key: cord-0039502-4l50i80a authors: Dumler, J. Stephen title: Molecular Pathology of Rickettsial Lung Infections date: 2010-05-21 journal: Molecular Pathology of Lung Diseases DOI: 10.1007/978-0-387-72430-0_38 sha: 7cb24a817a3653d80b192987fa593b60b3338521 doc_id: 39502 cord_uid: 4l50i80a Rickettsial infections of humans comprise a diverse group of infections caused by pathogens that are obligate intracellular bacteria with a genetic relationship, including the genera Rickettsia, Orientia, Ehrlichia, and Anaplasma. The host cells of these pathogens largely belie the systemic clinical manifestations, because Rickettsia and Orientia infect endothelial cells, and Ehrlichia and Anaplasma infect circulating leukocytes (monocytes and neutrophils, respectively). Thus, the predominant manifestations (fever, headache, myalgia, with or without rash) do not usually focus attention on the respiratory system; however, the underlying pathogenesis of these infections involves degrees of vascular compromise either by direct injury and inflammation or by the action of vasoactive proinflammatory molecules such as cytokines, chemokines, and prostaglandins. Given that the lung possesses the largest vascular bed in the human body, it is not surprising that pulmonary involvement is periodically identified and, when severely affected, is considered a potentially life-threatening complication.1,2 Rickettsial infections of humans comprise a diverse group of infections caused by pathogens that are obligate intracellular bacteria with a genetic relationship, including the genera Rickettsia, Orientia, Ehrlichia, and Anaplasma. The host cells of these pathogens largely belie the systemic clinical manifestations, because Rickettsia and Orientia infect endothelial cells, and Ehrlichia and Anaplasma infect circulating leukocytes (monocytes and neutrophils, respectively). Thus, the predominant manifestations (fever, headache, myalgia, with or without rash) do not usually focus attention on the respiratory system; however, the underlying pathogenesis of these infections involves degrees of vascular compromise either by direct injury and infl ammation or by the action of vasoactive proinfl ammatory molecules such as cytokines, chemokines, and prostaglandins. Given that the lung possesses the largest vascular bed in the human body, it is not surprising that pulmonary involvement is periodically identifi ed and, when severely affected, is considered a potentially lifethreatening complication. 1, 2 The precise microbial molecular pathogenetic mechanisms and the relative contributions of molecular proinfl ammatory responses toward pulmonary infection with these pathogens are in general poorly understood. However, recent years have seen signifi cant advances in understanding the principles of how these obligate intracellular pathogens interact with host cells to exert direct infl uence over cellular function and integrity and how the host immune system responds. The main purposes of this chapter are to briefl y describe the histopathologic and pathophysiologic alterations observed with two major rickettsial infections that impact the lung, Rocky Mountain spotted fever (Rickettsia rickettsii) and human monocytic ehrlichiosis (Ehrlichia chaffeensis) and to describe the molecular basis of cellular alterations in the host and pathogen virulence mechanisms that belie the pulmonary pathology with these diseases. believed to be due to the local or systemic release of proinfl ammatory vasoactive cytokines that impair endothelial integrity and lead to increased vascular permeability as observed in vasculitis. 7, 8 Of those Anaplasmataceae that are signifi cant causes of human disease, E. chaffeensis is the most frequent cause of infection in which lung structure and function are impaired and thus is the other main topic of this chapter. Clinical Disease and Pathophysiology Rocky Mountain spotted fever is an acute febrile illness that results after transmission of R. rickettsii into a human or animal host after the bite of competent vector ticks. The infection is limited to the Western hemisphere, but infections by related spotted fever group rickettsiae are documented on every continent except Antarctica. 9 After tick bite, the rickettsiae usually disseminate within hours to days via the blood or lymphatics. 10 These obligate intracellular bacteria attach to host endothelial cells in which they become internalized, escape the endocytic vacuole, and propagate within the cytosol. 