarities between sars-cov- and sars-cov) is that sars-cov- can induce a decrease in hypothalamic ace levels, thus contributing to hpa hyperactivity. (c) although pro-inflammatory cytokines classically increase the activity of the hpa axis, some cytokines (e.g., tgf-β) can decrease the activity of this neuroendocrine axis under specific conditions that remain unclear. this is another mechanism by which the sars-cov- infection, inducing an exacerbated inflammatory response, may lead to changes in the hpa axis, in this case, hypoactivity. continuous arrows: stimulation; dashed arrows: inhibition. response), under some conditions, tnf-α and transforming growth factor beta (tgf-β) may induce hpa axis hypoactivity ( ). therefore, it is possible that some cytokines that are increased in sars patients play a causative role in sars-associated hypocortisolism. as both hyperactivity and hypoactivity of the hpa axis are associated with depression ( , ) , hypocortisolism can also be associated with depressive symptoms that can be in sars survivors. in addition, due to the similarities between sars-cov- and sars-cov, it is possible that this mechanism involved in hpa axis hypoactivity can also be observed in covid- ( figure c) . thus, studies that simultaneously evaluate the axis hpa activity, cytokine levels, and psychiatric disturbances in patients and survivors of covid- will certainly improve the current knowledge. in the above context, it is noticeable that long-term survivors of the acute respiratory distress syndrome often report traumatic memories from the icu. interestingly, these patients displayed lower baseline cortisol levels and higher incidence of ptsd ( ) . such an information leads to questions related to the hypocortisolism observed in sars-cov infected patients, which may reflect an exhaustion of the adrenal cortex function, as a result of the viral infection or distress associated with hospitalization. clearly, future studies are needed to assess whether sars-cov- can affect the functioning of the hpa axis and whether this is involved in the association between sars-cov- infection and mental health outcomes. in clinical settings, it will be important to observe and measure the stress associated with hospitalization, as well as the presence of traumatic memories, as these factors may also be associated with changes in the hpa axis. a dysfunctional glucocorticoid-immune circuitry has been observed in schizophrenia. after a stress paradigm, while healthy patients experienced an increase in cortisol levels, negatively correlated to the subsequent changes in il- levels, patients with schizophrenia had elevated cortisol positively correlated to subsequent changes in il- levels, suggesting an inability to down-regulate inflammatory responses to psychological stress in this psychiatric condition ( ) . it is well-known that stressful life events may precipitate subsequent exacerbations of the illness ( ) . interestingly, elevated levels of circulating il- have been found in early episode psychosis patients ( ) . increased levels of stress or il- have also been described in sars or covid- patients (see supplementary table and table ). in addition, several studies reported symptoms of psychosis during the acute or long-term phase in sars patients (see supplementary table ). therefore, it is possible that sars-cov- infection and stressors related to hospitalization may increase the risk of psychosis by increasing levels of cytokines and/or by disrupting the glucocorticoid-immune circuits. since infections are associated with increased risk of developing schizophrenia ( ) , it seems important that future studies further assess the potential association between sars or covid- and the development of schizophrenia, as well as highlighting the importance of measures that prevent or reduce the impact of covid- on mental health. therefore, it is possible that increased pro-inflammatory cytokine levels in covid- lead to hypoactivity or hyperactivity of the hpa axis and, due to a dysfunction in the negative feedback between the hpa axis and the immune system, this neuroendocrine axis is not able to reduce the production of inflammatory mediators. in this sense, we hypothesize that such a dysfunction in the negative feedback between the hpa axis and production of pro-inflammatory cytokines may also be associated with mental health outcomes of the sars-cov- infection, thus conceptually corresponding to a psychoneuroendocrine-immune dysfunction. pre-clinical studies will hopefully provide more consistent clues to define a putative causal association between sars-cov/sars-cov- infection and behavioral deficits. in addition, animal models should allow a better control of variables that could also affect this association, such as the isolation of infected patients, since social isolation per se can also lead to both immunological and behavioral dysfunctions. in the current scenario, where social isolation measures are being strongly implemented worldwide, it is also important put into focus the potential damage to the mental health of isolated individuals, infected or not, applied the psycho-neuroendocrine-immune approach discussed herein. the exponential increase in the number of people infected with sars-cov- is leading to saturation of health services worldwide. to prevent human-to-human transmission and, in this way, slow down the growth of the pandemic, who has recommended that people avoid getting outside as much as possible ( ) . although such a measure is necessary to contain the advance of the pandemic, social isolation can cause negative impacts on mental health of individuals. studies on mental health outcomes of the quarantine during other epidemics, including sars and mers, revealed negative psychological effects, such as symptoms of ptsd, depression, stress, anxiety, and fear. some of the predictors of psychological impact included having a history of psychiatric illness, healthcare work, longer quarantine duration, infection fears, boredom, inadequate supplies, inadequate information, and financial resources ( ) . results of an online survey that assessed the levels of psychological impact and stress during the initial stage of covid- outbreak were recently reported ( ) . the responses of , subjects showed that . , . , and . % had moderate to severe stress levels, anxiety and depression symptoms, respectively. moreover, the general public with no formal education had a significant greater likelihood of depression during epidemic and higher satisfaction with the health information received was associated with a lower mental health impact of outbreak. people that presented sars-cov- -related symptoms like coryza, cough, dizziness, and myalgia or reported a history of chronic illnesses showed significant high levels of anxiety, depression, and stress. these results suggest an importance of accurate health information to reduce the impact of rumors and show the need for the media to provide, not only true information, but also information in simple language so that to support those people with less educational background during the epidemic ( ) . in addition, these data lead to the urgent need of psychological and psychiatric interventions, together with measures to prevent the spread of sars-cov- , so that to provide, as much as possible, well-being to both infected and non-infected socially isolated people. several studies show that living alone (vs. living with a family member) is associated with elevated levels of depressive symptoms ( ) ( ) ( ) , higher risk of depression ( ), and higher mortality ( ). yet, it has been emphasized the need for caution in arguing for a negative association between living alone and mental health ( ) . one reason is that other factors may influence the association between living arrangements and mental health, such as social networks ( , ) , social support ( , ) and neighborhood environment ( , , ) . in a study using data from more than , individuals in the united kingdom or england, it was shown that prevalence of common mental disorders was higher in people living alone vs. people not living alone. this association occurred regardless of age and gender but was largely mediated by loneliness. therefore, we believe that people living alone may be more vulnerable to the effects of quarantine on mental health than people living with a family member. accordingly, it would be interesting for future studies to assess the influence of different living arrangements on outcomes of quarantine on mental health. in this framework, loneliness has been associated with several psychiatric disorders, such as depression, anxiety, and suicide behavior ( ) . importantly, it has been showed that lonely people present several immune dysregulations, such as upregulated expression of pro-inflammatory cytokine genes ( ) . on the other hand, several studies have revealed that changes in the immune system play a key role in mental disorders ( ) . therefore, it is possible that changes in the immune system are involved in the negative impacts of loneliness on mental health. accordingly, it is conceivable that inflammatory mediators are also involved in the impact of quarantine on mental health, during covid- . studies with animal models have provided important clues on the neurobiological and the behavioral consequences of social isolation. in rodents, the stress of social isolation is able to lead to changes in several neurotransmitter systems (e.g., dopaminergic, adrenergic, serotonergic, gabaergic, glutamatergic, nitrergic, and opioid systems). indeed, the synthesis, release and even the corresponding receptor expression can be altered in several brain regions (e.g., hippocampus, cortex) of animals submitted to social isolation stress [for review, see ( ) ]. disturbances in neuroplasticity-related signaling pathways are also observed in these models ( ) . for instance, rats submitted to chronic social isolation stress displayed brain morphological changes such as decreased number of dendritic spines in the hippocampus and prefrontal cortex, as well as decreased brain-derived neurotrophic factor (bdnf) and phosphorylatedprotein kinase b (p-akt) in the dorsal hippocampus ( ) . the bdnf/trkb/pi k/akt pathway had already been described to be an important pathway in the maintenance of synaptic plasticity through translation and transport of synaptic proteins ( , ) . in this context, a metanalysis study reported a positive correlation between lower bdnf serum levels and depressive symptoms ( ) , and patients who present depressive symptoms may have reduced hippocampal volume ( ) , which supports the association between neuroplasticity and depressive disorders. the social isolation stress can also lead to hyperactivity of the hpa axis through an increase in corticosterone production and release in rodents ( ) . the abnormal levels of glucocorticoid have been related to depressive-like behavior and can affect the hippocampal neurogenesis ( ) . additionally, social isolation stress can lead to neuroinflammation, with higher levels of tolllike receptors, il- and tnf-α in the hippocampus ( ) , as well as increased plasma levels of tnf-α, il- , il- , and acth in isolated rats ( ) . a recent systematic review reported that social isolation and loneliness may be linked to systemic inflammation (i.e., high levels of c-reactive protein and il- ) in the general population ( ) . accordingly, it is conceivable that nervous, immune and endocrine systems can be interacting with each other, mediating neurobehavior impairments induced by social isolation stress. thus, these interactions may be part of the mechanisms by which social isolation during quarantine, via changes in neuroendocrine-immune circuits, can trigger damage to mental health. yet, future studies are needed to understand the mechanisms associated with the psychological damage caused by quarantine. although the whole population can be affected by the psychological impacts of covid- , some vulnerable groups may experience the same pandemic scenario differently. a recent study based on a multidisciplinary approach called attention for measures that can support the population susceptibilities such as ( ) older adults with multicomorbidities, ( ) children and women that stay at home and suffer domestic violence, ( ) people with preexisting mental health issues, ( ) people with learning difficulties, which might be affected by disruption to support and by loneliness, ( ) front-line health care workers that can be affected by the fear of infection, and ( ) groups that have hard socio-economic difficulties ( ) . as previously mentioned, financial problems may enhance the impact of social isolation on mental health during quarantine ( ) . interestingly, studies demonstrated that a worse socioeconomic status is directly related to higher systemic levels of inflammatory markers such as il- and c-reactive protein [for review, see ( ) ]. thus, it is possible that neuroimmune interactions may also be involved in the impacts of financial stress during covid- on mental health. this represents a novel possibility, that for sure requires future investigation. in addition, higher levels of inflammatory markers associated with worse socioeconomic conditions may also explain why lower social support is also associated with symptoms of anxiety and depression in patients infected with sars-cov- ( ). even though the biological mechanisms involved in the impact of socioeconomic status on mental health are still unclear, actions aiming at reducing socioeconomic inequalities should be a priority, in order to mitigate the impacts of covid- on mental health. finally, it is important to note that the evidence highlighted here does not contradict the need for the isolation measures that are necessary to control the pandemic. however, they call attention to the usefulness of strategies aiming at reducing the harmful effects of social isolation on mental health of the general public, including the improvement of psychological intervention and the reduction of socioeconomic inequalities. in summary, previous studies have reported psychiatric manifestations in patients infected with sars-cov- , such as anxiety, depression and ptsd symptoms ( , ). since increased levels of cytokines have been observed in covid- and in psychiatric disorders, we can place immune/inflammatory pathways as one of the mechanisms involved in mental health outcomes of covid- . changes in the hpa axis have also been observed in sars patients, indicating that alterations in neuroendocrine-immune circuits may be related to the psychiatric symptoms observed in these individuals. therefore, the hypothesis of the present article is that sars-cov- infection can lead to neuroinflammatory and endocrine changes, which in turn may reflect poor mental health. however, it is important to note that related biological factors (e.g., older age, female gender, and overfat), together with other factors inherent to covid- (e.g., social isolation, financial stress, and adverse effects of treatments) can influence psychiatric outcomes. accordingly, it is likely that the psychiatric symptoms observed in covid- patients are due to processes involved in the virus-host relationship, as well as to psychosocial and therapeutic issues associated with the pandemic. a further important aspect to be pointed out is the impact that the covid- pandemic can have on people who are isolated to prevent the transmission of the virus and to prevent health system overload. similar to possible mechanisms involved in the impacts of sars-cov- infection on mental health, social isolation may also be associated with dysfunctional psycho-neuroendocrine-immune interactions, which in turn can contribute to the development or the worsening of psychiatric disturbances (figure ) . it urges to put all ours efforts in understanding the pathophysiology of covid- , including cns infection and the risk of mental health compromise, but also the effects of this pandemic in the healthy isolated individuals, including children and adolescents, so that to prevent a "new generation" of groups in which the risk of developing mental disturbances, as anxiety or depression, could be increased. if nothing is done, we will probably be doomed to face a new mental health "pandemic" in the future. in terms of social aspects, a number of short term simple attitudes or initiatives, can comprise the encouragement to: (a) strengthen bonds using social media and start thinking positively ( ); (b) sleep properly and exercise regularly ( ); (c) balance the diet, regular daily routine, relaxation exercise and other healthy lifestyle measures ( ) . on the other hand, people should be avoid: substance use, eating too much fast food, excessive online activity, excessive watching television, and believing fake news ( ) . it is also important to look for strategies that mitigate the impacts of covid- on frontline healthcare providers. for instance, as recommended by ho et al. ( ) , healthcare organizations should introduce shorter working periods, regular breaks, and rotating shifts. individuals who experience moderate to severe and/or persistence distress should seek help from mental health professionals or in hospitals in cases of emergency situations ( ) . in addition, online consultation can be a potential alternative of delivering therapy ( ) . based on the similarity of sars-cov- and sars-cov, hematogenic or neuronal retrograde dissemination routes (via olfactory nerve) may be involved in the entry of the sars-cov- into the central nervous system (cns). in the cns (left) the virus can lead to increase in cytokines levels (e.g., il- , il- , tnf-α, il- β, inf-γ, and il- ) due to its local or peripheral (right) actions. increased cytokine levels are associated to neuronal death, synaptic plasticity impairments, dysfunction in the neurotransmitter metabolism and in the hypothalamic-pituitary-adrenocortical (hpa) axis. likewise, social isolation can also lead to these neuroendocrine-immune disturbances, for instance: increase in cytokine levels, changes in neurotransmitter systems, hpa axis hyperactivity and disturbances in neuroplasticity-related signaling pathways. through these common mechanisms, both sars-cov- infection and social isolation can lead to mental health impairments [e.g., impaired memory, depression, psychoses, anxiety and posttraumatic stress disorder symptoms (ptsd)]. il, interleukin; tnf-α, tumor necrosis factor alpha; inf-γ, interferon gamma. we also believe that art (especially music) can be an ally in the quest to improving mental health, whether for inpatients, health care workers, or isolated people. a meta-analysis study reported that music can modulate cytokine levels (including reducing il- levels), as well as neuroendocrine-immune responses triggered by stress, including physical stress caused by viral infection ( ) . in addition, it has been reinforced that music interferes positively in the immune system when subjected to acute stress (co stress test), also regulating the function of il- and the hpa axis ( ) . therefore, music therapy can be a further relevant and simple strategy that might be adopted on a largescale basis, for individuals in social isolation (also including medical staff). overall, it is important that political and health authorities pay attention to the mental health of infected and uninfected individuals during the pandemic, looking for prevention and treatment strategies, since poorer mental health can be associated with shorter life expectancy ( ) ( ) ( ) and high economic burden ( , ) . beyond the immediate and fundamental task of saving lives during sars-cov- pandemic, the due care of his mental health should be timely addressed. protocols aiming at minimizing mental problems during the infection as well as during recovering after hospitalization must be designed. in addition, studies that evaluate the impact of isolation during sars-cov- pandemic on mental health are important as they can guide new strategies to preserve population mental health in other critical situations that we can live in the future. finally, it is noteworthy that the approach applied herein, related to psychoneuroimmunology in covid- , should be convergent with a social sciences approach so that to better understanding and to better tackling this disease. hopefully, future studies may test the hypothesis outlined herein to better understand and consequently mitigate the impacts of covid- on mental health. the original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author. 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the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © raony, de figueiredo, pandolfo, giestal-de-araujo, oliveira-silva bomfim and savino. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -m jsu q authors: ortiz de landazuri, iñaki; egri, natalia; muñoz-sánchez, guillermo; ortiz-maldonado, valentín; bolaño, victor; guijarro, carla; pascal, mariona; juan, manel title: manufacturing and management of car t-cell therapy in “covid- ’s time”: central versus point of care proposals date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: m jsu q the covid- pandemic, caused by severe acute respiratory syndrome coronavirus- (sars-cov- ), has generated a significant repercussion on the administration of adoptive cell therapies, including chimeric antigen receptor (car) t-cells. the closing of borders, the reduction of people transit and the confinement of the population has affected the supply chains of these life-saving medical products. the aim of this mini-review is to focus on how the covid- pandemic has affected car t-cell therapy and taking into consideration the differences between the large-scale centralized productions for the pharmaceutical industry versus product manufacturing in the academic/hospital environment. we also review different aspects of car t-cell therapy and our managerial experience of patient selection, resource prioritization and some practical aspects to consider for safe administration. although hospitals have been forced to change their usual workflows to cope with the saturation of health services by hospitalized patients, we recommend centers to continue offering this potentially curative treatment for patients with relapsed/refractory hematologic malignancies. consequently, we propose appropriate selection criteria, early intervention to attenuate neurotoxicity or cytokine release syndrome with tocilizumab and prophylactic/preventive strategies to prevent infection. these considerations may apply to other emerging adoptive cell treatments and the corresponding manufacturing processes. the sars-cov- coronavirus has generated an unprecedented global impact on multiple aspects of society, economy and health. sars-cov- was reported as a new emerging zoonotic pathogen in wuhan (china) in december , and declared as a pandemic by the world health organization in march ( ) . its rapid human-to-human transmission has affected millions of people ( ) . indeed, the routine operation of medical systems has been significantly disrupted generating a health crisis in many countries due to limited resources such as hospital beds and personal protective equipment. in early june, the number of infected people had already risen to more than million globally and the number of new cases detected is increasing daily. there is not pre-existing specific immunity placing at risk humanity as a whole to infection by sars-cov- . taken together with the severe pulmonary and systemic inflammatory complications associated with the disease, it has caused governments to enforce isolation measures to prevent its rapid spread. hospitals have been forced to change their usual workflows to cope with the saturation of hospitalized services. the closing of borders, the reduction of the transit of people and the confinement of the population has affected the supply chains of products. access to life-saving drugs has also been affected, negatively impacting on patients with other life-threatening diseases ( ) . chimeric antigen receptor (car) t-cell therapy is a lifesaving bioengineered cell replacement therapy against leukemia. this adoptive antitumor immunotherapy, based on autologous t-cells transduced with a genetically engineered receptor for cd to redirect their cytotoxicity against native cd surface antigens expressed in tumor cells, has completely changed the management of patients with hematologic malignancies such as acute lymphoblastic leukemia (all) or non-hodking lymphoma (nhl) ( ) ( ) ( ) . tisagenlecleucel (kymriah, novartis) and axicabtagen ciloleucel (yescarta, kite/gilead) are both anti-cd car t-cell (cart ) commercial products that obtained food and drug administration (fda) approval in for the treatment of pediatric and young adult patients with cd + relapsed/refractory b-cell all, relapsed/refractory b-cell nhl [diffuse large b-cell lymphoma (dlbcl), primary mediastinal b-cell lymphoma and transformed follicular lymphoma]. other regulatory agencies like the european medicines agency (ema) have also recently approved these commercial cart use. therefore, cart is considered a potential curative therapeutic option for cd + hematologic malignancies with no response to conventional treatments. in addition, approval of other car t-cell products developed commercially against other molecular targets are expected as further options for treatment in the coming months. more than clinical trials are being carried out testing car-based products. some of them are financed by the industry and others are being developed in the academic ambit. the cohabitation of both is inevitable and necessary. industry production could supply car t-cell therapies to reach the whole population using good manufacturing practice (gmp) accredited large facilities to centralize the manufacturing process. www.clinicaltrials.gov on the other hand, academic centers should be dedicated to develop car t-cell therapies against less frequent diseases. independently, both have been seriously affected by the covid- pandemic despite the differences they present in product manufacturing and management processes ( ) . the management of car t-cell therapy is a complex time-consuming process that requires highly specialized personnel and coordinated work systems including medical management of the patient before and after the infusion due to feasible toxicities ( ) ( ) ( ) . owing to the doubtless relevance of car t-cell therapy for hematologic patients, it is important to have in mind some considerations within the context of the covid- pandemic. therefore, the aim of this mini-review is to focus on how the effects caused by the pandemic have affected this therapy taking into consideration the differences between the large-scale centralized production of car t-cells by the pharmaceutical industry versus the product manufacturing processes employed by the academic/hospital environment. we also review different aspects of car t-cell therapy, including patient selection and resource prioritization performed in our center during the covid- pandemic. chimeric antigen receptor t-cell therapy has elicited an unprecedented response against b-cell malignancies, but it is associated with significant toxicity, including prolonged cytopenia, cytokine release syndrome (crs), and neurotoxicity ( ) ( ) ( ) ( ) . toxicity is normally associated with the t-cell's inherent mechanism of action that has been well-described in academic and industry developed car t-cells ( ) . the use of cart cell therapy can result in prolonged b-cell aplasia and therefore inability to develop an antibody response necessary to respond against pathogens such as sars-cov- ( , ) . furthermore, these patients are usually managed as in-patients to facilitate intensive monitoring due to the severity and rapidity of crs, neurotoxicity and frequent need the intensive care unit (icu) ( ) . the covid- pandemic is a threat to interrupt any cell therapy. it is priority that each center carefully review the internal policies and procedures to adopt the recommendations needed according to their healthcare needs. hospitals have instituted measures to defer multiple medical interventions, including adoptive cell treatments or hematopoietic stem cell transplantation. another relevant consideration is the inconsistent uniformity of clinical cell therapy protocols. this hampers the establishment of standardized strategies during the covid- pandemic. a large proportion of these patients receive therapy in academic or pharmaceutical clinical trials and many stopped to preserve patient's safety. indeed, lymphodepletion before final cell-product infusion facilitates the expansion of car t-cells as it generates a suitable environment for in vivo modified t-cell expansion and survival ( ) . however, this generates a severe immunosuppression that can be seriously complicated by sars-cov- infection. nevertheless, delaying cell therapy as a consequence of the covid- pandemic could be fatal for the majority of patients with relapsed/refractory malignancies. alternative therapeutic strategies are also generally associated with significant immunosuppression and subsequent potential hospitalizations. in fact, car t-cells are potentially curative for patients with poor prognosis ( ) . not all centers will be affected in the same way during the covid- pandemic. therefore, a careful evaluation of resources and infrastructures should be performed between the department of hematology and the hospital emergency planning group. the availability of icu, hospital beds and mechanical ventilation equipment are limited, but required resources. moreover, staff shortages due to potential exposure and resource constraints on personal protective equipment affect the management of the patient before and after the treatment infusion. some practical aspects should be considered for the safe administration of car t-cell therapy during the pandemic ( table ) : • in the case of icu collapse: establish a triage algorithm to select only patients who are most likely to benefit with no alternative treatment options and in whom the risk of toxicity is lower. after the manufacturing process of car t-cells final cell product should be tested for sars-cov- by qpcr before infusion in patients with a positive qpcr prior to leukapheresis the manufacturing process would continue if a patient becomes infected after leukapheresis. the infusion should be postponed until patient's clinical improvement • guarantee the availability of personnel to perform the leukapheresis and the reception and sample processing in the adoptive cellular immunotherapy unit. • initiate lymphodepletion procedure only after receiving car t-cell products at the site to avoid potential obstacles on supply chain operations. • hospital bed availability should be ensured for the immediate weeks surrounding the treatment. there are few children admitted with covid- so young adult patients who may benefit from this therapy may be transferred to a pediatric center with greater availability of beds. • guarantee the availability of a member of the medical team with the capacity to respond to complications associated with covid- . • establish a specific workflow for patients infected with covid- . the covid- pandemic has altered inclusion algorithms for the proper selection of patients, that is now associated to ethical dilemma. it is imperative to outline criteria to identify optimal candidates who have the potential to achieve significant remission. the benefit-risk balance should be clear, taking into account the risk of delaying car t-cell therapy against the risk of progression of the underlying disease. therefore, a group of experts should evaluate the inclusion of patients considering, among multiple factors, the lack of avalability in the hospital and alternative treatments if complications appear. in our experience at hospital clínic de barcelona, a medium-sized academic institution, we have continued to administer our academic and other commercial car-t cell therapy against aggressive relapsed/refractory b-cell nhl and all ( , ) . during the first month of the covid- state of alarm declared in spain, every car t-cell operation was postponed and only one infusion was performed in our center. ultimately, no treatment was canceled, although the final product infusions were delayed due to the lack of availability of space in the icu. different supportive measures to mitigate the risk of covid- in our patients have been taken ( table ) . two young patients (aged and ) were treated in another major hospital of the city, sant joan de déu children's hospital center. after the first month of the pandemic, one bed at icu was reserved for car t-cell therapy. this allowed the treatment of nine patients at hospital clínic de barcelona. they were patients under the age of with few co-morbidities and - ecog functional status. altogether, six patients were treated with academic cart and one patient with car t-cell therapy targeting bcma ( ) . on the other hand, two patients were treated with car-t cells developed by the industry. the feasible impact of the delay on car t-cell treatment outcomes will be analyzed in the mediumlong term. sars-cov- viral detection tests have been performed in every patient treated with car t-cells at various time-points. before leukapheresis, patients were screened for sars-cov- by qpcr given that qpcr is the gold standard test to measure viral loads. the determining factor in the start of the cell therapy manufacturing process is the infection status of the patient and the potential ability to transmit the virus. serological tests report directly on humoral immunization but serology is not a specific marker of infection. a positive igm result in the serological test is not always equivalent to an acute infection since it could persist for months without viral load being detected in the patient. as with other infections, serological tests are very useful for screening but should only be used for diagnosis if a gold standard test is not available. our patients were assessed for specific symptoms during the whole process. in addition, in-person visits have been avoided as a preventive measure and patients were asked about their potential risky contacts. the major importance of extreme confinement measures has been therefore repeatedly emphasized. the use of epi facemasks in patient's environment was essential to maximally reduce infection risk. the final cell product should be tested for sars-cov- by qpcr before infusion in those patients with a positive qpcr prior to leukapheresis. the special committee formed in our center to evaluate sars-cov- -positive cases initially decided to avoid the car t-cell manufacturing process if covid- was diagnosed in order to reduce possible contagions among medical staff and contamination of manufacturing facilities. this decision was controversial considering the low risk of contamination in a closed-circuit manufacturing process and the low viral load in blood samples ( ) . currently, we have established to perform the leukapheresis days after a positive qpcr result to ensure that the viral load in patient's samples are virtually undetectable ( ). diverse complications that could emerge from sars-cov- infection could actually worsen car t-cell therapy outcomes. thorough monitoring of these patients is required in order to detect eventual neutropenia or other infection. post-infusion standard antiviral, antifungal and antimicrobial prophylaxis protocols were recommended. autologous car t-cell therapy requires a personalized manufacturing process based on several critical steps that demands good coordination between different medical disciplines. for those heavily treated patients, time is crucial and well-established workflows are essential for academic or commercial cell-based therapies. after the theoretical design of the synthetic chimeric receptor and the corresponding pre-clinical studies, the complete process for car t-cell therapy includes (i) obtaining the starting patient's cell population by leukapheresis followed by the (ii) ex vivo genetic introduction of the synthetic car into these cells using mainly lentiviruses or retroviruses, previously generated. these modified t-cells are then (iii) activated and expanded in bioreactors and the resulting product might be (iv) adequately prepared and cryopreserve in infusible media for the final (v) product re-infusion into the patient ( , ) . all these steps can be affected by the pandemic setting. along with inherent complexity in any cell therapy, limitations and obstacles in academic hospitals and pharmaceutical industry must be added. in this way, the standard manufacturing period of - days until the final product could be fatally extended (figure ). the main difference between academic and pharmaceutical car t-cells, regarding the timeline of the manufacturing process, is obtaining the "living drug." as for academic products, commercial cart are based on autologous t-cells and each patient requires his own t-cells. in the academic car t-cell manufacturing, all steps are carried out in the hospital facilities enabling a more flexible, personalized and coordinated "patient-friendly process." it allows short transfer times from the leukapheresis site to the production facilities and from the production center to the patient's bed, both being less than hour ( ) . in commercial manufacturing, the same process takes place in distant geographical locations and depends on delivery protocols established by pharmaceutical companies. logically, the transfer times from the leukapheresis site to large-scale production gmp facilities and from this production center to the patient's bed could be, at least, day ( ) . however, covid- has rapidly constrained travel and mobility, extending the delivery times of commercial car t-cell products. another side effect of reduced mobility are the potential disruptions in the resources supply line essential to the manufacturing process. this may affect both academic car t-cell products and those produced by industry. for example, the parallel process to obtain vectors with the car transgene like lentiviruses is a compendium of gmp skilled steps where different reagents are needed. similarly, other specialized products are essential for the activation and expansion of t-cells in a bioreactor like clinimacs prodigy ( , ) . these critical steps can be seriously aggravated in countries where the production of these reagents is scarce and are imported from other regions. however, the large-scale production that takes place by industrial facilities ensures a greater storage capacity and a robust supply chain. . patients have to be tested by qpcr for sars-cov before leukapheresis ( ); in case of a negative result, peripheral blood mononuclear cells (pbmcs) are collected from patient via leukapheresis. timing of delivery to manufacturing site and the susceptibility to mobility restriction varies between academic/point of care ( a) and commercial/centralized ( b) production. manufacturing process is affected by workforce reduction ( ) . t-cells are selected, activated, transduced with a viral or non-viral vector to express the desired car and expanded in a bioreactor. after that, resulting car t-cells are isolated and cryopreservated and assessed by quality control (qc) and quality assurance (qa) programs. finally, the patient's modified own cells are transferred to bedside. timing of delivery to bedside and the susceptibility to mobility restriction varies between academic/point of care ( a) and commercial/centralized ( b) production. before car t-cell product infusion, final product is tested for sars-cov- by qpcr before infusion in patients with a positive qpcr result prior to leukapaheresis ( ). the reduction of personnel due to possible contagions among the staff, the restructuring of the workforce to treat infected patients and the limited resources and protective equipment has been a cause for concern (table ). all of these factors can impact equally on the academic or pharmaceutical production modalities. the staff and the reagent shortage can affect the leukapheresis process and cell processing in the laboratory. there exist strict regulations associateds with cell therapy where personnel who develop "living drugs" must work under sterile and gmp conditions. for this reason, a contagion that could spread between qualified personnel and the consequent imposing quarantine that would be imposed on the rest of the personnel can become a bottleneck in the global production of the adoptive cell therapy. the stringent and immobile regulation has not been amended in the context of the covid- pandemic to ensure the safety of patients, medical staff and laboratory personnel. similarly, within the associated legislation, a series of quality controls are imposed and must be complied with before car t-cell product infusion. among them, the confirmation of the product sterility is essential. in addition, it must be ensured that the product is free of endotoxins, adventitious viruses and mycoplasma. there are other nonmicrobiological controls that must be carried out as the number of copies of the car per genome, flow cytometry experiments to determine the percentage of cells with car, among others. having these controls in place for each cell product requires precious time in covid- pandemic setting and is highly dependent on human work. therefore, any delay in verifying all of these control points may delay the infusion. once the manufactured car t-cell product is infused, a resulting crs is the widest described associated side effect. a compilation of different symptoms from hypotension to fever due to a massive pro-inflammatory cytokine release (il- , il- β, tnf-α) have been reported ( ) . serum il- level has been correlated with crs severity ( ) . hence, tocilizumab (tcz), an anti-il- receptor monoclonal antibody, has become the drug of choice for the management of moderate or severe crs ( ) . this il- cytokine release profile resembles the virally driven hyperinflammation (crs-like) suggested as predictor of fatality in covid- ( ) . this fact has led to the setting-up of several clinical trials with the aim of studying the impact of tcz administration in the evolution of the acute respiratory distress syndrome and the off-label use of this drug in symptomatic covid- patients. the management of crs is extremely relevant given that viral infections (not only covid- ) may promote crs ( ) . crs requires treatment with tcz or corticosteroids depending on severity of symptoms. the notable increase in tcz use has raised a concern about supply during covid- pandemic. however, availability of tcz doses for each patient is a mandatory condition before car-t infusion. two patients treated with car t-cell therapy have received tcz to treat crs in our center in the pandemic setting. tocilizumab administration protocol has not been reconsidered despite supply problems. this protocol consists on early tcz use at the onset of grade crs. we have dealt with tcz scarcity through the use of other drugs such as sarilumab (human anti-il- receptor) or siltuximab (chimeric anti-il- ) to face the crs-like complications. if icu is collapsed, crs management protocols should initiate earlier to reduce the probability of using icu. as a practical option, we propose tcz administration from grade crs (and not grade as is usually done), or even prophylactic tcz administration in those patients considered to be at high risk of severe crs, with high tumor burden. corticosteroids used as crs treatment when tcz has failed could promote potentially disadvantageous effects on covid- outcomes ( ) . nevertheless, corticosteroids should be cautiously used in covid- patients that suffer from crs after car t-cell infusion ( ) . in fact, the current pandemic has exposed us to an ethical dilemma regarding the prioritization of tcz. each case must be thoroughly evaluated to ensure the availability of this drug. tocilizumab could decrease the duration and severity of covid- symptoms allowing the weaning of the ventilatory support ( ) . on the other hand, tcz supply prior to infusion must be ensured for the hematologic patients during car t-cell therapy to mitigate the toxicity of an eventual crs complication. car t-cell therapy has been consolidated as a potential curative therapy for patients with refractory/relapsed hematologic malignancies. the covid- pandemic represents an unprecedented challenge to continue safely treating patients with this adoptive cell therapy. nevertheless, centers should continue offering this potentially curative treatment with car t-cell therapy for critical patients using appropriate selection criteria, early intervention to attenuate side effects like crs with standardized tcz protocols, and prophylactic/preventive strategies to prevent infection. for those heavily treated patients, time is crucial. mobility and personnel restrictions add obstacles to a truly personalized therapy in which multidisciplinary teams intervenes. this has affected both academic car t-cell therapies and those commercially available. there are several limitations related to delivery and the time-consuming manufacturing processes that create new concepts concerning the benefit of academic point of care proposals. also, some regulatory rules should be re-evaluated to become more flexible during this pandemic. mj and mp proposed, directed, discussed, and revised the development of the manuscript. gm-s, ne, and iol had equally contributed to the data gathering and writing of the review. vo-m reviewed and collaborated particularly on aspects related to the hematological issue. vb and cg had been in charge of analyzing the technical aspects related to the product manufacturing. all authors contributed to the article and approved the submitted version. this review was included as a work sponsored by grants pi / and ac / (ce_era net_nanomed ) from instituto de salud carlos iii with fondos feder. this work is also funded by the "fundació bancaria la caixa" ( - fcrb) and cellnex telecom ( - /cpo ). the funder bodies were not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication. the trinity of covid- : immunity, inflammation and intervention. natural review immunology immunology of covid- : current state of the science chimeric antigen receptor t cell therapy during the covid- pandemic chimeric antigen receptor-modified t cells for acute lymphoid leukemia chimeric antigen receptor t cells in refractory b-cell lymphomas tisagenlecleucel in children and young adults with b-cell lymphoblastic leukemia cart manufacturing process and reasons for academy-pharma collaboration chimeric antigen 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lymphoblastic leukemia covid- : consider cytokine storm syndromes and immunosuppression optimizing chimeric antigen receptor t-cell therapy for adults with acute lymphoblastic leukemia on the use of corticosteroids for -ncov pneumonia effective treatment of severe covid- patients with tocilizumab we thank dr. michael j. edel for proofreading the manuscript and montserrat riego for administrative tasks. key: cord- - bniy b authors: peteranderl, christin; herold, susanne title: the impact of the interferon/tnf-related apoptosis-inducing ligand signaling axis on disease progression in respiratory viral infection and beyond date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: bniy b interferons (ifns) are well described to be rapidly induced upon pathogen-associated pattern recognition. after binding to their respective ifn receptors and activation of the cellular jak/signal transducer and activator of transcription signaling cascade, they stimulate the transcription of a plethora of ifn-stimulated genes (isgs) in infected as well as bystander cells such as the non-infected epithelium and cells of the immune system. isgs may directly act on the invading pathogen or can either positively or negatively regulate the innate and adaptive immune response. however, ifns and isgs do not only play a key role in the limitation of pathogen spread but have also been recently found to provoke an unbalanced, overshooting inflammatory response causing tissue injury and hampering repair processes. a prominent regulator of disease outcome, especially in—but not limited to—respiratory viral infection, is the ifn-dependent mediator trail (tnf-related apoptosis-inducing ligand) produced by several cell types including immune cells such as macrophages or t cells. first described as an apoptosis-inducing agent in transformed cells, it is now also well established to rapidly evoke cellular stress pathways in epithelial cells, finally leading to caspase-dependent or -independent cell death. hereby, pathogen spread is limited; however in some cases, also the surrounding tissue is severely harmed, thus augmenting disease severity. interestingly, the lack of a strictly controlled and well balanced ifn/trail signaling response has not only been implicated in viral infection but might furthermore be an important determinant of disease progression in bacterial superinfections and in chronic respiratory illness. conclusively, the ifn/trail signaling axis is subjected to a complex modulation and might be exploited for the evaluation of new therapeutic concepts aiming at attenuation of tissue injury. interferons (ifns) are well described to be rapidly induced upon pathogen-associated pattern recognition. after binding to their respective ifn receptors and activation of the cellular jak/signal transducer and activator of transcription signaling cascade, they stimulate the transcription of a plethora of ifn-stimulated genes (isgs) in infected as well as bystander cells such as the non-infected epithelium and cells of the immune system. isgs may directly act on the invading pathogen or can either positively or negatively regulate the innate and adaptive immune response. however, ifns and isgs do not only play a key role in the limitation of pathogen spread but have also been recently found to provoke an unbalanced, overshooting inflammatory response causing tissue injury and hampering repair processes. a prominent regulator of disease outcome, especially in-but not limited to-respiratory viral infection, is the ifn-dependent mediator trail (tnf-related apoptosis-inducing ligand) produced by several cell types including immune cells such as macrophages or t cells. first described as an apoptosis-inducing agent in transformed cells, it is now also well established to rapidly evoke cellular stress pathways in epithelial cells, finally leading to caspase-dependent or -independent cell death. hereby, pathogen spread is limited; however in some cases, also the surrounding tissue is severely harmed, thus augmenting disease severity. interestingly, the lack of a strictly controlled and well balanced ifn/trail signaling response has not only been implicated in viral infection but might furthermore be an important determinant of disease progression in bacterial superinfections and in chronic respiratory illness. conclusively, the ifn/ trail signaling axis is subjected to a complex modulation and might be exploited for the evaluation of new therapeutic concepts aiming at attenuation of tissue injury. ( ) cell death induction, e.g., bcl- -associated x protein, caspase- , fas-associated protein with death domain, fas ligand, and tnf-related apoptosis-inducing ligand (trail) dsrna, polyi:c ( , ) iav ( , , , ) sendai virus ( ) trail virus control by apoptosis induction in infected cells iav ( , , ) tissue injury by apoptosis of both infected and non-infected alveolar epithelial cells, lung macrophages iav ( , , ) rsv ( ) necrosis of fibroblasts, dendritic cells, and epithelial cells iav ( , , ) increased cellular infiltration cov ( ) decreased expression of na,k-atpase, impaired epithelial fluid reabsorption iav ( ) introduction in , isaacs and lindenmann ( ) first recognized the potential of a soluble and probably cell-derived factor to combat influenza virus infection and named this factor interferon [(ifn) from latin interferre, to interfere]. since then, three subgroups of ifns have been defined, primarily by their differential receptor usage. while the groups of type i ifn and type iii ifn comprise largely agents directly limiting pathogen spread by improving cellular counter measurements, ifn-γ, the sole type ii ifn, has been mainly implicated in the modulation of innate and also adaptive immune responses ( , ) . accordingly, type i and iii ifns are key signaling molecules in viral control, and lack of both signaling pathways results in increased viral loads and disease severity. still, there is accumulating evidence that not only lack of an antiviral response but that also an unbalanced overshooting activation of ifns contributes to an exaggerated inflammatory reaction, tissue injury, reduced proliferative capacity, and thus enhanced disease severity ( table ) . especially in viral infections, this effect has not only been tracked down to ifn signaling in general but specifically to the exaggerated production of key effector ifn-stimulated genes (isgs) ( ) . a prominent example is the tnf-related apoptosis-inducing ligand (trail) that displays an ambivalent role in viral infection ( - ) ( table ) . whereas first identified as factor produced by immune cells in non-respiratory infection ( , ) , trail is now especially well studied in influenza a virus (iav) infection, where it is released in high amounts from bone marrow-derived macrophages upon pathogenassociated molecular patterns (pamps) recognition and type i ifn production ( ) . macrophage-released soluble trail, but also membrane-bound cell-associated trail, acts via distinct receptors on infected but also on non-infected, neighboring cells. in viral infection, its preliminary role is to drive infected cells into apoptosis to limit virus spread. however, studies performed within the last decade demonstrate that trail's antiviral activity seems to be outweighed by the functional and structural damage it induces not only in infected but also in bystander cells such as uninfected cells of the alveolar epithelium ( , ) . this process is not only relevant in promoting viral disease progression but has further implications in bacterial superinfection and probably also in chronic diseases. the recognition of the ambivalent role of ifn-driven signaling in vivo is a first important step to better understand disease progression and to envision novel treatment options for primary viral respiratory infection targeting distinct host-derived signaling mediators such as trail. it is a commonly accepted concept that-as janeway ( ) already proposed in -immune activation toward invading pathogens is mounted upon recognition of pamps. pamps are evolutionary conserved biomolecules such as proteins, lipids, nitrogen bases, sugars, and complexed biomolecules such as lipoglycans that are essential to the survival of a given pathogen ( ) . pamps are recognized by distinct pattern recognition receptors (prrs) that are germ-line encoded and-similar to pamps-usually show a high evolutionary conservation. the first recognized and probably most intensely studied family of prrs are the toll-like receptors [tlrs; reviewed in mogensen ( ) ; leifer and medvedev ( ) ]. in viral infection, both host cell membrane-localized tlrs (tlr , tlr , detecting viral envelope proteins) and endosomal tlrs (tlr , tlr , tlr , and tlr , nucleic acid sensors) initiate signal transduction cascades leading to ifn production (figure ). tlr activation results in either myeloid differentiation factor (myd ) or tir-domaincontaining adaptor protein-inducing ifn-β (trif) recruitment that both trigger various downstream signaling events, eventually leading to ifn regulatory factor (irf) , irf , and nfκb nuclear translocation as well as map kinase and activator protein (ap- ) activation ( , ) . similar to endosomal tlrs, the cytosolic retinoic acidinducible gene (rig)-i-like receptors (rlrs) are specialized to recognize viral nucleic acid contents and are central prrs relevant to mount an antiviral response, providing resistance to most rna (e.g., orthomyxoviruses) and some dna (e.g., reoviruses) viruses [reviewed in ref. ( , ) ]. both melanoma differentiation-associated gene (mda- ) and rig-i recognize dsrna, ′-triphosphate rna, or the synthetic analog to dsrna, polyi:c ( , ) . both drive the dimerization of the mitochondriaassociated adaptor protein ifn-β promoter stimulation (ips- ) (also named mavs, visa, cardif). a subsequently activated cascade including tradd (tnf receptor type -associated death domain protein), traf (tnf receptor-associated factor ), and tank (traf family member-associated nf-κb activator) induces the phosphorylation of irf and irf , resulting in type i ifn production ( ) . the third rlr, lgp , so far has primarily been implicated to regulate rig-i or mda- as a cofactor; however, a recent study by stone et al. ( ) demonstrated a novel, non-redundant, and independent role of lgp in west nile virus infection. another class of prr, the nucleotide oligomerization domain (nod)-like receptors (nlrs), has mainly been implicated in bacterial recognition ( ) , still several nlrs are activated as well upon virus infection. especially, nlrp is known to recognize rna of different viruses including hepatitis c virus, measles virus, influenza, and vesicular stomatitis virus (vsv) ( ) ( ) ( ) ( ) , resulting in inflammasome formation and caspase- -dependent activation of il- β and il- ( ) ( ) ( ) . in addition, virus infections are sensed also by a structurally diverse group of viral rna and dna sensors residing in the cytoplasm. these include the cyclic gmp-amp synthase that synthesizes the second messenger cgamp. cgamp in turn activates stimulator of ifn genes (sting), tank-binding kinase , and irf , triggering ifn production ( ) ( ) ( ) . moreover, sting itself acts as a prr and has been implicated in dna virus recognition including hsv, adenovirus, vaccinia virus, and papilloma virus and in sensing of retroviral rna-dna hybrids ( ) and rna viruses after being activated by rig-i ( , ) . another cytosolic nucleic acid sensor, pkr, is well known for its phosphorylation of eukaryotic initiation factor α (eif α) in response to viral dsrna. phosphorylation of eif α results in its deactivation, host translational shut-off, and the limitation of viral replication. of note, the pkr-eif α-driven inhibition of protein synthesis can contribute to an ips- -dependent ifn-β induction ( ) . furthermore, pkr has been implicated in efficient type i ifn activation by tlr in response to dsrna ( ) and can mediate-at least partially-activities of irf ( ) . in addition to type i ifns, also type iii ifns exert antiviral activity and are widely expressed after viral recognition, being produced by most cell types including epithelial, endothelial, fibroblast, and polymorphonuclear cells [reviewed in ref. ( , ) ]. like type i ifns, type iii ifns are induced in viral infection by the prr rig-i as well as tlr and tlr and rely on the activation of the same transcriptional activators, including irf , irf , and nfκb. these observations initially led to the conclusion that type i and type iii ifn comprised two completely redundant systems to induce isgs in response to pamp recognition. however, more recent data suggest distinct selection mechanisms for either type i or type iii ifn expression. as such, ips- specifically induces ifn-λ, but not type i ifn, when located at the peroxisomal membrane instead of the mitochondrial membrane in response to rig-i activation by reovirus, sendai virus, or dengue virus challenge ( ) . interestingly, type iii ifn induction is largely independent toward ap- translocation, which facilitates an instantaneous induction of ifn-λ after viral recognition, highlighting it as an important immediate factor driving innate microbial defense mechanisms. release of ifns upon pathogen recognition is a highly conserved mechanism-found from teleost fish to insects and mammals-to prepare the surrounding cells as well as the host defense against the invading threat ( , ). whereas often high-level ifn production relies on specialized sentinel cells such as macrophages or dendritic cells (dcs), mostly all cells of the multicellular organisms are able to respond to at least one type of ifn by expression of respective receptors. receptor binding then induces a signal transduction cascade relying on the janus kinase (jak) and signal transducer and activator of transcription (stat), which results in efficient transcription of a plethora of different isgs in infected as well as bystander cells ( , ) . ifns engage a classical canonical signal transduction cascade employing jak/stat molecules after binding to their respective receptors. herein, type i ifns ligate to their common heterodimeric receptor consisting of the ifn-α receptor (ifnar) and ifnar subunits, whereas type iii ifns act via a interleukin- receptor (il- r )/ifn-λ receptor (ifnlr ) heterodimer that to date has been reported to be restricted in its expression to epithelial cells ( ) . in type i ifn signaling, ifnar engagement leads to the activation of the receptor-associated protein tyrosine kinases jak and tyrosine kinase followed by the recruitment or repositioning of already associated but elsewise latent cytoplasmatic transcription factors stat and stat . consequently, stat /stat are phosphorylated on conserved tyrosine residues, they disassemble, undergo conformational changes enabling their heterodimerization as well as the exposure of a nuclear localization sequence. subsequently, the stat /stat heterodimer translocates into the nucleus where it interacts with the irf to form the trimeric ifn-stimulated factor (isgf ). isgf binds cognate dna sequences, the ifn-stimulated response elements (isre), finally leading to isg induction. also type iii ifn interaction with the il- r /ifnlr receptor complex triggers stat /stat heterodimerization, nuclear translocation, and isgf assembly ( ) . interferon signaling results in the induction of isgs evoking different cellular responses against viral infection, both in infected as well as in non-infected cells, including direct antiviral, immune-modulatory, or cell death-inducing effects to enable an immediate and robust response to a pathogen challenge. many isgs directly interfere with viral replication on an intracellular level. well-studied examples of antiviral isgs comprise ifn-induced transmembrane proteins (ifitms) effective in iav, west nile virus, and dengue virus infection ( , ) , the myxovirus resistance protein a (mxa) that interferes with vsv mrna production and binds the iav nucleocapsid to prevent nuclear translocation of viral genetic material ( ) ( ) ( ) , the - -oligoadenylate synthase (oas), which activates rnase l triggering viral rna degradation, or the prr pkr, which besides activating the ifn response has a major impact on viral protein translation by inhibiting the eif α ( ) . more recently identified isgs include the plasminogen activator inhibitor that blocks iav infection by inhibiting glycoprotein cleavage executed by extracellular airway proteases ( ) or the antiviral isg that limits iav viral replication via its exonuclease activity most likely by interfering with the viral np ( ) . accordingly, ifn pretreatment usually results in the establishment of an antiviral state that limits viral replication and spread from the start of infection and thus favors milder disease outcomes. ifn-α pretreatment has been demonstrated to limit viral spreading of seasonal iav strains and thus decrease morbidity and mortality in mice, guinea pigs as well as ferrets ( ) ( ) ( ) . as shown by a study by tumpey et al. ( ) , this effect can be attributed to the early induction of antiviral isgs including mxa. importantly, type i ifn pretreatment also dampens early replication of highly pathogenic avian influenza in ferrets ( ) . also in respiratory syncytial virus (rsv) infection, treatment with recombinant ifn-α results in significantly decreased lung viral titers, alveolar inflammatory cell accumulation, and clinical disease in rsv-infected mice ( ) . in addition, respiratory infections caused by emerging coronaviruses (cov) can be ameliorated by type i ifn pretreatment strategies. in an in vivo macaque model (macaca fascicularis) of severe acute respiratory syndrome (sars)-cov infection, it could be demonstrated that pretreatment with pegylated ifn-α significantly diminished cov replication and excretion and resulted in reduced pulmonary damage ( ) . macaques also serve as a preclinical model for middle east respiratory syndrome (mers)-cov, and similar to sars-cov, ifn-α in combination therapy with ribavirin reduces viral replication and severe histopathological changes ( ) . in line, genetic alteration leading to an enhanced type i ifn signaling has been demonstrated to limit iav-induced disease outcomes, as a recent study by xing et al. ( ) reported that deletion of trim , a negative regulator of nemo, which leads to nfκb induction and therefore enhanced type i ifn production, is protective in vivo in iav-infected mice. conversely, the genetic depletion of ifn signaling in ifn receptor-deficient mice can result in a lack of viral control, resulting in enhanced viral titers in different viral infections including rsv or iav ( , ) . still, it must be noted that this effect is often mild in ifnar-or ifnlr-deficient animals, which is probably related to a certain redundancy between type i and type iii ifn signaling in limiting viral spreading in epithelial cells ( ) . in contrast, ifnar/ ifnlr-double knockout or stat knockout animals that are deficient in both type i and type iii ifn signal transduction succumb more readily to infection due to excessive viral replication ( ) ( ) ( ) . vice versa, mutations in key isgs such as ifitm are associated with increased iav disease severity in mice and humans ( ) . however, ifn pretreatment and genetic loss-of-function approaches generally are not relevant to human respiratory virus-induced hospitalizations, where patients already present with ongoing respiratory infection and inflammation, and preclinical studies underline that type i ifn signaling in an already inflamed organ is rather detrimental and enhances tissue injury, and lack of type i ifn in vivo may even ameliorate disease outcome. accordingly, in cases where the antiviral defense was not compromised (e.g., in animals with efficient type iii ifn signaling) ifnar-deficient mice infected with sendai virus or iav were reported to be more resistant to infection-induced morbidity and mortality ( , ) . similarly, in sendai virus in vivo infection, wetzel et al. ( ) showed that increased ifn-β levels in the lung homogenate correlates to increased morbidity and mortality, and also for sars-cov, a recent study demonstrates that high type i ifn induction in an already ongoing viral infection contributes to mortality in sars-cov-infected mice ( ) . also for iav infection, type i ifn application after infection has been proven to drive disease severity ( ) . of note, the detrimental effects of type i ifns were especially pronounced in mice lacking central antiviral factors, namely the ifit protein in sendai virus infection and mxa in iav. interestingly, beilharz et al. ( ) demonstrated that application of low doses of ifn-α reduces viral load, which to a certain degree led to attenuated disease progression, whereas high dose application of type i ifn contributed to morbidity ( ) . in line, high expression levels of isgs have been shown to correlate to worse outcomes in ards patients ( ) . this observation corresponds to reports stating that the ifn threshold needed to induce antiviral isgs-showing a beneficial effect in acute respiratory viral infection-is by at least -fold lower than the ifn dose necessary to trigger isgs that show immunomodulatory, death-inducing, or anti-proliferative effects and thus can contribute to disease progression ( ) ( ) ( ) ( ) . altogether, these data demonstrate that ifns may significantly contribute to unbalanced inflammation and tissue injury during respiratory viral infection depending on expression levels and duration of ifn-related signaling events. to date, the underlying mechanisms leading to the ifndependent enhanced disease progression are not fully understood but often result from a dysregulated ifn signaling response. one mode of action of ifn and ifn-stimulated isgs is to stimulate negative feedback loops on ifn signaling. for example, suppression of jak or stat via specific phosphatases, expression of suppressor of cytokine signaling (socs) and socs , or ubiquitination and endocytosis of the ifn receptors ( ) ( ) ( ) ( ) desensitize cells to ifn signaling and allow recovery and the return to homeostasis after microbial challenge. as demonstrated by bhattacharya et al. ( ) , the lack of ifnar downregulation and thus the failure to initiate ifn-desensitization contributes to increased inflammatory signaling, extensive lung injury and, importantly, also impaired tissue regeneration ( ) . moreover, ifns are immunomodulatory and shape the specific responses of cells of the immune system, which has been implied to influence disease progression both positively and negatively. in a recent study, type i ifns have been associated in the regulation of innate lymphoid immune cells (ilc) in iav infection, where they-in concert with ifn-γ and il- -promote an ilc -dependent restriction of immunopathology ( ) . moreover, type i ifns play an important role in stimulating the immune response driven by dcs; they stimulate the expression of mhc molecules as well as the co-stimulatory ligands cd and cd and thus activate t cell responses ( , ) . additionally, ligand-driven activation of ifnar enhances the proliferation of cd positive t cells, especially early in infection. however, late in infection, type i ifns were also implied in decreasing t cell expansion upon sars-cov and arenavirus infection ( , ) , which might potentially be related to the above described desensitization upon prolonged ifn signaling and might be detrimental if initiated too early in infection. in line, pinto et al. ( ) reported an impairment of t cell responses upon type ifn induction in west nile virus infection. in b cells, the lack of ifnar has been demonstrated to result in enhanced release of neutralizing antibodies in iav infection ( ) implying a repressive role for type i ifn in b cell antibody production. however, immunization studies by le bon et al. ( ) reported the necessity of ifnar on b cells for efficient igm and igg production, underlining the need for further studies to understand the detailed effects of ifn-dosage and timing adaptive immunity activity upon respiratory viral infection. type i ifns additionally induce the production of high levels of pro-inflammatory cytokines that have been closely linked to worsened outcomes of acute respiratory viral infection. especially in iav, disease severity and disease progression are linked with an overshooting, ifn-driven inflammatory response, in which further exogenous supplementation with type i ifn in fact correlates with increased morbidity and mortality ( , ) . in non-human primates, iav infection with a highly pathogenic h n isolate evokes a strong induction of type i ifn, resulting in severe lung injury by a necrotizing bronchiolitis and alveolotis ( ) . ifn levels in turn have been demonstrated to cause elevated pro-inflammatory cytokine levels after in vivo iav infection and additionally, in human alveolar macrophages, the release of pro-inflammatory cytokines (e.g., mcp- ) are preceded by a robust type i ifn response ( ) . importantly, also in human infection with h n , levels of pro-inflammatory cytokines are strongly elevated in bronchoalveolar lavage fluid, and cytokine levels have been associated with organ damage and worsened disease outcomes ( , ) . still, it should be noted that due to strain differences in virus-elicited prr activation and, importantly, ifn antagonism by the iav non-structural (ns) protein, ifn levels and disease severity do not always directly correlate; actually, the extent to which ns can suppress the ifn response relates to prolonged viremia and thus can also be a determinant of virus pathogenicity both in human bronchial epithelial cells and in an in vivo model of iav infection ( , ) . alongside iav, also in rsv infection the induction of high levels of pro-inflammatory cytokines has been directly related to type ifn, as rsv-infected but ifnar-deficient mice presented with significantly diminished pro-inflammatory cytokine release, which translated into an attenuated disease course ( ) . also in sars-cov, the late phase type i ifn induction relates to accumulation of inflammatory macrophage populations and elevated lung cytokine levels ( ) . in addition to antiviral, immunomodulatory, and pro-inflammatory isgs, ifn signaling results in the transcription and translation of cell death-inducing isgs. in the context of viral infection, these factors provide a mode to block viral spreading and reinfection by killing those infected cells, in which the internal activation of antiviral isgs is not sufficient to restrict viral replication. thus, the infected cell is sacrificed to prevent the release of infectious progeny virions to limit viral spreading. however, especially in the lung, the disruption of the alveolar epithelial barrier by cell death of infected cells, but importantly also non-infected bystander cells induced by factors such as trail, significantly contributes to worsened disease outcomes. controlled cell death or apoptosis can be induced by intrinsic and extrinsic signals. the intrinsic apoptosis pathway is initiated by diverse intracellular stimuli that influence the expression and activation of b cell lymphoma (bcl)- family proteins that govern the permeabilization status of the outer mitochondrial membrane. once cytochrome c is released from the mitochondria, it binds to the intracellular adaptor protein, apoptotic peptidase activating factor , forming the so-called apoptosome that in turn recruits pro-caspase- ( ). caspases (cysteine-aspartic proteases) exert their action by cleaving other proteins and substrates. herein, initiator caspases such as caspase- and caspase- target other downstream caspases, whereas effector caspases, including caspase- , - , and - , directly cause apoptosis by cleaving and thus inactivating or disassembling a vast array of cellular integral proteins and complexes ( ) . the extrinsic apoptosis pathway relies on an extracellular signal exerted by ligands of the tumor necrosis factor (tnf) receptor (tnfr) superfamily, including trail, tnf-α, and fas ligand (fasl) ( ) . their ligation to their respective cell surface-expressed death receptors (dr) leads via the signal transmission by fas-associated protein with death domain (fadd) to the activation of the initiator caspases- or - , finally stimulating effector caspases including caspase- ( ) . to date, several type i and type iii ifn-induced, proapoptotic factors have been identified ( ) . both caspase- and caspase- have been shown to be upregulated upon type i ifn signaling ( , ); caspase- enhances the fadd-driven extrinsic apoptosis pathway, whereas the less-studied caspase- may promote pro-il- β cleavage and inflammasome-driven cell death (pyroptosis) in macrophages ( , ) . chattopadhyay et al. ( ) demonstrated that sendai virus infection and polyi:c treatment resulted in bcl- -associated x protein (bax) activation and apoptosis induction via one of the key transcription factors of ifn genes, irf . in addition irf was reported to enhance trail-dependent extrinsic apoptosis by nuclear translocation resulting in the translation of to date undefined factors that increase cell death upstream of caspase- activation ( ) . furthermore, both rlrs, rig-i and mda- , trigger the proteins puma and noxa that induce bcl and the ifn/trail signaling axis frontiers in immunology | www.frontiersin.org march | volume | article thus activate the intrinsic mitochondrial apoptotic cascade ( ) . also, pkr influences a cell's susceptibility to apoptotic signals, as it was demonstrated to sensitize to the fadd/caspase- apoptosis pathway upon type i ifn signaling after challenge with iav or dsrna ( ) and the oas-rnasel system has been suggested to contribute to ifn-α-related cell death induction, but the exact mechanisms remain to be elucidated ( ) . finally, also two classical initiators of the extrinsic apoptosis cascade are induced as isgs. both fasl and its receptor fas are upregulated on mrna levels by ifn-α ( ) , and fasl was reported to be induced by type i ifn in iav infection in the murine lung in vivo ( ) . also, the proapoptotic factor trail (or tnfsf , apo l) is induced by ifn-mediated and isgf -executed transcriptional activation, as has been shown by sato et al. ( ) , who revealed the presence of the isre sequence within the trail promoter region ( ) . in iav infection, trail is released in high amounts from infected alveolar macrophages depending on a pkr-and ifn-β-driven autocrine signaling loop. binding of ifn-β to macrophageexpressed ifnar activates a jak/stat-dependent release of trail, which then acts through its receptor dr on the alveolar epithelial cells ( , ) . however, certain prerequisites may decrease the ability of a cell to undergo apoptosis, including a shortage in pro-caspase- availability, expression of cellular fadd-like il- β-converting enzyme-inhibitory proteins (c-flips) that block fadd-driven caspase activation, inactivation, or degradation of fadd itself, or expression of cyld, which acts as a receptor-interacting serine/threonine-protein (rip) kinase de-ubiquitinase and thus stabilizes rip . however, in these cases ifn signaling can still promote a caspase-independent, programmed inflammatory cell death by activating the necroptosis pathway ( , ) . necroptosis is induced by a complex formation by rip and rip kinases that activate both poly-adp-ribose (par) polymerase (parp- ) and/or mixed lineage kinase domain-like (mlkl), leading to atp depletion, calpain activation, par polymer accumulation or cell membrane permeabilization, and release of damage-associated molecular patterns, respectively [reviewed in ref. ( , ) ]. both a type i ifn-dependent jak/stat-driven activation of pkr as well as signaling by the prr dai (dnadependent activator of irfs) initiates necroptosis via rip /rip activation, respectively ( , ) . importantly, the activation of proapoptotic and pro-necroptotic pathways in respiratory infection can result in a structural disruption of the airway and the alveolar epithelial barrier, which is a major hallmark of respiratory disease and its progression to the acute respiratory distress syndrome ( , ) . in virusinduced lung injury, especially expression of trail, which can initiate both apoptosis as well as necroptosis has been correlated with more severe outcomes. as described earlier, trail belongs to the superfamily of tnf ligands and has been reported to be inducible by both type i and type iii ifns. trail has been found to be present in various cells of the immune system, among them natural killer (nk) cells, t cells, nk t cells, dc subsets such as ifn-γ-producing killer dcs and macrophages, and can be displayed in large amounts on the cell surface or be shed upon ifn-and/or pro-inflammatory cytokine signaling ( ) ( ) ( ) . in addition to cells of the immune system, fibroblasts have been shown to produce trail after ifnγ treatment or viral challenge. also, club cells and the alveolar epithelium have been reported to produce trail ( ) ( ) ( ) ( ) . similar to other ligands of the tnf superfamily, trail is a homotrimeric type ii transmembrane protein with a conserved c-terminal extracellular domain that mediates receptor binding and can be cleaved by metalloproteinases to generate a soluble mediator ( ) . however, trail can induce cell death also in its membrane-bound form, that is, similar to trail expression levels and trail shedding, upregulated by type i ifn ( ) . direct cell-to-cell trail-dr interactions have been demonstrated to play a role in macrophage, nk as well as cd + t cell-mediated induction of cellular death ( , ) . in humans, five different binding partners for trail are present: the membrane-bound dr (trail-r ) and dr (trail-r ) that both induce a proapoptotic signaling cascade, the membrane-bound anti-apoptotic decoy receptors (dcr) and dcr , and the soluble interaction partner osteoprotegerin ( ) . in the murine system, only dr has been identified to ligate to trail ( ) . in the human respiratory compartment, both dr and dr have been demonstrated to be present under steady-state conditions ( , ) . however, upon viral infection, cell-sensitivity to trail-induced apoptosis is enhanced, which has been attributed to increased trail receptor expression especially on infected cells, as dr levels are markedly increased in iav-, adenovirus-, and paramyxovirus-infected cells in contrast to non-infected bystander cells ( , , ) . of note, studies on the dependency of dr upregulation upon type i ifn signaling after iav infection have yielded conflicting results in different strains of mice ( , ) , highlighting the complex interplay of ifn-induced cascades in a host-and tissue-specific context, whereas the exact virus-and host-specific mechanisms for dr regulation remain less well defined. moreover, previous assumptions that also dcr expression would correlate with cell-sensitivity to trail-induced cell death could not be experimentally verified ( ) . tumor necrosis factor-related apoptosis-inducing ligand ligation to the proapoptotic receptors dr or dr triggers a trimerization of the receptors. subsequently, depending on additional stimuli, presence or absence of adaptor molecules or inhibitory proteins, different signaling pathways can be activated (figure ) . in the classical trail-dependent extrinsic apoptosis induction, the proteins rip, tradd, and fadd are subsequently recruited to the dr cytoplasmic domain upon trail ligation ( , ) . these factors and the proapoptotic drs all share a cytoplasmic death domain (dd), which is lacking or truncated and thus inactive in the dcr. the dd plays a central role in the concerted formation of the death-inducing signaling complex (disc). disc formation exposes a second functional domain of fadd, the death effector domain that is directly able to recruit pro-caspase- figure | trail/dr -mediated cellular signaling pathways. in presence of rip , tradd, and fadd, trail ligation to dr results in apoptosis induction, which is initiated by recruitment of the pro-caspase- or - to fadd. these in turn activate the effector caspases- and - , which leads to dna fragmentation and apoptosis induction. in addition, tradd can trigger a traf -and jnk-dependent activation of bax and subsequent release of mitochondrial cytochrome c, inducing the pro-caspase- activation. in the presence of cyld, c-flip or absence of sufficient amounts of fadd or pro-caspase- , trail ligation to dr triggers the interaction of rip and rip kinase, which in turn cause cell death via induction of mlkl and/or parp- . in the presence of ciaps, fadd is not recruited to dr upon trail ligation, and tak is activated by tradd/traf interactions. tak induces nemo followed by iκb degradation and nfκb activation, as well as mkk and jnk activation leading to ap- nuclear translocation; both events promote the production of cytoprotective factors such as xiap, ciaps, and c-flip. additionally, tak triggers ampk activation and thus mtorc inhibition, which results in enhanced autophagic activity. abbreviations: ap- , activator protein ; tnf, tumor necrosis factor; trail, tnf-related apoptosis-inducing ligand; dr , death receptor ; rip , receptor-interacting serine/threonine-protein kinase ; tradd, tnf receptor type -associated death protein; fadd, fas-associated protein with death domain; traf, tnf receptor-associated protein; jnk, janus kinase; bax, bcl- -associated x protein; c-flip, cellular fadd-like il- β-converting enzyme-inhibitory proteins; rip, receptor-interacting serine/threonine-protein; mlkl, mixed lineage kinase domain-like; parp- , poly-adp-ribose (par) polymerase ; ciap, cytoprotective factors including inhibition of the autophagic machinery; xiap, x-linked inhibitor of apoptosis protein; ampk, amp-activated protein kinase; mtorc, mammalian target of rapamycin complex; traf , tnf receptor-associated protein ; mkk, mitogen-activated protein kinase. and pro-caspase- . how exactly disc formation induces caspase activation is still under debate. the most probable scenarios include either an autocatalytic cleavage of caspase-pro-domains enabled by the spatial proximity between pro-caspases (generated by their recruitment to disc), by pro-caspase dimerization, or by pro-caspase conformational stabilization ( ) . removal of the pro-domain of caspase- and caspase- results in the activation of the effector caspases- and - , which cleave dna fragmentation factor and lead to apoptosis ( , ) . moreover, trailbinding to dr and dr can induce the jnk either via caspase- or recruitment of tnf receptor-associated protein (traf ) to the disc complex, which results in the activation of the intrinsic apoptotic cascade by bax-dependent mitochondrial cytochrome c release ( ) . in addition, trail signaling is also able to induce necroptosis by both activating the rip /rip kinase downstream effectors parp- and mlkl, contributing to epithelial cell death and tissue injury ( ) ( ) ( ) . it has become apparent in recent years that trail signaling is closely linked to induction of autophagy, a process generally associated with the blockade of apoptosis and necrosis. indeed, autophagy has been reported to improve cellular survival in cell stress by catabolic removal of cytoplasmic long-lived proteins and damaged organelles. it also contributes to viral clearance and the transfer of viral material to endosomal-/lysosomallocated tlr or mhc class ii compartments for the activation of adaptive immunity ( ). several studies outline that trail ligation to dr can result in a traf -dependent activation of tak (map k ) that has been attributed a central role in trail-induced autophagy activation ( ) . tak modulates the ikk-dependent translocation of nfκb, and it also induces jnk activation via mitogen-activated protein kinase. both events lead to expression of autophagy-related factors including inhibition of the autophagic machinery (ciap) , ciap , x-linked inhibitor of apoptosis protein, and c-flip ( , ) . especially, c-flip has been associated with desensitization of cells to trail-induced apoptosis, favoring autophagy-related cascades ( ) . another study revealed that upon trail signaling the amp-activated protein kinase (ampk) is activated. ampk in turn inhibits the mammalian target of rapamycin complex that itself is an inhibitor of autophagy, thus the activation of the autophagic machinery is promoted ( ) . the decision if trail signaling results rather in necroptotic or apoptotic cell death or in activation of autophagy seems to be dependent on the presence of ciaps that promote rip kinase ubiquitination and degradation ( ) , but also on the balance between active caspases and autophagic proteins such as beclin- ( , ) . this suggests a scenario where autophagy is activated as cell protective mechanism until cell stress-as executed by enhanced trail signaling or additional viral infection-increases over a threshold to favor cell death induction. accordingly, as trail signaling is not restricted to infected cells, excessive cell death activation might be limited by autophagy induction in non-infected bystander cells. however, autophagy is not only related to cell survival but can also positively affect apoptosis and induce-even if the exact mechanisms are still under debate-autosis, the autophagy-related cell death, another mode of trail to trigger cell death ( , ) . of note, autophagy activation needs to be placed into its virus-specific context, as some viruses, including dengue virus, poliovirus, and coxsackie b virus ( ) , can exploit autophagic pathways for their own replication and thus promote apoptosis and tissue injury. as discussed above, trail is a potent activator of cell death. however, its signaling outcomes can differ largely depending on its delivered form (e.g., membrane-bound versus soluble), the availability of drs on the target cell membrane, alternate intracellular pathways that might be activated and finally the pathogen itself, as it might exploit trail-induced pathways for its own survival and replication. in acute respiratory infection, trail signaling is often part of an ifn-driven overshooting inflammatory reaction that promotes unspecific tissue injury and thus disease severity by increasing functional and structural changes in infected but also non-infected cells, as will be outlined below. the release and effects of trail have been especially well studied in iav infection in the last decade. earlier studies reported that within days after infection, bronchial, bronchiolar, and alveolar epithelial cells undergo apoptosis ( ) . this early induction of cell death is mainly attributed to direct apoptosis induction by the virus itself, as iav actively promotes apoptosis for efficient viral replication ( ) . herein, the viral ns and pb-f proteins not only play a crucial role ( , ) but also the viral m protein has been implicated in this process as it inhibits autophagy in infected cells ( ) . in addition, our own data revealed that later in iav in vivo infection, the recruitment of bone marrowderived macrophages via the cc chemokine receptor type (ccr )-cc-chemokine ligand (ccl ) axis significantly contributes to alveolar cell apoptosis and structural damage of the alveolar epithelium ( ) . studies by wurzer et al. ( ) had previously demonstrated that iav promotes the production of proapoptotic factors in an auto-and paracrine fashion via nfκb transcriptional activation by iav ( ) . subsequently, brincks et al. ( ) elucidated that human peripheral blood mononuclear cell treated with iav released trail and that increased trail levels correlated with type i as well as ii ifn induction. additionally, trail sensitivity was increased in influenza virusinfected cells. in line, our investigations could elucidate that iav triggers a pkr-dependent translocation of nfκb that results the production of type i ifns. these in turn induce, via ligation to the ifnar receptor complex, expression and shedding of trail by bone marrow-derived macrophages ( ) . in addition, davidson et al. ( ) demonstrated that type i ifn application to iavinfected mice increased morbidity and lung injury, which could be attributed to both dr and trail upregulation inducing epithelial cell apoptosis. importantly, högner et al. also reported that the iav strain used in these studies, a/pr (h n ), which is highly pathogenic for mice, induced an approximately -fold induction in macrophage trail expression, whereas the lower pathogenic virus a/x- (h n ) only stimulated trail by a factor of eight. of note, the relation between trail induction and iav strain-specific pathogenicity also translates to the highly pathogenic avian h n iav, causing severe pneumonia in mice as well as in humans ( , ) . moreover, human infection with both the highly pathogenic h n as well as the pandemic h n iav strains are characterized by a massive influx of mononuclear phagocytes into the alveoli, which is correlated with extensive alveolar epithelial cell apoptosis ( , ) . additionally, macrophages gained from bronchoalveolar lavages of patients presenting with ards caused by the pandemic h n / virus strain showed high surface expression and release of trail ( ) . another recent report demonstrates that in highly pathogenic avian influenza, in addition to macrophages also the alveolar epithelium might be involved in causing elevated levels of trail in the alveolar space ( ) . besides its role in apoptosis, trail signaling upon iav infection has also been implicated in the induction of necroptosis in fibroblasts, dcs, and lung epithelial cells ( , , ) . rodrigue-gervais et al. ( ) demonstrated that lack of cipa promotes rip kinase-mediated necroptosis in response to trail-but also the proapoptotic factor faslreleased from hematopoietic cells. this contributed to severe lung epithelial degeneration and increased mortality, even though viral control was not compromised. nogusa et al. ( ) further elucidated that iav-induced necroptosis depends on rip kinase activation of mlkl, and that rip kinase deficiency, similar to ciap -deficiency, increased iav-susceptibility in vivo. in iav infection, as mentioned earlier, dr expression is elevated on infected alveolar epithelial cells, but not in noninfected cells in vivo, which might impact on trail susceptibility to apoptosis induction ( ) . however, both infected as well as neighboring bystander cells were found to be targeted for apoptosis induction by macrophage-released trail. nonetheless, we could recently show that specifically in noninfected cells within the iav-infected lung, trail severely compromises the function of the ion channel na,k-atpase, which was mediated via induction of the stress kinase ampk ( ) , thereby potentially revealing a cross-link to trailinduced autophagic cell stress pathways in bystander cells both in vitro and in vivo. the trail-induced and ampk-mediated downregulation of the na,k-atpase, a major driver of vertical ion and fluid transport from the alveolar airspace toward the interstitium, resulted in a reduced capacity of iav-infected mice to clear excessive fluid from the alveoli. thus, trail signaling contributes to intensive edema formation, a hallmark of disease in virus-induced ards ( ) . notably, this effect of trail on na,k-atpase expression was induced independently of cell death pathways elicited by caspases, as treatment of cells and mice with a specific caspase- inhibitor diminished apoptosis in alveolar epithelial cells but still allowed for the reduction of the na,k-atpase ( ) . conclusively, treatment of iav-infected mice with neutralizing antibodies directed against trail or the abrogation of recruitment of trail + bone marrow-derived macrophages inhibited apoptosis of both non-infected and bystander cells. thus, lung leakage due to loss of alveolar barrier function was reduced, whereas alveolar fluid clearance capacity was enhanced, resulting in reduced edema, improved survival, and outcome upon iav challenge in vivo. however, trail has also been shown to be upregulated on nk, dc, and on cd + and cd + t cells after iav infection ( ) . studies by brincks et al. demonstrated that especially cd + t involved in cytotoxic t cell responses toward iav and drive iav-infected cells into apoptosis via trail, thus contributing to efficient virus clearance ( , ) . in addition, both fasl and trail are involved in dc-mediated ctl activation and cytotoxicity against iav-infected cells ( , ) . furthermore, studies showed delayed viral clearance upon neutralizing anti-trail antibody administration ( , ) . our data, however, demonstrate that the transfer of trail-deficient bone marrow into irradiated wild-type mice, resulting in loss of trail production by bone marrow-derived macrophages upon iav infection, does not impact on the capacity to fully clear viral particles from the lung at day after infection, suggesting that other compensatory mechanisms are recruited to guarantee viral clearance ( ) . taken together, in iav infection, trail acts both as an important mediator of infected cell killing but particularly as a detrimental factor contributing to tissue injury and impaired inflammation resolution when released in excessive amounts by recruited immune cells. respiratory syncytial virus is an important cause of respiratory tract infections especially in children worldwide. generally, there seem to be virus-elicited anti-apoptotic mechanisms active in the lung epithelium, as rsv-infected primary human airway cells show a minimal cytopathic effect ( ) . however, several cell lines including small airway cells, primary tracheal-bronchial cells, and a and hep- showed increased expression of trail and its ligands dr and dr in an in vitro rsv infection model ( ) . moreover, soluble trail released from leukocytes was elevated in the bronchoalveolar lavage fluid of patients with rsv-associated respiratory failure, suggesting that similar to iav, trail contributes to rsv-induced epithelial injury and disease progression ( ) . also in cov respiratory tract infection, trail levels, but less so fasl, have been reported to be markedly elevated ( , ) . for sars-cov that presents with a severe damage to both the upper and lower respiratory tract ( ) , especially dcs respond with a strong induction of trail production, which was suggested to correlate to increased cellular lung infiltrations present in sars-cov patients ( ) . interestingly, sars-cov infection drives cells into apoptosis by a pkr-driven but eif α-independent pathway ( ) , which might-similarly as seen in iav infection-suggest a pkr-induced and autocrine/paracrine executed activation of apoptosis. also mers-cov, which causes pneumonia and respiratory failure, has been demonstrated to induce profound cell death within h of infection, irrespective of viral titers produced by the infected cells. however, type i ifn expression is strongly reduced in mers-cov in comparison to seasonal human cov in in vitro infection models, including human monocyte-derived macrophages, calu- , and human lung fibroblasts ( , ) , which might also dampen downstream trail induction. therefore, the exact mechanism by which mers-cov promotes cell death remains to be investigated. recurrently, viral infections of the respiratory tract are followed by outgrowth of colonizing gram-positive bacteria that aggravates the course of illness. this is well documented for iav, where "super" infections with streptococcus pneumoniae and staphylococcus aureus are the most frequent and increase viral pneumonia-associated morbidity and mortality ( ) . during the iav pandemic, bacterial pneumonia was evident in most cases ( ) and also during the recent h n pandemic, coinfections were a relevant factor for severe disease in a young patient population without comorbidities ( ) . interestingly, virus-induced elevation of the type i ifn response levels might promote secondary bacterial outgrowth by several mechanisms [reviewed in ref. ( ) ]. in line, it has been repeatedly demonstrated that lack of type i ifn signaling results in better bacterial clearance and increased survival rates in iav-and s. pneumoniae-superinfected mice ( ) ( ) ( ) . herein, ifn-induced apoptosis induction as well as depletion or impaired recruitment of lymphocyte subsets necessary for bacterial control play a critical role ( , ) . bacterial clearance from the lung has been reported to rely on sufficient phagocyte generation, recruitment, and survival. type i ifn has been demonstrated to cause apoptosis in bone marrow-derived granulocytes, affecting the numbers of recruited neutrophils ( ) , but also to impair expression of the cytokines cxcl (or kc) and cxcl (or mip- ), thus inhibiting neutrophil recruitment to the lungs with severe effects on survival of superinfected mice ( ) . a recent report by schliehe et al. ( ) elucidated the mechanistic background for impaired cxcl expression and secretion and demonstrated that type i ifns activate the histone methyltransferase setdb , which in turn represses the cxcl promoter and thus impairs neutrophil recruitment and bacterial clearance. moreover, type i ifn production decreases ccl production, thus inhibiting macrophage recruitment, which as well has been reported to have detrimental effects on bacterial clearance and disease progression in bacterial superinfection after viral insult in vivo ( ) . in addition, type i ifns also impair γδ t cell function and il- release, which was shown to increase susceptibility to s. pneumoniae superinfection after iav challenge ( ) . also in s. aureus pneumonia, a robust type i ifn response is correlated to excessive morbidity and tissue injury ( ) . in a model of polyi:c, s. aureus (methicillinresistant strain, mrsa) superinfection, polyi:c treatment prior to bacterial infection enhanced type i ifn levels and decreased bacterial clearance and survival ( ) . furthermore, shepardson et al. ( ) demonstrated that late type i ifn induction rendered mice more susceptible to secondary bacterial pneumonia in a model of iav-mrsa superinfection. only limited data are available on a direct role of trail in respiratory disease progression due to bacterial superinfections. in a model of iav-haemophilus influenza infection, neither deficiency for cc chemokine receptor type , inhibiting bone marrow-derived macrophage recruitment, nor deficiency of fas or tnfr impacted outcome ( ) . yet, during s. pneumoniae single infection, early cell death of macrophages is thought to limit an exuberant inflammatory reaction and accordingly, a study by steinwede et al. ( ) revealed that neutrophil-derived trail limits tissue injury by inducing cell death in dr -epressing lung macrophages in bacterial mono-infection ( ) . in contrast, in the iav-s. pneumoniae superinfection mouse model, iavinduced trail has a detrimental effect on overall mortality ( ), as trail-induced epithelial injury enhanced bacterial outgrowth of s. pneumoniae-administered at day after iav infection-markedly. importantly, administration of anti-trail neutralizing antibodies enhanced bacterial control by the host organism. thus, the activation of ifn/trail-mediated signaling in viral infection has detrimental implication for outcome of secondary bacterial infection following viral insult, rendering the ifn/trail signaling axis an interesting therapeutic target not only in respiratory viral infections but also in complicating bacterial superinfection. an increasing number of reports connect progression of chronic respiratory disease to acute respiratory virus infection or proapoptotic signaling events. in fact, trail has been reported to be a critical determinant for promoting the development of chronic lung disease in early life ( ) ; targeting trail by genetic deletion or neutralizing antibody application in early-life respiratory infections ameliorated infection-induced histopathology, inflammation, as well as emphysema-like alveolar enlargement and lung function. furthermore, trail was also shown to play a role in the development of allergy and asthma. trail is not only elevated in the sputum of asthmatic patients but has also been reported to be highly expressed in an experimental mouse model of asthma, where it induces ccl secretion by bronchial epithelial cells, thus promoting th cell responses and airway hyperreactivity ( ) . in copd, acute exacerbations driven by viral and bacterial infection are a major factor increasing both mortality and morbidity, and both influenza and s. pneumoniae have been identified among the most common causes of copd exacerbations ( ) . indeed, primary bronchial epithelial cells isolated from subjects with copd show an impaired production of type i ifn ( ) , which has been implied in the enhanced susceptibility of copd patients to respiratory infections; however, even in absence of high ifn induction, both an abnormally elevated loss of alveolar epithelial cells due to apoptosis as well as elevated trail and dr levels were reported ( ) , implying a possible link between viral/bacterial induction of trail and acute exacerbations in copd. trail induction has also been directly linked to cigarette-smoke exposure, a common cause of copd, and trail deficiency resulted in decreased pulmonary inflammation and emphysema-like alveolar enlargement in vivo ( ) . moreover, increased levels of both trail and dr were associated to impaired lung function and increased systemic inflammation in human copd patients ( ) . while alveolar epithelial cell death is closely connected to idiopathic pulmonary fibrosis (ipf), trail and its receptors dr and dr in aec were shown to be upregulated in ipf lungs ( ) . also, in pulmonary arterial hypertension virus infection is considered to be a possible risk factor ( ) , and pulmonary hypertension has been reported to be a side effect of prolonged treatment with type i ifn ( , ) . in line, trail has been closely linked to disease progression in pulmonary hypertension. trail has been found to be increased within pulmonary vascular lesions of patients with pulmonary hypertension ( ) and also in a mouse model of hypoxia-induced pulmonary hypertension, levels of soluble trail correlated with right ventricular systolic pressure, right ventricular hypertrophy, and pathologic alterations ( , ) . importantly, neutralizing antibody-treatment against trail showed positive effects on survival while reducing pulmonary vascular remodeling ( ) . notably, the extent to which infection-induced trail release causes or exacerbates chronic lung disease or in how far trail production in chronic lung diseases affects susceptibility to respiratory viral and complicating bacterial infection remains to be elucidated. respiratory viral infections are major causative agents for lung injury and ards; however, in many cases antivirals are not sufficient to limit disease ( ). besides the fact that most viruses are subject to strong selective pressures that favor quickly evolving, drug-resistant virus variants, recent advances in understanding the processes that contribute to tissue injury and ards highlight a crucial role of immune-related, ifn-driven events. therefore, novel therapeutic strategies often aim to improve the outcome of severe respiratory infection by modulating host cell responses; however, to date, clinical trials trying to improve severe viral infections or ards outcomes by targeting host pathways have not resulted in approval of new drugs ( ) . of note, for establishment of such therapies it has to be considered that the timing and intensity of induction and amplification as well as of dampening and termination of the ifn-driven immune response needs to precisely match the pathogen-and organ-specific requirements of a given infection. a non-controlled regulation of these processes may lead to either an unrestricted pathogen spreading or, on the other extreme, to an overshooting inflammatory response, including the increased production of pro-inflammatory and proapoptotic mediators, elevated levels of recruited immune cells, and/or aberrant repair processes. notably, both too low and too high levels of ifn-induced effects facilitate disease progression with a possible increase of fatal outcomes in ards patients ( ) . accordingly, preclinical in vivo studies of ifn-directed therapies yielded seemingly adverse results, depending on the context, timing, and dosage of ifn modulation. however, in multiple settings of acute respiratory viral infection, studies demonstrate that an exaggerated signaling derived from type i ifn in an already inflamed tissue contributes to worsened outcomes, and importantly, might favor secondary bacterial superinfection [e.g., ref. ( , , ) ]. interestingly, davidson et al. ( ) demonstrated that type iii ifn release upon influenza challenge-in contrast to type i ifn induction-does not trigger an unbalanced inflammatory response that critically contributes to respiratory disease progression in vivo, highlighting it as a possible therapeutic option in iav-induced lung injury. most likely, this effect derives from the lack of the ifn-λr /il- r receptor complex, but presence of ifnar, on immune cells, including bone marrowderived macrophages. nonetheless, other reports identify ifn-λ as a driver of macrophage polarization to an inflammatory m phenotype ( ) that has been attributed to further promote an overshooting inflammatory response, highlighting the need for further studies of type iii ifn biology in pathogen-associated disease progression. as generally ifn-directed therapeutic approaches target various downstream signaling events that might both act beneficially as well as detrimentally on viral replication and pathogenesis, a further approach is to address specific isgs that primarily show detrimental effects on disease progression. as outlined above, trail or its downstream signaling events might comprise a suitable target for adjunct therapies in addition to antivirals. accordingly, our own data in a preclinical mouse model of iav infection demonstrate a clear benefit of the systemic application of neutralizing antibodies against trail at days and postinfection for lung injury, morbidity, and mortality ( , ) . targeting trail as a major determinant of disease severity in respiratory viral infections including iav, but also rsv and cov, may yield therapeutic approaches that are superior to ifn-directed strategies, as they seemingly do not bear the risk of compromising host defense. yet, it should be thoroughly excluded that blocking trail-induced cell death of infected cells will not lead to an overwhelming viral spreading, especially as reports on viral loads upon trail inhibition in preclinical models of iav are controversial ( , , ) . accordingly, additional studies are needed to understand how and to which extent virus-infected cells can be killed or viral spreading can be controlled by other means in the absence of trail. moreover, targeting pathways and signaling hubs downstream of trail/drs, such as ampk ( ), in a well-timed and lung compartment-specific way, may open new therapeutic avenues but requires more detailed preclinical studies on efficacies and side effects. a valid approach might be the use of a combination therapy of such a treatment together with a classical antiviral drug therapy limiting viral replication; however, exact dosage, timing, kinetics, and application routes remain to be defined. cp and sh performed bibliographic research and drafted the manuscript. this work was supported by the german research foundation (sfb-tr b , sfb c , kfo p /p , exc ), by the german center for lung research (dzl), and by the german center for infection research (dzif). virus interference. i. the 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apoptosis-inducing ligand (trail) reverses experimental pulmonary hypertension luks am. ventilatory strategies and supportive care in acute respiratory distress syndrome ifnλ is a potent anti-influenza therapeutic without the inflammatory side effects of ifnα treatment the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -z vxamvp authors: gagiannis, daniel; steinestel, julie; hackenbroch, carsten; schreiner, benno; hannemann, michael; bloch, wilhelm; umathum, vincent g.; gebauer, niklas; rother, conn; stahl, marcel; witte, hanno m.; steinestel, konrad title: clinical, serological, and histopathological similarities between severe covid- and acute exacerbation of connective tissue disease-associated interstitial lung disease (ctd-ild) date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: z vxamvp background and objectives: understanding the pathophysiology of respiratory failure in coronavirus disease (covid- ) is indispensable for development of therapeutic strategies. since we observed similarities between covid- and interstitial lung disease in connective tissue disease (ctd-ild), we investigated features of autoimmunity in sars-cov- -associated respiratory failure. methods: we prospectively enrolled patients with rt-pcr-confirmed sars-cov- infection and patients with non-covid- -associated pneumonia. full laboratory testing was performed including autoantibody (aab; ana/ena) screening using indirect immunofluorescence and immunoblot. fifteen covid- patients underwent high-resolution computed tomography. transbronchial biopsies/autopsy tissue samples for histopathology and ultrastructural analyses were obtained from / cases, respectively. results: thirteen ( . %) patients developed acute respiratory distress syndrome (ards), and five patients ( . %) died from the disease. ana titers ≥ : and/or positive ena immunoblots were detected in / ( . %) covid- patients with ards, in / ( . %) covid- patients without ards (p = . ) and in / ( %) patients with non-covid- -associated pneumonias (p = . ). detection of aabs was significantly associated with a need for intensive care treatment ( . vs. %; p = . ) and occurrence of severe complications ( vs. %, p = . ). radiological and histopathological findings were highly heterogeneous including patterns reminiscent of exacerbating ctd-ild, while ultrastructural analyses revealed interstitial thickening, fibroblast activation, and deposition of collagen fibrils. conclusions: we are the first to report overlapping clinical, serological, and imaging features between severe covid- and acute exacerbation of ctd-ild. our findings indicate that autoimmune mechanisms determine both clinical course and long-term sequelae after sars-cov- infection, and the presence of autoantibodies might predict adverse clinical course in covid- patients. coronavirus disease , caused by severe acute respiratory syndrome coronavirus (sars-cov- ), has caused or contributed to hundreds of thousands of deaths and led to almost complete shutdown of social and economic life in many countries ( ) . based on what is currently known about epidemiology, covid- is associated with a mortality rate between and % ( ) . major cause of death in covid- infections is acute respiratory failure (acute respiratory distress syndrome, ards), but the exact mechanism of how covid- leads to ards is unclear. in most reported morphological analyses, the authors describe diffuse alveolar damage (dad) with an early edematous phase followed by hyaline membrane formation, desquamation of pneumocytes, and an increased interstitial mononuclear infiltrate in severe sars-cov- infection ( ) . in one case, tian et al. report loose intra-alveolar fibromyxoid proliferation reminiscent of organizing pneumonia (op) ( ) . such combined histological patterns of (organizing) dad and op, summarized by some authors under the term acute fibrinous organizing pneumonia (afop), have also been observed in interstitial lung disease associated with systemic lupus erythematosus (sle), dermatomyositis, and progressive systemic sclerosis (pss) ( ) ( ) ( ) . this is of special relevance since both organizing dad as well as ctd-ild may evolve to pulmonary fibrosis, and long-term effects of covid- are so far unknown. only recently, upregulation of fibrosis-associated gene expression in covid- has been described ( ) . most ctds are defined by the presence of specific antinuclear autoantibodies (anas), several of which have been identified and summarized under the historic term extractable nuclear antibodies (enas), such as anticentromer antibodies (cenp-b), pm-scl, ss-b/la, jo- , and scl- ( ) . only recently, the presence of such autoantibodies has been described in cases of severe covid- , but the exact relevance of this finding remains unclear ( , ) . taken together, since available data suggests histomorphological as well as pathophysiological similarities between covid- associated ards and lung manifestations of autoimmune disease, we hypothesized that a dysregulated immune response upon sars-cov- infection might show similarities to acute exacerbation of ctd-ild which might shed some light on the mechanism of lung damage in covid- . in this prospective trial we consecutively included all patients with positive sars-cov- -rt-pcr (mucosal swab, pharyngeal or bronchoalveolar lavage) admitted to the bundeswehrkrankenhaus (armed forces hospital) ulm in march and april after obtaining informed consent. suspected cases without rt-pcrconfirmed sars-cov- infection were excluded from the study. a group of patients with non-covid- -associated pneumoniae served as control group for serological analyses (supplementary table ). patients or their relatives had given written informed consent to routine diagnostic procedures (serology, bronchoscopy, radiology) as well as (partial) autopsy in the case of death, respectively, as well as to the scientific use of data and tissue samples in the present study. this project was approved by the local ethics committee of the university of and conducted in accordance with the declaration of helsinki. we collected clinical information from electronic patient files. data included disease-related events, preexisting comorbidities, imaging, treatment approaches (supplementary table ) , and clinical follow-up. the "berlin definition'' was used to categorize ards ( ) . the horovitz quotient (pao /fio ) was assessed in all ards cases based on arterial blood gas analysis. during icu treatment, ventilation parameters, duration of invasive ventilation, catecholamine support, prone positioning, murray lung injury score and the need of additional temporary dialysis were continuously assessed (supplementary table ) ( ) . a profitable trial of prone positioning was defined by an increasing horovitz quotient of mmhg or more. one entire trial covered h of sustained prone positioning. blood samples for serology and monitoring of laboratory values were taken at hospital admission and during ward/icu treatment, respectively. laboratory values included possible predictors of outcome in covid- patients such as lymphocyte count, fibrinogen, d-dimers, ferritin, lactate dehydrogenase (ldh), and bilirubin. we also assessed troponin-t levels as a marker for cardiac events and infection-associated parameters (neutrophils, interleukin- (il- ), creactive protein (crp), and procalcitonin (pct)). cut-off values, median, and range for these parameters are summarized in supplementary table . ana/anca/ena testing initial screenings for ana and anca (p-anca, c-anca, x-anca, anti-pr , anti-mpo) were performed by iif using patient sera on hep- cells and primate liver tissue slides (ana) as well as ethanol-and formol-fixed granulocytes and purified pr- and mpo antigens (anca) on glass slides (euroimmun ag, lübeck, germany) according to the manufacturer's protocols ( , ) . in all cases, presence of specific anti-ena autoantibodies (anti-sm, anti-ss-a/ro, anti-ss-b/la, anti-scl- , anti-centromere, anti-jo , anti-mi- , anti-u -rnp, anti-ro- , anti-pm-scl, anti-cnp b, anti-pcna, anti-dsdna, anti-nucleosome, anti-histone, anti-ribosomal pprotein, anti-ama-m ) was assessed by semiquantitative immunoblot (anti-ena profile ; euroimmun ag, lübeck, germany) irrespective of the initial screening result. following previously published guidelines, ana titers ≥ : with or without positive ena immunoblot or ana titers of : with positive ena immunoblot were regarded as positive ( , ) . laboratory testing was performed by investigators who were blinded to patient status, and in cases with unclear/borderline results in either ana screening or ena subtyping, tests were repeated on a new sample, and results were verified by an external reference laboratory. quality and reliability of ana/ anca/ena testing in our institution have been evaluated through regular interlaboratory ring trials. imaging was performed on a somatom force scanner (dual source scanner * slices, siemens, erlangen, germany) in accordance to the guidelines of the german radiological society and our hospital's covid- guidelines, using low-dose ct (computed tomography) with high-pitch technology ( ) . the following parameters were used: tube voltage: kv with tin filtering, tube current: mas with tube current modulation. in two cases examination was performed as a non-contrast enhanced full-dose protocol because of suspected ild, in one case as a contrast-enhanced ct scan to exclude pulmonary thromboembolism. x-ray examinations were performed at the respective wards as bed-side x-ray examinations (mobilett mira max, siemens, erlangen, germany) as a single anterior-posterior view. the ct images were evaluated according to the expert consensus statement of the rsna and classified as typical, indeterminate, atypical, and negative appearance for covid- ( , ) . lung tissue specimens were obtained as transbronchial biopsies in four cases. in three deceased patients, partial autopsies were performed, and lung, heart, and liver tissues were sampled extensively. specimens were stained with hematoxylin-eosin (he), phosphotungstic-acid-hematoxylin (ptah), elasticavan-gieson (evg) and masson-goldner (mg). furthermore, immunohistochemistry for cd , cd , ck , cmv, and ebv was performed using prediluted antibodies on a ventana benchmark autostainer (roche tissue diagnostics, mannheim, germany) following routine protocols. lung, heart, and liver tissues were immersion-fixed with % paraformaldehyde in . m pbs, ph . . after several time washing in . m pbs, tissue was osmicated with % oso in . m cacodylate and dehydrated in increasing ethanol concentrations. epon infiltration and flat embedding were performed following standard procedures. methylene blue was used to stain semithin sections of . µm. seventy to ninetynanometer-thick sections were cut with an ultracut uct ultramicrotome (fa. reichert) and stained with % aqueous uranylic acetate and lead citrate. samples were studied with a zeiss em electron microscope (fa. zeiss) coupled to a megaview iii soft imaging system camera analysis ® software both from fa. (soft imaging system gmbh). descriptive statistical methods were used to summarize the data. medians and interquartile ranges were used to announce results. absolute numbers and percentages were employed to represent categorial variables. student's t-test was used for the comparison of continuous variables, while chi-square-test/fisher's test was used for categorial variables. all statistical analyses were conducted using graphpad prism (graphpad software inc., san diego, ca, usa). a p-value < . was regarded as statistically significant. baseline clinical characteristics of sars-cov- infected patients are briefly summarized in table , and we show a timeline of the complete study cohort with all relevant events in figure . clinical characteristics of non-covid- -associated pneumonia patients (controls) are summarized in supplementary table . median age at initial diagnosis was . years (range, - years). the majority of patients were male ( / cases; . %). most frequent preexisting comorbidities were cardiovascular risk factors ( / , . %) and established cardiovascular disease ( / , . %). preexisting rheumatic disease was present in / cases ( . %): one patient (# ) had rheumatoid factor-positive rheumatoid arthritis, the other patient (# ) had a history of rheumatic disease and associated treatment which could not be evaluated in more detail. treatment approaches and drug-related toxicities are summarized in supplementary table . bacterial superinfection was suspected in / ( . %) cases by clinical course, imaging, and laboratory values. antibiotic treatment approaches are summarized in supplementary table . there was an overall high rate of complications compared to regular (non-covid) ards patients (supplementary table ). one patient was temporarily transferred to another hospital because vv-ecmo (veno-venous extracorporeal membrane oxygenation) was required. after a median follow-up period of . days (range, - days), five patients ( . %) had died from the disease. thirteen of covid- cases ( . %) and / patients ( %) with non-covid- -associated pneumonia presented with or developed ards according to the "berlin definition'' ( ) , and intensive care unit (icu) treatment was required in / ( %) of covid- patients, and one patient with non-covid- associated pneumonia. covid- patients who developed ards were significantly older, and most of them were male (p = . and p = . , table ). furthermore, these patients presented with more preexisting comorbidities (p = . ). ards was significantly associated with icu treatment, occurrence of severe complications and (invasive) ventilation in covid- positive patients (p < . , p = . and p < . , respectively). the murray lung injury score was calculated for all patients who underwent invasive ventilation and revealed moderate or severe ards in / cases ( . %) ( ) . all five covid- -associated deaths occurred in the ards group. laboratory values for ldh, crp, and il- were significantly higher in the group of covid- -positive patients who developed ards compared to covid- patients with mild clinical course. however, ldh, crp, and il- values were not significantly different between covid- ards patients and patients with non-covid- -associated pneumonia (each p > . , table and supplementary table ). initial ana screening by iif showed titers ≥ : in all covid- ards patients ( %) but in / ( . %) covid- patients without ards. among the group of non-covid- -associated pneumonias, the initial ana screening by iif showed titers ≥ : in / patients ( %). anca screening was completely negative in all investigated covid- cases but positive in / patients ( %) with non-covid- -associated pneumonia. specific autoantibodies could be detected by ena immunoblot in / covid- ards patients ( %) but in / covid- non-ards patients ( . %) and / patients ( %) with non-covid- -associated pneumonia. one patient from the ards group (# , ana titer : ) showed borderline positivity for anti-rna polymerase iii (rp ) autoantibodies only after reference laboratory testing and was therefore classified as negative. the distribution and type of ana/ena among the different subgroups are shown in figure a , while representative images of iif and ib are shown in figure b . pm-scl was the most commonly detected autoantibody ( / cases) and could only be detected in the covid- ards group. taken together, applying established diagnostic criteria for ana/ena screening as described above ( ) detection of ana was associated with higher age and male sex, although not significant. ana positivity, however, was associated with a necessity of assisted/invasive ventilation, icu treatment (both p = . ) and occurrence of severe complications (p = . ). typical or atypical patterns in ct supplementary table ; **, case-related complications are summarized in supplementary table ; ***, patient was transferred to another institution because vv-ecmo was required; ****, patient w/chronic lymphocytic leukemia (cll) and antibody deficiency syndrome. rheumatoid factor was positive in this case. [ ] [ ] [ ] [ ] [ ] [ ] [ ] specific enas: anti-ss-b, anti-pm-scl, anti-jo, anti-cenp, anti-scl- , anti-nucleosome, anti-dsdna. imaging were not different between patients with and without anas. while there were no significant differences in serum levels of crp and il- , ldh levels were significantly higher in the ana+ group (p = . ). the association between ana status and disease-specific survival did not reach statistical significance (p = . ) ( table ) . "typical" radiologic covid- patterns were found in . % of patients (ards: . %/non-ards: %). these included ground glass opacities (all "typical" cases), consolidation and (c)op-like pattern ( figure ). atypical/negative patterns were found in . % of ards patients and % of non-ards patients. bronchoscopy with transbronchial biopsy (tbb) was performed in four patients (cases # , , and ) before (# ) and after (# , , ) an established diagnosis of covid- , respectively ( figure ). from three of these patients (# , , ) , additional post-mortem tissue samples were obtained during a partial autopsy procedure. in all samples, we observed reactive pneumocyte changes ("napoleon hat sign") consistent with viral infection (figure ) . however, there was a marked variance in the histologic appearance between different patients, between tbb and autopsy samples from the same patient and between autopsy samples from different regions of the lung. in addition to hyaline membrane formation consistent with diffuse alveolar damage (classic dad), there was also early septal thickening and intra-alveolar fibrinous plug formation with partial fibromyxoid change, reminiscent of acute fibrinous organizing pneumonia (afop) (# , figure a ). ultrastructural analyses of tissue samples from the same patient showed widening of alveolar septa with activated fibroblasts and early deposition of fine collagen fibrils. in patient # , where biopsies were obtained on day after initial diagnosis, there was a pattern of organizing dad with parenchymal collapse and entrapment of fibrin ( figure b ). tissue samples from autopsy from the same patient showed areas of beginning, patchy fibrosis with a foreshadowing of honeycombing. in all autopsy samples, there was capillary congestion with formation of microthrombi especially in late-stage disease (supplementary figure ) . in the present study, we found overlapping serological, clinical, radiologic, and histopathological features of severe covid- and lung manifestation of autoimmune disease (ctd-ild). we show that presence of anas is significantly associated with the development of ards, necessity for icu treatment and invasive ventilation as well as occurrence of severe complications in these patients; noteworthy, every patient in the present study who presented with or developed ards had detectable autoantibodies. with respect to baseline clinical characteristics, the investigated cohort is comparable to previous reports ( , ) . our result of autoantibodies in patients with severe covd- is in line with first results from other groups ( , , ) ; however, we are the first to put this observation into context with clinical, imaging, and histopathology findings. furthermore, we confirm the association between higher age, male sex, and elevated ldh with severe course of covid- in line with literature data ( ) . given the fact that only hospitalized patients were included, it is not surprising that the mortality rate ( . %) was higher compared to the general population. imaging and histopathological data in the present and in previous studies show that presentation of covid- in the lung is heterogeneous and evolves over time ( , ( ) ( ) ( ) . overall diversity of these changes, including (organizing) diffuse alveolar damage, fibromyxoid plugging and interstitial thickening are reminiscent of exacerbation of ctd ( , ) . however, it has to be clearly acknowledged that the histopathological spectrum of virus-induced dad is wide and also includes findings that have recently been described to be specific for covid- , such as endothelialitis and (micro-)thrombotic events. our finding that significant ana titers and/or detection of specific autoantibodies are found in most patients who develop ards raises the question if there is a comparable mechanism of lung damage between sars-cov- infection and exacerbating autoimmune disease. in / covid- patients with specific enas who developed ards, detected autoantibodies were anti-pm-scl or anti-scl- ; if the borderline positivity for rp in patient # was included, / specific enas in our cohort would be associated with a form of sclerosing ctd, as these autoantibodies (as well as similar hr-ct) patterns have previously been described in dermatomyositis, (progressive) systemic sclerosis and ctd-overlap syndromes ( , ) . of note, a significant proportion of anti-pm-scl-/anti-scl- positive patients develop pulmonary fibrosis, raising the question of long-term effects of severe covid- in these patients ( ) . the possibility of progressively evolving fibrosis would be supported by our findings from histopathology and electron microscopy, where we observed organization and pseudo-honeycombing as well as interstitial fibroblast activation with collagen deposition. another parallel between ctds and covid- are the vasculitis-like changes, vascular dysfunction or microangiopathies that have been described in a subset of patients ( ) ( ) ( ) . while thromboembolic complications occurred in only two patients in our cohort (both ana-positive), it would be of great interest to screen patients with more widespread vascular or cutaneous involvement for the presence of ana. anca screening, however, was completely negative in our cohort of covid- patients. since it is well-known that ana screening can be false positive in severely ill patients or patients who undergo icu treatment, a possible epiphenomenon has to be discussed very frankly. according to a recent publication, a there is no cross-reactivity between anti-sars-cov- igg/igm and ctdassociated autoantibodies ( ) . however, to enhance test validity in the present study, we a) doubled the recommended threshold for ana screening ( ) from : to : and b) figure b for histology) weeks after onset of the disease and ecmo therapy. in addition to diffuse ground glass opacities, a mixture of bronchiectasis, cysts and airtrapping is evident. additional pneumothorax and mediastinal emphysema are visible. performed additional immunoblot for specific ena in all patients, thus adding an independent methodological approach combining high sensitivity and specificity ( , ) . moreover, we included a non-covid- pneumonia control group in which ana titers ≥ : could be detected in two patients. specific autoantibodies against ama-m (associated with primary biliary cirrhosis) and ro (associated with sle) were detected in two additional patients. laboratory values in the pneumonia control group (ldh, crp, il- ) were not significantly different compared to covid- ards cases. while it is conceivable that ana titers rise and specific autoantibodies may appear in severely ill patients in general, we think that the observed clustering of high ana titers with specific, sclerosis-associated autoantibodies in the covid- ards group is reliable, raising the question of how these autoantibodies arise in the context of sars-cov- infection. a recent preprint suggests significant extrafollicular b cell activation with an excessive production of antibody-secreting cells (ascs) in critically ill sars-cov- patients ( ). this mechanism is highly similar to the development and progression of sle, and these asc might represent a possible source of the autoantibodies we report here ( , ) . we do not assume that these autoantibodies were already present in predisposed patients prior to infection, because only two patients in our cohort had any clinical history of rheumatic or autoimmune disease. however, in light of our results, there are interesting parallels between the reported epidemiology of severe covid- and the presence of autoantibodies in the general population. autoantibody titers above : and : can be detected in . and % of otherwise healthy individuals ( ) , reflecting reported proportions of severe ( %) and critical ( %) course of covid- ( ) . preliminary reports from the u.s. suggest that the covid- -associated death rate among african americans is significantly higher compared to the general population ( ) , while at the same time ana titers in african americans exceed those of americans with another ethnic background ( ) . it would be interesting to screen patients for class i and class ii major histocompatibility complex antigens to see whether it is possible to identify patients with an enhanced risk for development of autoantibodies and severe course of the disease. there is an ongoing debate with regard to a possible dysregulation of the immune system by sars-cov- , and it has been discussed whether anti-inflammatory drugs might be beneficial to prevent potentially harmful hyperinflammation ( ) . our hypothesis of sars-cov- -induced immune dysregulation closely correlates with results from the wuhan cohort reported by wu et al., in which methylprednisolone treatment was associated with a more favorable outcome among the patients who had already developed ards ( ) . a recent report from japan described high anti-ssa/ro antibody titers in two patients with severe covid- pneumonia, one of which responded well to corticosteroid therapy ( ) . moreover, first results from the uk recovery trial (eudract - - , press release from oxford university on june , ) indicate a significant benefit for dexamethasone, a drug that is also in use for the treatment of ssc, in mechanically ventilated patients. it would furthermore be interesting to evaluate if patients with sle-like ana pattern (anti-ds-dna) profit from hydroxychloroquine, while patients with an ssc-like ana pattern (anti-scl- , anti-cenp) might respond to figure a for imaging) days after admission shows septal thickening without fibrinous exudate. tissue samples from the autopsy ("a") of the same patient with reactive pneumocyte changes ("napoleon hat sign", arrowhead), ball-like fibrin (yellow circle) and alveoli with plug-like fibromyxoid organization (right). electron microscopy shows widening of alveolar septa with activated fibroblasts (asterisk) and deposition of collagen (silvery filaments in inter-alveolar septum). (b) transbronchial biopsies in a -year-old man (patient # , ana : , positive for scl- ; see figure d for imaging) show alveolar fibromyxoid plugs with entrapment of fibrin ("bx", left; arrowhead). other areas from the same sample show parenchymal collapse with granulation tissue around residual fibrin (yellow circle). ck immunohistochemistry highlights pneumocyte lining of collapsed alveoli. right: tissue samples from autopsy ("a") show interstitial fibroblast activation (arrowhead) and pseudohoneycombing. scale bar, µm. cyclophosphamide. in line with that, one case report described a mild clinical course of covid- in patient with established anti-scl- -positive ssc under treatment with the antiinterleukin (il) receptor blocker tocilizumab ( ) . the correct timing and dosing for any immunosuppressive or antifibrotic treatment approach in response to a viral infection however remains unclear. possible limitations of this study include its limited sample size and the lack of randomization. a further limitation of this study is the possibility of selection bias, which could not be ruled out on account of the study design. our observation of ctd-associated autoantibodies together with the ctd-like radiologic and histopathologic lung findings in severe cases of covid- point towards a possible dysregulation of the immune response upon sars-cov- infection that might fuel organizing pneumonia and trigger interstitial fibrosis, with deleterious effects on the functional outcome in long-term survivors. early detection of the reported autoantibodies might identify patients who profit from immunosuppressive and/or anti-fibrotic therapy to prevent the development of respiratory failure and fibrosis in covid- . all datasets presented in this study are included in the article/ supplementary material. the studies involving human participants were reviewed and approved by the ethics committee of the university of ulm (ref. no. - ). the patients/participants or their relatives provided their written informed consent to participate in this study. written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article. functional exhaustion of antiviral lymphocytes in covid- patients case-fatality rate and characteristics of patients dying in relation to covid- in italy pathological findings of covid- associated with acute respiratory distress syndrome pulmonary pathology of early-phase novel coronavirus (covid- ) pneumonia in two patients with lung cancer organizing diffuse alveolar damage associated with progressive systemic sclerosis acute fibrinous and organizing pneumonia in systemic lupus erythematosus: a case report and review of the literature fatal acute fibrinous and organizing pneumonia in a child with juvenile dermatomyositis pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid- international recommendations for the assessment of 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nuclear antigens critically ill sars-cov- patients display lupus-like hallmarks of extrafollicular b cell activation. medrxiv ( ) : epstein-barr virus infection, vitamin d deficiency, and steps to autoimmunity: a unifying hypothesis autoantibodies to killer cell immunoglobulin-like receptors in patients with systemic lupus erythematosus induce natural killer cell hyporesponsiveness range of antinuclear antibodies in "healthy" individuals characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention covid- and african americans risk factors for ana positivity in healthy persons covid- : risk for cytokine targeting in chronic inflammatory diseases? high levels of anti-ssa/ro antibodies in covid- patients with severe respiratory failure: a casebased review covid- in a patient with systemic sclerosis treated with tocilizumab for ssc-ild the authors would like to thank all patients and their families for their consent to the use of data and images in the present study. we further thank judith bauer, md and stephan opderbeck, md for providing clinical data. the authors are grateful for the outstanding quality of care of covid- patients provided by the team of the intensive care unit (icu) at the bundeswehrkrankenhaus ulm. the study was supported by the german registry of covid- autopsies (deregcovid). the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material key: cord- -jyk zphp authors: bonaventura, aldo; vecchié, alessandra; wang, tisha s.; lee, elinor; cremer, paul c.; carey, brenna; rajendram, prabalini; hudock, kristin m.; korbee, leslie; van tassell, benjamin w.; dagna, lorenzo; abbate, antonio title: targeting gm-csf in covid- pneumonia: rationale and strategies date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: jyk zphp covid- is a clinical syndrome ranging from mild symptoms to severe pneumonia that often leads to respiratory failure, need for mechanical ventilation, and death. most of the lung damage is driven by a surge in inflammatory cytokines [interleukin- , interferon-γ, and granulocyte-monocyte stimulating factor (gm-csf)]. blunting this hyperinflammation with immunomodulation may lead to clinical improvement. gm-csf is produced by many cells, including macrophages and t-cells. gm-csf-derived signals are involved in differentiation of macrophages, including alveolar macrophages (ams). in animal models of respiratory infections, the intranasal administration of gm-csf increased the proliferation of ams and improved outcomes. increased levels of gm-csf have been recently described in patients with covid- compared to healthy controls. while gm-csf might be beneficial in some circumstances as an appropriate response, in this case the inflammatory response is maladaptive by virtue of being later and disproportionate. the inhibition of gm-csf signaling may be beneficial in improving the hyperinflammation-related lung damage in the most severe cases of covid- . this blockade can be achieved through antagonism of the gm-csf receptor or the direct binding of circulating gm-csf. initial findings from patients with covid- treated with a single intravenous dose of mavrilimumab, a monoclonal antibody binding gm-csf receptor α, showed oxygenation improvement and shorter hospitalization. prospective, randomized, placebo-controlled trials are ongoing. anti-gm-csf monoclonal antibodies, tj and gimsilumab, will be tested in clinical trials in patients with covid- , while lenzilumab received fda approval for compassionate use. these trials will help inform whether blunting the inflammatory signaling provided by the gm-csf axis in covid- is beneficial. coronavirus disease is caused by severe acute respiratory syndrome coronavirus (sars-cov- ) with a clinical spectrum ranging from asymptomatic/paucisymptomatic forms to severe pneumonia leading to respiratory failure, need for mechanical ventilation, and death ( ) . to date, no specific treatment is approved for covid- , and management is supportive. severe covid- pneumonia seems to be mediated by a cytokine storm ( , ) . therefore, therapies that target hyperinflammation may be effective. in a recent report, patients with covid- needing intensive care unit (icu) admission showed a cytokine profile similar to that of secondary hemophagocytic lymphohistiocytosis with increased levels of several inflammatory cytokines [interleukin (il)- , il- , granulocyte-colony stimulating factor (g-csf), granulocyte-monocyte stimulating factor (gm-csf), interferon-γ-inducible protein (ip- ), monocyte chemoattractant protein (mcp- ), macrophage inflammatory protein -α (mip -α), and tumor necrosis factor-α (tnfα)] ( ). additionally, increased levels of ferritin and il- have been shown to correlate with a worse prognosis [ ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ; supplementary table ]. these observations underline that covid- is a complex disease capable to combine different patterns of inflammatory biomarkers. indeed, most of the infections can trigger the release of il- β from the inflammasome ( ) followed by the production of il- that increases the circulating levels of c-reactive protein, the prototypical acute-phase reactant ( ) . in viral infections, including covid- , elevated levels of the pro-inflammatory cytokine il- , that derives from the inflammasome as il- β, are found along with high levels of ferritin ( ) , thus replicating the events commonly observed in the macrophage activation syndrome ( ) . altogether, these findings support the hypothesis that a maladaptive hyperinflammatory response to the virus orchestrated by il- , il- β, and eventually gm-csf-referred to as cytokine storm-rather than the virus itself may drive the lung damage leading to hypoxia and acute respiratory failure. immunomodulation may be beneficial in the treatment of hyperinflammation-associated conditions. data supporting the role of hyperinflammation in sepsisrelated acute respiratory distress syndrome (ards) are derived from a sub-group analysis of a phase randomized controlled trial of il- receptor antagonist (anakinra), which showed significant survival benefit in patients treated with anakinra compared to placebo ( ) . il- β is an upstream pro-inflammatory cytokine that is released following activation of the inflammasome in response to infection and/or injury ( ) . il- is a pleiotropic cytokine that influences several processes, such as acute-phase protein generation, inflammation, and antigen-specific immune responses ( ) . in the innate immune response, il- is produced by myeloid cells [e.g., macrophages and dendritic cells (dcs)] following the recognition of sterile or non-sterile stimuli through toll-like receptors at the site of infection or tissue injury. in the adaptive immune response, il- is a critical modulator of plasma b-cell differentiation and antibody production ( ) . a deregulated il- expression is involved in the pathogenesis of several disorders, such as chronic inflammatory diseases, autoimmune diseases, and tumor development ( , ) . cytokine release syndrome (crs) represents an on-target effect of chimeric antigen receptor (car) t-cell therapy and consists of a systemic inflammatory response due to a massive cytokine release, including il- , gm-csf, and interferon-γ, following the in vivo activation of car t-cells ( , ) . the incidence of crs after car t-cell therapy ranges from to % with - % of patients having severe crs ( ) . tocilizumab, an il- receptor blocker, has been approved for the treatment of severe crs after car t-cell therapy in light of its association with a rapid improvement of clinical manifestations and a decrease in the aforementioned cytokines along with a low toxicity for car t-cells ( ) . different trials are recruiting patients with covid- pneumonia to test whether il- receptor blockers (tocilizumab, sirukumab, and sarilumab: chictr , nct , nct , nct ; nct , nct , and nct ) and an il- receptor blocker (anakinra, nct , nct , nct , nct , nct , nct , nct , and nct ) improve covid- pneumonia outcomes. the identification and treatment of hyperinflammation using existing therapies with understood safety profiles that are either in clinical development or approved for other indications represent a valid option to cope with the immediate need to reduce the rising mortality of covid- . in an attempt to approach hyperinflammation upstream of both il- and il- and to target neutrophils as well as macrophages, gm-csf may be considered as an appealing mediator. gm-csf is generally perceived as a pro-inflammatory cytokine and is produced by many cells, including macrophages, t-cells, fibroblasts, endothelial cells, epithelial cells, and tumor cells ( ) , with most of the production occurring at sites of inflammation ( ) . gm-csf signals are mediated by the gm-csf receptor (gm-csf-r) consisting of a specific ligand-binding α-chain (gm csf-rα) and a signal-transducing β-chain (gm csf-rβ) ( figure a) . downstream signaling of gm-csf-r includes janus kinase (jak )/signal transducer and activator of transcription (stat ), nuclear factor kappa-light-chain-enhancer of activated b cells (nf-κb), extracellular signal-regulated kinase (erk), and the phosphoinositide -kinase (pi k)-akt pathway ( ) ( ) ( ) ( ) . importantly, erk activity is responsible for gm-csf-mediated human monocyte survival in vitro ( ) . interferon regulatory factor (irf ) is a hemopoietic-specific transcription factor that has been involved in the induction of dc-like properties in monocytes treated with gm-csf ( , ) . recently, achuthan gm-csf is involved in the differentiation of alveolar macrophages, thus enhancing the clearance of respiratory microbes through an increase in phagocytosis and release of pro-inflammatory cytokines (il- β, il- , and tnf-α) in a feed-forward inflammatory loop. based on previous experiences, the early administration of a rhgm-csf, like sargramostim, may improve the initial response against viruses, including sars-cov- . (c) mavrilimumab prevents gm-csf from binding to the α-chain of its receptor, while gimsilumab, lenzilumab, and tj directly bind gm-csf with the final common result of blocking the intracellular signaling. based on the current knowledge, these agents can be used to reduce the hyperinflammation caused by sars-cov- in the course of the disease. differently from rh-gm-csf, these agents should be considered later in order to not negatively impact the favorable effects of gm-csf on the immune response. apc, antigen presenting cell; dc, dendritic cell; gm-csf, granulocyte-macrophage colony-stimulating factor; sars-cov- , severe acute respiratory syndrome coronavirus . this figure has been partially created using servier medical art templates, which are licensed under a creative commons attribution . unported license; https://smart.servier.com. et al. found that gm-csf is capable to up-regulate irf expression via jumonji domain-containing protein d (jmjd ) demethylase in monocytes/macrophages ( ) . increased levels of irf are responsible for the production of chemokine (c-c motif) ligand (ccl ), which is involved in inflammation and tissue remodeling, as occurs in arthritis ( ) . the gm-csf-irf signaling was also described to up-regulate major histocompatibility complex (mhc) class ii expression in mouse bone marrow cultures and macrophages ( , ) . gm-csf levels are low or undetectable in normal conditions; however, any immune trigger can rapidly increase concentrations, as it has been seen in the lungs of patients with asthma or within the synovial fluid of patients with arthritis ( ) . bacterial endotoxins and inflammatory cytokines (e.g., il- β, il- , and tnf-α) potently induce gm-csf ( ) . indeed, increased mrna expression for tnf-α, il- β, and il- were reported in monocytes/macrophages treated with gm-csf ( ) . these findings led to hypothesize that gm-csf is part of the inflammatory milieu of some inflammatory/autoimmune reactions. gm-csf would work as a co-regulator along with tnf-α, il- , and il- , as part of a positive feed-forward inflammatory loop involving monocytes/macrophages, fibroblasts, and endothelial cells ( ) ( ) ( ) , but also dcs and th cells ( ) ( ) ( ) . il- , however, was found to induce intestinal and splenic production of gm-csf ( ), thus promoting systemic effects, like an increase in splenic macrophage precursors. the importance of il- β and the il- receptor/myeloid differentiation primary response (myd ) signaling axis appears of importance in the regulation of gm-csf by cd + and γδ t cells ( ) . il- β, together with tnf-α, can also promote monocyte viability via gm-csf while not inducing any specific macrophage polarization ( ) . increased levels of gm-csf have been found in the bronchoalveolar fluid of patients with ards compared with healthy controls ( , ) . higher levels were observed in the early phases ( - days) with a progressive decrease in late stages (day ) ( ) . gm-csf may indirectly contribute to ards by the suppression of neutrophil apoptosis ( , ) as activated neutrophils play a major role in the microvascular damage contributing to lung damage ( , ) . limited evidence describes a regulatory role for gm-csf through the promotion of dc differentiation to a tolerogenic profile, thus increasing the number and function of regulatory tcells ( ) . this can also lead to t-cell hypo-responsiveness and/or anergy ( ). the mechanisms underlying pro-inflammatory and immunomodulatory phenotypes of gm-csf are not fully understood and need to be further investigated. these properties are hypothesized to depend on the dose and the presence of other cytokines in the setting of the immune response. at lower doses, gm-csf stimulates the tolerogenesis of myeloid cells involved in the regulatory t-cell homeostasis ( ) , while at higher doses gm-csf causes myeloproliferation, leading to a sustained immune response ( ) . gm-csf-derived signals are critically involved in the differentiation of macrophages and in the proliferation and activation of other immune cells. alveolar macrophages (ams) are essential to clear respiratory microbes ( , ) , and their depletion has been associated with increased disease severity in murine models of influenza infection ( , ) . therefore, several pre-clinical studies reported that the intranasal administration of gm-csf prior to inducing an experimental viral infection conferred resistance to respiratory pathogens through an increased proliferation of ams [( , ); figure b ]. this is probably due to an enhanced clearance of the virus, thus limiting the direct damage provided by the virus itself. recently, a subset of ams, the nerve-associated interstitial alveolar macrophages (nams), have been identified and characterized in human and murine lung ( ) . nams seem to originate from the yolk sac and, differently from the other ams, require colonystimulating factor (csf ) and not gm-csf for development and maintenance in adulthood. mouse models of influenza virus infection on selectively nam-depleted animals suggest a central role for nams in the negative regulation of virusinduced inflammation, whereas the other gm-csf-dependent ams display a pro-inflammatory profile ( ) . in addition, gm-csf receptor activation triggers stimulation of multiple downstream signaling pathways, including jak /stat , the mitogen-activated protein kinase (mapk), and the pi k, all fundamental in activation and differentiation of myeloid cells [( , ) ; figure a ]. along with its key role in inflammation, gm-csf is critical in lung physiology. this has been clearly highlighted by gm-csfdeficient and gm-csf receptor-deficient mice which develop pulmonary alveolar proteinosis (pap) because ams require gm-csf to differentiate ( ) . the poor differentiation of these macrophages is responsible for the accumulation of surfactant proteins, saturated phosphatidylcholine, and cholesterol, leading to pap. indeed, local expression of gm-csf in the lung is able to restore normal surfactant homeostasis and clearance in the setting of pap ( ) . additionally, gm-csf-deficient mice show a persistent, low-grade inflammation resulting from inappropriate responses to commensal microbes. this chronic inflammation predisposes mice to develop different kinds of tumors ( ) . to date, no function-altering gm-csf mutations have been identified in humans. however, an autoimmune form of pap can develop in humans and is associated with high levels of neutralizing gm-csf autoantibodies that inhibit gm-csf signaling ( ) . a congenital form of pap ending up with a complete inhibition of the macrophage clearance of surfactant has also been described and is caused by mutations in csf ra or csf rb, the genes encoding the gm-csf-rα and gm-csf-rβ chains ( ) . increased circulating levels of gm-csf have been recently described in patients with covid- compared to healthy controls ( ). a paper from china appearing on the preprint online platform biorxiv reported that in patients with covid- , especially those admitted to the icu, cd + t lymphocytes were rapidly activated in the lung to pathogenic t helper (th) cells and generated gm-csf and il- . this potent pro-inflammatory environment strongly induced cd + cd + monocytes, which also released gm-csf and il- , further worsening the cytokine storm. these aberrant and numerous gm-csf + -il- + cells may enter the lungs and explain the detrimental actions provided by hyperinflammation in the most severe and even fatal cases ( ). in light of the results in animal studies following the intranasal administration of gm-csf in the setting of respiratory infections, two human recombinant gm-csf (hrgm-csf), sargramostim and molgramostim, were investigated in humans ( ) ( ) ( ) . sargramostim was tested in a randomized, doubleblind, placebo-controlled clinical trial in patients with acute lung injury/ards ( ) . the drug was administered as an intravenous infusion once daily for days at a dosage of µg/m . the study showed no significant difference in the number of ventilator-free days, organ failure-free days, and -day mortality between the hrgm-csf and placebo groups; there was also no difference in the number of serious adverse events ( ) . a randomized, double-blind, placebo-controlled phase ii study tested the effects of low-dose hrgm-csf (molgramostim, µg/kg daily) for days in patients in addition to the standard of care in critically ill patients with severe sepsis and respiratory dysfunction ( ) . the study found that hrgm-csf was associated with an improvement in gas exchange and functional activation of pulmonary macrophages; however, there was no improvement in -day survival ( ) . in another randomized, double-blind, placebo-controlled clinical trial in patients with bacterial and fungal abdominal sepsis, molgramostim µg/kg daily for days was administered in addition to standard of care. the treatment group had a reduction in the rate of infectious complications and in the length of hospitalization ( ) . in the early phases of viral infections, gm-csf's role may be protective as it helps limit virus-related injury. for this reason, an inhaled formulation of sargramostim is being tested in patients with covid- -related acute hypoxic respiratory failure (nct ). in later stages of covid- , the severity of the illness appears to be driven by the inappropriate release of several cytokines, such as il- and gm-csf. these mediators are involved in the inflammatory lung injury, predisposing patients to respiratory failure and eventually ards. therefore, inhibition of gm-csf signaling may be a reasonable treatment in this stage of disease. this is supported by pre-clinical data in crs showing that gm-csf blockade reduced car t-cell therapy-related toxicity by preventing crs development without affecting its therapeutic activity ( ) . mavrilimumab is a high-affinity monoclonal igg antibody against gm-csf-rα [( ); figure c ]. the efficacy and safety of mavrilimumab have been studied in rheumatoid arthritis (ra) and showed promising results. in a phase b multicenter placebocontrolled study, patients with moderate-to-severe ra were randomized to receive different dose levels of mavrilimumab ( , , and mg subcutaneously every weeks) or placebo. mavrilimumab at a dose of mg subcutaneously every weeks was the most effective in improving clinical and laboratory disease activity ( ) . no substantial differences in adverse events or severe adverse events were observed between groups ( ). these results on safety and efficacy were confirmed in a phase double-blind randomized trial evaluating the use of mavrilimumab at a dose of mg subcutaneously every other week in long-standing ra patients ( ) . a post-hoc analysis of these studies has shown that the administration of mavrilimumab was associated with a significant downregulation of the macrophage-derived chemokine c-c motif chemokine ligand (ccl ) and il- ( ), related to a direct inhibition of the proinflammatory cytokine release from myeloid cells. mavrilimumab also showed a decreased expression of il- /il- -associated transcripts, the latter suggesting an indirect suppressive effect of mavrilimumab on t cell activation ( ) . moreover, a sustained suppression of serum markers of disease activity, such as c-reactive protein and il- , was observed in ra patients treated with mavrilimumab ( ) . mavrilimumab is currently under investigation for the treatment of giant cell arteritis (nct ). a prospective interventional single-center cohort study tested the efficacy and safety of mavrilimumab in patients with severe covid- pneumonia and evidence of hyperinflammation in italy ( ) . thirteen non-mechanically ventilated patients with severe covid- pneumonia and hyperinflammation were treated with a single intravenous dose of mavrilimumab mg/kg upon admission to the hospital. twenty-six non-mechanically ventilated patients with severe covid- pneumonia and hyperinflammation and with similar baseline characteristics were evaluated as a control-group. all patients received similar standard of care therapy, including antivirals and antibiotics. over the course of the -day follow-up period, mavrilimumabtreated patients experienced earlier and improved clinical outcomes than control-group patients, including earlier weaning from supplemental oxygen and shorter hospitalizations. death occurred in % (n = / ) of mavrilimumab-treated patients by day compared to % (n = / ) of control-group patients ( ) . these data are consistent with the hypothesis that excessive host immune response driven by t cells and monocytes may have a central role in the pathogenesis of covid- pneumonia. a randomized controlled trial is being designed and is now active (mavrilimumab in severe covid- pneumonia and hyperinflammation , nct ). five monoclonal antibodies targeting gm-csf (gimsilumab, otilimab, namilumab, lenzilumab, and tj ) are in development and are currently under investigation mainly for the treatment of ra. the principal clinical trials both completed and ongoing are described in table ( , ) . recently, tj (also known as tjm ) obtained the us food and drug administration (fda) clearance to start a clinical study for covid- associated crs (i-mab) . additionally, lenzilumab has received fda approval for compassionate use in covid- patients (fda) , while a phase study is ongoing. a clinical trial has also been approved for gimsilumab for the treatment of covid- and is now enrolling patients in the us (nct ) (figure c ). in addition, csl is a monoclonal antibody targeting the gm-csf-rβ, common to gm-csf, il- , and il- . a phase trial is evaluating the safety and tolerability of this drug in patients with asthma ( table ) . because gm-csf is a key mediator in pulmonary homeostasis, there is the theoretical concern that inhibition of gm-csf signaling by either binding to gm-csf or blocking the receptor may result in dysfunctional ams, leading to pap and development of new infections. fortunately, there has yet to be a case of pap reported with the use of anti-gm-csf monoclonal antibodies. this may be due to the fact that patients with autoimmune pap have to reach a "critical threshold" of neutralizing antibodies to develop the disease, and the doses currently being utilized in clinical trials may not reach this threshold ( ) . this may actually be true for the chronic use where the low level of lung penetration of the - mg subcutaneously every weeks may not provide the level of necessary inhibition ( ) . however, in the case of covid- pneumonia and hyperinflammation, the lung penetration of the drug may be critical. this is the reason why the dose has been increased from . - mg/kg subcutaneously to - mg/kg intravenously. this means that pap should not necessarily be an issue in the covid- treatment in that a single intravenous dose is being given and it will wear off in a month, while pap is a disease caused by chronic inhibition over years. as covid- pneumonia is likely to be aggravated by a cytokine storm, immunomodulation gained importance as a possible therapeutic strategy to this disease. a wealth of il- and il- blockade trials are ongoing and results are awaited. i-mab announces ind clearance from fda for tjm to treat cytokine release syndrome (crs) associated with severe coronavirus disease . available online at: https://www.biospace.com/article/releases/i-mab-announcesind-clearance-from-fda-for-tjm -to-treat-cytokine-release-syndrome-crsassociated-with-severe-coronavirus-disease- -covid- -/ (accessed april , ). however, an approach targeting hyperinflammation upstream of il- and il- as well as neutrophils and macrophages may be envisioned through gm-csf signaling. gm-csf is an immunomodulatory cytokine that may help to clear respiratory microbes by stimulating ams. a clinical trial with a hrgm-csf, sargramostim, will be conducted in covid- patients with the rationale that it may help clear the sars-cov- earlier in the disease course. however, in the later phase of covid- lung injury, the marked elevation in gm-csf levels as part of the cytokine storm during the onset of covid- pneumonia suggests that gm-csf may actually be deleterious at this stage of the disease. blocking gm-csf signaling could therefore be an effective therapeutic strategy by reducing the cytokine storm, which leads to the progression of acute respiratory failure in patients with hyperinflammation. multiple clinical trials with inhibition of the gm-csf pathway are either ongoing or under development. ab, av, and aa conceived the manuscript. ab and av drafted manuscript, figure, and tables. tw, el, pc, bc, pr, kh, lk, bv, ld, and aa critically revised the manuscript. all authors approved the final version. clinical characteristics of coronavirus disease in china cardiovascular considerations in treating patients with coronavirus (covid- ) covid- : consider cytokine storm syndromes and immunosuppression clinical features of patients 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anti-gm-csfralpha antibody enables therapeutic dosing that limits exposure in the lung the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material key: cord- -pfccsgco authors: gössling, katharina l.; schipp, cyrill; fischer, ute; babor, florian; koch, gerhard; schuster, friedhelm r.; dietzel-dahmen, jutta; wieczorek, dagmar; borkhardt, arndt; meisel, roland; kuhlen, michaela title: hematopoietic stem cell transplantation in an infant with immunodeficiency, centromeric instability, and facial anomaly syndrome date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: pfccsgco immunodeficiency, centromeric instability, and facial anomaly (icf) syndrome is a rare autosomal recessive genetic condition with severe immunodeficiency, which leads to lethal infections if not recognized and treated in early childhood. up-to-date treatment regimens consist of prophylactic and supportive treatment of the recurrent infections. here, we report the case of a -year-old boy of moroccan consanguineous parents, who was diagnosed at months of age with icf syndrome with a homozygous missense mutation in the dnmt b gene. he was initially admitted to the hospital with recurrent pulmonary infections from the opportunistic pathogen pneumocystis jirovecii (pj). further immunological workup revealed agammaglobulinemia in the presence of b cells. after successful recovery from the pj pneumonia, he underwent hematopoietic stem cell transplantation (hsct) from the hla-matched healthy sister using a chemotherapeutic conditioning regimen consisting of treosulfan, fludarabine, and thiotepa. other than acute chemotherapy-associated side effects, no serious adverse events occurred. six months after hsct immune-reconstitution, he had a stable chimerism with . % autologous portion in the peripheral blood and a normal differential blood cell count, including all immunoglobulin subtypes. this is one of the first cases of successful hsct in icf syndrome. early diagnosis and subsequent hsct can prevent severe opportunistic infections and cure the immunodeficiency. centromeric instability and facial anomaly remain unaffected. although the long-term patient outcome and the neurological development remain to be seen, this curative therapy for immunodeficiency improves life expectancy and quality of life. this case is meant to raise physicians awareness for icf syndrome and highlight the consideration for hsct in icf syndrome early on. immunodeficiency, centromeric instability, and facial anomaly (icf) syndrome is a rare autosomal recessive genetic condition with severe immunodeficiency, which leads to lethal infections if not recognized and treated in early childhood. up-to-date treatment regimens consist of prophylactic and supportive treatment of the recurrent infections. here, we report the case of a -year-old boy of moroccan consanguineous parents, who was diagnosed at months of age with icf syndrome with a homozygous missense mutation in the dnmt b gene. he was initially admitted to the hospital with recurrent pulmonary infections from the opportunistic pathogen pneumocystis jirovecii (pj). further immunological workup revealed agammaglobulinemia in the presence of b cells. after successful recovery from the pj pneumonia, he underwent hematopoietic stem cell transplantation (hsct) from the hla-matched healthy sister using a chemotherapeutic conditioning regimen consisting of treosulfan, fludarabine, and thiotepa. other than acute chemotherapy-associated side effects, no serious adverse events occurred. six months after hsct immune-reconstitution, he had a stable chimerism with . % autologous portion in the peripheral blood and a normal differential blood cell count, including all immunoglobulin subtypes. this is one of the first cases of successful hsct in icf syndrome. early diagnosis and subsequent hsct can prevent severe opportunistic infections and cure the immunodeficiency. centromeric instability and facial anomaly remain unaffected. although the long-term patient outcome and the neurological development remain to be seen, this curative therapy for immunodeficiency improves life expectancy and quality of life. this case is meant to raise physicians awareness for icf syndrome and highlight the consideration for hsct in icf syndrome early on. immunodeficiency, centromeric instability, and facial anomaly (icf) syndrome is a rare autosomal recessively inherited genetic condition. the majority of the affected individuals have mutations in the methyltransferase b gene (dnmt b, omim ) on chromosome leading to reduced dna methylation of the pericentromeric regions of chromosomes , , and ( , ) . epigenetic dysregulation rather than a single gene defect determines the clinical phenotype. although all body cells carry the same mutation, various tissues are differently affected due to varying degrees of dna methylation. this is especially seen in mitogen-stimulated lymphocytes where whole arm deletions, translocations, and multibranched chromosomes cause an abnormal gene regulation of b cell immunoglobulin isotype switching, lymphocyte activation, and migration ( ). icf patients suffer from recurrent gastrointestinal and pulmonary infections in early childhood due to the agammablobulinemia resulting in failure to thrive ( ) . an intrinsic t cell defect has also been linked to the high frequency of opportunistic infections from pathogens such as with pneumocystis jirovecii (pj), but the exact mechanism has not been elucidated ( ) . typical clinical characteristics include the eponymous facial anomaly of epicanthic folds, hypertelorism, and a flat nasal bridge, as well as a delay in psychological and cognitive development. treatment options are limited and consist primarily of supportive therapy such as substitution of immunoglobulins, prophylactic sulfamethoxazole-trimethoprim therapy, or antibiotic therapy ( ) ( ) ( ) . life expectancy of icf patients is poor and prognosis is dependent on the frequency and severity of infections. a high proportion of reported icf patients die at a young age ( ) . early igg replacement and antibiotic prophylaxis can significantly improve patient outcomes ( ). an early sustainable therapy for the immunodeficiency can dramatically better the disease course. the only curative treatment of the immune dysfunction is hematopoietic stem cell transplantation (hsct), as described in isolated case studies ( , ) . however, no long-term follow-up data are available to date. here, we report the case of a -year-old boy of moroccan consanguineous descent diagnosed with icf syndrome carrying a homozygous missense mutation in the dnmt b gene (ala thr) with hypogammagobulinemia, normal b cell count, facial anomaly, and failure to thrive. this variant has been previously described in icf syndrome by hansen et al. it consists of a point mutation (rs ) and an amino acid exchange from alanine to threonine. the variant has a minor allele frequency of a = . ( ) and a prevalence of less than / , , . hansen et al. described this variant among other mutations in the dnmt b gene, while control dnas from unrelated healthy caucasians in north america and the netherlands did not carry this mutation ( ). the boy was born with a body weight of , g ( th percentile) and length of cm ( th percentile) (cdc/who growth charts ). at the age of . months, his body weight was at , g ( th percentile) and the length was cm ( th percentile) (cdc/who growth charts ). at the age of months, the patient was admitted to the hospital for the first time with recurrent and prolonged respiratory infections of the lower and upper airways with hypoxemia. laboratory workup revealed a bilateral bronchopneumonia positive for parainfluenza virus type and and pj, as well as purulent conjunctivitis and purulent otitis media perforans positive for haemophilus influenzae. in the subsequent immunological diagnostics, a significantly reduced level of all immunoglobulin subclasses ( table ) and a normal b cell count with a lack of memory b cells were detected. t cell count and proliferation rate of cd + and cd + cells after mitogenic stimulation with phytohemagglutinin were normal. bronchoscopy revealed normal anatomic proportions of the airways. the pj pneumonia was treated with intravenous cotrimoxazole, corticosteroids, and oxygen supplementation. subsequent regular intravenous immunoglobulins treatment and prophylactic cotrimoxazole prevented further severe pulmonary infections. in addition, the patient has typical facial dysmorphisms consisting of hypertelorism, flat nasal bridge, epicanthic folds, and low set ears ( figure a -pictures of the face). icf syndrome was clinically suspected and confirmed with the cytogenetic analysis revealed whole arm deletions, translocations, and multibranched chromosomes due to the centromeric instability in chromosomes , , and (figure -karyogram) . the patient is the fifth child of consanguineous moroccan parents, who are first cousins (figure b-pedigree) . three healthy older siblings ( -, -, and -year olds) developed normally. icf in the siblings was excluded with normal blood levels of immunoglobulins and a normal karyogram. the oldest daughter of the family died of respiratory failure at the age of months in morocco, no further genetic or pathological diagnostics were performed post mortem. at the age of months, he presented with a body weight of , g ( th percentile) and a length of cm ( th percentile), failure to thrive was diagnosed (cdc/who growth charts ). at the age of months, the patient underwent hematological stem cell transplantation from the healthy -year-old sister with a hla match of / as donor. after the conditioning regimen consisting of thiotepa, treosulfan, and fludarabine, he received cd + cells ( . × cells/kg bodyweight). cyclosporine a, methotrexate, and antithymocyte globulin were given as prophylaxis for graft-versus-host disease (gvhd) (figure ) . patients with icf syndrome often display residual t cell function that may result in graft failure or rejection and are, therefore, subjected to reduced intensity conditioning ( ) . given the good performance status of our patient, a myeloablative conditioning with reduced toxicity was administered in order to achieve stable lymphohematopoietic engraftment with low risk of organ toxicity ( ) . he suffered from acute chemotherapeutic reactions in the mucosa (toxicity who grade ) and on the skin (toxicity who grade ), nausea and vomiting (who grade ), and renal clearance impairment (who toxicity grade ). with time, he recovered completely from all side effects. leukocyte and neutrophil engraftment was achieved on day + and on day + , respectively. the chimerism analysis of the blood showed an autologous portion at a maximum of . %, while the chimerism of the bone marrow was completely from the donor. without any infections or signs of gvhd, he was discharged from the hospital on day + . he continued to receive cyclosporine a until day + , which was stepwise reduced in the absence of any signs of gvhd. three months after transplantation, he was admitted again to the hospital with rsv infection that was successfully treated with ribavirin. four months posttransplantation, he was admitted again due to diarrhea, obstructive bronchitis, and fever. antibiotic treatment was started initially and discontinued when influenza, rsv, and corona virus were detected in the throat. without specific antiviral therapy, he recovered completely. five months after transplantation, he presented with diarrhea, subfebrile temperature, and a partially compensated metabolic acidosis. rotavirus was detected in the stool. iv-fluid therapy as an outpatient was sufficient to normalize the blood gases and the symptoms receded spontaneously. six months after transplantation, adenovirus was detected in blood, stool, and throat without any clinical symptoms. an intranvenous treatment with cidofovir was started until the virus cleared sufficiently. his differential blood counts increased steadily and were normal months after transplantation ( table ). the chimerism in the bone marrow showed an overall autologous portion of - %, with - % in the cd + and cd + fractions as well as - % in the cd + subset. the chimerism in the blood was with . % autologous cells stable over the last months. at year of age, his neurological development correlated approximately with a -month-old child. his body weight was , g ( th percentile) and the length cm ( th percentile) (cdc/who growth charts ). we report the successful allogeneic hsct of a -year-old boy with icf syndrome carrying a homozygous mutation in the dnmt b gene after receipt of bone marrow cells from the / hla-matched clinically healthy sister. during the -year followup, we observed a complete immune reconstitution without acute or short-term transplantation-related complications. immunodeficiency, centromeric instability, and facial anomaly syndrome is a rare and severe immunodeficiency disease. our patient is the fifth child of consanguineous parents and underwent hsct at the early age of months. the first daughter of the same parents died at months of age in in morocco due to respiratory failure of unknown origin. since no further diagnostics were conducted post mortem, we can only speculate that the same homozygous mutation in the dnmt b gene might have been the cause for her early death. while a gender bias for icf syndrome caused by a mutation in the zbtb gene has been observed recently, icf syndrome affects both genders equally making it even more plausible that the deceased daughter had the same genetic defect as our patient ( ) . this family history emphasizes the fact that icf patients have very poor clinical outcomes if they are not treated early and adequately. in case reports from the s, only patients on ig therapy survived to childhood, while others died within the first year of life from opportunistic infections ( ) as described in nine icf cases in france. recently, gennery et al. published the successful cure of the immunodeficiency by hsct in three icf patients, who received hsct at the ages of months, and years ( ). here, we report another case of successful hsct in icf syndrome that received hsct at months of age. hsct itself is associated with a high risk of mortality due to short-term opportunistic infections and gvhd. long-term side effects include chemotherapeutic toxicity such as liver or kidney dysfunction, growth retardation, infertility, and secondary malignancies ( ) . patients who had never received chemotherapeutic treatment beforehand usually have a better clinical outcome and can be transplanted successfully with few acute side effects as seen in patients with hemoglobinopathies or sickle-cell anemia ( , ) . since severe infections significantly worsen patient outcomes after hsct, we aim to perform hsct in icf syndrome as early as possible in an infection-free period. icf syndrome is invariably associated with low or absent levels of immunoglobulins given the defective peripheral terminal b cell differentiation. indeed, this could be treated with life-long igg replacement. however, a number of these patients also display functional t cell immunodeficiency, as evidenced in our case by pj pneumonia. furthermore, recent studies from rechavi et al. and weemaes et al. highlight the fact that a t cell proliferation defect develops over time ( , ) . as such, patients with icf syndrome suffer from multisystem disease leading to long-term morbidity and substantial mortality. this usually stems from severe infections (i.e., in the gi tract) associated with growth retardation and limited life expectancy. particularly in an advanced stage of the disease, hsct is associated with a higher risk of mortality due to preexisting infections. allogeneic hsct is, therefore, an attractive therapeutic approach in those patients with signs of t cell immunodeficiency without severe preexisting morbidity and is usually recommended when an hla-matched donor can be identified. although the conditioning chemotherapy is accompanied by high toxicity, we believe the advantages of hsct significantly outweigh the natural disease course in icf. hsct replaces all hematopoietic cells carrying the dnmt b mutation and can thereby cure the immunodeficiency in icf patients. weemaes et al. and hagleitner et al. examined the clinical features of icf patients, which can help to predict clinical outcomes after hsct ( , ) . recurrent infections and a higher risk for hemato-oncological malignancies are eliminated in our patient with hsct, while a delayed neurological development or cortical atrophy with seizures are features that remain ( , ) . during the immunosuppressive treatment and before reconstitution of the adaptive immune system, our patient suffered from recurrent respiratory and gastrointestinal infections. this can be attributed to the posttransplantation immunosuppression and are expected to improve with time. while failure to thrive can be attributed to the high frequency of infections, we expect our patient to catch up on weight gain within the coming months. dna methylation is a dynamic process that epigenetically regulates gene expression during development and other processes such as aging, cancerogenesis, or cell differentiation. the dnmt b mutation was described to lead to altered epigenetic modifications of genes regulating development, neurogenesis, and immune function ( ) . since the dna methyltransferase is an intracellular enzyme that is not secreted and taken up by the surrounding tissue, hsct is not able to compensate for the enzyme deficiency in all other cell types of the body. a recent study shows next to the hypomethylation of the pericentromeric regions of chromosomes , , and , a global loss of methylation in icf patients' fibroblasts and lymphoblastoid cell lineages leads to significantly upregulated gene expression of the differentially methylated positions ( ) . although of such regions were identified, it remains unclear, how and to which degree a given tissue is affected by the hypomethylation and to what extent this pattern changes during development. all these questions need to be answered before we are able to translate the molecular findings into the clinical phenotype. nevertheless, we can speculate that an overactive gene expression might have a great impact on genes that are highly epigenetically regulated, such as those responsible for neurological development or germ line genes important in preventing cancer development. both of which can still lead to delayed neurological development and a higher risk for malignancies in icf patients even after successful hsct. seeing the combination of a mild facial abnormality, agammaglobulinemia in the presence of normal b cell count and opportunistic infections in a patient, physicians must consider icf syndrome as a differential diagnosis. hsct is the only curative treatment option of the immune defect and should be carried out early to improve survival. however, developmental and neurological outcomes remain largely unchanged since the epigenetic dysregulation of non-hematopoetic cells cannot be corrected with hsct. the study was approved by the local ethics committee and carried out in accordance with the declaration of helsinki. the parents provided written informed consent to publish the report and the picture appearing in figure . kg first drafted the manuscript. cs and uf contributed to wes analysis. ab, fb, fs, gk, and rm cared for the child. jd-d and dw performed cytogenetic analysis. ab cared for the child and critically revised the manuscript for important intellectual content. mk cared for the child, designed, and supervised the project and critically reviewed and revised the manuscript for important intellectual content. all authors approved the final manuscript as submitted. the authors thank their patients and their family. the dnmt b dna methyltransferase gene is mutated in the icf immunodeficiency syndrome an embryonic-like methylation pattern of classical satellite dna is observed in icf syndrome hematopoietic stem cell transplantation corrects the immunologic abnormalities associated with immunodeficiency-centromeric instability-facial dysmorphism syndrome heterogeneous clinical presentation in icf syndrome: correlation with underlying gene defects t-cell apoptosis in icf syndrome immunodeficiency, centromeric region instability, facial anomalies syndrome (icf) clinical spectrum of immunodeficiency, centromeric instability and facial dysmorphism (icf syndrome) the health status and quality of life of adults with x-linked agammaglobulinemia icf syndrome (immunodeficiency, centromeric instability and facial anomalies): investigation of heterochromatin abnormalities and review of clinical outcome genetic, cellular and clinical features of icf syndrome: a french national survey treosulfan-based conditioning regimens for hematopoietic stem cell transplantation in children with primary immunodeficiency: united kingdom experience expanding the mutation spectrum in icf syndrome: evidence for a gender bias in icf hematopoetic stem cell transplantation in children allogeneic and autologous transplantation for haematological diseases, solid tumours and immune disorders: current practice in europe quantitatively different red cell/nucleated cell chimerism in patients with long-term, persistent hematopoietic mixed chimerism after bone marrow transplantation for thalassemia major or sickle cell disease a novel mutation in a critical region for the methyl donor binding in dnmt b causes immunodeficiency, centromeric instability, and facial anomalies syndrome (icf) dna methyltransferase b (dnmt b) mutations in icf syndrome lead to altered epigenetic modifications and aberrant expression of genes regulating development, neurogenesis and immune function genome-wide dna methylation analysis identifies novel hypomethylated non-pericentromeric genes with potential clinical implications in icf syndrome the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord- -barmkkwx authors: geginat, jens; nizzoli, giulia; paroni, moira; maglie, stefano; larghi, paola; pascolo, steve; abrignani, sergio title: immunity to pathogens taught by specialized human dendritic cell subsets date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: barmkkwx dendritic cells (dcs) are specialized antigen-presenting cells (apcs) that have a key role in immune responses because they bridge the innate and adaptive arms of the immune system. they mature upon recognition of pathogens and upregulate mhc molecules and costimulatory receptors to activate antigen-specific cd (+) and cd (+) t cells. it is now well established that dcs are not a homogeneous population but are composed of different subsets with specialized functions in immune responses to specific pathogens. upon viral infections, plasmacytoid dcs (pdcs) rapidly produce large amounts of ifn-α, which has potent antiviral functions and activates several other immune cells. however, pdcs are not particularly potent apcs and induce the tolerogenic cytokine il- in cd (+) t cells. in contrast, myeloid dcs (mdcs) are very potent apcs and possess the unique capacity to prime naive t cells and consequently to initiate a primary adaptive immune response. different subsets of mdcs with specialized functions have been identified. in mice, cd α(+) mdcs capture antigenic material from necrotic cells, secrete high levels of il- , and prime th and cytotoxic t-cell responses to control intracellular pathogens. conversely, cd α(−) mdcs preferentially prime cd (+) t cells and promote th or th differentiation. bdca- (+) mdc are the human homologue of cd α(+) mdcs, since they share the expression of several key molecules, the capacity to cross-present antigens to cd (+) t-cells and to produce ifn-λ. however, although several features of the dc network are conserved between humans and mice, the expression of several toll-like receptors as well as the production of cytokines that regulate t-cell differentiation are different. intriguingly, recent data suggest specific roles for human dc subsets in immune responses against individual pathogens. the biology of human dc subsets holds the promise to be exploitable in translational medicine, in particular for the development of vaccines against persistent infections or cancer. introduction human beings are constantly exposed to a myriad of pathogens, including bacteria, fungi, and viruses. these foreign invaders or cohabitants contain molecular structures that are sensed by the innate immune system, which mounts a first-line defense and also activates a pathogen-specific, adaptive immune response. the adaptive immune system is composed of b cells that produce specific antibodies, cd + t cells that can kill pathogen-infected cells, and cd + t cells that produce effector cytokines and coordinate the immune response. t cells express antigen receptors (t-cell antigen receptors, tcr) that recognize specific peptides presented on mhc molecules. cd + t cells recognize peptides presented by mhc class-i molecules that are ubiquitously expressed, whereas cd + t cells are activated by peptide-mhc class-ii complexes, which are largely restricted to antigen-presenting cells (apcs). dendritic cells (dcs) can express very high levels of mhc and costimulatory molecules, and it is generally accepted that they are the relevant cells to induce the activation ("priming") of antigen-specific "naive" t cells ( , ) and induce their differentiation into various types of effector t cells. the elimination or containment of different types of pathogens requires dedicated classes of adaptive immune responses ( ) . thus, pathogens like viruses or intracellular bacteria require cd + and cd + t cells that produce ifn-γ and kill infected cells (th and ctl, respectively). il- is the critical cytokine that induces this type of response, but il- production by dc is tightly controlled and requires several stimuli derived from pathogens and from cd + helper t cells ( ) ( ) ( ) ( ) ( ) ( ) . conversely, extracellular bacteria and fungi require a different type of response that can be mediated by th cells ( ) ( ) ( ) . these effector cells are induced by proinflammatory cytokines produced by dc and macrophages ( ) and attract neutrophils that in turn phagocytose extracellular bacteria ( ) . a third type of effector response is the th response, which is required to expel extracellular parasites such as helminths by activating eosinophils and basophils and by inducing antibodies of the ige class ( ) . il- is the critical cytokine that induces this response ( ) , but il- is normally not produced by dc ( , ) . finally, these different effector responses have to be controlled by specialized regulatory t cells, in particular by il- -producing t cells ("tr cells"), which are generated from effector cells and are important to avoid excessive tissue damage by adaptive immune responses ( ) ( ) ( ) ( ) . cytokines that promote this type of regulatory t-cell response are ifn-α, il- , and il- ( ) ( ) ( ) , and all these cytokines can be produced by dcs ( , ) . professional apcs have to present pathogen-derived peptides on mhc molecules to activate antigen-specific t cells. dcs are phagocytic in the immature state, i.e., under steady-state conditions and upon initial pathogen encounter, and can take up antigenic material by pinocytosis or by surface receptor-mediated internalization ( ) . proteins from pathogens are then shuttled to lysosomes where they are chopped to peptides and loaded on mhc class-ii molecules ( , ) . these peptide-mhc complexes are then transported to the plasma membrane to activate specific cd + t cells. the presentation of peptides derived from exogenous proteins on mhc class-i, a process called cross-presentation ( , ) , is a largely unique feature of dcs and is particularly important to activate cd + t cells in viral infections. virus-infected cells express viral proteins in the cytosol where they are degraded to peptides by the proteasome, translocated to the endoplasmic reticulum by tap proteins, and loaded on mhc class-i molecules ( ) . however, since dcs are not necessarily infected by viruses, they must be able to process virus-derived proteins also from external sources, such as virus-infected cells, to activate cd + t cells. the mechanism of cross-presentation is still incompletely understood, but two distinct pathways via vacuoles and peptide translocation from phagolysosomes to the cytosol have been described ( ) . it is believed that cross-presentation is the most important pathway leading to the induction of cytotoxic t-cell responses, and excellent reviews have been published on this relevant topic ( ) ( ) ( ) . naive t cells have a very high activation threshold ( ) , and only professional apcs that express high levels of mhc and costimulatory molecules such as dcs are able to induce proliferation of naive t cells ( ) . several receptor-ligand interactions contribute to naive t-cell activation ( ) ( ) ( ) , but cd costimulation is particularly important to amplify the signal transduced by the tcr ( ) . monocytes efficiently present peptides derived from extracellular proteins on mhc class-ii to activate antigen-experienced cd + t cells ( ) , and this capacity can be exploited to selectively expand antigen-specific memory t cells ( ) . however, monocytes have an approximately -fold lower capacity to prime naive cd + t cells as compared to dcs (nizzoli et al., under review) and home to non-lymphoid tissues in the steady state. however, upon inflammation, they can differentiate to inflammatory dcs ( ) and home to lymph nodes where they can activate t cells ( , ) . in addition, there is some evidence that cd + subsets of human blood monocytes might contain dcs ( , , ) . naive t cells constantly recirculate in the blood and migrate through secondary lymphoid organs ( ) , but are largely excluded from non-lymphoid tissues. in secondary lymphoid tissues, they migrate to the t-cell zone, where they encounter dcs ( ) . b cells are also present in secondary lymphoid organs and can potently present antigen to t cells when they internalize and process antigens that have specifically bound to their b-cell receptor ( ) . however, b cells are physically separated from naive t cells in lymph nodes and only following tcr activation naive t cells migrate to the b-cell zone where they interact with antigen-specific b cells to induce antibody production ( , ) . thus, antigen presentation by b cells appears to be important for the activation of antigen-experienced t cells rather than for naive t-cell priming. dendritic cells are generated from committed precursors in the bone marrow that are released into the circulation to seed peripheral organs ( ) ( ) ( ) ( ) ( ) . both monocytes and dcs can be derived from common myeloid progenitors (cmps), but committed precursors that selectively give rise to monocytes or dcs ( ) or even selected dc subsets ( , ) have been identified in humans and mice. dcs are poorly stimulatory in the immature state and can induce a partial t-cell activation, leading to deletion of autoreactive cd + t cells ( ) ( ) ( ) ( ) . in addition, they promote self-tolerance by inducing foxp + regulatory cd + t cells that suppress autoreactive t cells ( ) . pathogens induce the maturation of dcs that consequently acquire the capacity to produce polarizing cytokines and to prime pathogen-specific effector t-cell responses. pathogen-derived molecular patterns [pamps ( , ) ] are recognized by dcs and lead to the efficient presentation of antigens to t cells ( ) . there are different classes of pathogen-sensing receptors, including toll-like receptors ( , ) , nucleotide-binding oligomerization domain (nod)like receptors ( ) , retinoic acid-inducible gene (rig-i)-like receptors ( ) , and c-type lectins ( ) . tlrs recognize different pamps, including nucleic acids or cell wall components such as proteins and lipoproteins ( , ) . in the case of viruses, nucleic acids are sensed not only by different tlrs in endosomes but also by cytosolic receptors like rig-i ( , ) and induce a potent activation of dcs. importantly, subsets of dcs express different patterns of pathogen-sensing receptors and might thus preferentially respond to individual pathogens ( , ) . dna viruses such as cytomegalovirus (cmv) and herpes simplex virus (hsv) and also bacteria can activate dcs via unmethylated cpg-containing dna ( ) , which is sensed by tlr . double-and single-stranded rnas, which are generated by both dna and rna viruses, are sensed by dcs via tlr ( ) and tlr / ( , ) , respectively. of note, tlr is restricted to mdcs ( ) and induces cross-presentation capacities ( ) . viruses such as respiratory syncytial virus (rsv) and hepatitis c virus (hcv) can also activate dcs via tlr or tlr , which are expressed on the plasma membrane and recognize viral proteins ( ) . tlr is also involved in immune responses to fungi ( ) and gram-positive bacteria ( , ) while tlr recognizes lipopolysaccharide (lps) ( ), a cell membrane compound of gram-negative bacteria. many pathogens like viruses activate dcs via multiple tlrs ( ) . moreover, other immune cells, including t cells themselves, feed-back on dcs to regulate the ongoing response. in particular, cd stimulation by cd + helper t cells is crucial for cd + t-cell stimulation and il- production ( , ). moreover, ifn-γ ( ) and paradoxically also il- ( , ) that can be provided by t cells further enhance il- production ( ). surface tlrs such as tlr and tlr signal via the adaptor protein myd ( ) to induce the activation of map kinases and the nuclear translocation of the transcription factor nf-κb, which in turn induces the transcription of proinflammatory cytokines ( ) . endosomal tlrs , , and also signal via myd but activate irf , which in turn induces type- interferon production ( , ) . tlr is an exception since it does not signal via myd but utilizes trif ( ) to activate irf ( , ) or irf ( ) . how all these complex signaling pathways are integrated by dcs to induce the appropriate t-cell response is still incompletely understood ( ) ( ) ( ) . dendritic cells in mice can be subdivided into distinct subsets with specific functions. some dcs are stably resident in lymph nodes while others are positioned in non-lymphoid tissues to sense tissue-invading pathogens, but are migratory and are recruited via the lymph following pathogen encounter in a ccr -dependent manner ( , ) . in secondary lymphoid tissues, two major dc subsets are myeloid dcs (mdcs) and plasmacytoid dcs (pdcs; table ) ( , , ) . both pdcs and mdcs upregulate mhc and costimulatory molecules like cd and cd upon maturation ( ) that bind to cd and are required to induce full t-cell stimulation ( ) . however, pdcs are poorly phagocytic and have a different regulation of mhc class-ii turnover upon maturation as compared to mdcs ( ) . thus, mdcs stop phagocytosis and peptide loading on mhc upon pathogen recognition and stably present peptides from antigenic material they had acquired upon pathogen encounter ( , , ) . this maturation-induced stabilization of peptide-mhc complexes enhances the priming of pathogenspecific t cells by mdcs. in contrast, pdcs continue to present new peptides on mhc complexes even in the mature stage ( ) . on the one hand, this limits their capacity to stimulate pathogen-specific t cells; on the other hand, this enables them to present also late-expressed viral antigens when they are actively infected. this diverse regulation of mhc-peptide stability in mdcs and pdcs suggests that they present different antigens to t cells. n/a n/a n/a plasmacytoid dcs are present in lymph nodes and are largely absent from non-lymphoid organs, but they can be recruited upon inflammation ( ) . the role of pdc in t-cell priming is still debated ( ) . there is consensus that they are poorly stimulatory in their resting state ( , ), but while some groups proposed that they become potent apcs following tlr stimulation and prime cd + and cross-prime cd + t-cell responses ( ) ( ) ( ) ( ) , others concluded that also mature pdcs have only low priming and cross-priming capacities and might rather be tolerogenic ( ) . the rapid and abundant production of type- interferon by pdc suggests a pivotal role in viral infections, even if their capacity to prime virus-specific t cells directly appears to be limited. ifn-α can also be produced by other immune cells and by virus-infected cells, but the early and systemic ifn-α response is believed to depend on pdcs ( ) . consistently, in the case of hsv infections, it was shown that pdcs were important for systemic but not local protection ( ) . however, in several other viral infections in mice, including vesicular stomatitis virus (vsv), lymphocytic choriomeningitis virus (lcmv), rsv, and mouse cytomegalovirus (mcmv), pdcs do not seem to play a major role ( ) . in marked contrast, in mouse hepatitis virus (mhv) infection, the antiviral response against this coronavirus was largely pdc dependent ( ) (figure ) . finally, pdcs have been found by several groups to induce the production of the anti-inflammatory cytokine il- by cd + t cells, suggesting that they might be important to inhibit excessive t-cell responses. several proteins expressed by pdcs were found to promote il- induction in t cells, including the notch ligand delta-like ( ), icosl ( , ) , as well as ifn-α ( , nizzoli et al., under review). myeloid dcs are a heterogeneous population, and different mdc subsets can be identified that preferentially initiate different types of adaptive immune responses (figure ). in the spleen of mice, mdcs can be subdivided into cd α + and cd α − subsets ( table ) . cd α + mdcs produce high levels of bioactive il- p and efficiently cross-prime cd + t-cell responses ( ) . they express clec a, a c-type lectin, that enables them to take up antigenic material from dying cells, and their generation was shown to rely on the transcription factors batf and irf ( , ) . moreover, they express the chemokine receptor xcr that favors their colocalization with cd + t cells ( ) . altogether the present evidence indicates that cd α + dcs are specialized to induce th and ctl responses in response to intracellular pathogens ( , ) . notably, dcs in the gut that express cd have similar characteristics and are closely related to cd α + dc ( , ) . cd α − dcs express cd b and can be further subdivided into cd + and cd − cd − subsets. they preferentially prime cd + t-cell responses ( ) and promote th responses, but they can also induce th cells ( , ) . interestingly, cd b + dcs produce il- in the gut and are required for protection against citrobacter rodentium ( ) . their generation depends on the transcription factor irf , while klf expression is required for th , but not for th induction ( ) . notably, however, cd α − dcs and also pdcs can cross-prime cd + t-cell responses under certain conditions ( ) ( ) ( ) ) . moreover, it was shown that upon appropriate microbial stimulation all mdc subsets have the potential to promote either th or th responses ( ) . thus, although the proposed functional specialization of dc subsets is an intriguing and helpful concept, it might also be an oversimplification, since dc subsets have considerable plasticity and the induction of a specific type of immune response critically depends on the stimuli they receive from pathogens as well as from other immune cells ( ) . high numbers of human dcs can be generated in vitro by culturing monocytes with cytokines ( ) , and the large majority of studies on human dcs have been done with these monocytederived dcs. they are primary cells and show many behaviors of in vivo occurring dcs, including cytokine production as well as stable and potent antigen presentation upon maturation with tlr ligands ( ) . however, monocyte-derived dcs are not the appropriate model to study the role of specialized dc subsets in human immune responses. dendritic cells circulating at low frequency in human peripheral blood share several features with murine splenic dc subsets ( ) ( table ) . human pdcs have been identified more than years ago as the natural ifn-α-producing cells ( , ) . they express tlr and tlr and produce large amounts of ifn-α in response to cpg dna or influenza virus. similar to their murine counterparts, they are poorly stimulatory ( ) , express the c-type lectin bdca- ( ), and induce il- production in cd + t cells ( ) . in addition, subsets of mdcs can also be found in human blood and in tissues ( ) ( ) ( ) ( ) . as their murine homologues, they express cd c and potently prime cd + and cd + t-cell responses. the expression of cd c/bdca- and cd /bdca- identifies two subsets among human mdcs in peripheral blood ( ) and also in secondary lymphoid organs ( , , , ) . bdca- + "mdc " ( table ) are rare, but it could recently be demonstrated that they represent the human counterpart of murine cd α + dcs ( ) ( ) ( ) ( ) ( ) . thus, as cd α + dcs, they selectively express clec a and xcr and are dependent on the transcription factor batf ( , , , , ) . importantly, they can cross-present exogenous antigens on mhc class-i to cd + t cells and produce il- ( ) ( ) ( ) . cd c + "mdc " ( table ) are more frequent and share some features with cd α − dc, including cd b expression and il- production ( , , nizzoli et al., under review). also tlr expression in dc subsets appears to be similar in humans and mice, since it is expressed at high levels on cd α + dcs and mdc , at lower levels on cd α − dcs and mdc , and absent on pdc. surprisingly, tlr in mice is not required for immune responses against several viruses, including lcmv, vsv, mcmv, and reovirus, suggesting that tlr has not a pivotal role in antiviral immune defense ( ) . consistently, tlr deficiency in humans selectively leads to uncontrolled hsv infections in the central nervous system (cns) ( ) . different subsets of dc have also been identified in human non-lymphoid tissues where they are strategically positioned to recognize invading pathogens, in particular at barrier surfaces. these migratory dc subsets play a crucial role to transport antigenic material of pathogens that invade specific tissues to draining lymph nodes and thus to initiate a tissue-specific t-cell response ( , , ) . human langerhans cells were first described more than a century ago and reside in the epidermis and are thus the first dcs that encounter skin-invading pathogens. upon activation, they mature and migrate to draining lymph nodes to activate cd + and cd + t cells. in the dermis, different subsets of interstitial dcs are present and can be classified according to cd , cd a, and cd expression. dermal cd + cells might represent monocyte-derived macrophages rather then dcs ( ) , but cd a + and cd + dcs, respectively, resemble the cd c + and cd + dc subsets in peripheral blood ( ) . also in the lung and the liver, dc subsets that are related to cd c + and cd + dcs could be identified ( ) . finally, in the human intestine, dc subsets that express cd b and cd are similar to cd c + and cd + dcs, respectively, and these intestinal dc subsets are also largely conserved between humans and mice ( ) . although the similarities between human and mouse dc subsets are often emphasized, there are also some important differences in pathogen sensing by dcs in humans and mice ( ) . importantly, the expression of several relevant tlrs is not conserved (figure ) , presumably because humans and mice have evolved under the selective pressure of different pathogens. thus, in mice, tlr and tlr are expressed by both pdc and mdc subsets ( ) , whereas in humans, they are restricted to pdcs ( ) . also tlr expression is more restricted in human dcs, since it is expressed by mdc but not by mdc ( ) . moreover, tlr is not expressed by human pdcs ( ) , and some agonists of human tlr such as the resiquimod r do not activate murine tlr ( , ) . another relevant difference seems to be the role of the adaptor protein myd , which transduces signals from all tlrs with the notable exception of tlr . thus, mice deficient for myd are highly susceptible to several infections by bacteria, viruses, parasites, and fungi. conversely, myd -deficient patients are selectively affected by infections with pyogenic bacteria in childhood ( ) . finally, human cd c + dcs and also langerhans cells seem to have superior capacities to cross-present antigens and to induce ctl responses as compared to their murine homologues ( , , , ) . overall, these differences in pathogen sensing and t-cell activation between human and murine dcs are likely to have an important impact on their role in immune responses against specific pathogens. dendritic cell subsets in humans and mice express not only different patterns of toll-like receptors, but they have also partially distinct cytokine profiles (figure ) . in particular, human mdc have a complex and quite unique regulation of cytokine production. thus, while lps triggers only low levels of cytokine production by mdc , dual tlr stimulation with lps or poly-i:c (tlr ligand) in combination with r induces very high levels of a broad panel of cytokines, including tnf, il- , il- , il- , and il- (nizzoli et al., under review) . the very potent cytokine-producing capacity of mdc has been missed in several studies where mdc were activated with single tlr ligands ( , , ) . of note, single tlr stimulation is sufficient to induce antiviral cytokines by mdc and pdcs (see below) and proinflammatory cytokines by monocytes. although mdc can secrete several proinflammatory cytokines that promote th cell generation including il- ( ) , it is unclear if they are the physiological inducers of th cells or if monocyte-derived, inflammatory dcs do the job ( , ) . also the identity of the dc subset that induces human th responses is still enigmatic. it was originally proposed that mdcs induce th polarization and pdcs th , but later it was shown that also pdcs can drive th responses ( , ) . more recently, mdc but not mdc were found to induce th cells in an aberrant response to influenza virus ( ) . in apparent contrast to cd α − dcs, mdc can produce high levels of il- ( , ) , suggesting a relevant role in immune responses against intracellular pathogens. moreover, the production of the anti-inflammatory cytokine il- , which can be produced by all mdcs in mice, is largely restricted to mdc in humans (nizzoli et al., under review) . stimulation of mdc with the intestinal bacterium escherichia coli or with lps alone induces il- and was proposed to induce a tolerogenic state in mdc ( ) . although il- is indeed a tolerogenic cytokine and a well-established negative regulator of dc maturation and cytokine production ( ) , it can paradoxically also have positive effects, in particular on cd + t-cell responses ( , ) . consistently, we found that il- produced by mdc completely blocked the cross-priming of low-affinity ctl and enhanced the responsiveness of cd + memory t cells to the homeostatic cytokine il- . thus, mdc -derived il- appears to play an important positive role in ctl responses, since it selects high affinity cells upon priming and inhibits ctl memory attrition at the same time (nizzoli et al., under review) . while mdc can secrete a broad panel of pro-and antiinflammatory cytokines, mdc and pdc are largely dedicated to secrete high levels of antiviral cytokines. the subset-specific production of ifn-α by pdc ( ) and of ifn-λ by cd α + and mdc ( , ) appears to be largely conserved between humans and mice. the very potent ifn-λ-producing capacities of bdca- + dc ( , ) suggest that analogous to pdcs they might be the relevant source of early and systemic ifn-λ in viral infections. notably, ifn-λ has antiproliferative and antiviral activities similar to type-i interferon, but the expression of the ifn-λ receptor is much more restricted and found mainly on epithelial cells at barrier surfaces and in the liver ( ) . mdc can also secrete selected isoforms of ifn-α ( ) and some il- ( ) ( ) ( ) ) , consistent with the view that they play an important role in antiviral immune responses. as previously mentioned for murine pdcs, ifn-α is not only a powerful antiviral cytokine that activates several different types of immune cells, but it also induces il- production in cd + t cells, suggesting that pdcs induce tr -like regulatory t cells also in humans ( , , , nizzoli et al., under review). the more restricted expression of tlrs and the specific cytokineproducing capacities of human dc subset suggest that they play unique roles in immune responses against individual pathogens. the roles of human dc subsets in pathogen-specific immune response are however difficult to address directly because patients that selectively lack a dc subset of interest have not been identified so far. nevertheless, some interesting findings were reported. in particular, mdc appear to be highly relevant in hcv infection. single-nucleotide polymorphisms in the ifn-λ gene locus are strongly associated with spontaneous clearance and response to therapy in hcv patients ( ) . all dc subsets can secrete some ifn-λ ( , ) , but mdc produce much higher amounts. moreover, ifn-λ / are largely restricted to mdc , and importantly hcv induces ifn-λ production by mdc ( , nizzoli et al., under review) . thus, mdc appear to be a highly relevant source for protective ifn-λ in hcv infection ( ) . interestingly, an important role for mdc rather than for mdc was recently proposed in tuberculosis ( , ) . thus, mdc were more efficiently infected with the bacillus calmette-guérin (bcg) vaccine than other dcs and induced the activation of pdcs and cd + t cells. notably, mdc could not be replaced by mdc in this system, suggesting that mdc could play a non-redundant role in the defense against selected intracellular pathogens. mdc and mdc have also been suggested to play different roles in rsv infection ( , ) . thus, mdc subsets produced different cytokines in response to rsv, consistent with their different cytokine profiles upon stimulation with purified tlr ligands ( , nizzoli et al., under review) . moreover, they induced different classes of t-cell responses, with mdc inducing preferentially th cells and mdc inducing predominantly th and t-regulatory cells. similarly, mdc , but not mdc , were found to induce th response to influenza virus ( ) . also the capacity of pdcs to induce il- -producing regulatory t cells has been documented with a relevant pathogen, since pdcs were shown to induce ifn-γ and il- production in antigen-experienced cd + t cells specific for mumps virus ( ) . conversely, cd c + mdcs, which contain both mdc and mdc , induced ifn-γ and, surprisingly, il- . it is largely accepted that pdc-derived ifn-α is important to contain human viral infections. thus, stabilized pegylated ifn-α is a widely used therapy for hcv patients. ifn-λ appears to be similar effective, but is less toxic presumably because of the more restricted expression of its receptor ( ) . interestingly, the hcv glycoprotein e is a ligand for bdca- , which is specifically expressed on pdcs ( table ) and inhibits ifn-α production ( , ) . in this way, hcv might inhibit ifn-α production to establish chronic infection. finally, pdcs are also targeted by human immunodeficiency virus (hiv), but whether they play a protective or detrimental role is still unclear ( ) . vaccines have been a major breakthrough for human health. attenuated or killed pathogens are highly efficient to induce protective cellular and humoral immune responses, and the induced protective memory can last for a lifetime ( , ) . however, since these pathogen-based vaccines also have considerable side effects, proteins in combination with adjuvants that activate apcs are more often used. protein vaccines induce cd + t-cell responses and neutralizing antibodies, but they are poorly efficient in inducing cytotoxic t-cell responses and are also rather inefficient in inducing th cells ( , ) . frequently used adjuvants are alum, oil-in-water emulsions like mf , and more recently also monophosphoryl lipid a (mpl), a detoxified form of lps. in mice, different adjuvants were shown to induce different proinflammatory cytokines. thus, alum acts via uric acid on inflammatory dcs ( ) , which leads to nod-like receptor protein- (nalp )-dependent il- β production ( ) . conversely, mpl does not induce il- β ( ) but induces specific antibodies through an il- -dependent mechanism ( ), while mf- and alum act independently of il- ( ). however, the different tlr expression and cytokine production by human apc subsets should be considered when translating this knowledge from animal models to patients. a recent interesting report analyzed the response of apc subsets to different vaccines and concluded that different vaccines activate indeed different apc populations ( ) . more direct information on the effect of dcs was obtained by vaccinations with peptide-pulsed monocyte-derived dcs in cancer patients, which can induce tumor-specific cd + t cells ( ) , but the clinical responses were so far largely insufficient. mdcs might be more potent and are currently tested in clinical trials. nucleic acid-sensing tlrs are particularly potent to induce cd + t-cell responses in mice ( ) and have recently been employed as adjuvants in vaccines. examples are cpg-dna that stimulates tlr ( ) , and the tlr ligand imiquimod, which is used as a cream to stimulate dc locally in the skin, and was shown to induce cd + t-cell responses in situ ( ) . vaccines consisting of plasmid dna coding for relevant protein antigens are a novel approach that efficiently induces humoral and cellular immune responses in animals. however, in humans, these dna vaccines are often poorly immunogenic ( ) , presumably because they have only low adjuvant activity and stimulate mainly cytosolic dna sensors rather than tlr ( ) , which in addition is restricted to pdcs and b cells in humans. an alternative promising approach is the vaccination with mrna ( , ) , which delivers not only the antigenic protein directly to the cytosol, thereby bypassing the requirements for cross-presentation ( ) , but also induces mdc and pdc maturation and cytokine production via tlr / at the same time ( ) . indeed, intradermal injection of naked mrna results in local uptake and translation of the nucleic acid ( ) followed by the development of an adaptive immunity in mice ( ) and in humans ( , ) . since also lymph node-resident dcs are expected to be appropriate apcs to process antigens encoded by mrna, direct injection of nucleic acid into lymph nodes has also been evaluated. in animal models, intra-lymph node injections of mrna result in expression of the protein encoded by the mrna in dcs. furthermore, the injected mrna activated lymph node-resident apcs and induced potent cd + and cd + t-cell responses as well as prophylactic and therapeutic antitumor immunity ( ) . the approach is currently being evaluated through two clinical studies exploring the efficacy of intra-lymph node mrna vaccination in advanced melanoma patients. as a further development, systemic administration of a liposomal formulation of mrna that delivers the nucleic acids to apcs present in secondary lymphoid organs is also being evaluated. using the functional diversity of dcs in vivo, and their specific capabilities in generating appropriate adaptive immune responses, those systemic synthetic vaccines might recapitulate the natural mechanisms of immunity developed during pathogen infection and guarantee the development of therapeutically efficacious immune responses. dendritic cells continue to attract much interest of immunologists because they are the most potent apcs in the immune system and are the principal inducers of naive t-cell differentiation. intensive research in the last years has established that different subsets of dc exist in mice that have specialized functions and preferentially induce different types of immune responses. in humans, much has been learned from in vitro differentiated monocytederived dcs, and more recently, also different subsets of dc populating human tissues have been analyzed at the molecular and functional levels. it is fundamental to further define the biology of these in vivo occurring human dc subsets to understand and cure pathogenic immune-mediated processes in so different settings as autoimmunity, infections, and cancer. in particular, appropriate targeting of dc subsets by vaccines holds the promise to induce cytotoxic t-cell responses to eradicate persistent intracellular pathogens or tumors. acknowledgments jg, gn, and sa are supported by the cariplo foundation, sa is supported by the european research council, and the ingm is supported by the "romeo ed enrica invernizzi" foundation. the dendritic cell system and its role in immunogenicity immunobiology of dendritic cells the cd -centered universe of human t cell subsets ligation of cd on dendritic cells triggers production of high levels of interleukin- and enhances t cell stimulatory capacity: t-t help via apc activation high level il- production by murine dendritic cells: upregulation via mhc class ii and cd molecules and downregulation by il- and il- differential regulation of human blood dendritic cell subsets by ifns il- is a mediator of il- p induction by human th cells: reversal of polarized th phenotype by dendritic cells interleukin (il)- is a major regulatory cytokine governing bioactive il- production by mouse and human dendritic cells differential production of il- , ifn-α, and ifn-γ by mouse dendritic cell subsets interleukin (il)- and il- are coexpressed by th cells and cooperatively enhance expression of antimicrobial peptides deficiency of th cells in hyper ige syndrome due to mutations in stat surface phenotype and antigenic specificity of human interleukin -producing t helper memory cells interleukins beta and but not transforming growth factor-beta are essential for the differentiation of interleukin -producing human t helper cells induction, function and regulation of il- -producing t cells th and 'th -like' cells in allergy and asthma: pharmacological perspectives disruption of the murine il- gene blocks th cytokine responses group innate lymphoid cells are critical for the initiation of adaptive t helper cell-mediated allergic lung inflammation th , allergy and group innate lymphoid cells conventional t-bet(+)foxp (-) th cells are the major source of host-protective regulatory il- during intracellular protozoan infection a cd + t-cell subset inhibits antigen-specific t-cell responses and prevents colitis identification and characterization of il- /ifn-gamma-producing effector-like t cells with regulatory function in human blood th cells express interleukin- receptor and are controlled by foxp and foxp + regulatory cd + t cells in an interleukin- -dependent manner ifn-alpha and il- induce the differentiation of human type t regulatory cells il- is a key regulator of il- and il- production by human cd + t cells ifn-alpha and cd stimulation are associated with active lupus and skew natural t regulatory cell differentiation to type regulatory t (tr ) cells il- and il- producing dendritic cells capable of enhancing il- production of t cells are induced in oral tolerance differentiation of type t regulatory cells (tr ) by tolerogenic dc- requires the il- -dependent ilt /hla-g pathway dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class ii compartment: downregulation by cytokines and bacterial products the formation of immunogenic major histocompatibility complex class ii-peptide ligands in lysosomal compartments of dendritic cells is regulated by inflammatory stimuli transport of peptide-mhc class ii complexes in developing dendritic cells cross-presentation in viral immunity and self-tolerance cross-presentation by dendritic cells the biochemistry and cell biology of antigen processing and presentation enhanced responsiveness of human memory t cells to cd and cd receptor-mediated activation generation of antigen-specific cd + t cell lines from naive precursors cd and lfa- contribute to cyclosporin a-resistant t cell growth by stabilizing the il- mrna through distinct signaling pathways ox costimulation enhances interleukin- (il- ) expression at priming and promotes the differentiation of naive human cd (+) t cells into high il- -producing effectors analysis of - bb ligand ( - bbl)-deficient mice and of mice lacking both - bbl and cd reveals a role for - bbl in skin allograft rejection and in the cytotoxic t cell response to influenza virus accessory cell-derived signals required for t cell activation chemokine receptor expression identifies pre-t helper (th) , pre-th , and nonpolarized cells among human cd + central memory t cells efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin and downregulated by tumor necrosis factor alpha microbial stimulation fully differentiates monocytes to dc-sign/ cd (+) dendritic cells for immune t cell areas blood-derived inflammatory dendritic cells in lymph nodes stimulate acute t helper type immune responses human -sulfo lacnac-expressing dendritic cells are principal producers of early interleukin- and are controlled by erythrocytes functional specialization of human circulating cd and cd c myeloid dendritic-cell subsets naive and memory t cells show distinct pathways of lymphocyte recirculation dendritic cells in the t-cell areas of lymphoid organs antigen-specific interaction between t and b cells follicular b helper t cells express cxc chemokine receptor , localize to b cell follicles, and support immunoglobulin production shaping up adaptive immunity: the impact of ccr and cxcr on lymphocyte trafficking development and function of dendritic cell subsets in vivo analysis of dendritic cell development and homeostasis circulating precursors of human cd c+ and cd + dendritic cells restricted dendritic cell and monocyte progenitors in human cord blood and bone marrow identification of clonogenic common flt +m-csfr+ plasmacytoid and conventional dendritic cell progenitors in mouse bone marrow avoiding horror autotoxicus: the importance of dendritic cells in peripheral t cell tolerance dendritic cells induce peripheral t cell unresponsiveness under steady state conditions in vivo phenotypic and functional analysis of cd (+) t cells undergoing peripheral deletion in response to cross-presentation of self-antigen t cell fitness determined by signal strength cd + regulatory t cells: mechanisms of induction and effector function the immune system evolved to discriminate infectious nonself from noninfectious self a human homologue of the drosophila toll protein signals activation of adaptive immunity origin, maturation and antigen presenting function of dendritic cells a family of human receptors structurally related to drosophila toll nod-like receptors: versatile cytosolic sentinels rig-i: tri-ing to discriminate between self and non-self rna myeloid c-type lectin receptors in pathogen recognition and host defense toll-like receptors: a growing family of immune receptors that are differentially expressed and regulated by different leukocytes a toll-like receptor recognizes bacterial dna deficient signaling in mice devoid of double-stranded rna-dependent protein kinase toll-like receptor expression in murine dc subsets: lack of tlr expression by cd alpha+ dc correlates with unresponsiveness to imidazoquinolines specialization and complementarity in microbial molecule recognition by human myeloid and plasmacytoid dendritic cells recognition of double-stranded rna and activation of nf-kappab by toll-like receptor innate antiviral responses by means of tlr -mediated recognition of single-stranded rna species-specific recognition of single-stranded rna via toll-like receptor and toll-like receptor promotes cross-priming to virus-infected cells toll-like receptors and viruses: induction of innate antiviral immune responses fungal recognition by tlr and dectin- differential roles of tlr and tlr in recognition of gram-negative and gram-positive bacterial cell wall components toll-like receptor (tlr ) mediates astrocyte activation in response to the gram-positive bacterium staphylococcus aureus cutting edge: toll-like receptor (tlr )-deficient mice are hyporesponsive to lipopolysaccharide: evidence for tlr as the lps gene product myd is an adaptor protein in the htoll/il- receptor family signaling pathways irf- is the master regulator of type-i interferon-dependent immune responses interferon-alpha induction through toll-like receptors involves a direct interaction of irf with myd and traf cutting edge: a novel toll/il- receptor domain-containing adapter that preferentially activates the ifn-beta promoter in the toll-like receptor signaling irf mediates a tlr /tlr -specific antiviral gene program role of adaptor trif in the myd -independent toll-like receptor signaling pathway how toll-like receptors signal: what we know and what we don't know duration, combination and timing: the signal integration model of dendritic cell activation cutting edge: synchronization of irf , junb, and c/ebpbeta activities during tlr -tlr cross-talk orchestrates timely cytokine synergy in the proinflammatory response differential regulation of chemokine receptors during dendritic cell maturation: a model for their trafficking properties rapid and coordinated switch in chemokine receptor expression during dendritic cell maturation bdca- , bdca- , and bdca- : three markers for distinct subsets of dendritic cells in human peripheral blood characterization of human blood dendritic cell subsets developmental regulation of mhc ii expression and transport in human plasmacytoid-derived dendritic cells developmental regulation of mhc class ii transport in mouse dendritic cells capture and processing of exogenous antigens for presentation on mhc molecules evidence for recruitment of plasmacytoid dendritic cell precursors to inflamed lymph nodes through high endothelial venules antigen-presentation properties of plasmacytoid dendritic cells plasmacytoid dendritic cells: recent progress and open questions plasmacytoid dendritic cells: one-trick ponies or workhorses of the immune system? plasmacytoid dendritic cells efficiently cross-prime naive t cells in vivo after tlr activation antigen crosspresentation by human plasmacytoid dendritic cells human plasmacytoid dendritic cells efficiently cross-present exogenous ags to cd + t-cells, despite lower ag uptake than myeloid dendritic cell subsets similar antigen cross-presentation capacity and phagocytic functions in all freshly isolated human lymphoid organ-resident dendritic cells plasmacytoid dendritic cells contribute to systemic but not local antiviral responses to hsv infections control of coronavirus infection through plasmacytoid dendritic-cell-derived type i interferon cutting edge: plasmacytoid dendritic cells induce il- production in t cells via the delta-like- /notch axis icos is an inducible t-cell co-stimulator structurally and functionally related to cd plasmacytoid dendritic cells prime il- -producing t regulatory cells by inducible costimulator ligand the cd + dendritic cell subset dngr- is a specific and universal marker of mouse and human batf -dependent dendritic cells in lymphoid and nonlymphoid tissues transcriptional control of dendritic cell development selective expression of the chemokine receptor xcr on cross-presenting dendritic cells determines cooperation with cd + t cells cytokines regulate the capacity of cd alpha(+) and cd alpha(-) dendritic cells to prime th /th cells in vivo cross-presenting dendritic cells are required for control of leishmania major infection peripheral cd + dendritic cells form a unified subset developmentally related to cd alpha+ conventional dendritic cells dendritic cells in intestinal homeostasis and disease differential antigen processing by dendritic cell subsets in vivo cd alpha+ and cd alpha− subclasses of dendritic cells direct the development of distinct t helper cells in vivo notch -dependent classical dendritic cells orchestrate intestinal immunity to attaching-and-effacing bacterial pathogens klf expression in conventional dendritic cells is required for t helper cell responses constitutive versus activation-dependent cross-presentation of immune complexes by cd (+) and cd (-) dendritic cells in vivo the ability of murine dendritic cell subsets to direct t helper cell differentiation is dependent on microbial signals the instructive role of dendritic cells on t-cell responses human blood contains two subsets of dendritic cells, one immunologically mature and the other immature the nature of the principal type interferon-producing cells in human blood plasmacytoid monocytes migrate to inflamed lymph nodes and produce large amounts of type i interferon plasmacytoid dendritic cells induce a distinct cytokine pattern in virus-specific cd + memory t cells that is modulated by cpg oligodeoxynucleotides characterization of resident and migratory dendritic cells in human lymph nodes the number and distribution of blood dendritic cells in the epidermis and dermis of healthy human subjects cd c+ and cd + dendritic cells in peripheral blood, lymph nodes and tumor tissue of patients with non-small cell lung cancer human tissues contain cd hi cross-presenting dendritic cells with functional homology to mouse cd + nonlymphoid dendritic cells human cd c+ dendritic cells secrete high levels of il- and potently prime cytotoxic t cell responses human dendritic cell subsets from spleen and blood are similar in phenotype and function but modified by donor health status bdca- )+ dendritic cells (dcs) represent a unique myeloid dc subset that cross-presents necrotic cell antigens mouse cd alpha+ dcs and human bdca + dcs are major producers of ifn-lambda in response to poly ic characterization of human dngr- + bdca + leukocytes as putative equivalents of mouse cd alpha+ dendritic cells found in translation: the human equivalent of mouse cd + dendritic cells the xc chemokine receptor is a conserved selective marker of mammalian cells homologous to mouse cd alpha+ dendritic cells superior antigen cross-presentation and xcr expression define human cd c+cd + cells as homologues of mouse cd + dendritic