key: cord-0944702-ufr0l4v7
authors: Manik, Moumita; Singh, Rakesh K.
title: Role of toll‐like receptors in modulation of cytokine storm signaling in SARS‐CoV‐2‐induced COVID‐19
date: 2021-10-26
journal: J Med Virol
DOI: 10.1002/jmv.27405
sha: 4fa7b8ffa411f01e86545ec69721508ac3184bc8
doc_id: 944702
cord_uid: ufr0l4v7

Balanced immune regulation is crucial for recognizing an invading pathogen, its killing, and elimination. Toll‐like receptors (TLRs) are the key regulators of the innate immune system. It helps in identifying between self and nonself‐molecule and eventually eliminates the nonself. Endosomal TLR, mainly TLR3, TLR7, TLR8, and membrane‐bound TLR4, has a role in the induction of cytokine storms. TLR7/8 recognizes the ssRNA SARS‐COV‐2 and when it replicates to dsRNA, it is recognized by TLR3 and drives the TRIF‐mediated inflammatory signaling like NF‐κB, MAPK. Such signaling leads to significant transcription and translation of pro‐inflammatory genes, releasing inflammatory molecules into the systemic circulation, causing an imbalance in the system. So, whenever an imbalance occurs, a surge in the pro‐inflammatory mediators is observed in the blood, including cytokines like interleukin (IL)‐2, IL‐4, IL‐6, IL‐1β, IL‐8, interferon (IFN)‐γ, tumor necrosis factor (TNF)‐α. IL‐6 and IL‐1β are one of the driving factors for bringing the cytokine storm into the systemic circulation, which migrates into the other organs, causing multiple organ failures leading to the death of the individual with severe illness.

nucleic acid by the cell surface or cytosolic PRR. 10 TLRs are a class of membrane PRR that detect the microbes on the cell surface as well as in the cytoplasm. These receptors detect both the DNA as well as RNA of the invading pathogen. Each TLR is composed of leucine-rich repeats (LRR) ectodomain, a transmembrane domain, and a cytoplasmic domain, through which it mediates the downstream signaling. 11 A total of 11 types of TLRs have been found in human beings, among those some are membrane-bound and some are intracellular receptors. TLR3, TLR7, TLR8, TLR9 are endosomal in nature. 12, 13 These are expressed in alveolar and bronchial epithelial cells, whereas TLR2, TLR4, TLR6 are restricted to the cellular membrane. The TLRs can also be classified based on detecting PAMP. TLR4 detects glycoprotein, TLR7 and TLR8 detects viral single-stranded ribonucleic acid (ssRNA), TLR3 detects viral double-stranded RNA (dsRNA), while TLR9 detects viral deoxyribonucleic acid (DNA). 14 The known recognition of genetic material of SARS-COV-2 (i.e., ssRNA) occurs through TLR4, TLR7, or TLR8. Interaction of TLRs with PAMP stimulates NF-κB and IFN regulatory factor (IRF) leading to the production of Type I IFN, which activates the adaptive immune responses considered as the main antiviral response. 15 

TLR4 is a membrane receptor that regulates the inflammatory response by recognizing PAMP as well as DAMP. TLR4 remains attached to alveolar cells and bronchial epithelial cells in the lungs.

TLR4 is widely known for its function in recognizing bacterial LPS but their association in SARS-COV-2 pathogenesis has been found as well. SARS-COV-2 bind with TLR4 and activate transcription factors like AP-1, NF-kB, and IRF. TLR4 was also found to regulate IL-6 via NF-kB. Some researchers have found oxidized phospholipid (OxPL) mediated TLR4 response in SARS-COV-1 infection resulting in cytokine production and lung injury. 15 A few other research studies have found the involvement of TLR4 in SARS-COV-2 through molecular docking study revealing the interaction between spike protein and cell surface TLRs. The binding interaction of spike protein and TLR1, TLR4, TLR6 is found significant with the respective binding energy of −57.3, −120.3, and −68.4. 13, 16 The interaction of spike protein of SARS-COV-2 with TLR4 was found to be the highest than other TLRs. However, the reason behind such strong recognition of TLR4 by SARS-COV-2 having ssRNA was not found. SARS-COV-2 should be recognized by endosomal TLR7 and TLR8 because it has ssRNA; however, due to the concept that immune response always happens on the basis of host-viral interaction, it interacts with cell surface stimulating the downstream pro-inflammatory signaling. 9 TLR mediate their function by two pathways MyD88 and toll/IL-1 receptor/resistance protein domain-containing adapter-inducing interferon-β (TRIF)-dependent and MyD88 and TRIF-independent pathways ( Figure 1 ). TLR4 mediates through both the MyD88 and