1 During this process, endothelial cell functions are altered, leading to proinfl ammatory and procoagulant conditions that favor leukocyte infi ltration, focal thrombus formation and increase in vascular permeability. [11] [12] [13] Endothelial cells either release the rickettsiae into the bloodstream or the infected cell is lysed, also releasing bacteria for hematogenous spread into all organs and tissues, including the lung. In the extensive microvasculature of the lung, R. rickettsii infection may be widely spread. 3 Host infl ammatory response, usually manifest as some combination of interstitial mononuclear cell pneumonitis and edema, is observed in histopathologic preparations, leading to the occasional interstitial infi ltrative pattern observed with chest x-rays in RMSF. 14 The fact that pulmonary vasculature permeability increases at a time when multiorgan failure and hypotension are also observed sometimes leads to overly aggressive fl uid therapy that can precipitate or aggravate pulmonary edema. Prompt treatment with doxycycline can arrest and reverse many clinical manifestations, indicating that much of the infl ammatory process is initiated and maintained by the bacterium. The molecular mechanisms by which infection of endothelial cells results in vasculitis and altered vascular permeability in the lung is an area of active study. Initially, bacteria that are inoculated into the host replicate locally and then disseminate into the lymphatics. 10 Thereafter, the rickettsiae enter the bloodstream and interact with endothelial cells in the microvasculature of many organs including the lung. The initial rickettsia-endothelial cell interface is a receptor-ligand mediated event, dependent on rickettsial expression of outer membrane proteins A and B (OmpA and OmpB). [15] [16] [17] The host cell ligand for OmpA, only found in the spotted fever group rickettsiae, is not known. However, for both the spotted fever group rickettsiae Rickettsia japonica and Rickettsia conorii, OmpB ligation occurs via binding to host cell Ku70 that is recruited to host membrane lipid microdomains. 15 After OmpB binding, Ku70 is ubiquitinated by the protein tyrosine kinase adaptor protein Cbl, an event linked to internalization of the rickettsia-containing endosome because its inhibition blocks R. conorii entry. Similarly critical for internalization of rickettsiae is the recruitment of Arp2/3, c-Src, and p80/85 cortactin to binding sites that leads to localized actin cytoskeletal rearrangements in part mediated by Cdc42, phosphatidylinositol-3 kinase, and the Src family of kinases. 18 Spotted fever group rickettsiae also express RickA on one pole of the bacterium, a protein that mediates actin polymerization via Arp2/3 complex assembly, effectively creating an intracellular scaffold that allows propulsion through the host cell and occasionally through host membranes. 19 On entry, the endosomal membrane that contains the rickettsiae is rapidly degraded, presumably by the action of rickettsial phospholipase D or membrane hemolysin TlyC that are actively expressed during this interval. 20, 21 Rickettsial genomes encode an intact tricarboxylic acid cycle, but otherwise have only a limited capacity for energy generation, lacking genes for enzymes for carbohydrate, lipid, nucleotide, and amino acid metabolism. 5 These observations and the demonstration of active adenosine triphosphate/adenosine diphosphate translocases establish the concept of rickettsiae as energy parasites. The presence of an intact pathway for a type IV secretion mechanism underscores the potential importance of transporting rickettsial proteins into the host cytosol. 5 Rickettsia rickettsii infection of endothelial cells leads to membrane injury that can be antagonized by antioxidants, and this membrane injury leads to loss of cellular osmoregulation and eventually cell lysis, even in the absence of large organism loads. 22, 23 The degree of membrane injury in typhus group infections is much less substantial, and cytolysis is generally believed to be mechanical owing to the accumulation of large bacterial quantities within infected cells. 1 Endothelial cells infected by R. rickettsii undergo a number of transcriptional changes, including upregulation of proinfl ammatory cytokines and chemokines (interleukin [IL]-1α, IL-6, IL-8, monocyte chemoattractant protein 1), surface procoagulant activity and tissue factor expression, E-selectin upregulation, and release of von Willebrand factor multimers. 