cells human intestinal lamina propria cd c+ dendritic cells display an activated phenotype at steady state and produce il- in response to tlr / stimulation does toll-like receptor play a biological role in virus infections? tlr deficiency in patients with herpes simplex encephalitis human dendritic cells -stars in the skin cd + cd + dermal dendritic cells cross-present keratinocyte-derived frontiers in immunology | www.frontiersin.org antigens irrespective of the presence of langerhans cells human dermal cd (+) cells are a transient population of monocyte-derived macrophages regulation of dendritic cell function in inflammation comparative transcriptional and functional profiling defines conserved programs of intestinal dc differentiation in humans and mice experimental and natural infections in myd -and irak- -deficient mice and humans recognition of nucleic acid and nucleic acid analogs by toll-like receptors , and pyogenic bacterial infections in humans with myd deficiency modular expression analysis reveals functional conservation between human langerhans cells and mouse cross-priming dendritic cells antigen delivery to early endosomes eliminates the superiority of human blood bdca + dendritic cells at cross presentation human cd c (bdca- )+ myeloid dendritic cells secrete il- and display an immuno-regulatory phenotype and function in response to escherichia coli selected toll-like receptor agonist combinations synergistically trigger a t helper type -polarizing program in dendritic cells human inflammatory dendritic cells induce th cell differentiation reciprocal control of t helper cell and dendritic cell differentiation plasmacytoid dendritic cells activated by influenza virus and cd l drive a potent th polarization human cd + dendritic cells induce cd + t cells to produce type cytokines biology of interleukin- inhibitory and stimulatory effects of il- on human cd + t cells an interleukin- -interleukin- -stat pathway is critical for functional maturation of memory cd + t cells interferon-lambda and therapy for chronic hepatitis c virus infection cd + dendritic cells produce prominent amounts of ifn-alpha after dsrna recognition and can be targeted via dec- in humanized mice genetic variation in il b predicts hepatitis c treatment-induced viral clearance il- enhances ifn-lambda (il- ) production by plasmacytoid dcs via monocyte secretion of il- ra human type myeloid dendritic cells produce interferon-lambda and amplify interferon-alpha in response to hepatitis c virus infection human blood dendritic cell antigen (bdca )(+) dendritic cells are a potent producer of interferon-lambda in response to hepatitis c virus communication between human dendritic cell subsets in tuberculosis: requirements for naive cd (+) t cell stimulation crosstalk between human dc subsets promotes antibacterial activity and cd + t-cell stimulation in response to bacille calmette-guerin differential response of bdca- + and bdca- + myeloid dendritic cells to respiratory syncytial virus infection paramyxovirus infection regulates t cell responses by bdca- + and bdca- + myeloid dendritic cells hcv glycoprotein e is a novel bdca- ligand and acts as an inhibitor of ifn production by plasmacytoid dendritic cells bdca- , a novel plasmacytoid dendritic cell-specific type ii c-type lectin, mediates antigen capture and is a potent inhibitor of interferon alpha/beta induction plasmacytoid dendritic cells in hiv infection duration of antiviral immunity after smallpox vaccination yellow fever vaccine protein vaccines induce uncommitted il- -secreting human and mouse cd t cells, whereas infections induce more ifn-gamma-secreting cells the form of ny-eso- antigen has an impact on the clinical efficacy of anti-tumor vaccination alum adjuvant boosts adaptive immunity by inducing uric acid and activating inflammatory dendritic cells gout-associated uric acid crystals activate the nalp inflammasome a potent adjuvant monophosphoryl lipid a triggers various immune responses, but not secretion of il- beta or activation of caspase- interleukin- has differential influence on the ability of adjuvant formulations to potentiate antibody responses to a plasmodium falciparum blood-stage vaccine neither interleukin- nor signalling via tumour necrosis factor receptor- contribute to the adjuvant activity of alum and freund's adjuvant transcriptional specialization of human dendritic cell subsets in response to microbial vaccines harnessing dendritic cells to generate cancer vaccines differential ability of surface and endosomal tlrs to induce cd t cell responses in vivo clinical evaluation of cpg oligonucleotides as adjuvants for vaccines targeting infectious diseases and cancer topical rather than intradermal application of the tlr ligand imiquimod leads to human dermal dendritic cell maturation and cd + t-cell cross-priming vaccine adjuvant systems containing monophosphoryl lipid a and qs induce strong and persistent humoral and t cell responses against hepatitis b surface antigen in healthy adult volunteers dna vaccines: a simple dna sensing matter? rna-based vaccines the messenger's great message for vaccination human peripheral blood mononuclear cells transfected with messenger rna stimulate antigen-specific cytotoxic t-lymphocytes in vitro toll-like receptor-dependent activation of several human blood cell types by protamine-condensed mrna spontaneous cellular uptake of exogenous messenger rna in vivo is nucleic acid-specific, saturable and ion dependent in vivo application of rna leads to induction of specific cytotoxic t lymphocytes and antibodies results of the first phase i/ii clinical vaccination trial with direct injection of mrna intradermal vaccinations with rna coding for taa generate cd + and cd + immune responses and induce clinical benefit in vaccinated patients intranodal vaccination with naked antigen-encoding rna elicits potent prophylactic and therapeutic antitumoral immunity key: cord- -bhxm zua authors: nayak, tapas kumar; mamidi, prabhudutta; sahoo, subhransu sekhar; kumar, p. sanjai; mahish, chandan; chatterjee, sanchari; subudhi, bharat bhusan; chattopadhyay, soma; chattopadhyay, subhasis title: p and jnk mitogen-activated protein kinases interact with chikungunya virus non-structural protein- and regulate tnf induction during viral infection in macrophages date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: bhxm zua chikungunya virus (chikv), a mosquito-borne alphavirus, is endemic in different parts of the globe. the host macrophages are identified as the major cellular reservoirs of chikv during infection and this virus triggers robust tnf production in the host macrophages, which might be a key mediator of virus induced inflammation. however, the molecular mechanism underneath tnf induction is not understood yet. accordingly, the raw . cells, a mouse macrophage cell line, were infected with chikv to address the above-mentioned question. it was observed that chikv induces both p and jnk phosphorylation in macrophages in a time-dependent manner and p-p inhibitor, sb is effective in reducing infection even at lower concentration as compared to the p-jnk inhibitor, sp . however, inhibition of p-p and p-jnk decreased chikv induced tnf production in the host macrophages. moreover, chikv induced macrophage derived tnf was found to facilitate tcr driven t cell activation. additionally, it was noticed that the expressions of key transcription factors involved mainly in antiviral responses (p-irf ) and tnf production (p-c-jun) were induced significantly in the chikv infected macrophages as compared to the corresponding mock cells. further, it was demonstrated that chikv mediated tnf production in the macrophages is dependent on p and jnk mapk pathways linking p-c-jun transcription factor. interestingly, it was found that chikv nsp interacts with both p-p and p-jnk mapks in the macrophages. this observation was supported by the in silico protein-protein docking analysis which illustrates the specific amino acids responsible for the nsp -mapks interactions. a strong polar interaction was predicted between thr- (within the phosphorylation lip) of p and gln- of nsp , whereas, no such polar interaction was predicted for the phosphorylation lip of jnk which indicates the differential roles of p-p and p-jnk during chikv infection in the host macrophages. in summary, for the first time it has been shown that chikv triggers robust tnf production in the host macrophages via both p-p and p-jnk/p-c-jun pathways and the interaction of viral protein, nsp with these mapks during infection. hence, this information might shed light in rationale-based drug designing strategies toward a possible control measure of chikv infection in future. chikungunya virus (chikv), a mosquito-borne alphavirus belongs to togaviridae family, is transmitted through either aedes aegypti or aedes albopictus mosquito. chikv mediated disease is one of the global challenges due to its endemics in different parts of the world ( countries), such as tanzania ( ) ( ) ( ) , reunion island ( - ), india ( ) ( ) ( ) ( ) ( ) , italy ( , ) , and thailand ( ) ( ) ( ) ( ) . among alphaviruses, chikv is considered as one of the most successfully evolved virus. the arboviruses including chikv have been evolving and re-emerging from centuries and their emergence and dispersion are more rapid and geographically extensive. this might be due to increase in global communication, mass immigration, vector adaptation to urbanization and land perturbation ( ) . even though mortality due to chikv is very rare and restricted to children's (below year), old age (above years) or immune compromised patients, the pathogenesis (mainly inflammatory responses) may persist for very long periods of time both in humans and macaque model ( , ) . currently, arboviruses raise a serious threat to the global public health, due to unavailability of effective drugs or vaccines ( , ) . recent studies on chikv induced immune responses suggest that the host immune system is found to be both beneficiary in one hand by controlling viral infection, whereas deleterious on the other hand by promoting severe inflammatory responses ( ) ( ) ( ) ( ) ( ) . studies have shown that chikv induces different inflammatory cytokines/chemokines (tnf, il- β, il- , ifnγ, il- , and mcp- ) ( , ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , which might be associated with arthritis like pathogenesis during chikv infection. in different in vivo systems (both mouse and non-human primates), predominant cellular infiltration of macrophages, monocytes, nk cells and t cells to the site of inoculation and other tissues have been observed ( , ) . moreover, immunohistochemistry and flow cytometry based analysis of muscles and synovial biopsies revealed that macrophages are major infiltrating cells among mps (mononuclear phagocytic system) ( , ) . blood monocytes and tissue macrophages are the major immune cells infected by chikv ( , , ) . in macaque, synovial macrophages have been identified as the major host cell for longterm viral persistence ( ) . this productive infection of chikv in the host macrophages might be associated with arthritis like pathogenesis despite robust immune activation ( , ) . t cell immune responses specific to chikv is not clearly understood yet. teo th et al. have suggested that cd + t cells (but not cd + t cells) are essential for the development of chikv induced pathogenesis without affecting virus infection and dissemination in mice and this is independent of ifn-γ ( ) . flow cytometry based analysis of circulating lymphocytes in chikv patients confirms that there are both cd + and cd + t cell responses during early and late phases of infection, respectively. moreover, cd mediated apoptosis was also detected in cd + t cells after days of symptom appearance ( ) , which might be one of the strategies to evade host immunity. purified t cells (both cd + and cd + ) from the chronic and recovered patients from to la reunion islands showed immune activation when challenged with synthetic chikv peptides and inactivated virus particles ( ) . the dna vaccine based on the consensus sequences of e / and capsid protein (with several modifications) of chikv resulted in robust ifn-γ and igg production suggesting that chikv induces both t and b cell specific responses ( , ) . there are three major studied ser/thr kinases under the mitogen-activated protein kinase (mapk) family, such as p , jnk, and erk, which are known to regulate multiple cellular pathways such as cell proliferation, activation, inflammation, cytokine and chemokine productions and different pathological conditions ( ) ( ) ( ) ( ) ( ) ( ) ( ) . in addition, activation of mapks by different pathogens and other inflammatory diseases have been reported to induce pro-inflammatory cytokines such as tnf in the host cells ( - , , ) . the mapks have been shown to be activated by phosphorylation in specific positions (ser/tyr/thr) by several viral infections, such as coronavirus type , hepatitis c virus, rhinovirus and epstein-barr virus ( ) ( ) ( ) ( ) ( ) . chikv is also known to induce mapks during infection in various nonimmune cells and treatment of an alkaloid berberine, reduces viral infection and joint swelling in mice ( , ) . we have shown earlier that chikv triggers robust tnf production in the host macrophages, which might be a key mediator of virus induced inflammation ( ) and macrophages are identified as the major cellular reservoirs during the late stages of chikv infection in macaques ( ) . however, the precise role of mapk activation pathways in terms of chikv infection and associated robust tnf induction in macrophages (immune cell) remains largely unknown. hence, an attempt was made to understand the involvement of mapks in chikv infection and tnf induction in the host macrophages. the mouse anti-chikv-nsp antibody used in the current study was developed by us ( ) . anti-mouse cd antibody, anti-tnf antibody, anti-cd fitc, hrp linked anti-mouse, and hrp linked anti-rabbit secondary antibodies were purchased from bd biosciences (ca, usa). anti-mouse cd and cd . apc were procured from tonbo biosciences (ca, usa). the monoclonal antibodies for p , p-p , jnk, p-jnk, erk / , p-erk / , p-irf , and p-c-jun were purchased from cell signaling technology (ma, usa). the anti-mouse alexa fluor and anti-rabbit alexa fluor were purchased from invitrogen (ca, usa). the rabbit polyclonal antibodies against p-p and p-jnk used for immunoprecipitation were purchased from santa cruz biotechnology (tx, usa). mouse igg, rabbit igg isotype control, and anti-gapdh antibody were purchased from abgenex india pvt. ltd (od, india). saponin and bovine serum albumin fraction v were purchased from sigma-aldrich (mo, usa). sb (p-p inhibitor, sb), and sp (p-jnk inhibitor, sp) were purchased from merck millipore (ma, usa). mtt assay was performed to assess the cytotoxicity of sb and sp according to the methods described before ( ) . briefly, the raw . cells were seeded in well plates at a density of × cells per well before - h of drug treatment. then, the cells were washed in x pbs and incubated with different concentrations of drugs in triplicate. as both sb and sp were dissolved in the dimethyl sulfoxide (dmso), it was taken as solvent control. after h, the cells were incubated with the mtt reagent to a final concentration of % (v/v) in rpmi media. then, the cells were placed in the incubator for upto h for the formation of visible crystals. later, the media (containing mtt) were removed without disturbing the cells and µl of solubilization solution was added per well followed by incubation for min at room temperature (rt). the percent viable cells were calculated after taking the absorbance of the solution at nm by microplate reader (bio-rad, ca, usa). raw . cell line has been well-reported to study chikv infection, replication and associated altered host immune responses ( , ) . the raw . cells were seeded in six-well cell culture plates before - h of infection with around % confluency. the cells were infected with the drde- strain of chikv with multiplicity of infection (moi) as reported previously ( ) . briefly, after washing the cells in x pbs, the virus was added over confluent monolayer for h in the incubator with manual shaking at an interval of min. then, the virus inoculum was washed in x pbs to remove unbound viruses and the cells were maintained in the complete rpmi- media. the infected cells and the supernatants were collected at different time points and subjected to further processing according to the assay. sb and sp treatments were given as described before ( ) . briefly, cells were pretreated with the desired concentrations of sb, sp or dmso for h in serum free media (sfm). then the infection was carried out in the presence of either solvent control (dmso), sb or sp. the cells were washed thoroughly with x pbs after h and cultured in sfm containing the drug for h. then, serum was added to the cells and maintained in the incubator until harvesting ( ) . viral plaque assay was performed to determine the titer of chikv as described previously ( ) . in brief, after infecting the vero cells with different dilutions of cell culture supernatants (collected from chikv infected raw cells), the cells were overlaid with complete dmem containing methyl cellulose and maintained in the incubator. after the development of the visible plaques (usually - days), the cells were fixed in formaldehyde at room temperature, washed gently in tap water and stained with crystal violet. then, the numbers of plaques were counted manually under white light. flow cytometric assay was carried out as reported previously ( ) . briefly, both mock and chikv infected raw . cells were harvested and fixed in % paraformaldehyde for min at rt. then, the cells were re-suspended in facs buffer and stored at • c until staining. for intracellular staining (ics), the cells were permeabilized in freshly prepared x permeabilization buffer followed by blocking buffer ( % bsa in permeabilization buffer) for min at rt. then, the cells were incubated with different primary antibodies for min at rt, followed by washing with x permeabilization buffer twice. after that, the cells were incubated in alexa fluor r and alexa fluor r conjugated secondary antibodies followed by washing with x permeabilization buffer. the mouse igg and rabbit igg were taken as isotype control during ics. the fcr blocking reagent (miltenyi biotec, bergisch gladbach, germany) was used prior to the primary antibody incubation to prevent non-specific binding of antibodies to the fc receptors on macrophages. then, the cells were acquired by the bd facs calibur tm flow cytometer (bd biosciences, ca, usa) and analyzed by the cellquest pro software (bd biosciences, ca, usa). a total of approximately × cells were acquired per sample. sandwich elisa for cytokine analysis tnf production from the macrophage cell culture supernatants was quantified by the bd opteia tm sandwich elisa kit (bd biosciences, ca, usa) according to the manufacturer's instructions ( ) . the cytokine concentrations in the test samples were calculated in comparison with the corresponding standard curve prepared by using different concentrations of the recombinant tnf in pg/ml. western blot analysis was performed to assess the levels of different protein expressions according to the protocol mentioned before ( ) . in brief, both the mock and chikv infected cells were washed with ice-cold x pbs and the whole cell lysate (wcl) was prepared by radio immuno precipitation assay (ripa) lysis buffer. the protein concentration was quantified by the bradford reagent (sigma-aldrich, mo, usa). equal amount of protein was loaded in the % sds-page after mixing with x laemmli buffer ( : ) and blotted on to a pvdf membrane (millipore, ma, usa). then the transferred membranes were blocked with % bsa followed by overnight incubation with different primary antibodies. then, the membranes were thoroughly washed with tbst and incubated with the hrp conjugated secondary antibodies for h at rt. after washing with tbst, the blots were subjected to chemiluminescence detection by the bio-rad gel doc with the quantity one software (bio-rad, ca, usa). for band intensity quantification, western blot images were subjected to further analysis by the quantity one -d analysis software while normalizing to the corresponding gapdh loading control. raw cells were infected with chikv as described above and harvested at hpi. the cells were lysed with np- (nonidet p- ) lysis buffer ( mm nacl, mm edta, % glycerol, % np- , mm tris, ph . , supplemented with protease inhibitor and phosphatase inhibitor cocktail). the resultant whole cell lysates were subjected to immunoprecipitation by immunoprecipitation kit dynabeads r protein a (thermo fisher scientific, ma, usa) according to the manufacturer's instructions. briefly, both the mock and chikv infected whole cell lysates were incubated with primary antibodies overnight on the vertical rotor at • c. then, µl of dynabeads r protein a was added to the cell lysate and incubated for another h on the vertical rotor at • c. the dynabeads r -ab-ag complexes were washed three times in lysis buffer followed by elution with elution buffer supplied in the kit. then, the eluted complexes were resuspended in x laemmli buffer, boiled at • c for min and processed further for western blot analysis as described above. the protein-protein docking was performed using the cluspro . webserver ( , ) . this server performs three computational steps. in the first step, it does rigid-body docking using the piper. this docking program is based on the fast fourier transform (fft) correlation approach and uses pairwise interaction potential as part of its scoring function e = w e rep + w e attr + w e elec + w edars. while e rep and e attr represent the repulsive and attractive contributions, e elec denotes electrostatic energy term and edars refers to the pairwise structure-based potential ( , ) . in the second step, , lowest energy docked structures are clustered using pairwise interface rmsd (irmsd) ( , ) . based on the irmsd values the structure with the highest neighbors within a Å radius is defined as the center of the first cluster. further clustering is performed within the remaining structures to generate clusters. the energy minimization is done for the structures using the van der waals terms of the charmm potential in the third step ( , ) , following which the structures at the center of the most populated clusters are taken as the output. since there was no satisfactory template available in pdb to build the homologous model of nsp , the structure was generated earlier using the i-tasser algorithm ( ) . this was used as a ligand in the study. xray crystallographic structures of jnk (pdb id: elj) and p (pdb id: a u) were taken as receptors for the protein-protein docking. these structures were recovered from the protein data bank. the co-crystallized ligands were extracted and energy was minimized before submission of chain a of these structures as receptors. the output of docking generated four types of models using the scoring algorithms designated as balanced, electrostatic-favored, hydrophobic-favored, and van der waals+ electrostatic. amongst these, the balanced outputs were analyzed. the docking solution with largest members was taken for further visualization using the pymol software. mouse splenocytes isolation and splenic t cell purification from balb/c mice were performed as reported earlier ( ) . in brief, using a µm cell strainer the splenocytes were collected from mice spleens. after rbc lysis and washing with x pbs, cells were suspended in rpmi- media supplemented with % fbs. according to instructions given by the manufacturer's protocol, mouse splenic t cell purification was carried out using dynabeads untouched mouse t cells kit (invitrogen, ca, usa). tcr driven t cell activation was carried out with those purified t cells (cd . + ) in the presence of either chikv infected or uninfected (mock) macrophage culture supernatants ( . µm membrane filtered) to study the status of cd (a t cell activation marker) as described earlier for other infection model ( ) . for tnf neutralization, anti-tnf purified antibody (bd bioscience) was incubated for min with the chikv infected supernatant prior tcr stimulation. statistical analysis was performed by using the graphpad prism . software (graphpad software inc. usa). data were represented as mean ± sem. the comparison between the groups was performed by either one-or two-way anova with tukey or bonferroni post-hoc test, respectively. data presented here were representative of at least three independent experiments. p < . was considered as statistically significant difference between the groups. to determine whether any mapk (p , jnk, and erk) is activated during chikv infection in macrophages, raw cells were infected with the virus at moi and harvested at different time points ( - hpi). both the cells and cell culture supernatants were subjected to various downstream assays. as shown in figure a , the p-p and p-jnk expressions were increased significantly as compared to the corresponding mock cells. the p-p mapk expression was found to be increased around . -fold as early as and hpi, followed by approximately -fold increments toward hpi as compared to the corresponding mock cells. similarly, the expression of the p-jnk was found to be increased rapidly around -fold during early hours ( and hpi), whereas, it increased up to -fold with respect to the mock in later time points. the total p and jnk (t-p and t-jnk) expressions remain unaffected in both the groups. moreover, p-erk / and t-erk / (total-erk / ) expressions remain unchanged throughout all the time points as compared to the corresponding mock (figures a,b) . this data suggests that chikv induces activation of both p and jnk by phosphorylation in a time-dependent manner in macrophages. since chikv induces both p and jnk activation in the host macrophages, next we sought to assess whether these two mapks are crucial for the viral infection and replication in the macrophages. for that, pharmaceutical inhibitors of p (sb ) and jnk (sp ) were used. first, different concentrations of both sb ( . , . and . µm) and sp ( , and µm) were assessed for cytotoxicity in raw cells by mtt assay. it was observed that around % cells were viable in all the concentrations of sb, whereas up to and % cells were found to be viable at and µm concentrations of sp, respectively (figures a,e) . thus, both and µm concentrations of sp were selected for further experiments. as sb treatment with > µm concentration was known to inhibit phosphorylation and activation of pkb non-specifically ( ), both . and . µm concentrations of sb was used in the current study. raw cells were inoculated with chikv in the presence of sb, sp, or solvent control dmso as described above. at hpi both mock and chikv infected cells were harvested and the expressions of nsp , p-p , and p-jnk were assessed by flow cytometry. it was observed that the percent positive cells for nsp were reduced from . ± . (chikv+dmso) to . ± . (chikv+sb . µm) and . ± . (chikv+sb . µm), whereas the percent positive cells for p-p were reduced from . ± . (chikv+dmso) to . ± . (chikv+sb . µm) and . ± . (chikv+sb . µm) (figures b,c) . likewise, the mfi for both the p-p and nsp were reduced at hpi in the sb treated cells as compared to the dmso control ( figure d) . the inhibition of p-jnk by sp at . µm concentration did not affect nsp expression in the macrophages as compared to the dmso control (chikv+dmso; . ± . , chikv+sp µm; . ± . , p > . ), despite significant reduction in the p-jnk percent positive cells (chikv+dmso; . ± . , chikv+sp; . ± . , p < . ). however, sp at the comparatively higher concentration ( µm) did reduces nsp expression by around . -fold (figures f-h) . further, plaque assay of the cell culture supernatants revealed that sb treatment reduces the number of new viral progeny release around . -and . -fold at . and . µm, respectively. whereas, sp at µm concentration treatment reduces the number of new viral progeny release around . -fold as compared to the corresponding dmso control (figure i) . this result indicates that the activation of both p and jnk mapks might be crucial for the chikv infection and replication in the host macrophages with sb being more effective comparatively in controlling infection than sp. pharmaceutical inhibitors specific to p-p and p-jnk reduces chikv induced tnf production in the host macrophages activation of mapks by different pathogens has been shown to induce pro-inflammatory cytokines such as tnf in the host cells ( , ) . since, chikv triggers robust tnf production (a key mediator of inflammation) in the host macrophages ( , ) , it was interesting to investigate whether any mapks are involved in this pathway. accordingly, macrophages were treated with either sb or sp and infected with chikv as mentioned earlier. the cell culture supernatants were subjected to sandwich elisa for the detection of tnf at early ( hpi) and late ( hpi) time postinfection. it was observed that both sb and sp could suppress chikv induced tnf significantly at both the time points as compared to the corresponding dmso control. at hpi the tnf level for chikv+dmso was found to be ± pg/ml (mean ± sem), which was reduced to ± pg/ml (mean ± sem, p < . ) and ± pg/ml (mean ± sem, p < . ) in the presence of sb ( . µm) and sp ( . µm), respectively. similarly, at hpi, the tnf production was , ± pg/ml (mean ± sem) in the chikv+dmso sample, whereas it was reduced to ± pg/ml (mean ± sem, p < . ) for sb and ± pg/ml (mean ± sem, p < . ) for sp treatment (figure ) . taken together, this result suggests that chikv might induce tnf via p as well as jnk mediated pathways in the host macrophages. tnf, one of the potent inflammatory cytokine, which can enhance tcr-dependent t cell activation ( ) . we and others have previously reported that in vitro chikv infection in raw . cells leads to tnf production ( , ) . recent studies have shown a pathogenic role of t cells during chikv infection associated to host inflammatory responses ( , , ) . here we have investigated whether chikv infection induced macrophage derived tnf can facilitate mouse t cell activation associated with cell mediated immunity. for this, chikv infected culture supernatant of raw . cells were tested toward tcr driven resting t cell activation assay ( ) . we have found that chikv infected macrophage culture supernatant along with tcr activation facilitated the induction of cd level (around %) as compared to uninfected culture supernatant (around %). interestingly, when the chikv infected macrophage culture supernatant was treated with tnf neutralizing antibody, a sharp decrease of cd frequency (around %) was observed. beside this, sb and sp treated chikv infected raw . culture supernatant along with tcr stimulation also showed downregulation of cd frequency in t cells (figures a,b) . so, the above observations may underscore that the tnf present in chikv infected culture supernatant might be able to facilitate the induction of t cell activation. often, the viral infection is associated with the activation and localization of several transcription factors (e.g., irfs, c-jun, p ), which in turn regulates host responses to viruses ( ) ( ) ( ) ( ) ( ) ( ) . here, the expressions of key transcription factors involved mainly in antiviral responses (p-irf ) and tnf production (pc-jun) were assessed at different hpi by western blot analysis. it was observed that both p-irf and p-c-jun were induced significantly in the chikv infected macrophages as compared to the corresponding mock (figures a,b) . this data suggest that chikv infection in the raw cell line might be associated with the elevation of key antiviral and inflammatory transcription factors. it has been reported previously that tnf is one of the key mediators for arthritis or arthritis-like diseases in humans by promoting severe inflammation. although, several other inflammatory cytokines are elevated in ra (rheumatoid arthritis), anti-tnf therapy seems to be promising for the effective treatment against it ( ) . since chikv induces tnf via p /jnk map kinase pathways and phosphorylation of cjun is reported to be associated with tnf production in other inflammatory model system ( , ) , phosphorylation of c-jun in both mock and chikv infected macrophages was assessed by western blot analysis. surprisingly, the expression of p-cjun was reduced around . -and . -fold in the presence of sp at and µm indicating a plausible role of jnk toward cjun phosphorylation, whereas sb treatment at . µm did not affect p-c-jun expression significantly. however, both sb and sp treatment suppressed p-irf- expression which is induced by chikv as compared to the dmso control (figures a-d) . taken together, the current data depict that chikv may induce p-c-jun via jnk pathway whereas induction of p-irf- might be dependent on both p and jnk mapks. viruses are small obligatory intracellular pathogens utilizes the metabolic pathways of the host for replication. very often viruses also shut-off host translational process, which might be a strategic decision to contain antiviral responses ( , ) . the integration of complex proteomics studies including in silico protein-protein interaction predictions keeps on unraveling the complex network of interaction with the host cell proteins. throughout the course of replication, these pathways rely heavily on the dynamic and temporarily regulated virus-host protein-protein interactions which are crucial for the virus replication, pathogenesis, and viral subversion of host defense. the identification and characterization of these interacting partners also help in the delineation of the viral protein functions precisely and might be very helpful in designing rationale drugs for an effective treatment ( ) ( ) ( ) . the interaction of host mapk with viral protein has been shown earlier, which in turn regulates infection and replications ( ) . since chikv infection modulated the phosphorylation of host p and jnk, their interaction with the nsp protein was investigated. for that, raw cells were infected with chikv and harvested at hpi for further analysis. co-immunoprecipitation followed by western blot analysis showed that both the p-p and p-jnk proteins were pulled with the chikv-nsp protein in the host macrophages (figures a,b) . this result indicates that chikv-nsp interacts with both p-p and p-jnk and this might be playing a crucial role in the chikv infection and tnf mediated inflammatory responses. in order to unravel the amino acid residues responsible for the interaction of nsp -mapks, protein-protein docking was carried out as mentioned above ( , , ) . the balanced outputs were preferred from the docking results as this mode takes into account all possible modes of interactions. the most stable complex of nsp -jnk on visualization by using the pymol software suggested the possible involvement of different residues in the interaction (supplementary table s ). no interaction was found between the phosphorylation lip (thr- -x-tyr- ) of jnk and nsp (figure a ). this suggests a poor fit of jnk active site with nsp . nonetheless, ten polar interactions were observed within å (figure b) . some of these include the interactions of met- , arg- , arg- , val- , arg- , lys- , and glu- of jnk with cys- , arg- , gln- , gly- , asp- , gly- , and lys- of nsp , respectively ( figure b) . the most stable complex of nsp -p showed a close fit of the phosphorylation lip (thr- -x-tyr- ). in addition to that, a polar interaction was suggested between thr- of p and gln- of nsp ( figure c) . some of the polar interactions were also observed between lys- , ser- , asn- , ser- , ser- , asp- , glu- , arg- , lys- , and asp- of p and asn- , gly- , asp- , cys- , asp- , cys- , arg- , phe- , arg- , and thr- of nsp , respectively ( figure d and supplementary table s ) . thus, these results further suggest that chikv-nsp interacts with p as well as jnk mapks during viral infection in the host macrophages. moreover, the phosphorylation lip of p interacts more closely with the gln- of chikv-nsp , which supports the findings of ip experiments. the recent epidemics of chikungunya virus (chikv) with unprecedented magnitude and unusual clinical severity have raised a great public health concern worldwide, due to the absence of a vaccine or specific anti-chikv therapy. tnf is one of the robustly induced cytokine by chikv and in the current study, we have investigated the molecular mechanism involved in the induction of tnf in the host macrophages. our data suggested that chikv induces both p and jnk phosphorylation in macrophages in a time-dependent manner. moreover, p-p and p-jnk inhibition by sb and sp were found to reduce chikv infection. interestingly, sb mediated inhibition of chikv infection was found to be more effective even at lower concentration as compared to sp. further, inhibition of both p-p and p-jnk reduced chikv induced tnf in the host macrophages. moreover, chikv infected cell culture supernatant is found to facilitates t cell activation via tnf in tcr primed t cells. besides, it was observed that the expressions of key transcription factors involved mainly in antiviral responses (p-irf ) and tnf production (p-c-jun) were induced significantly in the chikv infected macrophages as compared to the corresponding mock cells. further, it was found that chikv mediated tnf production in the macrophages is dependent on p and jnk mapk pathways linking p-cjun transcription factor. interestingly, it was also noticed that chikv nsp interacts with host p-p and p-jnk mapks in the macrophages. this observation was supported by the in silico protein-protein docking analysis which illustrates the specific amino acids responsible for the nsp -mapks interactions and a strong polar interaction was predicted between thr- (within the phosphorylation lip) of p and gln- of nsp . however, no such polar interaction was predicted for the phosphorylation lip of jnk which indicates the differential roles of p-p and p-jnk during chikv infection in the host macrophages. the mapks have been shown to be activated by several viral infections ( ) ( ) ( ) ( ) . using the mouse macrophage cell line, raw . cells, we report for the first time that chikv induces both p-p and p-jnk significantly, however, the p-erk / expression remains unchanged. interestingly, the up-regulation of p-erk has been reported earlier during chikv infection in non-immune bhk cell lines ( ) . another report suggested that, the nuclear localization of erk / (un-phosphorylated form) in the uninfected microglia cells increases after chikv infection in astrocytes and this might be due to the release of some factor(s) from infected astrocytes in vitro ( ) . in this study, it was found that inhibition of p signaling by sb reduces nsp protein expression and new viral progeny release remarkably, whereas inhibition of jnk signaling by higher concentration of sp could reduce nsp moderately as compared to dmso control. this result indicates that both p and jnk play pro-viral role in chikv infection in the host macrophages and similar observations have been reported previously in case of other viral infections ( , ( ) ( ) ( ) . the encephalomyocarditis virus infection was suppressed in l cells by sb, mainly through the reduction of the viral protein synthesis ( ) . whereas, in the human enterovirus infection it was shown that the blockage of virus induced p-p leads to significant reduction in both viral protein and progeny release ( ) . further investigation can be carried out on other chikv proteins and rna synthesis to understand the pro-viral role of the p-p in viral replication in details. mapks are known to regulate tnf production via p-c-jun in other inflammation models ( ) . here, it was observed that the expression of p-c-jun is dependent on p-jnk pathway (as sp reduces p-c-jun expression in a dose dependent manner), whereas induction of p-irf is dependent on both mapks (p and jnk) during chikv infection in macrophages. therefore, it is quite possible that the p-jnk pathway induction by chikv leads to the activation of antiviral responses via p-irf and proinflammatory responses (tnf) via p-c-jun pathway. on the other hand, p-p is involved in activating both pro-viral and antiviral pathways (via induction of p-irf ) (figure ). it has also been observed during this investigation that pro-inflammatory tnf production was decreased significantly during sb treatment. this might be due to the marked inhibition of chikv infection, however the possibilities of the involvement of other factors cannot be ignored. tnf may promote the activation and proliferation of t cells and thereby regulate the overall t cell mediated effector function ( ) . in mouse model system, it has been demonstrated that host t cells are induced during experimental chikv infection and are associated with chikv mediated pathogenesis ( , , ) . in the present study, we found that chikv infected macrophage culture supernatant may facilitate tcr driven activation of resting t cells as compared to the mock supernatant. further, the use of neutralizing anti-tnf antibody towards the regulation of the t cell activation suggests that it could be mediated via chikv induced macrophage derived tnf. additionally, presence of either sb or sp in the chikv infected macrophage supernatant also able to reduce t cell activation in vitro, indicating an effect of macrophage derived tnf on t cell activation during chikv infection. except few cases, chikv is not fatal, however, the long-term polyarthralgia, arthritis-like symptoms along with severe inflammation remain a concern for most of the chronic patients ( , , ( ) ( ) ( ) ( ) . tnf is one of the key mediator of arthritis or arthritis-like diseases in humans by triggering severe inflammation. despite the elevation of several other inflammatory cytokines in ra, anti-tnf therapy holds a promise for the effective treatment against it ( , ) , which might be exploited against chikv pathogenesis in future. further, the co-immunoprecipitation analysis revealed that chikv-nsp interacts with both p-p and p-jnk upon infection in the host macrophages. this was also supported by the in silico analysis of the protein-protein interaction of chikv-nsp with p and jnk. the phosphorylation lip of p was found to interact with nsp due to the observed close fit model and a polar interaction between thr- of p and gln- of nsp . residues from the n-terminus of nsp were also suggested to have strong (< Å) polar interactions with the other residues around this active site. unlike this, the interaction of chikv-nsp showed poor fit with the phosphorylation lip of jnk and close (< Å) polar interaction was also observed for residues from n-terminus of nsp . these interactions might be one of the yet unknown strategies to utilize host signaling pathways through protein-protein interactions for effective viral infection ( ) ( ) ( ) , which can be explored further in details. viral proteins are found to be phosphorylated by various kinases, which in turn regulate its functions, stability and interactions with other cellular and viral proteins ( ) . however, the precise role of the viral protein phosphorylation (especially in alphavirus) has not been reported yet. in this investigation, nsp was found to interact with the phosphorylation lip of p , hence, in silico analysis was carried out using ptm prediction tools, gps (group-based phosphorylation scoring method) ( , ) and netphos . , to predict the target phosphorylation sites of nsp in a kinase specific manner ( ) . the gps is a groupbased phosphorylation algorithm, which predicts kinase-specific phosphorylation sites among different host protein kinase groups according to specific sequence pattern ( , ) . whereas, the netphos server is based on an artificial neuronal network (ann) that allows the users to choose between generic predictions based on the given protein sequence or kinase-specific predictions ( , ) . out of several predictions, both the softwares predicted t , s , and s sites in chikv-nsp with a high probability of phosphorylation by p (supplementary table s ). further, to elucidate whether positions of these amino acids in chikv-nsp is associated with any consensus regions of functional importance, the predicted peptides were searched in the expasy-prosite protein database. surprisingly, the peptide "fkedkayspevalne" with s (at the middle, red) showed a hit with alphavirus nsp protease domain belonging to the c cysteine protease family ( ) . since we have shown earlier that p interacts strongly with chikv nsp (with phosphorylation lip) and the inhibition of p activation strongly reduces chikv infection, it might be possible that, p phosphorylates either nsp directly or through the association of other client protein(s) which in turn may modulate its function. however, further studies are required to corroborate the chikv-nsp phosphorylation by host kinases and its functional consequences on infection and pathogenesis. in summary, for the first 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hellas; chieppa, marcello title: coronavirus disease (covid- –sars-cov- ) and nutrition: is infection in italy suggesting a connection? date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: b vmnwv novel coronavirus disease (covid- ) was declared a global pandemic on march , . the outbreak first occurred in wuhan, hubei, china, in december and hit italy heavily in february . several countries are adopting complete or partial lockdown to contain the growth of covid- infection. these measures may affect people's mental health and well-being but are necessary to avoid spreading the pandemic. there has been a gradual increase in studies exploring prevention and control measures, and we recommend paying close attention to nutrition, which may contribute to modulating some important consequences of covid- infection, as such pro-inflammatory cytokine storm. novel coronavirus disease spread to all regions of italy on january , . the northern region of lombardy was identified as the center of the two main italian clusters of cases. on march , , the director-general of the who declared covid- a global pandemic as the virus spread rapidly from china to the rest of the world, particularly europe ( ) . currently, the scientific community is sharing potentially useful data to treat patients and protect the population, but the details of covid- infection are still largely unknown, and thus options for risk assessment and pharmacological intervention have only been partially developed. by march , more than , confirmed cases of covid- had been reported, of which , were registered in italy, so far the country with the highest mortality rate ( . ) ( ) . four regions in italy have reported , cases of covid- , including , confirmed infections solely in italy's epicentral lombardy region, corresponding to more than a third of the total number of infected people in the entire country (ministry of health). italy's mortality rate may be partially explained by the country's relatively higher proportion of older people. similarly to what has been observed in china, the most common symptoms were fever, cough, and fatigue ( ) . acute respiratory distress syndrome (ards) was the main cause of death ( ) . older adults and people with chronic illness are most vulnerable to the worst effects of the disease. knowledge about novel covid- is based on a few months of observation and some similarities to severe acute respiratory syndrome (sars) and to middle east respiratory syndrome coronavirus (mers-cov). despite the lower case fatality rate than mers and sars, covid- has so far proven extremely contagious. moreover, a significant percentage of those who are infected require hospitalization in an intensive care unit. here we speculate on a possible link between nutritional status and covid- mortality, based on data emerging from the italian national health system. furthermore, while waiting for clinical trials to shed light on the clinical efficacy and beneficial effects of antibodies and anti-inflammatory cytokines, we highlight nutritionally derived products that may inhibit the inflammatory cytokine secretion caused by covid- infection. subjects with diabetes are at risk of infections and have severe disease when infected with a respiratory virus, showing an increased risk of mortality ( ) . data from mers-cov, which broke out in in saudi arabia, showed that the severity and length of the pulmonary pathology observed were increased in those affected by type diabetes, which likely deregulates immune response ( ) . data on covid- in patients with diabetes is limited at present ( ). the chinese centre for disease control and prevention published a report of , cases of covid- , showing an increased mortality rate in subjects with diabetes ( ) . diabetes is the most common comorbidity observed in covid- -positive deceased patients in italy after hypertension ( ) . available data on pre-existing comorbidities updated on april , , and extracted from clinical charts showed hypertension and diabetes before hospitalization in . % (n = , ) and . % (n = ) of covid- -positive deceased patients, respectively ( ). the same data showed that among , covid- -positive deceased patients, . % (n = ) presented three or more comorbidities that had been diagnosed before covid- infection, . % (n = ) had two, . % (n = ) had one, and only . % (n = ) had no pre-existing pathology ( ). diabetes is prevalent in our population, and most patients with diabetes type ii are overweight or affected by obesity ( ) . however, obesity is not included within the who "fiveby-five" framework of non-communicable diseases (ncds) and risk factors, and data on bmi are not collected in a standardized manner . unfortunately, we do not yet have weight, height, and waist circumference data for all patients with laboratory-confirmed covid- , and, therefore, we cannot disentangle the effects of adiposity on lung function and immune response to viral infection. excess body weight and increased visceral adiposity are habitually associated with metabolic alterations such as insulin dysregulation, high fasting glucose levels, hyperlipidemia, or systemic hypertension, which cause dysregulation of the immune system through mediation in various immune, metabolic, and thrombogenic responses. however, the clinical impact of this immune dysregulation on susceptibility to and severity and outcome of viral infections and on lung function is not yet clearly understood ( , ) . nevertheless, preliminary data from giviti (https://giviti. marionegri.it/covid- /) presented on march , , showed a high prevalence of obesity ( %) and overweight ( %) in italian patients, median age years, from different italian icus, confirming evidence available so far in the literature. recent data on patients with laboratory-confirmed covid- treated at an academic health institution in new york city, the epicenter of the covid- outbreak in the united states, between march , , and april , , with follow up through april , ( , ) showed that obesity, after age, was linked to more severe coronavirus cases, with a substantially higher odds ratio than any cardiovascular or pulmonary disease. obese and obese-diabetic subjects undergo modifications of the innate and adaptive immune response at different phases, characterized by a state of chronic, and low-grade inflammation and a high basal concentration of several pro-inflammatory cytokines such as alpha-tnf, mcp- , and il- , leading to a defect in innate immunity ( ). recent evidence indicates that obesity not only increases the risk of infection and of complications for the individual but also increases the chance of appearance of a more virulent viral strain, prolonging virus shedding, and eventually increasing the overall mortality rate of an influenza pandemic ( ) . hence, both diabetes and obesity impair the immune response to viral infections like influenza and influenza vaccination through alterations of the cellular immune system ( ) . studies so far suggest that diabetics, as well as subjects with obesity, are at a greater risk of hospitalization and increased complications from influenza ( , ) . compared with vaccinated healthy-weight adults, vaccinated obese adults have twice the risk of influenza or influenza-like illness despite equal serological response to vaccination ( ) . this should be considered one of the challenges to be overcome in vaccine development and/or medications to combat this virulent respiratory virus and prevent future epidemics similar to covid- . obesity is also associated with chronic low-grade inflammation, dysbiosis, and increased secretion of inflammatory cytokines, including interleukin (il- ) ( ) . elevated plasma levels of pro-inflammatory cytokines are observed in covid- infected patients; in particular, il- and ferritin release have been identified as predictors of fatality ( ) . several studies focusing on previous outbreaks of severe influenza confirmed that the mortality caused by organ injury could be reduced by immunomodulatory agents ( , ) . currently, a study on the safety and efficacy of tocilizumab is being conducted (clinicaltrials.gov identifier: nct ) ( ) to assess its capacity to suppress the virally driven hyperinflammation and acute respiratory syndrome caused by covid- . as mentioned previously, covid- -related deaths are due to acute respiratory distress syndrome (ards); nonetheless, data indicate that the covid- virus is detectable in stool of infected patients, suggesting systemic manifestations ( ) . in line with this observation, reports indicate abundant expression of ace in absorptive enterocytes of the gi tract ( ) and diarrhea as among the most frequent infection symptoms ( ) . inflammation of intestinal mucosa may result in increased intestinal permeability with a consequent cascade of events that cause persistent inflammation, worsening the infectionrelated symptoms. immune homeostasis is a dynamic process maintained by a complex interplay between the gut microbiota and host mucosal immune system ( ) . dysbiosis, defined as imbalances in gut microbial species, is now a wellrecognized factor in the pathogenesis of age-associated frailty ( ) . it is possible that covid- mortality is increased in older patients with comorbidities associated with intestinal dysbiosis, as this could support systemic chronic inflammation in the host. nonetheless, only future studies will clarify this aspect. ace -expressing epithelial cells are the primary targets of covid- . similarly to other pulmonary viral infections, following primary exposure, the progeny proliferate in the host cells, which consequently die and release their contents. covid- is now able to infect other cells, including alveolar macrophages ( ) . activated or infected immune cells secrete excessive pro-inflammatory cytokines and chemokines, fuelling a vicious circle leading to pulmonary tissue damage. data accumulating from covid- patients indicate that these patients might have a cytokine storm syndrome, with markedly higher levels of ifn-γ, ccl- , ccl- , tnf, and the aforementioned il- ( - ). for optimal functioning of the immune system, an adequate nutritional status is required. this is well-acknowledged from evidence linking nutritional deficiencies to the functionality of the immune system ( ) . poor nutrition leads to poor immune defense, and it is frequently associated with figure | deleterious effects of the anti-covid- strategy (red) can impact the base of the mediterranean diet pyramid. an increase in polyphenol uptake and substitution of meat with legume-derived proteins can help prevent chronic inflammation, and reduction of caloric intake may compensate for the reduction in physical activity. alcohol consumption is discouraged for healthy people and is detrimental for patients, even in the absence of clinical symptoms. impaired immunity and increased susceptibility to infection. nevertheless, nutrient inadequacies and deficiencies in our habitual diet are common ( ) , and immune function may be improved by restoring nutrients to recommended levels, increasing resistance to infection, and hastening recovery once infected ( ) . furthermore, studies have revealed inadequate micronutrient levels in patients who were hospitalized in the infectious disease department, including thiamine, selenium, zinc, and vitamin b deficiencies ( ) , which were associated with adverse clinical outcomes. early detection, prevention, and treatment should aim at decreasing the inflammatory response and avoiding the excessive post-inflammatory immune suppression, defined as compensatory response ( ) , that is observed in many such patients ( ) . various micronutrients are essential for immunocompetence, particularly vitamins a, c, d, e, b , b , and b , folic acid, iron, selenium, and zinc, as well as macronutrients likely omega fatty acids ( ) and bioactive components as polyphenols ( ) . during recent years, our groups, together with several others worldwide, demonstrated the ability of nutritionally derived bioactive compounds to suppress inflammatory cytokine release ( ) ( ) ( ) ( ) . in particular, the administration of several plant-derived polyphenols to in vitro cultured immune cells suppressed the release of inflammatory cytokines ( ) . traditional chinese herbal medicines for influenza treatment demonstrated potential antiviral activity ( , ) . polyphenol bioavailability has been long discussed, as many have observed health benefits, but few have observed circulating traces of bioactive compounds ( , ) . nonetheless, exposure of the epithelial barrier to a polyphenol-rich environment can efficiently activate local immune suppression and tissue repair mechanisms ( ) , and systemic benefits may be related to the release of circulating microrna, defined as small non-coding rna molecules, with anti-inflammatory effects ( ) . indeed, even if orally introduced, bioactive dietary factors induce mirna synthesis, these are packaged into exosomes and released into the bloodstream to act systemically ( ) . dietary factors are emerging as anti-inflammatory mirna promoters able to regulate metabolic functions, inflammation, and oxidation systemically ( ) ( ) ( ) ( ) . furthermore, herb extracts may combine antiviral, anti-inflammatory, and antioxidant activity, and tissue-repair properties ( , ) (figure ) . altogether, these observations suggest that people may benefit from a correct nutritional intake, particularly during this period of uncertainty. choosing dietary regimes that may potentially work as adjuvants for preventing undesired hyper-inflammation might be particularly useful for patients with mild signs of infection (figure ). general recommendations for healthy adults over years of age observing a period of lockdown and thus with limited options for physical activity should focus on healthy dietary patterns. these can be generally described as those rich in plant-based foods, including fresh fruits and vegetables, soya, nuts, good sources of antioxidants ( ) , and omega- fatty acids ( ) and low in saturated fats and trans fats, animalderived proteins, and added/refined sugars ( ) . moreover, mild energy restriction is recommended for obese and obese-diabetic patients ( ) . most of these dietary targets can be met in our country by means of the well-known and traditionally familiar mediterranean diet ( , ) , which is rich in polyphenols with immune-protective and anti-inflammatory activities, playing an adjuvant role in both prophylaxis and therapy ( ) . the scientific community is already discussing how to manage future epidemic outbreaks by learning from the current experience ( ) . future studies should also focus on the effects of nutrition on immune function, identifying target population subgroups with the most vulnerable immune systems, such as the elderly and those with comorbidities. hc and mc wrote the 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journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: hyz gmpz nan it is widely assumed that the spread of severe acute respiratory syndrome coronavirus- (sars-cov- ) infection in humans occurs through close contact with an infected person, short-range transmission through respirable droplets from an infected individuals' cough or sneeze, and aerosolized airborne droplets in long-range (over a few meters) transmission ( ) . large respirable droplets (> µm) rapidly settle out of the air, whereas virus-laden small droplets (< µm), often referred to as "droplet nuclei" remain longer in the air and propagate depending on air-flow ( ) . assuming that a fraction of aerosols remains infective, "droplet-nuclei" might contribute to airborne transmission of the virus, particularly in poorly ventilated and crowded indoor spaces. a recent commentary, and supported with > signatories, has further stressed the importance of inhalation exposure to viruses in respirable droplets at short to medium distances (up to several meters) ( ) . in contrast to the inhalation mode of viral transmission through airborne respirable droplets, here we speculate an additional role for settled and airborne particulate matter (pm) not only in viral transmission through inhalation and ingestion, but also in promoting immunity through antigen delivery, adjuvanticity and trained immunity. a number of recent studies have suggested some correlations between air pollution and coronavirus disease- (covid- ) cases and deaths ( , ) . for instance, a recent epidemiological study concluded that an increase of µg/m in long-term exposure to fine pm air pollutants (≤ . µm, pm . ) is associated with an % increase in covid- mortality rate in the united states ( ). another study involving municipalities in the netherlands has further shown that a µg/m increase in concentrations is associated with . more covid- cases, . more hospital admissions, and . more deaths ( ) . similarly, previous reports have also indicated that air pollution exposure increases severe outcomes during infectious disease outbreaks ( , ) . for instance, during an outbreak in in china, severe acute respiratory syndrome case fatality rates were dramatically higher in locations with a moderate to high long-term air pollution index in comparison to regions with a low air pollution index ( ) . while long-term exposure to air pollutants such as pm . and nitrous dioxide contributes to persistent inflammatory responses and cardiopulmonary diseases ( ) , which might increase vulnerability to covid- , it is also plausible that depending on the environment sars-cov- "hitchhiking" on airborne pm pollutants might be an additional mechanism for spreading the infection. a number of studies have shown that pm is a carrier of airborne pathogens ( , ) . for instance, a metagenomic study has confirmed the presence of bacteria, archaea, fungi and dsdna viruses on pm pollutants collected during a severe smog event in beijing ( ) . more recently, setti et al. ( ) detected the presence of sars-cov- rna on outdoor/airborne pm of . - µm (pm ) collected from an industrial site of bergamo province, which is known for high air pollution and some of the most severe cases of covid- . this study was limited to detection of viral nucleic acid, rather than infectious virus. however, it is likely that air samplers can severely damage pathogens making detection of intact microorganisms difficult. notwithstanding, the exact role of pm in transmission of pathogens remains to be elucidated, but will be dependent on the presence of bound/trapped infectious viruses. can infectious viruses survive on pm? in "droplet nuclei" sars-cov- remain stable for at least up to h, whereas on plastic and stainless steel surfaces the viral stability is increased dramatically and detectable up to h after application ( ) . viruses in aerosol droplets, originated from a cough or sneeze, are likely embedded in and covered by a "corona" of mucins/proteoglycans (figure ) , which may aid viral aggregation and preservation as well as viral adsorption to airborne pm and/or settled pm on surfaces. furthermore, pm, depending on its composition, shape, physicochemical properties and density might further aid viral clustering and preservation, and account for prolonged viral stability and persistence on surfaces and in the air. viral adhesion might also bridge pm-pm binding/agglomeration and enhance respirable properties of these structures. can pm-virus composites induce infection? viruses bound to pm seemingly resemble drug powders adsorbed to coarse carrier particles (e.g., lactose) in dry powder inhalers (dpi) in their behavior. in dpi, coarse carriers increase drug particle dispersion, improve flow and on inertial impaction in the back of the throat releases drug particles, which subsequently deposit to the lower respiratory tract regions by sedimentation ( ) . on inhalation, infectious virus clusters bound to pm (e.g., composites > µm) are expected to predominantly deposit in the upper respiratory airways due to greater inertia (figure ) . the binding target for sars-cov- is angiotensin-converting enzyme (ace ), which is not only expressed by lung epithelial cells in the lower respiratory tract ( ) , but also in the nose as reported recently ( , ) , as well as proximal and distal enterocytes ( ) . assuming that some bound viruses to pm remain intact and infectious, the bulk of inhaled virus-bound pm most likely corresponds to a very low viral titer. in spite of this, pm could increase viral persistence in the upper respiratory airways and through irritation and ulceration of the nasal epithelium ( , ) promotes viral spread particularly in individuals where mucociliary clearance is reduced (e.g., smokers, asthma, acute respiratory distress syndrome). it has been recently reported that inhalation of as few as viral rna copies are enough to effectively induce disease and this supports the notion that the inefficient nature of particulate inhalation could still achieve pathogenesis ( ) . accordingly, this mode of inhalation might eventually promote viral translocation to the lower respiratory tract and contribute to disease severity (figure ) . second, those inhaled large-sized virus-bound pm in the mouth and throat are susceptible to ingestion. it is also plausible that pm adsorption/embedding might enhance viral stability in the hostile environment of the gastrointestinal tract and eventually promotes infection through interaction with ace expressing enterocytes (or even through sampling by m cells in the gut-associated lymphoid tissue). third, considering the complex and composite nature of airborne pm (which is typically an agglomerate of carbonaceous combustion particles, coarse dust, fibers, microplastics, transition elements, secondary nitrates, sulfates, adsorbed gases, etc.) ( , ) , inertial compaction may de-agglomerate pm forming smaller viral-bound particles. virus-pm composites of < µm, depending on their shapes and densities, could distribute to primary, secondary and terminal bronchi as well as reach the alveoli by sedimentation. indeed, these are anatomical regions where significant inflammatory reactions have been observed in covid- cases ( ) . within the alveoli, instead of targeting ace expressing epithelial cells, virus-pm composites could be highly susceptible to phagocytic recognition and clearance by alveolar macrophages (am) through a plethora of endocytic and pattern-recognition receptors as well as ace (which is also expressed by am), where even a low dose exposure might result in macrophage infection. however, this still requires pathogen translocation from endolysosomal compartments to the cytosol and successful viral replication. earlier studies have established a cytoplasmic mode of entry for a predecessor coronavirus through a proteolysis-dependent endo-lysosomal pathway ( ) , which might apply to sars-cov- . although coronaviruses such as sars-cov replicate poorly in human monocytes/macrophages ( ), shedding of low viral titres from a small population of am might still be sufficient to spread infection through the regional ace expressing epithelial cells. furthermore, by considering the heterogeneous composition and proinflammatory nature of airborne pm pollutants, on phagocytosis, some pm might act synergistically with viruses to initiate the bystander alveolar macrophage-mediated damage to epithelial tissues. for instance, this could occur through a cytokine storm and coupled with down-regulation of cd r (a receptor which inhibits macrophage activation) and upregulation of tumor necrosis factor-related apoptosis-inducing ligand (trail) on the am surface ( ). subsequently, trail binding to death receptor (dr ) expressed on epithelial cells could induce apoptosis in the epithelium ( ) resulting in alveolar leakage and spread of virus out of the affected alveoli. furthermore, these disruptions through pm-virus alliance may further help with the recruitment and activation of monocytederived macrophages that is seen in patients with covid- ( ) . in summary, although long-term exposure to polluted air might increase vulnerability to covid- through prior adverse cellular effects of settled pm ( ), our proposed "hitchhiking" hypothesis offers an additional multi-mechanistic pathogenic process through delivery of low viral titres with diverse pm-virus composites and is applicable to both indoor and outdoor situations, where the pathogenic severity is dependent on pm concentration, composition, shape and size as well as the infectious viral load. for instance, the hypothetical concentration of viruses carried by pm . is expected to be much lower than those by pm , since the carrying capacity is proportional to the particle volume. accordingly, larger pm might play more significant roles in viral transmission. nevertheless, it is still plausible that during infectious disease outbreaks pathogen hitchhiking on pm . might be an additional, and yet, effective contributing factor to epidemic and disease pathogenesis in urban as well as rural hot spots (e.g., locations with intensive livestock farming, which is rich in pm . ) with poor local air pollution and particularly among high-risk individuals. contrary to the suggestions that long-term exposure to pm might increase vulnerability to sar-cov- infection, inhaled pm might promote some forms of immunity to the virus in some individuals. pm could be the carrier of spike, envelope, membrane and nucleocapsid protein fragments of coronaviruses and on inhalation could deliver accompanied antigens to a variety of dendritic cells (dcs) subsets located throughout the respiratory tract. since, a large number of dcs are associated with the large airways (e.g., in the respiratory epithelium of the nose, nasopharynx, trachea, and large bronchi) ( ) , these dcs might recognize and capture inhaled pm-antigen composites through receptor-mediated endocytic processes. depending on pm composition, physicochemical characteristics and antigen load they could undergo a maturation process, leave the lung, and migrate to draining lymphoid tissues, where they are capable of activating naïve t cells for the expression of acquired immunity. thus, pm could not only act as an antigen depot, but also as an adjuvant to initiate dc maturation. on the other hand, it is even more tempting to speculate that frequent exposure to particular types of pm/pm-antigen composites (e.g., as in areas with high air pollution) could trigger some forms of trained immunity (a de facto innate immune memory) presumably through epigenetic reprogramming of transcriptional pathways ( ), thus offering a plausible explanation as to why some sars-cov- infected individuals are asymptomatic. interestingly, evidence suggests that the asymptomatic covid- patients have a significantly lower virus-specific igg and neutralizing antibody levels relative to symptomatic patients in the early convalescent phase ( ) . furthermore, asymptomatic individuals exhibited lower levels of many pro-and anti-inflammatory cytokines ( ) . these observations may be indicative of trained immunity in these individuals. irrespective of the immunological outcomes, these possibilities are analogous to immunization strategies with engineered nano-and micro-particles ( ) . here, we propose a hypothesis and a working mechanism for the role of pm pollutants in binding, stabilization and delivery of low titres of sars-cov- to alveolar macrophages. perturbations of macrophage function by virus-pm composites may be an additional mechanism for spreading infection and contributing to covid- severity. contrary to this, some inhaled pm might promote immunity to sars-cov- through complex mechanisms. since, the role of pm in airborne transmission of pathogens and associated immune responses are poorly understood, research in this neglected area should be encouraged, resulting in understanding that 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key role for monocytes and macrophages does air pollution influence covid- outbreaks? atmosphere lung dendritic cells at the innate-adaptive immune interface defining trained immunity and its role in health and disease clinical and immunological assessment of asymptomatic sars-cov- infections the innate immune responses, adjuvants and delivery systems all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. the remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © farhangrazi, sancini, hunter and moghimi. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -xcqz d t authors: wang, jun; li, qian; yin, yongmei; zhang, yingying; cao, yingying; lin, xiaoming; huang, lihua; hoffmann, daniel; lu, mengji; qiu, yuanwang title: excessive neutrophils and neutrophil extracellular traps in covid- date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: xcqz d t background: cases of excessive neutrophil counts in the blood in severe coronavirus disease (covid- ) patients have drawn significant attention. neutrophil infiltration was also noted on the pathological findings from autopsies. it is urgent to clarify the pathogenesis of neutrophils leading to severe pneumonia in covid- . methods: a retrospective analysis was performed on covid- patients classified as mild (n = ), moderate (n = ), and severe (n = ) according to the guidelines released by the national health commission of china. trends relating leukocyte counts and lungs examined by chest ct scan were quantified by bayesian inference. transcriptional signatures of host immune cells of four covid patients were analyzed by rna sequencing of lung specimens and balf. results: neutrophilia occurred in of severe patients at – days after symptom onset, coinciding with lesion progression. increasing neutrophil counts paralleled lesion ct values (slope: . and . – . ), reflecting neutrophilia-induced lung injury in severe patients. transcriptome analysis revealed that neutrophil activation was correlated with neutrophil extracellular trap (net)-associated genes in covid- patients, which was related to innate immunity and interacted with t/nk/b cells, as supported by a protein–protein interaction network analysis. conclusion: excessive neutrophils and associated nets could explain the pathogenesis of lung injury in covid- pneumonia. as of early may , more than million cases of coronavirus disease (covid- ) have been confirmed worldwide, resulting in hundreds of thousands of deaths ( ) . according to the guidelines of the diagnosis and treatment of new coronavirus pneumonia (version ) published by the national health commission of china, covid- patients can be classified as mild, moderate, and severe cases. severe patients easily develop acute respiratory distress syndrome (ards) or multiple organ failure, with a - % death rate ( , ) it is not well-understood what drives the exacerbated host response involving a cytokine storm in severe covid- ( ) . specifically, it is unclear what initiates and propagates the cytokine storm. neutrophil infiltration was noted in three recent reports on the pathological findings from autopsied covid- patients ( ) ( ) ( ) . neutrophil infiltration in pulmonary capillaries, acute capillaritis with fibrin deposition, extravasation of neutrophils into the alveolar space, and neutrophilic mucositis were observed. similarly, increased neutrophil counts were reported to occur simultaneously in the peripheral blood of severe and non-surviving covid- patients ( , ) . neutrophilia predicts poor outcomes in patients with covid- , and our previous research also indicated the neutrophil-to-lymphocyte ratio (nlr) is an independent risk factor for severe disease ( , ) . recently, two serum markers of neutrophil extracellular traps (nets), myeloperoxidase (mpo)-dna, and citrullinated histone h (cit-h ) levels were found to be elevated in the serum of covid- patients ( ) . this suggested that neutrophilia and excessive nets may contribute to cytokine release and respiratory failure. as a contributor to pathological inflammation of pneumonia, excessive neutrophils lead to tissue injury by oxidative burst, phagocytosis, and the formation of neutrophil nets, known as netosis. nets are composed of extracellular webs of dna, histones, microbicidal proteins, and oxidative enzymes that are released by neutrophils to corral infections ( ) ( ) ( ) ( ) ( ) . the ability of nets to damage tissues is well-documented in infection and sterile disease. nets directly kill epithelial and endothelial cells ( , ) , and excessive netosis damages the epithelium in pulmonary fungal infection ( ) and the endothelium in transfusion-related acute lung injury ( ) . in the present study, first, the dynamics of neutrophil counts in covid- patients (n = ) during hospitalization were examined, together with the corresponding lung injury, to clinically define the relationship between lung injury and leukocyte counts. second, transcriptional signatures of host immune cells from covid- patients (n = ) were analyzed by rna sequencing of lung specimens or bronchoalveolar lavage fluids (balf). immune cell frequency was analyzed by mcpcouter. we used average expression of genes enriched in neutrophil degranulation and activation to screen highly correlated genes and further identified net associated genes in the correlated gene list to construct an interactive network from the string database. the study was approved by the ethics committee of the fifth people's hospital, wuxi (no. - - ). the confirmed covid- patients were enrolled in this retrospective study from january to march , . written informed consent was obtained from all patients from the fifth people's hospital, wuxi, china. the clinical handling of covid- patients was performed according to the guidelines of the diagnosis and treatment of new coronavirus pneumonia (version ) published by the national health commission of china. mild, moderate, and severe cases were defined by the following conditions: ( ) epidemiological history, ( ) fever or other respiratory symptoms, ( ) frequency of typical ct image abnormalities of viral pneumonia, and ( ) positive rt-pcr result for sars-cov- rna. in addition, mild cases were diagnosed if no typical ct image abnormality of viral pneumonia (# above) was seen and severe patients also met at least one of the following conditions: ( ) shortness of breath, respiratory rate (rr) ≥ times/min, ( ) oxygen saturation (resting state) ≤ %, or ( ) pao /fio ≤ mm hg. all medical records including epidemiological, demographic, clinical manifestation, laboratory data, radiological characteristics, treatment, and outcome data were reviewed and collected. laboratory confirmation of sars-cov- infection was performed by real-time rt-pcr (bojie ltd, shanghai, china) according to chinese cdc approval. five sets of rnaseq data from balf of two covid- patients were acquired from big data center (accession number cra ), and corresponding data of three healthy controls were from the ncbi sra database (accession numbers srr , srr , and srr ). four rna-seq data from lung specimens of two covid- patients and two healthy controls were acquired from the geo database (accession numbers gsm , gsm , gsm , and gsm ). all images were obtained on the ct system (somatom definition as+, siemens healthineers, germany) with patients in supine position. the main scanning parameters were as follows: tube voltage = kv, automatic tube current modulation (about mas), pitch = . mm, slice thickness = mm, field of view = mm × mm. all images were then reconstructed with a slice thickness of . mm with the same increment. two professional radiologists (y.m.y. and x.m.l.), who were blinded to the laboratory test data, reported chest ct features and assessed the ct features by consensus. the lesion ct values were assessed using the skyview pacs system. the region-of-interest was selected manually marking the area of highest intensity (most restricted area) of the lesion in ct images. kallisto was used to pseudoalign the rna-seq reads and perform bootstrap analysis using an index based on the ensembl grch homo sapiens release transcriptomes ( ) . gene expression levels were then calculated as transcripts per million (tpm). sleuth (version . . ) ( ) was used to perform differential gene expression (degs) analysis with the wald test. benjamini-hochberg-adjusted false discovery rate (q < . ) was used to correct for multiple comparisons. to compare lung and balf samples of covid- patients with healthy controls, differentially expressed genes were exhibited in a scaled heatmap using pheatmap ( ) . mcp-counter was used to characterize immune cell subpopulations ( ) . the mcp-counter scores obtained from the three underlying transcriptome platforms (affymetrix human genome u plus . , affymetrix a, and illumina hiseq) were used to estimate the expression of each cell population. functional enrichment analysis of the upregulated marker genes of neutrophils was conducted with metascape (http://metascape.org/) ( ) . gene set enrichment analysis (gsea) was performed in pre-ranked list mode with , permutations and weighted enrichment statistic ( ) . the gene interaction was analyzed by string ( ). gene interaction networks were visualized with examine ( ) . quantitative parameters are described as the median value followed by the inter-quartile range (iqr) in parentheses. principal component analysis was performed with r package "factominer" to identify those clinical parameters that contribute most to distinguishing severe, moderate, and mild cases of covid- ( ) . figures were produced with r package "ggplot " ( ) . logistic regression was conducted with r package "rstanarm" ( ) to identify associations of laboratory parameters with severity of cases. severe cases were typed as severe and others (moderate and mild cases) as non-severe. the generalized linear model was then used to calculate coefficients (mean value with %, % confidence interval) of all parameters for severe. finally, we used the function of exp [exp(x) = ex] for coefficients. the results were an odd's ratio (mean, - % credible interval). receiver operating characteristic curves (roc) were calculated by r package "proc." the area under the roc curve (auc) and cut-off values of selected parameters were used to distinguish mild and severe cases ( ) . numerical bayesian linear regression was carried out with stan using hamiltonian monte carlo (supplemental materials; supplementary figure ) ( ) . fifty-five confirmed covid- patients were hospitalized in the fifth people's hospital of wuxi from jan to mar , . the median age of patients was years (iqr - ), and ( %) were male. based on the previously described guidelines, ( %), ( %), and ( %) of the covid- patients were classified as mild, moderate, and severe cases, respectively. there were five patients with diabetes ( %), with hypertension ( %), eight with surgical history ( %), and two with co-infections ( %). the most common symptoms at onset were fever in cases ( %), sputum production in cases ( %), cough in cases ( %), and fatigue in cases ( %) ( table ) . the clinical handling and relevant time-points of patients including eight severe and moderate cases are shown in figure . the median time from the date of onset of symptoms to hospital admission, lymphopenia, ards, and neutrophilia was , , , and d, respectively. lymphopenia occurred in seven of eight severe patients and of moderate cases within d, ards occurred in all eight severe patients within d, and neutrophilia occurred in six of eight severe patients and one of moderate cases within d (figure ) . the laboratory test of each patient on the day of hospital admission showed that the median neutrophil count in severe covid- patients ( . , iqr: . - . ) was higher than in the moderate ( . , . - . ) and mild ( . , . - . ) groups. in contrast, lymphocyte and monocyte counts in severe covid- patients were lower than in the other two groups ( principal component analysis was performed to visualize the contribution of all mentioned clinical parameters on disease severity (figure a) . nine variables contributed most strongly. among them, higher crp, fib, neutrophil count, and nlr, and lower lymphocyte count were associated with increased disease severity. these parameters may therefore be used for prognosis. to assess the diagnostic value of the top two contributors, crp and lymphocytes, the auc and cut-off values from the roc curves were calculated for the severe and mild cases, respectively (supplementary figure b) . the cut-off values for severe patients were crp ( . ) and lymphocytes ( . ), and for mild patients the values were crp ( . ) and lymphocytes ( . ) (see dashed lines in figure b ). next, dynamic changes of neutrophil, lymphocyte, and monocyte counts in the peripheral blood of covid- patients were monitored (figure c ). dramatically increased neutrophil counts were found in severe covid- patients in comparison to the other two groups. in contrast, lymphocyte counts persisted at lower values in severe covid- patients. monocyte counts were lower in severe cases, although the monocyte count fluctuated over a wide range. timing of the occurrence of maximum neutrophil, minimum lymphocyte, and minimum monocyte counts, and the corresponding counts in covid- patients, during hospitalization are shown in figure d . from day to day after symptom onset, neutrophil counts erupted (> . × /l) and peaked in six of eight severe covid- patients. in contrast, only one moderate ( / ) covid- patient was found with neutrophilia. lymphopenia occurred in seven of eight severe patients but only in four mild ( / ) covid- patients. monopenia (< × /l) was found in three moderate ( / ) and four severe ( / ) covid- patients. overall, monitoring blood cell parameters revealed neutrophilia as a characteristic of severe covid- patients. neutrophilia and lymphopenia obviously occurred in severe covid- patients during hospitalization. here was a case of severe patient. the crp level remained low when neutrophilia occurred, and the d-dimer levels increased after neutrophilia. series of chest ct images exhibited enlarged patches and groundglass nodules in the sub-pleura area of both lungs during neutrophilia. interestingly, all observed lesions were reduced or gradually absorbed along with the return of neutrophils to normal levels after neutrophilia (figures a,b) . the ct value of lesions, reflecting lung lesions, was further demonstrated to have the same trend with neutrophils but the opposite trend with lymphocytes ( figure c) . to estimate the overall correlation of ct value with neutrophil and lymphocyte counts across patients with a visual inspection of possible trends, linear models were fitted to summarize the dependency of z-values of ct value (ctz, see figure d . overall, the results showed that the ctz value has no average trend with changing neutrophil and lymphocyte counts for moderate cases (green). however, for the severe cases (red), there are clear trends for ctz value with changing cell counts; specifically, ctz value increased for increasing neutrophil counts, whereas ctz value decreased for increasing lymphocyte counts (figure d ). immune cell transcriptional signatures were established from rna-seq data of balf and lung specimens of covid- patients and healthy controls. marker genes of neutrophils, t cells, monocytes, and b cells were identified from microenvironment cell populations-counter (mcp-counter). their representation in the rna-seq data were exhibited using a scaled heatmap by comparing both lung and balf samples of covid- patients to healthy controls ( figure a) . the results revealed that marker genes represented four immune populations: neutrophils ( genes), t cells ( genes), monocytes ( genes), and b cells ( genes). for lung white blood cell (× /l) . - . tissue, the most up-regulated marker genes were enriched in neutrophils, second in monocytes, and only a small proportion were enriched in b cells. marker genes of t cells were almost all lowly expressed. for balf, the most upregulated marker genes were similarly enriched in neutrophils, but more up-regulated genes in monocytes and b cells were observed in covid- patients compared to healthy controls, which is different from the lung samples. functional enrichment analysis of the upregulated marker genes of neutrophils were further conducted with metascape. the enrichment analysis revealed that five gene sets with lowest qvalue were related to neutrophil degranulation and activation ( figure b ) and there were marker genes involved. then, we calculated the average expression of these genes as an evaluating score for neutrophil activation (nas). to further assess the abundance of infiltrating immune cells of the lung and balf in covid- patients, the mcp-counter score was used to quantify the absolute abundance of immune cell subpopulations. notably, the neutrophil scores were higher and t cell scores were lower in lung samples of covid- patients. the higher abundance of cytotoxic t lymphocytes contributed for cell injury, not for anti-virus. due to the marker genes for cytotoxic t lymphocytes was klrc (killer cell lectin like receptor c ). for the balf samples, the score of neutrophils, cytotoxic lymphocytes, b cells, monocytes, and dendritic cells were found to be higher in one of the covid- patients compared to the three healthy controls (figure c ). to explore the outcome of neutrophil activation in covid- , we further analyzed the correlation of nas with , degs that overlapped in both the lung and balf samples. the spearman correlation was used separately for covid- patients and healthy controls. then, the r value for every single gene was acquired for covid- patients (r ) and healthy cases (r ). all degs were ranked based on r (r -r ). the "r value" of the top genes (r > ) in the two groups are displayed in figure a . of these genes, genes were nets associated genes ( figure b ; table ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) of the genes, lgals , hck, lcp , ceacam were involved in the cytokine-mediated signaling pathway. s a , lgals , and ctsc were involved in regulation of apoptotic signal by enrichment annotation from the metascape tool ( figure b ; table ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . to further investigate the role of nets in covid- , we generated a gene set termed "net-associated genes" based on genes coding for proteins enriched in nets released from human neutrophils with mass spectrometry (supplementary table ) . pre-ranked gsea by r resulted in significant enriched gene sets of "net-associated genes" (enrichment score = . ) and "regulation of inflammatory response" (enrichment score = . ) (figure c ). as known, the formation of nets could induce direct lung injury ( ). there were nets associated genes related with neutrophils activation in covid- patients. to further illustrate the interaction between these nets associated genes with other neutrophils activation related genes, we constructed a protein-to-protein interaction network from the string database (figure ) . we found that the nets interacted with stat induced interferon stimulated genes by il rg, implying that nets associated genes may be triggered by ifn signaling. besides, nets in turn may activate b cells via tnfsf b and inhibit the function of t and nk cells via lgas and ceacam , which are negative regulators for t and nk cells. lgas is a possible promoter of protein-arginine deiminase type (pad ). pad , a key nets associated gene, lies downstream of ros and promotes chromatin decondensation ( , ) . of note, we also observed ros related genes including hck, rac , and ncf among nets associated genes (figure ) . to annotate the function of nets associated genes, they were categorized as metabolic enzymes (rac , ncf ), structural proteins (lcp ), anti-microbial related (trem ), peroxisomal (sh bgrl ), and others (c qc, lgals , serpina , c qb, ccl , ccl , ceacam , hck, and cxcl ) ( table ) . thus, we speculate that nets may be activated by innate immunity such as ifn signaling, in covid- patients. nets may negatively regulate the immune function of t cells and nk cells via lgas and ceacam , respectively, leading to insufficient anti-viral immunity and injuring the lung tissue directly. in this study, a set of laboratory test parameters and the corresponding chest ct images of covid- patients were collected during hospitalization. among these variables, excessive neutrophils were associated with disease severity, as shown by principal component analysis. bayesian inference across patients quantified that the increased trend of pneumonia lung injury, as represented by ct values, was in accord with the increased trend in neutrophil counts. transcriptome analysis of lung specimens and balf from covid- patients also indicated the most up-regulated marker genes were neutrophil related. importantly, many neutrophil activation genes were categorized as net-associated genes. these genes were further assessed to interact with t and nk cells via negative regulatory functional enrichment analysis of these genes, of which genes were nets associated genes. (c) nets associated genes set (enrichment score, . ) and the go term of regulation of inflammatory (enrichment score, . ) by gsea with degs from pre-ranked by r. molecules in covid- patients leading to insufficient anti-viral response and lung injury (figure ) . our previous study also found an increased neutrophil-tolymphocyte ratio in the most severe disease cases ( ) . recently, neutrophil infiltration was also noted in the lung tissue of autopsied covid- patients ( - ). since neutrophilia predicts poor outcomes in patients with , we propose that the change in neutrophil counts in peripheral blood or tissues may be closely associated with pathological injury in covid- patients. we demonstrated here that the dynamics of neutrophil counts in covid- patients during hospitalization exhibited the same trend as the corresponding lung injury. nets, as confirmed contributors to pathological inflammation of pneumonia, can damage tissues by killing epithelial and endothelial cells ( , ) of pulmonary tissue in infection and sterile disease. recently, two elevated nets markers have been observed in serum from covid- patients, which suggests that neutrophilia and excessive nets may contribute to cytokine release and respiratory failure in covid patients ( ) . however, evidence is still lacking regarding netosis in lungs. we analyzed the differentially expressed genes in lung tissue and balf samples from covid- patient in comparison to healthy controls. among all up-regulated genes in neutrophil modules in covid- patients, we found genes derived from the neutrophil activation pathway were nets associated genes. thus, nets may be activated in the lung of covid- patients. it is also poorly understood how netosis induces the cytokine storm or modulates the host immune response. our string analysis suggests that nets associated genes could interact with t, nk, and b cells through regulation of lgals , ceacam , and tnfsf b expressions, respectively. we suspect that the progression of lesions in covid- patients may be induced by nets as well as nets-t/nk/b cell interactions. in conclusion, the clear trend of lung injury in accord with the trend of increasing neutrophils was quantified by bayesian inference analysis in covid- patients. the transcriptome signature of immune cells also indicated elevated neutrophil markers in the lung and balf samples of covid- patients. frontiers in immunology | www.frontiersin.org importantly, among the excessive neutrophil activated genes, were nets associated genes and these genes interacted with t cells and nk cells through negative regulation. therefore, we posit that netosis in lung tissue leads to an insufficient anti-viral response in covid- patients. we hope that future studies will investigate the predictive power of circulating nets in well-phenotyped longitudinal cohorts. the datasets presented in this study can be found in online repositories. the names of the repository/repositories and accession number(s) can be found in the article/supplementary material. the studies involving human participants were reviewed and approved by the ethics committee of the fifth people's hospital, wuxi (no. - - ). the patients provided their written informed consent to participate in this study. jw, yq, and ql conceived and designed the experiments. ql, jw, dh, and ml drafted and revised the manuscript. yy, yz, and xl carried out the data collection. jw, dh, and yc carried out the data analysis and interpretation. dh, yq, ml, and lh contributed reagents, materials, and analysis tools. all authors contributed to the article and approved the submitted version. this work was supported by the foundation of wuxi medical development discipline for infectious disease (fzxk ) and wuxi young medical talents (qnrc ), health and science bureau of wuxi (ms , cse n , q ). the funding source was not involved in the study design; in the collection, analysis, and interpretation of data, in the writing of the report, and 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probabilistic programming language rac is required for the formation of neutrophil extracellular traps haploinsufficiency of nadph oxidase subunit neutrophil cytosolic factor is sufficient to accelerate full-blown lupus in nzm mice trem- promotes survival during klebsiella pneumoniae liver abscess in mice neutrophil extracellular traps that are not degraded in systemic lupus erythematosus activate complement exacerbating the disease neutrophil extracellular traps protein composition is specific for patients with lupus nephritis and includes methyl-oxidized αenolase (methionine sulfoxide ) galectin- is a possible promoter of immunopathology in rheumatoid arthritis by activation of peptidyl arginine deiminase (pad- ) in granulocytes disruption of neutrophil extracellular traps (nets) links mechanical strain to post-traumatic inflammation cystic fibrosis sputum dna has netosis characteristics and neutrophil extracellular trap release is regulated by macrophage migration-inhibitory factor characterization of neutrophil function in papillon-lefèvre syndrome src family kinases and syk are required for neutrophil extracellular trap formation in response to β-glucan particles protective role of mincle in bacterial pneumonia by regulation of neutrophil mediated phagocytosis and extracellular trap formation neutrophil extracellular trap-associated ceacam as a putative therapeutic target to prevent metastatic progression of colon carcinoma siglec- and siglec- are polymorphic paired receptors that modulate neutrophil and amnion signaling responses to group b streptococcus histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation we are grateful to the doctors, nurses, disease control workers, and researchers for their fight against covid- under extreme conditions. some of them have lost their lives in this fight. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material key: cord- -f yd guj authors: tang, yujun; liu, jiajia; zhang, dingyi; xu, zhenghao; ji, jinjun; wen, chengping title: cytokine storm in covid- : the current evidence and treatment strategies date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: f yd guj severe acute respiratory syndrome coronavirus (sars-cov- ) is the pathogen that causes coronavirus disease (covid- ). as of may , the outbreak of covid- has caused , deaths around the world. the current evidence showed that severely ill patients tend to have a high concentration of pro-inflammatory cytokines, such as interleukin (il)- , compared to those who are moderately ill. the high level of cytokines also indicates a poor prognosis in covid- . besides, excessive infiltration of pro-inflammatory cells, mainly involving macrophages and t-helper cells, has been found in lung tissues of patients with covid- by postmortem examination. recently, increasing studies indicate that the “cytokine storm” may contribute to the mortality of covid- . here, we summarize the clinical and pathologic features of the cytokine storm in covid- . our review shows that sars-cov- selectively induces a high level of il- and results in the exhaustion of lymphocytes. the current evidence indicates that tocilizumab, an il- inhibitor, is relatively effective and safe. besides, corticosteroids, programmed cell death protein (pd)- /pd-l checkpoint inhibition, cytokine-adsorption devices, intravenous immunoglobulin, and antimalarial agents could be potentially useful and reliable approaches to counteract cytokine storm in covid- patients. in december , an outbreak of a novel coronavirus-based disease was reported in wuhan, china. on february , the world health organization (who) named this coronavirus "severe acute respiratory syndrome coronavirus " (sars-cov- ) and the disease that it caused "coronavirus disease " (covid- ) . as of may , sars-cov- has affected over countries, and about , , cases have been confirmed around the world, of which , people have died. the reason for these deaths is suspected to be the "cytokine storm" [also called "cytokine storm syndrome" (css)]. the international classification of diseases (icd) does not include the cytokine storm or css. cron and behrens bring the current knowledge of css ( ) . they define that "cytokine storm" is an activation cascade of auto-amplifying cytokine production due to unregulated host immune response to different triggers. the triggers involved infections, malignancy, rheumatic disorders, etc. another scholar described that cytokine storm is a systemic inflammatory response to infections and drugs and leads to excessive activation of immune cells and the generation of pro-inflammatory cytokines ( ) . a similar entity is termed "cytokine release syndrome" (crs), which is not defined in the textbook of css ( ) . crs is an acute systemic inflammatory syndrome characterized by multiple-organ dysfunction (mod). it has been reported that chimeric antigen receptor (car)-t-cell therapy could help to distinguish crs from a cytokine storm ( ) . of note, the textbook described the criteria of css based on hemophagocytic lymphohistiocytosis (hlh) and secondary hlh (shlh) associated with rheumatic disorders, such as macrophage activation syndrome (mas) ( ) . thus, it may be not applicable in covid- because the covid- is a contagious disease and relatively irrelevant to a genetic disorder. up to date, there is still a lack of clinical and laboratory criteria to identify the cytokine storm. in this review, we referred covid- associated cytokine storm as the patients who are severely ill along with a high concentration of pro-inflammatory cytokines. for patients with covid- , the number of white blood cells, neutrophils, as well as levels of procalcitonin, c-reactive protein, and other inflammatory indices, are significantly higher in the intensive care unit (icu) cases than in non-icu cases ( , ) . many studies showed that severely ill patients tended to have a higher concentration of pro-inflammatory cytokines, especially interleukin (il) , than moderately ill patients in covid- ( ) ( ) ( ) ( ) ( ) . the result of the bronchoalveolar lavage fluid (balf) cells, which tested by transcriptome sequencing, reveals excessive chemokines releasing caused by sars-cov- infection, such as cxcl and ccl ( ) . the high level of cytokines also indicates a poor prognosis in covid- ( , , ) . furthermore, the pathology of postmortem examination of the lung, from who was died of covid- , demonstrated the existence of acute respiratory distress syndrome (ards) and t-cell overactivation chen and subbarao ( ) ]. method of detection (number of patients studied) cba ( ) cba ( chemokines ( ) . this phenomenon is due to an increase in the number of t-helper (th) cells and the high cytotoxicity of the cd + t cells ( ) . the innate and adaptive immune responses activated by sars-cov- infection lead to uncontrolled inflammatory responses and ultimately cause the cytokine storm ( ) . the cytokine storm can lead to apoptosis of epithelial cells and endothelial cells, and vascular leakage and, finally, result in ards, other severe syndromes, and even death ( ) . to lower mortality due to cytokine storm, we summarized the clinical and pathology features of the coronavirus-related cytokine storm. we explored the efficacy and safety of potential treatments and their molecular mechanism. there is still lacking sufficient evidence supporting the regulation of cytokine expression may be beneficial to the mortality of covid- . the early-stage clinical characteristics of mers and sars are influenza-like symptoms ( ) ( ) ( ) : pyrexia, sore throat, dry cough, myalgia, and dyspnea. those symptoms are very similar to the characteristics of early covid- and progress rapidly to pneumonia ( , , ) . it has been found that the regulation of several cytokines is disordered in the peripheral blood of sars patients, as summarized by chen and colleagues ( ) and listed in table . table shows an increase in levels of cytokines and chemokines and a decrease in levels of anti-inflammatory cytokines such as il- . of note, the release of pro-inflammatory cytokines, especially interferon (ifn)-α and ifn-γ, is correlated with lethal sars ( , ) . the cytokines with increased levels in fatal sars are il- , il- β, ifn, and cxcl . these cytokines are secreted mainly by dendritic cells (dcs) and macrophages, indicating that innate immunity plays a pivotal part in lethal sars. ccr + ccr + th cells have many chemokine receptors and may share the same mechanism and function in cell-cell interactions in sars. cytokines secreted by dcs and macrophages induce the infiltration and recruitment of pro-inflammatory th cells. analyses of lungs from sars patients have revealed diffuse alveolar damage as a crucial feature. histopathological studies have shown lung consolidation and edema with pleural effusions and focal hemorrhage, all of which resemble covid- features ( , ) . besides, the lungs of sars patients are infiltrated extensively with neutrophils and macrophages, which are not observed in covid- . in peripheral blood, numbers of cd + and cd + t cells are reduced in cases of covid- and sars ( , ) and are associated with death in the latter ( ) . interestingly, unlike mers and sars, a high concentration of pro-inflammatory cc chemokine receptor (ccr) + ccr + th cells are found in covid- ( ) . the innate and adaptive immune system takes multiple measures to respond to virus infection. mers-cov infects human epithelial cells and leads to these cells inducing significant but delayed responses by ifn, pro-inflammatory cytokines (e.g., il- β, il- ) and chemokines (e.g., il- ) ( , ) . sars-cov infects airway epithelial cells and results in delayed release of chemokines such as ccl , ccl , ccl , and cxcl ( ) . besides, mers-cov infects hematopoietic cells such as monocytes, macrophages, and dcs, which is not seen in those cells upon sars-cov infection ( ) ( ) ( ) ( ) . mers-cov infects the cells mentioned above to induce delayed (but increased) levels of pro-inflammatory cytokines (e.g., il- ) and chemokines (e.g., ccl , ccl ) ( , ) . although sars-cov is abortive in macrophages and dcs, the virus induces an increase in levels of pro-inflammatory cytokines and chemokines ( , ) . sars-cov and sars-cov- infect cells using the same receptor: angiotensin-converting enzyme- ( ) . hence, it has been postulated that both viruses can affect the same spectrum of cells. in the aspects of murine models of coronavirus, infection with sars-cov in balb/c mice has been shown to induce an increase in the number of pathogenic inflammatory monocytemacrophages (imms) ( ) . through stimulation of ifnα/β receptors, the accumulating imms produce monocyte chemokines (e.g., ccl , ccl , ccl ) and pro-inflammatory cytokines [e.g., tumor necrosis factor (tnf), il- , il β], which results in further accumulation of pathogenic imms. targeting of ifn signaling, imms, or pro-inflammatory cytokines could offer protection from lethal sars-cov infection. in this way, the chemokines (produced by activated monocytes and macrophages) lead to the recruitment of neutrophils, monocytes, and t cells into the lungs ( ) . after chemotaxis, activated effector t cells migrate to the lungs and destroy pneumocytes/permissive cells due to response to the virus infection ( ) . the damage caused by neutrophils, monocytes, and t cells results in lung-parenchyma changes, such as diffuse alveolar damage, which leads to ards ( ) . in summary, the excessive cytokines and chemokines caused by lethal coronavirus infection involve mainly antigen-presenting cells (apcs) (such as macrophages) and t cells. however, cytokines secreted by immune cells are produced to eliminate viral infection, and deficiency of such cytokines may be harmful to the body. for example, virus titers are significantly higher in toll-like receptor (tlr) −/− , tir-domain-containing adapterinducing interferon-β (trif) −/− , and il- −/− mice compared with their wild-type counterparts, and are associated with severe lung damage ( , ) . in china, we classified the stage of covid- according to the guidelines ( ) issued by the national health commission of the people's republic of china (nhc). according to the instructions, nhc defines severe illness of covid- as one of the following conditions: respiratory rate ≥ breaths/min in the resting state; oxygen saturation ≤ %; arterial blood oxygen partial pressure (pao )/fraction of inspired oxygen concentration (fio ) ≤ mmhg. critical illness as one of the following conditions: respiratory failure and requiring mechanical ventilation; shock; complication of other organ failures, and needs intensive care. the most common symptoms of covid- were fever, cough, shortness of breath, fatigue, and myalgia ( , , , ) , and severe cases tend to be older with more basic diseases and suffer from dyspnea, more complications ( , ) . in covid- , % of patients progress to severe disease and % to critical illness ( ) . a prospective study reported that the computerized tomography (ct) of the lungs of covid- ( ) . the lung lesions increase and the scope expands as the disease progresses, and ground-glass opacity coexisted with consolidation or striated shadow. some severe patients showed diffuse lesions in both lungs. up to date, the inflammatory disorders (insufficient in chemokines) in covid- have been reported in many clinical studies. the covid- is inclined to cause a decrease of lymphocyte count and an increase of c reactive protein (crp), especially in severely ill patients ( ) ( ) ( ) ( ) ( ) ( ) . the major subsets of the t lymphocytes (t cell) (cd + cd + t cell and cd + cd + t cells) are reduced in the covid- and are significantly lower in the severe cases ( , , , , , ) ; however, controversial results are also reported in some studies ( , ) . the results of the other immune cells, the b cell and natural killer (nk) cell, have more inconsistency in recent researches. il- was observed increased in all studies, and only one study show il- was not elevated. about half of the studies we collected showed tnf-α was increased. only huang et al. ( ) inspected the multiple types of chemokines and found that severe patients had higher levels of g-csf, gm-csf, ip- , mcp- , mip- a, mip- b, rantes, and il- . the inflammatory disorders of covid- were summarized in table . comparison objects il- il- β il- tnf-α ifn-γ il- (r) the pathologic features of covid- showed the lungs were infiltrated with excessive ccr + th cells and high cytotoxicity of cd + t cells ( ) . but high cytotoxicity of cd + t cells does not mean they exert the normal function. the sars-cov- could lead to cytotoxic lymphocytes (mainly involving nk cells and cd + t cells) exhaustion, which is manifested as the upregulated exhaustion markers, such as nkg . the exhaustion markers return to normal in patients who have recovered or are convalescent ( , ) . balf cells were found extreme cytokine releases, such as ccl , cxcl , ccl , and ccl ( ). furthermore, xiong et al. ( ) use the transcriptome dataset approach to discover that sars-cov- can activate apoptosis and p signaling pathway (one of the pathways responsible for the survival of the cell) in lymphocytes. these results could provide some reasons for the cause of patients' lymphopenia. another team of chen and his colleagues studied the mechanisms for lymphopenia ( ) . their results demonstrate that sars-cov- infected the cd + macrophages in spleens and lymph nodes (lns), and lead to lymphoid tissue damage, such as splenic nodule atrophy and lymph follicle depletion, etc. the cd + macrophages express high fas and cause activation-induced cell death (aicd) through fas/fasl interactions. furthermore, sars-cov- selectively induced macrophages to produce il- , not tnf-α and il- β, to directly promotes lymphocyte necrosis. the analysis of peripheral blood mononuclear cells (pbmcs) revealed that non-structural protein (nsp) and nsp of sars-cov- target nkrf (nf-κb repressor) to promote il- /il- production ( ) . as a consequence, it recruits neutrophils and induces uncontrollable host inflammatory response. collectively, the clinical, immunological, and pathologic features of covid- have something in common with sars and mers. for example, all the viruses can cause lymphopenia and influenza-like symptoms in the early stage. sars and covid- do not lead to the upgrade of tnf-α, but the increase of il- and il- is more prevalent in covid- . the il- plays a crucial role in the pathologic of covid- , including the chemotaxis of neutrophils and lymphocyte necrosis. importantly, covid- is more able to cause cytotoxic lymphocytes exhaustion. tocilizumab (tcz) is a recombinant humanized anti-human il- receptor monoclonal antibody, preventing il- binding to its receptor to exert the immunosuppression promoted by il- . michot et al. ( ) reported that -year-old male suffering from respiratory failure due to sars-cov- infection. after days of tcz treatment, the crp decreased from to mg/l and ultimately clinically fully recovered. similarly, some case reports showed tcz is an efficacy and safety approach in covid- , even patients with other diseases combined, such as multiple myeloma, end-stage renal disease, and sickle cell disease ( ) ( ) ( ) . recently, a retrospective study ( ) found that tcz decreased crp in all patients (n = ) rapidly, but three of them, who are critically ill, still dead. the dead patients show continuously rising of il- even after the administration of tcz and methylprednisolone, indicating that repeat doses of tcz may be needed in covid- patients who are critically ill. another retrospective study ( ) demonstrated that tcz showed a quick control of severe covid- manifestation, such as fever, respiratory function. all patients (n = , two were critically ill), have recovered and have been discharged from hospital, and no adverse event was reported during the treatment. a prospective open-label, multicenter single-arm study manifests the pilot results of the off-label application of tcz in severe patients with covid- ( ) . the study involved patients with severe covid- , and tcz succeeded in improving respiratory and laboratory parameters, such as pa , fi , consequently, increased the likelihood of survival (the death rate of the study is %). it is worth mentioning that a cautionary case report by radbel et al. ( ) . two patients were diagnosed with covid- complicated by crs and treated with tcz. unfortunately, both patients progressed to severe hlh, and one developed to viral myocarditis. all the cytokines produced by immune cells are responsible for viral clearance. suppression of cytokine release at an early stage of disease as treatment is controversial. application of synthetic disease-modifying antirheumatic drugs (dmards) and biologic dmards to downregulate cytokine expression in ra increases the risk of infection ( , ) . the timing and the doses of the intervention still need to be inspected clearly. sars-cov- mainly causes a dramatic increase in il- and does not remarkably promote other pro-inflammatory factors, such as il- β and ifn-γ. although treating covid- with tcz is an off-label use, it may be relatively appropriate and safe in coping with covid- associated cytokine storm basing on the current evidence. it still needs more large samples and high-quality studies to evaluate the exact efficacy and safety in covid- . the ongoing trials of potential treatments and other treatments focus on inflammatory disorders in covid- are available in supplementary table . glucocorticoid therapy is used widely among critically ill patients with other coronavirus infections (e.g., sars, mers). corticosteroids have been administered to icu patients infected with sars-cov- ( , , ) . glucocorticoids exhibit pharmacologic effects at any therapeutically relevant dose through classic genomic mechanisms. some immunosuppressive effects are based on transactivation, and glucocorticoid induces gene transcription and protein synthesis of nf-κb inhibitors and lipocortin- . through inhibition of nf-κb signaling, glucocorticoids induce inhibition of synthesis of downstream proteins such as il- , il- , granulocyte-macrophage colony-stimulating factor, and inducible cyclooxygenase- ( , ) . glucocorticoids reduce the proliferation, activation, differentiation, and survival of t cells and macrophages ( ) . glucocorticoids proffer inhibitory actions on the transcription and action of various cytokines. the th and macrophage-based pro-inflammatory cytokines il- β, il- , il- , tnf-α, and il- are inhibited by glucocorticoids ( ) . however, it is controversial whether corticosteroids are beneficial in the treatment of severe covid- patients. a comment and a meta-analysis, which mainly bases on the evidence of sars and mers ( , ) , stated that corticosteroid would increase mortality and delayed clearance of viral in coronavirus infection diseases. thus, the corticosteroids should not be administrated for the treatment of sars-cov- induced lung injury or shock. newly published studies also indicate that the use of corticosteroids is not beneficial for covid- patients (not severe cases), and high-dose corticosteroids are associated with mortality ( , , ) . most covid- patients discussed in these studies are not severe cases. inspecting the studies included and analyzed by the meta-analysis, only one study ( ) described the numbers of patients with corticosteroids and non-corticosteroids treatment in the severe group and non-severe group. the study demonstrated the benefit of corticosteroids use in severe sars-cov infection. another comment ( ) , which was written by front-line physicians from china, showed corticosteroids might have some benefit for critically ill patients with covid- . systematic corticosteroid therapy could promote oxygen saturation and pao /fio . however, corticosteroids might not improve mortality in critical covid- patients. current evidence shows that sars-cov- induces an increase in a small range of cytokines. it might be overuse to administrate corticosteroids to counteract a wide range of cytokines. furthermore, sars-cov- causes relatively serious lymphocytopenia and lymphocytes exhaustion. glucocorticoidmediated stimulation of the "hypothalamic-pituitary-adrenal axis" might also exacerbate lymphocytopenia ( ) . thus, the use of corticosteroid is a double-edged sword in covid- . the dose, duration, and timing of corticosteroid therapy will be crucial if administrated to covid- patients. as stated above, lymphocytes exhaustion is one of the characteristics of covid- , and pd- checkpoint-inhibitor might some help in reversing the anergy of lymphocytes. up to may , no study of pd- checkpoint-inhibitor has been reported in the treatment of covid- . the pathway consisting of the receptor pd- and its ligands, pd-l and pd-l , play crucial parts in the maintenance of peripheral tolerance. treatments with antibodies targeting pd- /pd- ligands have elicited an increased response in different cancer types and, in tandem with antibodies targeting cytotoxic-tlymphocyte-associated antigen- , have changed cancer therapy radically ( ) . unfortunately, signaling regulated by the pd- /pd-l pathway is also related to substantial inflammatory effects (e.g., sepsis), as this pathway plays a role in balancing protective immunity and immunopathology ( ) . increased pd-l expression in monocytes is associated with mortality in patients with septic shock ( ) . a meta-analysis of checkpoint inhibitors showed that such therapy increased the chance of survival ( ) . nivolumab (anti-pd- ) and bms- (anti-pd-l ) had completed phase-ib randomized studies for severe sepsis. they revealed that giving a checkpoint inhibitor did not result in unexpected safety findings or indicate a cytokine storm ( , ) . also, cd + and cd + t cells were hyperactivated, as revealed by the high proportions of human leukocyte antigen-dr isotype and cd , in covid- ; cd + t cells harbored high levels of cytotoxic granules in covid- patients, in which the phenotype is similar to fatal h n disease ( , ) . those results suggest that lethal covid, along with h n , may be related to defective activation and exhaustion of t cells, which also suggest that checkpoint-inhibitor administration may reverse this status. cytokine adsorption involves using a method, such as extracorporeal membrane oxygenation (ecmo), to filter harmful substances directly. an extracorporeal cytokine hemoadsorption device called cytosorb r (cytosorbents, monmouth, nj, usa) has been reported to capture and reduce inflammatory mediators. bruenger and colleagues reported that the plasma level of il- and procalcitonin decreased in one patient with severe ards after treatment with ecmo using a hemoadsorption device ( ) . a -year-old patient with severe ards showed that venous arterial-ecmo combined with hemoadsorption therapy decreased plasma concentrations of il- and il- . moreover, hemodynamic stabilization, respiratory improvement, and a decline in capillary leakage can be achieved in combination therapy ( ) . two trials employing hemoadsorption therapy for infection-related cytokine storm are ongoing (nct , nct ). a similar therapy involves dialysis. the mainly water-soluble mediators are removed from plasma, and the hemofilters can have additional adsorptive properties ( ) . continuous venovenous hemofiltration and adsorption for severe septic shock are being tested in one clinical trial (nct ). neutralizing excessive cytokines with hemoadsorption devices might be relatively effective. the disadvantage is like corticosteroids: a wide range of cytokines would be adsorbed. thus, it would lead to the a lack of cytokines, which are at reasonable or even insufficient levels. we suggest treating the cytokine storm in covid- should base on the laboratory results of cytokines and chemokines. meanwhile, adjusting the parameters of the devices (e.g., treatment duration) for preventing overtreatment. ivig can elicit passive immunity, anti-inflammatory, and immunomodulatory effects that can improve treatment effects and increase survival in severe infection. an igg molecule binds to a specific target antigen through the humoral and cellular arms of the immune system. for example, igg molecule blocks the cellcell interactions mediated by cell-surface receptors (such as cd and cd ligand), neutralize the autoantibodies by anti-idiotypic antibodies, expanse the regulatory t (treg) cell populations via the blockade of immune complex binding to low-affinity fcγ receptors (fcγrs), to exert the functions of immunomodulation ( ). ma and colleagues detailed a severe case of glandular fever treated with ivig ( ) . levels of th cytokines (ifn-γ, il- , soluble tumor necrosis factor receptor (stnfr ), cxcl , cxcl , ccl ), and viral loads eventually recovered after the combination of prednisolone with ivig. a multicenter, doubleblind, randomized controlled trial for cases with severe influenza a (h n ) infection demonstrated that ivig reduced the serum concentration of cytokines, viral load, and reduced mortality ( ) . a meta-analysis of studies ( , participants) found igm-enriched polyclonal and standard ig molecules decreased mortality in adults with severe sepsis or septic shock. however, a meta-analysis did not reveal a benefit in adult mortality with polyclonal ivig using high-quality trials only ( ) . despite a lack of clinical evidence, the us gave emergency approval to hcq, a member of antimalarial agents, in covid- on march ( ) . a meta-analysis included the studies up to april ( ) and showed that four clinical trials and three observational studies are eligible for the study. unfortunately, the authors concluded that hcq has no clinical effect on patients with covid- . however, a randomized clinical trial published on april, which included the patients (n = ) with critically ill covid- (such as high respiratory rate, peripheral oxygen saturation lower than %, shock), indicated . % patients ( of ) have died in the low-dosage group (i.e., mg twice daily on day and once daily for days). the critically ill death rate is over %, as reported by who ( ) . thus, low-dosage of hcq could be beneficial for critically ill patients with covid- . the study also indicates high dosage hcq might not be suitable for critically ill patients because of its potential safety hazards. traditional chinese medicine (tcm) has an essential role in the latest sars epidemic. several studies ( ) ( ) ( ) ( ) ( ) ( ) have shown that the add-on of tcm to western medicine can shorten the duration of hospitalization, alleviate symptoms, reduce mortality (including for critically ill patients), and reduce the prevalence of adverse reactions in sars. compared with a control group (western medicine only), a combination of tcm with western medicine has shown advantages in terms of symptom alleviation and preventing covid- ( ) ( ) ( ) . however, the quality of the studies must be improved. the administration of tcm in a standard manner worldwide is complicated because of the different decoctions used and the matching of herbs. artemisinin can be obtained from artemisia annua, and one kind of antimalarial agents. hou and colleagues showed that extracts from artemisinin-family drugs could regulate cells from the innate and adaptive immune system, and lead to anti-inflammatory and immunomodulatory actions ( ). the scope of application for artemisinin-family medicines includes infectious disease and autoimmune diseases, and artemisininfamily shows a difference in immune regulation compared with hydroxychloroquine ( - ). as stated above, ali and aki are crucial mortality factors in infectious diseases. artesunate is a derivative of artemisinin and can lessen the pathologic changes and neutrophil infiltration in the lungs of ali patients, and decrease sepsis-induced mortality ( ) . by inhibiting expression of nf-κb signaling and enhancing heme oxygense- expression, the artesunate can lower the concentrations of tnf-α and il- in serum and bronchoalveolar lavage fluid. huang and colleagues discovered that dihydroartemisinin could attenuate lipopolysaccharide (lps)-induced ali through suppressing nf-κb signaling in a nuclear factor erythroid -related factor (nrf )-dependent fashion, thereby leading to a decrease in expression of the pro-inflammatory cytokines il- β, tnf-α, and il- ( ). hu and colleagues explored a new and efficacious approach for ali ( ) . "artesunate liposomes" were prepared using film dispersion and then lyophilized to obtain liposomal artesunate dry powder inhalers (ladpis). after treatment with ladpis, a rapid reduction in accelerated inhalation, ali syndromes, and levels of tnf-α and il- has been observed in rats. besides, kidney impairment in hospitalized covid- patients is associated with a high risk of in-hospital death ( ). cheng et al. ( ) observed that dihydroartemisinin lessened glomerular injury and relieving increases in the urine albumin: creatinine ratio and serum levels of creatinine. current evidence of pathologic changes of covid- suggests the dysregulation of the cytokines involves mainly macrophages/monocytes. in a burn-based sepsis model balb/c mice, concentrations of adhesion molecules and neutrophil infiltration in the lungs and heart, and mortality rate are significantly increased, but those phenotypes could be reversed by artemisinin ( ) . the authors discovered that artemisinin downregulates protein levels of nod-, lrr-and pyrin domaincontaining protein (nlrp ) and caspase in macrophages in burn-induced sepsis mice. also, a reduction in levels of the pro-inflammatory cytokines il- β and il- has been observed post-therapy. nlrp is a sensor component expressed mainly in macrophages and which undergoes transcription by nf-κb. nlrp is responsible for the maturation and secretion of il- β and il- ( ) ( ) ( ) . nf-κb also increases the level of il- in the macrophages infected by plasmodium falciparum, and artemisinin could reduce il- production in animal models ( ) , as well as in the clinic ( ) . two studies focused on the relationship among tlr, nf-κb, nucleotide-binding oligomerization domain-containing protein (nod) , and macrophages. tlr mainly locates outside the cell membrane of macrophages, dcs, and granulocytes, and recognizes bacteria ( ) . tlr induces nf-kb activation through recruitment of tir domain containing adaptor protein (tirap) and myeloid differentiation primary response (myd) in macrophages and dcs. in inflammatory monocytes, tlr is expressed within endosomes and induces the release of type-i ifns via interferon regulatory factor (irf ) and irf in response to viruses ( ) . artesunate increases survival of mice challenged with live staphylococcus aureus/methicillin-resistant staphylococcus aureus (mrsa) compared with antibiotics alone, and its protection may be associated with reductions in tnf-α levels. artesunate reduces the expression of tlr mrna and nod mrna that upregulated by s. aureus/mrsa and also inhibits the activation of nf-κb ( ) . kuang and colleagues found that the artesunate attenuated the release of tnf-α and il- from macrophages by inhibiting tlr -mediated autophagic activation ( ) . tlr also locates in the endolysosomal compartment, can recognize gram-negative bacteria and viruses ( ) , shares the same pathway as the activation of nf-κb, and induces the release of type-i ifns via the tnf receptorassociated factor (traf )-tank binding kinase (tbk )-irf axis ( ) . however, kuang and co-workers discovered that artesunate attenuates the cytokine release by the traf -beclin -class iii phosphatidylinositol -kinase (pi kc ) pathway. in a model of severe acute pancreatitis in rats, artesunate attenuates the release of il- β and il- via the tlr -nf-κb axis ( ) . in addition, dihydroartemisinin inhibited the activation of tlr and irf in the spleen cells of systemic lupus erythematosus (sle)-prone mrl/lpr mice, which lead to a decrease in levels of ifn-α and ifn-β ( ) . the mitogen-activated protein kinase (mapk) signaling pathway plays a vital part in the development, differentiation, proliferation, transformation, and apoptosis of cells ( ) . the extracellular signal-regulated kinase (erk), jnk/stressactivated protein kinases (sapk), and p mapk are the dominant members of the mapk family. the cascades can be summarized as the erk pathway (raf-mek-erk), jnk pathway (tak -mkk-jnk), and p pathway (tak -mkk-p ). proinflammatory cytokines such as il- and tnf-α, ifnα, and ifnγ can induce activation of the p pathway, and p can regulate nf-κb-dependent transcription after its nuclear translocation. meanwhile, nf-κb is a crucial transcriptor for il- , which could activate the il- -janus kinase (jak)-signal transducer and activator of transcription (stat) pathways ( ). wang and colleagues ( ) found that another artemisinin derivative, sm , suppressed generation of nitric oxide, tnf-α, il- β, and il- in lps-induced macrophages. the underlying mechanism was that sm reduced activation of p and erk, and jnk suppressed iκbα degradation. furthermore, they observed that nf-κb was inhibited correspondingly in sm -treated cells. in another lps-induced macrophage model, artemisinin has a property of prohibiting stat activation, and it leads to the reduction of no (an inflammatory-cascade inducer) in macrophages ( ) . except for stat , stat , and stat in the splenocytes of sle-prone mrl/lpr mice could be inhibited by sm , an artemisinin derivative ( ) . artesunate therapy has been shown to improve the survival of mice infected with the herpes simplex virus. artesunate can lower levels of il- β, il- , il- , ifn-γ, ccl , ccl , and ccl in these mice. these cytokines are produced primarily by apcs and th cells. previous studies have suggested that the artesunate can regulate th cells in virus infections. du and colleagues ( ) demonstrated that the artesunate downregulated the th response and reduced levels of ifn-γ, tnf-α, il- , il- , ccl , cxcl , and cxcl in an experimental model of cerebral malaria. ra is an autoimmune disease manifested by dysfunction of various immune cells (e.g., apcs, th , th ), which leads to a high concentration of il- , il- , tnf-α, and chemokines in plasma and tissues ( ) . in the experimental models of ra, the proliferation of th cells and the production of il- a and il- are inhibited by sm therapy and, correspondingly, the expression of retinoic acid receptor-related orphan nuclear receptor gamma t (rorγt) (a specific transcription factor for th cells) is also reduced ( ) . fan et al. ( ) demonstrated similar data and found that dc (an artemisinin derivative) can restore the t reg /th balance and reduce transcription of cxcl and cx cl . t reg can be anti-inflammatory, secrete anti-inflammatory cytokines (e.g., il- ), target th cells and macrophages, as well as reduce the concentration of il- , il- , tnf-α, and il- ( ) . the immunosuppressive mechanisms of artemisinin on t cells include inhibiting differentiation of th cells by regulating the figure | artemisinin-family drugs for cytokine storm in covid- . the dysregulation of the cytokine storm involves mainly apcs. tlr and tlr locate mainly outside macrophages, dcs, and granulocytes. also, they are expressed within endosomes, play a role in recognizing bacteria and viruses. through myd -dependent or trif-dependent pathway, tlr and tlr transmit signals for the activation of irf and nf-κb to induce the type i interferon and cytokines. besides, tlr leads to the activation of ap- , which is responsible for the transcription of inflammatory cytokines. the cytokines target at the naïve t helper cell, to result in the naïve t helper cell to differentiate to th cell and th cell, subsequently to secrete the inflammatory cytokines and chemokines. moreover, the il- , il- , and il- secreted by monocytes and macrophages could activate cytokines receptors (i.e., il- r, il- r), lead to the activation of jak-stat signaling pathways and cell migration. the artemisinin-family drugs target at a variety of molecules (red and blueness nodes) in the inflammatory networks, such as nf-κb, irf , erk (not shown in the figure), and rorγt, which inhibit the differentiation of inflammatory cells and the production of cytokines and chemokines. il- is an anti-inflammatory cytokine. it could be secreted by virtually all immune cells, including macrophages, dcs, nk cells, t cells, and b cells. at the moment, the high concentration of il- in severely ill patients with covid- is a mystery. on the one hand, it might play a role in antagonizing the biological function induced by il- . on the other hand, the high concentration of il- might contribute to the lymphocytes exhaustion. ap- , activating protein- ; ccl, c-c motif chemokine ligand; cxcl, c-x-c motif chemokine ligand; ikk, iκb kinase; ifn, interferon; irf , interferon response factor ; jak, janus kinase; jnk, jun n-terminal kinase; myd , myeloid differentiation primary response protein ; nf-κb, nuclear factor κ b; nlpr , nod-, lrr-and pyrin domain-containing protein ; mkk, mitogen-activated protein kinase kinase; smad , smad family member ; rorγt, retinoic acid receptor-related orphan nuclear receptor gamma t; stat, signal transducer and activator of transcription; tak , tgfβ-activated kinase; t-bet, t-box transcription factor (also known as tbx ); tlr, toll-like receptor; traf, tnf receptor-associated factor; tram, trif-related adaptor molecule; trif, tir domain-containing adaptor protein inducing interferon-β. il, interleukin. expression of rorγt and maybe also inhibition of activation of the erk pathway (ras-raf -erk / ) ( ). in the model of ra-fibroblast-like synoviocytes (fls), artesunate decreased the production of il- , il- , and il- β through preventing nf-κb translocation and iκbα degradation ( ) . artemisinin-family drugs have shown efficacy and safety in treating malaria. one study reported patients with severe malaria caused by plasmodium falciparum. ten patients suffered renal failure, eight had cerebral malaria, and had other causes of severe malaria. after artesunate treatment, concentrations of il- , and soluble il- receptor in plasma were normalized within h ( ). in recent years, artemisinin-family drugs have been shown to be beneficial against infection caused by the human cytomegalovirus, hepatitis-b virus, ebola virus, and human immunodeficiency virus ( ) . shapira and co-workers reported the first case of the treatment of hcmv infection with artesunate ( ) . germi and collaborators ( ) reported that the artesunate led to an effective response in three cases with mild hcmv infection but was not efficacious in two patients with severe hcmv infection. the elevations of il- and il- are highly consistent in covid- . il- targets the il- receptor, and the letter recruit jak, which transit cascade signal to activate signal transducer and activator of transcription (stat ) ( ) . some physicians suggest tofacitinib, a small molecule compound target jak and jak , could be applied in the treatment of covid- , and tofacitinib success in treating a covid- patient complicated with ulcerative colitis ( ) . il- , a cytokine with anti-inflammatory properties, could be secreted by virtually all immune cells, including macrophages, dcs, nk cells, t cells, and b cells ( ) . we might tend to regard the high levels of il- as negative feedback of counteracting the increase of il- because il- can block the activity of nf-κb to downregulate the production of il- ( ). however, an abundance of il- also inhibits the function and proliferation of immune cells (e.g., th , nk cells, and cd t cells), which delays the clearance of viruses ( ) . therefore, a mass of il- might be responsible for the normal levels (one study report low level) of ifn-γ (a cytokine for the clearance of viruses) and the exhaustion of lymphocytes. the il- inhibitor in the treatment of covid- also needs to be considered. even the combination of il- and il- inhibitor could be designed in future prospective studies. when using any method to regulate the dysregulation of cytokines, we might better closely monitor the laboratory index for preventing overtreatment. for example, if we use tcz to reduce the levels of il- , we could check il- levels once every days to keep it at a suitable concentration, which should be studied in the future. also, the dose and duration would be illuminated. the current evidence indicates that tcz, an il- inhibitor, is relatively effective and safe. based on the therapeutic mechanisms, we classified the remaining therapies, corticosteroids, pd- /pd-l checkpoint inhibition, cytokine-adsorption devices, intravenous immunoglobulin, and antimalarial agents, as "less potential treatments." no literature of covid- except for corticosteroids mentions the effectiveness and safety of the less potential treatments. the benefits, dose, duration, and timing of corticosteroids still in debate, and the other less potential treatments need clinical evidence to validate. although the experimental model of infectious disease (e.g., malaria and sepsis) and autoimmune disease (e.g., ra and sle) indicates that artemisinin-family drugs could target the inflammatory networks to decrease the levels of cytokines (e.g., il- and tnf-α) and chemokines (e.g., il- , cxcl ) (figure ) . the effect and safety of antimalarial agents still need to be validated in the high-quality clinical studies and the sars-cov- infection disease model. a precise definition of a cytokine storm is needed urgently. mehta et al. ( ) suggest that the criteria of shlh could be applied. moreover, the term needs to be placed in the icd code. the icd code would bring the standardization of disease names, the convenience of electronic medical records (emr) management, and the efficiency in information sharing. for example, the characteristic of cytokine storm would be more accessible to be collected for a retrospective study. yt: manuscript preparation and wrote the main part of the manuscript. jl: evidence collection, wrote the parts of the manuscript, and manuscript editing. dz: helped to perform the analysis with constructive discussions. zx: helped to revise the manuscript and gave many professional suggestions. jj: ideas, formulation of overarching research goals, and aims. cw: critically reviewed the manuscript, project funding, and study initiation. all authors approved the final version of the manuscript. we thank the charlesworth group (https://www.cwauthors.com. cn) for its linguistic assistance during the 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in five transplant recipients with disease caused by drug-resistant cytomegalovirus case report of a sars-cov- infection in a patient with ulcerative colitis on tofacitinib il- : a multifunctional cytokine in viral infections covid- : consider cytokine storm syndromes and immunosuppression key: cord- -gbmnoo authors: callender, lauren a.; curran, michelle; bates, stephanie m.; mairesse, maelle; weigandt, julia; betts, catherine j. title: the impact of pre-existing comorbidities and therapeutic interventions on covid- date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: gbmnoo evidence from the global outbreak of sars-cov- has clearly demonstrated that individuals with pre-existing comorbidities are at a much greater risk of dying from covid- . this is of great concern for individuals living with these conditions, and a major challenge for global healthcare systems and biomedical research. not all comorbidities confer the same risk, however, many affect the function of the immune system, which in turn directly impacts the response to covid- . furthermore, the myriad of drugs prescribed for these comorbidities can also influence the progression of covid- and limit additional treatment options available for covid- . here, we review immune dysfunction in response to sars-cov- infection and the impact of pre-existing comorbidities on the development of covid- . we explore how underlying disease etiologies and common therapies used to treat these conditions exacerbate covid- progression. moreover, we discuss the long-term challenges associated with the use of both novel and repurposed therapies for the treatment of covid- in patients with pre-existing comorbidities. the novel severe acute respiratory syndrome coronavirus (sars-cov- ), and subsequent sars-cov- induced coronavirus disease has spread on an unprecedented scale. according to the world health organization (who), as of the th july , there have been , , cases and , covid- related deaths worldwide. evidence from the global outbreak has clearly demonstrated that individuals with pre-existing comorbidities such as hypertension, cardiovascular disease, and diabetes are at a much greater risk of dying from covid- ( , ) . this is of great concern for individuals living with these conditions, and a major challenge for global healthcare systems and biomedical research. given that comorbidities are associated with high mortality among covid- patients, a better understanding of the biological mechanisms that underpin this risk are needed to enable development of appropriate preventative and therapeutic strategies. the immune system plays a vital role during covid- , and the degree of immune dysfunction correlates with disease severity ( , ) . severe covid- cases are associated with significant lymphopenia and an overactivated innate immune response resulting in hyperinflammation ( ) . many covid- associated comorbidities affect the function of the immune system, which in turn directly impacts the response to covid- . furthermore, the myriad of drugs prescribed for these comorbidities will also influence the progression of covid- and limit additional treatment options available for covid- . here, we review the current sars-cov- literature and explore how preexisting comorbidities adversely affect covid- outcome. furthermore, we discuss the long-term challenges associated with the use of both novel and repurposed therapies for the treatment of covid- in patients with pre-existing comorbidities. in december , several cases of an infectious pneumonia with an unknown etiology emerged in wuhan province, china. by january , a novel coronavirus termed sars-cov- was identified as the cause. the virus spread rapidly and was classified as a pandemic by the who on the th march . however, this is not the first pathogenic human coronavirus to emerge in the last decade. in , a severe acute respiratory syndrome (sars) coronavirus (sars-cov) with animal to human transmission was reported in guangdong province, china ( , ) . prior to sars-cov, four human coronaviruses belonging to the alpha and beta genera of the coronaviridae family had been identified: hcov- e, hcov-oc , hcov-nl , and hcov-hku ( ) . however, unlike the previously identified coronaviruses, sars-cov was phylogenetically distinct ( ) . furthermore, rather than causing upper respiratory tract infections with mild common cold symptoms, sars-cov caused severe lower respiratory tract infections, resulting in viral pneumonia and risk of developing acute respiratory distress syndrome (ards). the sars-cov outbreak lasted months, and infected , individuals across different countries, with a mortality rate of ∼ % ( ) . in , a second novel human coronavirus emerged in saudi-arabia and was termed middle east respiratory syndrome (mers) coronavirus (mers-cov). similar to sars-cov, infection with mers-cov can cause fatal pneumonia. to date, the who has reported , mers-cov cases, with a mortality rate of ∼ % ( ) . while the previous two coronavirus outbreaks were relatively well-contained, the unprecedented spread of the current pandemic has demonstrated increased infectivity of sars-cov- . early genome sequencing from china revealed that the k base-pair viral genome of sars-cov- shared . % sequence identity with sars-cov, whereas a bat coronavirus previously detected in rhinolophus affinis shared % sequence identity ( , ) . whilst mers-cov utilizes dipeptidyl peptidase- (ddp ) for cell entry, sars-cov, and sars-cov- share the same cell entry receptor; angiotensin converting enzyme ii (ace ) ( , ) . ace is recognized by the s -subunit of the spike protein and is ubiquitously expressed in the epithelia of the nasal cavity, airway tract and the alveolar space. notably, reports have shown that the receptor binding domain of sars-cov- s has a higher affinity for ace , which may be one contributing factor to the increased viral pathogenesis of sars-cov- ( , ) . the scale of the ongoing pandemic demonstrates the need for a more comprehensive understanding of the disease, and of the contributing factors such as pre-existing comorbidities, which are proving detrimental for disease severity and outcome. clinical presentation of covid- varies greatly. a metaanalysis of studies from countries ( , patients) reported . % of cases as mild, . % as severe and . % as critical ( ) . most healthy individuals are asymptomatic or present with mild/moderate respiratory illness ( ) . the majority of critical cases occur in older (≥ years) or comorbid individuals ( , , ) . covid- symptoms are typical of pathogenic human coronaviruses (figure ) ( ) , with fever and cough reported most commonly, however other common symptoms include dyspnoea, sore throat, sputum production, fatiguem and headache ( , , ) . more recently, olfactory dysfunction such as anosmia has been described in covid- patients ( , ( ) ( ) ( ) ( ) . furthermore, rare gastrointestinal symptoms such as nausea, diarrhea and vomiting have also been described ( , , ) . patients with severe covid- can develop serious and potentially fatal complications such as ards, thromboembolic events, septic shock and multiple organ failure (figure ) . due to the severity of these complications, many are associated with critical covid- patients who require intensive care ( , ) . due to underreporting and large inter-country variation, the fatality rate remains unclear. for instance, in italy the overall case-fatality rate has been reported as . % compared to . % in china ( ) . however, despite the variation, the evidence clearly demonstrates a higher fatality rate among those who develop severe covid- ( ) . the immune system plays a vital role during covid- , and the degree of immune dysfunction correlates with disease severity (figure ) ( , ) . during sars-cov- infection the immune system becomes activated, resulting in local inflammation, the recruitment of monocytes, dendritic cells (dcs), natural killer (nk), t and b cells. this response may manifest as mild/moderate disease resulting in a fever, cough and fatigue, however this will be followed by resolution of both the infection and inflammation. in severe covid- cases, severe lymphopenia and the accumulation of functionally exhausted t and nk cells result in an inability to mount an effective antiviral immune response to clear sars-cov- ( , ) . furthermore, interleukin (il- ) levels remain elevated over time, and are accompanied by high levels of il- , il- , il- , tumor necrosis factor-α (tnf-α), c-x-c motif chemokine (cxcl- ), monocyte chemoattractant protein- (mcp- ), and macrophage inflammatory protein- α (mip- α) resulting in systemic cytokine storm ( ) . this uncontrolled systemic hyperinflammation can cause the development of critical and potentially life-threatening complications such as severe pneumonia, ards, septic shock and multiple organ failure ( , , ) . both lymphopenia and hyperinflammation figure | prevalence of covid- symptoms and complications. data obtained from a quantitative meta-analysis of studies ( ) . it's important to note that % of all cases analyzed in the meta-analysis were hospitalized cases. therefore, the proportion of symptoms and complications are representative of this and the overall proportions with regards to all covid- cases will be much lower. the prevalence of anosmia ( ) and thromboembolic events ( ) were obtained independently from smaller cohorts and may therefore change as more data is published. figure | immune response in mild/moderate and severe covid- cases. mild/moderate covid- is characterized by local inflammation, the recruitment of monocytes, dcs, nk cells, t and b cells, followed by resolution of the infection and inflammation. severe covid- is characterized by severe lymphopenia, t cell exhaustion, and systemic hyperinflammation that can cause the development of critical and potentially life-threatening complication such as severe pneumonia, ards, septic shock, and multiple organ failure ( , , ) . are being reported in the majority of covid- cases admitted to hospital and is associated with a poor prognosis ( , , ) . more detailed examination has demonstrated negative effects on all lymphocyte subpopulations including cd + and cd + t cells, b cells, and nk cells ( ) . high-dimensional analysis of circulatory immune profiles in mild, moderate and severe covid- patients by mass cytometry revealed that proportions of naïve cd + t cells, tgfβ + cd − naïve cd + t cells, dcs, and macrophages are associated with mild cases, whereas a sharp decline in the proportion of cd + t cells and nk cells was observed in severe cases ( ) . interestingly, single-cell rna sequencing of pbmcs isolated from hospitalized covid- patients revealed a novel population of developing neutrophils, which appeared to be closely related to plasmablasts, in patients that had developed ards ( ) . as this was a small cohort of patients, further studies are needed to assess whether this novel subset of neutrophils plays a role in the development of ards and other covid- complications. functionally, cd + t cells and nk cells in severe covid- patients exhibited more signs of exhaustion than mild/moderate patients ( , ) . for example, elevated programmed cell death protein- (pd- ), cytotoxic t-lymphocyte-associated protein- (ctla- ) and t cell ig and itm domain (tigit) on cd + t cells and increased nkg a on nk cells ( , ) . as exhausted t and nk cells are less able to mount an effective antiviral immune response, it is unsurprising that these subsets are unable to eradicate sars-cov- and correlate with severe covid- cases. in addition to t cell changes, humoral immunity against sars-cov- is starting to come to light. evidence of sars-cov- specific antibodies was demonstrated in a study of hospitalized patients. igg and igm sars-cov antibodies were present in % of patients within week of onset and % by day ( ) . in another study, most patients developed robust antibody responses between and days, and although delayed, a stronger antibody response was observed in critical patients ( ) . these findings were also mirrored in a study of covid- patients, who all showed a positive igg response by day , followed by seroconversion to igm ( ) . interestingly, antibody titers were found to be higher among severe covid- patients ( ) , however the authors acknowledge that interpreting an association between antibody response and disease severity is difficult due to the small sample size of severe and critical patients in their study. a recent study reported low variable plasma antibody titers in convalescent individuals, however they found binding domain specific antibodies with potent anti-viral activity in all individuals ( ) . older individuals (≥ years) are more prone to severe covid- and have a higher mortality rate ( , , , ) . clinically, older patients have more pronounced immune dysfunction compared to younger patients, as lymphocyte counts are lower and pro-inflammatory cytokine levels higher ( ) . this is not surprising as aged immune systems are associated with immunosenescence and chronic low-grade inflammation, termed inflammaging ( ) . although immunosenescence affects all aspects of the immune system, much of the deterioration in protective viral immunity can be attributed to defective t cell immunity ( ) . the decline of naïve t cell output due to thymic involution ( ) and the accumulation of senescent t cells leads to reduced viral host immunity ( ) . in mice, cd + t cells were shown to be crucial against sars due to their important role in sars-cov clearance. this protection was lost in aged mice as senescent cd + t cells responded poorly to antigen ( , ) . moreover, in addition to inflammaging, the accumulation of senescent cd + t cells and b cells with distinct senescenceassociated secretory phenotypes ( , ) in older individuals results in elevated baseline inflammation, further increasing susceptibility to hyperinflammation and cytokine storm upon sars-cov- infection. in addition to age, biological sex and ethnicity have also been implicated in covid- outcomes. although no major sex differences exist when examining absolute number of covid- cases, disease incidence is higher in males when comparing older individuals (≥ years). furthermore, initial reports from china suggested a male bias in mortality ( , ) , which has now been reported in out of countries that have reported sex-disaggregated data, revealing a global male case fatality rate of . % compared to . % in females ( , ) . this finding is consistent with data obtained from the previous sars and mers epidemic ( ) ( ) ( ) . the predominant hypothesis to explain these biological sex differences is that estrogen plays a protective role against covid- . following the sars epidemic, studies in mice demonstrated that ovariectomy or pharmaceutical blocking of estrogen in female mice resulted in elevated immune cell infiltration in the lung and consequently a more severe disease outcome ( ) . in support of this, researchers in china reported that lower levels of estrogen were associated with more severe covid- cases in women ( ) . although the exact molecular mechanisms underpinning how estrogen protects against covid- are yet to be confirmed, the influence of estrogen on aging and immunity, ace levels, and sex-related risk factors for comorbidities have all been suggested ( , , ) . due to these benefits, researchers in the uk have commenced investigations into the effects of hormonal therapies such as the contraceptive pill and hormone replacement therapy, however no data has been published yet. more recently, data has emerged suggesting that black, asian and minority ethnic (bame) individuals are at a greater risk of acquiring sars-cov- and have worse clinical outcomes ( ) . for instance, in the uk two thirds of covid- fatalities among healthcare workers were bame individuals ( , ) . underlying comorbidities, which are more prevalent in bame individuals, in addition to cultural, behavioral and socio-economic differences have been proposed as possible causes ( ) . however, more data is needed in order to truly establish whether a relationship between covid- and ethnicity exists. evidence from the global outbreak has demonstrated that individuals with pre-existing comorbidities are at a much greater risk of dying from covid- ( , ) . however, a greater understanding of the biological mechanisms that underpin this risk is needed to develop appropriate preventative and therapeutic strategies. here we review the major comorbidities identified in a number of meta-analyses ( figure ) ( , ). after identification, independent searches were conducted in order to comprehensively assess the impact of each comorbidity and its associated therapies on sars-cov- risk, covid- progression and outcome, and future covid- therapy options. hypertension has been repeatedly reported as the highest pre-existing comorbidity in covid- patients ( , , , - ). retrospective analysis revealed that patients with hypertension have an increased risk for severe infection and mortality ( , ) . however, whether hypertension itself or the use of hypertensive therapies are responsible for these statistics is currently unknown. hypertensive patients are commonly treated with renin angiotensin system inhibitors, such as ace inhibitors (acei) and angiotensin-receptor blockers (arb). as acei and arb can significantly increase ace expression ( ), many speculate they are responsible for the increased risk to hypertensive patients ( ) . conversely, a retrospective review of hospitalized covid- patients, of which . % had underlying hypertensions, indicated that acei and arb may be protective effect against covid- , as the percentage of severe cases were lower in patients treated with acei/arb ( . %) when compared to those treated with other anti-hypertensive treatments such as calcium channel blockers, β-blockers, and diuretics ( %) ( ) . however, patients treated with non-aci/arb were also found to have a higher incidence of additional comorbidities ( ), which may have been responsible for the development of severe disease. due to the conflicting evidence and opinions among the scientific community, it remains unclear whether treatment with acei/arb has a positive or negative impact on covid- progression, however many are continuing to examine this. ( ). hypertension ( %), cardiovascular disease ( . %), and diabetes ( . %) were the most prevalent pre-existing co-morbidities. as this data is representative of hospitalized patients, the prevalence of these among mild/moderate cases may be different, and as more data is analyzed these may change. frontiers in immunology | www.frontiersin.org cardiovascular disease has also been highly reported among covid- patients and is associated with an increased mortality rate ( , , ) . furthermore, cardiovascular complications such as thromboembolic events, myocarditis, acute coronary syndrome, arrythmia, cardiogenic shock and heat failure, have been documented in covid- patients without prior cardiovascular disease ( ), demonstrating a significant impact of sars-cov- infection on the heart. in a case series of covid- patients, those with underlying cardiovascular disease had a mortality rate of . %, which was further increased to . % in a subset of patients who had both underlying cardiovascular disease and elevated troponin t levels, indicative of myocardial injury ( ) . two possible explanations for the increased prevalence and mortality among patients with comorbid cardiovascular disease have been proposed. firstly, cardiovascular disease is commonly treated with renin angiotensin system inhibitors as described above ( , ) , and secondly, ace is highly expressed in the heart ( ) . a recent study analyzing the cellular distribution of ace in human heart tissue obtained from covid- patients identified that ace was highly expressed in pericytes, cardiomyocytes and fibroblasts ( ) . furthermore, cd , an additional binding receptor for sars-cov, was specifically expressed in macrophages ( ) . due to the increased presence of macrophages in cardiovascular disease, the authors speculate that cd + macrophages may enhance viral entry into the human heart. collectively, this study indicated an intrinsic susceptibility to sars-cov- infection in the heart, which could explain the high susceptibility to covid- among cardiovascular disease patients and the higher incidence of acute cardiac injury in non-cardiovascular disease patients. according to numerous reports severe covid- patients are at heightened risk of thromboembolic events, with - % of critically ill covid- patients reported to have developed thromboembolic complications ( , ( ) ( ) ( ) ( ) . systemic inflammation and subsequent activation of coagulation are both contributing factors to this increased risk ( , ) . coagulation abnormalities have been reported throughout the covid- pandemic and the term covid- -associated coagulopathy (cac) has been used to describe patients displaying coagulation changes ( ) . elevated levels of prothrombin, fibrinogen and ddimer, in addition to elevated inflammatory markers such as c-reactive protein (crp) and il- , are markers of cac ( ) . in particular, increased d-dimer levels highly correlate with disease severity, as elevated d-dimer presenting at admission or over time are associated with increase mortality in covid- patients ( ) . due to the high incidence of thromboembolic events, the use of thromboprophylaxis for patients admitted to hospital with severe covid- has been suggested ( , ) , and cac should be monitored carefully in all hospitalized covid- patient, particularly those with pre-existing risk of thromboembolic events. diabetes is the third most prevalent underlying comorbidity in covid- patients ( , , , , ) . type diabetes is a multifactorial disease characterized by chronic inflammation and impaired metabolism and has become an increasing risk to human health. diabetic individuals have an increased susceptibility to infection ( ) , and are at a high risk of developing multiple comorbidities such as cardiovascular disease ( ) . one study on covid- found that diabetic patients were more likely to develop pneumonia and were responsible for . % of severe cases, but only % of mild/moderate cases ( ) . there are a number of reasons as to why people with diabetes are more likely to develop severe covid- . firstly, chronic inflammation in diabetic patients increases their susceptibility to hyperinflammation and the development of cytokine storm. this has been already reported in covid- patients, as il- and crp levels were found to be significantly higher in diabetic patients ( ) . secondly, it is well-documented that hyperglycaemia can impair the immune response, increase oxidative stress and is associated with the onset of premature senescence ( , ) . consequently, diabetic patients that are unable to control their blood glucose levels may have an even greater vulnerability to severe disease. finally, in addition to disease etiology, the treatment of diabetes may also impact covid- development. as previously discussed for hypertension and cardiovascular disease, the use of renin angiotensin system inhibitors may increase susceptibility to sars-cov- infection ( , ) . furthermore, dpp inhibitors commonly used to treat diabetes have an anti-inflammatory effect, resulting in reduced macrophage infiltration, which could impair the innate immune response during covid- ( ) . obesity is associated with most of the common covid- comorbidities such as hypertension, cardiovascular disease and diabetes ( , ) . the global prevalence of obesity varies greatly, for example obesity is more common in the united states and europe than it is in asian countries ( ) . consequently, covid- severity and mortality rates may also vary as a result of this. one study reported a higher bmi in patients with severe infection, and when comparing survivors vs. nonsurvivors, it was reported that . % of non-survivors had a bmi above kg/m , which was a significantly higher proportion than survivors ( ) . however, the cohort for this study was small (n = ) and further retrospective analysis of existing studies is needed to clarify the impact of obesity on covid- . following the previous h n pandemic, retrospective analyses reported that obesity was associated with increased risk of severe infection and mortality ( ) , which is in line with the increased risk of infection in obese individuals ( ) . in addition to increased risk of comorbidities, obesity is also linked to an impaired immune response ( ) , with evidence of impaired antibody ( ) and t cell ( ) responses. furthermore, expression of ace is upregulated in adipocytes of obese individuals and therefore may act as a potential target for sars-cov- ( ). the incidence of cancer as a covid- comorbidity has been low. in an extensive meta-analysis of , patients, malignancy accounted for just . % of comorbidities reported ( ). however, a nationwide analysis in china reported that . % of covid- cases had active cancer, and these patients had a higher proportion of serious events in comparison to individuals without cancer ( ) . at this current time, it is unclear whether cancer patients are at high risk of covid- due to an immunocompromised state linked to certain cancer therapies. in one particular study, patients that underwent recent chemotherapy or surgery had a higher risk of clinically severe events than those who did not. however, a limited number of just patients were included in this analysis ( ) , demonstrating the need for more thorough research. furthermore, not all cancer patients should be considered equally immunocompromised. cancer patients treated with immuno-oncology therapies such as immune checkpoint inhibitors (ici) could be more immunocompetent than patients undergoing chemotherapy ( ) . nonetheless, there are two major concern associated with the use of immuno-oncology therapies during covid- . firstly, there is potential overlap between covid- interstitial pneumonia and possible pneumological toxicity from anti-pd- /pdl- agents, which can be fatal. although this is a rare immune-related adverse event, it has been reported in . - % of patients treated with anti-pd- /pdl- monotherapy, and - % of patients treated with anti-pd- /anti-ctla- combination therapy ( , ) . secondly, there is a risk of cytokine release syndrome associated with t cell-engaging immunotherapy, such as chimeric antigen receptor (car) t cells. given that cytokine storm has been linked to a negative outcome in covid- due to the development of ards and multiple organ failure, ici and car-t cell therapies may exacerbate this hyperinflammatory state and increase mortality in these patients ( ) . overall, despite the lower incidence of cancer among covid- patients, those being treated with immunocompromising therapies such as chemotherapy, and those susceptible to immune-related adverse events in response to immuno-oncology therapies should be monitored carefully. as infection with sars-cov- results in an acute respiratory disease that can progress to ards, respiratory failure and potentially even death, it is reasonable to speculate that patients with pre-existing respiratory disease would be at increased risk of severe covid- . surprisingly, this risk is not as striking as one might anticipate, as the prevalence of asthma was just . % in a study of covid- patients in china ( ) . furthermore, the incidence of chronic obstructive pulmonary disease (copd) among hospitalized covid- patients was reported to be just . % in a meta-analysis of , patients ( ). conversely, the center for disease control and prevention (cdc) recently released data of us hospitalizations indicating that copd is present in . % of patients ( ) . a possible explanation for this global variation could be due to differences in therapeutics agents and disease management. for instance, a global survey assessing the severity and control of , asthmatic adults worldwide published that individuals in japan and asia-pacific regions reported less severe disease than those in europe and the united states ( ). the prevalence of chronic liver disease in covid- patients is estimated to be % ( , ) . however, liver damage has been repeatedly reported as a common complication in response to covid- ( , , ) . in a study of , patients in china, elevated levels of the liver enzyme aspartate aminotransferase (ast) were reported in % of mild/moderate covid- patients, and % of severe covid- patients ( ) . the same study also reported elevated levels of alanine aminotransferase (alt) in % of mild/moderate covid- patients and % of severe covid- patients ( ) . consequently, it has been proposed that liver damage associated with severe covid- patients is due to dysregulated innate immunity against sars-cov- , or hepatoxicity in response to treatments, rather than pre-existing liver disease. chronic kidney disease is associated with an increased risk of pneumonia, and elevated mortality from infectious diseases has been reported in patients with end stage renal disease ( ) . in an extensive meta-analysis, chronic kidney disease accounted for . % of comorbidities in covid- patients ( ). while the prevalence of chronic kidney disease among covid- patients is low, those with pre-existing kidney disease have been associated with severe disease and increased mortalities ( ) . ace expression in the kidney is elevated in chronic kidney disease ( ) . however, elevated ace expression in the kidney does not appear to correlate with increased susceptibility to sars-cov- in the same way as it does in the heart. nonetheless, chronic kidney disease is associated with persistent, low-grade inflammation which could exacerbate covid- symptoms. several factors contribute to this inflammation such as elevated cytokines including il- and crp, oxidative stress and impaired metabolism ( ) . therefore, the underlying pathogenesis of chronic kidney disease may increase vulnerability to hyperinflammation and cytokine storm upon sars-cov- infection, resulting in severe covid- . autoimmune diseases are conditions characterized by inappropriate immune activation and destruction of healthy cells. lymphopenia is common among autoimmune diseases such as type diabetes, rheumatoid arthritis and systemic erythematosus lupus ( ) , and since lymphopenia is regarded as a major risk factor for developing severe covid- , individuals living with an autoimmune condition may be perceived as high risk. however, unlike other comorbidities mentioned above, autoimmune diseases have not been reported as a risk factor in the current meta-analyses. one possible explanation could be that autoimmune diseases are often treated with drugs designed to restrict immune activation, many of which are now being repurposed for covid- and will be discussed below. furthermore, potential alterations in patient behaviors in order to shield themselves from infection could influence reporting of autoimmunity as a risk factor. as the pandemic continues to accelerate globally, so does the race to develop an effective therapy to protect against and treat covid- (figure ) . however, robust efficacy and safety assessments are needed, particularly with regards to their use in comorbid patients, as reviewed below. the sars-cov- genome and spike protein structure was discovered very rapidly ( ) , enabling focussed development of both rna-and protein-based vaccines. however, vaccine development is a challenging and time-consuming process. following the sars-cov epidemic, several vaccines were developed and assessed using animal models. vaccination with live virus was shown to cause complications in mice such as lung damage ( , ) , and although recombinant spike protein based vaccines were able to protect animals from sars-cov challenge, they were ineffective at inducing sterilizing immunity ( ) . other vaccines with inactivated sars-cov and mers-cov have demonstrated reduced viral titers, reduced morbidity and greater survival in animal models ( , ) . however, the rapid eradication of sars-cov reduced demand for development. some mers-cov vaccines are currently in pre-clinical and clinical development ( ) , however as mers-cov is less closely related, it is unlikely that these will cross-protect against sars-cov- . learnings from previous coronavirus outbreaks indicate that antibody responses are not particularly long-lived, with sars-cov antibodies lasting on average just years ( ) . this apparent lack of long-term protection exacerbates the need for an effective vaccine or vaccine programme to protect against potential recurrent seasonal sars-cov- infections. on the th july , the who reported candidate sars-cov- vaccines under development worldwide ( ) . some of the most promising include an mrna vaccine; mrna- ( ), a dna plasmid vaccine ( ) , and an adenovirus vaccine; chadox ncov- ( ) . although clinical evaluation is underway, outcomes are not available yet. furthermore, frontiers in immunology | www.frontiersin.org vaccine efficacy depends upon an individual's ability to mount a strong immune response against it. consequently, vaccine regimens that grant protection in healthy individuals may not be adequate for older individuals and those with comorbid conditions who have increased immunosenescence ( ) . for influenza, the use of adjuvant ( ) and high dose vaccines ( , ) have been developed and implemented to increase vaccine immunogenicity for older and high-risk comorbid adults. therefore, similar measures are likely to be needed to fine tune a sars-cov- vaccine once it becomes available. in the interim, rolling out a sars-cov- vaccine with proven efficacy in healthy individuals could result in herd immunity, preventing transmission to those more vulnerable and offer indirect protection. another therapeutic approach intended to prevent covid- is convalescent plasma therapy. previous use during the sars ( ) and mers ( ) epidemics, and h n pandemic ( ), demonstrated reasonable efficacy and safety. evidence from those outbreaks revealed that convalescent plasma contained neutralizing antibodies ( ) , and a meta-analysis from sars-cov and influenza studies, reported a significant reduction in mortality following convalescent plasma therapy ( ) . several convalescent plasma studies for sars-cov- have been reported. in china, a small pilot study was conducted to test convalescent plasma collected from recently recovered patients on severe covid- patients, four of which had underlying conditions including hypertension and cardiovascular disease ( ) . from the patients, had high neutralizing antibody titers of ≥ : . no serious adverse events were reported following transfusion, and all patients experienced improved symptoms within to days post-transfusion. however, there are many caveats to this study, the very small sample size and the use of concomitant treatments mean it is not possible to ascertain if clinical improvements were due to convalescent plasma. consequently, additional large scale, controlled, randomized trials are needed and are currently underway. despite no adverse events being reported in the small sample sizes currently being tested for covid- , plasma transfusions are not without risk. as with any transfusion, there is a risk of transfusion transmitted infections, albeit small ( ) . perhaps of more concern are the non-infectious risks such as allergic reactions, transfusion related acute lung injury characterized by acute hypoxemia and pulmonary oedema, and transfusion associated circulatory overload, characterized by hypertension, tachycardia, tachypnoea and dyspnea ( ). these are of particular concern for severe covid- patients with extensive lung damage, and for those with pre-existing hypertension, cardiovascular disease or renal failure ( , ). due to the increased prevalence of comorbidities among covid- patients, those predisposed to transfusion-related adverse events will need to be carefully evaluated. as transfusion volume and rate have both been identified as risk factors for transfusion associated circulatory overload ( ) , covid- trials should examine these parameters to ensure safety in comorbid patients. while many scientists attempt to develop novel therapies to prevent covid- , others are focusing their attention on repurposing drugs that are already on the market (figure ) . at present, the most contested repurposed drug is chloroquine and its analog hydroxychloroquine. following the sars-cov epidemic, researchers found that in vitro treatment of chloroquine could increase endosomal ph and impair terminal glycosylation of ace receptors on the cell surface, therefore inhibiting sars-cov-ace interactions and preventing virus entry ( , ) . similarly, after the mers-cov epidemic, researchers demonstrated that chloroquine could inhibit in vitro replication of mers-cov in well-established cell lines ( ) and primary mature antigen presenting cells ( ) . however, as sars-cov and mers-cov resolved relatively quickly, continued assessment of chloroquine remained limited. furthermore, although in vitro treatment can inhibit sars-cov- ( ), the use of chloroquine and hydroxychloroquine for covid- in the clinic is highly controversial. a series of early clinical trials conducted in china reported apparent efficacy of chloroquine phosphate in treating covid- ( ) . this was followed by a small, non-randomized study of just patients from france, which demonstrated a clinical benefit from the use of hydroxychloroquine, but lacked an appropriate control group and a long-term follow-up ( ) . however, an observational study of , patients treated with hydroxychloroquine report no sign of efficacy for covid- ( ) . this was further confirmed by results from the large randomized evaluation of covid- therapy (recovery) trial that examined , hospitalized covid- patients treated with hydroxychloroquine and reported no significant difference in mortality when compared to , standard of care patients ( , ) . lopinavir is a protease inhibitor often formulated with lowdose ritonavir and traditionally used to treat hiv patients ( ) . lopinavir/ritonavir was previously assessed in a small, nonrandomized study conducted in hong kong during the sars-cov epidemic. this study demonstrated that lopinavir/ritonavir treatment resulted in reduced viral load and milder disease, with less recurrence of fever, diarrhea, and improved chest radiographs when compared with those treated with ribavirin, an alternative antiviral ( ) . during the current outbreak, two independent case studies of covid- patients hospitalized in south korea, reported a reduction in viral load and subsequent recovery of patients following the administration of lopinavir/ritonavir ( , ) . however, a randomized control study in china of severe covid- patients demonstrated no difference in mortality, or in the amount of viral rna detected ( ) . furthermore, recent findings from the recovery trial comparing , patients randomized to lopinavir/ritonavir to , standard of care patients concluded no clinical benefit from the use of lopinavir/ritonavir ( , ) . moreover, protease inhibitors such as lopinavir/ritonavir have been associated with hepatotoxicity during hiv treatment ( , ) , and drugto-drug interaction (ddi) with certain statins, for example rosuvastatin, can increase the risk of myopathy ( , ) . therefore, administration to covid- patients with preexisting liver disease and those being treated with statins could prove detrimental. like other members of the protease inhibitor class, lopinavir/ritonavir has also been associated with metabolic changes that can result in hyperglycaemia ( ) , hyperlipidaemia ( ) , and insulin resistance ( ) . due to the lack of efficacy and increased risk of toxicity to certain covid- patients lopinavir/ritonavir could be prove more harmful to patients and should therefore be avoided. oseltamivir and favipiravir, two antiviral treatments traditionally used to treat influenza, have also been examined for use in covid- . oseltamivir is a neuraminidase inhibitor, which has been extensively used as a prophylactic against influenza. in a small, single-center study of patients with sars-cov- pneumonia in china, . % of patients received oseltamivir alongside anti-bacterial drugs such as moxifloxacin, ceftriaxone, azithromycin, and glucocorticoid therapy ( ) . the study concluded no effective outcomes based on oseltamivir ( ) . however, the small study size, lack of appropriate control and the fact that many patients remained hospitalized at the time of publications limits full interpretations. favipiravir is an antiviral with potent inhibitory activity against viral rna-dependent rna polymerase. experimentally, favipiravir demonstrated effective sars-cov- inhibition in vero e cells ( ) . furthermore, a small, open-label, non-randomized comparative study of patients in china, compared clinical outcomes of patients treated with favipiravir and lopinavir/ritonavir ( ) . the median time until viral clearance was days with favipiravir compared to days with lopinavir/ritonavir. at day , ct scans of the chest from patients treated with favipiravir demonstrated significant improvements. adverse events occurred in % of favipiravir treated patients compared to % of lopinavir/ritonavir treated patients ( ) . however, despite initial promise more robust clinical data is needed in order to establish the efficacy and safety of favipiravir as a treatment of covid- . another antiviral agent being examined for use in covid- is remdesivir. when metabolized into its active form, remdesivir inhibits viral rna polymerases, causing a decrease in viral rna production. remdesivir has been shown to inhibit sars-cov and mers-cov in human airway epithelial in vitro models ( , ) , and in combination with interferon beta, remdesivir has been shown to be superior to lopinavir/ritonavir in a mers-cov mouse model ( ) . despite some initial controversial findings ( ) , results from more robust clinical trials have demonstrated reasonable efficacy. for instance, preliminary results from the national institute of allergy and infectious diseases adaptive covid- treatment trial involving , patients, demonstrated that remdesivir accelerated recovered by % compared to placebo ( ) . furthermore, recent results from the phase simple trial investigating the use of remdesivir in patients with moderate covid- showed that patients receiving remdesivir treatment were % more likely to have improved clinically by day than standard of care patients ( , ). due to the success of these two trials, the use of remdesivir has been approved by the fda, ema, uk, and japan as a treatment for covid- . despite proven efficacy, adverse events in response to remdesivir have been reported in % of patients of which % were severe ( ) . these included septic shock, multiple organ dysfunction syndrome, acute kidney injury and hypotension. therefore, the use of remdesivir in comorbid patients with increased susceptible to these adverse events requires further evaluation. as severe covid- cases are characterized by hyperinflammation, the use of immune modulating and anti-inflammatory treatments to prevent severe lung injury and disease progression are being explored. due to their immunomodulatory properties, mscs are being clinically assessed to treat inflammatory conditions such as systemic lupus erythematosus ( ) and graft vs. host disease following allogeneic haemopoietic stem-cell transplantation ( ) . consequently, a pilot study was initiated to investigate the potential therapeutic benefit of mscs for covid- infected patients in china. the study involved just covid- patients who were monitored for days post-msc injection ( ) . msc treatment was well-tolerated and no adverse events occurred during treatment. furthermore, virtually all clinical symptoms subsided, with patients being discharged days post-msc injection ( ) . mass cytometry of pbmcs revealed that peripheral lymphocytes, regulatory cd + cd c + cd b mid dcs and il- increased. whereas, crp, tnf-α and overactivated cxcr + cd + /cd + /nk cells decreased to days following injection compared to placebo group. mscs were shown to be ace negative, meaning they were immune to sars-cov- infection ( ) . although this pilot study shows promise, more robust clinical data is required to validate therapeutic benefit and safety. moreover, the use of mscs would require clinical grade msc production and may not be a plausible solution for many healthcare systems. il- is considered the key cytokine responsible for the induction of cytokine storm during sars-cov, mers-cov and sars-cov- infections ( , , ) . consequently, the recombinant humanized anti-human il- receptor monoclonal antibody tocilizumab, currently used to treat rheumatoid arthritis (ra), has been examined for use during covid- . tocilizumab first demonstrated effectiveness in a small retrospective study of patients with severe covid- pneumonia in china ( ) . oxygen intake was reduced, and improved symptoms occurred in % of patients. lymphocyte levels returned to normal in % of patients and crp levels reduced significantly in . % of patients ( ) . several case studies have also demonstrated rapid clinical improvements following tocilizumab ( ) ( ) ( ) . although these initial studies are promising, the full clinical trial data is still unavailable. furthermore, although safety profiles for tocilizumab are well-established for intermittent use in ra, the safety of tocilizumab when used in combination with antiviral agents and other comorbid therapies has not been established. other anti-inflammatory treatments now being tested clinically for their use against covid- are janus kinase (jak) and bruton's tyrosine kinase (btk) inhibitors. the jak-signal transducer and activator of transcription (jak/stat) pathway mediates the signal transduction of numerous cytokines in a number of immune cells such as t cell, nk cells, and dcs ( ) . consequently, jak inhibitors have emerged as effective treatments for many autoimmune and immunemediated disease. at present, a number of jak inhibitors such as ruxolitinib ( ), baricitinib ( ) , and fedratinib ( ), are being assessed as a potential treatment for covid- . preliminary results from a pilot study evaluating hospitalized patients, of which were treated with baricitinib, demonstrated significant reductions in serum il- , il- β, and tnf-α, as well as a rapid recovery of circulatory t and b cell frequencies following baricitinib treatment ( ) . consequently, a phase adaptive covid- treatment trial (attc- ) has now been established in order to evaluation the use of baricitinib in combination with remdesivir compared to remdesivir alone ( ) . however, despite promising initial findings, as jak inhibitors block a wide range of cytokines including ifn-α, which is crucial during early innate immunity in response to viral infections, the impact of this on viral clearance needs to be evaluated. furthermore, ruxolitinib and baricitinib have both been associated with increased weight gain, cholesterol and albumin levels ( , ) . although no causal association has been reported yet, the issue should not be dismissed and covid- patients with metabolic and cardiovascular comorbidities should be carefully considered before use. bruton's tyrosine kinase (btk) is a key regulator of cell surface receptors expressed primarily in b cells, but also in monocytes/macrophages and neutrophils ( ) . currently, btk inhibitors are used to treat various b cell malignancies and chronic graft vs. host diseases ( ) . as btk can regulate il- , tnf-α, and mcp- , btk inhibitors are being tested in combination with car-t cells ( ) , to alleviate cytokine release syndrome. furthermore, in chronic lymphocytic leukemia, btk inhibitors have been shown to increase cd + and cd + t cell, and significantly downregulate pd- and ctla- ( ) , highlighting a potential reversal of t cell exhaustion, which could be beneficial in covid- . consequently, clinical trials to assess the use of btk inhibitors against covid- are underway. the use of corticosteroids to treat covid- remained largely uncertain until recently. although individuals being treated with long term corticosteroid were instructed to continue with their medication, the use of corticosteroids specifically to treat covid- was not recommended ( ) . this was largely due to the unknown impact of immune suppression on viral clearance and potential adverse outcomes. however, preliminary data from the recovery trial evaluating , patients randomly allocated to receive dexamethasone has proven significant clinical improvements ( ) . dexamethasone is a steroid used to reduce inflammation in a myriad of inflammatory conditions and now reported to reduce covid- related deaths among patients receiving respiratory support by one-third ( ) . in response to these findings the demand for dexamethasone to treat the most critical covid- patients has surged globally ( ) . whilst these findings are exciting, safety data detailing potential adverse events and the impact of co-medications, such as nonsteroidal anti-inflammatory drugs, has not yet been reported and thus dexamethasone should still be considered carefully prior to administration. patients with pre-existing comorbidities are at a greater risk of dying from covid- . however, not all comorbidities confer the same risk. by exploring the underlying disease etiologies and common therapies used to treat these conditions, we have discussed their impact on covid- . comorbidities closely associated with age, chronic inflammation and dysregulated metabolism such as hypertension, cardiovascular disease, and diabetes are the most prevalent comorbidities. however, many of these comorbidities are strongly associated with each other. consequently, many patients will have multiple comorbidities and therefore while we have discussed these individually, the reality is that a combination of factors will be at play. furthermore, as multiple drug use is inevitable for patients with pre-existing comorbidities, the impact of overlaying drugs on an already compromised state and the possibility of ddi leading to adverse events needs to be carefully considered. as the scale of this pandemic continues to accelerate globally, we hope this review provides healthcare professionals and biomedical researchers with a more comprehensive understanding of the impact of pre-existing comorbidities on covid- development and treatment. the authors lc and mc are fellows of the astrazeneca postdoc programme. figures were created with biorender.com. lc and mc wrote the first draft of the article. sb, mm, jw, and cb reviewed and edited the article before submission. all authors contributed extensively to the discussion of the content and researched data for the article. prevalence of underlying diseases in hospitalized patients with covid- : a systematic review and meta-analysis prevalence of comorbidities in the novel wuhan coronavirus (covid- ) infection: a systematic review and meta-analysis high-dimensional immune profiling by mass 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metabolic and nutritional parameters in patients with myelofibrosis from comfort-i baricitinib induces ldl-c and hdl-c increases in rheumatoid arthritis: a metaanalysis of randomized controlled trials bruton's tyrosine kinase (btk) inhibitors in clinical trials acp- ) in relapsed chronic lymphocytic leukemia the specific bruton tyrosine kinase inhibitor acalabrutinib (acp- ) shows favorable in vitro activity against chronic lymphocytic leukemia b cells with cd antibodies ibrutinib treatment improves t cell number and function in cll patients clinical evidence does not support corticosteroid treatment for -ncov lung injury effect of dexamethasone in hospitalized patients with covid- : preliminary report. medrxiv covid- : demand for dexamethasone surges as recovery trial publishes preprint august | volume | article key: cord- - enteev authors: brisse, morgan; ly, hinh title: comparative structure and function analysis of the rig-i-like receptors: rig-i and mda date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: enteev rig-i (retinoic acid-inducible gene i) and mda (melanoma differentiation-associated protein ), collectively known as the rig-i-like receptors (rlrs), are key protein sensors of the pathogen-associated molecular patterns (pamps) in the form of viral double-stranded rna (dsrna) motifs to induce expression of type interferons (ifn ) (ifnα and ifnβ) and other pro-inflammatory cytokines during the early stage of viral infection. while rig-i and mda share many genetic, structural and functional similarities, there is increasing evidence that they can have significantly different strategies to recognize different pathogens, pamps, and in different host species. this review article discusses the similarities and differences between rig-i and mda from multiple perspectives, including their structures, evolution and functional relationships with other cellular proteins, their differential mechanisms of distinguishing between host and viral dsrnas and interactions with host and viral protein factors, and their immunogenic signaling. a comprehensive comparative analysis can help inform future studies of rig-i and mda in order to fully understand their functions in order to optimize potential therapeutic approaches targeting them. rig-i (retinoic acid-inducible gene i) encoded by the ddx gene in the human genome ( , ) and mda (melanoma differentiation-associated protein ) encoded by the ifih gene ( , ) are known as important protein initiators of earliest immune responses to viral infection. a relatively large body of work has focused on understanding their roles in triggering the same innate immune pathway as they indeed share many similarities at a structural and functional level. however, it is becoming increasingly clear that there are unique differences between rig-i and mda , such as their activation mechanisms and contextual functionalities, that need to be considered in order to fully appreciate their individual function. a comprehensive analysis of multiple aspects of rig-i and mda from their evolutionary origins and behavior among different species to their structures and molecular signaling will allow for a more nuanced understanding of their functional purposes. the innate immune response is a combination of non-specific defense mechanisms by the host that are critical for early detection and inhibition of pathogen growth before the adaptive immune response has time to produce proper cell-mediated immunity, such as the development of antibodies and cytotoxic t-lymphocyte responses (ctl) against the invading pathogen and/or the pathogen-infected cells ( ) . cells of the innate immune arm, such as leukocytes and epithelial cells, are able recognize general components of the microbes (e.g., viruses) that are shared among related microbes. these microbial structures are called pathogen-associated molecular patterns (pamps) (e.g., viral dsrna) that are specifically recognized by the cellular pattern recognition receptors (prrs) (e.g., rig-i, mda , or tolllike receptors tlrs) which are then activated (figure ) . the specific signaling mechanisms of rig-i and mda activation will be discussed in detail below. here, the cascade of event leading to ifn production is briefly summarized. upon binding to pamp (e.g., dsrna), the activated rig-i and mda interact with the mitochondrial antiviral signaling proteins (mavs), which forms a multilayered protein complex contain several different proteins ( ) ( ) ( ) ( ) . the mavs complex then catalyzes the interaction of inhibitor of nuclear factor kappa-b kinase subunit epsilon (ikkε) and the serine/threonine-protein kinase (tbk ) ( ) ( ) ( ) , which phosphorylate the transcription factors interferon regulatory factors and (irf and irf ) ( ). phosphorylated p-irf ( ) and -pirf ( ) factors then dimerize and translocate into the nucleus, where they activate the expression of the type interferon genes (ifn : ifnα and ifnβ). ifn proteins are then exported out of the cell to activate ifn signaling cascade by binding to their receptor (the ifnα/β receptor or ifnar) either on the same cells or neighboring cells in an autocrine or paracrine fashion. this results in the production of more ifn (in a positive feedback loop) and a variety of interferon-stimulated genes (isgs), which mediate vasodilation near the site of the pathogen infection and uptake of fluid, recruitment of innate immune cells, such as macrophages, neutrophils, and dendritic cells to the site of the infection that is aided by chemokine gradients to mediate innate immune cell-mediated killing of the infected cells ( ). rig-i and mda appear to differentially induce ifn in response to different viral pathogens ( ), with rig-i generally responding most potently to negative-strand rna viruses, such as influenza viruses ( , ) , bunyaviruses ( , ), filoviruses ( ), and rhabdoviruses ( , ) as well as the positive-stranded japanese encephalitis virus ( ), while mda is activated during infection by positive-strand picornaviruses ( , , ) and arteriviruses ( , ) as well as by hepatitis d virus ( ), kaposi's sarcoma-associated herpesvirus (kshv) ( ). rig-i and mda may also play a role in recognizing non-viral pathogens, as mda has been found to respond to malaria ( ) (figure ) . neither are individually critical in reovirus ( ) and in dengue virus infection ( , ) but the presence of either in combination with toll-like receptor (tlr ) is critical to have effective anti-viral repsonses ( ). each serves an additive role during west nile virus infection ( ), which is likely mediated by the production of multiple pamp species in the infected cells ( ). indeed, rig-i and mda have also been shown to recognize different sections of the same viral genome due to their differing preferences for rna binding ( ), illustrating how rig-i and mda can act both independently and synergistically. this has also been shown functionally in viruses where both rig-i and mda have been found to be essential to induce the necessary levels of ifnβ signaling for antiviral control against paramyxovirus ( , - ) and rotavirus infections ( ). while rig-i and mda participate in the ifn signaling pathway ( ), it is clear from animal modeling that they might be functionally distinct. while c bl/ mda ko mice exhibit no obvious phenotypes ( ), c bl/ rig-i ko have high embryonic lethality as they don't live past weeks of birth and experience growth retardation and liver degeneration ( , ) . furthermore, when rig-i ko mice are back crossed onto the more genetically flexible s strain ( ), these mice can spontaneously develop colitis symptoms ( ). clinical cases with mutations in rig-i and mda have distinct autoimmune presentations, with rig-i mutations being associated with atypical singleton-merten syndrome, while mda mutations have been linked to classical singleton-merten syndrome, aicardi-goutières syndrome, systemic lupus erythematosus, type diabetes and graves disease ( , ) (figure ). there is growing evidence that overt innate-immune interferon signaling plays a critical role in the development of other forms of autoimmune conditions ( ). taken together, this suggests that rig-i and mda may differ significantly in their roles during development as well as in responding to different types of viral infection that is partially dependent on the pamps that are available in any given context. there is also increasing evidence that rig-i and mda have additional distinct molecular functionalities in immune signaling ( ). it is well-established that the interferon regulatory factor (irf) and innate immune nfκb cytokine signaling pathways have many areas of cross-regulation and expression ( ). accordingly, both rig-i and mda have been shown to activate nfκb signaling during rsv infection, but only rig-i appears to act upstream of the canonical iκbα-nfκb pathway ( , ) (figure ) . while both are known to activate nfκb mediated expression of il- and pro-il- β through the interaction of card with bcl ( , ), the independence of mda from the iκbα pathway suggests that it influences nfκb signaling in other as yet uncharacterized ways ( ). a possible explanation for mda 's independence from the iκbα pathway may be that mda -mediated nfκb (but not irf) signaling requires trim , which activates rig-i by ubiquitination (to be discussed in detail below). this potentially implicates trim in other mechanisms besides activating rig-i ( , ). rig-i (but not mda ) also induces inflammasome assembly-mediated cleavage and maturation of pro-il- β by caspase ( , , ) . finally, rig-i has been shown to inhibit rnai complexes mediated by the endoribonuclease dicer, which is encoded by the dicer gene and cleaves dsrna and pre-micro rna into short single-stranded rna fragments known as small interfering rna (sirna) and microrna figure | rig-i/mda signaling pathway rig-i and mda are first activated by recognition of pamp dsrna, which causes them to interact with mavs. following the activation of mavs by rig-i/mda , a molecular cascade involves the interaction of ikkε and tbk , which is followed by phosphorylation of the transcription factors irf and irf , ensure to translocate the phosphorylated p-irf and p-irf into the nucleus, where they dimerize and bind to transcription factor binding sites of the ifnα and ifnβ genes to activate their transcriptions. expression and exportation of these genes into the cellular milieu trigger the ifn signaling cascade in an autocrine or paracrine fashion to induce expression of hundreds of interferon stimulated genes (isgs) and inflammatory genes to confer antiviral resistance. rig-i and mda also activate the nf-κb pathway. rig-i appears to act upstream of the canonical pathway, which results in the translocation of the two functional nf-κb units (p and p ) into the nucleus, while mda appears to affect nf-κb expression independently from this pathway. figure created using biorender software. ( ), by interacting with the probable atp-dependent rna helicase dhx (also known as the laboratory of genetics and physiology lgp protein), which inhibits dicer ( ) as well as the dicer-complex protein trbp ( ) . lgp has been shown to exhibit conflicting effects on rig-i and mda signaling ( ) ( ) ( ) , and future studies are needed in order to clarify these regulatory mechanisms. rig-i and mda are expressed in all cell types ( ) , but are most well-known for their functions inside innate immune cells, such as macrophages, neutrophils, and dendritic cells, as well as in other cells like mucosal epithelial cells. they are classified as atp-dependent dexd/h box rna helicases. their structure figure | venn diagram comparing the signaling and functional similarities and differences between rig-i and mda . is highly helical and consists of two caspase activating and recruiting domains (card) at the n terminus of ∼ amino acids each, followed by a flexible hinge region and the helicase domain that consists of the reca-like hel and hel domains with an atp binding and hydrolyzing domain at their interface ( figures a,b) . in particular, the structure of the atp binding site distinguishes rig-i and mda from other helicase proteins, such as dicer. unlike other dexd/h box helicases where rna binding catalyzes the atp binding site to become structurally organized, the atp binding site in rig-i and mda remains comparatively open and structurally dynamic following rna binding. this is aided by the atp binding site being formed by an interface between the two hel domains, which are relatively far apart ( ) . these structural features are connected by another flexible hinge region to the unique and predominantly β-sheet c terminal domain (ctd), which recognizes and binds to rna ( ) . the ctd in rig-i and mda contains a zinc binding domain that is related to those of the gdp/gtp exchange factors ( ) . each protein also contains a positively charged groove within this domain that recognizes dsrna and this groove is structurally unique in each protein, potentially explaining their different rna binding preferences ( ) . rig-i primarily recognizes short double-stranded rna with ′ triphosphate groups ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , while mda primarily recognizes long double-stranded rna ( ) ( ) ( ) ( ) (to be discussed in detail below.) it is notable in this regard that the hel-ctd motifs adopt different orientations relative to dsrna in rig-i and mda . specifically, the rig-i hel-ctd domain is tilted relative to dsrna with the ctd interacting with the ′ and ′ ends of the dsrna ( ), whereas the mda hel-ctd domain runs parallel to the rna strand (figures c,d) . the series of steps required for rig-i and mda activation have been described in depth elsewhere ( ) ( ) ( ) ( ) ( ) . briefly summarized, these proteins endogenously exist in the cytoplasm of the cell in a phosphorylated and inactivated conformation when they are not activated by pamp (dsrna) ( ) ( ) ( ) (figures a,f) . phosphorylation is mediated at the n terminal card domains (s and t ) of rig-i by pkc-α/β ( , ) and at the c terminal rna interaction domain (s , s , and t ) by ckβ ( ) . on the other hand, mda is phosphorylated at s by riok ( ) as well as by other yet unknown kinases ( , ) . rig-i is also acetylated at k in its c terminal domain that requires deacetylation by hdac to be able to recognize rna in its activated form ( ) . upon recognition of pamp (dsrna), rig-i unfolds into an open and activated state that is mediated by the flexible hinge regions between the card domains and the helicase domain, and between the helicase and the c terminal domain ( , , ( ) ( ) ( ) ( ) (figure b) . on the contrary, there is evidence to suggest that mda has a more dynamic structure ( ) . unlike a model of rig-i activation described above, mda exists in a conformational equilibrium between close and open forms, with close forms favored in the dsrna unliganded state. while not yet formally demonstrated, it is possible that mda may be inhibited in the absence of the dsrna ligand by its structural dynamics, which may prevent strong protein-protein interactions ( figure f) . however, upon binding to dsrna ligand, mda adopts an open and activated form, which is perhaps more conducive for protein-protein interactions (figures g,h) . once the c terminal domains have been de-phosphorylated, the e ubiquitin ligase riplet attaches ubiquitin peptides onto the c terminal domain of rig-i at residues k and k ( , ) . it was previously shown that ubiquitination by riplet was necessary for opening rig-i and for ubiquitination of the card domain ( ) . however, in-situ studies found that dsrna was sufficient to weaken the interaction between purified rig-i c terminal domain and rig-i card domains ( ) and that dsrna was necessary for riplet ubiquitination ( ) , calling into question the sequential order for rig-i activation ( figure c ). following de-phosphorylation of the card domain by the phosphatase pp -α/γ ( ) , this domain is polyubiquinated at k by the e trim ubiquitin ligase ( ) , which itself is activated by caspase ( ) (figure d ). trim interacting with rig-i may also be mediated by their mutual interactions with certain host long non-coding rna (lncrna), which occurs outside of the dsrna recognizing domain in the ctd of rig-i ( ) . a recent study showed that riplet rather than trim was primarily responsible for ubiquitinating and activating rig-i ( ). however, there are several factors to take into consideration with this study. these recent results were obtained using ko t and mouse embryonic fibroblast (mef) cells and that it was not clear whether k ubiquitination occurred at other known lysine sites in rig-i. the question remains whether riplet can ubiquitinate other lysine residues in the absence of trim . additionally, in-situ experiments comparing rig-i ubiquitination by riplet and trim utilized an e enzyme ( ) that had been found to be specific for riplet ( ) . while the e that utilizes trim has not yet been identified, trim has been shown to ubiquitinate rig-i in-situ when a general mixture of e proteins was used ( ) . the protein levels of trim may also have to be at a certain level in order for it to productively ubiquitinate rig-i, as the ubiquitin protease usp deubiquitinates trim at later time points in viral infection ( ) . finally, trim has been found to be essential for rig-i activation and ifn signaling in-vitro and in-vivo. for the former, sirna-mediated knock-down ( , ) , cellular knockout ( ) and inhibition by viral protein ( , ( ) ( ) ( ) ( ) conditions for trim in multiple cell types have been shown to change rig-i cellular localization ( ) and to negatively affect rig-i k ubiquitination, association with mavs and ifn signaling [when the constitutively active rig-i card domain was overexpressed ( , ( ) ( ) ( ) ( ) ( ) or during viral infection ( , , ) ]. viral inhibition of trim may even be a source of a positive selection during the evolution of certain viruses, as ns iav proteins have been found to interact with species specific trim ( ) . for the latter, mefs from trim ko mice have significantly downregulated ifn production upon viral infection ( ) and ko mice for nlrp , which is a competitive interactor with trim to rig-i, show increased interferon production and more resistance to viral infection ( ) . the known contributions of trim to innate immunity have recently been summarized elsewhere ( ) . it is clear that both riplet and trim can mediate k linked polyubiquitination. however, it has also been found that in-situ incubation of purified rig-i card domains with ubiquitin can be activated by free and unlinked k polyubiquitin chains ( ) , calling into question whether trim only attaches k -linked ubiquitin motifs to rig-i-card or if it also catalyzes the formation of unlinked k polyubiquitination chains ( ) . a possible explanation for these differing results is that rig-i has been shown to be covalently k ubiquitinated by trim when analyzed by mass spectrometry from cells ( ) , while experiments that demonstrate non-covalent k ubiquitination are those involve primarily interactions with purified proteins. it has also been recently found that rig-i is k ubiquitinated at k and that it may be functionally redundant to k ( , ) , with their ubiquitination possibly upregulating the k ubiquitination of the other lysine residues in rig-i ( ). however, it is unknown whether trim ubiquitinates k or any of the other rig-i lysine residues. notably, these additional lysine residues in the card and c terminal domains of rig-i and mda are known to be k and k ubiquitinated [which are associated with degradation of rig-i ( , ) and mda ( ) ], but the four listed above appear to be the essential residues for activation of rig-i ( , ) . the presence of k ubiquitin modifications on mda is more controversial. independent studies have found that mda is ( , ) or is not ( ) k polyubiquitinated. it has also been independently found that trim does not affect ubiquitination of mda (without distinguishing between k and k polyubiquitination) ( ) and for trim to increase k ubiquitination ( ) , the only apparent difference in the experimental models being the usage of hek t ( , ) vs. hek ( ) cells. trim has also been recently found to be essential for mda activation by k polyubiquitination at k ( ) . it is clear that additional studies are needed in order to clarify the ubiquitination mechanisms of mda . upon binding to pamp (dsrna), rig-i oligomerizes with other rig-i/dsrna complexes to form helical oligomers ( ) in a : complex using the purified rig-i protein ( ) , where the activating ubiquitin motifs serve as a scaffold to link the oligomers together ( ) . these oligomers have been found to be necessary under normal conditions to activate rig-i. this may be due to the helical structure of the rig-i oligomers closely matching those formed by mavs ( ) , which is known to form filaments in-vitro ( , ) mediated by its own card domains ( , ) . a structural model of mavs activation by rig-i has been proposed of stacking mavs card domains on top of rig-i card domains to extend the rig-i helix ( ) . the minimum length of dsrna found to activate rig-i is base pairs, which is equivalent to the minimum length to facilitate the formation of a -rig-i/dsrna dimer ( ) . that being said, shorter (∼ bp) ′ ppp stem loop dsrna complexes that have previously been used to obtain x-ray crystallographic structures of rig-i interacting with dsrna ( , , ) (figure c ) can also activate ifnβ signaling in cells ( , ) and in mice ( ) . furthermore, a cells that were transfected with rig-i plasmid h prior to rna transfection had a minimum dsrna length of only - bp required for activation ( ) . this indicates that rig-i oligomerization may not be necessary for activation of the ifnβ pathway under some experimental conditions, which need to be further investigated. mda has also been shown to oligomerize to form long rnaassociated filaments in vitro ( , , ) ( figures d, h) , which may be aided by chaperone proteins ( ). given that the k residue found to have been ubiquitinated by trim ( ) is located on the surface of hel , it is possible that k ubiquitin residues may also help stabilize mda -dsrna filaments ( ). however, mda also spontaneously forms filaments and induce mavs to form filaments independently of ubiquitin in-situ. it is also thought that the formation of longer filaments by mda may be mediated by a longer linkage region between card and hel than in rig-i by amino acids (the length of which is wellconserved across species), allowing for the association of more card domains in an oligomer ( ) . the formation of longer filaments by rig-i has been more controversial, giving rise to two alternate models of rig-i activation: formation of individual single unit of rig-i with short dsrna monomers (leaving a free dsrna end, such as a hairpin loop), which then oligomerizes via card tetramerization that is linked by their ubiquitin chains, or filamentation on longer dsrna. like mda , rig-i can form filaments in-situ independent of ubiquitin ( , ) and induces mavs to also form filaments ( ) , and mavs is known to form filaments in-vitro ( , ) mediated by its own card domains ( , ) . however, rig-i filamentation on an rna template (forming "beads on a string") as opposed to smallerscale oligomerization hasn't yet been shown to occur in-vitro. part of the reasons for the suggestion that rig-i was strongly activated by shorter dsrna was based the comparison on mass equivalents of rna species as there were less ′ triphosphorylated ends for longer dsrnas with greater mass than shorter dsrnas with more ′ triphosphorylated ends ( ) . however, when rna species were normalized by molar equivalence, dsrna length appeared to be positively correlated with rig-i signaling ( ) ( ) ( ) , which became insignificant at around bp ( , ) . it is significantly shorter than the length of dsrna that activates mda , which forms filaments on , bp dsrna ( ). the kinetics of rig-i and mda interacting with dsrna (which will be discussed in detail below) might possibly explain the decrease in dsrna length efficiency to activate rig-i as compared to mda , as rig-i seems to first recognize the ′ ppp end before sliding down the length of the dsrna ( ), whereas mda dynamically associates and disassociates along the length of long dsrna ( ). meanwhile, it is still unclear whether rig-i can preferentially be activated by longer dsrna independently of its unknown ability to form filaments in-vitro ( ) . once fully activated and oligomerized, the rig-i card domain can then interact with mavs ( ) ( ) ( ) ( ) (figures e,i) , which is part of a protein complex containing a variety of other cellular proteins ( ) ( ) ( ) ( ) . while the mda card domain has much weaker direct association with mavs than the rig-i card domain, it is sufficient to lead to its activation and potentiates activation of mavs by rig-i ( ), the mechanisms of which have yet to be determined. the activated mavs complex then initiates a molecular cascade which eventually results in expression of ifn ( ) (figure ) . interestingly, full length rig-i, when overexpressed, has been found to associate with mavs in the absence of activating dsrna and the interaction can be ablated by phosphorylation at s and t ( ) , suggesting that the card phosphorylation sites function at least in part to prevent association of the inactive form of rig-i with mavs. furthermore, the crystal structure of the interaction between the rig-i card and mavs card domains shows the rig-i card domain interacting with mavs card domains on the outside of the tetramer and the rig-i card ( - ) domain facing toward the center of the tetramer ( ) (figure e ). nmr solution structures of rig-i card also shows that t (which is required for dephosphorylation by pp -α/γ) is largely buried within the card domain in a section that would be in closer contact with the helicase domains, suggesting that dephosphorylation of t affects an interaction domain between card and the c terminus ( ) . furthermore, nmr of a c terminal construct of rig-i with the card domain shows stable interactions of card and the c terminal domain ( ) . what all this may mean is that, while the card domain of rig-i is somewhat exposed in its inactivated form and therefore can be shown to interact with mavs, full exposure and engagement of both rig-i card domains (card and card ) with the card domain of mavs is necessary in order to induce ifn signaling. the card domains of rig-i also appear to be generally structurally stable, as electron microscopic structures have been obtained of the full length rig-i bound to blunt-ended dsrna showing both card domains exposed ( ) . on the contrary, the card domains of mda may be comparatively more flexible than those of rig-i in order to mediate long mda -dsrna filament formation ( ) . the activated mavs complex induces association of the inhibitor of nuclear factor kappa-b kinase subunit epsilon (ikkε) and the serine/threonine-protein kinase (tbk ) ( - ), which collectively phosphorylate the interferon regulatory factors and (irf and irf ) ( ) (figure ) . ikkε and tbk also interact with a number of other co-factors ( , ) , such as the deadbox helicase (ddx ) ( ) . the activated p-irf ( ) and p-irf ( ) then translocate into the nucleus and dimerize, where they then act as the primary transcription factors for ifnα and ifnβ, respectively. existing evidence suggests that ifnα is more primarily produced in the earliest time points following rig-i/mda activation, while ifnβ is produced later and is responsible for more robust anti-viral control throughout the innate immune response period ( ) . there is also a distinction between innate immune cell types for ifn production, as cells like fibroblasts and conventional dendritic cells produce ifnα and ifnβ ( , ), while neutrophils only produce ifnβ ( ) and plasmacytoid dendritic cells only produce ifnα primarily through the tlr signaling pathways ( , ). signaling through rig-i is also known to be essential for the process of tlrmediated phagocytosis by macrophages ( ) . interferons are then secreted out of the cell, where they bind to their own receptor (ifnar) and activate the janus kinase/signal transducer and activator of transcription proteins (jak/stat) signaling pathways, which result in a positive feedback signaling loop to further increase rig-i/mda expression and activation ( ) and ifn production ( , ) . expression levels of rig-i and mda have consistently been found to be upregulated downstream of type i ( , ) and type ii ( , ) ifn signals. mda upregulation has additionally been found to occur independently of cytokine expression at least during picornavirus infection ( ). one of the most obvious distinctions between rig-i and mda is in the rna species to which they bind for activation (figures , ) . rig-i has the highest affinity for short dsrna that is tri-phosphorylated at the ′ end ( - ), with rig-i having been found to directly interact with the ′ tri-phosphate group of the dsrna ( , ) . while rig-i can bind to ss- ′ tri-phosphorylated rna ( ), rig-i cannot be activated by it ( , , ) , likely due to a conformational need to recognize double-stranded rna. as a result, rig-i is greatly attenuated by a ′ overhang as well as those with a ′ overhanging the ′ tri-phosphate end ( ) . in fact, a single unpaired ′ triphosphorylated nucleotide is sufficient to competitively inhibit rig-i, which has been exploited by rna viruses to evade rig-i recognition and ifn signaling ( ) . the unique preference of rig-i for ′ tri-phosphorylated rna can be explained by the specific orientation that the rig-i c terminus adopts when directly interacting with the ′ tri-phosphate group of the ′ tri-phosphorylated dsrna ( , ) as compared to unphosphorylated blunt-ended dsrna ( ) . the minimally required and exclusionary features of the ′ and ′ dsrna ends for rig-i activation have proven to be complex. certain studies suggest that a ′ diphosphate group is the minimum feature required for rig-i binding and activation, with ′ monophosphate dsrna failing to productively activate rig-i as compared to ′ di and tri-phosphate dsrna ( ) . frontiers in immunology | www.frontiersin.org additionally, rig-i poorly distinguishes between dsrnas with either ′ tri-phosphate and ′ diphosphate group. when the free energies of each interaction are calculated, the affinity for ′ triphosphate being lowered by disassociation of magnesium from the rig-i/dsrna complex. both are significantly more favorable for binding rig-i monophosphate dsrna ( ) . this similarity in affinity appears to be important in the context of infection with viruses that produce ′ diphosphate rnas, such as reoviruses ( ) . likewise, the difference of energic binding between monophosphate dsrna and bi-and triphosphate dsrnas is likely important for distinction between self (host) and non-self (foreign) rna, the mechanisms of which will be discussed in detail below. the atp hydrolysis functions of rig-i have been shown to drive rapid disassociation from certain rna features, such as ′ monophosphate dsrna ( , ) and ′ oh rna ( , ) , which is particularly important for ′ monophosphate dsrna because it is found in mrna after decapping during the mrna degradation process ( ) . on the other hand, other studies have shown that rig-i can interact with monophosphate dsrna to a certain degree, as has been found to be the case for short synthetic dsrna with a ′ and ′ monophosphate group ( ), poly(i:c) digested with rnase iii ( ) [which generates ′ mono-phosphate/ ′ -oh dsrna ( ) ] and hcv rna ( ) and mitochondrial rna [in the p deficient mice ( ) ] digested with rnase l [which produces ′ oh and ′ mono-phosphate dsrna at subnanomolar levels ( ) , as has been found to be the case for hcv rna ( ) .] it appears that the ′ monophosphate is the determinate feature for rig-i activation independently of the ′ or ′ oh group in all these cases. a possible explanation for the discrepancy between the studies was that higher order rna structures might compensate for the less optimal ′ and ′ ends, as monophosphate dsrna that did not contain stem-loop structures did not activate rig-i and rna regions repetitive in certain nucleotides had been found to be critical for rig-i activation ( ) . future studies are required to further characterize the behavior of rig-i with these rna species. as previously mentioned, mda preferentially associates with long dsrna ( ) ( ) ( ) ( ) . the crystal structure and molecular modeling of mda /dsrna complex suggest that it can recognize the entire first turn of the blunt-ended dsrna ( ) in a similar way as lgp can ( ) . like rig-i and mda , lgp belongs to the atp-dependent dexd/h box rna helicases ( ) , which is structurally similar to rig-i and mda but lacks the card domains at the n terminus ( ) . mda has also been found to be activated by the digested products of rnase l specifically from parainfluenza virus ( ) . the presence of certain repetitive rna elements appears to be another contributing factor in determining interaction of rna with rig-i and mda , which has recently been described in detail elsewhere ( ) . while rig-i and mda are mostly implicated in the immune response to rna viruses, it has also been found to be activated by ′ tri-phosphorylated dsrna intermediates generated by cellular rna polymerase iii from at-rich dna sequences ( ) and during infection with epstein-barr virus (a dna virus) ( ) . rig-i has additional binding preferences for certain nucleotide motifs, such as uridine-rich ′ tri-phosphorylated hairpin rna ( ) , synthetic au-rich hairpins ( ) and those naturally found in the genomes of sendai virus defective-interfering (di) particles ( ) , measles ( ) , influenza a virus (iav) ( ) and in kshv rna transcripts ( ) , and poly (u/uc) regions ( ) and poly (a/ag) regions ( ) in the antisense hepatitis c virus (hcv) genome. it is of particular interest that the poly (a/ag) hcv regions are located significantly downstream of the ′ triphosphate group ( ) , thus potentially implicating other parts of rig-i (e.g., helicase domain) as potential rna interacting domains. repetitive rna elements may also be important in allowing for interaction of inhibitory rnas that do not have ′ or ′ features needed for full activation of rig-i, as has been shown to be the case with ga-rich regions in circular longnon-coding rna lnc-lsm b ( ) . these specific interactions explain their primary role as anti-viral receptors, as these viral motifs are mostly not found in cellular rnas ( ) . rig-i and mda have been particularly implicated in their response to rna genomes of viral defective interfering (di) particles, as these defective viral genomes (dvgs) have originally been found to induce interferon signaling ( ) . di particles are produced by many viruses during infection, and while they are similar in many regards to standard viral particles, such as in appearance and composition, they cannot productively infect cells ( ) . this is largely thought to be due to the presence of large and deleterious deletions in the dvg of di particles ( ) . some dvg rnas have also been noted to have "copy-back" motifs in which one end of the genome can base pair with an inverted copy at the opposite end of the genome, which may be due to stalled and aberrant replication ( , ) . copy-back rna motifs specifically seem to be important for rlr activation in that they tend to contain hairpin motifs and ′ tri-phosphate groups, as has been found for sendai ( ) ( ) ( ) , measles ( , ), and chikungunya ( ) dvg rnas in activating rig-i. in the case of iav, dvg rnas might even be more potent activators of rig-i than the full-length viral genome. cells that were blocked from viral protein synthesis experienced rig-i mediated ifn expression when infected with iav stocks grown in chicken embryonic eggs (which produced higher relative quantities of di particles with dvg rnas) but not with iav grown in cell culture, indicating that rig-i activation by the genomes from primarily non-di iav particles may require active viral rna synthesis ( ) . a potential explanation to this observation is that rig-i appears to be activated by the full viral genome via its panhandle structure, the affinity of which is lowered by the presence of mismatched and unpaired nucleotides in this region of the viral genome that is conserved across influenza virus strains ( ) . however, the overall panhandle structure is conserved between dvgs ( ) and the full length viral genome ( ) , and deletions within dvgs are monogenic and internal ( ) . the specific molecular mechanisms of enhanced rig-i signaling by iav dvgs have yet to be elucidated, although the level of exposure of the panhandle may play a role. while the full extent of mda interacting with di rna is currently unknown, mda appears to be more predominantly activated by dvg rna than rig-i specifically in dendritic cells early in the viral infection cycle ( ) , which may be a contributor toward the phenomenon of di particles enhancing dendritic cell maturation ( ) . the comparative abilities for di particles vs. infectious virions to activate rig-i and mda have important implications for understanding viral pathogenesis and for vaccine development. there is a burgeoning interest in this regard, especially in populations which are typically more challenging to achieve successful preventative vaccination, such as elderly populations with iav vaccination ( ) . elderly populations in general do not develop as strong of memory immune responses to vaccines as their younger counterparts ( ) ( ) ( ) ( ) and have been found to have decreased rig-i mediated ifn signaling ( ) . correspondingly, the influenza vaccine has been shown to decrease in effectiveness in older populations as the influenza season progresses ( ) . a di-vaccine that strongly activates innate immune cells and increases the adaptive immune response could therefore potentially boost the immune responses to vaccines in more vulnerable populations. additionally, di particles have shown to be an important contributor of viral persistence ( , , ) . this raises the question of whether a viral infection may alternate between producing primarily infectious virions which eventually activates the innate immune response and producing primarily di particles which requires less cellular activity but may initiate an even stronger innate immune response ( ) ( ) ( ) . taken altogether, di particles provide yet another layer of distinction between rig-i and mda in terms of how each recognizes different species of dsrna. the preference for specific rna species by rig-i and mda allow for them to distinguish between viral rna and host rna in most circumstances ( ) , although the specific mechanisms of distinction are not as clear for mda as for rig-i. studies from clinical cases of mda mutations provide contradictory models, with certain mutations found in aicardi-goutières syndrome (ags) increasing mda avidity for self rna ( ) with alu retroelements found to be significantly enriched for interaction with ags mda mutations ( ) . the modification of dsrna by host cells may be a primary inhibitor of mda activation by host rna as knockout of adenosine deaminase (adar ), which weakens dsrna structures, allows wild-type mda to be activated by alu retroelements ( ) . however, other mda mutations decrease affinity for known mda ligands and atpase activity, yet still demonstrate increased ifnβ expression ( , ) . for rig-i, a highly conserved residue in the c-terminal rna binding pocket (h ) has been found to sterically exclude canonical self-rna by the means of the n - ′ o-methyl self-rna motif, also known as cap rna ( , ) . this results in a low binding affinity of rig-i to cellular cap rna and decreased atpase activity as compared to pamp (dsrna) ( , ) . flaviviruses take advantage of this precise discrimination by encoding a viral ′ -o-methyltransferase capable of n - ′ o-methylating its positive-strand rna genome in order to evade rig-i recognition and ifn activation ( ) . conversely, the mutations e a and c f found in the rig-i protein in patients with auto-immune disorder singleton-merten syndrome confer the ability of the protein to recognize cap rna and become activated by atp dependent and independent mechanisms, respectively ( ) . furthermore, the e q mutation of rig-i, which was designed to constitutively bind atp, was found to increase the affinity of rig-i with ribosomal rna ( ) . it is noteworthy that host rna contains additional internal rna modifications and non-watson-crick base pairing which can inhibit activation of the other known dsrna-sensing protein, the interferon-induced double-stranded rna-activated protein kinase (pkr) ( ) , and it is known that synthetic ′ triphosphorylated rna containing pseudouridine, -thiouridine or ′ -o-methylated uridine has significantly decreased ability to activate rig-i ( ), which has been demonstrated to occur by preventing rig-i filament formation in-situ ( ) . n- -methyladenosine (m a) nucleotides, which are well-known nucleotide modifications among viruses ( ) , have also been found to ablate dsrna binding to rig-i ( ) . it has been demonstrated that certain rna-dna hybrid constructs with ribonucleotides at positions and of the dna strand can bind to rig-i and activate its atpase activity ( ) . atpase activity is necessary for full activation of rig-i and expression of ifnβ ( , ) , so the minimum requirement of a motif not found in host rna for atpase activity has significant implications for the distinction between self and non-self rnas. expanding on this observation, exogenous atpase activity may also be sufficient to potentiate rig-i and mda , as lgp atpase mutant mice are significantly more susceptible to viral infection even in the presence of functional rig-i and mda ( ) . however, this model is further complicated by certain rna-dna hybrids that are able to bind rig-i and activate atpase activity, but don't induce ifnβ expression ( ) . it is currently undetermined whether such hybrids can sterically inhibit rig-i due to the presence of mostly dntps or whether they inhibit rig-i in a yet undescribed way. recent kinetic studies of rig-i and mda activation by pamp (dsrna) help illustrate how atpase activity is critical for their function and distinction between host (self) and foreign (non-self) rna. rig-i binding to atp is sufficient for interaction with dsrna ( , ) . rig-i atpase activity is inhibited in the absence of pamp (dsrna) by a helical arm that blocks the atpase site ( ) . upon interaction with pamp (dsrna), the helical arm shifts and the two helicase domains are brought together to form an active atpase site ( ) . rig-i then catalyzes atp to break the ′ ppp dsrna interactions within seconds. atp is then rapidly hydrolyzed to facilitate translocation of rig-i to the opposite dsrna end, after which the rig-i oligomers can form ( ) . on the other hand, atp hydrolysis drives rapid disassociation of rig-i from host rna features. these features include dsrna with a ′ monophosphate group ( , ) that is