7, 11, 12, 24, 25 The net result of the intracellular infection is an increased proinfl ammatory and procoagulant endothelial cell phenotype. These changes are mediated in part by direct rickettsial activation and nuclear mobilization of nuclear factor (NF)-κB via a mechanism involving activation of inhibitory-κB kinase α and β and phosphorylation-proteolysis of the inhibitor protein IκBα. 26, 27 This phenomenon is abrogated by the bacterial protein synthesis inhibitor doxycycline, implying a contribution of bacterial proteins toward NF-κB proinfl ammatory gene activation. The triggers for the transcriptional alterations are not completely defi ned but appear to involve interactions with protein kinase C isoforms, and are modulated in part by p38 mitogen-activated protein (MAP) kinase. 28, 29 Interestingly, this process also inhibits apoptosis, prolonging survival of infected cells, an obvious advantage for the bacterium. Intracellular rickettsial infection also yields upregulated expression of heme oxygenase 1, a host defense against oxidative injury and a critical regulator of the cyclooxygenases, including cyclooxygenase-2, that are upregulated with rickettsial infection and is a key enzyme that governs prostaglandin production, increased release of prostaglandins I 2 and E 2 , and, indirectly, vascular tone and integrity. 13 The upregulated presence of E-selectin and procoagulant molecules on infected endothelial cells promotes infl ammation and focal thrombosis, features considered typical of rickettsial vasculitis. 11, 12 Although large-vessel thrombosis is atypical for RMSF, fi brin clots are not infrequently detected as eccentrically localized lesions among vessels in which only focal infection is demonstrated (Figure 38 .1). When rickettsial infection occurs within the confi nes of the pulmonary parenchyma, capillaries are the dominant vascular structure and support the heaviest burden of rickettsiae. 3 As a consequence of infection in these small-caliber vessels, interstitial infl ammatory cell infi ltration is the dominant feature and appears as widened, hypercellular alveolar septae, some of which may be edematous; capillaries that are dispersed within the alveolar septae are often surrounded by mononuclear cells, chiefl y lymphocytes and macrophages ( Figure 38 .2). Host response to rickettsial infection is dominated by the infi ltration of tissues and vessel walls by lymphocytes and macrophages. Immunophenotyping methods of rickettsial infl ammatory lesions have identifi ed a polymorphous mixture of CD4 and CD8 T lymphocytes, admixed with scattered B lymphocytes, macrophages, and occasional neutrophils. 1, 30 Infection by rickettsiae leads to substantial chemokine production by endothelial cells, including IL-8, monocyte chemoattractant protein 1, and fractalkine (CX3CL1), and it is likely that these signals in part recruit and retain infl ammatory cells and primed immune cells that participate in the localized tissue vasculitis. 30 As anticipated, effective antirickettsial immunity is predominantly dependent on cellular immunity, especially on expansion of adaptive immunity via CD4 and CD8 cells that produce interferon (IFN)-γ and mediate cytotoxic responses. However, recent investigations provide evidence that antibody plays a more important role than previously ascribed. 30 The infl ammatory and immune response to the presence of rickettsiae in cells is at fi rst heralded by expansion of natural killer cell populations, and depletion of these cells allows greater rickettsial propagation and reduced production of suppressive IFN-γ. Depletion of both CD4 and CD8 T lymphocytes also adversely impacts survival in murine models, and the most dramatic effect implicates a role for major histocompatibility complex I-mediated, perforin-dependent adaptive immune responses. 1, 30 The molecular mechanism of rickettsial restriction by immune cells appears to be dependent on the synergistic effects of IFN-γ, tumor necrosis factor (TNF)-α, IL-1β, and CCL5 produced from natural killer cells, CD8 T cells, and macrophages; critical effectors include both nitric oxide and hydrogen peroxide, accentuated by tryptophan starvation after enhanced host cell degradation of this amino acid essential for bacterial propagation. Interestingly, there appears to be a critical balance between benefi cial and deleterious immune responses, because adoptive transfer of immune CD8 T cells into rickettsia-infected naïve animals accelerates death if introduced during early phases of infection. 1 In the context of RMSF lung involvement, it is likely that many of the events are simultaneously occurring, with outcome dependent on degree of pulmonary microvascular infection and compromise and the degree to which a rapid and protective immune response is induced. 