found in mrna after decapping during the mrna degradation process ( ) and ′ overhang rna ( , ) found in mirna ( ) as well as other rna motifs, such as ′ oh rna ( , ) found in bacteria ( ). furthermore, an impaired atpase functionality increases the promiscuity of rig-i binding these host rna motifs ( , , ) . similar atpase functions have been found during mda filamentous formation. the c terminus of mda is critical to form organized helical filaments ( ) and atp binding drives association and hydrolysis and disassociation from foreign dsrna [with little coordination being observed between neighboring mda proteins ( )] in a manner that involves mda twisting along its flexible and hydrophobic interface domains ( ) . taken together, atpase activity may be directed toward rapid disassociation from host dsrna and degradation of rna-dna hybrids, but primarily act on the translocation pathway upon interaction with pamp (dsrna). it is also possible that host and hybrid dsrnas could inactivate rig-i independently of their ability to bind the c-terminus and activate atpase activity. this has been shown, for example, for a hybrid rna that has one strand consists mostly of dna except at positions and , which appears to bind rig-i and activate its atpase activity but doesn't activate ifn signaling ( ) . future studies are needed in order to determine these differential interaction mechanisms. contrary to the traditional paradigm, there is increasing evidence to suggest that rig-i and mda interact with certain host rna motifs, resulting in auto-activation or auto-inhibition of the irf pathway ( figure ) . one of the most strongly supported models is activation by host and viral circular rnas (circrna). originally found in a variety of pathogen genomes, circrnas in eukaryotic cells were first thought to be byproducts of the pre-mrna splicing process. however, they have later been found to be produced by a non-canonical "backsplicing" process and there is increasing evidence to suggest that they play some important regulatory roles ( ) , suggesting that they may have specifically evolved for this purpose. rig-i was first found to interact with circrna produced in situ ( ) . interestingly, the minimum component required for rig-i activation is an intron of pathogenic origin to be spliced out during the circularization process. as human introns have been found to be associated with many rna binding proteins, it is speculated that these proteins may have prevented circularization of this particular synthetic circrna used in this study ( ) and that host rna binding proteins normally prevent endogenous circrnas from being detected by the innate immune system. nevertheless, some viral infections can potentially expose these endogenous circrnas for immune detection, as has recently been found to be the case for a novel host-derived circrna (lnc-lsm b) that is ifn-inducible and shows a down-regulation of its binding to host proteins during viral infection and therefore appears to compete with viral dsrna as an inhibitor of the rig-i signaling feedback loop ( ) . similar inhibitory mechanisms have also been noted for rna products of the exonuclease skiv l ( ) . finally, recent studies have found that hepatitis c virus (hcv) infection increases the expression of certain cellular rnas that can inhibit rig-i function. hcv infection increased the mrna levels of hepatic selenoprotein, which was able to bind to rig-i through a hairpin structure and inactivated it during viral infection ( ) . infection by hcv, vesicular stomatitis virus (vsv), or sendai virus, or direct exposure of cells to type and interferons increases expression of the cellular long non-coding rna (lncrna), namely lncatv, which similarly inhibits rig-i function by directly interacting with it in order to promote virus replication ( ) . in addition to the greatly increased implications of rig-i and mda modulation, these findings also have significant implications in characterizing new biomarkers of disease, as increased serum selenoprotein level has been found to significantly associate with treatment failure of anti-viral drugs in hcv patients, and can possibly explain the increased prevalence of type diabetes in hcv patients ( ) . cellular rna has also been found to activate rlr signaling during viral infection. vault rnas, which are transcribed from four genes and are normally found in large ribonucleoprotein complexes in cytoplasmic "vaults, " are significantly enriched for binding to rig-i during infection with kshv ( ). this may be due partly to viral infection-induced reduction in the level of cellular triphosphatase dusp , which dephosphorylates the ′ ppp group on the vault rnas, as they could only be immunogenic (in the absence of viral infection) by the addition of the ′ ppp group. rig-i and mda have also been found to be activated by rna microparticles produced in situ by rolling circle transcription, generating tandem repeat rna strands ( ) . retrotransposons may also be able to activate both rig-i and mda , as both can be activated by line rna independently of dna sensing mechanisms and retrotransposition ( ) . viral infections can also induce recognition of host rnas. herpes simplex virus (hsv ) infection, for example, has been shown to induce translocation of the host pseudogene rna sp ribosomal rna into the cytosol to bind to rig-i. knockdown of rna sp decreased cytokine signaling during infection with hsv and ebv as well as influenza a virus (iav) ( ) . rig-i has also been found to be activated by hairpin rna structures generated by cleavage of rna by rnase l, which has been demonstrated to occur during hcv infection ( ) as well as from mitochondrial dsrna produced in p deficient mice ( ) . the mitochondria, in particular, may be an important source of immunostimulatory host dsrna. viral infections are well-known to cause mitochondrial damage ( ) . knockdown and hepatocyte-specific conditional ko of mitochondrial rna degrading enzymes resulted in the increase of cytoplasmic mitochondrial dsrna which was able to activate mda ( ) . additionally, extracellular vesicles (ev) secreted by apoptotic endothelial cells were found to contain long interspersed nuclear element (line) and short interspersed nuclear element (sine) rnas that are products of rna polymerase iii and were able to activate rig-i signaling ( ) . collectively, these findings demonstrate the many unique ways by which cellular rnas can modulate rig-i and mda functions as well as the potential implications of rig-i activation by pharmaceuticals as an anti-viral or generalized immunotherapy, though much caution and studies would still be needed to determine the appropriate levels of rig-i and mda activation. given that rig-i and mda are critical for activating expression of ifn during viral infection, there is much interest in studying the interactions of these cellular proteins with viral factors (rnas or proteins), as the ability to modulate interferon expression is a major evolutionary driving force in viral evolution ( , ) . there are many mechanisms viruses have evolved to evade rig-i and mda signaling, which have been discussed at length elsewhere ( , ) . such mechanisms are of particular importance to segmented rna viruses, providing potentially more dsrnas for rig-i and mda activation ( ) . iav and the other orthomyxoviruses are unique in that they replicate in the nucleus of the cells ( ) , preventing the viral rna from being detected by the prrs. however, recent preliminary evidence seems to suggest that rig-i may also endogenously be present in the nucleus and performs similar viral rna binding and activation of the ifn pathway ( ) , yet this finding has yet to be replicated by other laboratories. there is also increasing evidence to suggest that rna processing is another mechanism of immune modulation. certain bunyaviruses can cleave the ′ tri-phosphate group from their genomic rna ( ) in order to avoid immune detection. rig-i has also been found to be subjected to negative modulation by rnai during iav infection ( ) . on the contrary, nucleoproteins from the sendai virus ( ) regulate the number of di particles being produced, and iav nucleoproteins also regulate the production of abortive replication rna ( ) , mini viral rnas ( ) and dvg rna ( ) , all of which are immunostimulatory. the semliki forest virus (sfv) polymerase has even been found to convert host rna into ′ -ppp dsrna to induce ifn expression ( ) . this raises an intriguing possibility that induction of ifn may actually benefit some viruses under certain circumstances despite ifn signaling negatively regulating viral replication. the viral rna levels and localization throughout the viral life cycle might also play an important role in immune evasion ( ) . control of viral rna levels by viral exoribonucleases in particular illustrates the complicated balance between viral production and immune evasion for optimal viral propagation, as has found to be the case for arenaviral nucleoproteins (nps) ( , ) and nonstructural proteins found in coronaviruses ( , ) . finally, viral infection has the capability to disrupt processes of the cell's basic functions, such as transcription and translation, thereby affecting viral replication and immune signaling in complicated ways ( ) . one of the most significant ways viruses modulate rig-i and mda signaling is through their viral proteins ( ) (figure ) . the respiratory syncytial virus (rsv) non-structural protein (ns ) protein and the z matrix proteins of pathogenic arenaviruses interact with the rig-i card domains to block its interaction with mavs ( , ) . the hsv deamidase ul specifically targets rig-i through its helicase domain, abrogating its ability to bind to rna ( ) . the iav polymerase components also interact directly with rig-i ( ), though their biological significance has yet to be determined as they don't significantly affect ifn production. on the other hand, rna binding appears to be an important bridge between the interaction of rig-i with other viral proteins, as the nucleoproteins (nps) of iav ( ) and arenaviruses ( , ) both interact with rig-i through viral rna. the ns protein of rotaviruses targets rig-i for degradation that is independent of proteasomes ( ) . the v protein of paramyxoviruses inhibits mda ( ) by targeting a unique feature of the atp binding pocket in mda ( ) and by inhibiting mda card dephosphorylation ( ) , but can also inhibit rig-i by interacting with the card domain to prevent its ubiquitination by trim ( ) . finally, the us protein of hsv ( ) and the arenaviral z matrix proteins ( ) directly interact with and inhibit rig-i and mda in a similar fashion. there are also many other viral proteins that can regulate proteins in the rig-i and mda pathways, which have been discussed in detail elsewhere ( , , , , , ) . it is important to consider the different regulatory mechanisms of rig-i and mda when considering their different functionalities (figures , ) . one of the key differences between these proteins is in their post-translation modifications ( ) . ubiquitination of rig-i is necessary for its activation ( ) and is a point of negative regulation by host proteins ( , , ) , viral proteins ( , , ) and ubiquitin mimics ( ) as well as positively regulated by influenza b ns protein ( ) and another ubiquitin mimic ( ) . on the contrary, mda is more well-known to be negatively regulated by ubiquitination ( ) , with positive regulation by k ubiquitination being more controversial. while the deubiquitinase usp inhibits mda as well as rig-i, it is thought that this may be due to usp directly binding the mda card domain to prevent rna filamentation ( ) . this raises the question of how rig-i can maintain its stability outside of the proteasome, as ubiquitination at other lysine residues in rig-i besides k induces proteasomal degradation ( ) ( ) ( ) . this proteasomal degradation may be mediated by a p autophagic complex that associates with lrrc /isg ( ) and sqstm ( ) and also mediates mitophagy and downregulation of mavs signaling during measles virus infection ( ) . one key observation is that, while both rig-i and mda are cleaved during picornavirus infection, this cleavage is mediated by the viral proteinase c pro ( ) and is independent of the proteasome ( ) for rig-i, whereas it is mediated by cellular caspases and the proteasome for mda ( ) . mda is also cleaved by caspases during apoptosis ( ), though it hasn't been shown whether this is mediated by mda 's ubiquitination sites. the ubiquitin linkage site may be a determinate of function, as the ubiquitin ligases rnf ( ) and stub ( , ) have been shown to negatively regulate rig-i catalyzed k linked ubiquitination as opposed to the known k -linked ubiquitination at the k , k and k activating sites, and rnf has also been proposed to k ubiquitinate rig-i ( ) (though it hasn't been shown directly) ( ) . trim has also been shown to negatively regulate rig-i and mda by k and k ubiquitination ( ) . substantiating the possibility that k ubiquitination on rig-i may be functionally distinct from its other ubiquitination sites by protecting it from degradation is the finding that the ns protein of west nile virus (wnv) targets both rig-i and mda for degradation by proteasomes. additionally, ns inhibited k ubiquitination of rig-i, but mda was not found to be k ubiquitinated ( ) . heat shock protein -alpha (hsp ) has been found to protect rig-i from proteasomal degradation, but it is unknown which type of ubiquitination that is inhibited by hsp ( ) . taken together, the experimental evidence suggests that rig-i may be protected from proteasome degradation despite its activating ubiquitin moieties ( ) . this warrants further studies for mechanistic elucidation. rig-i and mda additionally interact with different cellular co-factors, contributing to their differential regulations of function. rig-i is well-known for being potentiated by proteins that also bind dsrna, such as (pact) ( , ) , which was first discovered as a protein activator of pkr, the serine/threonineprotein kinase (tbk ) ( ) ( ) ( ) ( ) ( ) and the oligoadenylate synthetase l (oasl) ( ) . pact in particular has some functional similarities to rig-i, as they each contain three distinct rna binding domains ( ) and interact with many of the same cellular co-factors, such as pkr ( ) and dicer ( , ) . because of the important role of pact in augmenting rig-i function, it is a prime target for inhibition of rig-i signaling by several viral proteins from diverse families of viruses ( ) ( ) ( ) , the molecular mechanisms of pact inhibition by these viral proteins can vary and still need to be characterized in detail in future studies. similarly, the host ribonucleoprotein raver can increase affinity of mda for dsrna ( ) , and the zinc-finger protein zcchc has recently been found do so for both rig-i and mda ( ) in similar mechanisms to the other known rna-binding proteins. on the contrary, the human hemoglobin subunit beta (hb) has recently been suggested to decrease mda signaling by competing for long dsrna, while hb can enhance rig-i signaling by increasing k ubiquitination on rig-i ( ) . several host factors interacting with rig-i and mda do so by yet undescribed mechanisms. pkr [which is also activated by pact ( , ) and is sequestered by the cellular helicase dhx protein to form stress granules ( , ) along with rig-i ( , ) and trim ( )] appears to have a novel and yet uncharacterized function in enhancing mda -dependent mavs signaling that is dependent on the kinase activity of pkr ( ) . additionally, the porcine interferon-inducible oligoadenylate synthetase-like protein (poasl) has also been found to interact with and inhibit mda by an unknown mechanism ( ) . the rig-i card domain interacts with mavs to induce interferon signaling, so proteins that disrupt this interaction [as it has been proposed for the atg and atg autophagy proteins ( ) ] can specifically inhibit rig-i signaling. however, other cellular proteins, such as the complement protein gc qr ( ) and tarbp ( ) that interact directly with mavs, inhibit both rig-i and mda . lactate and hexokinase have also recently been found to inhibit rig-i and mda by interacting with mavs, which may be significant in explaining the interplay between metabolism and immune signaling as glycolysis was found to be greatly decreased upon rlr signaling ( ) . likewise, cellular proteins, such as nlrc ( ) that interacts with the rig-i and mda card domains have been shown to block interaction of both rig-i and mda with mavs. contrarily, dhx has been identified as a rig-i cofactor that interacts with the rig-i card domains and with pamp (dsrna), thereby increasing rig-i atpase activity ( ) . additionally, adp-ribosylation factor proteins can block rig-i and mda from interacting with pamps and thereby inhibit their activation ( , ) . lastly, the green tea molecule egcg has also been shown to inhibit the atpase function of rig-i ( ) . the similarities and differences between rig-i and mda modulations and signaling are complex and will need to be elucidated further in future studies. despite their structural and mechanistic differences, it is important to emphasize that existing phylogenetic analysis indicates that rig-i and mda come from a common origin that is also shared among several other protein families (figure ) . the linkage of the helicase and dexd/h box protein appear to be ancient, as orthologs of these proteins are found in the archaea kingdom ( , ) . mda orthologs are found in most vertebrates ( ) , while rig-i orthologs are only found in mammals, ducks, geese and some selected fish and reptiles ( , ( ) ( ) ( ) ( ) ( ) ( ) ( ) (figures , ) . it is therefore likely that mda evolved first, perhaps from a common ancestor with the closely related lgp helicase family ( ) , which is structural similar to rig-i and mda but lacks the card domains at its n terminus ( ) . lgp orthologs are also only found in vertebrates while the next closest related family of proteins (dicer) are more ancient proteins. it has therefore been proposed that the rig-i helicase-dexd/h complex may have been duplicated from mda in the common ancestor of vertebrates ( ) . the association of the two card domains appears to have followed, as individual card domains are found in a variety of vertebrates that also encode caspases ( , ) , but only rig-i, mda , and certain members of the nacht family of ntpases ( ) have two card domains. phylogenetic analysis has shown that the helicase-dexd/h and card have strong co-evolution history ( , ) , while card has evolved more independently ( ) . card appears to have been grafted onto the rig-i helicase-dexd/h complex first, with the card -mda being duplicated from this event. finally, card was grafted onto the card -helicase-dexd/h complex in separate events for rig-i and mda ( ) . in mammals, positive selection can be seen in the flexible hinge region connecting the card domains to the helicase in rig-i and mda . rig-i contains an additional site of positive selection within the hel structural motif (n ), while most of the unique positive selection sites for mda are in regions specific to it, including a amino acid insertion in hel ( ) . while rig-i and mda may both originate from common ancestors of vertebrates, there is increasing evidence to suggest , and related dexd/h-box helicases are shown as a phylogenetic tree, along with their lowest level of biological taxonomy that these proteins are found in present day. in short, the precursor of the mda helicase-ctd likely originated from a common ancestor with the precursor for lgp , which was then duplicated to create the helicase-ctd precursor of rig-i in the common ancestor of vertebrates. card was then grafted onto the helicase-ctd protein, and this protein was duplicated to create the card -helicase-ctd precursor of mda . finally, card was grafted onto these proteins in separate events, forming the modern-day rig-i and mda . that proteins with similar functions may have evolved separately in other species from ancient helicase-dexd/h proteins, implicating rna-mediated defense responses as a potentially universal biological function. a rig-i homolog has recently been found in a planarian that is able to activate downstream inflammatory genes in the absence of the traditional card domains ( ) , and a similar homolog in caenorhabditis elegans has been proposed to mediate anti-viral rnai by complexing with dicer and catalyzing their translocation on the viral genome ( ) . additionally, insects have been found to primarily respond to rna viruses by rnai mediated by dicer proteins ( ) . dicer may potentially mediate dsrna-activated anti-viral signaling pathways that is independent of rnai pathways, as has been found to be the case for the expanded cag-repeat dsrna ( ) . pattern recognition receptors (prrs) that respond to viral rna have not yet been found outside of the animal kingdom, as rlrlike proteins in prokaryotes do not have card domains and the prrs in plants found so far are surface-receptor kinases that respond to external molecular elements of bacteria ( ) [similar to the mammalian toll-like receptors (tlrs)]. however, rna silencing has been demonstrated to be an important anti-viral strategy in plants ( , ) and certain arabidopsis mutants appear to be more susceptible to infection by rna viruses ( ) . rig-i ( ) and mda ( , ) are known to influence antiviral signaling in zebrafish (danio rerio) and other fish species ( , ( ) ( ) ( ) through the canonical mavs signaling pathway. fish rig-i like receptors (rlrs) have been shown to be regulated by the expression of alternate splicing isoforms ( , ) , which have also been found to occur with a dominant-negative splice variant of the human rig-i ( ). rig-i and mda have also been found to participate in anti-viral signaling in ducks ( ) ( ) ( ) ( ) and geese ( , , ) , and mda alone in chickens ( ) ( ) ( ) and other birds ( ) . the observation across species of rlr's performing compensatory mechanisms when a function or a pathway protein is absent is reiterated in birds, as mda has been found to sense short and long dsrna in chickens ( ) and in the chinese shrew ( ) , both of which lack rig-i. additionally, trim activates rig-i in ducks ( ) and in the chinese goose ( ) in the absence of the k activating ubiquitin binding site that is conserved in primates and some rodents ( ) . finally, the rainbow trout (oncorhynchus mykiss) has been found to express a lgp variant in addition to the canonical lgp that contains an incomplete c-terminal domain of rig-i ( ). the differential presence of prrs may also influence viral evolution. a mutation in the iav polymerase subunit pb found in avian-adapted h n strains decreases the inhibition of human rig-i function by iav nucleoproteins, which may indicate a differential selective pressure for viruses that propagate in species that don't contain rig-i ( ) . the evolutionary pattern and compensatory mechanisms of rlrs across species implicate them as critical for anti-viral function, and that evolutionary forces drive the available pathway proteins to meet these functional needs. future studies need to be done to further differentiate rlr function among the different species, as this will provide critical information concerning the various methods of disease control by targeting the pathogen by these important host proteins. there is also increasing evidence for other rna-sensing dexd/h helicases serving important roles in anti-pathogen immune sensing, which have recently been reviewed elsewhere ( ) . some rna helicase (ddx) proteins appear to serve as complex proteins upon interacting with viral rna. ddx is a well-known example, being suspected of being a transcription factor for ifn-β ( ), associating with spliceosomes and the stress-induced p-bodies to influence mrna splicing and decay, respectively ( , ) , and interacting with the mavs complex during viral infection conditions ( , ) . in particular, ddx associating with mavs has been found to be important for anti-viral control against several viruses ( ) ( ) ( ) , and since the two ddx homologs are found on the x and y chromosomes, they may contribute to immunological differences between genders ( ). this is a repeated theme, as dhx ( ), dhx ( ) , and a complex consisting of ddx /ddx /dhx ( ) have also been found to associate with the mavs complex to enhance ifn signaling, while dhx interacts with mavs independently of viral infection ( ) . ddx proteins can also activate other proteins in the irf pathway. multiple ddx proteins can interact with ikkε, with ddx being phosphorylated by ikkε to induce irf interaction with the tbk -ikkε complex ( ) , and ddx blocking this interaction to inhibit ifn signaling ( ) . similar control mechanisms have been demonstrated for ddx interacting with viral proteins. for example, ddx has recently been found to associate with arenaviral nps to increase viral rna synthesis and ifn expression ( ) . additionally, the np of the h n iav pandemic strain has been shown to target ddx for degradation as a potential mechanism of virulence ( ) . dhx ( ) and dhx have also been found to activate nfκb and mapk signaling pathways. finally, ddx has been shown to act as a cofactor for rig-i ( , ) and dhx for mda ( ) . taken altogether, these cellular proteins have likely evolved to regulate rig-i and mda signaling from their common dexd/h helicase predecessors. as our capacity to study the molecular mechanisms and to purposefully modulate immune responses increases in specificity, so will our needs to characterize the differences between related immune signaling proteins. the concept of personalized medicine derives from the idea that we can therapeutically intervene in a situation that is designed around the individual's unique characteristics. while this is an achievable realm of medicine in the future, an immediate step is to determine the functions of some critical proteins, such as the rig-i and mda of the innate immune arm. examining their structural and functional similarities and differences at multiple levels will allow for a deeper level of appreciation of these proteins, which may be exploited therapeutically to differentially modulate rig-i and mda signalings by different rna ligands ( , , , ) or other pharmaceutical compounds ( ) rig-i, a human homolog gene of rna helicase, is induced by retinoic acid during the differentiation of acute promyelocytic leukemia cell retinoic acid-inducible gene-i is induced in endothelial cells by lps and regulates expression of cox- mda- : an interferon-inducible putative rna helicase with double-stranded rnadependent atpase activity and melanoma growth-suppressive properties overexpression of helicard, a card-containing helicase cleaved during apoptosis, accelerates dna degradation interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures mechanisms of mavs regulation at the mitochondrial membrane mavs coordination of antiviral innate immunity assembly of the whip-trim -ppp c mitochondrial complex promotes rig-i-mediated antiviral signaling zyxin stabilizes rig-i and mavs interactions and promotes type i interferon response. sci rep ikkε and tbk are essential components of the irf signaling pathway multiple functions of the ikk-related kinase ikke in interferon-mediated antiviral immunity trim in the regulation of the antiviral 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signaling by targeting mavs nlrc negatively regulates the nf-kappab and type i interferon signaling pathways dhx is a coreceptor for rlr signaling that promotes antiviral defense against rna virus infection arflike protein (arl ) inhibits rig-i by binding with its c-terminal domain in a gtp-dependent manner negative regulation of melanoma differentiation-associated gene (mda )-dependent antiviral innate immune responses by arf-like protein b green tea catechin, epigallocatechin gallate, suppresses signaling by the dsrna innate immune receptor rig-i crystal structure of a dead box protein from the hyperthermophile methanococcus jannaschii unlocking the dead-box: a key to cryptococcal virulence? sensors of infection: viral nucleic acid prrs in fish association of rig-i with innate immunity of ducks to influenza pathogen recognition receptors in channel catfish: ii. identification, phylogeny and expression of retinoic acid-inducible gene i (rig-i)-like receptors (rlrs) goose rig-i functions in innate immunity against newcastle disease virus infections innate immunity of finfish: primordial conservation and function of viral rna sensors in teleosts comparative study on pattern recognition receptors in non-teleost ray-finned fishes and their evolutionary significance in primitive vertebrates genomic analysis and adaptive evolution of the rig-i-like and nod-like receptors in reptiles nod-lrr proteins: role in host-microbial interactions and inflammatory disease a phylogenetic and functional overview of inflammatory caspases and caspase- -related card-only proteins nacht-lrr proteins (nlrs) in bacterial infection and immunity molecular evolution of glutamate receptors: a primitive signaling mechanism that existed before plants and animals diverged the origin of polynucleotide phosphorylase domains rig-i-like receptors evolved adaptively in mammals, with parallel evolution at lgp and rig-i identification and characterization of an atypical rig-i encoded by planarian dugesia japonica and its essential role in the immune response caenorhabditis elegans rig-i homolog mediates antiviral rna interference downstream of dicer-dependent biogenesis of viral small interfering rnas nucleic acid-induced antiviral immunity in invertebrates: an evolutionary perspective non-self ' mutation: double-stranded rna elicits antiviral pathogenic response in a drosophila model of expanded cag repeat neurodegenerative diseases plant pattern-recognition receptors rna silencing and its suppression: novel insights from in planta analyses the immunity regulator bak contributes to resistance against diverse rna viruses svcv infection triggers fish ifn response through rlr signaling pathway alternative splicing transcripts of zebrafish lgp gene differentially contribute to ifn antiviral response inducible microrna- feedback inhibits the rig-i-dependent innate immune response to rhabdovirus in teleost fish by targeting mavs/ips- microrna- participates in regulating rig-i signaling pathway via targeting duba in miiuy croaker after poly(i:c) stimulation mda and lgp acts as a key regulator though activating nf-κb and irf in rlrs signaling of mandarinfish higher antiviral response of rig-i through enhancing rig-i/mavs-mediated signaling by its long insertion variant in zebrafish roles of rig-i n-terminal tandem card and splice variant in trim -mediated antiviral signal transduction activation of duck rig-i by trim is independent of anchored ubiquitin expression of immune genes rig-i and mx in mallard ducks infected with low pathogenic avian influenza (lpai): a dataset duck rig-i restricts duck enteritis virus infection in vivo cellular and molecular study on duck spleen infected by duck tembusu virus identification and expression profiling analysis of goose melanoma differentiation associated gene (mda ) gene goose mavs functions in rig-i-mediated ifn-β signaling activation characterization of chicken mda activity: regulation of ifnβ in the absence of rig-i functionality chicken cells sense influenza a virus infection through mda and cardif signaling involving lgp chicken mda senses short double-stranded rna with implications for antiviral response against avian influenza viruses in chicken cloning, expression and bioinformatics analysis of a putative pigeon melanoma differentiation-associated gene loss of rig-i leads to a functional replacement with mda in the chinese tree shrew trim identification in the chinese goose: gene structure, tissue expression profiles, and antiviral immune responses in vivo and in vitro expression and functional characterization of the rig-i-like receptors mda and lgp in rainbow trout (oncorhynchus mykiss) influenza virus adaptation pb - k modulates nucleocapsid inhibition by the pathogen sensor rig-i multiple functions of ddx rna helicase in gene regulation, tumorigenesis, and viral infection rna helicase ddx : at the crossroad of viral replication and antiviral immunity hiv- blocks the signaling adaptor mavs to evade antiviral host defense after sensing of abortive hiv- rna by the host helicase ddx the rna helicase ddx x is an essential mediator of innate antimicrobial immunity dhx pairs with ips- to sense double-stranded rna in myeloid dendritic cells dhx senses doublestranded rna in myeloid dendritic cells ddx , ddx , and dhx helicases form a complex with the adaptor molecule trif to sense dsrna in dendritic cells the interaction between the helicase dhx and ips- as a novel pathway to sense double-stranded rna and rna viruses in myeloid dendritic cells ddx inhibits type i interferon production by disrupting tbk -ikkε-irf interactions and promoting tbk and ikkε degradation ddx suppresses type i interferons and favors viral replication during arenavirus infection codegradation of interferon signaling factor ddx by pb -f as a basis for high virulence of pandemic influenza the deah-box rna helicase dhx activates nf-κb and mapk signaling downstream of mavs during antiviral responses ddx , a dexd/h box helicase, is a novel antiviral factor promoting rig-i-like receptor-mediated signaling ddx is involved in rig-i-dependent and independent antiviral responses, and its function is attenuated by virus-induced egfr activation dhx functions as an rna cosensor for mda -mediated emcv-specific antiviral immunity activation of rig-i signaling to increase the pro-inflammatory phenotype of a tumor immunotherapeutic effects of intratumoral nanoplexed poly i:c rig-i-like receptors as novel targets for panantivirals and vaccine adjuvants against emerging and re-emerging viral infections the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © brisse and ly. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -fl l b authors: daryabor, gholamreza; atashzar, mohamad reza; kabelitz, dieter; meri, seppo; kalantar, kurosh title: the effects of type diabetes mellitus on organ metabolism and the immune system date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: fl l b metabolic abnormalities such as dyslipidemia, hyperinsulinemia, or insulin resistance and obesity play key roles in the induction and progression of type diabetes mellitus (t dm). the field of immunometabolism implies a bidirectional link between the immune system and metabolism, in which inflammation plays an essential role in the promotion of metabolic abnormalities (e.g., obesity and t dm), and metabolic factors, in turn, regulate immune cell functions. obesity as the main inducer of a systemic low-level inflammation is a main susceptibility factor for t dm. obesity-related immune cell infiltration, inflammation, and increased oxidative stress promote metabolic impairments in the insulin-sensitive tissues and finally, insulin resistance, organ failure, and premature aging occur. hyperglycemia and the subsequent inflammation are the main causes of micro- and macroangiopathies in the circulatory system. they also promote the gut microbiota dysbiosis, increased intestinal permeability, and fatty liver disease. the impaired immune system together with metabolic imbalance also increases the susceptibility of patients to several pathogenic agents such as the severe acute respiratory syndrome coronavirus (sars-cov- ). thus, the need for a proper immunization protocol among such patients is granted. the focus of the current review is to explore metabolic and immunological abnormalities affecting several organs of t dm patients and explain the mechanisms, whereby diabetic patients become more susceptible to infectious diseases. the metabolic syndrome is defined by the presence of metabolic abnormalities such as obesity, dyslipidemia, insulin resistance, and subsequent hyperinsulinemia in an individual ( ) . dyslipidemia, the main characteristic of metabolic syndrome, is defined by decreased serum levels of high-density lipoproteins (hdls) but increased levels of cholesterol, free fatty acids (ffas), triglycerides (tg), vldl, small dense ldl (sdldl), and oxidized ldl (ox-ldl) ( table ) ( ) . individuals with the metabolic syndrome are much more likely to develop type diabetes mellitus (t dm), cardiovascular diseases (cvds), and fatty liver disease ( ) ( ) ( ) . t dm, the most common form of diabetes (∼ %), is characterized by a systemic inflammatory disease accompanied by insulin resistance (ir) or decreased metabolic response to insulin in several tissues, including the adipose tissue, liver, and skeletal muscle, as well as by reduced insulin synthesis by pancreatic beta cells ( , ) . studies on immunometabolism have indicated that the metabolic states and immunological processes are inherently interconnected ( ) . in this scenario, metabolites derived from the host or microbiota regulate immunological responses during health and disease ( ) . accordingly, in obese individuals, expanded adipose tissue at different locations, by initiating and perpetuating the inflammation, induces a chronic low-level inflammatory state that promotes ir ( ). every organ system in human body can be affected by diabetes, but the extent of organ involvement depends largely on the severity and duration of the disease (figure and table ). during the progression of diabetes, hyperglycemia promotes mitochondrial dysfunction and induces the formation of reactive oxygen species (ros) that cause oxidative stress in several tissues such as blood vessels and pancreatic beta cells ( ) ( ) ( ) . accumulating damage to the mitochondria, as well as several macromolecules, including proteins, lipids, and nucleic acids by ros promotes the process of aging ( ) . as a result, pancreatic β cells that require functional mitochondria to maintain insulin synthesis fail to generate high enough levels of insulin ( , ) . in the absence of compensatory mechanisms, stress-responsive intracellular signaling molecules are activated and cellular damage occurs. elevated intracellular levels of ros and subsequent oxidative stress play an important role in the pro-atherosclerotic consequences of diabetes and the development vascular complications ( , ) . moreover, the non-enzymatic covalent attachment of glucose and its toxic derivatives [e.g., glyoxal, methylglyoxal (mgo), and deoxyglucosone] to the biological macromolecules such as nucleic acids, lipids, and proteins leads to the formation of advanced glycation end products (ages) ( , ) . accumulated ages block the insulin signaling pathway and promote inflammation ( , ) . in addition, the attachment of ages to their receptors [e.g., cd , galectin- , scavenger receptors types i (sr-a ), and ii (sr-a )] on the surfaces of immune cells in the circulation and tissues activates the expression of pro-inflammatory cytokines and increases free radical generation ( ) . furthermore, due to the chronic exposure of cells to high glucose levels in untreated t dm patients, glucose toxicity might occur in several organs. this will figure | effects of t dm on body organs. t dm is an inflammatory state that affects circulatory system, gastrointestinal tract, pancreatic beta cells, liver, and skeletal muscles and makes them dysfunctional. nfald, non-alcoholic fatty liver disease; nash, non-alcoholic steatohepatitis; er, endoplasmic reticulum. eventually lead to nephropathy, cardiomyopathy, neuropathy, and retinopathy. gut microbiome dysbiosis is another important factor that can facilitate the induction and progression of metabolic diseases such as t dm ( ) . the gut microbiome dysbiosis, by altering the barrier functions of intestine and the host metabolic status, promotes the insulin resistance in diabetic patients ( ) . diabetes also impairs the immune system and increases the susceptibility of patients to serious and prolonged infections ( ) . this is likely to be the case with the severe acute respiratory syndrome coronavirus (sars-cov- ), as well ( , ) . in the current paper we will review recent research to explore the impairment of body organs in t dm patients and explain how diabetic patients become more susceptible to certain infectious diseases. vascular homeostasis is an important function of the endothelium. under homeostatic conditions, the ecs maintain the integrity of blood vessels, modulate blood flow, deliver nutrients to the underlying tissues, regulate fibrinolysis and coagulation, control platelet adherence and patrol the trafficking of leukocytes (figure a ) ( ) . normal ecs also internalize high-density lipoproteins (hdls) and its main protein part apolipoprotein a-i (apoa-i) in a receptor-mediated manner to activate endothelial cell nitric oxide (enos) synthase and promote anti-inflammatory and antiapoptotic mechanisms ( figure b ) ( ) . hdl receptors on the surfaces of ecs include: the atp-binding cassette (abc) transporters a and g , the scavenger receptor (sr)-b and the ecto-f -atpase ( ) . according to the epidemiological studies, diabetes mellitus is considered as one of the main risk factors for cvd (figure ) ( ) . from the beginning of t dm, the functions of ecs are impaired, which is the main cause of disease-related side-effects ( ) . ecs can initiate and perpetuate the inflammatory milieu during the pathogenesis of diabetes. due to the negative impacts of hyperglycemia and subsequent oxidative stress, cvds are more common among diabetic patients ( ) . it has been observed that incubation of human aortal endothelial cells (haecs) with a medium containing high glucose concentrations (hg, mm) increases the intracellular levels of mgo and glycated proteins that in turn activate the unfolded protein response (upr) and trigger inflammatory and prothrombotic pathways ( ) . glycated apoa-i, which is formed during hyperglycemia, modifies its structure, decreases its lipid-binding ability, prevents cholesterol efflux from macrophages and impairs its anti-inflammatory function ( , ) . vaisar et al. have shown that hdls from diabetic patients have a reduced capacity to trigger enos production and suppress tumor necrosis factor-α (tnf-α)mediated inflammatory responses within ecs ( ) . diseases such as t dm that induce high levels of vascular injury are accompanied by an elevated number of circulating endothelial cells (cecs) ( ) . t dm-related risk factors such as dyslipidemia, hyperglycemia, and hyperinsulinemia as well as other conditions (e.g., inadequate physical activity, smoking, and high blood pressure) facilitate the formation of atherosclerotic plaques/lesions ( ) . dyslipidemia, due to the elevated flux of ffa from insulin-resistant tissues and spillover from entry (c) blood vessels in t dm patients. during the progression of the disease, red blood cells become glycated, while activated ecs synthesize elevated levels of adhesion molecules and chemokines that facilitate monocytes recruitment, adhesion, and transmigration across the endothelium toward the subendothelial region. monocytes are then differentiated into macrophages and eventually, by excess lipid uptake, generate foam cells. subsequently, further immune cell infiltration into the atherosclerotic lesion occurs, where their inflammatory cytokines promote platelet activation, ec apoptosis, and increased generation of ros and ox-ldl. (d) interactions between oxldl and its receptor aggravate ros generation, nf-κb activation and inflammation. ec, endothelial cell; rbc, red blood cell; plt, platelet; hdl, high-density lipoprotein; ox-ldl, oxidized low-density lipoprotein; ros, reactive oxygen species; enos, endothelial nitric oxide synthase; no, nitric oxide; lox- , lectin-type oxidized ldl receptor . into adipocytes, is considered as an important risk factor for developing cvd among diabetic patients. this is because dyslipidemia promotes inflammation, endothelial dysfunction, and platelet hyperactivation ( , ) . during the progression of atherosclerosis, lipids, immune cells, and extracellular matrix accumulate in the arterial intima or subendothelial regions ( figure c ) ( ) . advanced plaques can impede blood flow and cause tissue ischemia or might become disrupted and generate a thrombus that stops the blood flow of important organs. vascular complications of diabetes engage either tiny or large blood vessels (micro-and macroangiopathy, respectively). microangiopathies, which can be seen in the kidneys, vasa nervorum and eye tissues, cause nephropathy, neuropathy, and retinopathy. macroangiopathies, by inducing atherosclerosis in the coronary, carotid, and peripheral arteries, increase the risk of myocardial infarction (mi), stroke and peripheral artery disease (pad). macrovascular complications due to ec dysfunction are considered as an important cause of mortality and morbidity among diabetic patients ( ) . oxidative stress has an essential role in the induction of vascular complications during the course of diabetes ( ) . ec dysfunction (e.g., delayed replication, dysregulated cell cycling, and apoptosis), as well as enhanced ox-ldl formation are some consequences of oxidative stress. it has been well-established that sdldl and ox-ldl have an enhanced atherogenic ability and are more useful biomarkers than total ldl for predicting cvd ( , ) . sdldl particles have a smaller size than other ldl particles. thus, sdldl particles are more easily oxidized, and their atherogenic potential is enhanced. during oxidative stress, levels of ox-ldl increase by the excess action of reactive oxygen species (ros) ( ) . subsequently, ox-ldl interaction with scavenger receptors, including cd , sr-a /cd , sr-b , and lectin-like ox-ldl receptor- (lox- ) on the surface of ecs activates the nadph oxidase that in turn increases the expression of ros and activates the transcription factor nf-αb ( ) . afterwards, the expression of lox- , adhesion molecules (e.g., selectins and integrins) and the secretion of pro-inflammatory cytokines and chemokines are increased, while no synthesis is decreased in ecs ( figure d ) ( ) ( ) ( ) . ec-derived chemokines bind to their cognate receptors on the surfaces of monocytes and recruit them toward the inflamed endothelium. following this, selectin-based rolling and integrin-based attachment of monocytes to the ecs cause their migration toward the subendothelial region, where they develop into lipid-laden macrophages or foam cells later on ( ) . the scavenger receptor lox- plays an important role in the uptake of ox-ldl during atherogenesis. it is strongly expressed on the surfaces of ecs, but has an inducible pattern of expression on the surface of macrophages and smooth muscle cells ( ) . the accelerated uptake of ox-ldl by macrophages accounts for their transformation into foam cells, the initial hallmark of atherosclerosis ( , ) . besides, diabetes leads to both quantitative and qualitative defects in circulating angiogenic progenitor cells (capcs) that take part in the repair of injured endothelium ( ) . it has been shown that humans or mice with decreased numbers of cd + cd + cd + cd dim sca- + flk- + capcs have an increased prevalence of t dm, elevated hba c levels and aggravated cvd risk scores ( , ) . in diabetic patients, despite elevated serum levels of proangiogenic molecules, like angiopoietin- / , epo, and vegf-a, angiogenesis is impaired. this is mainly due to the decreased expression levels of vegfr and cxcr on the surfaces of capcs, which makes them unresponsive to the angiogenic factors ( , ) . it has also been shown that circulating proangiogenic granulocytes composed of eosinophils and neutrophils are also impaired in diabetic patients ( ) . besides, elevated levels of ages in t dm cause ec dysfunction and vascular inflammation ( ) . ren et al. have shown that incubation of human coronary artery endothelial cells (hcaecs) with ages causes decreased expression (at both mrna and protein levels) and enzymatic activity of enos, increased levels of ros, diminished mitochondrial membrane potential and declined activity of catalase and superoxide dismutase in treated cells ( ) . another study by lan et al. has shown that ages in the pancreas decrease ec viability and induce their apoptosis in an nfκb signaling-related manner ( ) . however, apigenin ( ′ , , trihydroxyflavone) can protect ecs against oxidative stress and subsequent inflammatory reactions mediated by ages ( ) . apigenin binds to methylglyoxal (mgo) and forms a complex that inhibits age formation. chettab et al. have shown that the expression of icam- as well as the production of il- , are significantly increased in huvecs cultured in hg medium compared to cells cultured in normal glucose (ng, . mm) conditions ( ). bammert et al. found out that incubation of huvecs with hg media promotes the generation of endothelial microparticles (emps) that, when added to normally cultured huvecs, downregulate the expression of anti-apoptotic microrna mir-let a, but enhance the synthesis of active caspase- and cause cell apoptosis ( ) . several micrornas, including mir- , mir- a, mir- , mir- a, mir- , mir- , mir- a, mir- , and mir-let d, regulate vascular homeostasis. it has been shown that the expressions of mir- a and mir- are significantly reduced in circulating mps isolated from diabetic patients compared with normal individuals. this could be involved in making diabetic individuals more susceptible to coronary heart disease ( ) . moreover, hg media upregulate the expression of nadph oxidase that will induce the generation of ros. this leads to subsequent apoptosis of the huvecs through a ros-dependent caspase- pathway ( ). su et al. have demonstrated that argirein medication, by inactivating nadph oxidase, can prevent endothelial cell apoptosis in a rat model of t dm and hence attenuate vascular dysfunction ( ) . hg further increases the permeability of the huvecs in a protein kinase c (pkc)-dependent manner ( , ) . hassanpour et al. showed that incubation of endothelial progenitor cells with the serum of t dm patients inhibits their migration toward bfgf, increases their expression of vegfr- , but reduces their expression of vegfr- and induces their apoptosis ( ) . however, humanin (hn), a mitochondriumderived peptide, is cytoprotective against apoptosis during pathological conditions, such as diabetes mellitus ( ) . it has been demonstrated that simultaneous incubation of h c cells, a line of rat cardiac myoblasts, with h o and hn decreases the intracellular levels of ros, preserve mitochondrial function/structure and decline cellular apoptosis ( ) . wang et al. have indicated that the treatment of huvecs with hn before their incubation with hg medium increases the expression of enos, while decreasing the expression of endothelin (et- ), vcam- , tnf-α, il- β, and e-selectin in a krüppellike factor (klf )-dependent manner. such changes in the expression of integrins prevent the attachment of monocytes to huvecs ( ) . accordingly, hn might be used to prevent the development of hyperglycemia-associated ec dysfunction in t dm. ec activation and expression of adhesion molecules also facilitate activation and adhesion of platelets. this will increase the risk of thrombosis and promote the development of thrombotic angiopathy, typical for diabetic patients. platelets are tiny anucleated cellular fragments generated from megakaryocytes in the bone marrow. they circulate in the blood for ∼ - days and play essential roles in hemostasis and in controlling vascular integrity ( ) . circulating inactive platelets move in the proximity of vessel walls (figure a ) and rapidly get activated in response to vascular injury. at the end of their life, platelets are cleared from circulation with the action of the liver and spleen-resident macrophages. platelets have an essential role in the initiation and progression of inflammation. platelet hyperactivation that occurs during inflammatory states (e.g., t dm) facilitates the pathogenesis of cvds ( figure c ) ( , ) . it has been shown that elevated levels of resistin, an adipokine, in diabetic patients enhances oxidative stress, promotes endothelial dysfunction and facilitates platelet activation ( ) . activated platelets with an increased mean volume [mean platelet volume (mpv)] secrete microparticles (mps) and soluble adhesion molecules (e.g., sp-selectin and scd l) that in turn activate endothelial and immune cells ( ) ( ) ( ) . higher levels of platelet-derived mps, which correlate positively with fasting blood sugar and glycated hemoglobin, have been shown in newly diagnosed t dm patients compared to healthy individuals ( ) . in t dm patients thrombotic microangiopathies can lead to the development of cvds ( ) . platelets in the patients adhere to ecs and aggregate more rapidly than in healthy individuals thereby increasing the risk of thrombosis. in a mouse model of t dm, zhu et al. have shown that ages interact with cd , a member of the type scavenger receptor family, on the surfaces of murine platelets to activate them and induce a prothrombotic state ( ) . elevated levels of the p y receptor on the surface of platelets in t dm expose diabetic patients to a prothrombotic condition. this receptor has an essential role in platelet activation ( ) . zhou et al. have shown that long non-coding rna (lncrna) metallothionein pseudogene (mt p ), which is markedly upregulated in megakaryocytes of t dm patients, enhances the expression of p y receptor in platelets ( ) . they indicated that this is due to the inhibitory action of mt p on mir- . virtually all parts of the human digestive system, including the gastrointestinal tract, pancreas, and the liver are affected by diabetes. the git is populated with a myriad of microorganisms, including principally bacteria but also archaea, viruses, fungi, and protozoans that dynamically influence the health status and homeostasis of the host. the physiological functions of the git resident microbes improve gut integrity, protect against microbial pathogens and regulate immune responses ( ) . mucosal barriers, such as intestinal epithelial cells (iecs) and the mucus layer, spatially isolate the host immune system and gut microbiota to prevent unnecessary immune activation and intestinal inflammation. they also facilitate the uptake of nutrients through receptors and transporters. however, hyperglycemia, in a glut -dependent manner, can influence the mucus and alter the integrity of adherence and tight junctions between intestinal epithelial cells of diabetic mice. this will enhance the permeability of the intestinal barrier leading to so called "leaky gut." subsequently, hyperglycemia may facilitate the dispersal of an enteric infection into a systemic infection (figure ) ( ) . interestingly, the reversal of hyperglycemia, conditional deletion of glut from the iecs and inhibition of glucose metabolism will fix the barrier dysfunction and prevent the spread of bacteria ( ). xu et al. have shown that faecalibacterium prausnitzii, one of the most frequent commensal bacteria in normal individuals with essential roles in gut homeostasis, generates anti-inflammatory molecules that enhance the expression of tight junctions and improve intestinal integrity during diabetes ( ) . however, in some cases, gut microbiota dysbiosis or altered microbial composition of the intestines could induce t dm and lead to its progression ( ) . of interest, the widely used antidiabetic drug metformin can improve barrier integrity and restore the healthy microbiota composition of the gut in diabetic patients ( ) . the intestinal commensal bacterium akkermansia muciniphila can also act as a sentinel to reduce microbial translocation across the gut and prevent the subsequent inflammation in patients with t dm ( ) . hyperglycemia can further decrease the intracellular levels of glutathione (gsh) but increase inos activity and no production in the iecs ( ). zhao et al. have found out that hyperglycemia in a pkcα-dependent manner inhibits the ubiquitination, internalization and degradation of the divalent metal transporter (dmt ) present on the microvillar membranes of iecs. subsequently, intestinal iron uptake is enhanced and accumulated iron ions aggravate diabetes-related complications and increase mortality ( , ) . the pancreas consists of the exocrine and endocrine compartments. the endocrine part is made of different cell types, including α, β, δ, and ε cells that secrete glucagon, insulin, somatostatin, and ghrelin hormones, respectively. these cells are aggregated into specialized structures called islets of langerhans, which play an important role in controlling blood glucose levels through the secretion of insulin and glucagon. in t dm, despite normal levels of β-cell replication and islet formation, β-cell apoptosis is increased so that the number of cells declines by ∼ % (figure ) ( ) . during the progression of t dm, the insulin-resistant state forces β-cells to compensate for the lack of insulin by elevating its synthesis to restore the normal blood glucose level. however, in severe diabetic patients, β-cell exhaustion, and subsequent persistent hyperglycemia occur ( ) . furthermore, chronic elevated serum levels of free fatty acids, seen in obesity and t dm, induce lipotoxicity in beta-cells and suppress their insulin secretion ability ( ) . to alleviate chronic inflammation, overcome insulin resistance (ir) and to prevent β-cell apoptosis, stem cells or stem cell derivatives such as insulin-producing cells (ipcs) and exosomes have been suggested ( ) ( ) ( ) ( ) . their effects are believed to be mainly due to their anti-inflammatory activities. secretagogin (scgn) is predominantly expressed by pancreatic β-cells protecting their normal functions. scgn also acts as an insulin binding protein to make it more stable, avoid its aggregation, improve its functions and enhance its secretion ( , ) . in t dm patients, due to the islet cell dysfunction and endoplasmic reticulum (er) stress, serum levels of scgn are elevated reflecting stress and dysfunctional islet cells ( ) . moreover, in patients with t dm, islet amyloid polypeptide (iapp or amylin), a peptide hormone and one of the main secretory products of pancreatic β-cells, tends to deposit in the islets of langerhans, form insoluble fibrils and impair secretory functions of β-cells ( ) . iapp is costored with insulin in the secretory granules of pancreatic β cells. in steady-state conditions it regulates food intake, insulin secretion, and glucose metabolism ( ). ribeiro et al. have noted that pancreatic extracellular vesicles (evs) from healthy individuals, but not from t dm patients, directly bind to iapps and prevent amyloid formation within the pancreatic islets ( ) . the authors showed that the altered protein-lipid composition of the evs is the main reason for this discrepancy ( ) . however, chatterjee et al. have shown that β-cells from t dm patients have a dysfunctional proteasome complex that fails to degrade pancreatic iapp, whereby amyloid formation is induced ( ) . furthermore, in t dm patients, lipids accelerate the formation of fibrillary iapp, which aggravates islet cell damage ( ) . dhar et al. have demonstrated that chronic use of mgo in sprague-dawley rats increases the expression of nf-αb, mgo-derived ages and their receptors in pancreatic β cells. mgo can also induce apoptosis of islet β cells, increase fasting plasma glucose levels and impair glucose tolerance ( ) . in t dm patients the plasma level of mgo directly correlates with fasting blood sugar and hba c levels ( ) . bo et al. further showed that mgo in a dose-based manner impairs insulin secretion of pancreatic β-cell lines min and ins- through increased generation of ros and by induction of mitochondrial dysfunction ( ). robertson et al. have found out that elevated levels of ros in pancreatic β-cells inhibit the pancreas duodenum homeobox- (pdx- ) transcription factor that is needed for insulin synthesis ( ) . it has been shown that chronic use of mgo in animals could induce t dm, while simultaneous use of alagebrium, which breaks age compounds, attenuates the disease ( ) . it has also been reported that during the course of diabetes dedifferentiation and conversion of β-cells into αand δ-"like" cells occurs ( ) . in conclusion, the pancreatic β cell function is progressively reduced during the progression of t dm. the liver is by far the most important metabolic organ with essential roles in regulating homeostasis and mediating glucose and lipid metabolism. metabolic activities of the tissue are precisely controlled by the actions of metabolic substrates, including free fatty acids (ffas) and hormones ( ) . t dm patients usually suffer from a chronic liver condition called non-alcoholic fatty liver disease (nafld). it is characterized by steatosis that means ectopic fat storage in hepatocytes and subsequent insulin resistance (figure ) ( ) . lipid accumulation in hepatocytes leads to impaired biogenesis of mir- that facilitates insulin signaling and prevents lipogenesis ( ) . several factors such as obesity, increased serum levels of fatty acids, and insulin resistance can increase the risk of fatty liver disease. p y receptor, through the induction of the c-jun n-terminal kinase (jnk) and prevention of insulin signaling, can promote insulin resistance in hepatocytes in t dm ( ) . in some cases, nafld may progress into an aggressive form of inflammatory fatty liver disease called non-alcoholic steatohepatitis (nash), which might cause liver cirrhosis and organ failure ( ). dang et al. have indicated that exosomes released from the adipose tissues of obese mice due to the smaller mir- - p content can promote insulin resistance in the murine hepatocyte cell line aml (alpha mouse liver ) ( ) . the adipokine visfatin that is released from the adipose tissue of obese individuals has also been shown to activate the pro-inflammatory stat signaling pathway and nf-κb in the human liver cell line hepg and promote their insulin resistant state ( ) . nevertheless, the hepatocyte growth factor (hgf) can alleviate the insulin resistance of hepatocytes and control their triglyceride and cholesterol contents ( ) . skeletal muscle (sm) is the main tissue that releases glucose after insulin stimulation. hence, insulin resistance in sm has a pivotal role in the metabolic dysregulation of t dm. insulin resistance in sm is the primary defect of t dm that facilitates the progression of fatty liver disease, deposition of fat in the liver (figure ) ( ) . skeletal muscle from diabetic patients expresses less genes related to insulin signaling and metabolic pathways, but more apoptosis and immune-related genes ( ) . this inflammatory milieu is mainly due to the proinflammatory actions of obesity-related adipose tissue mediators, which are released into the circulation and promote inflammation within the sm ( ). furthermore, obesity causes intermyocellular and perimuscular adipose tissue expansion that acts like adipose tissue depots to enhance sm inflammation ( ) . it has been shown that human skeletal muscle cells (hsmc), isolated from diabetic patients, after a -h culture generate significantly more tnf-α, il- , il- , il- , monocyte chemotactic protein (mcp)- , growth-related oncogene (gro)-α, and follistatin compared to non-diabetic individuals ( ) . this altered secretion of myokines (e.g., cytokines secreted by sms) is an intrinsic feature of sm during the progression of t dm. in sm, glut- , which is quickly translocated to the cell surface, facilitates glucose uptake in response to insulin hormone as well as muscle contraction. pinto-junior et al. have shown that the use of age-albumin in rats increases the expression of the inflammatory molecule nf-κb within the sm. nf-κb binds to the promoter of the glut- gene and suppresses its expression (at both mrna and protein levels) ( ) . accordingly, glut- levels on the surfaces of sm decrease and subsequently, whole-body ir develops. the immune system is generally classified into two main arms, innate and adaptive (or acquired) immunity. adaptive immunity is mediated by b cells, which produce antibodies and t cells, which are classified into cd + helper cells and cytotoxic cd + cells. a considerable literature has discussed the dysfunctional immune responses in diabetic patients ( table ) ( ) ( ) ( ) ( ) ( ) ( ) . abnormal immune cell activation and subsequent inflammatory environment has an essential role in the progression of t dm ( ) . in this regard, chronic inflammation due mainly to the activation of the myeloid cell lineage (e.g., macrophages and neutrophils), is directly related to the induction of ir ( , ). their numbers are elevated, are larger and more granular, express diminished levels of antioxidant genes but elevated levels of pro-apoptotic and pro-inflammatory genes. complement system attachment of c-type lectin proteins to mannose residues is decreased, lectin pathway is impaired, cd activity is reduced, mac deposition in vascular walls is increased. dendritic cells (dcs) their numbers and activity are reduced. their cholesterol efflux is decreased, generate foam cells, have dysfunctional efferocytosis. are activated, constitutively release nets, produce high levels of mpo, ros, and calprotectin (s a /a ), are more susceptible to apoptosis, their migration, phagocytosis and microbial killing are impaired. nk cells their numbers are increased but are usually dysfunctional, express high levels of glut but decreased levels of nkg d and nkp , have reduced degranulation capacity, are more susceptible to apoptosis. nkt cells their numbers are increased, produce high levels of ifn-γ, il- , and il- , express high levels of nkp , nkg d, and nkp but low levels of nkg a and b. innate lymphoid cells (ilcs) ilc s are increased and produce high levels of ifn-γ. humoral immunity (b cells) germinal centers are reduced, ab production and isotype switching is defective, abs become glycated, abs fail to activate complement. functions of osteoclasts, which are bone-resident innate immune cells ( ) . this may affect bone structure and delay bone healing. defects in the innate, as well as adaptive immunity, are supposed to be the main cause of diabetic individuals' susceptibility to infections ( ) . furthermore, some microorganisms, especially bacteria, in hyperglycemic conditions are better nourished and become more virulent, while also having a better milieu to cause infections. complement system the complement system is a first-line defense mechanism against invading microorganisms. it acts via different but interconnected classical, alternative, and lectin pathways ( ) . ilyas et al. have shown that under high glucose conditions, the attachment of ctype lectin proteins to high-mannose containing glycoproteins is substantially decreased in a dose-dependent manner. these carbohydrate-binding proteins include mannose-binding lectin (mbl), surfactant protein d (sp-d), dendritic cell-specific intercellular adhesion molecule- -grabbing non-integrin (dc-sign, cd ), and dc-sign-related (dc-signr) protein ( ) . reduced binding of mbl in the presence of high levels of sugar causes a significant reduction in the lectin pathway activity, but does not influence classical or alternative pathway activity ( ) . nevertheless, barkai et al. did not find significant differences in the function of classical or mbl pathways between t dm and healthy individuals ( ) . however, significantly decreased activity of ficolin- -mediated lectin and alternative pathways, as well as decreased levels of c d and soluble complement c b- (sc b- ) were seen in diabetic patients with escherichia coli-mediated urinary tract infections ( ) . this may be linked to a reduced ability of diabetics to protect themselves against bacterial infections. the lipopolysaccharides of certain gram-negative bacteria, like salmonella serotype o , as well as the cell walls of fungi, are rich in mannose. possibly, because of this, in addition to additional provision of nutrients, an increased prevalence of fungal infections is seen in t dm patients ( , ) . patel et al. found a significantly higher prevalence of oral candida carriage in diabetic patients compared to healthy controls ( ) . they found that candida albicans was the most commonly isolated species followed by c. tropicalis, but uncommon species such as c. lusitaniae and c. lipolytica were also isolated ( ) . another study by jhugroo et al. showed that c. albicans is the predominant yeast isolated from oral mucosal lesions of diabetic patients, followed by. c. tropicalis and c. krusei dendritic cells (dcs) are a heterogeneous population of specialized and professional antigen-presenting cells (apcs) that create a crucial link between the innate and adaptive immune responses ( , ) . some studies have shown that the numbers of dcs are reduced in both type and diabetes ( , ). seifarth et al. have found that t dm patients with poor metabolic control have decreased numbers of both myeloid and plasmacytoid dcs compared with healthy controls. this could make them more susceptible to opportunistic infections ( ) . in the case of good blood glucose control, the reduction in dc numbers was less prominent but still significant, especially for myeloid dc (mdc ) cells ( ) . another study by blank et al. demonstrated that women with t dm and poor glycemic control (hba c ≥ %) have fewer numbers of circulating plasmacytoid dcs (pdcs) compared to diabetic women with good glycemic control (hba c < %) or to healthy women ( ). montani et al. have recently shown that hyperglycemic medium and hyperglycemic sera derived from t dm patients prevent the maturation of monocytes into effective dcs and their activation in vitro ( ) . interestingly, quercetin, a flavonoid with antiinflammatory and antioxidant characteristics, prevented such effects ( ) . macrophages are important immune cells that play critical roles through all stages of the pathogenesis of t dmrelated atherosclerosis ( ). swirski et al. have shown a significantly elevated number of pro-inflammatory monocytes in the circulation of apoe −/− mice, an animal model of atherosclerosis, compared to control mice ( ) . modifications of the lipoproteins in the arterial walls of diabetic individuals make them pro-inflammatory and activate the overlying endothelium. in response, monocytes are recruited into the subendothelial region, differentiate into macrophages and internalize the accumulated lipoproteins. finally, cholesterol-laden foam cells are generated. they promote inflammation and progression of the disease through the synthesis and secretion of cytokines, chemokines, ros, and matrix metalloproteinases (mmps) (figure c ) ( ) . foam cells lose their migratory potential, die by apoptosis and generate a necrotic core within the atherosclerotic plaque ( ) . it has been demonstrated that the use of mesenchymal stem cells in apoe −/− mice reduces the numbers of monocytes/macrophages at the site of inflammation, decreases lipid deposition and diminishes plaque size ( ). ma et al. have studied the effects of long-term hyperglycemia in diabetic mice and found out that compared to non-diabetic control mice, the numbers of f / + macrophages isolated from spleen (spms), as well as from peritoneal exudates (pems) of diabetic mice are significantly decreased ( ) . subsequently, sun et al. showed that stimulation of pems from diabetic mice in vitro with ifn-γ and lipopolysaccharide (lps) significantly decreased the expression of intercellular adhesion molecule (icam- or cd ), cd , tnf-α, and il- , while it increased the production of nitric oxide (no) ( ) . they further showed that stimulation of pems isolated from diabetic mice with il- caused an enhanced arginase activity ( ) . kousathana et al. have demonstrated that circulating monocytes isolated from diabetic patients produce higher levels il- , while having an impaired activation of the nlrp inflammasome and subsequently reduced il- β production ( ) . however, they showed that proper glycemic control would restore such modifications. poor inflammatory responses in circulating monocytes, as well as in macrophages, are responsible for elevated susceptibility to infections and their severity in patients with t dm. macrophages play a critical role in tissue repair. early in wound healing, they are pro-inflammatory to clear pathogens and debris but later, they resolve inflammation and promote tissue repair. in pathological conditions, failure to transform from pro-inflammatory to the anti-inflammatory proliferative phase can cause chronic inflammation in the affected tissue ( ). have shown that an impaired wound healing process in animals with t dm is due to high levels of nlrp inflammasome activity, which promotes the generation of il- β and il- in macrophages ( , ) . efficient skin wound healing process is mediated by the up-regulation of the peroxisome proliferatoractivated receptor (ppar)-γ in macrophages that convert their pro-inflammatory phenotype into healing-related. pparγ suppresses cytokine production by macrophages and hence is upregulated in inflamed tissue-resident macrophages. however, in t dm, pparγ expression is down-regulated in skin-resident macrophages that enhance the activity of nlrp- inflammasome and cause chronic inflammation. using myeloid-specific pparγ −/− mice, it has been shown that the absence of ppar-γ in macrophages is sufficient to delay the healing process and extend tissue inflammation ( ) . in t dm patients, chronic hyperglycemia and hyperlipidemia trigger the secretion of a damage-associated s a molecule (calgranulin a) from pancreatic islets that in turn increase macrophage infiltration ( ) . westwell-roper et al. have shown that iapp aggregates in t dm patients polarize islet-resident macrophages toward the m -like f / + cd b + cd c + phenotype that produces pro-inflammatory cytokines, including tnf-α, il- β, and il- . furthermore, m cells promote islet inflammation, cause β-cell malfunction and apoptosis ( ) . in t dm, excess phagocytosis of apoptotic β-cells by macrophages induces their lysosomal permeabilization, generation of ros, inflammasome activation, and pro-inflammatory cytokines secretion ( ) . collectively, these observations reveal that the functions and plasticity of macrophages are compromised during the progression of t dm. neutrophils are the most prevalent circulating leukocytes and one of the main components of innate immunity. they are recruited to the sites of infection through chemotaxis following complement activation, most importantly by c a. activated neutrophils bind via their surface receptors to induced ligands on the surfaces of inflamed endothelial cells to migrate to tissues. there they phagocytose and kill invading microbes with lysosomal enzymes, antimicrobial peptides and by the generation of ros ( ). neutrophils from patients with t dm, but not from healthy individuals, are activated and produce elevated levels of ros. so, it could increase the risk of random organ injury ( ) . in diabetic patients, the plasma levels of homocysteine are elevated, which is mainly due to its impaired clearance rate ( ) . this will induce neutrophils to constitutively release neutrophil extracellular traps (nets) that can cause vascular damage and delays in wound healing ( , ) . it has been shown that the circulating level of hydrogen sulfide (h s) is significantly reduced in fasting blood of patients with t dm compared with healthy individuals as well as in streptozotocin-induced diabetic rats compared with controls ( ) . h s is produced from cysteine by the action of several enzymes. it acts as a regulator of cell signaling and homeostasis ( ) . it is essential to maintain balanced levels of antioxidants and protect tissues from oxidative stress ( ) . the use of h s or the endogenous l-cysteine in vitro blocks the production of il- and monocyte chemoattractant protein- (mcp- ) in the human u monocyte cell line incubated in high-glucose medium ( ) . yang et al. have shown that h s treatment decreases netosis and enhances the healing process of diabetic wounds by preventing ros-dependent erk / and p activation ( ) . it has been shown that the levels of net components, including histones, elastase and proteinase- , are elevated in the sera from patients with diabetic foot ulcers ( ) . wang et al. have recently indicated that hg dramatically enhances nadph oxidasedependent net generation in diabetic rats and humans. it was proposed that this could have a role in the induction of diabetic retinopathy ( ) . indeed, patients with t dm have elevated plasma levels of mgo, which can induce the production of proinflammatory cytokines like tnf-α, il- , and il- by neutrophils and make them more susceptible to apoptosis ( ) . myeloperoxidase (mpo), which is abundantly produced by neutrophils, but only to a small extent by monocytes and macrophages, might be useful as an early biomarker of inflammation in diabetic individuals ( ) . binding of mpo to endothelial cells increases its half-life. thereby, more proinflammatory oxidant hypochloric acid (hclo) is generated that extends the damage to blood vessels ( ) . in t dm patients, neutrophil activities, including migration, phagocytosis and microbial killing are impaired. this makes diabetic individuals more susceptible to infections ( ) . it has been welldocumented that neutrophils isolated in animal models of t dm have an impaired tlr signaling pathway. this is reflected as a diminished cytokine and chemokine production, possibly as a consequence of reduced phosphorylation of nfκb and iκbα ( ) . the half-life of these neutrophils as well as their in vivo migration and myeloperoxidase activity are decreased. during hyperglycemia, neutrophils produce calprotectin (s a /a ), which interacts with the receptor for advanced glycation end products (rage) on the surface of hepatic kupffer cells and promotes the synthesis of il- ( ). subsequently, il- stimulates hepatocytes to increase the generation of thrombopoietin that in turn attaches to its receptor on the surfaces of bone marrow precursor cells and megakaryocytes to enhance their proliferation and expansion. this results in reticulated thrombocytosis, which means elevated megakaryocyte activity and thrombopoiesis. interestingly, diabetes-related thrombocytosis and subsequent atherothrombosis can be reduced by lowering blood glucose, depleting kupffer cells or neutrophils or by preventing the binding of s a /a to rage using paquinimod ( ) . thom et al. have shown that the incubation of human and murine neutrophils with hg medium would cause their cytoskeletal and membrane instability. this will induce the generation of . to µm diameter microparticles and activate the nlrp inflammasome ( ) . microparticles, which are potently pro-inflammatory, are found in the circulation of healthy individuals, but their generation is increased during cell activation in several diseases, including t dm and cardiovascular diseases ( , ) . furthermore, serum levels of soluble fasl (sfasl) are increased in patients with t dm thereby activating neutrophils and aggravating the inflammatory milieu ( , ) . the proinflammatory roles of sfasl are mediated through increased amounts or activity of nfκb, il- β, caspase- , cd b/cd , and ros ( ) . caspase- activation prevents the sfasl-dependent apoptosis of neutrophils and inhibits their expression of fas and caspase- ( ) . accordingly, hyperglycemia disturbs the normal functions of neutrophils and increases the susceptibility to infections by pathogenic microorganisms. the expression level of nkg d is negatively correlated with hba c levels implying that chronic hyperglycemia would cause nk cell dysfunction ( ) . also, hyperglycemia increases the expression of unfolded protein response (upr) genes in nk cells and induces their apoptosis ( ) . nkt cells express simultaneously markers of both t cells (tcr and cd ) and nk cells [cd , cd , cd (nkg d), and cd (nkp )]. nkt cell subsets produce a broad range of cytokines, including gm-csf, ifn-γ, tnf-α, il- , il- , il- , il- , il- , il- , il- , and il- ( ) . they recognize lipids and glycolipids presented by cd d molecules. phoksawat et al. have shown that the frequency of cd + cd + cd null cd + nkg d hi nkt cells, which produce high levels of il- , are increased in diabetic patients and their numbers are directly correlated with hba c levels ( , ) . lv et al. have recently shown that the numbers of cd + cd + nkt cells are higher in diabetic patients compared to healthy individuals ( ) . they further showed that such cells are mostly cd + , produce elevated levels of ifn-γ and il- and express high levels of nkp , nkg d, and nkp but low levels of inhibitory receptors nkg a and b ( ) . the co-culture of these cells with huvecs significantly decreased their proliferation and migration abilities that were mainly il- dependent ( ) . taken together these studies show that diabetic individuals appear to have elevated levels of inflammationpromoting nkt cells. ilcs are critical effectors of innate immunity that produce both regulatory and pro-inflammatory cytokines to promote tissue repair, immunity, and inflammation ( ) . mature ilcs lack the tcrs. based on their cell surface markers, cytokine production as well as expression of transcription factors the ilcs are classified into types , , and ( ) . these correspond to the different types of cd + t helper cells: th , th , and th , respectively. ifn-γ is the cytokine signature of ilc s, while type cytokines (e.g., il- and il- ) are mainly produced by ilc s and the main product of ilc s are il- and il- . regarding transcription factors, t-bet is mainly expressed by ilc s, gata and rorα are mostly expressed by ilc s and rorγt is predominantly expressed by ilc ( ) . in t dm, the numbers of circulating as well as adipose tissue-resident ilc s are increased compared with normal individuals ( , ) . the frequency of circulating ilc s is positively correlated with fasting plasma glucose (fpg), hba c, homeostasis model assessment for insulin resistance (homa-ir), serum-free fatty acids (ffas) and adipose tissue insulin resistance index (adipo-ir) ( , ) . it has also been shown that patients with increased numbers of ilc have an elevated risk of developing t dm ( ) . a study by wang et al. indicated that adipose tissue-resident ilc s, via the production of ifn-γ, promote tissue fibrosis and induce diabetes in obese individuals ( ) . liu et al. have demonstrated that the numbers of ilc s as well as serum cytokine levels of il- , il- , and il- are significantly elevated in diabetic kidney disease patients and have a positive correlation with disease severity ( ) . they further demonstrated that ilc s, through the tgf-β signaling pathway, are involved in renal fibrosis seen in diabetic kidney disease ( ) . however, galle-treger et al. indicated that the engagement of the glucocorticoid-induced tumor necrosis factor receptor (gitr/or tnfrsf ) on the surface of activated ilc s promotes their secretion of il- and il- , ameliorates glucose homeostasis, protects against the onset of and improves established insulin resistance ( ) . the protective role of ilc s during acute metabolic stress has also been well-documented by dalmas et al. ( ) . humoral immunity (b cells) elevated levels of blood glucose generate covalent sugar adducts with several proteins through non-enzymatic glycation. this can impair humoral immunity in many ways, e.g., by modifying the structure and functions of immunoglobulins (igs) ( ) ( ) ( ) ( ) ( ) ( ) . such modifications in the structure of igs can be determined using matrix-assisted laser desorption ionization (maldi) mass spectrometry ( , ) . the molecular mass of igs in diabetic patients is higher than in normal subjects ( ) . this can lead to reduced efficiency of vaccines that stimulate humoral immunity in these patients. it has been shown that immunization with influenza (flu) vaccines in diabetic patients induces normal or even elevated levels of flu-specific antibodies compared with normal individuals ( ) ( ) ( ) ( ) . however, the ability of the dysfunctional glycated antibodies to neutralize viruses is impaired, which will increase the susceptibility to infections. farnsworth et al. have shown that in t dm, class switch defects in the assembly of antibody genes are also present ( ) . in a model system, mice with t dm have decreased amounts of specific anti-staphylococcus aureus antibodies (total as well as igg), which will increase the risk of infection and morbidity of diabetic mice. however, the levels of igm were elevated, but inefficient in protecting against infection, possibly because of their inability to directly promote phagocytosis. in another study, farnsworth et al. have demonstrated that defects in humoral immunity, as shown by decreased levels of total igg and anti-staphylococcus aureus antibody, aggravate foot infections in a murine model of t dm ( ) . this was due to a reduced germinal center induction and decreased numbers of t and blymphocytes within the germinal centers. this causes failures in antibody generation and class-switch recombination ( ) . mathews et al. have shown that the protective levels of antibodies against streptococcus pneumoniae surface protein a are lower in diabetic patients compared to non-diabetic individuals. these antibodies also have a reduced potential to trigger complement activation on the surface of pneumococci, whereby phagocytosis of the bacteria becomes compromised ( ) . they showed that hyperglycemia reduces both the antibody titers as well as the ability to deposit complement on the bacteria. the abovementioned changes in the ability to protect against s. aureus and s. pneumoniae are important, because these bacteria belong to the most common infection-causing pathogens in diabetic patients. another major group is constituted by gram-negative bacteria that commonly cause e.g., urinary tract infections. many studies have shown that t-cell functions are impaired in individuals with t dm ( ) ( ) ( ) ( ) . elevated levels of activated cd + cd + t helper cells, cytotoxic t-cells, and th cells have been observed in obese diabetic patients compared to nonobese ones ( , ) . nevertheless, pbmcs isolated from obese diabetic patients produced smaller amounts of il- , il- , and tnf-α after stimulation with phytohemagglutinin (pha) ( ) . martinez et al. indicated that diabetic patients have reduced pathogen-specific memory th responses as well as decreased numbers of cd + t cells in response to stimulation with streptococcus pneumoniae ( ) . th cells are critical for the recruitment of neutrophils to the infection site and improve the phagocytosis of invading bacteria and yeast ( ) . moura et al. have shown that diabetic patients, particularly those with foot ulcers, have reduced levels of naive t-cells, but an elevated number of effector t cells and a reduction in the tcr-vβ repertoire diversity ( ) . the observed changes are mainly due to an abnormal amount of inflammatory cytokines (e.g., ifn-γ and tnf-α) produced during infection and to subsequent robust stimulation of t-cells. leung et al. have reported that ischemic tissues of t dm patients contain elevated numbers of tnf-α and ifn-γ producing th cells but diminished numbers of regulatory t cells (tregs), which suppress angiogenesis and decrease vascular density ( ) . the high rate of infectious diseases in t dm patients might also be linked to a reduction in the mitochondrial dna function that causes downstream lymphocyte dysfunction and subsequently increased susceptibility to infection ( ) ( ) ( ) ( ) . in support, we have recently shown that the numbers of ifn-γ producing cells against cytomegalovirus (cmv), epstein-barr virus (ebv), and influenza virus are fewer in t dm patients compared to normal controls ( ) . kumar et al. have also investigated the functions of cd + t cells and nk cells in the whole blood of t dm patients infected with mycobacterium tuberculosis (m.tb). compared to controls, the patients exhibited a reduction in cytokine production (ifn-γ, il- , il- a/f, and tnf-α) and decreased expression of cytotoxic molecules (perforin, granzyme b, and cd a) ( , ) . these studies conclude that the functions of both cd + and cd + t-cell are defective in t dm patients. t dm is usually associated with an elevated risk of asymptomatic bacteriuria, urinary tract infections (utis), pyelonephritis and non-sexually transmitted genital infections, such as balanitis and vulvovaginal infections ( ) ( ) ( ) . the incidence of infections with a complicated course is significantly higher in diabetic patients compared to healthy controls ( table ) . it seems that it is principally defects in the innate immune responses of diabetic individuals that are responsible for the increased susceptibility and prevalence of infections ( , ( , ) cd + tcells mycobacterium tuberculosis ( , ) susceptible to the causative pathogen of lyme disease, borrelia burgdorferi ( ) . the disease is mainly due to the ability of the bacteria to escape complement opsonization and attack, which leads to an impaired uptake and killing of bacteria by neutrophils ( ) . neutrophil dysfunction also increases the susceptibility of diabetic animals to staphylococcus aureus ( ) ( ) . during the progression of t dm in human subjects, the basal phenotype of macrophages is altered so their capacity to control mycobacterium tuberculosis is diminished ( ) . martinez et al. have indicated that alveolar macrophages isolated from diabetic mice express decreased levels of macrophage receptor with collagenous structure (marco) and cd that are engaged in the recognition of trehalose , '-dimycolate, a bacterial cell wall component ( ) . diabetes increases the severity of tuberculosis (tb) and enhances the risk of progression to the active form in latent infections ( , ) . diabetic tb patients have elevated frequencies of th and th cells as well as increased serum levels of inflammatory cytokines, including ifn-γ, tnf-α, il- β, il- , il- , il- a, and il- but decreased levels of il- compared to non-diabetic tb patients. this can contribute to dysfunctional immune responses and poor immune control of a tb infection ( ) . a positive correlation between the serum levels of ifn-γ, tnf-α, il- , and il- a with hb-a c levels was also observed. this indicates an association between impaired control of diabetes and the proinflammatory milieu. tripathi et al. have demonstrated that serum levels of il- were significantly decreased in tb-infected t dm mice and humans compared to non-diabetic tb-infected mice and humans ( ) . they revealed that the treatment of tb-infected diabetic mice with recombinant il- or ilc s (cellular source of il- ) increased the survival of mice, prevented the accumulation of neutrophils near alveoli, diminished the generation of neutrophil elastase (ela ) and prevented epithelial cell damage ( ) . tan et al. have shown that b. pseudomallei and m. tuberculosisinfected pbmcs of diabetic patients fail to produce il- . this leads to a decreased ifn-γ production, poor bacterial killing and elevated intracellular bacterial loads ( ) . an impaired il- production is mainly due to decreased intracellular glutathione (gsh) concentrations within the infected cells of diabetic individuals ( ) . such a combination of an inflammatory microenvironment and dysfunctional immune responses enhances the bacterial load and can subsequently amplify lung injury and fibrosis in diabetic tb patients. chellan et al. have further shown that infections caused by enterococcus faecalis, staphylococcus aureus, and pseudomonas aeruginosa are more prevalent in the wounds of diabetic patients ( ) . t dm patients are more susceptible to utis caused by antibioticresistant escherichia coli, proteus spp., klebsiella spp., coagulasenegative staphylococci, enterobacter spp., and enterococci ( , ) . diabetic patients are also more susceptible to helicobacter pylori (h. pylori) infections ( ). cui et al. have recently reported that t dm patients have an increased risk of infection with kaposi's sarcoma-associated herpesvirus (kshv or hhv- ) ( ) . they further showed that the viral load and antibody titers are positively correlated with blood glucose levels ( ) . diabetic patients also have been shown to have an increased risk of infection with the severe acute respiratory syndrome coronavirus (sars-cov) ( ( ) . the influenza virus that usually causes self-limiting infections can induce severe forms of the disease in diabetic patients ( , ) . following the h n influenza pandemic, diabetic individuals suffered from more severe infections compared to non-diabetic people ( , ) . diabetic patients have also a higher prevalence of chronic cytomegalovirus (cmv), herpes simplex virus (especially hsv- ), and varicellazoster virus infections ( ) ( ) ( ) . accordingly, it seems that the immune response against viruses is impaired in diabetics, and these patients need more care during viral infections. coronavirus virions are enveloped positive-strand rna spherical viruses with a diameter of ∼ nm characterized by spike proteins projecting from their surface and with an unusual large rna genome ( ) . the spike (s) protein of the virus binds to its receptor on the surface of cells by which intracellular proteases are induced ( ) ( ) ( ) . subsequently, the s protein priming and cleavage occurs that allow viral fusion to the plasma membrane and entrance of viral genome into the cells ( ) . sars-cov and sars-cov- use angiotensin-converting enzyme (ace ) as their receptor while mers-cov uses dipeptidyl peptidase- (dpp ) to enter the cells ( , ) . ace is strongly expressed in blood vessels, pancreas, intestine, brain, lungs, heart, and testis ( ) . interestingly, nasal epithelial cells, especially goblet, and ciliated cells express the highest levels of ace and the intracellular protease transmembrane serine protease (tmprss ) that facilitates the entrance of the sars-cov- ( ) . furthermore, the expression of ace is significantly up-regulated in diabetic patients and those treated with ace inhibitors ( ) . coronaviruses cause respiratory, enteric and central nervous system (cns) diseases in various animal species except rats and mice ( ) . most coronavirus infections are mild, but major outbreaks of deadly pneumonia have been caused by sars-cov, mers-cov, and sars-cov- in , , and - , respectively ( ) . on march , , the world health organization (who) announced the pandemic of sars-cov- , the etiologic agent of coronavirus disease- (covid- ) ( ) . the novel coronavirus pandemic, which has emanated from wuhan, china, promotes symptoms similar to those caused by the sars-cov outbreak in . the viral pandemic, which has put the world on alert, has caused over . × confirmed human cases and at least × deaths throughout the world (https://www.worldometers.info/coronavirus/) by june , . most of the infected people experience only mild to moderate respiratory disease and recover soon without the need for special treatment. however, aged individuals and those with health problems, including diabetes, obesity, cardiovascular disease (cvd), hypertension, immune deficiency, and chronic respiratory disease are more likely to develop serious illness (https://www.who.int/health-topics/coronavirus#tab= tab_ ). patients death is mainly due to the acute respiratory distress syndrome, disseminated intravascular coagulation, hemorrhage, coagulopathy, acute organ (e.g., kidney, heart, liver) injury, multi-organ failure, and secondary bacterial infections ( ) . elevated levels of adipose-tissue derived adipokines, interferon, and tnf-α in diabetic patients may impair immune-responses against sars-cov- ( , ) . it has been shown that diabetic patients have impaired clearance of sars-cov- from their circulation ( ) . accordingly, diabetic patients due to the diminished viral clearance, impaired t cell function, and accompanied cardiovascular disease are more susceptible to the coronaviruses infection and subsequent cytokine release syndrome (crs) ( , ) . in support, elevated levels of il- β, il- , il- , il- , il- , il- , ifn-γ, interferon gamma-induced protein (ip- ), granulocyte colony-stimulating factor (g-csf), macrophage inflammatory protein α (mip α), serum ferritin, fibrinogen, plasminogen, c-reactive protein (crp), and d-dimer have been observed in patients with covid- ( , , , ) . covid- patients, especially those requiring intensive care unit (icu) have decreased total lymphocytes (lymphopenia), t cells (both cd + and cd +), b cells, and nk cells ( , ) . it should be noted that most of the surviving t cells in such patients have an exhausted phenotype ( ) . consequently, disease severity is mainly because of the host immune response to viral infection. current evidence about the relationship between pathophysiological mechanisms of diabetes and covid- are limited and further research is still needed. patients with t dm have an elevated risk of infection with plasmodium falciparum ( ) , toxoplasma gondii ( ), opisthorchis viverrini ( ), strongyloides stercoralis ( ), cryptosporidium parvum ( ), blastocystis hominis ( ), ascaris lumbricoides ( , , ) , and giardia lamblia ( ) . interestingly, diabetic patients who were treated with metformin had less p. falciparum infections compared to untreated patients ( ) . omaña-molina et al. have shown that in a mouse model of t dm the animals have an increased susceptibility to granulomatous amoebic encephalitis (gae) caused by trophozoites of acanthamoeba culbertsoni ( ) . the possible reasons for the increased risk of diabetics for parasitic infections are metabolic abnormalities and immune dysregulation. chellan et al. have shown a higher prevalence of fungal infections in the wounds of diabetic patients ( ) . the prevalence correlated with the levels of hba c. the most widely observed fungal isolates were c. albicans, candida parapsilosis, c. tropicalis, trichosporon asahii, and aspergillus species. some of them were resistant to antifungal medications ( ) . al mubarak et al. have also demonstrated that diabetic patients with periodontitis are more susceptible to infection with c. albicans, c. dubliniensis, c. tropicalis, and c. glabrata ( ) . the incidence of candidiasis was significantly increased in patients over the age of with hba c > ( ). it has also been shown that diabetic patients are more susceptible to utis caused by c. albicans ( ) . hyperglycemia impairs the normal functions of the circulatory system, gastrointestinal tract, pancreatic beta cells, liver as well as of skeletal muscles to boost systemic insulin resistance. a hyperglycemic environment also leads to immune cells dysfunction. it increases intestinal permeability, which subsequently enhances the risk of infections in t dm patients. accordingly, further research is still needed to find missing links between impaired physiological/immunological mechanisms and increased susceptibility to infections in t dm patients. the information would be important for better therapy and the design of much more effective vaccination strategies in diabetic patients. gd 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the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -hcbs a authors: ifergan, igal; miller, stephen d. title: potential for targeting myeloid cells in controlling cns inflammation date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: hcbs a multiple sclerosis (ms) is characterized by immune cell infiltration to the central nervous system (cns) as well as loss of myelin. characterization of the cells in lesions of ms patients revealed an important accumulation of myeloid cells such as macrophages and dendritic cells (dcs). data from the experimental autoimmune encephalomyelitis (eae) model of ms supports the importance of peripheral myeloid cells in the disease pathology. however, the majority of ms therapies focus on lymphocytes. as we will discuss in this review, multiple strategies are now in place to target myeloid cells in clinical trials. these strategies have emerged from data in both human and mouse studies. we discuss strategies targeting myeloid cell migration, growth factors and cytokines, biological functions (with a focus on mirnas), and immunological activities (with a focus on nanoparticles). myeloid cells play critical roles in the health and diseases of the central nervous system (cns). for example, myeloid cells constitute a significant proportion of the cells found within perivascular infiltrates in cns lesions of multiple sclerosis (ms) and its animal model, experimental autoimmune encephalomyelitis (eae) ( ) ( ) ( ) . myeloid cells are also critically involved in the secondary damage in spinal cord injury (sci) and traumatic brain injury (tbi) ( ) ( ) ( ) . these myeloid cells have the ability to attract other immune cells, release neurotoxic factors, phagocytose proteins and debris and promote the expansion, and polarization of antigen-specific t cells in the cns. in addition to their capacity to induce and sustain inflammation, myeloid cells are also critically involved in communication with glial cells and neurons, as well as in promoting and maintaining peripheral tolerance ( - ). ms is an inflammatory autoimmune disease wherein cells of the immune system initiate an attack against myelin in the cns that supports axonal conduction. the immune response in ms is thought to be mediated by autoreactive t lymphocytes that recognize myelin peptides. typically, demyelination is associated with an accumulation of t lymphocytes (lymphoid component of infiltrates) and monocytes/ macrophages/ dendritic cells (myeloid cells component of infiltrates) that arise from the migration of peripheral blood immune cells across the cns microvascular endothelium ( - ).as they infiltrate the cns, encephalitogenic t lymphocytes require the presence of these blood-derived antigen-presenting cells (apcs) to further sustain lymphocyte proliferation and cytokine polarization in the cns compartment ( - ). the role of these peripherally-derived myeloid cells in cns inflammation will be the focus of the present review. experimental autoimmune encephalomyelitis is a commonly utilized mouse model of ms that recapitulates many aspects of the human disease such as the cns inflammation, encephalitogenic t cell infiltration, and attack of oligodendrocytes resulting in demyelination. although not perfect, eae has allowed uncovering some of the molecular pathways governing the pathogenesis of ms such as elucidating the pathogenic role of t h lymphocytes. in addition, eae models were critical in identifying and testing new therapeutic agents such as glatiramer acetate (ga) and natalizumab ( ). although myeloid apcs play a prominent role in the pathogenesis of ms, there has been little consideration given to targeting these cells as an ms therapy. some of the current ms disease-modifying therapies may act on myeloid cells even if these cells were not the original intended targets ( ). however, interfering directly with myeloid cell has proven to be efficacious in other diseases including psoriasis with multiple drugs targeting il- (guselkumab, risankizumab, and tildrakizumab) or il- and il- (ustekinumab) ( ), crohn's disease and ulcerative colitis targeting il- and il- (ustekinumab) ( ), rheumatoid arthritis targeting il- (anakinra) ( ), systemic juvenile idiopathic arthritis targeting il- β (canakinumab) ( ), and many others. there are ongoing clinical trials in rheumatoid arthritis, stroke, atherosclerosis, and cancer using agents that target myeloid cells and their products. biber et al. have provided a recent comprehensive review of drugs in clinical trials targeting myeloid cells in cns diseases such as alzheimer's disease, brain tumors, and inflammatory pain, as well as for other cns diseases ( ). as we will discuss in this review, multiple tools have been developed in the eae models of ms demonstrating significant regulation of disease progression by various approaches blocking myeloid cell activation and effector function, but to date, these approaches have not been tested for therapeutic efficacy in ms patients. the first strategy we will discuss is interference with peripheral myeloid cell migration to the cns. the blood-brain barrier (bbb), composed of tightly bound endothelial cells (ecs), regulates the entry of blood-borne molecules and immune cells into the cns. under physiological conditions, a limited number of peripheral blood immune cells gain access to the cns, a process called immune surveillance ( ). during an inflammatory process, meningeal, and bbb-ecs amplify the migration of immune cells into the cns parenchyma, in a multistep process that involves selectins, chemokines and cell adhesion molecules ( ). bbb-ecs express cell adhesion molecules such as intercellular adhesion molecule (icam)- , vascular cell adhesion molecule (vcam)- , activated leucocyte cell adhesion molecule (alcam), and melanoma cell adhesion molecule (mcam) which mediate at least in part, the adhesion process and the transmigration of leucocytes to the cns through their interaction with integrins αlβ [leucocyte function-associated antigen (lfa)- ], α β [very late antigen (vla)- ], cd , and mcam respectively ( - ). interfering with immune cell trafficking across the bbb by targeting adhesion molecules has proven to be beneficial in reducing clinical disease activity and pathological indices in ms ( ). indeed, natalizumab, which blocks vla- , the ligand of vcam- , is reported to reduce migration of most leukocyte subtypes, including myeloid cells, into the brain. more recently, a new adhesion molecule expressed by bbb-ecs called nerve injury-induced protein (ninjurin)- was described ( ). ninjurin- is a membrane protein known to interact in a homophilic manner through an extracellular residue-binding motif ( ). on immune cells, ninjurin- was weakly expressed by lymphocytes, but highly expressed by peripheral myeloid apcs including monocytes, macrophages and dendritic cells (dcs), in humans and mice. interestingly, ninjurin- was also found to be expressed in ms lesions. ninjurin- neutralization specifically abrogated the adhesion and migration of human monocytes across a monolayer of bbb endothelial cells, without affecting lymphocyte recruitment. moreover, ninjurin- blockade during the course of eae reduced infiltration of peripheral myeloid cells and reduced clinical disease activity and histopathological indices of eae ( ). another adhesion molecule involved in the migration of peripheral myeloid cells is junctional adhesion molecule (jam)-like (jaml). jams are type i transmembrane proteins differentially expressed at the junctions of ecs, epithelial cells, and on various leukocytes ( ). similarly to ninjurin- , jaml can interact in a homophilic manner ( ). it was observed that jaml is expressed by bbb-ecs, and has an increase expression in ms lesions compared to normal appearing white matter ( ). in addition, human monocytes and cd + t cells were found to express jaml, and its level was significantly increased on rrms patients when compared control subjects: vs. % for monocytes, and . vs. . % and for cd + t cells. these data reveals that jaml might be a more important adhesion molecule for monocytes than for cd + t cells. however, migratory capacity of both cell types was significantly compromised when jaml was blocked. chemotactic cytokines (chemokines) are secreted proteins that regulate the migration of leukocytes. chemokine receptor signaling plays a central role in cell migration during inflammatory responses in autoimmune and infectious diseases as well as in cancer. there are ∼ chemokines and receptors known at this time. blockade of ccr and ccr have been the two majors targets in a half dozen ms clinical trials ( ). the chemokines ccl (macrophage inflammatory protein- α-mip- α) and ccl (regulated on activation, normal t cell expressed and secreted-rantes) bind to ccr , while ccl (monocyte chemoattractant protein -mcp- ) binds to ccr . both lymphoid and myeloid cells express ccr and ccr , with monocytes/macrophages/dcs the cells where these chemokine receptors are most abundant ( - ). in animal models of ms, it was shown that ccr -deficient animals developed a less severe disease ( ), while ccr -deficient mice were completely resistant to disease induction ( , ), highlighting the importance of signaling through these chemokine receptors for disease initiation. in addition, it has been shown that ccr + ly- c hi monocytes are rapidly recruited to the inflamed cns in eae and are crucial for the effector phase of disease. selective depletion of this specific monocyte subpopulation through engagement of ccr significantly reduced disease severity ( ). ccr + and ccr + macrophages were both found in active ms lesions ( , ). the role of chemokines and their receptors are now well-characterized in ms and other inflammatory diseases. the potential for a therapy targeting this signaling pathway is well-recognized. however, none of the chemokine-directed ms clinical trials has shown robust clinical efficacy. similar lack of clinical responses have also been reported in therapeutic trials targeting chemokines in other diseases such as rheumatoid arthritis, psoriasis, asthma, and many others ( ). the issue may lie in the redundancy of chemokine/chemokine receptor action, in which case, it may be beneficial to develop strategies employing multiple antagonists simultaneously. as innate cells, myeloid cells express pattern recognition receptors (prrs). prrs include toll-like receptors (tlrs), rig-i-like receptors, nod-like receptors, and c-type lectin receptors (clrs) ( ). selectins, which are part of the c-type lectins family, are known to play a crucial role in the control of leukocyte trafficking and homing to sites of inflammation ( ). selectins are particularly important for the rolling of cells on endothelial cells, an important component of migration of myeloid cells into tissue sites of inflammation ( ). more recently, it was uncovered that clec a, a clr, was involved in facilitating binding and transmigration of dcs across the bbb in response to ccl chemotaxis ( ). in eae, clec a −/− mice displayed delayed disease onset and significantly reduced disease severity. additionally, in a chronic model of eae, anti-clec a antibody treatment initiated at disease initiation also delayed onset and lessened disease severity. anti-clec a antibody administration to mice undergoing relapsing-remitting eae after disease onset, resulted in less severe disease relapse ( ). although the ligand of clec a is currently unknown, it was suggested that the ligand is present on bbb endothelial cells ( ). abundance of immune cells as well as cytokines, chemokines and immunoglobulins in ms plaques and their accumulation in the cerebrospinal fluid (csf) of ms patients, support the notion that ms is an inflammatory disorder. these observations lend support to the idea that immune cell products, especially cytokines, have an important role in both the induction and progression of ms. targeting cytokines has been a successful strategy used in therapy of other inflammatory diseases. for example, blockade of tumor necrosis factor (tnf) has shown positive results in rheumatoid arthritis and crohn's disease ( , ) . as of december , tnf inhibitors were the world's leading drug class, with sales of more than us $ billion and used in more than seven million patients ( ) . in ms, the first treatment approved for rrms was interferon (ifn)-β, thus showing that cytokines manipulation is potentially a good strategy. current data suggests that ms, and its animal model, eae, are driven by both t h lymphocytes, producing ifn-γ, interleukin (il)- and tnf, and t h lymphocytes, producing il- , il- , il- , and granulocyte-macrophage colony-stimulating factor (gm-csf also known as csf- ). surprisingly, ifn-γ, il- , il- a, il- f, il- , and il- have all been shown to be dispensable for the development of eae [reviewed in ( ) ; discussed here ( ) ]. however, in , the cns pathogenicity of t h cells was reported to be primarily associated with their production of gm-csf ( , ) . gm-csf production by t cells has been correlated with pathogenesis in several autoimmune diseases, including ms, rheumatoid arthritis, and myocarditis. it was reported that il- β-and il- -induced production of gm-csf by cns-infiltrating cd + t cells is essential for the induction of eae ( , ) . gm-csf is a hematopoietic growth factor produced by a number of hematopoietic and non-hematopoietic cell types including activated cd + t cells, monocytes/macrophages, b cells, nk cells, endothelial cells and epithelial cells. gm-csf has a wide array of functions, notably the survival and activation of myeloid cells, the ability to induce differentiation of dendritic cells (dcs), the polarization of macrophages toward a pro-inflammatory m phenotype, enhanced antigen presentation, the induction of complement-and antibody-mediated phagocytosis, and the mobilization of monocytes and other myeloid populations from bone marrow to blood ( ) ( ) ( ) . the gm-csf receptor (gm-csf rc) is a heterodimer comprised of a specific low-affinity α chain (cd ; gm-csf rα) and a common β chain (cd ; gm-csf rβ) that is shared by il- and il- ( ) . the gm-csf rc is expressed in multipotent myeloid progenitor cells and continues to be expressed throughout myeloid development on monocytes, dcs, macrophages and neutrophils ( ) ( ) ( ) . it is not expressed by t and b lymphocytes ( ) . thus, most of the suspected direct effects of the gm-csf in diseases are focused on myeloid cells (peripheral and cns resident). findings related to the function of gm-csf signaling in eae pathology have been recently reviewed ( ) . in brief, in eae, gm-csf is necessary for disease as gm-csf kos were found to be resistant to disease induction ( ) . disease can be rescued by the administration of recombinant gm-csf. adoptive transfer using cytokine-deficient mice showed that wild-type, il- a −/− , and ifnγ −/− t cells induced eae with similar kinetics. by contrast, gm-csf −/− t cells were incapable of inducing eae and invading the cns ( ) . due to the variety of cells gm-csf can stimulate, it became important to determine the cell population in which signaling was necessary for disease. a bone marrow chimera study determined that peripheral myeloid cells, but not microglia, are key responders ( ) . this corresponds with earlier observations that gm-csf administration stimulated cd b + ly c hi inflammatory monocytes into the circulation ( ) . circulating ly c hi monocytes traffic across the blood-brain barrier, upregulate pro-inflammatory molecules, and differentiate into central nervous system dcs and macrophages ( ) . these data were confirmed recently using conditional gene targeting in which the β chain of the gm-csf receptor (csf rb) was deleted in specific subpopulations throughout the myeloid lineages ( ) . it was found that deletion of csf rb in ccr + ly c hi monocytes phenocopied the eae resistance seen in complete csf rb-deficient mice. in humans, gm-csf levels in the csf are higher in patients with active ms than in patients in remission ( ) . also, untreated ms patients had significantly greater numbers of cd + gm-csf + t cells and cd + gm-csf + t cells in peripheral blood compared with healthy controls and with ifn-β-treated ms patients ( ) . in addition, ifn-β significantly suppressed gm-csf production by t cells in vitro. more recently, the canadian b cells in ms team uncovered a subset of memory b cells producing gm-csf ( ) . in vitro, gm-csf-expressing b cells efficiently activated myeloid cells in a gm-csf-dependent manner, and in vivo, b cell depletion therapy resulted in a gm-csf-dependent decrease in pro-inflammatory myeloid responses of ms patients. in light of the critical role of gm-csf in the pathogenesis of ms and other inflammatory diseases, multiple tools have been developed targeting either the cytokine or the receptor. first tested in eae, it has been shown that blocking antibodies against gm-csf in chronic (c)-eae ( ) or antibodies against gm-csf rα in c-eae and relapsing-remitting (rr)-eae ( ) were able to prevent disease if given at the time of eae induction (day ). mice treated with anti-gm-csf after disease onset completely recovered within days of treatment in a model of c-eae ( ) . therapeutic treatment with anti-gm-csf rα ameliorated progression of c-eae and resulted in a significant reduction of the relapse severity of rr-eae ( ) . blockade of the gm-csf rα led to a reduction of activated mdcs, and reduced pro-inflammatory cytokine production by cd b + ly c + inflammatory monocytes. additionally, anti-gm-csf rα altered the expression of chemokine receptors, leading to the possibility that antibody treatment may impede cell migration ( ) . logically, the next step is to test the therapeutic potential of gm-csf targeting in humans. a review of tools developed for clinical trials can be found here ( ) . at this time, gm-csf blocking antibodies have been tested in rheumatoid arthritis and have shown promising results. as for ms, only one drug has been tested in clinical trials: mor- (also known as gsk or otilimab), a human antibody to gm-csf. the results of a phase ib clinical trial employing mor- in patients with relapsing-remitting or secondary-progressive ms have shown the drug to be safe and well-tolerated, although with modest efficacy ( ) . at this moment, there are no ongoing clinical trials targeting gm-csf or gm-csf receptor in ms. another important growth factor regulating myeloid cell function is macrophage colony-stimulating factor (m-csf also known as csf- ). m-csf is ubiquitously produced in the steady state by a variety of cells, including endothelial cells, fibroblasts, osteoblasts, smooth muscle, and macrophages, and can be detected in plasma at ∼ ng/ml ( ) ( ) ( ) . the levels of circulating m-csf are upregulated in pregnancy ( ) as well as in many different pathologies including cancer, autoimmune diseases and chronic inflammation ( ) ( ) ( ) ( ) ( ) . m-csf stimulates progenitor cells from bone marrow and plays an important regulatory role in the survival, proliferation (in mice), differentiation, phagocytosis, and chemotaxis of myeloid cells, including monocytes, macrophages, dcs, and microglia ( ) ( ) ( ) . the effects of m-csf are mediated by signaling through the type iii tyrosine kinase transmembrane receptor csf- r (cd ), which is encoded by the c-fms proto-oncogene ( ) . il- is also able to bind csf- r with similar outcomes as to m-csf binding ( ) . however, m-csf and il- present differences in their spatiotemporal expression patterns, and thus seem to play complementary roles in their biological activities on target cells ( , , ) . csf- r is expressed by myeloid cells such as monocytes, macrophages, dcs, and microglia, as well as by trophoblasts, neural progenitor cells and epithelial cells ( , ) . there is ongoing debate about whether m-csf is a pro-inflammatory or pro-repair cytokine. m-csf seems to be essential for the survival and renewal of tissue-resident macrophages, but not for circulating myeloid cells. indeed, in the osteopetrotic csf op /csf op mouse, which harbor an inactivating mutation in the coding region of the csf- gene and are m-csf deficient, the functions and numbers of several tissue macrophage populations are altered while there is no difference in monocyte populations in the blood ( ) . these findings were later confirmed in mice deficient for a specific enhancer for csf- r gene, the fms-intronic regulatory element (fire) ( ) . csf r fire/ fire mice present a deficit in tissue resident macrophages in the brain (microglia), skin, kidney, peritoneal, and heart without significant differences in blood monocytes. during inflammation, the presence of monocytes in inflamed tissue is critical for proper immune responses, notably due to their capacity to traffic to draining lymph nodes and their ability to present antigens to t cells ( , ( ) ( ) ( ) ( ) ( ) ( ) ( ) . while tissue resident macrophages also participate in inflammatory processes, their role in promoting tissue repair and regeneration is critical ( , ) . for example, m-csf favors kidney and liver repair after acute injury ( ) ( ) ( ) . moreover, m-csf is used to drive human and in mouse macrophage differentiation in vitro into an anti-inflammatory (m ) phenotype ( ) ( ) ( ) . in eae, it was shown that peritoneal apcs treated with m-csf and pulsed with mog − , the disease initiating peptide, were able to suppress ongoing eae when injected at the time of disease initiation or significantly reduce the severity of the disease when injected at day post-immunization ( ) . these m-csf activated apcs were demonstrated to induce a treg profile from cd + t cells (cd + foxp + ) with increased secretion of il- and decreased secretion of il- , ifn-γ, and tnf ( ) . however, as mentioned earlier, elevated levels of m-csf are also observed in different pathologies. there are multiple publications linking m-csf/il- and csf- r signaling in models of arthritis ( ) ( ) ( ) ( ) , diabetes ( ) , systemic lupus erythematosus ( , ) , cancer ( ) ( ) ( ) , amyotrophic lateral sclerosis ( ), parkinson's disease ( ) , and alzheimer's disease ( ) ( ) ( ) . in an effort to determine the role of m-csf/il- and csf- r signaling in ms, different groups used potent cfms tyrosine kinase inhibitors, which block m-csf signaling. ki ( ) , imatinib ( ) , gw ( , ) , sorafenid ( ) , and plx ( ) are all tyrosine kinase inhibitors that have shown to effectively treat c-eae. gw has the greatest apparent specificity for csf- r vs. the other kinase inhibitors ( ) . amelioration of eae using ki was associated with the suppression of myeloid cell expansion in the spleen and reduction in mog-specific t-cell proliferation ( ) . gw and sorafenib suppressed tnf-α production by macrophages whereas imatinib and sorafenib both abrogated pdgf-induced proliferation of astrocytes ( ) . plx effect was associated with microglia and macrophage ablation from the white matter ( ) . however, in the cuprizone model of cns demyelination, which allows study of the remyelination process with little involvement of the peripheral immune cells ( ), injection of m-csf reduced demyelination by boosting microglia activity ( ) . tamoxifen-induced conditional deletion of the csf- r in microglia from cuprizone-fed mice caused aberrant myelin debris accumulation and reduced microglial phagocytic responses ( , ) . these data indicate that m-csf plays an important role in ability of microglia to clear myelin debris and to support proper remyelination, and suggest m-csf functions as a critical factor in tissue repair. these divergent results exemplify the various functions of m-csf/il- and csf- r signaling on cells. the possible contribution of m-csf signaling to both inflammatory and repair processes suggest that targeting m-csf in ms may be problematic. however, although there is an increase of myeloid cells in ms lesions, the expression of csf- r is lower in ms lesions when compared to normal appearing white matter ( ) . it is thus possible to hypothesize that a therapeutic treatment targeting m-csf in ms would primarily target peripheral myeloid cells rather than those in the cns. there are now multiple tools targeting m-csf signaling approved for human therapy, especially for cancer. imatinib was the first tyrosine kinase inhibitor approved for the treatment of chronic myelogenous leukemia ( ) . imitanib is also now in clinical trials for the treatment of different pathologies, such as rheumatoid arthritis, type i diabetes and asthma, for which positive results of a phase clinical trial were recently published ( ) . sorafenib is approved for the treatment of primary kidney cancer and advanced primary liver cancer ( ) . although there are side effects related to these inhibitors, an important advantage of tyrosine kinase inhibitors is the fact they can be administered orally to the patients. in september , a phase clinical trial for rrms was started testing the efficacy of evobrutinib, a bruton's tyrosine kinase inhibitor. although this is not a csf- r inhibitor, it shows: ( ) the desire to develop oral treatments in ms, and ( ) the possibility of targeting tyrosine kinases in ms. bruton's tyrosine kinase are critical for b cell receptor signaling and is also involved in tlr signaling as well as inflammasome activation in myeloid cells ( ) cytokines as mentioned earlier, blockade of myeloid specific cytokines il- β, il- , and il- have proven to be efficient therapies in multiple diseases such as crohn's disease, ulcerative colitis, rheumatoid arthritis, psoriasis, and systemic juvenile idiopathic arthritis. these cytokines are all involved in cd + t lymphocytes differentiation. while il- is critical for t h induction ( ), il- β and il- are both involved in t h differentiation and promote the encephalitogenic capacity of these cells by inducing gm-csf expression ( , , ) . in eae, mice lacking il- β, or the receptor, il- r, developed a milder disease than wt animals ( , ( ) ( ) ( ) ( ) . moreover, specific ablation of il- r on cd + t cells resulted in significantly reduced disease severity ( ) , confirming the importance of il- β signaling on t cells to induce a full eae. in addition, rats treated with an il- receptor antagonist (il- ra), which blocks the biological activity of il- β, developed milder signs of eae compared to control animals ( ) . as il- β secretion is the result of inflammasome activation, mice treated with a blocking agent for the inflammasome component nlrp exhibited decreased eae severity ( ) . in ms, it was shown that il- r expression is significantly higher in cd + t cells from rrms patients than from healthy controls ( ) . il- β expression was also found to be significantly increased in ms lesions when compared to tissue from other neurological diseases ( ) . interestingly, multiple treatments used in ms [e.g., ifn-β, glatiramer acetate, and natalizumab] have shown to increase il- ra expression and/or to decrease il- β production ( , ) ]. multiple tools have been developed to block il- β activity: the recombinant il- ra anakinra used for rheumatoid arthritis, the neutralizing il- β antibody canakinumab used for systemic juvenile idiopathic arthritis as well as cryopyrin-associated periodic syndrome, and the soluble decoy il- receptor (rilonacept) also use for cryopyrin-associated periodic syndromes ( ) . at this time, anakinra is the only il- β-targeting drug in clinical testing for ms. this phase i/ii clinical trial just started a few months ago, and at this time, it is still in the recruitment phase (nct ). il- and il- are heterodimeric cytokines that share a common subunit il- p . the other subunit needed to form il- is il- p , while the other subunit to form il- is il- p . il- signals through the il- receptor (il- r) composed of the il- rβ and il- rβ subunits, while il- signals through il- r and il- rβ ( ) . thus, il- rβ is required for biological response to both il- and il- . when specific gene ablation was tested for the different receptor chains of il- and il- , it was found that il- rβ −/− mice were completely resistant to eae ( ) . however, il- rβ −/− mice developed severe eae, extensive inflammation and demyelination, and higher production of pro-inflammatory cytokines than wt animals ( ) . finally, similar to il- rβ −/− mice, il- r −/− mice were completely resistant to eae induction ( ) . as for the cytokines, mice deficient for the subunits il- p or il- p were resistant to eae. by contrast, mice in which the subunit il- p was deleted were highly susceptible to eae ( ) . in addition, treatment with anti-il- p antibodies inhibited both murine and primate models of eae ( ) ( ) ( ) . treatment with anti-il- p antibodies reduced the clinical severity and prevented relapsing eae by inhibiting epitope spreading ( ) . these results led to the conclusion that il- was a more critical factor than il- in the inflammatory response observed in eae. nevertheless, there are multiple studies linking both cytokines to ms pathology. it was demonstrated that peripheral blood monocytes from progressive ms patients produced increased amounts of il- compared to controls and that il- production correlated with disease activity ( ) . another study showed an augmented level of il- mrna-expressing cells in the peripheral blood and the csf of ms patients when compared to controls ( ) . there was also elevated levels of il- p detected in plasma from ms patients compared to healthy individuals. a more recent report showed that both rrms and secondary progressive ms patients had increased levels of il- p mrna compared with controls during the development of active lesions ( ) . il- p and il- p have also been detected in human ms lesions ( , ) . based on this and other data, there was hope that ustekinumab, an il- p neutralizing antibody, would be efficacious for treatment of ms. however, disappointingly no clinical improvement in the treatment group compared to the placebo was found ( ) . possible reasons for the failure of ustekinumab are the broad range of ms patients in the trial, many having very severe symptoms and long-standing disease. also, there may be weak bioavailability of the drug as ustekinumab may be inefficient in crossing the bbb ( ) . at this time there are no ongoing trials targeting il- /il- in ms despite the impressive results in the various animal models of the disease. micrornas (mirnas) are small non-coding rnas of - nucleotides that regulate gene expression by inducing mrna degradation or by interfering with translational machinery of mrnas ( ) . it is predicted that more than % of protein-coding genes are regulated by mirnas ( ) . they are key regulators of various biological processes including immune cell lineage commitment, differentiation, maturation, and maintenance of immune homeostasis and normal function [reviewed in ( ) ]. extensive evidence demonstrates that mirnas play crucial roles in the development, differentiation, and function of different immune cells, such as b and t lymphocytes, dcs and macrophages ( ) ( ) ( ) ( ) . in the last few years, mirnas have drawn a lot of interest due to their involvement in the pathogenesis of cancer, inflammatory and autoimmune diseases [reviewed in ( ) ]. in ms patients, expression studies using whole blood ( ), pbmcs ( ) , as well as brain sections ( ) identified multiple deregulated mirnas. of these mirnas, three were consistently upregulated across multiple studies and directly affecting myeloid cell functions: mir- , mir- and mir- a. mir- is induced by the myeloid transcription factors pu. and ccaat/enhancer-binding protein-β (c/ebpβ) ( ) . mir- expression is mainly confined to myeloid cells and is induced during the lineage differentiation of myeloid progenitor cells. it was shown to negatively regulate both the proliferation and activation of neutrophils ( ) . moreover, mir- −/− macrophages exhibited enhanced pro-inflammatory m , but decreased regulatory m responses ( ) . it was later described that mir- is required for efficient m -associated phenotype and function ( ) . moreover, a low functional level of the mir- is essential for monocyte differentiation. in ms patients, mir- was found significantly increased in blood, pbmcs and active ms lesions compared with control subjects ( , ) . during eae development, the expression level of mir- is dramatically increased in myeloid cell populations, but not in other cell types, and was maintained at comparable levels between disease onset and peak of disease ( ) . surprisingly, although mir- expression is associated with m macrophages and microglia ( ) , it was shown that mir- ko mice present a milder course of eae than wt mice ( ) ( ) ( ) . reduced disease severity was also observed in adoptive transfer eae induced by transfer wt t lymphocytes into mir- ko recipient mice compared to transfer into wt recipient mice, demonstrating the importance of mir- on the apcs side rather than on the t cells side ( ) . our group demonstrated that while m -like macrophages were upregulated in ko mice, dcs showed a reduced inflammatory profile characterized by increased pd-l expression and decreased expression of il- β, il- , and il- , all cytokines involved in differentiating and sustaining a t h profile ( ) . significantly, apcs from mir- ko mice have a comparable ability to drive t h cells, but possess a reduced capacity to drive t h cells ( ) . moreover, it was shown that monocytic-myeloid-derived suppressor cells (mo-mdscs) isolated from mir- −/− suppressed t cell proliferation and cytokine production in vitro and regulated eae more efficiently than mo-mdscs derived from wt animals ( ) . the enhanced suppressive function of mir- −/− mo-mdscs was associated with higher expression of arg and stat , which are mir- target genes ( ) . interestingly, stat controls the expression of pd-l on apcs ( ), consistent with the previous observation of pd-l upregulation on dcs in mir- −/− animals. although these results point to mir- as a potential therapeutic target in ms, it is important to note that in a model of lysolecithin-induced demyelination, the absence of mir- was demonstrated to lead to impaired cns remyelination and myelin debris clearance ( ) . the impaired capacity of m polarization by macrophages and microglia is likely a significant factor contributing to the decreased remyelination capacity in mir- ko mice. in particular, microglia adopting an m profile are critical for proper remyelination ( , ) . thus, when targeting mir- in ms, it is important to keep in mind the different implications of such therapy. mir- has drawn a lot of attention for its possible role in ms as detailed in a recent review ( ) . mir- has been shown to be upregulated in active ms lesions ( ) as well as in cd + monocytes isolated from the blood of rr-ms patients compared to control donors ( ) . while mir- expression is limited to myeloid cells, multiple immune cell populations express mir- such as b cells, t cells, macrophages and dcs ( ) . mir- is found at low levels in both myeloid and lymphoid cells, but its expression is upregulated following cellular activation via antigen, toll-like receptor (tlr) ligands, and inflammatory cytokines. an important target of mir- is src homology (sh )-domain containing inositol- ′phosphatase (ship- ) ( ) . ship- is an enzyme that inhibits phosphoinositide -kinase (pi k) activity, which governs cellular responses to multiple stimuli, cell proliferation and cell survival ( ) . thus, it is believed that mir- dysregulation would have critical consequences. indeed, forced expression of mir- in hematopoietic stem cells by a retroviral vector leads to severe splenomegaly as well as increased myeloid cell populations in the bone marrow and in circulation ( ) . in addition, it has been reported that in absence of mir- , mice displayed altered immune responses to infectious agents, due to defective functions of b cells, t cells, and dcs ( ) . focusing on myeloid populations, it was shown that dcs lacking mir- are less competent at inducing antigen-specific t cell activation ( ) . more recently, it was demonstrated that overexpression of mir- in dcs is a critical event that is alone sufficient to break selftolerance in an animal model of diabetes, and promote a cd mediated autoimmune response in vivo ( ) . human cd + monocytes and macrophages overexpressing mir- exhibit increased production of pro-inflammatory cytokines, including il- β, il- , and tnf, and decreased production of the antiinflammatory cytokine il- ( ) . mir- -deficient mice display a delayed course and reduced severity of clinical symptoms of eae ( , ) . decreased disease severity in mir- −/− mice was associated with reduced t h and t h responses. in addition to the direct effect on t cells, it was also shown that the decreased ability of mir- ko mice to mount inflammatory t cell responses was linked to dcs secreting less cytokines critical for driving t h and t h responses, mainly il- β, il- , il- , il- , and tnf ( ) . mir- is induced in macrophages and dcs after exposure to a variety of inflammatory cytokines such as ifnβ, ifn-γ, and tnf-α ( , ) . it is thus possible to speculate that following the first wave of inflammation, these myeloid apcs upregulate mir- leading to an accentuation of the inflammatory response. in addition, more recently, it has been demonstrated that mir- plays an essential role in driving the inflammatory phenotype of m macrophages ( ) , which would also impact the severity of the disease. lastly, treatment with a mir- inhibitor after eae onset reduced the clinical disease severity ( ) . considering the important role of mir- in driving inflammatory responses in general, and specifically in myeloid apcs, fine tuning the expression of this mirna in ms would most certainly prompt beneficial results in terms of slowing the inflammatory loop. it is noteworthy that mir- is the most consistent mirna found to be upregulated in ms being reported in eight independent studies ( ) . a third mirna that has been shown to regulate myeloid cell activation is mir- a. like mir- , mir- a is upregulated following cell stimulation and its induction is nf-κb dependent ( ) . however, contrary to mir- and mir- , mir- a represses inflammatory responses by targeting two adapter proteins, tnf receptor-associated factor (traf ) and il- receptor-associated kinase (irak ), that are crucial for pro-inflammatory signaling ( ) . mir- a ko mice develop a spontaneous autoimmune disorder, characterized by splenomegaly, lymphadenopathy, and multiorgan inflammation ( , ) . in addition, mir- a ko mice display excessive production of myeloid cells and develop flank tumors in their secondary lymphoid organs. consistent with the repression of inflammation, mir- a expression promotes m -macrophage polarization by targeting notch- ( ) . multiple studies have indicated that mir- a plays pivotal roles in the pathogenesis of several autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, and sjögren's syndrome ( ) . in ms, mir- a is upregulated in active lesions ( ) , as well as in pbmcs of rrms patients ( , ) . expression of mir- a is reported to be significantly downregulated in glatiramer acetate treated rrms patients ( ) . logically, upregulation of this mirna would seem to be beneficial in reducing the ongoing inflammation observed in ms patients leading to the possibility that the upregulation observed in ms patients is the result of the ongoing inflammation rather than a pathological expression. however, when studied in animal models of ms, there was no consensus on the suppressive effects of mir- a. one study using the cuprizoneinduced demyelination model found that mir- a-deficient mice displayed reduced inflammatory responses, demyelination, axonal loss, and numbers of infiltrating macrophages compared to wt controls ( ) . however, a second study found that mir- a-deficient mice developed more severe eae characterized by exaggerated t h responses ( ) , going along the possible beneficial effect of upregulation of this mirna in ms. more recently, it was shown that mir- a mimic treatment of mice with rr-eae at day improved neurological function, increased the number of newly generated oligodendrocytes, which may facilitate remyelination in the cns ( ) . in addition, the treatment increased the number of regulatory m macrophages while reducing the number of pro-inflammatory m macrophages ( ) . currently, targeting mirnas is a challenge since they control a myriad of immune and non-immune related functions. however, there is a strong interest in pursuing this approach, not only in ms, but also in many different diseases. identification of technology to target mirnas in a cell specific manner would appear to be the desired way to safely and effectively employ this targeting strategy. in the meantime, an abundance of researchers are also exploring the use of mirnas as biomarkers of diseases pathogenesis and therapy. the final strategy we will discuss is the use of nanoparticles to target myeloid cells for disease therapy which has been pioneered in our laboratory. in the recent past, many studies have focused on characterizing the ability of nanoparticles to modulate immune responses and ultimately to be used as potential therapeutics for immune-related diseases. here we will focus on the "carboxylated" poly(lactic-co-glycolic acid) (plga) nanoparticles, which are particles without any protein or peptide attached to the surface or encapsulated inside. phagocytic cells have the extraordinary ability to engulf dead cells, invading microbes and other particles, and this property of phagocyte cells led to the idea of using carriers such as apoptotic cells ( ) ( ) ( ) , liposomes ( ), extracellular vesicles ( ), or nanoparticles ( ) ( ) ( ) ( ) ( ) to deliver molecules to modify the immune response. we will restrict our discussion to the use carboxylated plga nanoparticles for the modulation of inflammatory monocytes for treatment of cns inflammation for multiple reasons. firstly, they can be easily manufactured under gmp conditions. secondly, they more specifically target inflammatory monocytes by their affinity of binding via the macrophage receptor of collagenous structure (marco) ( ) as compared to liposomes and extracellular vesicles. thirdly, they directly carry out immune-modulatory effects on monocytes without the need for add-on agents such as would be required with liposomes. lastly, they have been proven to be safe and efficacious for use in celiac disease patients treated via intravenous infusion of gliadin encapsulating plga nanoparticles for induction of immune tolerance in a phase / a clinical trial ( ) . nanoparticles have diameters between and , nm. smaller particles (< nm) are able to cross tissue barriers and traffic directly to the lymph nodes. larger particles (> nm) require uptake by phagocytic cells ( ) . nanoparticles administered subcutaneously or intradermally may be taken up by tissue resident apcs or their precursor cells and are ultimately transported to the draining lymph nodes. systemic administration of nanoparticles favors accumulation in the organs such as the spleen and liver ( ) . also, the shape of nanoparticles dictates efficiency of uptake by phagocyte cells. for example, phagocyte cells internalize spherical-shaped nanoparticles more easily than stretched-shaped structures ( ) . and although positively charged particles are taken up more avidly, negatively charged particles have been shown to exhibit lower toxicity ( ) ( ) ( ) ( ) . nanoparticles can be made from different materials, metallic (e.g., silver, gold, and copper), magnetic (e.g., iron) (useful for imaging), ceramic, carbon-based, silica, lipid-based, or polymeric such as poly(amino acids), polysaccharides and poly(alpha-hydroxy acids). our group was one of the first to test the ability of nm non-biodegradable carboxylated polystyrene (ps) particles to modulate immune responses in inflammatory settings in vivo. intravenous infusion of ps nanoparticles led to a reduction in trafficking of ly c hi inflammatory monocyte into the cns and increased survival in a mouse model of west nile virus (wnv) encephalitis ( ) . it was discovered that these inflammatory monocytes were redirected to the spleen of treated animals and resulted in a dramatic reduction of mortality in wnv-infected mice by preventing the release of a pro-inflammatory "cytokine storm" in the cns. robust antiinflammatory effects induced by infusion of ps nanoparticles were also observed in other inflammatory diseases such as peritoneal inflammation and inflammatory bowel disease. to enhance the clinical relevance of the nanoparticle targeting approach, we next investigated the potential of biodegradable carboxylated plga nanoparticles for regulation of myeloid cell-dependent inflammation. plga is one of the best characterized and most used biodegradable polymers. the hydrolysis of plga leads to metabolite monomers, lactic acid and glycolic acid. the two monomers are endogenous and easily metabolized by the body via the krebs cycle. there is minimal systemic toxicity associated with the use of plga ( , ) . because plga is a safe material, it has been approved by the united states food and drug administration (fda) and european medicine agency (ema) in various drug delivery systems in humans. indeed, plga can be engineered to deliver, alone or in any combination with small-molecule drugs, proteins, peptides, dna, mirnas, and even clustered regularly interspaced short palindromic repeat (crispr) ( ) . we have shown that administration of negatively charged nm plga nanoparticles resulted in reduced inflammatory monocytes accumulation and overall robust beneficial effects in disease severity in multiple mouse models of inflammatory disease such as eae ( ) , sci ( ), tbi ( ), myocardial infarction ( ) , and herpes simplex virus infection of the cornea ( ) . the exact mechanisms behind immunomodulatory effects of plga therapy are still under investigation. however, in all these models, it has been shown plga particles are selectively recognized and bound by inflammatory monocytes. these monocytes undergo sequestration and eventual apoptosis in the spleen, culminating in reduced immune pathology at sites of inflammation. phenotypic changes were also observed on dcs and macrophages in the inflammatory sites, showing decreased expression of activation markers such as mhc ii and cd . in the sci study, plga nanoparticle administration led to reduced m macrophage polarization. while our group has also shown that antigen (ag)-coupled or encapsulated plga nanoparticles can have important immunomodulatory effects ( , , , ) , other strategies using plga nanoparticles have also been shown to regulate eae ( ) . for example, cappellano et al. showed that simultaneous subcutaneous injection of plga nanoparticles loaded with either mog − or il- ameliorated the course of eae ( ) . tgf-β, another immunoregulatory molecule, coupled to the surface of plga nanaopartlces containing plp − peptide were shown to improve the tolerogenic effect of ag-plga nanoparticles ( ) . another example is maldonaldo et al. using plga nanoparticles loaded plp − together with rapamycin, an inhibitor of the mtor pathway, and demonstrating that a single dose of these particles injected at the peak of disease were able to protect from relapses ( ) . also, pei et al. aimed to develop plga nanoparticles which function as a direct modulator of t cells, without the involvement of apcs ( ) . for that purpose, tgf-β encapsulated nanoparticles were coupled with target antigens for cd and cd t cells (mog − /h- d b -ig dimer and mog − /i-a b multimer), regulatory molecules (anti-fas and pd-l -fc) and a "self-marker" cd -fc ( ) . these particles were injected in eae mice on day , , , and after immunization with mog − , and induced a significant reduction in eae symptoms that lasted for more than days. moreover, the authors observed a decrease of t h and t h mog − -specific cells as well as t c and t c mog − specific cells, an increase of regulatory t cells, inhibition of t cell proliferation and augmentation of t cell apoptosis in the spleen ( ) . in addition to regulating the immune response, plga nanoparticles have also been used as a transporter to help in the remyelination process. indeed, rittchen et al. encapsulated leukemia inhibitory factor (lif), which is a cytokine known to promote oligodendrocyte maturation thus favoring remyelination ( ) . to specifically target oligodendrocytes, the lif-plga nanoparticles were coupled with anti-ng antibodies. the authors showed that intra-lesion delivery of lif-plga nanoparticles improved cns remyelination increasing the percentage of remyelinated axons and their thickness ( ) . in conclusion, the mechanism(s) of action of plga nanoparticles are still incompletely understood, but studies in multiple models have shown their capacity to limit inflammatory events by targeting inflammatory monocytes. plga nanoparticles can also be used as delivery vectors, like liposomes and extracellular vesicles. however, a critical advantage of carboxylated plga nanoparticles, as compared to liposomes and extracellular vesicles, is their ability to act directly to modulate the function and trafficking of inflammatory monocytes based on their ability to engage the marco scavenger receptor. because of the safety record of plga nanoparticles, they can be easily translated into clinical use. in fact, cour pharmaceuticals successfully completed a phase iia clinical trial for celiac disease showing the safety and efficacy of systemic infusion of plga nanoparticles encapsulating gliadin for inducing gluten-specific immune tolerance in celiac disease patients undergoing oral gluten challenge. takeda pharmaceuticals has acquired the exclusive license for future development of this therapy for celiac and other gi diseases. cour pharmaceuticals is currently developing antigen encapsulating plga nanoparticle-based tolerance clinical programs for treatment of ms and peanut allergy and clinical programs using carboxylated "naked" plga nanoparticles targeting inflammatory monocytes for treatment of acute respiratory distress in covid- infection and treatment of tbi. the importance of peripheral myeloid cells in ms pathology is profound. there is an extensive presence of these cells and their products in ms lesions as well as in the csf of ms patients. studies in animal models of ms have clearly demonstrated the beneficial effects in targeting peripheral myeloid cells for the different forms of the disease. multiple tools have now been developed targeting these cells including blockade of their migration to the cns, their activation and cytokine production, their biological functions and their immunological activity (figure ) . however, contrary to other inflammatory disorders, no drug is currently approved targeting specifically these cells in ms. multiple pro-inflammatory cytokines including gm-csf, il- β, il- , il- , m-csf all represent potential ms therapeutic targets. treatments targeting these cytokines have been shown to be well-tolerated and safe in patients for different diseases. additionally, non-specific blockade of leukocyte entry to cns using natalizumab is beneficial in ms, however this carries the risk of severe side-effects from infections. however, specifically impeding the migration of myeloid cells would limit such adverse effects. clec a, ccr , ccr , jam-l, and ninjurin- represent interesting options to inhibit cns migration of peripheral myeloid cells. altering the biological functions of myeloid cells via through mirna modulation is an appealing strategy for treating ms and other chronic inflammatory diseases. mir- a, mir- , and mir- are all upregulated on myeloid cells from ms patients. lastly, nanoparticles represent one of the most exciting new tools for regulating myeloid cell functions. the biodegradable plga particles are particularly interesting due to their approval by the fda and ema for use in humans, as well as their ability to regulate many different inflammatory disorders, even those that take place in the cns. ii and sm provided intellectual contribution to the work. ii wrote the manuscript. sm provided guidance, edited, and reviewed the manuscript. both authors 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and improves cognition in xtg-ad mice pharmacological targeting of csf r inhibits microglial proliferation and prevents the progression of alzheimer's-like pathology early long-term administration of the csf r inhibitor plx ablates microglia and reduces accumulation of intraneuronal amyloid, neuritic plaque deposition and pre-fibrillar oligomers in xfad mouse model of alzheimer's disease the selective m-csf receptor tyrosine kinase inhibitor ki suppresses experimental autoimmune encephalomyelitis tyrosine kinase inhibitors ameliorate autoimmune encephalomyelitis in a mouse model of multiple sclerosis cytokine and chemokine alterations in tissue, csf, and plasma in early presymptomatic phase of experimental allergic encephalomyelitis (eae), in a rat model of multiple sclerosis csf r inhibition attenuates experimental autoimmune encephalomyelitis and promotes recovery inhibition of colony-stimulating-factor- signaling in vivo with the orally bioavailable cfms kinase inhibitor gw cellular and molecular neuropathology of the cuprizone mouse model: clinical relevance for multiple sclerosis mcsf-induced microglial activation prevents myelin loss and promotes its repair in a mouse model of multiple sclerosis the relative number of macrophages/microglia expressing macrophage colonystimulating factor and its receptor decreases in multiple sclerosis lesions adverse event oncotarget kinase inhibit kit inhibition by imatinib in patients with severe refractory asthma preclinical overview of sorafenib, a multikinase inhibitor that targets both raf and vegf and pdgf receptor tyrosine kinase signaling bruton's tyrosine kinase: an emerging key player in innate immunity development of th cd + t cells through il- produced by listeria-induced macrophages a crucial role for interleukin (il)- in the induction of il- -producing t cells that mediate autoimmune encephalomyelitis generation of pathogenic t(h) cells in the absence of tgf-beta signalling the induction of eae is only partially dependent on tnf receptor signaling but requires the il- type i receptor inflammasomederived il- beta regulates the production of gm-csf by cd (+) t cells and gammadelta t cells myeloid cell transmigration across the cns vasculature triggers il- beta-driven neuroinflammation during autoimmune encephalomyelitis in mice experimental priming of encephalitogenic th /th cells requires pertussis toxin-driven il- beta production by myeloid cells critical regulation of early th cell differentiation by interleukin- signaling interleukin- receptor antagonist suppresses experimental autoimmune encephalomyelitis (eae) in rats by influencing the activation and proliferation of encephalitogenic cells a small-molecule inhibitor of the nlrp inflammasome for the treatment of inflammatory diseases activated il- ri signaling pathway induces th cell differentiation via interferon regulatory factor signaling in patients with relapsing-remitting multiple sclerosis the adhesion molecule and cytokine profile of multiple sclerosis lesions new insights into the role of il- beta in experimental autoimmune encephalomyelitis and multiple sclerosis involvement of the il- system in experimental autoimmune encephalomyelitis and multiple sclerosis: breaking the vicious cycle between il- beta and gm-csf treating inflammation by blocking interleukin- in humans novel p protein engages il- p to form a cytokine, il- , with biological activities similar as well as distinct from il- role of il- receptor beta in regulation of t cell response by apc in experimental autoimmune encephalomyelitis induction of experimental autoimmune encephalomyelitis in il- receptor-beta -deficient mice: il- responsiveness is not required in the pathogenesis of inflammatory demyelination in the central nervous system cutting edge: il- receptor gfp reporter mice reveal distinct populations of il- -producing cells interleukin- rather than interleukin- is the critical cytokine for autoimmune inflammation of the brain anti-il- antibody prevents the development and progression of multiple sclerosis-like relapsing-remitting demyelinating disease in nod mice induced with myelin oligodendrocyte glycoprotein peptide modulation of susceptibility and resistance to an autoimmune model of multiple sclerosis in prototypically susceptible and resistant strains by neutralization of interleukin- and interleukin- , respectively prevention of experimental autoimmune encephalomyelitis in common marmosets using an anti-il- p monoclonal antibody anti-il- therapy inhibits multiple inflammatory pathways and ameliorates autoimmune encephalomyelitis elevated interleukin- in progressive multiple sclerosis correlates with disease activity and is normalized by pulse cyclophosphamide therapy interleukin- and perforin mrna expression is augmented in blood mononuclear cells in multiple sclerosis decreased interleukin- and increased interleukin- p mrna are associated with disease activity and characterize different disease stages in multiple sclerosis expression of costimulatory molecules b - (cd ), b - (cd ), and interleukin cytokine in multiple sclerosis lesions increased il- p expression in multiple sclerosis lesions and its induction in microglia repeated subcutaneous injections of il / p neutralising antibody, ustekinumab, in patients with relapsingremitting multiple sclerosis: a phase ii, double-blind, placebo-controlled, randomised, dose-ranging study why did il- /il- antibody therapy fail in multiple sclerosis? micrornas: genomics, biogenesis, mechanism, and function most mammalian mrnas are conserved targets of micrornas micrornas and the immune response micrornas of the immune system: roles in inflammation and cancer micrornas: key components of immune regulation physiological and pathological roles for micrornas in the immune system microrna regulation of inflammatory responses multiple sclerosis: microrna expression profiles accurately differentiate patients with relapsing-remitting disease from healthy controls microrna and mrna expression profile screening in multiple sclerosis patients to unravel novel pathogenic steps and identify potential biomarkers microrna profiling of multiple sclerosis lesions identifies modulators of the regulatory protein cd an evolutionarily conserved mechanism for microrna- expression revealed by microrna gene profiling regulation of progenitor cell proliferation and granulocyte function by microrna- a novel regulator of macrophage activation: mir- in obesityassociated adipose tissue inflammation mir- promotes regenerative myeloid cell phenotype and function in the demyelinated central nervous system cutting edge: microrna- regulates myeloid dendritic cell-driven th responses in experimental autoimmune encephalomyelitis microrna promotes pathogenic t-cell development and autoimmune inflammation in central nervous system in mice mir- regulates the number and function of myeloid-derived suppressor cells in multiple sclerosis and experimental autoimmune encephalomyelitis pd-l expression on tolerogenic apcs is controlled by stat- m microglia and macrophages drive oligodendrocyte differentiation during cns remyelination the pro-remyelination properties of microglia in the central nervous system mir- dysregulation and therapeutic intervention in multiple sclerosis mir- as a multiple sclerosis-relevant regulator of myeloid cell polarization physiological roles of mir- inositol phosphatase ship is a primary target of mir- inhibitor and activator: dual functions for ship in immunity and cancer sustained expression of microrna- in hematopoietic stem cells causes a myeloproliferative disorder requirement of bic/microrna- for normal immune function mir- upregulation in dendritic cells is sufficient to break tolerance in vivo by negatively regulating ship microrna- as a proinflammatory regulator in clinical and experimental arthritis silencing microrna- ameliorates experimental autoimmune encephalomyelitis microrna- promotes autoimmune inflammation by enhancing inflammatory t cell development microrna- is induced during the macrophage inflammatory response control of the inflammatory macrophage transcriptional signature by mir- small non-coding rnas as important players, biomarkers and therapeutic targets in multiple sclerosis: a comprehensive overview nf-kappab-dependent induction of microrna mir- , an inhibitor targeted to signaling proteins of innate immune responses mir- a is a significant brake on autoimmunity, myeloproliferation, and cancer in mice nf-κb dysregulation in microrna- a-deficient mice drives the development of myeloid malignancies mir- a modulates macrophage polarization by inhibiting notch pathway in raw . macrophages association of microrna- a with autoimmune diseases expression and genetic analysis of mirnas involved in cd + cell activation in patients with multiple sclerosis glatiramer acetate treatment normalizes deregulated microrna expression in relapsing remitting multiple sclerosis experimental demyelination and axonal loss are reduced in microrna- a deficient mice mir- a modulates autoreactive th cell differentiation and regulates organspecific autoimmunity mir- a promotes oligodendrocyte progenitor cell differentiation and enhances remyelination in a model of experimental autoimmune encephalomyelitis tolerance induced by apoptotic antigen-coupled leukocytes is induced by pd-l + and il- -producing splenic macrophages and maintained by t regulatory cells antigen-fixed leukocytes tolerize th responses in mouse models of allergy pathogenesis of nod diabetes is initiated by reactivity to the insulin b chain - epitope and involves functional epitope spreading advances and challenges of liposome assisted drug delivery extracellular vesicles for drug delivery microparticles bearing encephalitogenic peptides induce t-cell tolerance and ameliorate experimental autoimmune encephalomyelitis a biodegradable nanoparticle platform for the induction of antigen-specific immune tolerance for treatment of autoimmune disease expanding antigen-specific regulatory networks to treat autoimmunity biodegradable antigen-associated plg nanoparticles tolerize th -mediated allergic airway inflammation pre-and postsensitization an antigen-encapsulating nanoparticle platform for th / immune tolerance therapy therapeutic inflammatory monocyte modulation using immune-modifying microparticles tak- (timp-glia) prevents gluten challenge induced immune activation in adults with celiac disease nanoparticles target distinct dendritic cell populations according to their size harnessing nanoparticles for immune modulation influence of particle geometry and pegylation on phagocytosis of particulate carriers intracellular dynamics of cationic and anionic polystyrene nanoparticles without direct interaction with mitotic spindle and chromosomes multifunctional, self-assembling anionic peptidelipid nanocomplexes for targeted sirna delivery differential bioreactivity of neutral, cationic and anionic polystyrene nanoparticles with cells from the human alveolar compartment: robust response of alveolar type epithelial cells cationic nanoparticles directly bind angiotensin-converting enzyme and induce acute lung injury in mice biodegradable polymeric nanoparticles based drug delivery systems plga-based nanoparticles: an overview of biomedical applications intravenous immune-modifying nanoparticles as a therapy for spinal cord injury in mice intravenous immunomodulatory nanoparticle treatment for traumatic brain injury monocytes prime autoreactive t cells after myocardial infarction murine corneal inflammation and nerve damage after infection with hsv- are promoted by hvem and ameliorated by immunemodifying nanoparticle therapy tolerogenic ag-plg nanoparticles induce tregs to suppress activated diabetogenic cd and cd t cells promising nanotechnology approaches in treatment of autoimmune diseases of central nervous system subcutaneous inverse vaccination with plga particles loaded with a mog peptide and il- decreases the severity of experimental autoimmune encephalomyelitis conjugation of transforming growth factor beta to antigen-loaded poly(lactide-co-glycolide) nanoparticles enhances efficiency of antigen-specific tolerance polymeric synthetic nanoparticles for the induction of antigenspecific immunological tolerance direct modulation of myelin-autoreactive cd (+) and cd (+) t cells in eae mice by a tolerogenic nanoparticle co-carrying myelin peptide-loaded major histocompatibility complexes, cd and multiple regulatory molecules myelin repair in vivo is increased by targeting oligodendrocyte precursor cells with nanoparticles encapsulating leukaemia inhibitory factor (lif) co and also a member of the scientific advisory board and consultant for cour pharmaceutical development co as well as a shareholder.the remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © ifergan and miller. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -xf glr h authors: tian, bin; cai, dongjie; he, tianqiong; deng, liyao; wu, liping; wang, mingshu; jia, renyong; zhu, dekang; liu, mafeng; yang, qiao; wu, ying; zhao, xinxin; chen, shun; zhang, shaqiu; huang, juan; ou, xumin; mao, sai; yu, yanling; zhang, ling; liu, yunya; cheng, anchun title: isolation and selection of duck primary cells as pathogenic and innate immunologic cell models for duck plague virus date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: xf glr h duck plague virus (dpv) is a representative pathogen transmitted among aquatic animals that causes gross lesions and immune inhibition in geese and ducks. the mechanism of organ tropism and innate immune evasion of dpv has not been completely deciphered due to a lack of cell models to study the innate immune manipulation and pathogenicity of aquatic viruses. in the present study, we isolated five types of duck primary cells [duck embryo fibroblasts (defs), neurons, astrocytes, peripheral blood mononuclear cells (pbmcs), and monocytes/macrophages] to identify appropriate cell models for dpv, using tropism infection and innate immunologic assays. cells responded differently to stimulation with dna viruses or rna virus analogs. dpv infection exhibited broad tropism, as the recombinant virulent strain (chv-gfp) infected defs, neurons, astrocytes, and monocytes/macrophages, but not the pbmcs, as the expression of egfp was negligible. the basal levels of innate immunity molecules were highest in monocytes/macrophages and lower in defs and astrocytes. conversely, the titer and genomic copy number of the attenuated virus strain was higher in defs and astrocytes than in neurons and monocytes/macrophages. the titer and genomic copy number of the attenuated virus strain were higher compared with the virulent strain in defs, neurons, and astrocytes. the innate immune response was not significantly induced by either dpv strain in defs, neurons, or astrocytes. the virulent strain persistently infected monocytes/macrophages, but the attenuated strain did so abortively, and this was accompanied by the phenomenon of innate immune inhibition and activation by the virulent and attenuated strains, respectively. blockage of ifnar signaling promoted replication of the attenuated strain. pre-activation of ifnar signaling inhibited infection by the virulent strain. the selection assay results indicated that induction of innate immunity plays an essential role in controlling dpv infection, and monocytes/macrophages are an important cell model for further investigations. our study provided practical methods for isolating and culturing duck primary cells, and our results will facilitate further investigations of organ tropism, innate immune responses, latent infection, and the effectiveness of antiviral drugs for treating dpv and potentially other aerial bird pathogens. duck plague virus (dpv) is a representative pathogen transmitted among aquatic animals that causes gross lesions and immune inhibition in geese and ducks. the mechanism of organ tropism and innate immune evasion of dpv has not been completely deciphered due to a lack of cell models to study the innate immune manipulation and pathogenicity of aquatic viruses. in the present study, we isolated five types of duck primary cells [duck embryo fibroblasts (defs), neurons, astrocytes, peripheral blood mononuclear cells (pbmcs), and monocytes/macrophages] to identify appropriate cell models for dpv, using tropism infection and innate immunologic assays. cells responded differently to stimulation with dna viruses or rna virus analogs. dpv infection exhibited broad tropism, as the recombinant virulent strain (chv-gfp) infected defs, neurons, astrocytes, and monocytes/macrophages, but not the pbmcs, as the expression of egfp was negligible. the basal levels of innate immunity molecules were highest in monocytes/macrophages and lower in defs and astrocytes. conversely, the titer and genomic copy number of the attenuated virus strain was higher in defs and astrocytes than in neurons and monocytes/macrophages. the titer and genomic copy number of the attenuated virus strain were higher compared with the virulent strain in defs, neurons, and astrocytes. the innate immune response was not significantly induced by either dpv strain in defs, neurons, or astrocytes. the virulent strain persistently infected monocytes/macrophages, but the attenuated strain did so abortively, and this was accompanied by the phenomenon of innate immune inhibition and activation by the virulent and attenuated strains, respectively. blockage of ifnar signaling promoted replication of the attenuated strain. pre-activation of ifnar signaling inhibited infection by the virulent strain. the selection assay results indicated that induction of innate immunity plays an essential role in controlling dpv infection, and monocytes/macrophages are an important cell model for further investigations. our study provided practical methods the innate immune response is the first line of host defense against microbial pathogens. various pattern recognition receptors (prrs) play an essential role in detecting pathogenassociated molecular pattern (pamp) associated with invading pathogens. pamp recognition initiates an innate immune response, characterized by the production of type i interferon (ifn), proinflammatory cytokines, and ifn-stimulated genes (isgs) ( , ) . prrs comprise multiple family members, including toll-like receptors (tlrs), retinoic acid inducible gene i (rig-i)-like receptors (rlrs), nucleotide oligomerization domain (nod)-like receptors, c-type lectin receptors, and cytosolic dsdna sensors (cdss). rlrs primarily recognize ′ -phoshorylated rnas, including ssrna or dsrna produced during the replication of rna or dna viruses ( ) . although rig-i predominantly recognizes rna viruses, the rig-i/mitochondrial antiviral-signaling protein (mavs) pathway is also activated during infection with several dna viruses, including herpes simplex virus (hsv- ), epstein-barr virus, and kaposi's sarcoma-associated herpesvirus ( ) ( ) ( ) . during infection with dna viruses, rna polymerase iii recognizes at-rich dsdna and transcribes the dsdna into dsrna containing a -triphosphate moiety that activates the rig-i/mavs pathway to induce ifn-β production ( ) . dna from viruses or bacteria can be detected by cyclic gmp-amp synthase (cgas) and potentially rlr, which potentially activate the endoplasmic reticulum-resident adaptor protein, stimulator of interferon genes (sting), to translocate from the endoplasmic reticulum to the golgi, where it activates tbk -irf and -nf-κb, resulting in robust induction of type i ifn and inflammatory cytokine production ( ) . ifn-i binds to ifnar and activates r -associated tyk protein tyrosine kinase and the ifnalpha/beta r -associated jak protein tyrosine kinase, which subsequently regulate the phosphorylation and activation of different stat proteins; the activated stat proteins homoor heterodimerize and translocate to the nucleus, where they promote the expression of numerous target genes. binding of stat proteins to either isres or gas sites regulates the expression of several hundred isgs, which mediate the anti-viral, anti-proliferative, and apoptotic effects of type i ifns. duck plague (dp), also known as duck viral enteritis (dve), is caused by anatid herpesvirus type (ahv- ) or duck plague virus (dpv), which is an enveloped, dsdna virus of the herpesviridae family, subfamily alpha-herpesvirinae ( , ) . first reported in the netherlands in , dp spread rapidly around the world ( , ) . although typically an acute or sometimes chronic and highly contagious disease, dp is characterized by high mortality rates (up to %) among domestic ( ) and wild ducks, swans, geese, and other waterfowl of different ages. to prevent dp outbreaks on duck farms, attenuated dpv vaccines have been widely used; in china, use of these vaccines is compulsory, with billions of doses administered annually ( , ) . dpv is the only herpes virus circulating in aquatic animals identified to date. infection with virulent dpv strains causes gross lesions in ducks in most tissues, including the heart, liver, spleen, bursa, and brain ( , ) , where the virus has been detected ( , ) . upregulation of prrs and isgs expression has been reported, indicating that dpv exhibits broad organ tropism and activates the innate immune system ( , ) . differing basal and induced levels of prrs and isgs among different cell types and organs are important factors in determining the organ tropism of viruses such as poliovirus, reovirus, and murine coronavirus ( ) ( ) ( ) . recently published data indicated that expression of rig-i, galectin- , mavs, sting, and irf is induced in dpv-infected ducks, demonstrating the strong capacity of the innate immune response to restrict dpv infection via over-expression of these factors in defs, although it is difficult to detect changes in these factors in defs infected with a high titer of dpv ( ) ( ) ( ) ( ) ( ) . according to a previous study, tlr , irf , isg , isg , and isg (ifits) are missing in birds, chickens also lack rig-i and riplet ( ) , and the immune system of birds is different from that of mammals. development of a suitable cell model for in-depth investigations of the mechanism of the innate immune response to dpv and the virus's ability to evade that response is thus an important priority. in the present study, therefore, we isolated and cultured five types of duck primary cells in vitro and then compared the basal and innate immune responses to dna and rna virus analogs. the cell tropism of dpv and changes in innate immune signaling induced by dpv infection and the antiviral effect of ifnar signaling against dpv infection were also investigated. the isolation and characterization of different types of duck primary cells could facilitate elucidation of the mechanism governing the organ tropism of dpv and the relationship between dpv infection and host antiviral innate immune responses. all animal experiments were conducted in accordance with approved guidelines. one-month-old peking ducklings were purchased from a dpv-free farm where vaccination against dpv was not implementation. all the ducks were housed in the animal facility at sichuan agricultural university, chengdu, china. the study was approved by the committee of experiment operational guidelines and animal welfare of sichuan agricultural university (approved permit number xf - ). nine-day-old duck embryos were cleaned with % ethanol and placed on a -well plate. the head, wings, legs, and viscera were removed, and the muscle tissues were washed with hbss, cut into -mm pieces, and then digested with . % trypsin for min at room temperature (rt). after digestion, the trypsin was removed via centrifugation at , rpm for min, and the digested tissues were dissociated by repeated pipetting (∼ times), after which the mixture was filtered through autoclaved medical gauze. single cells were collected and plated in cell culture plates or dishes and cultured in mem supplemented with % fetal bovine serum (fbs; gibco-brl, carlsbad ca, usa). usually, defs were cultured to % confluence for h and then passaged and sub-cultured for use in subsequent assays. duck neurons were isolated according to our previously described method for isolating mouse neurons ( ) . briefly, the brain was collected from -day-old duck embryos, the meninges were peeled away and carefully removed, and the cortex was transferred into a new dish filled with hbss, and then cut into -mm pieces by using scissors; the shears were transferred into . % trypsin diluted in hbss and digested for min at rt. the trypsin was removed by transferring the brain cells into a new tube with proper dmem, and then dnase i was added and treated for min at rt. the dnase i was removed and the cells were collected by centrifugation at , rpm for min at rt. then, the collected cells were resuspended with dmem and dissociated by repeated pipetting (< times). the cells were passed through a -nm nylon mesh (corning, ny, usa) to separate the single cells, washed once in hbss, and then cultured in d-polylysine-pretreated plates in dmem supplemented with % fbs and % penicillin-streptomycin for h. finally, the cells were washed once with phosphate-buffered saline (pbs), and the medium was replaced with serum-free, neural-basal medium supplemented with % b- plus (gibco-brl, ny, usa), and the cells were incubated for days to form a monolayer. primary duck astrocytes were isolated primarily according to the procedure described above for isolating duck neurons, with some differences. brain cells passed through a -nm nylon mesh were cultured in dmem supplemented with % fbs and % penicillin-streptomycin for h, washed once with pbs, and then cultured in the same medium for days to form a monolayer. the medium was changed every days. similar to defs, astrocytes readily agglomerated and detached from the plates or dishes once they began to overgrow. duck pbmcs were isolated from the -month-old anticoagulation whole blood by density gradient centrifugation using a duck leukocyte isolation kit (tbdscience, tianjin, china), according to the manufacturer's instructions and our previous study ( ) . briefly, whole blood was collected from the jugular vein of mature ducks and placed in anticoagulant-containing tubes. the blood was then diluted with sample dilution buffer and slowly added onto a layer of duck lymphocyte isolation buffer (density: . ± . g/ml) to avoid mixing and then centrifuged at g for min at rt. the second grayishwhite layer was transferred into a new tube, and resuspended in ml washing buffer and the mixed cells were collected by centrifugation at g for min at rt. the collected cells were resuspended in ml of red blood cell lysis buffer for min and then ml of washing buffer was added into the cells. then, the cells were collected by centrifugation at g for min at rt. the remaining cells were adjusted to × cells/ml and ml of cells was plated in one well of the -well plate, and cultured in rpmi medium supplemented with % fbs and % penicillin-streptomycin for h to allow for attachment to the plate; unattached cells were removed by washing twice with pbs, followed by addition of fresh medium. the isolated pbmcs were then ready for use in assays. duck monocytes/macrophages were isolated according to a reported method for isolating human macrophages ( ) . briefly, duck pbmcs were prepared as described above. adherent cells were enriched among pbmcs by adherence on plastic culture plates for h. non-adherent cells were removed via vigorous washing three times using pre-warmed pbs; the adherent cells were digested with trypsin for cell count. duck monocyte-derived macrophages were differentiated from adherent monocytes in rpmi medium supplemented with l-glutamine ( mm), sodium pyruvate ( mm), % heatinactivated fbs, % penicillin-streptomycin, and ng/ml human m-csf (novoprotein, shanghai, china). the medium was changed every days, and duck macrophages formed a monolayer by day . in our present isolation method, only adherent cells are able to differentiate into macrophages under the induction of m-csf; lymphocytes in pbmcs (mainly including t cells, b cells, and nk killer cells) continue to die due to inability to differentiate under induction of m-csf and are removed by constantly replacing fresh medium. the virulent strain of dpv, chv, was isolated and characterized by our lab ( ) . the recombinant virulent strain of dpv, bac-chv-egfp (chv-gfp), was constructed by our research center ( ) . the attenuated vaccine strain of dpv, cha, was retrieved from storage at our research center. the duck tambusu virus (dtmuv) was stored at our research center. each dpv and dtmuv strain was propagated on defs. the rabbit antihuman map and -human gfap polyclonal antibody and the goat anti-human β-actin monoclonal antibody were purchased from abclonal (wuhan, china). mouse anti-duck cd (gene id: ) and cd (gene id: ) antibodies were generated by our research center. ruxolitinib, poly(da:dt), and poly(i:c) were purchased from invivogen (hong kong, china). the cells were lysed with ripa buffer ( mm tris, ph . ; mm sodium chloride; % triton x- ; . % sodium deoxycholate; . % sds) containing protease inhibitors (roche), and the protein concentrations were measured using a dc protein assay kit (bio-rad). equal quantities of protein were resolved by % sds-page and then transferred to polyvinylidene difluoride (pvdf) membranes (bio-rad), which were blocked with % non-fat milk before being incubated with primary antibodies against cd , cd , or β-actin and then probed with the appropriate secondary antibodies. the blots were then visualized using ecl reagent (ge, pittsburgh, pa, usa) and detected under an intelligent dark box ii (ge, pittsburgh, pa, usa). duck primary defs, neurons, astrocytes, pbmcs, and monocytes/macrophages were treated with the dna and rna virus analogs poly(da:dt) or poly(i:c), respectively, at a dose of µg/ml for h. the cells were then lysed in trizol reagent for rna isolation to assess the innate immune response to the stimulators using quantitative real-time polymerase chain reaction (qrt-pcr). primary duck neurons, astrocytes, or monocytes/macrophages were cultured for , , or days, respectively, to form a confluent monolayer. the cells were then fixed with % neutral buffered paraformaldehyde for min, permeabilized with . % triton x- for min, blocked with % bsa dissolved in pbs for min, and then incubated with primary antibodies against map , gfap, cd , and cd at • c overnight. the cells were incubated with alexa fluor -conjugated goat anti-rabbit ormouse secondary antibodies or alexa fluor -conjugated goat anti-rabbit or -mouse secondary antibodies for h at rt. images were acquired using fluorescence microscopy. for virus infection, the required dose of virus was diluted in the medium used to culture the various types of duck primary cells ( × cells in a -well plate) and incubated with cells at • c for h. the cells were then washed twice with pbs and maintained in the corresponding medium supplemented with % fbs and % penicillin-streptomycin. the culture supernatant of virus-infected cells was collected and titrated to determine the tissue culture infectious dose (tcid ) on defs using -fold serial dilutions. viral dna was extracted using a hipure viral dna mini kit (magen, guangdong, china) according to the instructions provided by the manufacturer. dpv genomic dna in infected cells was quantified using an absolute q-pcr method as previously described ( ) using primers specific to the sequence of dpv ul (primers are listed in table ). a standard curve was generated from serially diluted plasmids harboring the entire coding sequence of ul and using the same pcr procedure as used for cell samples. dpv copy number in infected cells was calculated according to the standard curve and normalized to µg of total dna. rna was extracted from cells using trizol r reagent (invitrogen) according to the manufacturer's instructions; the genomic dna was removed and cdna was synthesized by using novoscript r plus all-in-one st strand cdna synthesis supermix (gdna purge) (novoprotein, shanghai, china), and qrt-pcr analysis was performed as described previously ( ) . briefly, µg of rna from cells was transcribed into cdna according to the instructions of the superscript iii reverse transcription kit. a total of µl of cdna was mixed with µl of iq sybr green mix (biorad, hercules, ca, usa), µl of double-distilled water, and . µl each of forward and reverse primer. the cdna was amplified and the cycle threshold (cq) values were recorded. the cdna concentration, primer sequence, and q-pcr procedure applied for each gene in each cell type were the same. the basal expression level of each gene in each cell type was compared by directly determining from the cq values according to the method used in the previous study ( ) . the relative mrna expression of each gene in each cell type was normalized to the expression level of the s mrna gene ( ct ct [prr or isg]/ ct [ s]). expression levels of induced mrnas are presented as the fold change relative to mock-infection levels according to the − ct method. all primer sequences are listed in table . in order to examine the impact of ifnar signaling on the dpv replication in each cell type, the ifnar-specific inhibitor, ruxolitinib, was applied. the cell toxicity of ruxolitinib at a dose of , , , and µm/ml on each cell type was detected by mtt method. duck defs, neurons, astrocytes, pbmcs, and monocytes/macrophages were pretreated with µm/ml of ruxolitinib for h and then infected with chv or cha at a moi of . for h. the cells were then washed twice with pbs, and the same concentration of ruxolitinib was added to the culture medium; dmso was used as a mock control. the cell culture supernatant in each cell type was collected to detect the tcid as above described. the cell was scraped in pbs from the plate to detect the viral genomic copy number. for the preventative antiviral assay, duck monocytes/ macrophages were pretreated with µg/ml poly(da:dt) or poly(i:c) for h, sterile water was used as control, and then infected with chv or cha at a moi of . . the cells were then washed twice with pbs, and the same concentration of poly(da:dt) or poly(i:c) was added to the culture medium and incubated for h. for the therapeutic antiviral assay, duck monocytes/ macrophages were pretreated with or µg of poly(da:dt) or poly(i:c) for h and then infected with chv or cha at a moi of . . after infection, the analogs were added at the same concentration and incubated for h. at hpi, the cell culture supernatants were collected for tcid determination, and the cells were washed with pbs, scraped from the dishes, and collected for viral genomic copy number determination as described above. data are expressed as the mean and standard error of the mean (sem), and the significance of differences between groups was evaluated using the student's t-test or one-way analysis of variance followed by tukey's post-hoc test. asterisks indicate the level of statistical significance ( * p < . ; * * p < . ; * * * p < . ; * * * * p < . ). all experiments were repeated at least three times individually. graphs were plotted and analyzed using graphpad prism software, version . (graphpad software, la jolla, ca, usa). defs have been cultured in our lab using sophisticated methods for many years, and pbmcs were isolated using a duck leukocyte isolation kit; these two cell types were applied in our previous studied ( ) . hence, we strove to isolate and culture duck neurons, astrocytes, and monocytes/macrophages according to previous methods used for mouse and human cells. first and foremost, we isolated pbmcs from the duck whole blood cells (wbc) through density gradient centrifugation, the wbcs and pbmcs were stained with wright strain (figure a) , and the results showed that there are mainly erythrocyte and little partial of thrombocytes and leucocytes in the wbcs. the leucocytes were above % in the pbmcs, and the monocytes were about % among the total pbmcs ( figure a) . since there is no duck-derived m-csf on sale, we blast the duck's m-csf protein sequence ( - aa) with the human's m-csf protein sequence, and found that the duck's m-csf protein sequence has the same functional domain as the human m-csf protein sequence ( - aa) (data did not show); thus, we tried to stimulate the duck-derived pbmcs with human m-csf, we observed and collected the time course and found that it can induce duck pbmcs to differentiate into macrophages, which were mainly composed of the wheel and spindleshaped cells (figure b) . in our present method, the survival time of the pbmcs was not more than h without m-csf, so we believe that the obtained monocytes/macrophages were induced by human m-csf. since the antibodies (see below) we used to identify the macrophage-like cells we have obtained were against the surface molecules that are found in both monocytes and macrophages, we term the cells as monocytes/macrophages (mm). under specific culture conditions, we successfully cultured duck primary neurons, astrocytes, and monocytes/macrophages and identified the cells using an ifa with a rabbit polyclonal antibody against map for neurons ( figure c) , a rabbit polyclonal antibody against gfap for astrocytes (figure d) , and a mouse polyclonal antibody against cd and cd for monocytes/macrophages (figure e) . to further identify the characteristics of monocytes/macrophages, we examined the expression of cd and cd through western blot; the relative molecular weight was and kd, respectively ( figure f) . the basal expression levels of tlr , tlr , cd , and cd in these five types of cells were detected by q-pcr and found that they were highly expressed in monocytes/macrophages (figure e) . these results indicated that our methods can successfully isolate duck neuronal, astrocytes, and monocytes/macrophages. duck primary cells exhibited differing innate immune responses to dna and rna viruses we then examined whether the five types of duck primary cells we isolated were able to respond to dna and rna viruses by stimulating the cells with the dna and rna virus analogs, poly(da:dt) or poly(i:c), respectively, at a dose of µg/ml. at h post-treatment, the expression of ifn-β (figure a) was analyzed, and the data indicated that poly(da:dt) induced significantly higher expression of ifn-β in astrocytes, pbmcs, and monocytes/macrophages than did poly(i:c) or mock treatment. poly(i:c) induced significantly higher levels of ifnβ expression in pbmcs and monocytes/macrophages compared with mock-treated cells. the expression level of mx, an isg induced by ifn or pathogens, was examined in each of the five cell types, and the data showed that poly(i:c) induced significantly higher expression of mx in all tested cells (defs, neurons, astrocytes, pbmcs, and monocytes/macrophages) than did poly(da:dt) or mock treatment (figure b) . under poly(da:dt) stimulation, significant upregulation of mx expression was observed only in astrocytes, pbmcs, and monocytes/macrophages. the expression of il- , an inflammatory factor, was also determined after stimulation (figure c) , and the data showed that poly(i:c) induced il- expression in all five types of duck cells. although poly(da:dt) induced il- only in astrocytes, pbmcs, and monocytes/macrophages, the levels were higher than those induced by poly(i:c). taken together, these data indicated that the five types of duck primary cells we examined are competent to respond to dna and rna viruses and that innate immune signaling is initiated. upon stimulation with dna viruses, monocytes/macrophages exhibit higher levels of ifn-β, isg, and inflammatory cytokine expression than defs, neurons, astrocytes, and pbmcs. to elucidate the mechanisms associated with the different responses of the five types of duck primary cells to dna and rna virus analogs, the basal levels of innate immune factors were compared. we examined a variety of representative factors, including cgas, sting, rig-i, mda , irf , ifnβ, mx, and il- . the basal levels of cgas were comparable among the different duck primary cells (figure a) . the basal levels of sting, rig-i, mda , irf , ifn-β, mx, and il- were highest in monocytes/macrophages and lowest in astrocytes and defs (figures b-h) . these results indicated that monocytes/macrophages mount a greater innate immune response than the other cell types after stimulation with dna or rna virus analogs. dpv, the only herpes virus circulating in aquatic animals, exhibits multi-tropic infection, and the virus can be detected in nearly every major organ, including the brain, lung, spleen, intestines, and liver. in order to investigate the tropism of dpv in duck cells, duck defs, neurons, astrocytes, pbmcs, and monocytes/macrophages were infected with a recombinant virulent virus strain, chv-gfp, at a low moi of . (figure ) . virus proliferation and morphology of the primary cells cultured in vitro were assessed by monitoring the expression of gfp. as the data demonstrate, at h post-infection, gfp was clearly expressed in duck defs, neurons, astrocytes, and monocytes/macrophages, and all four types of virus-infected cells were radially enlarged at h post-infection. no significant gfp expression was observed in dpv-infected pbmcs. these data demonstrated that dpv infects different types of duck cells in vitro and exhibits multi-tropic infection, with possible replication in many organs and tissues, such as muscle, brain (neurons and astrocytes), and spleen (monocytes/macrophages). to investigate the growth dynamics of dpv in the five types of duck primary cells, the cells were infected with a virulent dpv strain (chv) or an attenuated vaccine strain (cha) at a moi of . . the viral titer in the cell culture supernatant was determined based on the tcid (figures a-c) , and the intracellular viral genome copy number was also determined (figures d-f) . cha produced higher virus titer and genomic copy number than chv in neurons and astrocytes at , , and hpi. the viral titer and genome copy number of cha were comparable to chv at hpi but higher than chv at and hpi in pbmcs, although the values were near the detection limit. cha produced higher viral genome copy number in defs at hpi (figure d ), but the viral titer was comparable at , , and hpi (figures a-c) . these data indicated that the attenuated strain of dpv replicates faster and produces more virus particles than the virulent strain in duck defs, neurons, astrocytes, and pbmcs. an intriguing finding was that the titers of both virus strains were comparable in monocytes/macrophages at hpi ( figure a) . the genomic copy number of cha was significantly higher than that of chv at hpi ( figure d ). the genomic copy number and viral titer of cha slowly decreased between and hpi. in contrast, the genomic copy number and viral titer of chv increased over this time period (figures b,c,e,f) . the copy number and viral titer of cha was significantly lower at and hpi compared with chv (figures b,c,e,f) . these data demonstrate that the attenuated strain (cha) abortively infects duck monocytes/macrophages, whereas the virulent strain (chv) persistently infects duck monocytes/macrophages, we speculate that the monocytes/macrophages may be able to kill the virus and it is an active immune response that is responsible for the decrease in viral titer of cha strain, as we demonstrated in the experiments below. to determine whether the different growth dynamics exhibited by the virulent and attenuated strains of dpv affected the innate immune response in duck primary cells, the expression levels of cgas, sting, rig-i, mda , irf , ifn-β, mx, and il- were analyzed in the five types of duck primary cells infected with either the chv strain or cha strain at a moi of . . there were no significant differences in the expression of these molecules in the chv-or cha-infected defs or neurons at , , and hpi, but there was a slight increase in expression in astrocytes infected with cha (data did not show). in pbmcs, infection with the chv strain induced cgas, sting, mda , irf , ifn-β, mx, and il- expression early, at hpi, but expression decreased by and hpi (data did not show). infection with the cha strain induced a slight increase in the expression levels of these factors at hpi, and the expression continued to increase with time and was significantly higher compared with chv-infected cells or mocktreated cells. however, the chv strain may not be able to infect pbmcs observed from figures , , and the virus level of the cha strain infected with pbmcs is slightly higher than that of the chv strain, so comparing the innate immune responses caused by the two in pbmcs are complicated. the expression levels of cgas, sting, rig-i, mda , irf , ifn-ifn-β, mx, and il- were also examined in chv-and chainfected duck monocytes/macrophages. similar to the changes observed in dpv-infected pbmcs, infection with chv induced significant increases in the expression of cgas, sting, rig-i, mda , irf , ifn-β, and mx in monocytes/macrophages early, at hpi, but expression rapidly decreased by , , , and hpi (figure ) , a trend opposite to the growth curve on monocytes/macrophages (figure ) . infection with the cha strain induced significantly increased expression of cgas, rig-i, mda , irf , ifn-β, and mx in monocytes/macrophages at hpi, and these levels were higher than those observed in mock-treated cells but lower than those in cells infected with chv strain. the expression of cgas, sting, rig-i, mda , irf , ifn-β, mx, and il- decreased slightly at and hpi, but then increased markedly at and hpi (figure ) . these data indicated that the virulent dpv strain activates the innate immune system in pbmcs and monocytes/macrophages during the early stages of infection, but then the innate immune response is downregulated once the virus begins to replicate. the attenuated strain (cha) activates the innate immune system primarily during the later stages of infection, and this could be the reason why this strain abortively infects monocytes/macrophages and has been attenuated. to further investigate how dpv infection affects the innate immune response in the five types of duck primary cells, ifnar signaling was blocked using the specific inhibitor ruxolitinib. we first examined whether ruxolitinib functions in the duck cells by pretreating defs with ruxolitinib for h and then infecting the cells with a duck rna virus, dtmuv, which belongs to the flavivirus family and was demonstrated to induce strong innate immune response in def cells ( ) . after infection, rucolitinib was added at the same concentration and the cells were incubated for h. dtmuv induced significant expression of ifn-β, mx, and il- in defs (figures a-c) , but the expression levels of these factors were clearly reduced in cells treated with ruxolitinib, indicating that this inhibitor can be used to block ifnar signaling in duck cells. the cell toxicity of ruxolitinib on defs (figure d) , neurons, astrocytes, pbmcs, and monocytes/macrophages was determined (data did not show), and no obvious cell viability was changed by ruxolitinib at a dose of µm/ml on these five cell types. ruxolitinib was used to treat duck defs, neurons, astrocytes, pbmcs, and monocytes/macrophages in further assays. viral titer was examined at various time points in the culture supernatants of cells infected with either the chv or cha dpv strain, with and without ruxolitinib treatment. after ruxolitinib treatment for hpi, the titer of cha in the astrocyte culture supernatant was significantly increased, by ∼ -fold; however, at this time point, there were no obvious changes in cha titer in the cell culture supernatants of defs, neurons, pbmcs, or monocytes/macrophages (figure e) . at hpi, no change in chv titer was detected in any of the five duck primary cells (figure e) . at hpi, the titer of cha was significantly increased in the cell culture supernatants of astrocytes, pbmcs, and monocytes/macrophages (figure f ). there was a certain increase observed in the culture supernatant of astrocytes infected with chv. no changes were observed in the titers of either chv or cha in the supernatants of neurons and defs treated with ruxolitinib (figures e,f) . taken together, these data indicated that blockage of ifnar signaling enhances the replication of the attenuated dpv strain (cha) in duck astrocytes, pbmcs, and monocytes/macrophages. infection with cha activates the type i ifn response in these cells to a greater degree than does infection with the chv strain. infection with the chv or cha strains of dpv had a differential effect on the innate immune response in duck primary cells. both strains of dpv induced the most significant changes in the innate immune response in monocytes/macrophages (figure ) , and therefore monocytes/macrophages were chosen as a cell model to investigate the antiviral role of ifnar signaling. monocytes/macrophages were pretreated with poly(da:dt) or poly(i:c) at a dose of µg/ml for h and then infected with the chv or cha strain at a moi of . . the viral titer and genomic copy number were determined at hpi. the viral titer and genomic copy number of the chv strain were significantly decreased in monocytes/macrophages pretreated with poly(da:dt) or poly(i:c) (figures a,b) , but there was no change in either viral titer or genomic copy number of the cha strain (figures c,d) . to assess the therapeutic antiviral effect of the agonists, monocytes/macrophages were pretreated with poly(da:dt) or poly(i:c) at dose of or µg/ml for h and then infected with either the chv or cha strain at a moi of . . after infection, the same concentration of poly(da:dt) or poly(i:c) was added and the cells were incubated until the time point of examination. at hpi, the viral titer and genomic copy number were determined. as the data showed, treating monocytes/macrophages with poly(da:dt) at a dose of µg/ml had no effect on viral titer or copy number of the chv strain, but a significant reduction of viral titer and genomic copy number was observed at a dose of µg/ml (figures e,f) . significant reductions in both viral titer and genomic copy number of the chv strain were observed with monocytes/macrophages treated with poly(i:c) at a dose of either or µg/ml (figures e,f) . for monocytes/macrophages infected with the cha strain, treatment with poly(da:dt) or poly(i:c) at a dose of either or µg/ml resulted in a reduction in viral genome copy number (figure h ) but had no effect on viral titer (figure g) . taken together, these data indicate that activation of ifnar signaling via a prr agonist restricts the virulent strain of dpv. prr agonists thus exhibit preventative and therapeutic potential against dpv infection. in the current study, we isolated and cultured duck primary defs, neurons, astrocytes, pbmcs, and monocytes/macrophages. to our knowledge, this is the first report of the culture of duck neurons, astrocytes, and monocytes/macrophages in vitro. these five types of duck primary cells were used to investigate the tropism of dpv and the innate immune response to dpv infection. we found that dpv persistently infected and replicated in defs, neurons, astrocytes, and monocytes/macrophages, indicating that dpv exhibits wide in vivo organ tropism in ducks ( ) . indicative of the potential of dpv to evade the innate immune response, and infection of defs, neurons, and astrocytes, there were slight changes in prr and isg expression. upon infection of pbmcs and monocytes/macrophages at a high moi, the chv strain only upregulated the expression of some prrs and isgs at hpi, but expression of these innate immunity factors was downregulated at later time points by the virulent dpv strain. a particularly intriguing finding was that the expression of prrs and isgs in pbmcs and monocytes/macrophages was consistently upregulated by infection with the attenuated vaccine strain (cha). monocytes/macrophages represent an ideal cell model for investigating the relationship between dpv infection and the innate immune response; as the virulent strain of dpv persistently infects monocytes/macrophages, these cells may play a critical role in the pathogenicity of the virus. the current lack of methods for culturing duck primary cells significantly hinders investigations of the role of each cell type in the infectious cycle, tropism, and pathogenicity of aquatic bird viruses, such as dpv, avian influenza virus, novel duck reovirus (ndrv), and dtmuv, which cause major economic losses in the duck industry ( ) ( ) ( ) ( ) . as opposed to rna viruses such as avian influenza, ndrv, and dtmuv, dpv is a dsdna virus that does not readily induce an innate immune response in defs. hence, in order to investigate the mechanism by which the virus evades the innate immune response, a better primary cell model is needed. numerous types of mammalian primary cells have been isolated from the tissues and successfully cultured, but there are few such reports regarding the culture of primary cells of aquatic animals such as ducks and geese. in the present study, we isolated and cultured duck neurons, astrocytes, and monocytes/macrophages using methods developed for mouse or human cells. our data indicated that the synapses of neurons, the classic morphology, formed at days post-induction. the expected star shape was clearly observed for duck astrocytes isolated from brain tissue and cultured in dmem containing % fbs. it was surprising that we could induce the differentiation of wheel-shaped monocytes/macrophages from duck pbmcs using human or mouse m-csf at a concentration of ng/ml, and no living cells were detected at days post-seeding in the absence of human or mouse m-csf. as a herpesvirus, dpv sometimes exhibits chronic or latent infection in the duck trigeminal ganglion (tg) ( ) , similar to hsv- ( ) . studies of the molecular mechanism of hsv- latency typically use mouse or rabbit animal models, but not humans. however, the research using an in vitro latency model in neurons would be a preferable approach prior to in vivo studies. infection of stem-derived neurons with a low viral dose of wildtype hsv- in the presence of the antiviral agent acyclovir and interferon-alpha results in the establishment of a latent, nonproductive infection. in this state, viral replication and expression of late viral gene markers are not detected, but there is an accumulation of viral latency-associated transcript rna ( ) . in our current research, we isolated and cultured duck neurons, and both the virulent and attenuated strains of dpv were able to infect neurons in the absence of treatment with any antiviral drugs. this would be a good cell model for further studies of the molecular mechanism underlying latent dpv infection in neurons or investigations of how dpv invades the cns to cause fever and intracranial swelling. astrocytes are basal, functional cells of the cns that form and maintain the permeability of the blood-brain barrier and restrict pathogen invasion ( , ) . the tlr in astrocytes plays a critical role in containing the replication and transmission of hsv- in the cns; deficiency of tlr causes astrocytes to become permissive to hsv- infection, thus facilitating infection of the entire cns by hsv- ( ) . ifnar signaling in astrocytes exhibits regional differences that act to restrict the invasion of neurotropic viruses; loss of ifnar signaling decreases the survival of mice after west nile virus infection ( ) . in our present study, we isolated and cultured duck astrocytes. the basal levels of innate immune factors in astrocytes were the lowest among the five types of duck primary cells we examined. both strains of dpv infected astrocytes, and the titer of the attenuated strain (cha) in astrocytes was comparable to that in defs. blockage of ifnar signaling increased dpv replication in astrocytes. thus, astrocytes may play a critical role in containing dpv infection in the cns and might therefore be a useful model. further investigations of the role of astrocytes in dpv invasion and latent infection in the cns are warranted. persistent infection with hsv- is a prerequisite to this virus's pathogenicity, and hsv- has been reported to infect sensory neurons, the corneal epithelium, lymphocytes, and macrophages ( ) . in the sensory ganglia, macrophages infiltrate the tg and produce tnf-α and inos to control the primary hsv- infection ( ) . in human primary macrophages, knockdown of mda and mavs strongly inhibits the expression of ifn and tnf-α induced by hsv- entry and replication, indicating that the early innate recognition of hsv- involves mda-/mavs-dependent pathways ( ) . the expression of chemokines such as cxcl and ccl in human monocytederived macrophages induced by hsv- infection involves ifi dependent and ifi -independent pathways ( ) . activation of ifnar signaling promotes high isgs expression in macrophages infected with hsv- , in which samhd inhibits hsv- propagation by limiting viral dna synthesis ( ) . abortive infection of viruses in astrocytes or macrophages appears to be essential to induce innate and adaptive immune responses to restrict or clear vesicular stomatitis virus, rabies virus, or influenza virus ( , , ) . in our current study, monocytes/macrophages were infected by both strains of dpv, and the viral titer and copy number of the virulent strain (chv) increased from to hpi at a moi of . . in contrast, the viral titer and genomic copy number of the attenuated strain (cha) began to decrease early in the infection (figure ) . from these data, we inferred that the virulent strain (chv) persistently infects the monocytes/macrophages, whereas the attenuated strain (cha) causes an abortive infection in monocytes/macrophages. we further verified these observations by examining the innate immune response induced by these two strains. in duck monocytes/macrophages, the expression of prrs and isgs were induced by both strains at hpi but gradually decreased in chv-infected cells after hpi ( figure ). however, in cha-infected monocytes/macrophages, the expression of prrs and isgs were induced at hpi and declined slightly at and hpi before rapidly increasing between and hpi (figure ). in the later time points of infection, the titer of the cha strain was significantly lower than that of the chv strain (figure ) . in the previous studies on dpv and mdv viruses, researchers observed the slight up-regulation of the innate immune molecules around hpi in the fibroblasts, and the rapid down-regulation of these molecules from hpi ( , ) . these results are consistent with the data we obtained in this study; we hypothesized that dpv virulent strains have multiple, highly potent proteins that inhibit innate immunity, and the mechanism of suppressing innate immunity will be further studied in the future. blockage of ifnar signaling in monocytes/macrophages led to a marked increase in the titer of the cha strain, but not that of the chv strain (figure ) . priming monocytes/macrophages with poly(da:dt) or poly(i:c) selectively restricted the replication of the chv strain (figure ) . the growth dynamics and innate immune response were similar in pbmcs. these data indicated that the chv and cha strains exhibit different replication patterns in monocytes/macrophages based on activation or inhibition of the innate immune response. this could explain why the cha strain has been attenuated and provides protection from infection with the virulent dpv strain in ducklings. monocytes/macrophages thus represent a promising cell model suitable for further studies of dpv pathogenicity and the mechanism of innate immune response evasion by dpv. in a recent study, the authors found that monocytes/macrophages are important target cells for dtmuv infection ( ) . dtmuv successfully infects macrophages by subverting the innate immunity of monocytes/macrophages and transmits in the body using monocytes/macrophages as cell carriers. in that study, the authors separated lymphocytes and monocytes/macrophages by washing the pbmcs at and h after plating and then used the cells for assays. though the detection with antibodies against the hypothetical duck cd protein showed that the purity of monocytes/macrophages were greater than %, we think that it is difficult to obtain large numbers of macrophages from pbmcs if the monocytes are not induced with m-csf or gm-csf. therefore, the duck monocytes/macrophages isolated in our study will provide an important cell manipulation platform for similar researches and obtain more direct experimental results and conclusions. in conclusion, we isolated and cultured primary duck defs, neurons, astrocytes, pbmcs, and monocytes/macrophages. these five types of duck primary cells were able to respond to dna and rna virus stimulators and exhibited upregulated expression of prrs and isgs, thus demonstrating that they are useful cell models for deeper investigations of the induction/evasion of the innate immune response by aquatic animal viruses such as dpv. the virulent and attenuated strains of dpv infected these cells with differential replication dynamics, accompanied by differences in the innate immune response. blockage of ifnar signaling enhanced the replication of the attenuated strain of dpv in astrocytes, pbmcs, and monocytes/macrophages. priming the ifnar signaling pathway specifically reduced the titer of the virulent strain of dpv, indicating that the ifnar signaling pathway plays a key role in limiting dpv infection. persistent infection of monocytes/macrophages coupled with inhibition of the innate immune response represents an important junction in the pathogenicity of dpv. all datasets generated for this study are included in the 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source of interferon beta in the virus-infected brain inhibition of dnasensing pathway by marek's disease virus vp protein through suppression of interferon regulatory factor activation avian flavivirus infection of monocytes/macrophages by extensive subversion of host antiviral innate immune responses the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © tian, cai, he, deng, wu, wang, jia, zhu, liu, yang, wu, zhao, chen, zhang, huang, ou, mao, yu, zhang, liu and cheng. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -ey kc zj authors: degauque, nicolas; brouard, sophie; soulillou, jean-paul title: cross-reactivity of tcr repertoire: current concepts, challenges, and implication for allotransplantation date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: ey kc zj being able to track donor reactive t cells during the course of organ transplantation is a key to improve the graft survival, to prevent graft dysfunction, and to adapt the immunosuppressive regimen. the attempts of transplant immunologists have been for long hampered by the large size of the alloreactive t cell repertoire. understanding how self-tcr can interact with allogeneic mhc is a key to critically appraise the different assays available to analyze the tcr vβ repertoire usage. in this report, we will review conceptually and experimentally the process of cross-reactivity. we will then highlight what can be learned from allotransplantation, a situation of artificial cross-reactivity. finally, the low- and high-resolution techniques to characterize the tcr vβ repertoire usage in transplantation will be critically discussed. being able to track donor reactive t cells during the course of organ transplantation is a key to improve the graft survival, to prevent graft dysfunction, and to adapt the immunosuppressive regimen. the attempts of transplant immunologists have been for long hampered by the large size of the alloreactive t cell repertoire. understanding how self-tcr can interact with allogeneic mhc is a key to critically appraise the different assays available to analyze the tcr vβ repertoire usage. in this report, we will review conceptually and experimentally the process of cross-reactivity. we will then highlight what can be learned from allotransplantation, a situation of artificial cross-reactivity. finally, the low-and high-resolution techniques to characterize the tcr vβ repertoire usage in transplantation will be critically discussed. keywords: tcr repertoire, transplantation, cross-reactivity, alloreactivity, tcr, mhc, t cell understanding the cross-reactivity shaping the t lymphocyte receptor repertoire through evolution, numerous processes have been selected to generate a diverse repertoire of tcrαβ able to protect mammalian from pathogenic insults (figure ) . highly similar genes recombine to form functional genes and generate a highly diverse tcr repertoire. tcrβ chains are encoded by distinct variable (v; trbv), diversity (d; trbd), and joining (j; trbj) genes, whereas tcrα chains are encoded by distinct sets of v and j genes (trav and traj). junctional diversification further extends the combinatorial diversity by either trimming gene ends or adding nucleotides between the recombining genes ( ) . in contrast to the ighv (v genes of immunoglobulin heavy chain) germline dataset compiled by the immunogenetics (imgt) group that greatly benefit from the advanced of deep-sequencing technologies, the human tcr germline has been only minimally changed since the complete sequencing of the tcr gene loci in ( , ) . the functional genes, orfs, and pseudogenes have been reported for the trbv, for the trav and for the trbd dataset. the analysis of the tcr cdr is still a very challenging process. the identification of the trbd genes figure | understand the cross-reactivity of a highly diverse tcr repertoire. a highly diverse tcrαβ repertoire is generated by iterative processes selected through evolution. combinatory diversity results from the selection of variable (v; trav and trbv), diversity (d; trbd) and joining (j; traj and trbj) genes. junctional diversification further extends the combinatorial diversity by either trimming gene ends or adding nucleotides between the recombining genes. finally, the association of the tcrα and tcrβ chain constitutes the final steps of the numerous iteration processes that lead to the generation of a highly diverse tcr repertoire, which is able to efficiently protect individuals from pathogenic stimulations. tcrαβ adopts a stereotype docking geometry atop the mhc/peptide complex. this orientation leads to a spatial interaction between the germline-encoded cdr and cdr of the tcrα and β chains and the edges of the peptidegroove of mhc. the accumulation of reported crystallographic structures has challenged the stereotypic view of the angle of the tcr docking. however, the recognition of conserved motifs on the side of mhc molecules by cd / cd co-receptor constrained the tcr docking geometry. despite the high diversity of the tcr repertoire, a high degree of cross-reactivity has been reported that could be explained by the "natural" ability of tcr to interact with mhc molecules (mhc focus model) as well as the interaction of tcr to a limited number of amino acids of the peptide bound to the mhc peptide groove. march | volume | article frontiers in immunology | www.frontiersin.org cannot be performed due to the high degree of similarities of the trbd at their ′ ends, the short length of the two genes, and the presence of g-rich n nucleotides at the ′ ends that could be also added by the tdt enzyme. it is misleading to estimate the combinatory diversity by simply multiplying together the number of v, d, and j genes ( ). rather than a random combination of the tcr genes, studies have shown that tcr genes are highly biased in their usage, and that only part of the theoretical diversity is selected ( , ) . chromosomal recombination patterns can be explained by variations in enhancers and recombination signal sequences (rss) and organization of the trbj genes (a block of six and seven genes located respectively downstream from the trdb and trdb gene) that leads to a bias in d-j pairing. the diversity of the tcr repertoire is further broaden during the rearrangement process first by the addition of p nucleotides (palindromic nucleotides) thanks to recombination activating gene- and - (rag and rag ) ( ) that form hairpin loops at the gene end and then by the addition of n nucleotides (with a biased toward g nucleotides) by the terminal deoxynucleotidyl transferase (tdt) ( ) . insertions of nucleotides have a profound impact on the diversity of the complementary-determining regions (cdr ) sequences and contribute to most ( %) of this diversity ( ) . the coding ends of the genes can be also trimmed by exonucleases. however, given the limited number of amino acids, the removal of nucleotides by exonucleases is constrained to generate a productive codon and therefore limits the contribution of exonuclease trimming to the diversity of the tcr repertoire. finally, the association of the tcrα and tcrβ chain constitutes the final steps of the numerous iteration processes that lead to the generation of a highly diverse tcr repertoire, which is able to efficiently protect individuals from pathogenic stimulations. six cdr will engage the peptide/mhc complexes, endogenous and exogenous peptides being presented respectively by mhc class i and ii molecules. mhc class i grooves constrain the length of the presented peptides ( - amino acids length) while the open nature of peptide-binding cleft of mhc class ii molecules allow a broader range of peptides to be presented. the hla locus is the most polymorphic region of the human genome, with more than , variant alleles ( , hla class i alleles and , hla class ii alleles according to the imgt/hla). the high diversity of hla conferring an almost unique signature of hla for mankind is further extended by the combinatory diversity resulting from the association of six hla class i (two alleles of hla-a, -b, and cw) and six hla class ii molecules (two alleles of hla-dr, -dp, and -dq). the high mutation level of the hla loci is preferentially focused on the peptide-binding cleft that clustered most of the variability of the amino acid sequence. the focus of mutations underlines the function of the hla molecules, namely being able to display a very large array of peptides. garcia et al. were the first to report the crystallographic structure of a murine tcr c bound to peptide/mhc class i (h- k b -dev ). the cytotoxic t cell clone c is one of the most well-characterized tcr and has been initially isolated from a balb/b mouse as an allospecific t cell that recognized l d on the mastocytoma p . beside its primary antigen (peptide p c), the c tcr can bind to different antigens, including the dev ( ) and siyr ( ) . they also showed that tcrαβ adopts a ° diagonal orientation to the long axis of the peptide ( ) . this orientation leads to a spatial interaction between the germlineencoded cdr and cdr of the tcrα and β chains and the edges of the peptide-groove of mhc (figure ) . the highly diverse cdr region is facing the central portion of the bound peptide. the multiple crystallographic structures of tcr/peptide mhc complexes [more than of crystallographic structures tcr repertoire in transplantation frontiers in immunology | www.frontiersin.org have been obtained ( ) ] have revealed that the docking angle of the tcr is conserved with a stereotype position of a ° diagonal orientation to the long axis of the peptide ( ) . the conserved binding model has lead to the concept that tcr and mhc are hardwired to interact, resulting from a coevolution selection of conserved regions (codons) to lock in tcr onto mhc molecule. the stereotyped orientation of tcr atop mhc molecule is however more flexible than initially proposed, with the accumulation of crystal structures. the median docking angle of tcr is . ° (min-max - °) with mhc class i and . ° (min-max - °) with mhc class ii ( ) (figure ) . different theories have been postulated to explain the hardwire of tcr to mhc molecules ( ) , including the key role of co-receptors of cd and cd that imposed steric requirements for concurrent associations of tcr, cd , cd /cd , and mhc complexes allowing the appropriate signaling events to occur. indeed, the main role of co-receptor cd and cd is to recruit the src tyrosine kinase p lck (lck) via its association with the cytoplasmic tail of cd or cd . lck concentration promotes phosphorylation of immunoreceptor tyrosine activation motifs (itams) in the cytoplasmic tails of cd subunits and then initiates the cascade of signaling events leading to the full activation of the t lymphocyte. given the key role of co-receptor cd and cd in process, it was assumed that their ability to bind, respectively, the membrane-proximal α and β domains of the mhc class ii molecule and the protruding loop in the α domain of the mhc class i molecule will constrain the docking geometry of the tcr to the pmhc (figure ) . a recent report by beringer et al. ( ) had challenged the consensus idea of a highly stereotype docking of tcr atop mhc molecules ( ) . crystallographic structures of two tcr binding to proinsulin peptide presented by hla-dr (hla-dr proinsulin ) have been obtained from two clones of induced regulatory cd t cells. the ternary complexes revealed a ° polarity reversal compared to all other tcr-peptide-mhc complex structures. it remains to be address whether this singular observation could be generalizable, whether the reverse docking is a unique feature of regulatory cells and whether the potential signaling differences may influence the phenotype and the function of the t cells. cross-reactivity can be defined by the ability of a given tcr to interact with more than one pmhc complex with different presented peptides or mhc molecules. this new concept has been presented as early as by matzinger and bevan ( ) . an alloreactive t cell clone was derived by owens et al. in with three h -e reactivity (allo-e k specific, h -e k , dba/b h -e d , and self h -e d ) ( ) . since then, numerous reports have provided evidence of cross-reactivity. for instance, mouse c tcr can interact with syngeneic mhc h- k b presenting dev ( ) and siyr ( ) and with allogeneic h- k bm presenting dev /k bm ( ) and allogeneic h- l d -p ca ( ) . the study by birnbaum et al. is an elegant attempt to quantify the cross-reactivity of a given tcr ( ) . using five different cd tcr clones (three from mouse origin and two from human origin), high throughput screening of yeast libraries and deep sequencing, the authors demonstrate that a single tcr can interact with more than different peptides. jerne et al. postulated in the mid- that each cell exhibits a unique clonotype able to recognize only one antigen ( , ) . don mason has been among the first to challenge the validity of this clonal selection theory ( ) showing that the immune system will be highly incompetent to protect an individual from external insult if one and only tcr was able to recognize a single peptide presented in a given hla context. more than t cells, which would weigh more than kg, would be needed to provide efficient coverage of the potential foreign peptides. this clearly stated that the immune system could not efficiently protect individual if one tcr interacts with a single antigen. unlike the affinity maturation of b cell receptor, the protein sequence of tcr is fixed and naive t cells are required to recognize foreign antigens not encountered before. the number of potential antigens to be recognized is huge given the variability induced by the high diversity of peptide-binding groove of hla class i and ii molecules. from the proteinogenic amino acids and given that peptides from -to -mer can be presented, an incredibly high number of peptides can be potentially generated (> peptides) ( ) . the diversity can be further extending by the posttranslational modifications of amino acids. in a opinion paper, andrew sewell elegantly presents the necessity of the cross-reactivity ( ), as the number of potential foreign peptide-mhc complexes that t cells might encounter dwarfs the number of tcrs available [the number of unique tcrαβ is estimated to be in the magnitude of ( , ) ]. the mechanisms described previously to generate a diverse tcrαβ have to be envisioned at the population level. given the relatively limited number of genes encoding for tcr chain α and β and the requirement of tcr to recognize the highly diverse hla molecules, the necessity of each t cell to recognize a large array of peptides is expected ( ) . before presenting the experimental approach aiming to quantify the number of peptides recognized by a single tcr, we would like to present clear evidences of the cross-reactivity involving memory t cells without previous antigen encounter. it has been described few years ago that cd t cells with a memory phenotype can be found in mice ( ) ( ) ( ) . cd t cells specific for ovalbumin and viral antigens (hsv, vaccinia) could be detected in mice despite their germ-free environment ( ) . despite the absence of previous antigen encounter, these pre-existing memory cd t cells harbor traits of memory cells such as the ability to rapidly proliferate upon stimulation and to secrete rapidly pro-inflammatory cytokines. homeostatic proliferation, aging, and cross-recognition of alternate ligands have been postulated to drive the accumulation of these memory-like naive cd t cells ( , ) . this observation has been extended to human settings in which cd t cells specific for hiv- , cmv, and herpes simplex virus (hsv) epitopes were identified in healthy volunteers that had never been infected with these viruses ( ) . again, these cells exhibit not only memory markers but also memory-associated features (rapid proliferation and cytokine secretion). the acquisition of memory characteristics could be a consequence of homeostatic proliferation ( ) or a consequence of the cross-reactivity to other antigens in the environment. to support the latest hypothesis, su et al. have shown that hiv- specific t cells can recognize environmental peptides present in the gut and soil, bacteria and ocean algae, and plants. of interest, t cells specific for hiv- can even be purified from cord-blood ( ) , demonstrating thereby the presence of t cells able to recognize self and non-self antigens in newborns. of interest, the phenotype of cross-reactive t cells was different between newborns and adults, with a naive and a memory phenotype, respectively. the concept of clonal deletion that occurred in the thymus is challenged by the aforementioned reports and compelling evidences suggest that from an evolutionary perspective, the necessity to protect an individual against pathogens is far more important than to limit the autoreactivity. a recent study from davis team further sustained this claim ( ) . the frequency of cd t cells specific of a y chromosome specific antigen (equivalent to hy peptide) is only threefold lower in man as compared to women ( ) . of interest, whereas cd t cells purified for their specificity regarding a pool of six self peptides do not proliferate after stimulation with the same set of self peptides, cd t cells specific of a pool of six non-self peptides exhibit a potent proliferative response ( ) . the absence of response reported for self-specific cd t cells and not for foreign antigen-specific cd t cells has been linked to a different genetic programing as compared to the clones purified from woman, with a lower expression of il- r, il- r, and bcl-xl ( ) . thus, evolution has favored the absence of hole over autoimmune disease (about % of incidence). it may seem awkward that the evolution has favor the escape of antiself specific t cells from thymic selection over a more stringent deletion of all anti-self t cells. a heavier burden of maintaining tolerance is needed to prevent the development of autoimmune diseases. however, the need to defend the immune system against pathogens, especially during childhood, is far greater than the need to prevent autoimmunity as for population's survival. by limiting the deletion of self-reactive t cells and thanks to the large cross-reactivity of t cells, the holes in the t cell repertoire that pathogens might take advantage of are constrained. the analysis of the immune system in monozygotic twins is enlightened in many aspects as such studies allow the dissociation between the inborn and the acquired contributions. the team of davis has recently showed that the heritability of t and b cells parameters declines very rapidly with age ( ) . at the age of years, the heritability explained less than % of the variation in t and b cell parameters. cmv infection is a protypical example of the influence of non-heritable factor on the whole immune system. indeed, % of all parameters measured in discordant twins were influenced by cmv infection ( ) . the environment carves the immune system of each single individual, with each past immune response heavily imprinting the (present) immune system. since the initial observation that immunity against cowpox protects individual from smallpox ( ) , numerous examples of cross-reactivity had been reported in mice and in human ( ) . for instance, infections with bcg, influenza a virus (iav), lymphocytic choriomeningitis virus (lcmv), and murine cytomegalovirus (mcmv) all confer a level of protective immunity against vaccinia virus ( ) ( ) ( ) . the benefit of cross-reactivity as for pan-virus protection is more difficult to assess for obvious reasons. nevertheless, the numerous example of a single tcr able to recognize different antigens [bcg and poxviruses ( ) ; papillomavirus and coronavirus ( ) ; influenza virus and epstein-barr virus ( ) ]. the large cross-reactivity of t cells confers a more efficient protection cover using a limited number of t cells that need to screen an incredibly large array of peptides that can be presented by mhc molecules. beside the efficient use of limited t cell resource, cross-reactivity confers a spatiotemporal advantage to the immune system to scan any infected cells. cross-reactivity could also be envisioned as an evolution strategy to limit the immune recognition escape. recipient immune system can interact with foreign hla molecules under two very different circumstances: pregnancy and transplantation. thanks to evolution and adaptation of the maternal immune system to the presence of hla mismatch fetuses, allorecognition during pregnancy is not harmful and could even be beneficial as for mammalian sexual reproduction. immunological tolerance toward allogeneic fetus is obtained through a complex network of regulatory mechanisms including the lack of expression of classical mhc class i molecules by the placental trophoblast and the expression of non-classical mhc class i hla-e and hla-g. more surprisingly, hla mismatches have been proposed to be beneficial for pregnancy outcome. in the s, billington reports that the placenta is larger in h- incompatible mouse as compared to compatible fetuses ( ) . hla compatible fetuses (i.e., similar to maternal hla) have been shown to be more prone to be aborted ( ) . in contrast, recipient immune system will potently eliminate an allogeneic graft in the absence of immunosuppressive therapy. despite the absence of thymic central selection ( ) of potential graft-recipient t cells by allogeneic mhc motifs regarding their ability to recognize allogeneic potential hla, a large pool of t cells can be activated by donor hla molecules either through the direct pathway (i.e., donor hla presenting donor peptides) or the expected processing of foreign mhc molecules, coined as the "indirect pathway" (i.e., recipient hla presenting donor peptides) in transplantation immunologist jargon. the direct allorecognition pathway represents a unique example of functional and efficient cross reactivity. two main hypothesizes have been postulated to explain the basis of alloreactivity, emphasizing the role of either mhc molecule or peptide. the polymorphism between donor and mhc molecules could act as an "innate focus" that leads to the activation of unprimed recipient t cells or the allopeptide could be recognized as foreign antigen while allogeneic and self-mhc molecules exhibit a high degree of similarity (figure ) . according to the mhc centric model, the peptide plays only a minor role in the process, and alloreactive tcrs recognize structural determinants on the mhc helices of syngeneic or allogeneic mhc. the bias of tcr to interact with mhc molecules supports this theory. crystal structures of allo-pmhc complexes such as c tcr with allogeneic h- k bm presenting dev /k bm ( ) or bm . tcr with allogeneic pbm -h- k b ( ) have shown that alloreactive tcrs interact with allogeneic mhc in a similar fashion as with syngeneic mhc. to further support the role of mhc in alloreactivity, it has been reported that some hla mismatches between donor and recipient are associated with worse graft survival than others, leading to the notion of taboo mismatches based on shape rather than sequence differences ( ) . for instance, despite a single amino acid in an hla class i antigen, mismatches between hla-b* and hla-b* is associated with transplant rejection ( ) and acute graft-versushost disease ( ) . the peptide repertoire bound to hla-b* or hla-b* have been shown to be very similar ( ) . however, a recent report challenges this observation ( ) . the single amino acid mismatch induced the presentation of more unique peptides by hla-b* than