1, 30 Fundamental studies have identifi ed several novel targets for intervention at the level of the bacterium (inhibition of OmpB-Ku70 interaction, inhibition of type IV secretion system activity, inhibition of phospholipase D or hemolysin activity) and at the level of the host (inhibition of ubiquitination of Ku70, inhibition of induced actin cytoskeletal rearrangements or signaling via protein kinase C, p38 MAP kinase, cyclooxygenase-2, and NF-κB nuclear translocation). When involvement of the lung by RMSF becomes evident, it is usually during the course of increasing decompensation, hypotension, and multiorgan failure. Diagnosis of the infection at this interval is often too late to prevent signifi cant morbidity, long-term sequelae, or death, prompting physicians to have a low threshold for empirical doxycycline therapy at earlier times. Rocky Mountain spotted fever is best diagnosed during the active phase of infection by skin biopsy of petechial lesions followed by immunohistochemical or in situ hybridization demonstration of R. rickettsii in the tissue. 31 Antibodies are infrequently present during active infection, but demonstration of seroconversion or a fourfold titer change in convalescence can retrospectively confi rm a clinical diagnosis. Molecular diagnostic methods are less often used and generally focus on polymerase chain reaction (PCR) amplifi cation of R. rickettsii nucleic acids from whole blood samples obtained during the active phase of infection, although this is not currently considered highly sensitive. 32 Additional data suggest that PCR on freshly obtained skin biopsies or other tissues may also work well because the rickettsiae live predominantly within tissue-bound endothelial cells. 33 Immunohistochemistry is often used to establish a postmortem diagnosis, and PCR methods should be excellent adjuncts to this approach. 34 Human monocytic ehrlichiosis (HME) is a febrile illness with many similarities to rickettsial infections such as RMSF. 35 Some authors have used the terminology "spotless" spotted fever to indicate the clinical and historical similarity of HME to RMSF. After tick bite, E. chaffeensis gains access to the blood and may be visualized in peripheral blood smears from some patients after an incubation period of 7-10 days. The infection has been wellcharacterized to occur only in North America, although increasing evidence suggests that the pathogen and infection may be worldwide in distribution. 35 Unlike the situation for R. rickettsii, E. chaffeensis infects almost exclusively mononuclear phagocytes in both tissues and blood. It has been detected in blood, bone marrow, lymph node, liver, spleen, and many tissues and organs that possess mononuclear phagocyte populations or acquire these cells via infl ammatory cell infi ltration. 2, 6 Presumably, E. chaffeensis infects mononuclear phagocytes at the site of tick bite or passes via lymphatics to draining lymph nodes where initial infection occurs. Once E. chaffeensis attaches to and enters the mononuclear phagocyte, it accumulates in an endosomal vacuole that is arrested in maturation at the early endosome stage. 36 The bacteria replicate within this vacuole to form an intracytoplasmic inclusion called a morula that is occasionally visualized in peripheral blood monocytes on Romanowsky-stained blood smears. The infected cells undergo substantial alterations in function that presumably diminish innate and adaptive immune recognition and response; however, cells that manage to ingest E. chaffeensis via opsonophagocytosis generate considerable proinfl ammatory cytokine responses that could drive the underlying infl ammatory cell infi ltration and tissue necrosis observed in some cases. 2, 37 This response does not occur via typical lipopolysaccharide or peptidoglycan-mediated Toll-like receptor signaling because E. chaffeensis, like other Anaplasmataceae, lacks biosynthetic pathways for both of these bacterial components. 38 The majority of clinical infections present with fever and myalgias accompanied by thrombocytopenia, leukopenia, anemia, and evidence of mild to moderate hepatic injury supported by elevated liver transaminase activities in serum. 2, 35 Although E. chaffeensis has no known predilection for the respiratory system, when circulating mononuclear phagocytes become activated for proinfl ammatory function during passage through the pulmonary microvasculature, the result would be increased vascular permeability and infl ammatory cell infi ltration of the interstitial spaces-the typical interstitial pneumonitis observed with HME in severe cases. 6, 39, 40 Infection can precede diffuse alveolar damage (Figure 38. 3), even in the absence of large numbers of bacteria and a vigorous infl ammatory cell infi ltrate. In advanced stages, lung involvement can appear as macrophage-rich intraalveolar infi ltrates, again absent substantial quantities of bacteria (Figure 38.4) . 6 The clinical and histopathologic sequelae of lung involvement can take days or weeks to resolve. This process often occurs in the context of systemic infl ammatory response with fulminant E. chaffeensis infections in patients with preexisting immune compromise such as with human immunodefi ciency virus and immune suppression for organ transplantation or for autoimmune diseases. 2, 41, 42 Very often the presentation is septic-like or toxic shock-like and includes a component of acute respiratory distress syndrome. 39, 43, 44 Despite clinical similarities to RMSF and vasculitis, histopathologic investigations do not provide any support for vasculitis as a component of HME. 6, 41 Although a rapid clinical response is often demonstrated even after 1 to 2 days of doxycycline treatment, given the mononuclear phagocyte niche, it is difficult to explain pancytopenia and hepatic or pulmonary injury based on direct bacterial injury. 2 Most data now support a role for E. chaffeensis triggering of host proinfl ammatory response as a major pathogenetic feature in HME. 8, 45 Early Events in the Ehrlichia-Mononuclear Phagocyte Interaction During the initial encounter with a mononuclear phagocyte, E. chaffeensis binds to E-and L-selectins probably via the bacterial membrane glycoprotein gp120. 46, 47 Interestingly, there are two morphologic forms of E. chaffeensis, analogous to the situation with the Chlamydiae-a lower metabolic activity dense core form that expresses the gp120 adhesin and a more substantially metabolic reticulate form that expresses lower gp120 quantities and undergoes active binary fi ssion. 47 The initial internalization leads to interactions via glycosylphosphatidylinositolanchored proteins and caveolin in host cell membrane lipid rafts that is followed by intracellular calcium fl uxes and changes in tyrosine phosphorylation, activation of phospholipase Cγ2, and inositol 1,4,5-triphophate production. 48 The emerging parasitophorous vacuoles accumulate only early endosomal markers, including an increasing amount of transferrin receptor, a characteristic of recycling endosomes diverted from the phagosomelysosome fusion pathway. 36 Likewise, E. chaffeensisinfected THP-1 cells show downregulated transcription of RAB5A, SNAP23, and STX16, critical components of vesicular transport and fusogenic events. 49 Intracellular entry also occurs in the absence of significant proinfl ammatory cell activation and triggering. 37 Although pretreatment of macrophages with IFN-γ leads to restriction of ehrlichial growth, infection is associated with inhibition of IFN-γ-inducing pathways such as JAK/ STAT, and induction of important cytokines for maturation of Th1 and immune responses, such as IL-12, IL-15, IL-18, Toll-like receptors 2 and 3, and CD14. 48, 49 The IFNγ-mediated restriction occurs via its action in reducing expression of transferrin receptors, thereby reducing accessible free iron for bacterial growth, because E. chaffeensis lacks effective siderophores. 48 Similar to R. rickettsii, E. chaffeensis infection inhibits apoptosis of infected cells, presumably via its action on transcription of cell cycle proteins and inhibition of NF-κB activation. 48, 49 Cellular and Tissue Injury Cytolysis of cells infected by E. chaffeensis in vitro is the usual outcome. However, most in vivo examinations dem-onstrate only meager quantities of bacteria, out of proportion to the degree of histopathologic injury, cytopenias, and hepatic injury in nonfulminant cases. 2 Because no good animal model of E. chaffeensis infection exists, data extrapolated from murine models of infection by related Ehrlichia species provides additional evidence that most tissue injury results from the induction of aberrant and dysfunctional immune responses. 8 Humans infected with E. chaffeensis have a marked expansion of CD8 T lymphocytes in lymph nodes and presumably other tissues, and this feature is associated with a high frequency of hemophagocytic macrophages, suggesting activation of macrophages as a component of the pathogenesis. 50 Similarly, a dose-and route-dependent induction of CD8 T lymphocyte overproduction of TNF-α has been implicated as a mechanism of severe tissue injury in murine models. 8, 45 Infection of TNF receptor-defi cient mice substantially abrogates manifestations of shock in murine models, yet depletion of TNF-α from mice does not alter shock manifestations. 