hla-b* , consistent with the stronger t cell alloreactivity observed toward hla-b* compared with hla-b* ( ) . this observation supports the notion of a peptide focus tcr allorecognition, in the same line as molecular mimicry. allorecognition could also involved cross-reactivity between mhc class i and mhc class ii or even xeno mhc ( , ) . in , schilman et al. reported that cd t cell clone could be activated by both mhc class i (h- d b ) and mhc class ii (i-e k ) molecules ( ). by-directional recognition of t cells between mhc class i and mhc class ii have been reported later ( ) ( ) ( ) . these observations may have important implication in the attempt to minimize hla mismatches during the process of organ allocation. using a mixed lymphocyte reaction ( ) , it has been shown that - % of t cell in peripheral blood can be activated ( ) . as mentioned before, the number of hla mismatches between donor and recipient is a primary driving force that mobilized a larger fraction of t cells than nominal antigens. whether alloreactive t cells are activated by the high number of new antigens presented by donor hla or by the large number of different allo-phla complexes (or both) is still under debate, and the two hypotheses are not mutually exclusives. the indirect pathway further enhances the reactivity of recipient t cells toward allogeneic graft. indeed, peptides presented by mhc molecules derived predominantly from mhc-related molecules ( ) ( ) ( ) . the introduction of donor hla molecules will thus lead to the introduction of great pool of new peptides that can mobilized a large fraction of recipient t cells. it is now also well accepted that memory t cells generated prior transplantation constitute a major hurdle for long-term graft acceptance. chronic viruses such as ebv and cmv induce the generation of a large pool of memory t cells. for instance, % of both the cd and cd memory compartments in blood are reactive to hcmv ( ) . the cross-reactivity between virus-specific t cells and allogeneic hla has been extensively documented ( ) . ebv or cmv specific cd t cells exhibit frequently a crossreactivity toward allogeneic mhc class i complexes ( ) ( ) ( ) ( ) ( ) . similar observations have been reported for cd t cells specific for ebv or cmv ( ) ( ) ( ) . virus-specific t cells that cross-react with alloantigens have been shown in experimental models to proliferate in response to a transplanted allograft in vivo ( ) . for instance, lcmv-specific cd t cells generated after infection of mice with armstrong strain of lcmv are able to vigorously proliferate in vivo after skin transplantation and ultimately to mediate skin graft rejection ( ) . tracking anti-donor response by the investigation of tcr vβ repertoire: from low resolution technique to high throughput sequencing given the size of anti-donor t cell pool, great efforts have been paid to track the immune-response using the analysis of tcr vβ repertoire and to correlate specific usage of tcr vβ repertoire with graft status or graft outcome. before presenting the available reports, it is necessary to present the two major methods used to investigate tcr vβ usage; a low resolution (spectratype alone or tclandscape when combined with quantitative analysis) and, more recently, a high resolution (deep-sequencing of tcr vβ region) approach (figure ) . the low-resolution technique is based on the analysis of the length of the cdr region whereas the high-resolution technique identifies the sequence of each tcr vβ and later quantifies the abundance of the different t cell clones. each tcr vβ family is composed of t cells with various lengths of their cdr region. the distribution of the cdr length can be assessed by spectratype ( , ) . a broad spectrum of profiles can be identified ranging from a gaussian-like profile to a highly restricted profile, highlighting the absence of selection of t cell, or the expansion of t cell clones, respectively. different analytic tools have been used to characterize the cdr length distribution ( ) ( ) ( ) ( ) . the qualitative assessment of the tcr vβ repertoire can be complemented by the quantification of the different vβ families at the mrna level using qrt-pcr ( ) ( ) ( ) or at the cellular level using flow-cytometry ( ) . such techniques still offer several benefits over higher resolution techniques such as their cost, the short time frame to obtain results, and the generation of a reasonable amount of data can be also displayed as "visible" pattern as an "x-ray" of the global tcr alteration in a specific pathological context ( ) ( ) ( ) ( ) . a rapid survey of the usage of the tcr vβ repertoire can be efficiently performed, guiding further investigations focused on targeted tcr vβ families. at the other range of the resolution spectrum, deep-sequencing of tcr vβ obtains a full picture of the usage of t cell repertoire with deep or ultra-deep resolution. the availability of all tcr vβ sequences allows for the precise appraisal of the distribution of the different t cell clones especially across different biological compartments ( ) . furthermore, with a complete tcr vβ sequencing, researchers can investigate the similarity of t cell figure | characterization of the tcr vβ repertoire by low resolution and high resolution technique. immune challenge leads to the selection of t cells harboring specific tcrαβ among the highly diverse tcrαβ repertoire. antigen-specific t cells could be identified by low-resolution techniques (e.g., spectratype) or high-resolution techniques (e.g., ngs). low-resolution techniques are aiming to identify vβ families that exhibit monoclonal distribution of their cdr length distribution using vβ specific pcr and spectratyping. the clonality of the identified vβ families needs to be confirmed by the sequencing of the pcr product. vβ-specific t cell purification enables later to perform functional assay or to reconstruct the tcrαβ in order to identify the recognized antigen. deep-sequencing of tcr vβ region identify the sequence of each tcr vβ and intensive bio-informatic process is needed to quantify the abundance of the different t cell clones. given the burden of data generated, the next-generation sequencing is well-fitted to track t cell clones in time or across different anatomic sites. march | volume | article frontiers in immunology | www.frontiersin.org sequences between biological compartments or individuals and take advantage of public repository databases to assess the specificity of a sequence and potentially to reconstruct the tcr in order to search for the recognized peptides. however, the amount of data generated using this technique is extremely high and efficient bio-informatics tools specifically devoted to the analysis are needed to identify meaningful information in the ocean of data. the accessibility of deep-sequencing is likely to be broaden in the near future thanks to the advances in bio-informatics tools and the reduction of the cost. tcr repertoire in transplantation frontiers in immunology | www.frontiersin.org low-resolution techniques have been used to investigate the usage of tcr vβ repertoire in kidney transplant recipients with various clinical outcomes or at various time points posttransplantation ( ) ( ) ( ) . using the combination of spectratyping and quantitative assessment of the tcr vβ transcript, we have been able to define direct or indirect allorecognition patterns in an experiment model of allograft in congenic rats ( , , ) . using the same approach, we reported that patients with biopsy-proven chronic antibody-mediated (camr) rejection exhibits strong alterations of their tcr vβ repertoire correlating with the level of graft lesions classified with banff classification ( ) . in contrast, operationally tolerant patients [i.e., patients off-immunosuppression for more than months with a wellfunctioning graft ( ) ( ) ( ) ] exhibit a polyclonal tcr vβ repertoire ( ) . a large cohort of patients with stable graft function for more than years post-transplantation had been prospectively recruited in our center with stringent clinical and demographic inclusion criteria in order to obtain a homogeneous population. nevertheless, we could highlight that the usage of tcr vβ repertoire is highly heterogeneous ranging from the absence of clonal selection (similar to operational tolerance) to an accumulation of selected t cells (as for camr rejection) ( ) . the presence of altered tcr vβ repertoire has been previously reported in a rat model of camr ( ) in which similar cd clones could be identified in the blood and in the graft ( ) . in a large prospective study of kidney transplant recipients with a stable graft function for more than years, we show that the altered tcr vβ repertoire was due to an accumulation of temra (t cell effector memory re-expressing cd ra; cd ra + ccr − ) cd t cells with an activated profile (cd − cd − ), a high expression of cytotoxic molecules, perforin (perf) and granzym b (gzm-b) , t-bet, and cd and the ability to secrete tnf-α and ifn-γ ( ) . of interest, stable patients who have an increase in differentiated temra cd t cells have a twofold higher risk of long-term graft dysfunction ( ) . of note, using a similar strategy, kim et al. recently reported that clonal cd t cell could be evidenced in human transplanted hand, with several tcr clonal selections persisting at least days (among the days of surveillance) ( ) . collectively, these data highlight that a low-resolution technic provides key features as for the accumulation of selected t cell clones that can be used to monitor the kidney transplant recipients. a major drawback of spectratype-based method is its intrinsic low resolution as multiple t cell clones could share the same cdr length. it is necessary to sequence the tcr vβ chain with an altered cdr length distribution to assess the clonality of a given vβ family. however, we recently compared spectratype or next generation sequencing (ngs) techniques to characterize the tcr vβ repertoire in the blood, the cerebral spinal fluid (csf), and the central nervous system (cns) of patients with multiple sclerosis ( ) . both methods were as efficient to highlight the similarity of tcr vβ repertoire between csf and cns (≈ % of tcr vβ clones identified in the cns were also found in the csf) and to identify ≈ % of the tcr vβ clones using blood cd sample ( ) . as previously discussed, the size of donor-reactive t cell repertoire is large and constitutes a limitation to the use of deep-sequencing approach. it may thus be a naive approach to perform ngs on unfractioned t cells with the aim to identify t cell clones specific to a given situation, such as kidney transplantation or viral infection; a two-step approach is needed. the first step is to purify the t cell population of interest based on the expression of phenotypic (using tetramer for instance) or functional (e.g., cytokine secretion, proliferation) markers. the in-depth characterization of tcr vβ of t cell population of interest allows for the definition of a signature that can be later used as a tag when unpurified samples are analyzed. this approach has been used to track cmv-or bk-specific t cell clones ( ) or alloreactive t cells ( , ) . the first report hypothesizing such an approach in the transplant context has been published by the group of leventhal ( ) . using healthy volunteers, this study aimed to assess breadth, clonal structure, and dynamics of the alloreactive t cell repertoire. after days of mlr, the proliferating t cells were purified according to the dilution of cell division dye. by comparing the number of clones before culture and in the proliferated mlr responder, two types of alloreactive clones were identified, low-(i.e., unobserved in pre-culture sample and ≥ t cells after mlr) and high-abundance pre-culture clones (i.e., present in pre-culture sample and ≥ × enriched after mlr). more than , low-abundant clones and more than , high-abundant clones were detected in the different experiments. these data provide new evidences of the large size of the alloreactive t cell pool. this approach was used recently to track donor-reactive t cells in kidney transplant recipients ( ) . the fingerprint of donorreactive t cell repertoire was established before transplantation by deep-sequencing of proliferating cd and cd t cells after days of mlr. the fingerprint of donor-reactive t cells was monitored later after transplantation without the need to perform mlr. the team of sykes provides evidences that tolerance induction protocol based on combined kidney and non-myeloablative bone marrow transplantation results in a reduction of donor-alloreactive t cell clones. however, this decrease was neither observed in the patient that failed to respond to the tolerant inducing protocol nor in patients with standard immunosuppressive regimens. pretransplant identification of donor-reactive t cell clones before transplantation could thus be a means to track the activation of the immune system by allogeneic graft. the studies of emerson ( ) and morris ( ) showed that the anti-donor fingerprint is stable over-time in healthy volunteers. given the design of the assay, only pre-existing clones could be tracked. it would be of great value to compare the anti-donor clone repertoire before and after transplantation, starting each time from a direct mlr assay to investigate if new anti-donor t cells arise after transplantation. indeed, infections that occurred frequently after transplantation could generate virus-specific t cells with an allogeneic crossreactivity potential ( ) . moreover, not all proliferating cells after days of mlr are per se donor-specific as proliferation of t cells could also be linked to bystander stimulation ( ) . will transplant immunologists be able to track the rise and the expansion of donor-specific t cells and would this approach be widely available and useful to the clinical management are still open questions. high-through put techniques that have recently emerged are certainly an important step forward. nevertheless, the high cross-reactivity of t cells is a major hurdle to identify the trigger of the expansion of donor-reactive t cells, as donor antigen, viral peptides, and other environmental antigens can lead to the selection of donor-specific t cells. while promising, the study of tcr alteration has not overcome the double difficulties of offering an accessible technical presentation of the data and a validated correlation with clinical outcomes. therefore, longitudinal studies to test the reactivity of recipient t cells against donor antigens at different time points are needed. nd, sb, and j-p s wrote the review. this work was realized in the context of the labex igo program supported by the national research agency via the investment of the future program anr- -labx- - and the labex transplantex [anr- -labx- _transplantex], in the context of the ihu-cesti project which received french government financial support managed by the national research agency via the "investment into the future" program anr- -ibhu- and in the context of a centaure foundation. the ihu-cesti project is also supported by nantes metropole and the pays de la loire region. this work was also supported by the fp visicort project that has received funding from the european union's seventh framework program for research, technological development and demonstration under grant agreement no . we thank dr. yap and dr. haspot for their fruitful discussion. somatic generation of antibody diversity the complete -kilobase dna sequence of the human beta t cell receptor locus analysis of the . -mb human alpha/delta t-cell receptor locus with bacterial artificial chromosome clones estimating the diversity, completeness, and cross-reactivity of the t cell repertoire different tcrbv genes generate biased patterns of v-d-j diversity in human t cells exhaustive t-cell repertoire sequencing of human peripheral blood samples reveals signatures of antigen selection and a directly measured repertoire size of at least million clonotypes junctional sequences of t cell receptor gamma delta genes: implications for gamma delta t cell lineages and for a novel intermediate of v-(d)-j joining most alpha/beta t cell receptor 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leucocyte antigen d-related ) mice: a mouse model of human allogeneic graft-versus-host disease characterization of an lyt- + alloreactive cytotoxic t cell clone specific for h- db that cross-reacts with i-ek restricted mhc-peptide repertoire predisposes to autoimmunity how the t cell repertoire becomes peptide and mhc specific a single t cell receptor bound to major histocompatibility complex class i and class ii glycoproteins reveals switchable tcr conformers quantitative assay of antigenic disparity at hl-a-the major histocompatibility locus in man quantifying the frequency of alloreactive t cells in vivo: new answers to an old question predominant naturally processed peptides bound to hla-dr are derived from mhc-related molecules and are heterogeneous in size specificity and promiscuity among naturally processed peptides bound to hla-dr alleles sequence analysis of peptides bound to mhc class ii molecules broadly targeted human cytomegalovirus-specific cd + and cd + t cells dominate the memory compartments of exposed subjects advances in direct t-cell alloreactivity: function, avidity, biophysics and structure t cell receptor repertoire for a viral epitope in humans is diversified by tolerance to a background major histocompatibility complex antigen cross-reactivity of herpesvirus-specific cd t cell lines toward allogeneic class i mhc molecules an alloresponse in humans is dominated by cytotoxic t lymphocytes (ctl) cross-reactive with a single epstein-barr virus ctl epitope: implications for graft-versus-host disease cross-reactive memory t cells for epstein-barr virus augment the alloresponse to common human leukocyte antigens: degenerate recognition of major histocompatibility complex-bound peptide by t cells and its role in alloreactivity cross-recognition of hla dr alloantigen by virus-specific cd + t cells: a new paradigm for self-/nonself-recognition ebv-specific cd + t cell clones exhibit vigorous allogeneic responses cross-recognition of human alloantigen by cytomegalovirus glycoprotein-specific cd + cytotoxic t lymphocytes: implications for graft-versus-host disease allo-hla reactivity of virus-specific memory t cells is common allografts stimulate cross-reactive virus-specific memory cd t cells with private specificity circulating t cell repertoire complexity in normal individuals and bone marrow recipients analyzed by cdr size spectratyping. correlation with immune status molecular detection and in vivo analysis of the specific t cell response to a protein antigen statistical analysis of cdr length distributions for the assessment of t and b cell repertoire biases expanded cd t-cell sharing between periphery and cns in multiple sclerosis the blood of healthy individuals exhibits cd t cells with a highly altered tcr vb repertoire but with an unmodified phenotype t cell repertoire alterations of vascularized xenografts direct recognition of foreign mhc determinants by naive t cells mobilizes specific vbeta families without skewing of the complementarity-determining region length distribution highly altered v beta repertoire of t cells infiltrating long-term rejected kidney allografts immune responses elicited in tertiary lymphoid tissues display distinctive features improved assessment of t-cell receptor (tcr) vb repertoire in clinical specimens: combination of tcr-cdr spectratyping with flow cytometry-based tcr vb frequency analysis blood t-cell vbeta transcriptome in melanoma patients serial blood t cell repertoire alterations in multiple sclerosis patients; correlation with clinical and mri parameters serial evolution of tcr beta chain transcript mobilization in hiv type- -infected patients following vaccine immune stimulation and haart interruption operationally tolerant and minimally immunosuppressed kidney recipients display strongly altered blood t-cell clonal regulation analysis of the peripheral t-cell repertoire in kidney transplant patients expansion of highly differentiated cytotoxic terminally differentiated effector memory cd + t cells in a subset of clinically stable kidney transplant recipients: a potential marker for late graft dysfunction identification of a peripheral blood transcriptional biomarker panel associated with operational renal allograft tolerance the natural history of clinical operational tolerance after kidney transplantation through twenty-seven cases clinical operational tolerance after kidney transplantation indirect cd + th response, antidonor antibodies and diffuse c d graft deposits in long-term recipients conditioned by donor antigens priming functional compartmentalization following induction of long-term graft survival with pregraft donor-specific transfusion clonal cd + t cell persistence and variable gene usage bias in a human transplanted hand tcr repertoire analysis by next generation sequencing allows complex differential diagnosis of t cell-related pathology defining the alloreactive t cell repertoire using high-throughput sequencing of mixed lymphocyte reaction culture tracking donor-reactive t cells: evidence for clonal deletion in tolerant kidney transplant patients high-resolution characterization of cytokine-producing alloreactivity in naive and allograft-primed mice the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - mazd eu authors: beeraka, narasimha m.; sadhu, surya p.; madhunapantula, subbarao v.; rao pragada, rajeswara; svistunov, andrey a.; nikolenko, vladimir n.; mikhaleva, liudmila m.; aliev, gjumrakch title: strategies for targeting sars cov- : small molecule inhibitors—the current status date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: mazd eu severe acute respiratory syndrome-corona virus- (sars-cov- ) induced coronavirus disease - (covid- ) cases have been increasing at an alarming rate ( . million positive cases as on june ), causing high mortality ( , , deaths as on june ) and economic loss (a . % shrink in global economy in ) across countries globally. the clinical manifestations of this disease are pneumonia, lung injury, inflammation, and severe acute respiratory syndrome (sars). currently, there is no vaccine or effective pharmacological agents available for the prevention/treatment of sars-cov infections. moreover, development of a suitable vaccine is a challenging task due to antibody-dependent enhancement (ade) and th- immunopathology, which aggravates infection with sars-cov- . furthermore, the emerging sars-cov- strain exhibits several distinct genomic and structural patterns compared to other coronavirus strains, making the development of a suitable vaccine even more difficult. therefore, the identification of novel small molecule inhibitors (nsmis) that can interfere with viral entry or viral propagation is of special interest and is vital in managing already infected cases. sars-cov- infection is mediated by the binding of viral spike proteins (s-protein) to human cells through a -step process, which involves angiotensin converting enzyme- (ace ) and transmembrane serine protease (tmprss)- . therefore, the development of novel inhibitors of ace /tmprss is likely to be beneficial in combating sars-cov- infections. however, the usage of ace- inhibitors to block the sars-cov- viral entry requires additional studies as there are conflicting findings and severe health complications reported for these inhibitors in patients. hence, the current interest is shifted toward the development of nsmis, which includes natural antiviral phytochemicals and nrf- activators to manage a sars-cov- infection. it is imperative to investigate the efficacy of existing antiviral phytochemicals and nrf- activators to mitigate the sars-cov- -mediated oxidative stress. therefore, in this review, we have reviewed structural features of sars-cov- with special emphasis on key molecular targets and their known modulators that can be considered for the development of nsmis. covid- is a devastating disease caused by a coronavirus related to the one that caused outbreaks of severe acute respiratory syndrome (sars) in the year ( , ) . middle east respiratory syndrome (mers)-related coronavirus is an infamous member of this cohort. covid- , which is caused by the sars-cov- infection, was detected in wuhan, china in december . the world health organization (who) declared this infection a pandemic on march due to its severity and rapid spread across the globe. as of june , sars-cov- had infected . million individuals, and caused , , deaths across countries worldwide ( table ) . coronaviruses (cov) belongs to a family of single-stranded rna viruses (+rna) that can infect a variety of mammals such as bats and humans ( ) . sars-cov- contains rna of , nucleotide length, which codes for , amino acids ( ) . the rna has a ' cap and ' poly-a tail and produces a poly-protein a/ ab (pp a/pp ab) in the host ( ) . sars-cov- belongs to beta cov category and appears in a crown shape with a size of ∼ - nm (figure ) . gene sequencing data revealed that sars-cov- has and % sequence similarity with bat sars-like-cov-zxc and human sars-cov, respectively ( , ) . the spike (s) proteincoding gene mutation in the nsp and nsp regions results in the replacement of glycine (g) with serine (s) at position (g s), and an isoleucine (i) replaced with proline (p) at amino acid position (i p). due to these mutations, the invading potential of sars-cov- has increased significantly toward host tissues. this virus can also be transmitted through the respiratory droplets from coughs and sneezes of infected individuals ( ) . this mode of aerosol transmission is possible, especially, when protracted exposure occurs in closed areas ( ) . the incubation time of the virus varies significantly from individual to individual. in general it takes about days from the day of infection to the first appearance of symptoms. however, in a few cases the symptoms may appear only after weeks ( ) . members of coronaviridae are known to induce respiratory complications in humans ( , ) . at first, sars-cov, mers-cov, and sars-cov- varieties were transmitted from animals to humans which triggered severe respiratory diseases ( ) ( ) ( ) . however, subsequent transmission occurred among humans primarily due to physical contact. hence, conventional preventive measures such as physical isolation were implemented to avoid propagation of early infection across the human population ( , ) . similar to the sars-cov, the pathological manifestations of sars-cov- could induce lung malfunction in humans as indicated by the severe acute respiratory syndrome and pneumonia ( ) . recent studies reported that sars-cov- infection can induce mild, moderate, and severe illness in infected patients ( ) . clinical manifestations of this infection include chronic pneumonia, sepsis, septic shock, fever, and dry cough ( ) . a progressive respiratory failure during this infection may lead to sudden death ( ) . mild illness resulting from a sars-cov- infection is characterized by the presence of malaise, headache, low fever and dyspnea. in the case of moderate illness from sars-cov- , the complication is manifested by the presence of cough and mild pneumonia. severe illness from sars-cov- is associated with chronic pneumonia, cough, sars, hypoxia, and tachypnea (in children) followed by respiratory, and cardiovascular system failure ( ). the autopsy and biopsy reports of sars-cov- patients revealed severe edema with pulmonary tissue exudates, focal reactive hyperplasia, damage to pneumocytes as well as alveolar macrophages, and patchy cellular infiltration ( ) . coronavirus-induced lung damage has been demonstrated experimentally by several investigators in animal models ( ) . for instance, the sialodacryoadenitis virus and parker's rcov were shown to induce damage to alveolar type-i cells through the expression of pro-inflammatory cytokines, and chemokines such as cinc- , cinc- , lix, mip- α, and fractalkines ( ) ( ) ( ) ( ) ( ) ( ) ( ) . for example, fractalkine promotes the infiltration of cytotoxic lymphocytes in the alveolar epithelium thereby inducing a severe inflammatory response ( , ) . similarly, mip- α confers the chemotaxis of immune cells via il- β and tnf-α inflammatory mediators ( , ( ) ( ) ( ) ( ) . therefore, these animal models could be used to develop effective pharmacological agents against sars-cov- infections. studies from several laboratories have demonstrated that the entry of sars-cov- into human cells is facilitated by ace- ( ) . ace- is a member of the renin-angiotensin system (ras), which plays a vital role in cardiovascular and renal homeostasis. ace- and tmprss facilitates the entry of the virus into host cells during sars-cov- infection ( ). in addition, there are other proteases such as aminopeptidase n (apn) which plays a prominent role for the entry of hcov-nl and hcov- e into host cells ( ) ( ) ( ) ( ) . apn is a membrane-bound glycoprotein that mediates the zincdependent protease activity during the entry and or replication of coronavirus strains into host cells ( , , ) . hence, the ace- receptor's down-modulation may prevent sars-cov- viral entry/replication ( ) . the s-protein of sars-cov and other coronavirus strains are different in their structural and functional domains ( ). s-protein can bind to the n-terminus of ace- receptors on the outer surface of host cells including respiratory epithelium of the lungs ( ) ( ) ( ) . identifying the key amino acid residues in s-protein of the sars-cov- strain may benefit virologists and medical scientists to develop better therapeutic agents. however, to date these details are not known, hence, there is an immediate requirement to identify the amino acids involved in binding s-proteins to ace- receptors on host cell surfaces. furthermore, investigations should also focus on establishing the structural similarities of s-protein motifs that are interacting with the ace- receptors of other coronavirus strains ( ) ( ) ( ) ( ) ( ) . these investigations might help in deciphering molecular strategies to target receptor binding sites of ace- proteins with sars-cov- using novel therapeutics and vaccines to avoid membrane fusion process and viral entry ( ) . the tmprss protease can foster the entry of the sars-cov- virus by activating the s-protein for virus-host cell membrane fusion, consequently enhancing viral replication in the host cells ( , ( ) ( ) ( ) ( ) ( ) . tmprss plays a vital role in generating inflammatory cytokines and chemokines in lung epithelial cells by cleaving s-protein during coronavirus infections including sars-cov- . hence, tmprss is another potential therapeutic target to consider for the novel drug development against sars-cov- ( ) ( ) ( ) . prevention and treatment of sars-cov- infections are achieved at different levels ( ) . the primary approach involves physical isolation to prevent the spread of virus from individual to individual; the second approach involves inhibiting the entry of virus into human cells and the third method includes treating the infected individuals to minimize inflammatory reactions and blocks cathepsin l required for sars-cov processing note: yet to be examined against sars-cov- infection blocks sars-cov interaction with ace- note: yet to be examined against sars-cov- infection [n-( , -dioxo- , -dihydroanthracen- yl)benzamide] blocks sars-cov fusion to host cell membrane note: yet to be examined against sars-cov- infection ( ) nct numbers were obtained from https://clinicaltrials.gov/. pulmonary damage. although physical isolation is the ideal way of limiting the spread, in reality this approach is difficult to execute, hence, many pharmacological companies are actively involved in developing small molecule inhibitors to prevent the entry of the virus into human hosts ( , ) . in this regard several nsmis have been investigated to treat sars-cov; but, significant breakthroughs are yet to come for treating sars-cov- ( ) ( table ) . adedeji et al. ( ) reported the discovery and characterization of novel inhibitors to block sars-cov replication via different mechanisms. one mechanism uses screening of small molecule inhibitors using "hiv- pseudotyped with sars-cov surface glycoprotein s (sars-s)" ( , ) . "ssaa e " is a novel small molecule inhibitor, which blocks the interaction of cov sars-s with ace- receptors, thus blocking the viral entry ( ) . another nsmi is "ssaa e " reported to be involved in blocking the cathepsin l, which is required for cov-sars-s processing to mediate viral entry into the host cell ( ) . ssaa e is another nsmi, which can block the fusion of viral membranes with host cell surfaces ( ) (figure ). since the pathological aspects and genomic similarity of sars-cov- virus with sars-cov, the above strategies of inhibition may figure | molecular pathogenesis of sars-cov- in human lung cells. binding of s-protein of sars-cov- to the ace- receptors triggers the processing of ace- through adam- /tnf-α-converting enzyme and induces the "ace- shedding" into the extracellular space and facilitates uptake of sars-cov- followed by the development of sars. alternatively, the entry of sars-cov- by membrane tmprss serine protease'/hat (human airway trypsin-like protease)-mediated cleavage of ace can facilitate sars-cov s-glycoprotein-mediated virus entry. even though, several nsmis targeting these processes were described and their mode of action against coronavirus were delineated, their efficacy against sars-cov- is yet to be tested. be considered for developing potent pharmacological agents to prevent sars-cov- infections ( ) . however, the prospective research should address the efficacy of these inhibitors against sars-cov- infections. cytokine storm was predominantly reported during sars-cov- infection. targeting cytokine-mediated inflammatory responses induced by sars-cov- is another viable approach for mitigating the complications of viral infection. in this regard, chang et al. ( ) , documented the inflammatory cascades mediated through intracellular signaling pathways conferred by the sars-cov in both lung epithelial cells and fibroblasts. authors of this study have reported that s-protein of sars-cov efficiently mediate the il- release in the infected lung cells by activating mapkinases, and activator protein- (ap- ) without intervention of nf-kb cascade ( ) . this study suggested a promising lead for novel rational drug design through the identification of a "specific sequence motif of sprotein functional domain, " which is responsible for inducing il- -mediated inflammatory response in lungs ( ) . baricitinib is a pharmacological agent, which was reported to block the sars-cov- viral entry and inflammation through the inhibition of ap -associated protein kinase (aak ), cyclin g-associated kinase, and janus kinase- and ( ) . chloroquine (cq) and hydroxychloroquine (hcq) were reported to be effective in mitigating the coronaviral load ( , ) . cq and hcq not only inhibit the entry of sars-cov- but also change the ph of acidic intracellular organelles such as endosomes and lysosomes thereby preventing membrane fusion reactions. however, many contradictions and queries prevail pertaining to the use of hcq for the treatment of covid- . at the time of the submission of this review, results of many clinical trials are yet to be announced, hence, the efficacy of hcq for inhibiting sars-cov- infection is still a possibility. prospective studies should focus on testing the fda approved inhibitors of "abl- kinases, " "pi k/akt/mtor" signaling, and "mapkinase" pathways against sars cov- . since these pathways are involved in cell survival, inflammatory cytokines production, and proliferation of cells, targeted downregulation of these pathways is likely to mitigate the exacerbations induced by coronavirus. in this direction, many of these inhibitors are currently being tested against sars-cov- ( table ) ( ) ( ) ( ) . for instance, sorafenib, which inhibits raf, is being experimented in preclinical models and early clinical trials ( ) . likewise, the efficacy of il- receptor antagonists and tnf-α receptor antagonists for blocking the rat coronavirus-mediated chemokine production was already proven effective in animal models ( , , ) . further studies testing the safety and efficacy are warranted before considering these inhibitory agents for treating individuals infected with sars-cov- ( ). however, the concept of "one drug to treat all" should be followed to combat several devastating viral infections ( , ) . for instance, the ebola, marburg, and sars-cov- are undoubtedly devastating viral pathogens, which can induce high mortality as they transmit rapidly via air and body fluids ( ) . outbreaks of these viruses occur sporadically and currently there are no clinically approved nsmis available to combat these viruses. a recent report by taylor et al. ( ) demonstrated the efficacy of a synthetic adenosine analog, bcx in blocking a broad spectrum of viral species viz., "coronaviruses, paramyxoviruses, and bunyaviruses" as these viruses could induce sars, measles, and mumps. bcx could efficiently block both ebola and marburg viral titers in non-human primate models by targeting viral rna polymerase ( , ) . hence, this molecule should be tested for further studies against sars-cov infections in humans. targeting the membrane protease involved in viral s-protein processing and the viral entry into host cells is another approach in mitigating sars-cov- . the host cellular proteases viz., "trypsin, ", "miniplasmin, " "human airway trypsin like protease, " "tryptase clara, " and "tmprss " could cleave the ha glycoprotein located in influenza a virus and thereby promote viral entry into lung cells ( ) . the usage of serine protease inhibitors such as camostat and aprotinin significantly blocked the replication of influenza virus in epithelial cells of lungs and bronchioles ( ) . in addition, these nsmis could block the release of inflammatory mediators such as cytokines, il- and tnf-α, during this infection ( ) . tmprss is a key protein involved in the pathogenesis of several seasonal viral infections including influenza, h n , h n , and h n ( ) ( ) ( ) ( ) . tmprss cleaves the s-protein of coronavirus to produce unlocked, fusion-catalyzing viral forms and binds to the host cell surface thereby enhancing rapid viral entry ( , , ( ) ( ) ( ) ( ) ( ) . both sars-cov and mers-cov could rapidly enter into the host cells as tmprss can facilitate viral binding to the cell surface ( , , , , , ) . tmprss also plays a vital role in the immuno-pathology of coronavirus infections including sars-cov- across lungs by inducing lung fibrosis ( ) . hence, the emerging research should promote the development of nsmis to target these proteases thereby hindering the entry of sars-cov- into host cells. a proof-of-concept study by iwata-yoshikawa et al. ( ) reported that sars-cov failed to replicate in the bronchioles and lungs of tmprss knockout mice. authors of this study reported elevated expression of tlr -mrna expression in the lungs of "sars-cov-inoculated tmprss -deficient mice" and showed enhanced tlr- mediated localization of dsrna into endosomes ( ) . in this study, tmprss knockout has resulted in downregulation of inflammatory cytokines and chemokine expression, which are involved in the bronchiolitis obliterans organizing pneumonia (boop), sars, and pulmonary fibrosis in sars-cov infection ( , , ) . the intricate sars-cov- pathogenesis is similar to that of sars-cov. studies have reported the efficacy of ifns to block sars-cov in cell line models but not against sars-cov- . among ifnα/ -β/ and -γ, the ifn-β was reported to be the most potent blocker of sars-cov growth ( , ( ) ( ) ( ) ( ) . furthermore, ifn-β and-γ have a synergistic effect in blocking sars-cov viral replication ( , ) . however, the effect of this combination against sars-cov- is not yet reported. therefore, future studies should focus on determining the efficacy of ifnα/ -β/ and -γ against sars-cov- infections. unlike small molecule inhibitors, sirnas are specific and can be designed to mitigate sars-cov associated structural proteins by targeting orf ( , ), orf ( , ), orf a ( , , ) , and orf a ( , ( ) ( ) ( ) . for example, sirnas sisc and sisc have shown success in cultured cells as well as in preclinical mouse models in inhibiting the sars infection without causing toxicity ( ) . several other reports have also recently demonstrated the efficacy of sirnas to inhibit the expression of sars-cov genes coding for cl protease in cell line models ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the activity of sars-cov cl protease is essential for viral replication as this protein is involved in the processing of viral proteins ( ) . selective optimization and screening of hexa-chlorophene analogs can be "active cl protease inhibitors" during a sars-cov infection ( ) . hence, the pharmacological agents/sirnas targeting these pathways may likely produce effective clinical outcomes in sars-cov- infections. however, clinical studies should test the utility of these agents/sirna in reducing the burden of infections caused by sars-cov- ( ). the genome of coronaviruses is reported to be significantly involved in coding both structural proteins, and non-structural proteins (nsp's) for the effective viral replication ( ) . the nsp's (nsp c and nsp ) are required for novice cov viral particle formation through viral orf ab polyprotein processing ( ) . several nsmis were reported to target these non-structural proteins in coronavirus infections to treat sars ( ) . for instance, grl , a bendioxolane derivative, could target papain-like proteinases like nsp ( , , , ) , whereas choloropyridinyl indolecarboxylate targets nsp ( , ( ) ( ) ( ) and a "combination of zinc derivatives with pyrithione" targets nsp ( , ) ; ranitidine bismuth citrate targets nsp ( ) ( ) ( ) ( ) ( ) . monoclonal antibodies; cr ( ), mab- ( ), mdef- ( ) , ampligen ( ), polyiclc ( , ) , stinging nettle lectin ( ) , and tapi- (a tace-inhibitor) ( ) are anticoronaviral agents tested in vivo models of sars. for instance, a study showed that amiodarone (a known anti-arrhythmic agent) effectively targets coronaviral spreading in in vitro models ( ) . working in a similar fashion, / humanized antibodies can neutralize coronaviruses thereby reduce the complications caused by viral infections ( ) . however, the above nsmis should be tested against sars-cov- viral associated proteins and against the activity of nsp's to derive an effective therapeutic intervention. prospective research must focus on the development of novel "helicase inhibitors, viral attachment inhibitors, and activity of rhesus θ -defensin" that block sars-cov- infection using in vitro, in vivo, and clinical studies ( ) . hence, the development of nsmis to target the synthesis of nsp's in sars-cov- may deliver cellular antiviral responses by blocking their replication in host cells ( , , ) . repurposing existing drugs is another strategy widely under consideration to target key proteins involved in the sars-cov- infection. in this regard, the existing nsmis viz., antivirals (umefenovir, remdesivir, nitazoxanide, favipiravir, ritonavir, lopinavir, ifns), anticytokines, antimalaria drugs (chloroquine, hydroxychloroquine), and passive antibody therapies are currently being evaluated to improve clinical outcomes in sars-cov- infected patients ( , , , , ) . however, these agents require additional experimental and clinical validations before being tested in sars-cov- infections. for example, hydroxychloroquine (anti-malarial drug) and the tocilizumab (immunosuppressive drug) are preferred currently to mitigate viral entry and cytokine production in the sars-cov- infection. these drugs are being tested in ongoing trails in china and italy ( , ) . priming the spike (s)-protein of coronavirus by host cells using membrane proteases is a necessary process for viral entry and replication, which further determines zoonotic potential of coronaviruses ( ) . a recent report by markus hoffmann et al. ( ) investigated the protease dependence of sars-cov- for its entry into cells. for example, sars-cov- uses the tmprss protease for its priming ( ) . inhibition of tmprss using camostat mesylate retarded the viral entry into caco- cells ( ). camostat mesylate could be recommended as an nsmi for human clinical trials to combat the sars-cov- virus ( ). this report delineated the ability of neutralizing antibody responses against s-protein to block the sars-cov- entry into host cells ( ) . the serum antibody responses raised to combat the "sars-s protein/ace- interface" during the sars-cov- infection indicates that the vaccination strategy may be an effective therapeutic modality against the covid- infection ( ). ace- catalytic efficacy is significantly higher than ace for angiotensin-ii ( ) . several compounds, such as mln- , were screened according to structure-based/substrate-based studies through virtual screening for inhibiting ace- activity ( ) ( ) ( ) ( ) . ace- is predominantly expressed in lungs, brain, heart, blood vessels, and renal organs ( , ) . ace- is essential for cardiovascular homeostasis, and cns homeostasis as ace- confer redox homeostasis by mitigating ang-ii-induced oxidative stress ( ) . however, in covid- , ace- acts as receptor on human respiratory epithelial cells for sars-cov- binding ( ). a recent report by markus hoffmann et al. ( ) provided evidence that the sars-cov- strain use its spike (s)-protein to bind to ace- . authors of this paper have also demonstrated the efficiency of tmprss in sars-cov- viral strain priming in host cells ( ) . therefore, targeting ace- could be a viable strategy to prevent the entry of sars-cov- into the human system. however, a recent report by guan et al. ( ) cautioned that the administration of ace inhibitors significantly induced adverse clinical outcomes in covid- patients due to severe hypertension, coronary artery disease, and chronic renal failure; hence, further use of ace inhibitors to treat covid- infections was halted ( ) ( ) ( ) . in another report diaz ( ) hypothesized that covid- patients receiving i.v. infusions of aceis and arbs (at -receptor blockers) are at a higher risk of attaining severe disease pathogenesis. hence, they supported the development of nsmis such as "tmprss inhibitors to treat sars-cov- infections ( )". the failure of disease management and lack of selective therapies could be due to the intricate covid- pathogenesis induced by the sars-cov- infection. hence, the early recognition of disease is essential for effective management of covid- ( ) . although, several reports delineated the efficacy of certain nsmis viz., ribavirin, promazine, and imp dehydrogenase inhibitors to inhibit in vivo models of sars-cov replication, later, they were proven ineffective ( , ( ) ( ) ( ) . a report by reghunathan et al. ( ) showed that the immune response produced against sars-cov may be different from other viral infections as indicated by the lack of upregulation in mhc-i genes, cytokines, and ifns or complementmediated cytolysis in peripheral blood mononuclear cells (pbmcs). the failure in the development of a vaccine is due to antibody-dependent enhancement and th- immunopathology ( ) ( ) ( ) . pegylated ifn-α inhibits viral replication of sars-cov and offers protection against type i pneumocytes in lungs ( ) . a significant reason for the failure or lack of selective therapies against sars-cov- -induced sars is the intricate immune system mediated pathophysiology ( ) . other reports by law et al., also detailed similar mechanisms ( , ) . sars-cov can evade host ifn-mediated viral growth inhibition by activating ifn-regulatory factor ( ) . furthermore, sars-cov could induce apoptosis in lymphocytes in vitro using "orf a, orf a, and orf b, e protein, and n protein" ( ) ( ) ( ) ( ) . for instance, the sars-cov can evade immunity as indicated by the decline in cd and cd t cells ( ) . therefore, it is necessary to uncover the complement-based cytolysis in human patients in response to the sars-cov- strain as this virus executes unusual mechanisms to evade the human immune system consequently inducing pathogenesis and mortality. the prospective research should focus on this viral-mediated immune signaling with respect to sars-cov for developing effective nsmis. intravenous (iv) hyperimmune globulin therapy is one of the immunotherapies known to downmodulate pro-inflammatory cytokines and mitigate the severity of infection in covid- patients. iv infusion of immunoglobulins composed of a high dose of antibodies, which can bind to a number of inhibitory receptors viz., fc gamma receptor iib (fcγriib) ( , ) and fcγriic ( ) and confer anti-inflammatory responses against sars-cov- (completed clinical trials: hyperimmune plasma nct ). oxidative stress is significantly induced by several viral infections inside the lungs through the downregulation of redox regulator nuclear factor-erythroid related factor (nrf- ) ( ). nrf- is a leucine-zipper transcription factor ( ) expressed predominantly in nasal epithelium, epithelial cells of lungs, and alveolar macrophages ( , ) . disruption of nrf- and keap interaction triggers the activation of the anti-oxidant defense mechanism ( ) . for instance, nrf- activation offers protection against inflammation and lung injury induced by influenza viral infections and respiratory syncytial virus (rsv) through the anti-oxidant defense pathway ( ) . several viral proteins in the host cells can foster optimum levels of ros-mediated oxidative stress to facilitate viral metabolism and the viral replication cycle without killing host cells ( , ( ) ( ) ( ) ( ) . recent seminal studies described the active role of viruses in inhibiting the nrf- pathway ( ) ( ) ( ) . for instance, the positive regulation of nrf- in modulating the thiol redox system and oxidative stress for the survival of infected astrocytes was observed in moloney murine leukemia virus and hiv virus ( ) . the hcv virus could induce the downregulation of nrf- dependent nqo , gclc, and gpx and modulate oxidative stress ( , ) . an rsv infection mediates proteasomal degradation, deacetylation, and sumoylation of nrf- consequently causing the downregulation of nqo , cat, and sod gene expression ( ) . hence, nrf- activators are potential anti-viral agents, which can be tested against the sars-cov- infection ( ) . future research is highly imperative in unraveling the underlying activity of nrf- for emerging sars-cov- survival by analyzing nrf target genes nqo , gclc, and gpx. in addition, the sars-cov- mediated expression of serine and cysteine proteases in different cell lines should be investigated in relation to nrf- activation, which is a beneficial strategy to combating sars-cov- pathogenesis. however, in the case of certain viral infections, it is imperative to develop nrf- inhibitors to protect the host cells ( ) . for instance, the marburg virus (a causative agent for lethal hemorrhagic fever) can modulate oxidative stress by activating nrf- dependent signaling through the blockade of "vp- viral protein" binding to keap ( ) . therefore, vp- dependent nrf activation can mediate the upregulation of genes ho (heme oxygenase)- , nqo , and gclm ( ) . in the case of dengue virus, the viral particles could induce er stress and activate nrf- signaling, which then lead to tnf-α secretion ( ) . in this scenario, it is crucial to uncover any underlying mechanisms of emerging sars-cov- survival through the modulation of oxidative stress via nrf- signaling in different cells of different organs including lungs ( ) . the prospective lindera erythrocarpa makino ( ) celastrol (quinone methide triterpene) inhibits tat-induced hiv- infection tripterygium wilfordii ( ) bakuchiol (phenolic isoprenoid) psoralea corylifolia l. ( ) rupestonic acid (sesquiterpene) artemisia rupestris l. (continued) frontiers in immunology | www.frontiersin.org research studies should focus on the development of nrf- modulators against sars-cov- . natural products were proven to offer protection against virusinduced oxidative stress by modulating anti-oxidant defense pathways ( , ) . for instance, the administration of egcg has mitigated viral replication of "influenza a/bangkok/ / infection" by activating nrf- to attenuate virus-induced oxidative stress, inflammation, and apoptosis in lung cells ( ) . similarly, the cytoprotective and antioxidant efficacy of nrf- was reported against pr influenza-a viral infection in at-i and at-ii cells ( ) . prospective research should focus on testing the efficacy of several natural products to block sars-cov- viral replication by ascertaining nrf- mediated antioxidant responses. studies have also shown the activation of host cellular transmembrane proteases (for example, serine proteases, cysteine proteases), which can further foster a prompt viral entry and viral replication in host cells by reducing nrf- expression ( ). decline in proteolysis of the above proteases can actuate the propagation of several human viruses viz., influenza, hiv, nipah, ebola, and coronaviruses (sars-cov, mers-cov, sars-cov- ) ( , , ( ) ( ) ( ) . in this scenario, similar to influenza-a virus ( ) , it is highly important to unravel the influence of nrf- expression on tmprss , and human airway trypsin-like protease during sars-cov- -mediated inflammatory conditions and oxidative stress in lungs. the downregulation of the nrf- gene is correlated to serine protease activity and consequent influenza viral entry ( ) . recent studies have demonstrated the efficacy of natural nrf- activators viz., egcg and sulforaphane (sfn) for blocking viral entry/viral replication as well as promoting antiviral mediators rig-i, ifn-β, and mxa ( ) . in this context, it is essential to demonstrate the effects of nutritional interventions like sfn and egcg against sars-cov- induced oxidative stress by modulating nrf- signaling. evidence has demonstrated the use of naturally occurring nrf activators for mitigating viral infections/post-viral infection induced complications. for example, α-luminol is a natural nrf- activator, which confers the protection of astrocytes against the momul virus ( ) . egcg enhances nuclear nrf- levels during tat-induced hiv- infection and offers protection against virus induced oxidative stress ( ) . tanshinone ii a can induce upregulation of nrf- expression and mitigates ros production during tat-induced hiv- infection via modulating ampk/nampt/sirt signaling in host cells ( ) . sfn enhances the phagocytic function of "hiv-infected alveolar macrophages in lungs" by activating nrf- signaling, which further induces downstream antioxidant cascades ( ) . lucidone is effective for the nrf- mediated blockade of dengue virus by inducing heme oxygenase- ( ) ; rographolide could induce nrf- induced antioxidant defenses against influenza a in lung cells ( ) ; celastrol can mediate nrf- induced antioxidant defenses against hiv- tat-induced inflammation ( ) . broccoli sprouts containing sfn acts as a nrf- activator to reduce influenza-induced infection in lung cells ( ) . bakuchiol and rupestonic acid are phytoconstituents that confer nrf- activation thereby promoting nqo gene expression and ho- -mediated interferon activity to enhance antioxidant response against influenza virus in lung cells ( , ) . curcumin is another significant compound that can modulate nrf- signaling and enhance the generation of ifn-β to offer protection against the influenza virus ( ) . curcumin can mitigate this viral infection by modulating tlr / , p /jnk mapk, and nf-κb pathways ( ) . however, studies are required to decipher the activity of these phytochemicals against sars-cov- ( table ) . a recent report by drȃgoi ( ) hypothesized that the potent natural nrf- activators viz., resveratrol, sfn, curcumin, and asea redox should be evaluated in different combinations with conventional drugs against sars-cov- infection in both in vitro and in vivo models and to further deduce a correlation between nrf- activity and sars-cov- viral entry/replication. sars-cov- is progressively inducing a high mortality rate across the globe due to the lack of selective therapeutic interventions or vaccination. recent reports by lu et al. ( ) and xu et al. ( ) delineated that the s-protein of sars-cov- and sars co-v exhibit similar -d pharmacophore in the receptor binding domain (rbd) of ace- of human cells. covid- patients are characterized by the severe viral pathogenesis due to extensive cytokine storm viz., tnf-α, il- β, il- , ifnγ, and mcp- in infected lung tissues ( ) . a report by chen and du ( ) hypothesized that the phyto-constituents such as "baicalin, scutellarin, hesperetin, nicotianamine, and glycyrrhizin" may deliver anti-sars-cov- effects. hesperetin glycoside abundant in citrus fruits, which can inhibit the sars-cov clpro ( ) . the activity of this molecule must be examined against serine/cysteine proteases, which support sars-cov- viral entry/replication. traditional citrus flavonoids were frontiers in immunology | www.frontiersin.org reported to have a potential to act against sars-cov- as studied by molecular docking studies. molecular docking simulations, lc-ms studies described the efficacy of citrus flavonoids (ex. naringenin) in binding to ace- , and mitigating inflammationinduced lung injury by the sars-cov- virus ( ) . further studies should evaluate these compounds in preclinical models to determine the safety and efficacy against the sars-cov- infection. natural products such as di/tri-terpenoids, lignoids were proven to inhibit the viral replication of coronaviruses in vitro ( ) ; griffithsin could block coronaviral entry by binding to the sars-cov spike glycoprotein ( ) . tsl- can block coronaviral entry/replication; leaf extracts of toona sinesis roem effectively blocked sars-cov replication ( ) . betulinic acid, savinin can act as competitive inhibitors against sars-cov cl protease to block viral entry ( ) . the research gap must be filled to develop nutritional therapeutic interventions by investigating the efficacy of these phytochemicals against sars-cov- viral entry. seeds of psorelia corylifolia exhibit inhibitory effects against the sars-cov papain-like protease required for coronavirus entry/replication. the efficacy of these molecules should be examined against sars-cov- ( ) ( table ). the active site pockets of main proteases such as lu and gtb in sars-cov- are reported to be involved in conferring viral entry/fusion; hence, these sites should be considered as the potential drug targets against sars-cov- ( ) . a molecular docking study by khaerunnisa et al. ( ) reported the predicted efficacy of bioactive compounds against above sars-cov- main protease (mpro) sites viz., "nelfinavir, lopinavir, kaempferol, quercetin, luteolin- glucoside, demethoxycurcumin, naringenin, apigenin- -glucoside, oleuropein, curcumin, catechin, epicatechin-gallate, zingerol, gingerol, and allicin" ( table ) . the life-threatening consequences of the covid- pandemic remain high due to lack of selective targeted therapies and vaccination strategies. this is primarily due to extreme genomic variability of rna viruses as well as variations in the host-cell invading mechanisms. hence, this review benefits virologists, medical scientists, and cell biologists to ascertain and develop nsmis, nrf- modulators, and clinically viable vaccines to combat this devastating sars-cov- strain. however, many more preclinical and clinical studies are required to uncover the therapeutic efficacy of potential phytochemicals, natural nrf modulators, and several nsmis against the 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influenza etc.) based on their extrapolated cytoprotective antioxidant effects (especially on vital organs), including the cytoprotection offered by ars on the cardiac muscle of dmd patients which can be extrapolated to the lungs -a very short medical communication genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding anti-sars coronavirus c-like protease effects of isatis indigotica root and plant-derived phenolic compounds citrus fruits are rich in flavonoids for immunoregulation and potential targeting ace specific plant terpenoids and lignoids possess potent antiviral activities against severe acute respiratory syndrome coronavirus broad-spectrum in vitro activity and in vivo efficacy of the antiviral protein griffithsin against emerging viruses of the family coronaviridae phenolic phytochemical displaying sars-cov papain-like protease inhibition from the seeds of psoralea corylifolia baicalin, a metabolite of baicalein with antiviral activity against dengue virus dual effect of glucuronidation of a pyrogallol-type phytophenol antioxidant: a comparison between scutellarein and scutellarin co-occurrence of nicotianamine and avenic acids in avena sativa and oryza sativa azetidine- -carboxylic acid derivatives from seeds of fagus silvatica l. and a revised structure for nicotianamine betulin and betulinic acid: triterpenoids derivatives with a powerful biological potential the griffithsin dimer is required for high-potency inhibition of hiv- : evidence for manipulation of the structure of gp as part of the griffithsin dimer mechanism research & consulting llc.the remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © beeraka, sadhu, madhunapantula, rao pragada, svistunov, nikolenko, mikhaleva and aliev. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - ttrx lf authors: cunha, lucas leite; perazzio, sandro felix; azzi, jamil; cravedi, paolo; riella, leonardo vidal title: remodeling of the immune response with aging: immunosenescence and its potential impact on covid- immune response date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: ttrx lf elderly individuals are the most susceptible to an aggressive form of coronavirus disease (covid- ), caused by sars-cov- . the remodeling of immune response that is observed among the elderly could explain, at least in part, the age gradient in lethality of covid- . in this review, we will discuss the phenomenon of immunosenescence, which entails changes that occur in both innate and adaptive immunity with aging. furthermore, we will discuss inflamm-aging, a low-grade inflammatory state triggered by continuous antigenic stimulation, which may ultimately increase all-cause mortality. in general, the elderly are less capable of responding to neo-antigens, because of lower naïve t cell frequency. furthermore, they have an expansion of memory t cells with a shrinkage of the t cell diversity repertoire. when infected by sars-cov- , young people present with a milder disease as they frequently clear the virus through an efficient adaptive immune response. indeed, antibody-secreting cells and follicular helper t cells are thought to be effectively activated in young patients that present a favorable prognosis. in contrast, the elderly are more prone to an uncontrolled activation of innate immune response that leads to cytokine release syndrome and tissue damage. the failure to trigger an effective adaptive immune response in combination with a higher pro-inflammatory tonus may explain why the elderly do not appropriately control viral replication and the potential clinical consequences triggered by a cytokine storm, endothelial injury, and disseminated organ injury. enhancing the efficacy of the adaptive immune response may be an important issue both for infection resolution as well as for the appropriate generation of immunity upon vaccination, while inhibiting inflamm-aging will likely emerge as a potential complementary therapeutic approach in the management of patients with severe covid- . elderly individuals are the most susceptible to an aggressive form of coronavirus disease , caused by sars-cov- . the remodeling of immune response that is observed among the elderly could explain, at least in part, the age gradient in lethality of in this review, we will discuss the phenomenon of immunosenescence, which entails changes that occur in both innate and adaptive immunity with aging. furthermore, we will discuss inflamm-aging, a low-grade inflammatory state triggered by continuous antigenic stimulation, which may ultimately increase all-cause mortality. in general, the elderly are less capable of responding to neo-antigens, because of lower naïve t cell frequency. furthermore, they have an expansion of memory t cells with a shrinkage of the t cell diversity repertoire. when infected by sars-cov- , young people present with a milder disease as they frequently clear the virus through an efficient adaptive immune response. indeed, antibody-secreting cells and follicular helper t cells are thought to be effectively activated in young patients that present a favorable prognosis. in contrast, the elderly are more prone to an uncontrolled activation of innate immune response that leads to cytokine release syndrome and tissue damage. the failure to trigger an effective adaptive immune response in combination with a higher pro-inflammatory tonus may explain why the elderly do not appropriately control viral replication and the potential clinical consequences triggered by a cytokine storm, endothelial injury, and disseminated organ injury. enhancing the efficacy of the adaptive immune response may be an important issue both for infection resolution as well as for the appropriate generation of immunity upon vaccination, while inhibiting inflamm-aging will likely emerge as a potential complementary therapeutic approach in the management of patients with severe covid- . in december , a novel coronavirus, severe acute respiratory syndrome coronavirus- (sars-cov- ), was discovered as the causative agent of an outbreak of viral lower-respiratory tract infections centered in wuhan (china) ( ) . since then, sars-cov- has caused a widespread outbreak of severe acute respiratory syndrome throughout china, with exported cases occurring in other continents, including the united states, in a worldwide pandemic ( ) . interestingly, a strong age gradient in the risk of death was observed among patients with coronavirus disease (covid- ) ( ) . in this scenario, the remodeling of immune response that is observed among the elderly could be a possible explanation for the higher lethality of covid- noted on this population. the immune response is dynamically remodeled with aging, a phenomenon denominated as immunosenescence. this phenomenon increases susceptibility to a myriad of clinical conditions such as infections, autoimmune disorders, and malignancies. recent data had shed light on the physiological aspects of immunosenescence, which is now considered an immune adaptation to the aged microenvironment rather than merely a collapse of the system ( ). both the innate and adaptive immunity is affected by aging. some individuals experience a sustained innate immune system activation, inducing proinflammatory cytokines secretion and innate immune cells' recruitment ( ). innate immunity hyperactivation may be detrimental and impair global functionality, causing a clinical phenotype known as frailty syndrome. frailty syndrome is defined as a state of cumulative decline in several physiological systems with a disproportionate vulnerability to stressor events ( ) . frailty syndrome prevalence increases with age, it is multifactorial in etiology, and the physical component of frailty can be objectively assessed by the fried frailty score (phenotype score) and the frailty index (deficit accumulation index) ( ) . likewise, adaptive immunity remarkably changes as age increases, which can be summarized into two main topics: ( ) bone marrow reorganization and hematopoietic stem cell pool differentiation into myeloid lineage, outnumbering lymphoid compartment; and ( ) physiological thymic involution, compromising naïve t cells generation. the sum of these two factors can help explain the prior known impairment of the regenerative capacity of lymphocytes compared to myeloidderived cells in the elderly ( ) . infectious diseases are more prevalent among the elderly. when compared to younger counterparts, the elderly more frequently present with respiratory and urinary tract infections, and those patients usually have a worse prognosis ( , ) . it is possible that the impaired barrier function of mucosae and diminished adaptive immune response (both cellular and humoral) are the reasons for the increased susceptibility to infectious microorganisms among the elderly ( ) . in addition, the natural killer (nk) cell senescence may affect the homeostasis of the immune system in the elderly, leading to an increased risk of cancer and additional risk of viral infections ( ) . lastly, age-related cell dysfunctions leading to an exhausted phenotype are also an important characteristic of the immune system remodeling with aging, which might accelerate tissue damage and disable modulatory mechanisms ( ) . herein, we review the state of the art research on senescence-induced immune dysregulation, focusing on innate and adaptive cell functional analysis and its potential impact in viral immune responses, such as in covid- . currently, the concept of immunosenescence refers to a comprehensive remodeling of the immune system and its microenvironment, involving both innate and adaptive compartments that occur with aging ( , ) . many physiological phenomena have been proposed to explain the immune response remodeling over time, including chronic exposure to antigens, impaired telomerase activity, mitochondrial dysfunction, defective autophagy, endoplasmic reticulum stress, defective ubiquitin/proteasome system, and age-related changes in the composition of gut microbiota ( ) ( ) ( ) ( ) . probably, a melting pot of diverse factors differently contributes to the final phenotype of the adapted and experienced immune system, named immunosenescence. aging of the immune system is characterized by an imbalance between stimulatory and regulatory mediators, such as cytokines and acute phase reactants, toward a sub-clinical chronic proinflammatory state called inflamm-aging. inflamm-aging is thought to be caused by a low-grade inflammation secondary to continuous antigenic stimulation ( ) , whose source may be exogenous, like a pathogenic microorganism infection ( , ) , or endogenous ( ) ( ) ( ) ( ) , like post-translational-modified macromolecules ( ) . population studies incorporate the notion that the immune response depends on environmental exposure and how it interacts with endogenous variables. in fact, diet, exercise, xenobiotic exposure, and other environmental factors may epigenetically affect the metabolic health of immune cells ( ) . lifestyle factors, such as exercise and favorable dietary habits, positively affect the immune system ( ) , while poor nutrition and reduced muscle mass may predispose an individual to a proinflammatory condition ( ) . age-related remodeling of innate immunity modifies the homeostasis of nk cells, neutrophils, and monocytes/macrophages ( ) . nk cells from the elderly exhibit impaired perforin release upon stimulation and granule exocytosis ( , ) . it reduces the elimination of senescent cells, which, in turn, promotes senescent cell accumulation in aged tissue. moreover, aging reduces the frequency of circulating nk p + cells, a modulatory cell subset involved in the resolution of inflammation and elimination of effector cells ( , ) . neutrophils and macrophages are classically classified as part of innate immunity and possibly comprise the most important effector cells against bacterial infections. it is thought that age is accompanied by a decline in production and secretion of most chemokines, including those responsible for neutrophil and monocyte chemoattraction ( ) . the absolute number of neutrophils seems to be maintained while the number of monocytes increase with age ( , ) . however, the function of these cells may be impaired among the elderly ( ) . the final consequence is that the delayed resolution of inflammation may be associated with age-related remodeling of neutrophils and macrophages ( ) . in addition to their phagocytosis' capabilities, neutrophils are capable of releasing a mesh-like structure under specific circumstances, called neutrophil extracellular traps (net), in an attempt to physically delimitate the pathogenic agent, mainly microorganisms, and facilitate its contact with microbicidal peptides and enzymes ( ) . net is composed of a decondensed chromatin meshwork imbedded with granule proteins with antimicrobial properties. net may also work as a physical path for immune cell migration to the inflammatory site ( ) . neutrophil function is impaired in both animal models and humans with aging. hazeldine et al. ( ) observed that older adults have less il- production, lps-induced net release, and cell migration compared to younger counterparts, probably secondary to an impaired signal transduction. microbicidal killing, phagocytic activity ( ) , and degranulation capacity ( ) of neutrophils are also reduced in the elderly. in addition, the same group investigated the migration pattern of neutrophils obtained from older compared to young adults. they observed that neutrophils from older subjects migrated with less accuracy than those from younger subjects. by inaccurately meandering among healthy tissues, neutrophils from the elderly inadvertently release more neutrophil proteinase that may contribute to tissue damage and systemic inflammation. reactive oxygen species (ros) are free radicals produced after oxidative bursts in phagosomes, which are pivotal for the microbicidal function of phagocytes ( ) . in fact, ros do not just directly contribute to the bacterial clearance, but additionally can trigger net formation. the free radical ros production by neutrophils in older adults is decreased ( , ) . interestingly, polymorphonuclear leucocytes from the elderly are less capable of modulating the triggering receptor expressed on myeloid cell- (trem- )-induced oxidative bursts, suggesting that trem- signal transduction altered with aging may be one of the mediators of the decrease in microbicidal potential of innate immune cells in older adults ( ) . animal models of premature immunosenescence have also shed some light into age-related remodeling of the immune system. guayerbas et al. ( ) described a mouse model of premature immunosenescence based on the demonstration of early decline of immune parameters and behavioral tests in swiss outbred mice. mouse model-derived peritoneal leukocytes exhibited reduced proliferative response, impaired nk activity, and increased in vitro tnf-alpha production compared to control mice ( ) . in addition, mouse modelderived macrophages of premature models were less functional with a striking loss of microbicidal activity ( ) . the mice model of premature immunosenescence was refined and new models were developed as well ( , ) . apparently, the key phenomenon are the oxidative and inflammatory stresses, which, not without reason, are associated with several non-communicable chronic diseases prevalent among the elderly ( , ) . in fact, spleen and thymus cells from prematurely immunosenescent mice models have decreased antioxidant defenses and significantly increased oxidants and pro-inflammatory cytokines production ( ) ( ) ( ) . interestingly, the antioxidant vs. oxidant imbalance observed in prematurely immunosenescent mice was similar to the one observed in old wild-type animals ( , ) . hence, lab tests determining the oxidative burst profile of phagocytes (e.g., nitro blue tetrazolium test, dihydrorhodamine oxidation, o − and h o − production by chemoluminescence, etc.) may be useful for assessing inflammaging features ( ) . the state of chronic inflammation has to be counter-balanced by anti-inflammatory molecules ( ) . when not under control, the low-grade inflammation loses its defense role and turns into a damaging state to the whole organism ( ) . the practical consequence is that inflamm-aging is deleterious to human health, predicts frailty, and is associated with higher mortality rates ( ) ( ) ( ) . remodeling of the adaptive immune response also occurs with aging. thymic involution and hematopoietic stem cell insufficiency play important roles in immunosenescence of adaptive immunity ( ) . in general, elderly individuals are less able to respond to neo-antigens, due to the reduction of new thymus-emergent t cells, though homeostatic proliferation can partially sustain the richness of the tcr repertoire ( , ) . moreover, peripheral t cells usually present a reduced absolute number in aged individuals with an inverted cd :cd ratio and expansion of terminally differentiated effector memory t cells ( , ) , associated with impaired proliferation ability, telomerase activity, and intracellular signaling ( , ) . furthermore, most adult regulatory t lymphocytes are a terminally differentiated highly suppressive apoptosis-prone population with a limited capacity for self-renewal ( ). this finding might explain, at least in part, the occurrence of agerelated autoimmune conditions. in addition, the imbalance between innate and adaptive immunity may disturb the fine regulation of the effector immune response, leading to a severe acute pro-inflammatory state that may lead to organ rejection in transplanted patients ( , ) . while naïve t and b cells become dysfunctional with aging, memory t and b cells' function is relatively maintained ( ) ( ) ( ) . in fact, naïve t lymphocytes obtained from the elderly present impaired cell binding of the immune synapse ( ), reduced signal transduction ( ), dysregulation of cytoskeletal function ( ), defective protein glycosylation and activation ( ) , and insufficient il- production ( ). some authors advocate that age-related t-cell dysfunction is different from t cell exhaustion, a state of low cell responsiveness mediated by chronic conditions, such as viral infections and malignancies (figure ) ( ) . constant antigen stimulation figure | t cell exhaustion vs. t cell senescence. in conceptual terms, the t-cell dysfunction observed in the elderly is different to the one reported as t-cell exhaustion. persistent viral and cancer stimulation leads to the remodeling of many t cells, which upregulate the expression of co-inhibitory receptors (e.g., pd- , ctla- , lag- , icos, tim- , and klrg- ), all of them hallmarks of t cell exhaustion. the co-inhibitory receptors downregulate the tcr-stimulated intracellular signal, and t-cells become hyporesponsive and develop responsiveness impairment. however, the immunosenescence is marked by similar levels of pd- and tim- and tiny elevations of ctla- and lag- in t cells from the elderly compared to those in younger groups. t-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (itim) domain (tigit) is a co-inhibitory receptor that is expressed on senescent t cells, which exhibited a marked terminal differentiated phenotype. interestingly, tigit-positive t cells from the elderly seems to retain some proliferative capacity, but produced significantly lower amounts of tnf-alpha, ifn-gamma, and il- . progressively exhausts t cells by gradually upregulating the expression of inhibitory checkpoint receptors (e.g., pd- , ctla- , lag- , icos, tim- , and klrg- ) on cd + t cells ( ) , which, in turn, downmodulate tcr-induced intracellular signaling ( ) . interestingly, despite this conceptual difference between immunosenescence and t cell exhaustion, most of those cell exhaustion surface hallmarks are observed on dysfunctional immunosenescent cells, suggesting that these two phenomena share many mechanisms ( ) . exhausted t-cells accumulate over time ( , ( ) ( ) ( ) ( ) . shimada et al. ( ) demonstrated both gene and protein hyper expression of pd- and ctla- in cells from old male c bl/ mice compared to young controls. most pd- + t cells were quiescent and presented an anergic effector memory phenotype with impaired proliferative response to mitogens ( ) . similarly, lee et al. ( ) reported the accumulation of tim- + murine t cells with impaired proliferative capacity with aging. literature discussing t cell exhaustion and immunosenescence in humans is scarce, though. song et al. ( ) described an elevated number of tigit + cd + t cells from old adults, another hallmark of cell exhaustion apparently associated with immunosuppressant features in neoplasm or chronic infection mouse models ( , ) . tigit + cd + t cells from old individuals seem to retain a proliferative capacity, although they impaired tnf-alpha, ifn-gamma, and il- in vitro production and increased susceptibility to apoptosis ( ) . therefore, we hypothesize that evaluation of the proliferative response to mitogens and in vitro cytokine production may be indirect ways to assess age-related remodeling of the immune system. in regards to b cell compartment, vaccine trials suggest that b cell repertoire abridge over time, foremost observed in frail patients ( , ) . in addition, b cells from the elderly present both impaired antibody production and class switch recombination ( ) . class switch recombination and immunoglobulin somatic hypermutation are crucial for humoral immune response and occur in mature b cells mediated by activation-induced cytidine deaminase, amongst other mediators ( , ) . similarly, activated b cells from old mice have less activation-induced cytidine frontiers in immunology | www.frontiersin.org deaminase expression and reduction of class switched antibodies ( , ) . interestingly, in vivo activated cd + t cells from oldaged individuals showed increased dual-specific phosphatase (dusp ) transcription, which, in turn, negatively correlated with antigen-specific b cells' expansion. silencing of dusp restored cd + t cell-induced b-cell differentiation, suggesting that b cell dysfunction observed with aging is t cell-dependent. table summarizes the main physiologic modifications of the immune system in the elderly. coronaviruses are a large family of viruses that cause upper and lower-respiratory tract illnesses in humans. sars-cov- is transmitted predominantly via respiratory droplets. clinically, patients frequently present with fever, cough, myalgia, and fatigue ( ) . in a subset of patients, mainly elderly individuals, sars-cov- was shown to lead to bilateral pulmonary diffuse alveolar damage that may progress to acute respiratory distress syndrome ( , ) . following the pulmonary phase, patients with poor outcome frequently evolve a life-threatening cytokine storm syndrome, characterized by bursts of proinflammatory cytokines and chemokines in the serum ( , ) . the uncontrolled systemic inflammation causes endothelial injury and activation of coagulation cascade. the consequence is an explosive process of disseminated intravascular coagulation and consumption of coagulation factors that leads to organ damage and death. the innate immune response is the first level of response in the detection and clearance of a viral infection. in sars-cov- , the spike protein (s) mediates the attachment, fusion, and entry of the virus in human cells ( ) . the protein s strongly binds to angiotensin-converting enzyme receptor leading to the attachment of the virus to the host cell ( ) . the successful entry needs the priming of the s protein by tmprss , a human cellular serine protease ( ) . once in the host cell, sars-cov- can be detected by macrophages, which orchestrate the production of a pro-inflammatory microenvironment that inhibits viral replication, stimulates adaptive immunity, and recruits other immune cells to the site of infection. macrophages from elderly lungs may have a more pronounced production of il- and other pro-inflammatory cytokines in response to stimuli ( ) . it is possible that il- has a critical role in the immune response of the elderly that mounts against sars-cov- ( ). il- helps the differentiation of th lymphocytes, but inhibits the production of interferon-γ, which is necessary for the activation of cd + cells ( ) . in addition, il- contributes to a pro-inflammatory microenvironment at the lung that impacts the integrity of the air-blood barrier ( ) . patients with severe covid- have a higher il- /interferon-γ ratio than those who present with a moderate disease, which could be related to the cytokine storm leading to lung injury ( ) ( ) ( ) ( ) . indeed, patients with severe covid- frequently have lower absolute numbers of interferon-γ producing cd + t cells compared to patients with moderate disease ( ) . then, when patients with covid- enter the immune dysregulation phase, the increase in il- leads to a relative immunoparalysis that may impair the clearance of sars-cov- ( ). elderly patients with covid- often present with a severe dysregulation of proinflammatory cytokines, such as il- and il- β, which may result in worse outcome ( ) . drugs that uncouple il- β/il- r signaling (anakinra) or il- /il- r signaling (tocilizumab) may have an immunomodulatory potential and are hypothesized to attenuate the dysfunctional immune response during the hyperinflammatory phase of covid- ( , ) . in fact, some reports suggest that infusion of anakinra ( , ) and tocilizumab ( ) may improve the disease course in patients with severe covid- presentation. neutrophils have traditionally been considered the primary immune cells active in the defense against bacterial infections. more recently, neutrophils' role in viral infection has emerged based on observations of its correlation with viral infection severity and neutrophils' biological ability to recognize viruses (via viral pamps) and respond to them with specific effector functions ( ) . patients with severe covid- more frequently present with a high neutrophil-to-lymphocyte ratio ( ) , in part driven by the relative lymphopenia or lymphocyte exhaustion. in addition, patients with severe covid- are more susceptible to a greater burst of systemic inflammation and secondary bacterial infection that can lead to the increment of neutrophils. it is unclear if changes in neutrophils are only a reflection of the overall immune activation in covid- or if they play a direct pathogenic role. lastly, nk cells are less functional in the elderly, and studies have shown that severe covid- patients have further depleted peripheral nk cell counts in comparison with mild cases and healthy controls ( ) ( ) ( ) . generally, nk cells are capable of recognizing infected cells and of triggering direct cell toxicity. further studies are needed to clarify how sars-cov infected cells interact with nk cells and if any apoptosis or downmodulation occurs and prevents the effective elimination of infected cells. the airway epithelium is a physical barrier to pathogens ( ) . the integrity of the air-blood barrier is essential for the maintenance of lung homeostasis and represents an important branch of innate immunity ( ) . the invasion of the airway epithelial by sars-cov- may break the barrier integrity, triggering a vicious cycle of inflammation and tissue injury that is more pronounced among the elderly ( ) . presumably, the same remodeling process that occurs in the immune system also happens at the lung microenvironment with aging ( ). data from animal models suggest that senescent lungs are more susceptible to settle a pro-inflammatory response when injured ( ) . in fact, bronchoalveolar lavage obtained from elderly patients with acute respiratory distress syndrome present with higher pro-inflammatory cytokine levels when compared to younger counterparts, suggesting that the lung may represent a small fraction of the inflamm-aging that occurs at the systemic level ( ) . this local phenomenon may help to explain why elderly patients with covid- are more susceptible to a more severe lung injury that implies loss of lung function and respiratory failure ( ) . the initial inflammation in covid- is propitious to the activation and differentiation of cd + and cd + t cells. the ideal final output is the development of an effective and specific immune response, involving both the production of anti-sars-cov- antibodies and the deployment of a large number of viral-specific cytotoxic lymphocytes that will ultimately eliminate the virus and achieve clinical recovery. in fact, when compared to severe h n disease, reduced pro-inflammatory cytokines and chemokines were found in covid- patients with good prognosis, reinforcing the idea that adaptive immunity is a key factor for a favorable outcome ( ) . thevarajan et al. ( ) described a kinetic of the immune response in a -year-old woman with covid- who presented a favorable outcome. they evidenced a persistent increase in antibody-secreting cells, follicular helper t cells, activated cd + and cd + t cells, and immunoglobulin m (igm) and igg antibodies that bound to sars-cov- . the peak of both antibody-secreting cells and follicular helper t cells was markedly higher in the patient compared to healthy controls and both cell subsets were persistently increased during convalescence (day ). the experience from the sars epidemic of showed that convalescent sars patients present with neutralizing antibodies against s protein ( ) . the sera stored from convalescent patients from the sars epidemic of can cross-neutralize the s protein-mediated sars-cov- entry in patients with . this data raises the possibility that the s protein could be an important antigen to vaccine protocols. in fact, in analogy to the sars epidemic of , convalescent patients with sars may present igg and neutralizing antibodies peaking at months after the disease and detectable up to years afterwards, suggesting that memory b cells can be elicited during coronavirus infection ( ) . cellular immune response may play a critical role in the adaptive immune response in patients with covid- . thevarajan et al. ( ) observed the emergence and rapid increase in activated cd + t cells at days - after infection preceded the resolution of symptoms of one young patient with a good prognosis. conversely, elderly patients and those requiring intensive care unit support presented a dramatically reduced number of cd + and cd + t cells ( ) . lower total amounts of t cells, cd +, and cd + t cells negatively correlated with patient survival ( ) . diao et al. ( ) noted that t cell absolute counting were negatively correlated to serum il- , il- , and tnf-α concentration in patients with covid- , suggesting that the failure of the adaptive immune response and the increase of pro-inflammatory cytokine may be associated with worse survival. it is also possible that increased il- leads to a reduction in cd + t cells and nk cells in patients with covid- and immune dysregulation ( ). in fact, some proinflammatory cytokines, such as il- , may block the antiviral immune response by favoring t cells' exhaustion ( ). diao et al. further characterized the exhaustion status of patients with covid- . they noted an increasing pd- and tim- expression on t cells as patients progressed from prodromal to overtly symptomatic stages ( ) . whether this reflects the emergence of exhaustive t cells with a defective capacity to eliminate the virus or a normal evolution of the immune response against the virus remains to be determined. if greater severity of disease is seen in patients with a higher frequency of exhausted t cells, a potential therapeutic approach could be attempted to block those inhibitory receptors, unleashing the t cell response against the virus. among children, a mild-symptom disease usually occurs. they frequently crush the viral infection through an effective adaptive immune response. however, the remodeling of the immune system that happens with aging may lead to modifications in both adaptive and innate immunity. the final result of these changes may trigger a maladaptive immune response against sars-cov- . in fact, the elderly are an at-risk group to a more aggressive disease that includes cytokine release syndrome, disruption of intrinsic lung defense, secondary bacterial pneumonia, endothelial injury, and end organ damage. the diminished naïve t cell repository observed among the elderly may dramatically affect the adaptive immune response against sars-cov- , since fewer naïve t cells will be capable of responding to new infections ( , ) . furthermore, there is also a reduction in the number of regulatory t cells with aging, which help keep the immune system under tighter control ( ) . since the elderly frequently present with a remodeled adaptive immune response, they may fail to enhance antibody production. instead, a proinflammatory tone characteristic of inflamm-aging may convert the immune response of patients with covid- in a lifethreatening cytokine storm. on the contrary, young patients usually present with an enormous number of naïve t cells that had never encountered a virus. then, naïve t lymphocytes are rapidly primed and innate immunity does not overwhelm the adaptive immune response. this may explain, at least in part, the favorable prognosis observed among young subjects. figure shows the possible relationship between immune response in patients with covid- and the remodeling process that takes place in the immune system with aging. the immune system faces a complex adaptation over time, culminating in functional and phenotyping alterations. the influence of age-related remodeling of the immune system is clinically observed within elderly features (e.g., frailty syndrome) that can be assessed by lab tests. despite several promising experimental methods, none are clinically validated so far, but certainly shed some light on the pathophysiology of immunosenescence. novel mechanisms of inflamm-aging may rise in the near future, leading to new potential therapeutic targets for age-related disorders. different from the chronological age, the "immune age" obtained by population studies may accurately reflect the molecular and cellular changes that occur over time ( ) . immunosenescence may explain the lethality amongst the elderly with covid- with a combination of ineffective t cell response, failed antibody production against sars-cov- , and inflamm-aging that terribly collapses the homeostasis, leading to severe organ dysfunction. the biomarkers that are hallmarks of 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patients with coronavirus disease ageing and life-long maintenance of t-cell subsets in the face of latent persistent infections the aged lymphoid tissue environment fails to support naïve t cell homeostasis the impact of aging on regulatory t-cells a clinically meaningful metric of immune age derived from high-dimensional longitudinal monitoring the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © cunha, perazzio, azzi, cravedi and riella. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - tfg h authors: dong, rong; chu, zhugang; yu, fuxun; zha, yan title: contriving multi-epitope subunit of vaccine for covid- : immunoinformatics approaches date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: tfg h covid- has recently become the most serious threat to public health, and its prevalence has been increasing at an alarming rate. the incubation period for the virus is ~ – days and all age groups may be susceptible to a fatality rate of about . %. covid- is caused by a novel single-stranded, positive (+) sense rna beta coronavirus. the development of a vaccine for sars-cov- is an urgent need worldwide. immunoinformatics approaches are both cost-effective and convenient, as in silico predictions can reduce the number of experiments needed. in this study, with the aid of immunoinformatics tools, we tried to design a multi-epitope vaccine that can be used for the prevention and treatment of covid- . the epitopes were computed by using b cells, cytotoxic t lymphocytes (ctl), and helper t lymphocytes (htl) base on the proteins of sars-cov- . a vaccine was devised by fusing together the b cell, htl, and ctl epitopes with linkers. to enhance the immunogenicity, the β-defensin ( mer) amino acid sequence, and pan-hla dr binding epitopes ( aa) were adjoined to the n-terminal of the vaccine with the help of the eaaak linker. to enable the intracellular delivery of the modeled vaccine, a tat sequence ( aa) was appended to c-terminal. linkers play vital roles in producing an extended conformation (flexibility), protein folding, and separation of functional domains, and therefore, make the protein structure more stable. the secondary and three-dimensional ( d) structure of the final vaccine was then predicted. furthermore, the complex between the final vaccine and immune receptors (toll-like receptor- (tlr- ), major histocompatibility complex (mhc-i), and mhc-ii) were evaluated by molecular docking. lastly, to confirm the expression of the designed vaccine, the mrna of the vaccine was enhanced with the aid of the java codon adaptation tool, and the secondary structure was generated from mfold. then we performed in silico cloning. the final vaccine requires experimental validation to determine its safety and efficacy in controlling sars-cov- infections. in december , covid- , caused by the severe acute respiratory syndrome coronavirus (sars-cov- ) was first discovered in china and has rapidly spread across the world. as of : noon on june , a total of , , confirmed cases of covid- have been reported globally, including , deaths. the prevalence of the disease has been increasing at an alarming rate. there were , , cases in the united states, , in brazil, , in russia, , in the united kingdom, and , , in a number of other countries ( ) . the incubation period for the virus is ∼ - days, and all age groups are susceptible to a fatality rate of about . %. the most common clinical manifestations are low-grade fever, dry cough, fatigue, and gastrointestinal symptoms ( ) . about half of all patients with covid- develop shortness of breath, and severe cases may rapidly develop sars, septic shock, difficult-to-correct metabolic acidosis, and coagulation disorders ( ) . covid- may also affect other organs, most commonly the heart and kidneys ( ) ( ) ( ) . some patients may have mild symptoms, without fever, and may recover after - weeks ( ) . other patients may show signs of serious illness and some may die; however, most patients show favorable progress ( ) . male individuals with the disease and aged patients have the worst prognosis. in children, the disease is relatively mild ( ) . covid- is caused by a novel single-stranded, positive (+) sense rna beta coronavirus, which is a pathogen of the coronaviridae family, named sars-cov- ( ) . the full-length genome sequences revealed that sars-cov- has the greatest genetic similarity to bat coronavirus, ∼ - % similarity to severe acute respiratory syndromerelated coronavirus (sarsr-cov), and a smaller similarity of - % to the middle east respiratory syndrome-related coronavirus (mers-cov) ( ) . thus, a bat might be the original host of sars-cov- , but the intermediate host remains undiscovered ( ) . the genes of sars-cov- encode structural proteins and non-structural proteins. four structural proteins are absolutely vital for viral assembly and invasion of sars-cov- . spike protein homotrimers constitute the spikes on the viral surface, and these spikes are responsible for attachment to host cells by binding to their receptors ( ) . the m protein has three transmembrane domains, which determine the shape of the virion, facilitate membrane curvature, and bind to the nucleocapsid. the e protein plays an important role in virion assembly and release, as well as involved in viral pathogenesis. the n protein has two different domains, both of which bind to the viral rna genome via totally different mechanisms. in addition, some reports have shown that non-structural proteins are essential for the replication of coronaviruses ( ) . vaccination is a vital tool for the control and elimination of the virus, and the development of a vaccine for sars-cov- remains an urgent need ( ) . traditional methods of vaccine development are time-consuming and very labor-intensive ( ). the realm of immunoinformatics tools considers the mechanism of the host immune response to yield additional methodologies in the design of vaccine against diseases are cost-effective and convenient, as in silico predictions can reduce the number of experiments needed ( , ) . dozens of studies have generated epitope-based peptide vaccine of sars-cov- . baruah and bose ( ) used immunoinformatics tools to discover cytotoxic t lymphocyte (ctl) and b cell epitopes for the spike protein of sars-cov- . then, abraham et al. developed a multi-epitope vaccine that was designed using immunoinformatics tools that potentially trigger both cd + and cd + t-cell immune responses ( ) . although there are many vaccines generated by immunoinformatics tools, most of these are based on spike protein. the spike protein is responsible for attachment to host cells by binding to angiotensin-converting enzyme (ace ) ( ) . a vaccine based on the spike protein could induce antibodies to block sars-cov binding and fusion or neutralize virus infection ( ) . but there are still many obstacles, spike protein-based sars vaccine may induce harmful immune responses that cause liver damage of the vaccinated animals ( ) . other virus proteins are considered as the candidates for designing vaccine with protective and less harmful immune responses ( ) . vaccine-based on structural and non-structural proteins of the virus is revealed potential vaccine inducing protective immune responses ( , ) . pandey et al. reported the more scientifically rigorous strategy of multi-epitope subunits based on multiple proteins against parasitic and viral diseases, such as malaria, visceral leishmaniasis, and hiv ( ) ( ) ( ) . in this present, we employed immunoinformatics to predict multiple immunogenic proteins from the sars-cov- proteome and thereby design a multi-epitope vaccine. these proteins included non-structural and structural sequences of sars-cov- , their reference sequences were retrieved from the national center for biotechnology information (ncbi) database. the proteins of the sars-cov- have been reported and reference could get from ncbi ( , ) . the reference sequences of sars-cov- proteins were retrieved from ncbi protein database (https://www.ncbi.nlm.nih.gov/protein) and accession numbers in table , then we stored the reference sequences as a fasta data type. the proteins with < amino acid sequences which are too short to predict epitopes were excluded, the remaining proteins were used for further analysis. vaxijen is the first server for alignment-independent prediction of protective antigens, which overcome the limitations of alignment-dependent methods ( ) . to identify the potential antigenicity of sars-cov- proteins, an online prediction server, vaxijen v . (http://www.ddg-pharmfac.net/vaxijen/ vaxijen/vaxijen.html) was used to predict the antigenic values of each protein ( ) . this identification was applied according to the default parameters of the server. proteins having antigenicity were sorted according to an antigenic score of ≥ . (threshold for this model is . ) and were selected for further structural modeling ( ) . there are no available experimental structures of sars-cov- proteins, phyre provide model regions trough a new ab initio folding simulation with no detectable homology ( ) . the sars-cov- proteins were modeled by phyre server (http://www.sbg. bio.ic.ac.uk/phyre /). because the sars-cov- proteins with no detectable homology protein to finish the modeling, we chose the intensive search and output the accurate alignment by the alignment of hidden markov models. modrefiner was used by the galaxyrefine server (http:// galaxy.seoklab.org /cgi-bin/submit.cgi?type=refine) ( ) . the structure assessment was performed by the swiss-model workspace (https://swissmodel.expasy.org/assess) ( ) . the three dimensional ( d) models were used for the conformational (discontinuous) b-cell epitope predictions while the sequences were utilized in linear b-cell and t-cell epitope predictions. netctl- . is demonstrated to have a high predictive performance ( ) . the netctl . server (http://www.cbs. dtu.dk/services/netctl/) was applied to predict ctl epitopes for the sars-cov- at the threshold value of . with high sensitivity and specificity ( ) . to cover ∼ % of the world's population, three supertypes (a , a , and b ) were selected based on artificial neural networks, to predict mhc class i binding epitopes ( ) . the best candidates for the sars-cov- vaccine construction were sorted for further prediction, based on a half-maximal inhibitory concentration (ic ) < nm and an integrated score. the ic < nm represents epitope has a high affinity to receptor. the integrated score indicated the transporter of antigenic peptides (tap) transport efficiency, class i binding, and proteasomal cleavage prediction ( ) ( ) ( ) . then the specific treg epitopes were screened and excluded by the epitoolkit (https://epivax.com/). for mhc class ii t cell epitope predictions, the immune epitope database server predicted binders based on the percentile rank or mhc binding affinity ( ) . the immune epitope database server (iedb; http://tools.iedb.org/mhcii/) was used to predict helper t lymphocyte (htl) epitopes ( ) . we chose the combinatorial approach which recommended by iedb to predict htl epitopes. the combinatorial approach combined nn-align, smm-align, comblib, sturniolo, and netmhciipan methods ( ) ( ) ( ) ( ) ( ) . the alleles of the human leukocyte antigen (hla) were selected for the prediction at α and β chains, separately ( ) . for final construction, epitopes were selected based on their scores (low scores indicated favorable binding), the release of interferongamma (ifn-γ), induction of emergent properties, and the ic < nm. the ifn-γ cytokine makes a major contribution to antiviral mechanisms. it excites both native and specific immune responses by activating macrophages and natural killer cells ( ) . further, ifn-γ augments the response of mhc to antigens. the ifn-γ epitope server (http://crdd.osdd.net/raghava/ifnepitope/ scan.php) was used to recognize ifn-γ epitopes ( ) . we entered the htl epitopes with low scores into the ifn-γ epitope server. positive ifn-γ induction was predicted based on the support vector machine (svm) hybrid approach. the final htl epitopes were determined based on ifn-γ induction and mhc class ii binding, both of which facilitate the stimulation of t-helper cells ( ) . the abcpred (http://crdd.osdd.net/raghava/abcpred/) and bepipred linear epitope prediction (http://tools.iedb.org/bcell/ result/) servers were utilized to predict linear b cell epitopes. the abcpred server is based on an artificial neural network (ann) ( , ) . the linear b cell epitopes of the sars-cov- protein were predicted at a threshold of . . the bepipred linear epitope prediction server is based on seven methods: ( ) ( ) ( ) ( ) ( ) ( ) . we used these seven methods separately to predict the average threshold. the overlap between abcpred and bepipred severs was selected to determine the candidate epitopes for the sars-cov- vaccine construction ( ) . unlike t-cell epitopes that are linear continuous stretches of residues, b-cell epitopes are generally conformational (discontinuous) ( ) . in this study, the ellipro servers (http://tools.iedb.org/ellipro/) were applied to predict the conformational b-cells epitopes ( ) . the server predicts epitopes based on pi (protrusion index) value. the epitope with pi = . would include % of residues with % being outside the ellipsoid, discontinues b-cells epitopes with the top pi value was selected for vaccine designing ( ) . to develop the final vaccine, epitopes determined by various immunoinformatics software were linked together with the aid of separate linkers. the ctl epitopes were linked by the aay linker, htl epitopes by the gpgpg linker, and b cells were linked by the kk linker ( , , ) . to increase the vaccine immunogenicity, the β-defensin ( mer) amino acid sequence was adjoined to the n-terminal of the vaccine with the help of the eaaak linker ( ). the β-defensin peptides provoke innate immunity cells and recruit naive t cells through the chemokine receptor- (ccr- ) ( ). the pan-hla dr binding epitopes ( aa) as well as added to the n-terminal of the vaccine with the aid of the same linker ( ) . the pan-hla dr binding epitopes in vaccine construct facilitating binding to many different types of mouse and human mhc-ii alleles to induce cd -helper cell responses ( ) . to enable the intracellular delivery of the modeled vaccine, a tat sequence ( aa) was appended to c-terminal ( ) . linkers (ayy, kk, and gpgpg) play vital roles in producing an extended conformation (flexibility), protein folding, and separation of functional domains, and therefore, make the protein structure more stable ( ). the allergenic proteins induce a harmful immune response, allergenicity of the vaccine should be non-allergic ( ) . the non-allergic character of the vaccine sequence was evaluated by the algpred server (http://www.imtech.res.in/raghava/algpred/) ( ) . we predicted allergenicity of vaccine sequences choosing a hybrid approach (svmc+ige epitope+arps blast+mast) with the highest accuracy and sensitivity ( ) . the vaxijen v . server (http://www.ddgpharmfac.net/ vaxijen/vaxijen/vaxijen.html) was applied to evaluate the antigenicity of the vaccine ( ) . the antigenicity prediction method was solely based on the physicochemical properties of proteins without recourse to sequence alignment. the precision rate of the server ranged from to %. to determine immune response profile of this multi-epitope vaccine, computational immune simulations were performed by the c-immsim online server at (http://kraken.iac.rm.cnr.it/ c-immsim/) ( ) . the c-immsim utilizes the celada-seiden model for describing both humoral and cellular profiles of a mammalian immune system against designed vaccine. as per the literature, three injections were administrated at different intervals of month. the simulation was performed with default parameters. the vaccine sequence was administered weeks apart. the simulation volume was , , simulation steps was , , random seed was , , and the vaccine injection with no lps ( ). the protparam tool (http://web.expasy.org/protparam/) was used to evaluate the physicochemical properties of the final vaccine protein ( ) . the physicochemical properties included the number of amino acids, molecular weight, theoretical isoelectric point (pi), amino acid composition, atomic composition, formula, extinction coefficients, estimated half-life, instability index, aliphatic index, and grand average of hydropathicity (gravy) ( ) . the molecular weight and theoretical pi were computed by user-entered sequences. the amino acid and atomic compositions were self-explanatory. the extinction coefficient of a protein was based on information about its amino acid composition. the instability index of a protein indirectly indicated the stability of the protein. if the computed instability index of protein was < , it was regarded as a stable protein, while values > were regarded as unstable. in vivo half-life evaluation of proteins was based on the principle of the "n-end rule." furthermore, gravy is a measurement of the hydrophobic nature of the protein, which is calculated by determining the total hydropathy of all amino acids divided by the number of amino acid residues in the protein. to avoid inducing pathogenic priming and autoimmunity, the sequence homology of the final vaccine to human protein was screened by blastp online server (https://blast.ncbi.nlm.nih. gov/blast.cgi) ( ) . an ideal vaccine should have non-sequence to human proteins. the designed vaccine was a reconstructed protein with no detectable homology ( ) . phyre incorporates an ab initio folding simulation to model regions of proteins with no detectable homology. the phyre server (http://www.sbg.bio. ic.ac. uk/phyre /) was used to predict the three-dimensional structure of the designed vaccine. the server generates a fulllength d model of a protein sequence by employing both multiple template modeling and simplified ab initio folding simulation ( ) . to enhance the overall and partial structural quality of the protein, the output d structure of the final vaccine from the phyre server was further refined by the galaxyrefine server (http://galaxy.seoklab.org/cgi-bin/submit.cgi?type=refine) ( ) . the galaxyrefine server predicted five refined models of our developed vaccine construct, in which model was made by the structural perturbation based simply on the clusters of the side chains; whereas, models - were generated by deeper perturbations of loops and secondary structural elements ( ) . for the assessment of the tertiary structure of the final vaccine protein, a ramachandran plot was performed by the swiss-model workspace (https://swissmodel.expasy.org/ assess) ( ) . the ramachandran plot illuminates favored regions for backbone dihedral angles against amino acid residues in protein structure ( ) . the structure assessment page shows the most relevant scores provided by molprobity and help we easily identify where residues of low quality lie in their model or structure ( ) . then, prosa-web (https://prosa.services.came. sbg.ac.at/prosa.php) was employed in the final vaccine protein structure validation. a positive z-score commonly means an erroneous or erratic section found in the generated d protein model ( ) . to revealing the binding affinity between the vaccine construct and antigenic recognition receptors of toll-like receptor- (tlr , a z) and major histocompatibility complex (mhc-i, wuu, and mhc-ii, c j) present on the surface of immune cells ( ) . docking analysis was performed using the cluspro server (https://cluspro.bu.edu/login.php?redir/queue. php). tlr act as receptors for antigenic recognition. the cluspro server computed the models based on electrostatic interactions and desolvation energy ( ) . to reconfirm the binding affinity of the designed vaccine construct between these receptors, the patchdock server (https://bioinfo d.cs.tau.ac.il/ patchdock/) was used for docking ( ) . the server predicted the potential complex with the help of three algorithm-molecular shape representations, surface patch matching, filtering, and scoring ( ) . after the acquisition of the output from the patchdock server, the complexes were refined by the firedock algorithm, which predicted the optimal complex with the aid of energy functions ( ) . the pdb file of vaccine protein and receptor complex (tlr , mhc-i, and mhc-ii) were used to start the molecular dynamic (md) simulations. the complexes were placed in a octahedron box of water molecules represented by the threepoint charge spc model, whose boundary is at least Å from any protein atoms. the solvated protein was subsequently neutralized by chloridions. covalent bonds involving hydrogen atoms were constrained using the lincs algorithm, and longrange electrostatic interactions were treated with particle-mesh ewald employing a real-space cutoff of Å. the system was first briefly minimized with backbone atoms restrained to the initial coordinates to remove close contacts, and the restrained system was gradually heated to k under constant volume conditions in ps. each system was equilibrated for ns using the constant isothermal-isobaric ensemble at atm and k without any restraints. the parrinello-rahman barostat and a v-rescale thermostat were used with an integration time step of fs. production run md simulations were performed for ns with coordinates recorded every ps. all simulations were performed using gromacs . along with the gromos a force field ( , ) . for the purpose of cloning, codon adaptation of the designed vaccine was performed for analyzing the codon usage by the prokaryotic organism (escherichia coli, e. coli). the java codon adaptation tool (http://www.jcat.de/) was used to optimize codon ( ) . then the secondary structure of mrna was predicted by mfold (http://unafold.rna.albany.edu/? q=mfold) ( ) . for raising the expression efficiency of the final vaccine protein, the e. coli k strain was chosen. for the valid translation of the vaccine gene, we proofread and avoided rho-independent transcription termination, prokaryote ribosome binding site, and cleavage site of restriction enzymes. restriction endonuclease sites xhoi and bamhi were appended to n and c terminals of vaccine, respectively. then, it was inserted into the pet a (+) vector between the xhoi and bamhi. the flow chart of the designed work is shown in figure . the strategy of vaccine construction is presented in figure . the proteome of sars-cov- was retrieved, which comprised proteins. the reference sequences of those proteins were retrieved in the fasta format and their details are presented in table . five proteins with < amino acid sequences are too short to predict epitopes (orf protein, orf protein, orf b protein, nsp , and envelope protein) were excluded. in order to develop a subunit vaccine, it is critical to identify candidate proteins that are important for inducing a protective immune response ( ) . the remaining proteins sequence were relayed to the vaxijen server to determine their antigenicity based on the antigenic scores ( table ) . proteins with antigenic scores > . were selected for further analysis ( ) . nine proteins, namely orf a protein, orf protein, nsp , nsp , nsp , endornase, orf a protein, membrane glycoprotein, and nucleocapsid phosphoprotein were finally selected for further epitope prediction. there is no available experimental structures of these nine proteins, we predicted homology models for the nine proteins applying the normal mode of phyre online server. the most suitable templates for the nine proteins were identified to be the pbd entries ( table s ). all of the modeled structures were showed over % residues in the ramachandran favored region figure s and table s . the prediction of ctl epitopes ( mer) was performed by the netctl server. the binder sites were determined based on three supertypes (a , a , and b ), with a % coverage rate of the world's population. nine proteins were selected based on antigenicity. one epitope of each supertype was selected based on the highest