51 In contrast, at least one severe infection in a human occurred while receiving the TNF-α inhibitor etanercept, seeming to contradict the murine model data. 52 Despite these advances, little investigation of the specifi c effectors of tissue injury, whether immunologic or not, has been conducted. Ehrlichia chaffeensis subverts many innate immune responses via interactions with nonopsonophagocytic macrophage receptors (L-selectin or E-Selectin) and by its early downregulation of infl ammation-and immuneinducing signals, receptors, and signaling pathways. 48, 49 Classic cellular immune pathways appear important for restriction of E. chaffeensis infection. 53 In mice that ordinarily are not susceptible, infection persists when devoid of Toll-like receptor 4 and major histocompatibility complex II. 54, 55 Murine models of monocytic ehrlichiosis generally employ the related species Ehrlichia muris or an Ehrlichia species isolated from Ixodes ovatus ticks. 8, 56, 57 In this model, infection and severity are decreased with low infectious doses and intradermal inoculation, and resistance to challenge depends on CD4 but not CD8 T lymphocytes and requires IL-12, IFN-γ, and TNF-α but not IL-4. 8, 45, 51, 58 Likewise, intact adaptive immunity is critical because severe combined immunodefi ciency mice cannot resist infection. 53, 59 Despite the evidence that cellular immunity, chiefl y Th1-polarized responses, are critical for control of Ehrlichia infections, a role for antibody has also been demonstrated with passive immunization using anti-E. chaffeensis or Ehrlichia monoclonal antibodies or with infection of animals devoid of Fc receptors or effector pathways such as phagocyte oxidase (gp91 phox knockout mice). 53, 57, [60] [61] [62] In the context of HME lung involvement, it is most likely that host immune response is a critical determinant of pathologic injury and outcome. The tissue injury that is disproportionate to bacterial load and the lack of any direct evidence of Ehrlichia-mediated cellular injury argue that the predominant pathologic force is an overly aggressive or misdirected host infl ammatory or immune response triggered by active infection. 2, 41, 45, 50 Why some infections are asymptomatic yet others are fatal is not understood, although studies with mouse models are yielding important clues regarding infectious load and genetic background. 8, 53, 58 If pathologic injury is driven primarily by host response, the most prudent approach as a supplement to antimicrobial treatment involves strategies to dampen overly aggressive production of proinfl ammatory cytokines or strategies that dampen vigorous Th1 responses culminating in excessive macrophage activation. That fulminant infection occurs with defects in Tcell immunity dictates that this approach must be carefully evaluated and implemented with great caution. 6, 41, 42, 45, 52 Molecular tools that could interfere with Ehrlichiamediated host transcriptional changes or that interfere with Ehrlichia-initiated signal transduction events and apoptosis delay might provide adjunctive treatments, especially among immunocompromised patients with fulminant infections. 2, 49 Diagnosis Approximately 20% of patients with HME demonstrate cough or other respiratory manifestations; however, signifi cant respiratory disease is relatively infrequent, presenting as acute respiratory distress syndrome, and usually accompanied by other severe systemic manifestations such as multiorgan failure, a shock syndrome, and meningoencephalitis. 2, 41 Even at this late stage, a diagnosis may prevent and reverse adverse outcomes by prompting doxycycline treatment. Although examination of peripheral blood smears will identify bacterial inclusion vacuoles in circulating monocytes in less than 10% of cases, and antibodies will be present in a small minority of infected persons, approximately 60% or more will have E. chaffeensis DNA demonstrable in peripheral blood by PCR. 2, 52 Tissue examination by immunohistochemistry and in situ hybridization is useful in some cases, although these methods may lack sensitivity. 63, 64 Polymerase chain reaction has also been applied as a diagnostic tool for E. chaffeensis on tissues including lung, although no careful evaluation of this approach has been conducted. Many current advances in understanding the molecular pathogenesis of RMSF and HME have been facilitated by the availability and improved annotations of rickettsial genomes; however, these efforts continue to be undermined by the lack of effective gene ablation methods for these pathogens. 5, [65] [66] [67] In time even this research bottleneck will be circumvented, and rickettsial organisms will release their unique secrets for the molecular and cellular perturbations that allow their intracellular survival and pathogenicity. 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