score and an ic value < nm. then the specific treg-inducing epitopes were excluded by epitoolkit. a total of epitopes were selected from nine proteins as the candidates for the construction of the vaccine ( table ) . the htl epitopes ( mer) were evaluated for three hla supertypes: hla-dr (drb * : , drb * : , drb * : , drb * : , drb * : ); hla-dq (dqa * : /dqb * : , dqa * : /dqb * : , dqa * : /dqb * : , dqa * : /dqb * : , dqa * : /dqb * : , dqa * : /dqb * : ); and hla-dp (dpa * /dpb * : , dpa * : /dpb * : , dpa * : /dpb * : , dpa * : /dpb * : , dpa * : /dpb * : ). we sorted the top epitopes with the lowest scores (low scores indicated the highest binding capability) from three supertypes. the best candidate was then selected based on positive ifn-γ induction and an ic < nm. then the specific treg-inducing epitopes were excluded by epitoolkit. thus, a total of epitopes were selected for vaccine design ( table ) . we used the abcpred and bepipred servers to identify the line b cell candidate epitopes. all predicted epitopes from both servers were compared, and only the overlapping epitopes were selected for the development of the vaccine. the line epitopes identified by abcpred had prediction scores ranging from . to . , and line epitopes identified by bepipred had prediction scores ranging from . to . among these line epitopes, only ( mer) were found to be common or partly common in both servers ( table ) . these line epitopes were selected for vaccine construction ( table ) . the non-continuous b cell epitopes were predicted by the ellipro severs, a total number of non-continuous b cell epitopes were generated from ellipro. amino acid residues, sequence location, the number of residues, and the pi scores of the predicted conformational epitopes are shown in table and the graphical depiction of these epitopes can be seen in figure s . twenty-four epitopes were excluded because it added the allergenicity of vaccine, three epitopes were marked red and selected for vaccine construction. the best candidate epitopes were used for the construction of the vaccine. a total of ctl epitopes, htl epitopes, the half-maximal inhibitory concentration (ic ) value was > nm, which ensured a higher binding capability of the selected epitopes to mhc molecules. linear, and three non-continuous b cell epitopes were fused together with the aid of linker sequences. the ctl epitopes were linked by ayy (the aay liner helps the epitopes produce suitable sites for binding to tap transporter and enhances epitope presentation), the htl epitopes were combined with the aid of gpgpg (the gpgpg linker stimulate htl responses and conserve conformational dependent immunogenicity of helpers as well as antibody epitopes), and b cell epitopes were merged with the aid of kk. the final to enhance vaccine immunogenicity, the human β-defensin- sequence ( aa) and pan-hla dr binding epitopes (the pan-hla dr binding epitopes in vaccine construct facilitating binding to many different types of mouse and human mhc-ii alleles to induce cd -helper cell responses.) was added to the nterminal of the vaccine with the aid of the eaak linker. to enable the intracellular delivery of the modeled vaccine, a tat sequence ( aa) was appended to c-terminal. the vaccine was developed to be amino acids in length ( figure s ) . the sequence homology of final vaccine protein to human protein sequence shown that there were no significant alignments ( figure s ). the allergenic character of the vaccine was determined by the algpred server and was based on the hybrid approach (svmc + ige epitope + arps blast + mast) with a . % coverage. the vaccine was non-allergen with % accuracy and . % sensitivity at threshold value was − . . similarly, the antigenic the half-maximal inhibitory concentration (ic ) value was < nm, which ensured a higher binding capability of selected epitopes to mhc molecules. nature of the vaccine construct was evaluated and showed that the protein was a favorable antigen with a global prediction score of a protective antigen of . (probable antigen). the default threshold value for antigenicity was . in the virus model. moreover, the vaccine constructs contained amino acids, and its molecular weight was . kda. the theoretical pi was predicted to be . . the vaccine contained negatively charged residues and positively charged residues. the vaccine construct was composed of , atoms, and its chemical the immune response profile in silico immune simulation the immune stimulation of the final vaccine was performed using c-immsim online server, which gives the immune profiles of the designed vaccine. the proliferation in the secondary and tertian immune response were identified by igg + igg and igm, as well as, the decreasing of the antigen count igg + igm showed the proliferated (figure a) . the stimulation result revealed the development of immune response after immunization. b cell population was highly stimulated upon immunization ( figure b) . similarly, the cytotoxic and helper t cell levels were proliferated that suggested the development of secondary and tertian immune response (figures c,d) . during the exposure time, it was also observed that the production of ifn-γafter immunization ( figure e) . these results were significant for the immune response against sars-cov- . hence, the tertiary structure of the full-length vaccine sequence was predicted by phyre , and it was applied for refinement and further analysis. twenty-five templates were employing modeling as figure s shown. there were three templates from human defensin which were we added in to enhance the immunogenicity, others from virus ( figure s ) . the immune epitopes were not structural homology to human proteins that could avoid inducing autoimmune. the secondary structure of the predicted model contained % alpha-helix, %tm helices % beta-sheets, and % disordered figure s . to optimize the d structure of the modeled protein, the initial model was refined in the galaxyrefine server. the galaxyrefine server-generated five models based on the rootmean-square deviation (rmsd) and molprobity algorithm. the details of the five models are shown in table s . model with the top ramachandran favored, therefore selected for docking purposes (figure ) . a model with more residues in the ramachandran favored region, less in outliers region and rotamer region was considered as a more ideal one. the initial model generated from phyre server and refine model from galaxyrefine were evaluated with the aid of the swiss-model workspace. the initial model was . % of residues in the ramachandran favored region, . % in the ramachandran outliers region, and only . % in the rotamer region (figure ). the refine model was . % of residues in the ramachandran favored region, . % in the ramachandran outliers region, and only . % in the rotamer region (figure ) . other favorable parameters of the refined model were as follows: gdt score of . , rmsd value of . , molprobability of . , clash score of . , and poor rotamers totaling . ( table s ). the quality and potential errors in the final vaccine d model were verified by prosa-web. the z-score indicates overall model quality, the model with a lower z-score was considered as the higher quality one. the z-score of the initial model was − . , refine model is − . (figure ). to further evaluate the binding affinity between the developed vaccine construct and the relative antigenic receptors (tlr , mhc-i, and mhc-ii), molecular docking was performed. the server yielded candidate models with different binding energies. twenty-nine model complexes of tlr and covid- vaccine were determined, from which just one complex with the lower binding energy score of − . was selected to show ( table and figure ) . a total of model complexes of mhc-i and the covid- vaccine were discovered, and the lowest binding energy score was − . ( table and figure ) . a total of complex models of mhc-ii and the covid- vaccine were predicted, among which, one model complex with the lowest binding energy score of − . was chosen to show ( table and figure ) . further, the vaccine construct was evaluated using the patchdock server, which identified different models and produced a score table. the top complexes identified were refined by the firedock algorithm. among those top models, the model with the lowest binding energy was further selected to show in this paper. the refinement outcomes of tlr and the vaccine complex was solution number with global energy of − . , attractive van der waals energy (vdw) of − . , repulsive (vdw) of . , and atomic contact energy of − . ( table and figure ). the complex of mhc-i and the vaccine was ranked number nine, with global energy of − . , attractive vdw of − . , repulsive vdw of . , and atomic , g , t , t , l , k , e , p , c , s , s , g , p , h , p , l , a , d , n , k , c , c , p , d , g , v , r , s , v , s , p , k , l , f , i , r , e , e , e , t , c , g , q , q , q , t , t , l , k , g , k , k , g , v , q , i , p , c ,t , c , g , k , q , a , t , k , y , l , v , q , q , e , s , p , f . k , p , q , v , n , g , l , t , w , a , d , n ,n , c , l , s , a , p , p , a , q , y , e , l , k , h , g , t , f , t , e , y , t , g , n , y , q , c , g , h , k , t , s , k , e , t , l , y , c , i , d , g , a , l , l , t , k , s , s , e , y , k , g , p , i . d , d , t , l , v , e , f . k , e , n . endornase s , l , e , n , v , a , f , n , v , v , n , k , g , h , f , d , g , q , q , g , e , v , p , v , s , i , i , n , n , t , v , y , t , k , v , d , g , v , d , v , e , l , e , n , k , t , t , l , p , v , n . e , g , s , v , k , g , l , g , e , a , v , k . l , p , s , m , i , d , l , e , l , a , m , d , e , f , i , e , r , y , l , e , g , y , a , f , e , h , i , y , g , d , f , s , h , s , q , l , g , k , r , f , k , e , s , p , e , f , t , d , a , q , t , g , s , s , k , c , k , s , q , d , l , s , v , v , s , k , v , m , l , w , c , k , d , g , h , v , e . t , i , g , c , s , m , t , d , i , a , k , k , p , t , e , t , i , c , a , p , l , t , g , r , v , d , g , v , d , l , f , r , n , a , r , n , k , v , d , g (table and figure ). to accomplish the estimate of the stability of the vaccinereceptor complex, we performed the simulation of the docked complexes (vaccine and tlr- , mhc-i, and mhc-ii) with the help of gromacs. then, various analysis like energy minimization, pressure assessment, temperature, and potential energy calculations were performed. the temperature and pressure of the simulation system during the production run was around k and atmosphere, respectively, indicating a stable system and successful md run. the temperature and pressure of the three simulation systems (vaccine and tlr- , mhc-i, and mhc-ii complexes) during the production run were around k and atmosphere, respectively, indicating the stable systems and successful md run (figures a-f) . the complex root mean square deviation (rmsd) plot represents the structural fluctuation of the overall structure of the complex of vaccine and immune receptor. the rmsd of vaccine-tlr complex has large fluctuation during - ns simulation. after ns, the rmsd value was kept around . nm, indicating that the conformation of this complex was stable ( figure g) . otherwise, the rmsd of vaccine-mhc-i and -mhc-ii complexes has large fluctuation during - ns simulation. after ns, the rmsd value were kept around nm, indicating that the conformation of the two complexes were stable (figures h,i) has low rmsf value, indicating these residues has low structural flexibility. by contrast, residue - and - has relatively higher rmsf value, indicating the larger flexibility during those regions (figures j-l) . to fuse the final vaccine to an expression vector, codon conversion of the vaccine protein was performed by the java codon adaptation tool. restriction site xhoi and bam hi were added to n and c terminals of the codon sequence, then was inserted into the pet a (+) vector between the xhoi and bamhi (figure ) . the rna secondary structure using the mfold program was generated foldings contain , base pairs out of . % in the energy dot plot. mfold predicted an identical secondary structure of , bp formed by nucleotide fragments (figure s ). sars-cov- is characterized by high infectivity and high transmission speed; thus, a prophylactic vaccine is needed ( ) . the availability and advantages of the multi-peptide vaccine developed by immunoinformatics methods have been confirmed by previous studies ( , ) . ojha et al. used the immunoinformatics methods to develop a multiepitope subunit vaccine to epstein-barr virus-associated malignancy ( ) . in recent studies, genomics and proteomics information of sars-cov- have been retrieved, stored, and utilized ( , ) . in the present research, we tried to develop a multi-epitope subunit prophylactic vaccine of sars-cov- , with the help of immunoinformatics tools. a line of research have tried to develop the vaccine of sars-cov- by immunoinformatics tools. baruah and bose ( ) used immunoinformatics tools to discover cytotoxic t lymphocyte (ctl) and b cell epitopes for the spike protein of sars-cov- . then, abraham et al. developed a multi-epitope vaccine that was designed using immunoinformatics tools that potentially trigger both cd + and cd + t-cell immune responses ( ) . most of those research just focus on the spike protein-based vaccine. a vaccine based on the spike protein could induce antibodies to block sars-cov- binding and fusion or neutralize virus infection ( ) , as well as induce harmful immune responses that cause liver damage ( ) . other proteins should be ideal candidates for designing vaccines. in the present report, we selected nine proteins with positive antigenicity for further epitope prediction. all proteins from sars-cov- with < amino acid sequences were excluded, and the antigenic nature of the remaining proteins was evaluated. this method can facilitate the discovery of potential antigens of sars-cov- when the precise immunity mechanisms are unknown. to design an effective vaccine, we selected the sars-cov- protein through the above-mentioned methods for epitope prediction. in recently, asaf et al. reported that identify multiple epitopes for cd + and cd + t cells based on muti-protein ( ) . their protein list was the same as this in our research. in asaf 's report, they just predicted the t cell epitopes, non-b cell, b cell peptide was not predicted ( ) . the b cell epitopes are antigenic determinants from the antigen that are recognized by the b cell surface membrane receptor and evoke the production of specific antibodies. the persistent challenge in immunological prediction tools is the prediction of epitopes to a higher level of accuracy ( ) . to determine accurate linear b cell epitopes from the antigenic proteins, we used two bioinformatics tools based on different algorithms of prediction. we identified nine overlapping linear b cell epitope candidates from two different bioinformatics tools. this method was superior to the prediction of epitopes from a single tool ( ) . moreover, we also have predicted the noncontinue b-cell epitopes. the b cell immune response is preferred in the design of a vaccine. however, t cells may also elicit a strong immunoreaction. the vaccine that activates both ctls and htls should be more effective than a vaccine that only targets ctl responses ( ) . to generate a more effective vaccine, we predicted both ctl epitopes and htl epitopes. the t cell epitopes were decomposed fragments from the antigen presented by the mhc molecules of t cells and stimulated the production of effector t cells, immunological memory t cells, and ifn-γ. the cellmediated immune response induced by ctls plays a vital role in the defense against viral infections through the recognition of intracellular viral pathogens by mhc class i molecules. in the present report, mhc-i binding epitopes were predicted by choosing a , a , and b alleles, which cover ∼ % of world's population. we selected ctl epitopes. the htls play a vital role in the antiviral immune response by producing ifn-γ. moreover, htls are able to induce and maintain ctl responses. furthermore, htls epitopes were chosen based on both the binding capability and ifn-γ induction. bhattacharya et al. also used the spike protein sequence predicted for mhc-i and mhc-ii epitopes of sars-cov- , but not predicted capability of producing ifn-γ ( ). the t cell epitopes enhanced ifn-γ inducing capability, which evokes both the native and specific immune responses by activating macrophages and natural killer cells, and augmenting the response of the mhc to the antigen ( , ) . in this study, the immunogenic epitopes from b cells, ctls, and htls were chosen to develop a more valid, reliable, and effective vaccine against sars-cov- . a multiepitope approach was used by splicing together epitopes with the aid of their respective linkers. to improve the immunogenicity of this multiepitope vaccine, an adjuvant β-defensin and pan-hla dr binding epitopes ( aa) were fused to the n-terminal with the aid of an eaaak linker, then a tat sequence ( aa) was appended to c-terminal with the added of kk. the final vaccine constituted amino acids. the allergenicity, antigenicity, and stability of the designed vaccine constructs were then evaluated. the tertiary structure of the generated vaccine was predicted by using the phyre server and then refined by the galaxyrefine server. the binding affinity of complexes of the developed vaccine and receptors, in which tlr- , mhc-i, and mhc-ii (present on the surface of the immune cell) were confirmed by the cluspro server was based on molecular docking. furthermore, to ensure the translation efficiency of the designed vaccine in a specific expression system, the mrna of the vaccine was enhanced with the aid of the java codon adaptation tool. the restriction enzyme cutting sites of xho? and bamh? were then appended to the n and c terminals, respectively. the vaccine sequence was subsequently cloned in pet a (+), the expression vector. further experimental validation of the safety and efficacy of the designed vaccine for sars-cov- is warranted. all datasets presented in this study are included in the article. rd and zc performed the experiments. rd and yz wrote the paper. yz and fy edited the final version. all authors participated in the experimental design, data analysis, and agreed with the final version of the paper. available online at clinical characteristics of patients infected with sars-cov- in wuhan severe acute respiratory 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coronavirus (sars-cov- ): immunoinformatics approach the use of databases, data mining, and immunoinformatics in vaccinology: where are we? role of allograft inflammatory factor- in the pathogenesis of diseases we extend the sincerest appreciation to nhc key laboratory of pulmonary immunological diseases, and guizhou provincial people's hospital, for their technical assistance. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © dong, chu, yu and zha. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -wfakzb w authors: trovato, maria; sartorius, rossella; d’apice, luciana; manco, roberta; de berardinis, piergiuseppe title: viral emerging diseases: challenges in developing vaccination strategies date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: wfakzb w in the last decades, a number of infectious viruses have emerged from wildlife or re-emerged, generating serious threats to the global health and to the economy worldwide. ebola and marburg hemorrhagic fevers, lassa fever, dengue fever, yellow fever, west nile fever, zika, and chikungunya vector-borne diseases, swine flu, severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), and the recent coronavirus disease (covid- ) are examples of zoonoses that have spread throughout the globe with such a significant impact on public health that the scientific community has been called for a rapid intervention in preventing and treating emerging infections. vaccination is probably the most effective tool in helping the immune system to activate protective responses against pathogens, reducing morbidity and mortality, as proven by historical records. under health emergency conditions, new and alternative approaches in vaccine design and development are imperative for a rapid and massive vaccination coverage, to manage a disease outbreak and curtail the epidemic spread. this review gives an update on the current vaccination strategies for some of the emerging/re-emerging viruses, and discusses challenges and hurdles to overcome for developing efficacious vaccines against future pathogens. since the start of this century, a certain number of new or neglected pathogens have emerged from wildlife reservoirs and spilt over into human populations, causing severe diseases ( - ). factors such as urbanization, globalization, travels, international commerce, aging, and climate changes have contributed to favor emergence, spread, and transmission of pathogens. contacts among humans and potential zoonotic reservoirs are increasing, the number of travelers and their movements is growing, the aged population are more susceptible to infections, and the geographic distribution of pathogens within a previous endemic zone is changing ( , ) . during the last decades, the global community faced several outbreaks of emerging and reemerging infectious diseases, with high threats to the health security, biodefense, and economy worldwide ( , ) . the occurrence of significant disease outbreaks-such as sars (severe acute respiratory syndrome) originating in china in ( ) , the h n swine flu pandemic from mexico ( ) , mers (middle east respiratory syndrome) that occurred in saudi arabia in ( ) , the west african outbreak of ebola virus (ebov) in late ( ) , the zika virus (zikv) outbreak originating in brazil in ( ) , the health emergence in nigeria caused by lassa virus ( ) , and the ongoing coronavirus disease (covid- ) pandemic ( ) -has renewed interests in developing strategies to faster prevent, treat, and/or control emerging and re-emerging viruses with high epidemic potential. usually, there is little or no knowledge about identity, epidemiology, and pathogenesis of a new infectious agent appearing for a first time in a certain geographic area (as in case of novel coronaviruses or new influenza variants), as well as the potential to spread out from the zoonotic reservoir, making hard to predict if, where, and when a disease outbreak will occur. the world health organization (who) and the national institutes for allergy and infectious diseases (niaid) published a list of pathogens to be prioritized for research and development, given their epidemic potential. this non-exhaustive list comprises viruses, bacteria, protozoa, and fungi, causing diseases for which efficient countermeasures do not actually exist, or require new therapeutics ( , ) . as proven by historical records, vaccination has played a pivotal role in reducing morbidity and mortality from devastating infectious diseases, successfully leading to disease eradication (i.e., smallpox), and generally decreasing infectious disease burdens. even in presence of therapeutic options, vaccines are the valuable means to prevent infections and overall represent the much wanted achievement. however, even with worldwide efforts, getting a vaccine to the public takes time, and side effects, dosing issues, and manufacturing problems can all cause delays. thus, we have to use this time with great concern. generally speaking, in case of newly emergent diseases, conventional strategies might raise some issues. the unpredictable identity of largely unknown emerging pathogens, the lack of appropriate experimental animal models, and the time and costs for faster developing, producing, licensing, and globally distributing effective vaccine candidates are some of the major challenges to overcome in case of pandemic threats. hence, new and/or alternative approaches in vaccine design and development are required to rapidly face outbreak situations ( ) . this review will discuss the current vaccination strategies for some of the emerging and re-emerging viruses, as well as the approaches that might be suitable in face of global pandemic threats. emerging and re-emerging pathogens represent a constant epidemic threat to humanity not only for the public health consequences but also for the economic, social, and political effects they may globally provoke. therefore, a major public awareness and preparedness would be fundamental in fighting emerging infectious diseases. the terms "emerging and reemerging infectious diseases" mainly refer to two major categories of infectious diseases: newly emergent infections, caused by novel pathogens; and re-emerging infectious diseases, caused by microbes reappearing after previous control, and/or eradication ( ). almost % of emerging infectious diseases are zoonoses, with the great majority of them originating in wildlife, and the number is constantly increasing. climate changes have been related to the emergence of vector-borne diseases in severe environmental conditions, but this is a most debated issue, as well as the contribution of agricultural practices ( ) . in addition, the chances of infectious disease spreading could also include livestock/wildlife animal markets and consumption of those. a study where australia was used as a model of urbanization has proposed a relation among increasing pandemic threats and urbanization: it ascribes the increased threat of pandemic to the high number of major city residents, the exponential intensification of international air traffic, and the commuter mobility network ( ) . basically, an epidemic is an event that occurs when there is an increase, often sudden, in the frequency of a disease above what is normally expected in that population, in that area; while pandemic (from: παν = all, and δεµoσ = people) refers to an epidemic that spreads over several countries or continents at the same time, usually affecting a large number of people ( ). in the last decades, a certain number of viruses came to light for the first time or reappeared, giving rise to significant epidemics and pandemics (figure and table ) . epidemic outbreaks of viral diseases were mostly caused by flaviviruses generally transmitted by vectors, including west nile virus (wnv) ( , ) , zikv ( , ) , yellow fever virus (yfv) ( , ) , and dengue virus (denv) ( , ) . vector-borne diseases, including chikungunya fever caused by chikungunya alphavirus (chikv) ( , ) , are extremely difficult to eradicate because viruses are maintained in nature by propagation among vectors and hosts, without human-human contact. moreover, dry and hot climate conditions seem to foster mosquitoes to bite humans than animals, increasing the risk of spreading diseases with a devastating impact ( ) . today, most areas of the world are endemic for at least one flavivirus, with denv being the most prevalent, and approximately - million people are infected each year. among viral hemorrhagic fevers, lassa fever (lf) is a rodent-borne acute disease caused by lassa virus (lasv) ( ) , endemic in many west african countries, including nigeria that experienced a high mortality rate in the outbreak ( ) . ebola virus disease (evd) and marburg virus disease (mvd) are caused by members of the filoviridae family, ebov ( ), and marburg virus (marv) ( ), respectively. the - ebola outbreak in west africa was the largest since the virus was first discovered in , with a case fatality rate for zaire ebolavirus of % ( ) , while the largest recorded mvd outbreak occurred in angola in ( ) . concerning pandemics, flu pandemics were reported three times during the twentieth century; genome analysis of pandemic influenza viruses dated (h n ), (h n ), and (h n ) demonstrated that all viral strains fully or partially originated from non-human reservoirs, and that the ultimate origin of ha (hemagglutinin) genes are from avian influenza viruses ( ) , with the strain likely being the ancestor of the subsequent epidemic variants. hence, the influenza pandemic has been called the mother of all pandemics ( nhps, non-human primates. frontiers in immunology | www.frontiersin.org during the first months, after the first reported case ( ) . the age of deceased people was below years in almost % of cases, a peculiarity compared with the seasonal influenza epidemic. the mortality rates observed in and flu were of . % and - % due to h n and h n , respectively, and ranging from . and . % in ( ) . the h n pandemic has been commonly referred to as swine flu for the swine origin of the virus, first isolated in mexico and united states in april . the viral genome sequencing indicated that the virus contains a combination of genes never reported in swine or humans before. it has been demonstrated that the swine has become a reservoir of h viruses with the potential to cause future pandemics ( ) . a (h n )pdm virus monovalent vaccine was produced in late ( ) , but the virus has not been eradicated and it continues to circulate as a seasonal variant, causing hospitalization, and death ( ) . in november , a first case of sars was reported in guangdong (china), and after months, the coronavirus (cov) causing the disease, named sars-cov, spread in countries, giving rise to lower respiratory tract infections, with a poor outcome in % of cases ( , ) . middle east respiratory syndrome-cov, the causative agent of mers, was isolated in . the coronavirus has caused isolated mers outbreaks thereafter, becoming endemic in arabian peninsula, with a case fatality rate of . % ( ) ( ) ( ) . on march , , who has declared the coronavirus disease (covid- ) outbreak a global pandemic. the disease is caused by a novel coronavirus, known as sars-cov- , that shares almost % of the genome with that of sars-cov ( , ) . actually more than . million (as of august , ) of people are infected, with an overall case fatality rate of . - . % ( ) . in case of global public health emergencies, governmental and private organizations, vaccine developers, and regulatory authorities should all massively collaborate in selecting and funding the most suitable vaccine platform and strategy to quickly act and curtail disease outbreaks. at the outset of a disease outbreak, gaps in knowledge of identity, pathogenesis, epidemiology of the new emerging pathogen, time required to study the immune responses correlating with the outcome of the viral infection, and the lack of appropriate preclinical models susceptible to infection for testing a vaccine candidate pose several barriers and impediments to expedite vaccine design and development, and thus to ensure global vaccination coverage in time. in the fight against newly emergent viruses, vaccine design might benefit from a range of platform technologies, including nucleic acid vaccines, viral-vector vaccines, and recombinant protein-based vaccines (likely to be administered with adjuvants) ( , ) . compared with conventional vaccines, such as live attenuated and inactivated vaccines, molecular-based platforms might offer a more versatile tool against new emergent viruses, allowing a more fast, low-cost, and scalable vaccine manufacturing. essentially, these platforms rely on the use of a system to deliver and present a new antigen (or a synthetic gene) to rapidly target an emergent pathogen. theoretically, once a platform has previously met safety and efficacy requirements to be moved and advanced into the market, a candidate vaccine against a new virus might profit from the same system, production, and purification protocols, only replacing the disease target antigen (or inserted gene), thus streamlining the vaccine discovery. in inactivated vaccine, the virus is rendered uninfectious using chemicals, such as formaldehyde or heat. this technology, conceived in the nineteenth century, is used for few vaccines still in use (i.e., inactivated polio, whole cell pertussis, and hepatitis a) ( ) . live attenuated vaccines are obtained by passing the virus through animal or human cells until it picks up mutations that make it unable to cause the disease (i.e., measles, mumps, chickenpox, etc.); the attenuated smallpox was used for the massive vaccination campaign that successfully eradicated the infection ( ) , and currently, attenuated influenza viruses are used as vaccines against the seasonal influenza ( ). the advantages of live attenuated vaccines are the intrinsic adjuvant properties, the ability to infect cells (figure ) , and to activate the innate immune response. interestingly, a safe sars-cov- inactivated vaccine (picovacc) has been recently described as being able to induce specific neutralizing antibodies (nabs) in experimental animal models ( ) , and a phase iii clinical trial (nct ) will soon assess efficacy and safety of this candidate in health care professionals ( table ) . nucleic acid vaccines include either mrna or plasmid dna (pdna) vaccines (figure ) . two types of mrna vaccines were developed: conventional non-replicating mrna vaccines and self-amplifying vaccines (or viral replicons). the in vitro enzymatic transcription (ivt) of a dna template plasmid, containing the promoter sequence for the dna-dependent rna polymerase, provides a mature mrna molecule, with the open reading frame that encodes the target antigen, the and flanking untranslated regions (utrs), the cap, and the terminal poly(a) tail. self-amplifying rna (sam) vaccines are commonly based on alphavirus genomes, where genes coding for the structural proteins are replaced with that encoding the target antigens, while the rna replication machinery sequences are conserved, allowing intracellular antigen-encoding rna amplification and higher antigen expression levels than the conventional mrna vaccines ( , ) . once the mrna vaccine is delivered to the host cells and reaches the cytoplasm, it is translated in vivo by the host cellular machinery, providing the corresponding posttranslationally modified antigen (figure ) , thus mimicking the in vivo natural infection. mrna vaccines activate the innate immune system, triggering host immune sensing receptors, and successively promoting adaptive immune responses ( ) . several figure | platforms for vaccine manufacturing: a graphical overview. nucleic acid, viral-vector, protein-based, live attenuated and inactivated vaccines are schematically illustrated. nucleic acid vaccines: conventional non-replicating mrna vaccine, containing the target gene sequence, can be encapsulated into a delivery system to aid its cellular uptake. once released from endosome into the cytosol, it is translated by the host cellular machinery into the target antigen. a pdna carrying a gene target reaches the nucleus to achieve transcription and translation into the cytosol. pdna can be internalized by somatic cells (i.e., myocytes) and then the secreted antigen can be taken up by apcs or naïve b cell, priming immune responses. viral vectored vaccines: defective viral vector, carrying a transgene cassette, can be employed as a system to deliver a transgene and allow the expression of the heterologous antigen within the infected cell. a recombinant replicating viral vector retains the ability to replicate and produce progeny virus particles that can then infect cells, leading to transgene expression and ag processing and presentation. protein-based vaccines: recombinant subunit vaccine or a vlp can be taken up by apcs for mhc presentation and b-cell recognition through bcr. virus vaccines: compared with an inactivated virus, a live attenuated virus retains the ability to replicate and infect cells, mimicking the natural infection. apcs, antigen-presenting cells; mhc, major histocompatibility complex; ag, antigen; pdna, plasmid dna; ep, electroporation; bcr, b-cell receptor; and vlp, virus-like particle. technological innovations have allowed to overcome some of the concerns associated with instability, half-life, inefficient in vivo delivery, and high innate immunogenicity of mrna platform ( ) . mrna vaccines do not produce infectious particles and potentially do not integrate into the host genome, reducing safety issues, and no anti-vector immunity is elicited. they can be quickly produced (likely within the time required to get genomic information from the new emergent virus), saving time and cutting costs. thus, the mrna platform offers a promising attractive alternative to conventional vaccines, should a disease outbreak occur. no rna vaccine has been yet licensed for humans, but encouraging results from preclinical and human clinical trials have shown that mrna vaccines are able to induce safe and long-lasting immunity against different infectious viral diseases, including zika ( ), influenza ( - ), ebola ( ), dengue ( ) , and other viral diseases ( ) . a sars-cov- mrnabased vaccine entered clinical phases just months after the identification of the viral genome sequence (nct ), and a phase iii study (nct ) will assess its effectiveness to prevent covid- ( ) ( ) ( ) . a clinical study (nct ) is currently evaluating a similar vaccine in healthy adults ( table ) . the dna-based strategy, like the mrna-based technology, offers a valuable platform to design and deliver any target of choice, due to safety profile, stability, ease of gene manipulation, and large-scale vaccine manufacturing, in short ( , ), on cellular uptake and in vivo long-term gene expression, potentially providing advantages over mrna vaccines in terms of protein coding capacity, and amount and extent of antigen production. unlike mrna, pdna needs to cross both plasma and nuclear membranes to enter into a cell target, reach the nucleus, and achieve transcription (figure ) . advances in pdna delivery devices (i.e., use of gene gun; in vivo electroporation, ep), and delivery systems (i.e., encapsulation in lnps; adsorption to polymers), have greatly enhanced molecular stability, delivery efficiency, uptake, and antigen expression. in addition, the use of optimized pdna formulations and encoding molecular adjuvants, to be administered in prime-boost strategies or simultaneously with other vaccine platforms, has generally improved the low protective immunestimulatory profile of pdna ( ) . however, some potential safety concerns should be considered, including long-term persistence upon administration, which could eventually lead to genomic integration events, antibodies against bacteria-derived plasmids that could potentially trigger autoimmune diseases, and unwanted side effects due to encoded and co-delivered molecular adjuvants ( , ) . even though no dna vaccine has been yet licensed for use in humans (four for veterinary use), this platform has shown great promise for several emerging viral diseases, including ebola and marburg ( ), mers ( ), west nile ( ), dengue ( ), chikungunya ( ) , and other viral diseases ( ) , and more recently for covid- ( ) . currently, dna-based vaccine candidates, encoding the s protein from sars-cov- , have moved into clinical phase i/ii development ( , , ) (table ). recombinant viral vector-based platform employs either live replicating often attenuated or non-replicating viruses as vector vaccines (figure ). viral vector vaccines represent the biotechnological evolution of live attenuated and inactivated vaccines: a viral backbone devoid of the replication machinery to be used as a shuttle to express in vivo the chosen target antigen. several viral backbones have been exploited to generate viral-vector vaccines. targeted deletion of replication genes represents the non-empirical way of virus attenuation, allowing the generation of a wide array of viral vectors, engineered by insertion of a transgene cassette. the modified virus ankara (mva) is an attenuated form of the vaccinia virus (vacv), derived from more than passages in chick embryo fibroblasts, a method that empirically modifies the viral genome, without affecting the immunogenicity ( ) . it is able to infect multiple cell types but cannot replicate inside the infected cells, ruling out the safety concerns related to the use of live vaccines. one of the drawbacks in the use of a viral vector vaccine is that multiple immunizations lead to the host response against the structural viral proteins, limiting the efficacy of vaccination, as demonstrated in a study based on cellular immune response. to overcome this limitation, the heterologous prime-boost regimen has been introduced in several clinical trials, where two different viral vectors or a pdna prime-viral vector boost were tested ( ) . risks of integration into the host genome do potentially exist, as some viral vectors enter to the nucleus of cells to achieve transcription and replication. a major restrain in the production of viral vector vaccines is the time-consuming manufacturing; several attempts to accelerate vaccine production are in development, like selecting cell lines with higher yield or choosing the best promoter for transgene expression to reduce vaccine doses ( ) . among the available viral vectors, the adenoviruses are the most used in priming the immune response, being able to induce humoral and cellular responses ( ) . a pre-existing anti-vector immune response jeopardized the vaccine response in adenoviral-based clinical trials ( ) . to avoid pre-existing immunity, adenoviral vectors of non-human origin or rare serotypes have been used as vaccine platform. the use of chimpanzee adenoviral vectors proved to be safe and effective in clinical trials conducted against ebola ( ) and respiratory syncytial virus (rsv) ( ) . vesicular stomatitis virus (vsv), a single-stranded negative sense rna virus that naturally infects livestock, represents an attractive safe alternative over other viral vectors due to low risk of pre-existing immunity, lack of dna molecules during replication, and ability of vsv-based vaccines to induce effective humoral responses ( ) . for humans, two viral-vector vaccines are available: imojev, a japanese encephalitic virus (jev) vaccine; and dengvaxia, a dengue vaccine (both from sanofi pasteur). both are produced using the chimeric yfv as vector: two of the yfv genes have been replaced by genes encoding the pre-membrane (prm) and the envelope (e) protein of jev or denv, and the chimeric viruses are propagated in cell culture ( , ) . conversely, several viral vector vaccines have been licensed for veterinary use because of the less stringent regulatory requirements ( ) . to face covid- , an adenovirus type vector expressing sars-cov- s protein (ad -ncov) has been advanced into phase ii trial (nct ), while a phase iii study (isrctn ) is currently investigating the chimpanzee adenoviral vector (chadox ncov- ), expressing the same protein ( , , ) ( table ) . recombinant protein-based vaccines consist of immunogenic proteins from the target pathogen. once identified, recombinant proteins can be produced on a large scale, in bioreactors, using heterologous expression systems, like bacteria, yeast, plants, insect, or mammalian cell lines, depending on the posttranscriptional pattern of modification required ( ) . vaccines based on recombinant proteins represent a safe platform because they do not contain pathogen-derived genetic information, and the manufacturing does not require manipulation of live pathogens. they might represent a platform of choice when a fast response to an epidemic is on demand, as the vaccine production can start once the genome of the new virus has been sequenced, even before the virus isolation. protein-based vaccines can be obtained producing recombinant virus subunits (suvs) that can be administered in combination with adjuvants to improve the host immune response against the recombinant viral antigens ( ) . recombinant proteins derived from viral capsid can selfassemble into virus-like particles (vlps), high ordered and repetitive structures devoid of the viral genome. vlps display antigenic epitopes in their original conformation in high copy number, they retain the size and geometrical organization of the original virus (mainly icosahedral or rod shape), preserving the viral immunogenicity due to the ability to crosslink b cell receptor on b cell surface ( ) , and to be taken up by antigenpresenting cells (apcs) ( , ) (figure ) . several strategies have been proposed to improve dendritic cell (dc) uptake, by expressing targeting molecules such as antibodies directed against endocytic receptors, and to augment immunogenicity, through simultaneous delivery of maturation stimuli, like tlr agonists ( , ). when not able to self-assemble into a vlp, the selected antigen can be expressed as chimeric protein: several vlp platforms are available for the display of heterologous antigens on the viral coat proteins. recombinant vlps from plant virus, like tobacco mosaic virus ( ) , or alpha mosaic virus ( ) , are easily produced, competing for speed and cost of production with vlp platform based on mammalian viruses ( ) . the most used vlp platform is the hbcag-vlp, the core antigen from hepatitis b virus (hbv) ( ) . it is also possible to chemically attach the heterologous antigen to a preformed vlp by using conjugation methods ( ) . although this strategy could increase the manufacturing costs, it might be suitable when the expression of recombinant antigens affects the vlp assembly. to table ) . it is worth mentioning that kim and colleagues designed and developed a sars-cov- subunit vaccine within weeks of the identification of sars-cov- s protein n-terminal domain s sequence. delivery of recombinant subunit vaccines by microneedle array resulted in potent antibody response in mice ( ) , and vaccination with a sars-cov- spike s -fc fusion protein induced antibody responses in small animal models and nabs in monkeys ( ) . in table are listed the vaccine candidates that currently moved into clinical trials for preventing the viral infectious diseases discussed in the following section. west nile virus includes five lineages; among them, lineage was classified as the most virulent, while lineage is considered more attenuated. however, during a serious outbreak in hungary in , the sequencing of lineage showed some genetic mutations that demonstrated the increased virulence of this strain and its explosion throughout the central europe ( , ) , causing renewed interest in the development of a vaccine against wnv. years after the epidemic that hit the united states, no wnv vaccine has been yet released for human use, while four vaccine formulations are on the market for veterinary use, three based on the whole inactivated virus (wn innovator, vetera wnv, and prestige wnv), and one on recombinant vaccine expressing wnv prm/e into a canarypox backbone (recombitek equine wnv) ( , ) . these vaccines completely protect horses from viral infection but require subsequent administrations and several booster doses overtime. for the development of a vaccine for humans, many different platforms were used in preclinical studies, and many of them entered into phase i/ii trials, including hydrogen peroxideinactivated whole virus (hydrovax) vaccine (nct ) ( ), a recombinant truncated form of wnv e protein ( ), recombinant chimeric live attenuated viral vectors, employing yfv ( ), or mva ( ) delivering wnv prm/e proteins (nct ), pdna vaccines encoding prm/e (nct ) ( , ) . all the envelope-based vaccines induced nabs against both wnv lineages and , but some candidates are unable to generate long-lasting antibody responses, requiring multiple administrations ( , , ) . thus, further improvements are needed for the development of next-generation vaccines ( ) . recently, a wnv replicationdeficient vaccine candidate with a deletion of the non-structural protein ns has been shown to protect mice from a highly lethal viral challenge, after a single dose, without adverse effects ( ) . during the outbreak in brazil, an abnormal microcephaly number and other birth defects in newborns were reported ( ) . for this reason, vaccination of pregnant and of reproductiveage women became an urgency. shan and colleagues developed a candidate vaccine, using a live attenuated viral strain containing a deletion in the region of the virus genome. this vaccine induced strong and protective antibody response, after a single injection in mice and macaques, and reduced viral rna in placental and fetal tissues in infected mice ( ) . the immunized mice also developed a robust t-cell response ( ) . although promising, this attenuated virus-based formulation does not meet the safety standards required to be used to vaccinate pregnant women, whose prophylaxis requires a vaccine that fulfill higher safety standards. a number of different replication-deficient viral vectors have been recently developed and are currently under evaluation. immunization of mice with a vaccine based on mva delivering the zikv prm and the e structural proteins (mva-zikv) elicited nabs and potent zikv-specific cd + t-cell responses, mainly with an effector memory phenotype ( ) . a rhesus adenovirus serotype vector (rhad ), expressing zikv prm and e proteins, induced high titer of zikv-specific antibodies after the first prime, offering complete protection against subcutaneous zikv challenge, in mice ( ) , and rhesus monkeys ( ) . these adenoviral-based vaccines induced antibodies that were also maternally transmitted ( ) . in addition, abbink et al. using the rhesus macaque model demonstrated that a complete anti-zikv immunity can only be achieved through vaccination with a combination of different vaccine platforms ( ) . zikv vaccine candidates currently in phase i clinical trials include inactivated and live attenuated vaccines, mrna and pdna vaccines, and recombinant viral-vectored vaccines, mainly targeting the prm and e proteins ( ) . a dna-based vaccine encoding the prm signal sequence from jev and zikv e proteins moved into phase ii (nct ), showing immunogenicity and safety in humans ( ) . a protective and efficacious vaccine against yfv is currently available. to date, the main type of yf vaccine produced on a large scale is based on the live attenuated d virus vaccine. this vaccine is obtained after numerous passages of asibi virus strain in mouse and chicken embryo that generate a strain with accumulated mutations in the envelope protein. these mutations affect the virus binding to the host receptor, reducing its neurotropism and vicerotropism, and mosquito transmissibility ( ) . because the vaccine is produced in chicken embryo, there are issues related to manufacturing costs and vaccine availability. the interruption of vaccination coverage against yf in endemic countries has caused major outbreaks in africa and south america in and , which exhausted the d vaccine stockpiles leading to the use of an emergency "fractional dose" campaign in the democratic republic of congo ( ) . thus, the fluctuating demand for doses during outbreaks makes the accessibility to the vaccine still a problem to be solved. the need for a vaccine against denv has become an urgency only in recent decades. dengue fever is caused by four distinct virus serotypes, denv - , able to circulate simultaneously in endemic areas, making extremely difficult the development of a broad protective vaccine. recently, the food and drug administration approved the first dengue vaccine by sanofi-pasteur, named cyd-tdv or dengvaxia ( , ), a tetravalent live attenuated virus vaccine on yfv backbone, whose release has generated controversy due to evidence that the administration can increase the risk of a more severe form of the illness in people with a pre-existing immunity toward other denv strains ( , ) . for this reason, the use of dengvaxia is strictly limited, depending on age (between and ) and serostatus of recipients to vaccinate (exclusively individuals who had a previous denv infection), generating concerns about its costbenefit balance. studies for the development of a safer vaccine are still ongoing, and candidate vaccines include a tetravalent dengue purified inactivated virus vaccine, currently in phase i/ii clinical trial (nct ), and two live attenuated tetravalent chimeric tdv (denvax), and tvd / (tetravax-dv) vaccines, currently in phase iii clinical trials (nct ; nct ) ( ). no vaccine is actually available to prevent chikv infection. among the candidates in ongoing studies, two of them achieved and completed phase i or ii trials: vla and mv-chik vaccines. vla candidate (by valneva) is a live chikv (la réunion isolate lr opy ) attenuated by a partial deletion of the gene encoding the non-structural replicase complex protein. this vaccine induced immunity lasting over months after a single shot immunization (nct ). mv-chik vaccine is a live attenuated measles-vectored chikv vaccine that induced chikv-specific nabs and shown to be well tolerated by all the participants (nct ) ( ) . recently, moderna therapeutics tested a vaccine based on engineered mrna encoding chikv structural polyproteins (mrna- ) in a phase i clinical trial. as shown in preclinical studies, this formulation induced strong immune responses after one single injection, totally protecting mice from developing the disease ( ) . several vaccine platforms have been tested in preclinical animal models and shown to be able to protect animals from marv infection and to induce both humoral and cellular immune responses. these include vlps ( ) , dna vaccines ( ), recombinant adenoviral vectors ( ) , and rvsv ( , ) . many works have emphasized the use of a multivalent vaccine formulation to achieve protection against different filoviruses. vaccination with a single dose of a trivalent formulation based on rvsv expressing glycoproteins from ebov, sudan ebolavirus (sudv), and the angola strain of marv elicited antibodies specific for the three glycoproteins in non-human primates (nhps) and a balanced t-cell response sufficient to protect against the viral challenges ( ) . similarly, vlps delivering a trimeric hybrid glycoprotein from marv, ebov, and sudv fully protected vaccinated animals from marv challenge, inducing specific nabs ( ) . using an enhanced dna-based platform encoding the envelope glycoprotein from marv and ebov, shedlock and colleagues showed that a polyvalent-filoviral vaccine candidate, delivered by in vivo ep, elicited in preclinical models robust nabs and cytotoxic t cells, completely protecting animals from the viral challenge, after a single dose administration ( ) . actually, a multivalent phase i study (nct ) is evaluating safety and immunogenicity of two heterologous and two homologous prime-boost regimens using a mva multi-filo and ad zaire ebola (ad .zebov) vaccines ( ) in healthy volunteers, with the aim to analyze the protective response to different filoviruses. coronaviruses are a group of single-stranded rna viruses that have been present in humans for at least - years and all originated in bats ( , ) . earlier than , six coronaviruses had been known to cause diseases in humans: hcov- e, hcov- , hcov-nl , hcov-hkn , sars coronavirus (sars-cov), and mers coronavirus (mers-cov) ( ) . in late and early , a novel coronavirus was discovered to be the cause of a rapidly spreading outbreak of respiratory disease, including potentially fatal pneumonia, in wuhan, china. the virus, provisionally designated -ncov and later given the official name sars-cov- , owing to its similarity to sars-cov (then named sars-cov- ), was isolated and the viral genome sequenced. sars-cov- was characterized as a beta-coronavirus ( ) . the disease caused by the virus was officially named coronavirus disease (covid- ) by who. coronaviruses are capable of adapting quickly to new hosts through the processes of genetic recombination and mutation in vivo. point mutations alone are not sufficient to create a new virus. however, this may occur when the same host is simultaneously infected with two coronavirus strains, enabling recombination of genomic fragments of hundreds or thousands of base pairs long and thus making a new virus ( , ) . this susceptibility enabled the emergence, in approximately two decades, of three new human coronavirus species with epidemic potential: sars-cov- , mers-cov, and sars-cov- . coronaviruses enter cells via binding to a host receptor followed by membrane fusion. the angiotensin-converting enzyme (ace ) was identified as the cell receptor for sars-cov ( ) , and recently also for the new sars-cov- ( ), while mers-cov binds the dipeptidyl peptidase (dpp ) receptor, also known as cd ( ) . the s protein is used for virus-cell receptor interaction during viral entry ( ) . transmission of the virus during the viremic stage of disease is primarily via respiratory secretions (droplets) or direct contact. sars-cov- is extremely contagious, with an estimated basic reproduction number (r ) of . - . ( ) . in contrast, the r for both sars-cov- and mers-cov is less than ( ). it soon became apparent that infected individuals might be capable of transmitting the virus during the prodromal period ( ) . social distancing strategies (quarantine and community containment) represent the only efficacious means of controlling coronavirus spread in the absence of effective drugs or vaccine against the pathogens. of importance, for preventing the spread of the disease caused by contact with patients or contaminated fomites, hygiene measures are also mandatory, such as washing hands with soap and water or with alcohol-based preparations. indeed, coronaviruses are able to survive on various surfaces for few days but can be inactivated by disinfection ( ) . finally, because it has been demonstrated that the overlap between human and animal ecosystems have given to coronaviruses the opportunity to cross the species barrier, to prevent future zoonotic diseases, a coordination with veterinary experts as well as stricter laws governing the trade of wild animals would be necessary. humans are extremely exposed to these pathogens because these viruses had not previously circulated in the human population, as testified by the absence of antibodies against coronavirus in healthy people. in addition, the innate immune response has demonstrated to be insufficient in controlling coronavirus infection because decreases in viral load are coincident with the specific antibody response ( , ) . in this context, vaccines represent a much expected resource. a hopeful premise is represented by the successful containment of coronavirus epidemics in farm animals by vaccines, based on either killed or attenuated virus ( ) , and concerning sars-cov- by the finding that specific antibodies are detectable in % of patients with covid- , - days after symptom onset ( ) , and that the magnitude of antibody titers positively correlated with viral neutralization potency ( ) . after the sars outbreak, several vaccines were formulated based on various strategies, as recombinant s protein-based vaccines, attenuated and whole inactivated vaccines, as well as vectored vaccines. pre-clinical data showed animal protection from challenge with sars-cov- . however, sterilizing immunity was not always achieved ( ) . in few cases, the use of live virus as a vaccine resulted in complication including lung damage, eosinophil infiltration, and liver damage in animal models. moreover, a study of vaccination with inactivated sars-cov- in nhps reported enhancement of disease caused by specific epitopes on the s protein [reviewed in ( ) ]. another issue is related to the length of a protective immune response. both humoral and cellular responses have been found important for lasting protection. in long-term studies of recovered sars patients, antibody responses waned after approximately years, while t-cell responses persisted, suggesting that the latter is required for long-lasting immunity. concerning mers-cov, the vaccines proposed target the s protein ( ) ( ) ( ) , including mucosal vaccine for intranasal administration ( ) . however, cases of enhanced lung diseases were also reported in preclinical models of vaccination in mice ( ) . new mers-cov vaccines in development also include live attenuated, protein subunit, and dna vaccines ( , ) . recently, a small animal model that replicates mers-cov transmission has been developed ( ) and will help the preclinical studies. following the alarming data and casualties provoked by covid- , a strong effort by the research community is going on at the moment, and who has been informed of dozens of vaccines in preparation using different platforms, as mentioned in section "vaccine platforms." some of these candidate vaccines are already in phase i/ii clinical trials, while others have been advanced to phase iii studies ( , , ) (table ). however, it is possible that a sars-cov- vaccine will not be available for another - months. recently, a rhesus macaque model that recapitulates sars-cov- infection has been developed to study immunopathogenesis and test vaccine candidates ( , ) . therapy based on passive administration of anti-coronavirus antibodies, isolated from patient sera, also represents a much wanted option for the treatment of coronavirus diseases ( ) , and a global effort is pursued in this direction to treat patients before the achievement of a validated vaccine. in addition, researchers are trying to produce in laboratory specific and protective anti-coronavirus antibodies. in the case of sars outbreak, a monoclonal antibody (mab) with neutralizing activity, being able to block receptor association, was identified and described ( ) . moreover, neutralizing mabs have also been produced to fight mers-cov infection. in a collaborative study by us and chinese researchers, mabs targeting the receptor (cd /dpp ) binding domain of mers-cov spike glycoprotein were reported ( ) . japanese researchers have also investigated anti-cd mab for mers-cov and have identified the humanized mab ys as a promising candidate ( ) . finally, in the case of sars-cov- outbreak, dutch researchers claimed the identification of a human mab named d able to block sars-cov- infection ( ). recently, a mab able to cross-neutralize sars-cov- has been identified from memory b cells of a sars-cov-infected individual. the antibody, named s , engages the s receptor-binding domain, recognizing a highly conserved protein/glycan epitope distinct from the receptor-binding motif ( ) . more recently, other potent neutralizing antibodies were isolated by different research institutions ( ) ( ) ( ) . amidst the gamut of high-affinity antibodies with the potential to neutralize human pathogenic viruses, single-domain antibodies, referred to as nanobodies or nbs ( kda), and nanobody-based human heavy chain antibodies ( kda) derived from camelids might be harnessed as useful therapeutics for the ongoing covid- pandemic ( ) . camelid heavy-chainonly antibodies (hcabs) are composed of two heavy chains with a single variable domain (vhh) as the target-binding module. recombinant vhhs, devoid of the effector domains, act as single-domain antibodies and harbor advantageous features over conventional antibodies (higher thermal and chemical stability, higher solubility, smaller size, lower susceptibility to steric hindrances, ease of manufacturing, and simple structure) to have been recently proposed as prospective therapeutic candidates against various infectious pathogens ( ). vhhs isolated from a llama subcutaneously immunized with perfusionstabilized sars-cov- and mers-cov s proteins have been recently characterized and shown to be able to neutralize s pseudotyped viruses in vitro, interfering with the host cell receptor binding ( ) . interestingly, sars-cov- s-directed vhh cross-reacted with sars-cov- receptor binding domain (rbd) and neutralized sars-cov- s pseudoviruses in vitro as a bivalent human igg fc-fusion format, underscoring the potential of vhhs to treat coronavirus diseases ( ) . because of the global spread of diseases caused by flaviviruses, understanding the cross-reactivity of anti-viral immunity among these viruses is of crucial importance for predicting the evolution of viral disease outbreaks. recently, the analysis of pbmcs isolated from individuals infected by denv or vaccinated with denv tv or yf d vaccines, and pulsed with a pool of antigens from autologous and heterologous flaviviruses, indicated that both cd and cd t-cell responses were specific, with little or no cross-reactivity, despite the high level of homology ( ) . individuals preexposed to denv infection developed t-cell responses against non-structural zika proteins rather than structural envelope protein, suggesting that previous flaviviral infections biased the t-cell response toward more cross-reactive non-structural epitopes ( ) . studies enrolling mothers who gave birth to microcephalic babies after zikv infection, showed serological evidence of a pre-existing anti-dengue response, suggesting that vaccination against denv does not protect against zikv microcephaly ( ) . however, cross-reactive antibodies between zikv and denv have been described, mainly targeting the structural dimeric envelope protein ( , ) . the antigenic sequences are both linear and quaternary, with nabs mainly recognizing the latter. the high-conserved e protein fusion loop induces cross-reactive but weak nabs that can be a marker of worst outcome during subsequent flaviviral infections ( ) . a research concerning zikv-specific b-cell responses in three denv-experienced donors showed that months after the infection, the pool of antibodies comprised both poorly nabs derived from pre-existing denv-induced memory b cells, associated with an enhanced zikv infection in vitro, and potent zikv-specific antibodies originated de novo ( , ) . the possibility that wnv-specific antibodies may drive the infection by other flaviviruses is still controversial, even if cross-reactivity was demonstrated. plasma samples from convalescent human wnv patients were shown to enhance zikv infections by antibody-dependent enhancement (ade) phenomenon ( ) ; conversely, mice previously infected with zikv and challenged with wnv showed enhanced protection toward the second infection ( ) . the immunological flavivirus cross-reactivity, the ade phenomenon (discussed below), genetic mutations that increase the virulence, potential pre-existing immunity concerns, combined with the necessity to increase cost-effectiveness of marketable products are among the issues that have limited the development of successful vaccines until now. the use of t-cell inducing vaccines or proteins with mutations into conserved envelope fusion-loop epitopes might be useful to overcome the cross-reactivity hurdle ( ) . known as ade of viral infection, ade is a phenomenon occurring when antibodies facilitate virus entry into the host cells, driving viral replication and increasing infectivity, with subsequent severe outcomes. among the several stumbling blocks in realizing a safe vaccine, ade is a phenomenon largely underestimated, but that can produce severe adverse effects, rendering vaccinated individuals more predisposed to develop harsh symptoms after infection ( ) . the first report of ade dates ( ) . the molecular mechanisms disclosed the involvement of fcγr ( ) and complement receptors ( ) . when an antiviral antibody (induced by vaccination or viral infection) with no neutralizing or sub-neutralizing activity is produced, it can act like a bridge between the virus and the fcγr expressed on the surface of immune cells, leading to viral uptake (figure ) , as demonstrated for denv, zikv, wnv, influenza, sars-cov, mers-cov, and ebov ( ) . the role of complement receptor has been demonstrated in ebov response: two antibodies directed against epitopes in close proximity bind the c q, forming an immune complex able to enhance the virus entry into a target cell ( ) , whereas in an animal model of mers-cov, c a and c protein level increase was observed after passive immunization ( ) . the first licensed vaccine against denv (cyd-tdv-dengvaxia) caused hospitalizations in two large multicenter phase iii trials; after result revision, it has been estimated that in seronegative individuals, it can produce adverse effects ( ) , and who recommendations are to vaccinate only seropositive individuals in endemic areas of age older than years. using a mathematical model of denv transmission to formulate hypothesis on vaccine trial results, it was speculated that "seronegative recipients gain transient protective cross-reactive immunity akin to that observed for natural infection, " increasing the risk of severe disease after infection, while vaccination of seropositive subjects results in boosting the immune response, producing a protection comparable with the one obtained in individuals who has had two natural infections ( ) . the most severe adverse effect after vaccination was registered when a formalin-inactivated vaccine against rsv produced an increase of severe illness in vaccinated infant (hospitalization: % rsv vaccinated vs. % vaccinated against parainfluenza) and two deaths ( ) . afterward, a role for the th response was hypothesized in generating the rsv-mediated ade ( ) , and it was demonstrated that the formalin-inactivated virus produced ade in monkeys ( ) , suggesting that the carbonyl groups on formaldehyde-inactivated rsv were responsible for the th response in mice ( ) . moreover, the observation that formalin inactivation produced an alteration of antigens, leading to the production of non-nabs, whose avidity did not mature, and the activation of complement were also reported for a measles vaccine ( ) . the low-avidity non-nabs are produced in absence of tlr activation (and affinity maturation), and they figure | antibody-dependent enhancement on dengue infection. antibodies generated from a previous denv infection can recognize but do not neutralize another denv serotype and can lead to antibody-dependent enhancement (ade) of entry of the latter virus into host cells. the pre-existing non-(or sub-) neutralizing antibodies bind denv through the fab domains and mediate viral entry into fcγr-expressing cells. on engagement by the fc domains, the virus-antibody immune complex is internalized by the activating fcγriia within the endosome. co-ligation of fcγriia and lilrb (leukocyte immunoglobulin-like receptor-b ) to opsonized denv drives the inhibitory signal cascade via immunoreceptor tyrosine-based inhibition motif (itim) pathway, abrogating the expression of isgs (interferon stimulated genes). ligation of fcγriia to immune complex also increases th cytokine production and reduces ifnγ, inhibiting the jak/stat signaling pathway, overall resulting in the suppression of the antiviral response and increase of viral replication. nabs, neutralizing antibodies; and itam, immunoreceptor tyrosine-based activation motif. trigger complement activation ( ) , enhancing viral infection. to induce potent nabs, the tlr activation has been obtained using a th -polarizing adjuvant ( ) , in association with the candidate vaccine exposing the epitopes of interest. antibody-dependent enhancement has been reported also in many studies focusing on the development of sars and mers vaccines, demonstrating that vaccination with the whole s glycoprotein can increase the susceptibility to viral infection with a mechanism not linked to the virus receptor expression on the host cells ( ) , and especially when antibodies are induced with low titer ( ) . while for many flaviviruses the mechanism of ade has been explained through evidences that antibodies developed during a primary infection can enhance entry of a heterologous virus via fc-receptor during a secondary infection, for mers-cov and sars-cov, it has also been proposed that nabs that strongly bind the rbd region of the s surface protein can induce conformational changes that enhance the virus entry via canonical viral-receptor-dependent pathways, mimicking viral receptor binding ( , ) , and antibodies targeting a specific region of the s protein enhanced the viral infection in a sars model of nhps ( ) . the high sequence homology and the similarity in structure shared among sars-cov, mers-cov, and sars-cov s glycoproteins raises reasonable concerns about the development of covid- vaccines based on the s protein. in potential pandemic settings, the clinical development of vaccines is the main aim. however, apart from technical reasons, the vaccine production might be delayed also for economic considerations and safety issues. other strategies may be based on self-disseminating vaccines and induction of trained immunity. to control zoonosis, the formulation of self-disseminating vaccines acts at the level of animal, insect, or environmental reservoir, to directly interfere within the animal-to-human transmission ( ) . they are essentially based on replicating viral vectors engineered to express the disease antigen and to target a certain animal population ( ) . global vaccination of animals could be achieved to effectively contain an emerging pathogen within the wildlife reservoir, avoiding its global spread. feasibility concerns, costs, and safety issues should be considered when using this strategy to control reservoirs linked to the emergence of high-risk pathogens. in addition, which animal pathogen will cause a human disease is generally unpredictable. it is interesting to underline that a vaccination of great apes with an engineered specific cmv-based vector has been proposed as a strategy to potentially interrupt (or at least decrease) the zoonotic transmission of ebola virus to humans, being able to protect animals from the lethal viral challenge ( , ) . trained immunity-based vaccines (tibv) might be formulated to stimulate broader anti-infectious responses compared with conventional vaccines for their capacity to increase innate immunity and enhance adaptive responses ( ) . this strategy exploits the ability of innate immune cells (monocytes, macrophages, nk cells) to undergo extensive metabolic and epigenetic reprogramming, following certain vaccinations or infections, and to become primed for a quite long period of time to respond more potently to autologous or heterologous re-infection, mounting the so-called "innate immune memory." triggering of pattern recognition receptors (prrs) by microbial effector stimuli results in increased production of pro-inflammatory cytokines and/or reactive oxygen species, and in enhanced immune responses, regardless the primary stimulation ( ) . many infectious stimuli are considered potent activators of trained innate immunity, including β-glucan and chitin (components of fungal cell wall), lps (a component of the cell wall of gram-negative bacteria), and the bacille calmette-guérin (bcg) vaccine ( ) . thus, tibv should contain pathogen-associated molecular patterns (pamps) to target prrs and subsequently induce trained immune cells. bcg vaccine, vacv, and live attenuated influenza vaccines, together with immunostimulants, could be ascribed to this category of vaccines ( ) . it is worth mentioning that a whole-cell killed bacterial vaccine might have played a role in preventing pneumonia and mortality during the influenza pandemic ( ) . recently, a work by berg and colleagues showed that bcg vaccination is associated with the flattening of the curve in the spread of covid- , suggesting that bcv vaccine might serve as a protective factor against the disease ( ). however, it should also be noted that an enhanced immune response mediated by reprogrammed immune cells could contribute to the development or maintenance of inflammatory, neuroinflammatory, and chronic metabolic disorders ( ) . the phenomenon of "trained immunity" occurring in the brain is known as microglial priming. exposure of primed microglial cells to a second stimuli can cause an augmented inflammatory response, leading to neuroinflammation and production of neurotoxic molecules. the hyperglycemia condition that characterizes type diabetes could long term affect the cellular metabolism of monocytes and macrophages, leading to increased cytokine production and subsequent diabetes complications, including atherosclerosis. an augmented activation of innate cells may also result in the induction and maintenance of chronic inflammatory disorders, including rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, or sarcoidosis ( ) . the covid- pandemic experience, combined with the previous viral disease outbreaks, should give blueprints for rapidly responding to the emergence of high-risk pathogens in the future. it is a common belief that vaccines would be the only means of providing long-term immunity and preventing viral diseases. despite the great progress made in vaccine research, we are still unable to produce successful vaccines in a timely manner. human trials take a long time and given a huge list of vaccine candidates, it is hard to choose the most promising one. while the who proposed a solidarity vaccine trial to test all the candidates in rolling trial until they fail, to increase the chances of succeeding, some vaccine stakeholders are considering extreme alternatives for emergency use: intentionally infect young healthy volunteers at low risk in controlled "human challenge trials" to define which vaccine will work ( ) . although these approaches are already used for studying influenza ( ) and dengue diseases ( ) , it is hard to ethically accept this option without a validated therapy. vaccines go through regulatory pathways before the final approval and licensure. in epidemic or pandemic settings, we need to carefully develop a vaccine, as quickly as possible, that adequately proved to be safe and effective ( ) . scientists need to fill the gaps in understanding the epidemiology of novel viruses, to identify potential zoonotic reservoirs or spill-over hosts, and the way of transmission of pathogens. once the pathogen is identified, preclinical models need to be developed to study virus-host interactions and early test vaccine candidates, defining the immune correlates of protection. pathogen-specific epitopes need to be identified to guide structure-based vaccines that will elicit protective antibodies, minimizing the induction of non-or weakly nabs that would promote ade of viral infection ( ) . moreover, data sharing and collaboration among academia, government, and companies will be essential to coordinate a strategic approach in face of next pandemic threats ( ) . all authors equally contributed to this work and read and approved the final manuscript. this work was supported by prin "nanotechvax tackling biological barriers to antigen delivery by nanotechnological vaccines" prot. zeccm, and consiglio nazionale delle ricerche (cnr), italy: laboratori congiunti bilaterali internazionali (scienze biomediche), project: "new vaccines against poverty-related and neglected tropical diseases." mt was supported by postdoctoral fellowship from cnr, italy: laboratori congiunti bilaterali internazionali (scienze biomediche), project: "new vaccines against poverty-related and neglected tropical diseases." references . nii-trebi ni. emerging and neglected infectious diseases: insights, advances, and challenges infectious disease threats in the twenty-first century: strengthening the global response the epidemic of -novel-coronavirus ( -ncov) pneumonia and insights for emerging infectious diseases in the future major factors affecting the emergence and re-emergence of infectious diseases climate change and multiple emerging infectious diseases infectious disease and economics: the case for considering multi-sectoral impacts emerging infectious diseases: a proactive approach 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memory in health and disease infectious agents as stimuli of trained innate immunity efficacy of whole-cell killed bacterial vaccines in preventing pneumonia and death during the influenza pandemic mandated bacillus calmette-guérin (bcg) vaccination predicts flattened curves for the spread of covid- trained innate immunity not always amicable scores of coronavirus vaccines are in competition -how will scientists choose the best? the future of flu: a review of the human challenge model and systems biology for advancement of influenza vaccinology challenge accepted: human challenge trials for dengue tortoises, hares, and vaccines: a cautionary note for sars-cov- vaccine development rational vaccine design in the time of covid- a strategic approach to covid- vaccine r&d a dictionary of epidemiology the authors declare that the research was conducted in the key: cord- - mqmpzw authors: qian, wei; wei, xiaoqin; guo, kelei; li, yongtao; lin, xian; zou, zhong; zhou, hongbo; jin, meilin title: the c-terminal effector domain of non-structural protein of influenza a virus blocks ifn-β production by targeting tnf receptor-associated factor date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: mqmpzw influenza a virus non-structural protein (ns ) antagonizes interferon response through diverse strategies, particularly by inhibiting the activation of interferon regulatory factor (irf ) and ifn-β transcription. however, the underlying mechanisms used by the ns c-terminal effector domain (ed) to inhibit the activation of ifn-β pathway are not well understood. in this study, we used influenza virus subtype of h n to demonstrate that the ns c-terminal ed but not the n-terminal rna-binding domain, binds tnf receptor-associated factor (traf ). this results in an attenuation of the type i ifn signaling pathway. we found that the ns c-terminal ed (named ns / - ) inhibits the active caspase activation and recruitment domain-containing form of rig-i [rig-i(n)]-induced ifn-β reporter activity, the phosphorylation of irf , and the induction of ifn-β. further analysis showed that ns / - binds to traf through the traf domain, subsequently decreasing traf k -linked ubiquitination. ns / - binding also disrupted the formation of the mitochondrial antiviral signaling (mavs)–traf complex, increasing the recruitment of ikkε to mavs; ultimately shutting down the rig-i(n)-mediated signal transduction and cellular antiviral responses. this attenuation of cellular antiviral responses leads to evasion of the innate immune response. taken together, our findings offer an important insight into the interplay between the influenza virus and host innate immunity. of caspase activation and recruitment domains (card). as the cascade continues, different residues in rig-i are ubiquitinated by the e ligases trim ( ) and riplet ( ) , resulting in rig-i oligomerization. subsequently, rig-i interacts with the adaptor protein mitochondrial antiviral signaling (mavs) via their card-card association, which in turn results in mavs oligomerization. through tnf receptor-associated factor (traf ) or traf , this leads to activation of the kinase complexes containing tbk or ikkε and ikkα/β/γ. through several final phosphorylation steps, these kinases ultimately elicit antiviral and pro-inflammatory responses through interferon regulatory factor (irf ) and nuclear factor κb (nf-κb), respectively ( , ) . influenza a virus belongs to the orthomyxovirus family, containing eight negative-sense rna segments in an enveloped viral particle encoding or proteins ( ) . this array of proteins contributes to virulence; including the proteins associated with viral rna-dependent rna polymerase ( ) and the nonstructural protein (ns ). ns consists of - amino acids and comprises two functional domains: an n-terminal rnabinding domain (rbd) (aa to ) and a c-terminal effector domain (ed) (aa -end) ( ) . the ns protein plays a crucial role in regulating the host antiviral response through various mechanisms. one important function of the ns protein involves inhibition of ifn production. the mechanism of this inhibition includes activation of the transcription factors irf ( ) , nf-κb ( ) , and ap- ( ) , thus blocking ifn production. this efficient inhibitory effect is associated with an rig-i signaling pathway through the ns -rig-i complex ( ) ( ) ( ) . previous studies have indicated that ns is also related to two positive factors of rig-i, the e ligases trim ( ) and riplet ( ) . the residues e / e of ns mediate their interaction with the coiled-coil domain of trim , thus blocking both trim multimerization and rig-i card domain ubiquitination. this subsequently induces lower levels of ifn-β ( ) . ns can also interact with riplet preventing the activation of rig-i, although e /e are not involved in that inhibition ( ) . the dsrna binding ability of ns could also be playing a role in the pre-transcriptional inhibition of the interferon pathway by sequestering the pathogenassociated molecular patterns (pamps) that rig-i recognizes. two residues, r and k , are required for the dsrna binding activity of ns ( ) , thus highly impairing its ability to block interferon production. in another similar pathway, ns has been shown to inhibit host mrna synthesis by binding a cellular ′ end-processing factor, the kda subunit of the cleavage and polyadenylation specificity factor (cpsf ), thus attenuating type i interferon (ifn-α/β) and other interferon stimulated gene (isg) mrnas that are involved in the antiviral response ( ) . the ns proteins encoded by the seasonal h n , h n , h n , and avian h n viral subtypes strongly bind to cpsf ( ) , whereas pr , pandemic h n , and novel h n virus do not efficiently bind cpsf ( ) . it is noteworthy that cells infected with viruses expressing ns proteins in seasonal h n and h n viruses do not inhibit irf activation. however, activation is blocked in cells infected with viruses expressing ns proteins in some, but not all, seasonal h n viruses, pandemic h n , and avian h n viruses. trim was previously reported to interact with each of these ns proteins, whether or not they block irf activation, indicating that binding of trim by the ns protein does not necessarily lead to blocking of irf activation ( ) . hence, binding of the ns protein to dsrna, rig-i, and trim has not established that these ns interactions are responsible for inhibiting the activation of irf and ifn transcription. in this case, one or more host factors may participate in the ns blocking of irf activation. in view of several yet undetermined roles of ns in the inhibition of interferon, we conducted a study aimed to determine the importance of the function of the n-and/or c-terminal domains of the ns protein in immune evasion. by using the luciferase reporter assay, we were able to demonstrate that the c-terminal ed (aa to , named ns / - ) of the ns protein was sufficient to inhibit the production of ifn-β driven by rig-i(n). mechanistically, ns / - was found specifically to interact with traf , to dissociate mavs-traf complex, and to decrease k -linked polyubiquitination of traf . this was shown to result in reduced irf -dependent production of ifn-β, with subsequent enhancement of virus replication. these data reveal a novel mechanism for how the influenza a virus ns protein induces inhibition of the host ifn production and may provide a potential target for antiviral drug development. the hpai h n virus strain, a/duck/hubei/hangmei / (h n ; designated h n /hm) was isolated from a duck. influenza a virus (strain a/puerto rico/ / h n ), a/ pr/ / , was grown in our laboratories and stored until use. rns -sd was constructed as previously described ( ) . influenza virus stocks of h n /hm, pr , and rns -sd strains were amplified using -day-old embryonic chicken eggs and then titrated by determining log tcid /ml values in mdck cells. all cell experiments with h n virus were performed in an animal biosafety level laboratory (bsl- ). this study was carried out in accordance with the recommendations of bsl- , huazhong agricultural university (hzau). the protocol was approved by the bsl- of hzau. the recombinant vesicular stomatitis virus (vsv) encoding green fluorescence protein (vsv-gfp) was a gift from the harbin veterinary research institute (harbin, china). sendai virus (sev) was grown in -day-old embryonic chicken eggs and titrated using a hemagglutination assay as previously described ( ) . human embryonic kidney t cells and hela cells were purchased from atcc (manassas, va, usa) and cultured at °c with % co in roswell park memorial institute- medium (hyclone, china) supplemented with % fetal bovine serum (fbs) (pan-biotech, germany), containing u/ml penicillin, and mg/ml streptomycin (gnm cell lysates and the immunoprecipitates were resolved by - % sds-page and transferred to pure nitrocellulose membranes (ge). the membranes were blocked in % bovine serum albumin (bsa) in tbst buffer for h at room temperature and probed with indicated primary antibodies for - h at room temperature. after hybridizing with either goat anti-rabbit or goat anti-mouse secondary antibodies at a dilution of : , in tbst buffer, the membranes were washed with tbst buffer for four times ( min each) before visualized with ecl reagents (advansta). hela cells were plated onto coverslips in -well plates and transfected with the indicated plasmids. at h post transfection, cells were washed once with phosphate-buffered saline (pbs) and fixed in % paraformaldehyde for min. cells were permeabilized with . % triton x- for min and blocked for h at room temperature with % bsa in pbs, followed by incubation with primary antibody for h. after three washes with pbs containing . % tween , cells were incubated with fitc or cy -conjugated secondary antibodies for h at room temperature and then incubated with ′, -dapi for min. finally, the coverslips were washed extensively and fixed onto slides. images were taken under a zeiss lsm meta confocal microscope (carl zeiss, zena, germany). quantitative real-time pcr (qrt-pcr) total rna was isolated from cells using trizol reagent (invitrogen) following manufacturer's instructions and cdna was prepared by using avian myeloblastosis virus reverse transcriptase (takara). cdna was used for quantification of the indicated mrna copy number on an abi viia pcr system (applied biosystems, usa) by using sybr green master mix (rox). to detect and validate the specific amplification of pcr products, dissociation curve analysis of the products was conducted at the end of each pcr. transcript levels of each gene were normalized with the expression of β-actin, and the −∆∆c t method was used to analyze gene expression in the samples ( ) . the primers used in qrt-pcr are listed in table . three sirna oligonucleotides against traf and the corresponding negative control sirna were obtained from genepharma. sequences are as follows: si- , ′-ccacuggagag augaauau- ′; si- , ′-guugugcagagcaguuaau- ′; and si- , ′-cugguuacuuuggcuauaa- ′. transfection of sirna into t cells was performed by lipofectamine according to manufacturer's instructions. t cells were seeded into -mm dishes and transiently transfected with the indicated plasmids. thirty-six hours after transfection, cells were harvested and the lysates were prepared in a % np- lysis buffer supplemented with a commercially available . % protease inhibitor cocktail and a mm deubiquitinase inhibitor n-ethylmaleimide (sigma-aldrich). samples were immunoprecipitated with µg anti-flag antibodies along with µl protein a/g plus-agarose. polyubiquitination was detected using anti-ha antibodies. influenza a virus infection of a cells a cells were transfected with the indicated plasmids for h at °c. cells were washed twice with f medium and then infected with h n /hm or pr at an indicated moi ( or . , the results are expressed as means ± sd. statistical analyses were performed on data from triplicate experiments by using twotailed student's t-test. a p-value of less than . was considered significant and a p-value of less than . was considered highly significant. the h n ns protein inhibits the rigi(n)-mediated activation of ifn-β via its c-terminal ed in an rna bindingindependent manner with the aim of elucidating the mechanism by which iav ns protein counteracts the host innate immune responses, we generated four truncated h n ns proteins, ns / - , ns / - , ns / - , and ns / - ( figure a) . in order to investigate the function of wtns , and its truncated peptides, we assessed its effect on ifn-β promoter activity using a luciferase reporter assay in t cells. our results showed that wtns , ns / - , and ns / - significantly decreased the ifn-β reporter activities driven by rig-i or rig-i(n). conversely, ns / - did not change the activity of ifn-β reporter, and ns / - only slightly increased the activity compared to an empty vector control ( figure b) . in addition, we found that wtns and all truncated peptides had inhibitory effects on ifn-β reporter activities induced by sev or rns -sd virus infection or transfection of poly(i:c) ( figure b) . this suggests that ns n-terminal rbd alone is sufficient to inhibit the activation of ifn-β only in the presence of dsrna; the c-terminal ed of ns could inhibit the activity of ifn-β reporter in all tested conditions. driven by rig-i(n), ns / - caused a dose-dependent inhibition of ifn-β promoter activity and ifn-β transcription ( figure c) . previous studies indicated that iav ns sequesters dsrna and binds rig-i at its rbd, subsequently inhibiting the activation of irf and preventing the induction of ifn-β ( , ) . our findings reveal that c-terminal ed of ns (ns / - ) blocked rig-i(n)-mediated ifn-β induction in an rna binding-independent manner. transcription factor irf is a key innate immune system component that mediates ifn-β induction. once irf is phosphorylated, it forms a dimer, translocates into the nucleus from the cytoplasm, and induces the expression of ifn-β and isgs through specifically binding to their promoter regions ( ) . in order to further investigate how ns / - inhibits the signaling that mediates type i ifn production, we used an irf -luciferase reporter plasmid, allowing for the measurement of irf activation. as shown in figure a , irf -luciferase reporter activation by rig-i(n) was blocked in t cells overexpressing ns / - or wtns . we next addressed whether ns / - affected the dimerization and nuclear localization of endogenous irf mediated by rig-i(n). non-reduced sds-page and immunoblot analysis of cell lysates after rig-i(n) transfection showed that ns / - or wtns expression produced a considerable reduction in the activated dimer form of irf (figure b) . similarly, rig-i(n)induced phosphorylation of irf was strongly repressed by ns / - or wtns and that phosphorylated irf was largely distributed in nuclear fractions ( figure c) . elisa assays of ifn-β in the medium of cells transfected with plasmids encoding ns / - or wtns along with rig-i(n) showed that both ns / - and wtns inhibited the production of ifn-β ( figure d) . together, these data indicate that ns / - inhibits the expression of type i ifn induced by rig-i(n) through blocking the phosphorylation of irf . previous studies have shown that a recombinant vsv-gfp system can be used as a strategy to screen proteins possessing ifn-antagonizing activity ( ) . in the present study, we employed recombinant vsv-gfp to investigate if ns / - serves as an antagonist of ifn production. when t cells expressed ns / - , a high level of vsv-gfp replication was present, consistent with wtns protein (figure e) , suggesting that the inhibitory effect of ns / - on ifn production is also present during actual viral infection. in order to test the effect of ns / - on various components of the rlr pathway, ns / - and expression (figure a) . by contrast, wtns significantly decreased the rlr adaptor-mediated ifn-β promoter activity. as expected, ns / - exhibited a decrease in the ifn-β mrna level induced by rig-i(n) or mavs ( figure a ). in addition, the secretion of ifn-β and the transcription level of isgs triggered by tbk or irf ( d) in the presence of ns / - were tested. ns / - induced nearly a complete loss of the inhibition of ifn-β and isgs, including oasl, pkr, and mx , whereas wtns strongly blocked the production of ifn-β and isgs mrna expression (figures b,c) . together, these data indicate that ns / - significantly inhibits the cellular antiviral response at the level between mavs and tbk . in the rlr-mediated signaling pathway, traf serves as a critical link between the adaptor mavs and downstream regulatory kinases that are essential for irf activation ( , ) . thus, we hypothesized that traf is the target of ns / - . co-ip experiments revealed that ns / - interacted selectively with traf but not other components (figures a,b) . in another experiment, wtns and ns / - also interacted most potently with traf ( figure c ). this association was confirmed under physiological conditions in an experiment that detected this interaction by overexpression of flag-traf in infected a cells, where rig-i served as a positive control ( figure d) . furthermore, pr ns also interacted with traf in infected a cells (figure e ). to address whether the wtns protein physically interacts with endogenous traf , we performed endogenous ip assays on h n /hm or pr -infected cell lysates. our results showed that traf could be co-precipitated by the ns antibody ( figure f ). in addition, the ns proteins of the strain a/shanghai/ / (h n ) and other avian the promoter activities were detected by the dual-luciferase assay system. all luciferase assays were repeated at least three times, and the data shown are mean ± sd from one representative experiment. significance was analyzed with a two-tailed student's t-test (*p < . or **p < . , ***p < . ). h n strain did not bind traf ( figure s in supplementary material). these results demonstrate that ns / - or wtns interacts with traf in a strain-specific manner. based on the findings that ns / - or wtns interacted with traf , we next asked whether the two molecules co-localize in cells. confocal microscopy revealed the co-staining of ns / - or wtns and traf in cells, suggesting the co-localization of the two proteins ( figure g) . normally, expression of wtns in hela cells resulted in a nuclear localization with minor cytoplasmic staining. traf overexpression led to a marked increase in the cytoplasmic localization of wtns or ns / - . these results indicate that ns / - or wtns colocalize with traf in cells and traf overexpression led to a marked increase of ns / - or wtns cytoplasmic localization. traf is essential for ns / - to downregulate ifn-β the above data showed that ns / - blocks ifn-β induction and interacts with traf . hence, we used traf sirna to determine whether traf is essential for the regulatory function of ns / - in t cells. knockdown of traf by sirna diminished ifn-β promoter activity triggered by rig-i(n) (figures a,b) . in cells with silenced traf , after transfection of rig-i(n), the induction of ifn-β was greatly reduced and the inhibitory effect of ns / - was markedly attenuated. nevertheless, this inhibitory effect was rescued successfully with a traf expression plasmid ( figure c) . these data suggest that traf is necessary for ns / - to decrease ifn-β activity. it is well-known that traf is modified with a polyubiquitin chain to provide a scaffold for complex formation. thus, in order to study the effect of ns / - on traf ubiquitination, co-ip experiments were conducted. flag-traf , pub-ha, and ns / - along with rig-i(n) were cotransfected into t cells and the ubiquitination level of traf was monitored. compared to the control, ns / - reduced the rig-i(n)-induced ubiquitination of traf (figure , left) . to explore the type of traf ubiquitin chains, flag-traf was transfected into t cells with ubiquitin mutants, including the pub-k -ha or pub-k -ha expression plasmid. the results showed that ns / - could decrease the k -linked ubiquitination of traf but not the k -linked ubiquitination of traf (figure , right) . in summary, these results demonstrate that ns / - suppresses the k -linked ubiquitination of traf that is important for the recruitment of the tbk -ikkε kinase complex. expressing aa to and containing the ring domain, the zinc-fingers domain, the isoleucine zipper domain, and flag-traf -td, expressing aa to and containing the traf domain ( figure a ). co-ip assays showed that the traf domain of traf was found to be the crucial region responsible for the association of traf with ns / - ( figure b ) as well as with wtns (data not shown). previously, it has been reported that the tim domain of mavs interacts with amino acid residues y and q within the traf domain of traf ( ) . to examine whether ns / - affected ifn signaling at the level of mavs-traf interaction, mavs-traf association was determined in the presence of ns / - . expression of mavs led to an interaction with traf and was increased by rig-i(n) transfection. however, ns / - and wtns markedly disrupted this interaction by . -and . -fold, respectively ( figure c) . in h n /hm infection of a cells, the mavs-traf complex was decreased by . -fold compared to the control ( figure d) . as reported previously, ikkε is recruited to the c-terminal region of mavs following sev or vsv infection, mediated by lys -linked polyubiquitination of mavs at lys , resulting in the inhibition of downstream ifn signaling ( , ) . therefore, it was necessary to test whether wtns or ns / - affect this process to accomplish its negative regulatory role in ifn-β production after sev infection. the interaction of mavs and ikkε was readily detected by co-ip, while sev infection resulted in a significant decrease in the mavs-ikkε interaction. interestingly, the interaction of mavs and ikkε was remarkably increased when cotransfected with ns / - or wtns (figure e) , indicating that ns / - or ns promotes the recruitment of ikkε to mavs. furthermore, we measured the secretion of ifn-β in cell supernatants. results showed that ns / - or ns inhibited the production of ifn-β mediated by mavs-traf or mavs-ikkε (figure f) , implying that ns / - functions in downregulating ifn expression. taken together, these results indicate that the association of the mavs-traf complex is disrupted by ns / - , which, in turn, blocks ifn-β production. to determine whether the replication of iav is enhanced by the ns / - protein, a cells were transfected with ns / - , or an empty vector, then infected with different titers of h n /hm or pr virus. upon infection with h n /hm or pr virus, ns / - could facilitate transcription of np of both viruses (figure a) , resulting in an increase in np and ha proteins observed in the ns / - group (figure b ). this was confirmed by the titers of h n /hm or pr virus, which significantly increased by -and -fold, respectively, in ns / - overexpressing cells compared with control cells (figure c) . these results demonstrate that ns / - enhances the capacity of iav to replicate in cells. prrs of host cells recognize a pamp and subsequently initiate a series of signaling cascades. the final step is activation of irf and nf-κb, thus inducing the transcription of ifn-β ( ) . given the cascade of responses triggered by the host in response to infection, influenza viruses adapted different strategies to escape the ifn response. this survival tactic has proven successful in order for virus proliferation and infection. both pb and pb -f limit ifn production by associating with mavs ( , , ) . other structural proteins, such as pb , pa, np, and even the genomic rna itself, also contribute to impairing rig-i-mediated antiviral responses ( ) . moreover, ha (ha ) was recently shown to drive the degradation of the ifn receptor chain ifnar , thereby suppressing ifn-triggered jak/stat signaling ( ) . the most effective weapon influenza a viruses have at their disposal is ns protein. the ns protein acts as an antiviral antagonist protein capable of limiting ifn production. rig-i recognizes and binds dsrna structures with ′-triphosphates upon infection to initiate the host antiviral response. during the course of viral infection, the ns protein of iav inhibits host ifn responses either by sequestering viral dsrna or by binding to rig-i and trim or riplet proteins required for rig-i activation and ifn signaling pathways ( , , , ) . in this study, we found that the ns c-terminal ed (aa to ) of h n virus inhibits the activation of ifn-β pathway. to achieve a negative regulatory function in the cellular antiviral response, ns / - associates with traf to remove the lys -polyubiquitin chains on traf and to disrupt the mavs-traf complex. ns / - also increases the recruitment of ikkε to mavs, releasing traf from the mitochondria. this further decreases the level of k linked ubiquitination of traf , impairing irf phosphorylation and reducing the production of ifn-β (figure ) . interestingly, our study has shown that the ed of ns protein possesses the ability to suppress ifn response in the absence of rna. typically, the rbd of ns mediates the inhibition of ifn synthesis, and the ed of ns induces the inhibition of gene expression, together with its known interactors ( ) . in this study, we have found that the ns c-terminal ed (ns / - and ns / - ), but not the rbd (ns / - and ns / - ) block ifn-β reporter activity induced by rig-i(n). expression of ns / - resulted in the inhibition of irf activation, indicating that the ns protein blocks ifn-β activation through an rna-independent manner. it was demonstrated in previous studies that influenza a viruses tx/ and a/viet nam/ / expressing c-terminally truncated ns proteins of , , or amino acids were attenuated. the resultant reduced growth correlated with a high level of ifn-α/β induced by these mutant viruses ( , ) . in addition, in both influenza b and c viruses, the c-terminal domains of the ns proteins were found to possess ifn antagonist activity ( , ) . more importantly, the n terminus-truncated ns proteins encoded by pr , which was to elucidate the mechanism of how the ns ed inhibits ifn-β activation, we speculated that the ns ed contacts its counterparts in rig-i signaling leading to inhibition. for this purpose, we examined a step within the signaling pathway that ns / - targets and found that ns / - acted downstream of mavs and upstream of tbk . the co-ip assays showed unexpectedly that ns / - binds to traf , which interacts with mavs forming a platform for rna virus signaling. we also tested the binding of traf to the full-length ns protein and found that this interaction exists, and co-localized in the cytoplasm. the ns / - protein mainly localized in the cytoplasm. traf expression led to a marked increase in the ns / - cytoplasmic localization, suggesting that ns / - inhibits the activation of the ifn-β pathway. although all types of influenza virus ns proteins interact with trim , only part of ns prevents irf activation, indicating that trim is not required for the inhibition of irf activation. in this study, we did not observe the interaction between ns / - and rig-i, consistent with the results described in a previous publication ( ) . consequently, rig-i seems to be non-essential for the optimal inhibition of ifn production in iav-infected cells. however, our study demonstrated that the influenza a virus ns ed targets traf , subsequently inhibits ifn production, implying that traf is a key factor involved for iav to escape host innate immune responses. the mavs-traf complex is a focal point of rlr-directed signaling response ( , ) . traf localizes to the endoplasmic reticulum (er) and needs to be recruited to mitochondrial mavs in order to activate tbk complexes ( ) . many viral proteins, accessory and non-structural proteins in particular, hijack traf or the traf complex to mediate immune evasion. sars coronavirus m protein or open reading frame- b prevents the formation of traf -tank-tbk /ikkε complex or mavs-traf /traf signalosome to evade host innate immunity ( , ) . sars-cov papain-like protease interacts with and disrupts sting-traf -tbk complex, it also inhibits the tlr -mediated innate immunity through removing lys linked ubiquitin chains of traf and traf ( , ) . herpes simplex virus ubiquitin-specific protease ul deubiquitinates traf then counteracts the ifn-β pathway ( ) . over the past years, there have been major advances in understanding how influenza a viruses successfully escape the surveillance of the immune system. the current report furthers this research revealing the surprising finding that ns / - acts by targeting traf ; specifically, ns / - targets the traf domain of traf . traf links the upstream ifn signaling responses of mavs to tbk relying on the traf domain. this report also shows that a specific interaction between traf and mavs was observed when traf and mavs were co-expressed in t cells. however, the interaction between traf and mavs was disrupted in the presence of ns / - . interestingly, the interaction between mavs and ikkε was markedly increased in ns / - -expressing cells. it has been previously demonstrated that, after sev infection, k -linked polyubiquitination at lys of mavs recruits ikkε to the mitochondria, functionally causes release of traf from mavs initiating the signal to shutdown the ifn response ( ) . the mavs-ikkε complex was enhanced when ns / - was present, indicating that ns / - can utilize this process to shut down further activation of ifn pathway. taken together, these data indicate that ns / - impedes the interactions between components of mavs-traf complex, preventing the phosphorylation of irf , where it would activate the ifn-β response. ubiquitination has emerged as a key posttranslational modification that controls induction and shutdown of the interferon response. traf , serving as a crucial functional link, is modified with a polyubiquitin chain providing a scaffold for complex formation, and, not surprisingly, many viruses encode proteins that inhibit ubiquitination processes to overcome host innate responses. previous studies showed that nairoviruses and arteriviruses encode for ovarian tumor domain-containing proteases that hydrolyze ubiquitin chains from host proteins ( , ) . in this report, we have shown that ns / - suppresses the k linked ubiquitination of traf . it is likely that ns / - works through recruiting a deubiquitinase to cleave the traf ubiquitin chain since it has been shown that ns / - does not belong to any known deubiquitinase family. for example, duba, a member of the otubain (otub) family, has been shown to deubiquitinate traf and negatively regulate tlr -and rig-i/ mda -mediated ifn induction ( ) . it was also shown that two otub deubiquitinating enzyme family members, otub and otub , can deubiquitinate traf and traf , leading to the inhibition of virus-induced ifn-β expression and cellular antiviral responses ( ) . therefore, whether these proteins, or other dub proteins, are involved in this regulation, and the detailed regulatory mechanism of traf activity triggered by ns / - remains to be discovered. interestingly, strain-specific targeting of traf was demonstrated by specific interaction of ns proteins encoded by pr or avian h n but not novel h n or avian h n viruses. this difference may be associated with strain-specific sequence variations. the ns protein most often occurs as a residue peptide, including ns of seasonal h n virus and avian h n virus ( - residues have been deleted since ), which were used in this study. however, premature stop codons or, alternatively, suppression of the genuine stop codon (codon ) resulted in length variations at ns 's c-terminus. abdelwhab et al. analyzed ns protein sequences of all aiv subtypes in birds from to to study the prevalence and distribution of carboxyl terminal end truncation (Δcte). they found that ns proteins lacking amino acids - were the most prevalent form ( %). this truncation is prevalent in lpaiv of non-h /h subtypes; particularly h n , h , and h viruses that are known to be widespread and mostly (semi)endemic in land-based poultry ( ) . similar truncations have also been observed in swine influenza viruses, which harbor a c-terminally truncated ns and have also been found in human h n viruses that have been in public circulation since the pandemic ( ) . hence, whether the interaction of ns and traf are associated with the Δcte requires further investigation. in summary, the present study demonstrated that traf is a target of the c-terminal ed (aa to ) of h n ns protein, revealing a novel function of the ns protein in modulating host innate immunity and possibly facilitating iav infection. the physiological significance of the ns ed in iav replication and its pathological role in flu diseases warrant further investigation to probe the potential value of this molecule as a therapeutic and/or disease prevention target. viral rna detection by rig-i-like receptors sensing viral invasion by rig-i like receptors rig-i-mediated antiviral responses to single-stranded rna bearing '-phosphates rig-i detects viral genomic rna during negative-strand rna virus infection trim ringfinger e ubiquitin ligase is essential for rig-i-mediated antiviral activity riplet/rnf , a ring finger protein, ubiquitinates rig-i to promote interferon-beta induction during the early phase of viral infection the roles of tlrs, rlrs and nlrs in pathogen recognition regulation of rig-i-like receptor signaling by host and viral proteins molecular mechanisms enhancing the proteome of influenza a viruses: an overview of recently discovered proteins the pb subunit of the influenza virus rna polymerase affects virulence by interacting with the mitochondrial antiviral signaling protein and inhibiting expression of beta interferon the multifunctional ns protein of influenza a viruses activation of interferon regulatory factor is inhibited by the influenza a virus ns protein influenza a virus ns protein prevents activation of nf-kappab and induction of alpha/beta interferon the influenza a virus ns protein inhibits activation of jun n-terminal kinase and ap- transcription factors ns protein of influenza a virus inhibits the function of intracytoplasmic pathogen sensor, rig-i inhibition of retinoic acid-inducible gene i-mediated induction of beta interferon by the ns protein of influenza a virus ifnbeta induction by influenza a virus is mediated by rig-i which is regulated by the viral ns protein influenza a virus ns targets the ubiquitin ligase trim to evade recognition by the host viral rna sensor rig-i species-specific inhibition of rig-i ubiquitination and ifn induction by the influenza a virus ns protein rna binding by the novel helical domain of the influenza virus ns protein requires its dimer structure and a small number of specific basic amino acids influenza virus ns protein interacts with the cellular kda subunit of cpsf and inhibits ' end formation of cellular pre-mrnas influenza a virus strains that circulate in humans differ in the ability of their ns proteins to block the activation of irf and interferon-beta transcription a single amino acid substitution in the novel h n influenza a virus ns protein increases cpsf binding and virulence effect on virulence and pathogenicity of h n influenza a virus through truncations of ns eif gi binding domain multiple anti-interferon actions of the influenza a virus ns protein analysis of relative gene expression data using realtime quantitative pcr and the (-delta delta c(t)) method immune signaling by rig-i-like receptors mutations in the ns protein of swine influenza virus impair anti-interferon activity and confer attenuation in pigs critical role of traf in the toll-like receptor-dependent and -independent antiviral response traf : uncovering the real but restricted role in human a functional c-terminal traf -binding site in mavs participates in positive and negative regulation of the ifn antiviral response ubiquitinregulated recruitment of ikappab kinase epsilon to the mavs interferon signaling adapter pathogen recognition and innate immunity the influenza virus protein pb -f inhibits the induction of type i interferon at the level of the mavs adaptor protein influenza virus protein pb -f inhibits the induction of type i interferon by binding to mavs and decreasing mitochondrial membrane potential to conquer the host, influenza virus is packing it in: interferon-antagonistic strategies beyond ns hemagglutinin of influenza a virus antagonizes type i interferon (ifn) responses by inducing degradation of type i ifn receptor live attenuated influenza viruses containing ns truncations as vaccine candidates against h n highly pathogenic avian influenza the n-and c-terminal domains of the ns protein of influenza b virus can independently inhibit irf- and beta interferon promoter activation influenza c virus ns protein counteracts rig-imediated ifn signalling role of n terminus-truncated ns proteins of influenza a virus in inhibiting irf activation triggering the innate antiviral response through irf- activation ikkepsilon and tbk are essential components of the irf signaling pathway proteomic profiling of the traf interactome network reveals a new role for the er-to-golgi transport compartments in innate immunity severe acute respiratory syndrome coronavirus m protein inhibits type i interferon production by impeding the formation of traf .tank.tbk /ikkepsilon complex sars-coronavirus open reading frame- b suppresses innate immunity by targeting mitochondria and the mavs/traf /traf signalosome sars coronavirus papain-like protease inhibits the type i interferon signaling pathway through interaction with the sting-traf -tbk complex sars coronavirus papain-like protease inhibits the tlr signaling pathway through removing lys -linked polyubiquitination of traf and traf herpes simplex virus ubiquitin-specific protease ul inhibits beta interferon production by deubiquitinating traf viral evasion mechanisms of early antiviral responses involving regulation of ubiquitin pathways viral otu deubiquitinases: a structural and functional comparison duba: a deubiquitinase that regulates type i interferon production regulation of virustriggered signaling by otub -and otub -mediated deubiquitination of traf and traf prevalence of the c-terminal truncations of ns in avian influenza a viruses and effect on virulence and replication of a highly pathogenic h n virus in chickens stop-codon variations in non-structural protein ns of avian influenza viruses the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.the reviewer, bn, and handling editor declared their shared affiliation, and the handling editor states that the process nevertheless met the standards of a fair and objective review. no use, distribution or reproduction is permitted which does not comply with these terms.