Endothelial cells are loaded with ACE2 receptors and their density depends on the tissue/organ in which they are present. The interaction of ACE2 with SARS-COV-2 may mediate the viral entry. Toll-like receptor 4 (TLR4) is a cell-surface receptor, whereas TLR3, TLR7, TLR8 are endosomal. TLR4 will induce JAK/STAT, NF-κB inflammatory signaling, whereas endosomal TLRs also induce similar inflammatory signaling via different adapter molecules. For example, TLR7/8 via MyD88 and TLR3 via TRIF adapter molecule (TRAM) TRIF pathways, TLR3 signals through TRIF, and all the other TLRs mediate through the MyD88 pathway. Activation of MyD88 leads to activation of type-1 IFN responses and increases the expression of pro-inflammatory cytokine. Both the pathways contribute to the augmented cytokine in body, directly or indirectly. 13, [17] [18] [19] 2.3 | TLR7/8 receptor The virus may get recognition by endosomal-located TLR7 and activate the MyD88-dependent MAPK -NF-κB pathway leading to the production of TNF-α, ILs, especially IL-6. The level of IL-6 is found to be significantly elevated in the serum of SARS-COV-2 patients during cytokine storms. 15, 20 IL-6 inactivates cell-mediated antiviral response by inhibiting the expression of cytotoxic T-cells, SOCS-3 signaling, and increase in expression of PD-1 cells. 21 However, the recognition of the ssRNA fragments in SARS-COV-2 by TLR7/8 has also been reported. 13 TLR7 and TLR8 are considered phylogenetically and structurally similar, but different TLR7 and TLR8 agonists produce different types of cytokines.

The elevated level of pro-inflammatory cytokines is also found to be mediated by TLR8, proved by bioinformatic analysis. 13 receptor-mediated infection. 24 The expression of TLR9 may increase due to predisposition of genetic factors, gender, and carboxy-alkylpyrrole protein adducts (CAPs)-mainly released by damaged host cells in response to oxidative stress and known to induce TLR9/ Myd88 pathway. 16 in turn activate pro-IL-1β into IL-1β ( Figure 2 ). This activates a process of cell pyroptosis. IL-1β is the main activator of IL-6 and contributes to the aggravation of cytokine storms. 15, [29] [30] [31] TLRs are solely responsible for mediating innate immune signaling. It is no wonder that blocking of TLR could suppress IFN-related signaling, which could possibly increase the viral load, but in cases of preexisting chronic autoimmune diseases, where the immunity system is hyperactive like cancer, SLE, rheumatoid arthritis, and so forth, 4 TLR7, 8, 9 antagonists may prove to show their benefits. As SARS-COV-2 targets TLR3, 4, 7, 8, 9 receptors to enter the host system, hence TLR blockers may prove to be beneficial in this deadly infection. 8 There are some drugs like hydroxychloroquine under Phase III clinical trial (NCT04448756) that has shown the endosomal TLR signaling (TLR3, 7, 8, 9) inhibition, which possibly could delimit the viral entry into the host; whereas M5049 developed by Merck, Germany, is under Phase II trial have also shown the inhibition of TLR7 and TLR8 by recognizing the genomic RNA of invading virus. 32 Hence, by scrutinizing both the limitations as well as the benefits of 36, 38, 39 : first, due to the high binding of SARS-COV-2 with ACE2 receptor in host cells, which was not observed earlier; and second, the presence of four amino acid residues between two subunits of spike protein (S): S1 and S2, which leads to the introduction of a novel "furin cleavage site." The exact function of this cleavage site is yet to be known, but due to the expression of the furin proteases, it facilitates the processing of the spike protein subunit, which activates the binding of SARS-COV-2 with the ACE2 receptor. 4, 35 These two reasons have a major impact on the expansion of SARS-COV-2 in other tissue or organ contributing to organ damage. Also, multiple organ failure has been observed in male patients more widely than that of females, particularly in aged individuals. This is because ACE2 is encoded by a gene expression on X-chromosomes. Females possess a pair of X-chromosomes; hence a much stronger immune mechanism is found in them, even if one of the X-chromosomes stays inactivated. The expression of TLR7 is also higher in females, providing them antiviral response by producing a large amount of antibodies by TLR7, as a result of which they possess F I G U R E 2 NF-κB activation occurs due to toll-like receptor (TLR)-mediated inflammation results in DAMP production. The ligation of the DAMP with nod-like receptor (NLR) produces inflammasomes, which activate the pro-caspase I into active caspase I, ultimately activating interleukin (IL)-1β F I G U R E 3 The cytokine storm originates in the lungs and is released into the bloodstream. After it reaches the systemic circulation, it produces hyperinflammation and imparts damage to the vital organs. In critically ill patients such inflammation spread into various other organs as well, may cause multiple organ failure a much stronger immunity than males, and can fight against singlestranded SARS-COV-2. 5 However, due to a single X-chromosomes male are more prone to the ssRNA SARS-COV-2. In a recent study through single-cell analysis, it has been found that Asian men have higher expression of ACE2 receptors in the lungs than women, hence making them more prone to SARS-COV-2 infection and disease severity. The expression of ACE2 increases with age, which is one of the possibilities why old people are more affected than young people. 5 However, a case report presented a frequent visitor young man of China seafood market, who was found to be severely affected by SARS-COV-2 infection and suffered multiple organ damage. 40 The patient was a nonsmoker and had no history of any chronic disease. The renin-angiotensin-system (RAS) and its receptor, ACE2 are highly correlated with invasion and initiation of inflammation associated with COVID-19 in the lungs. 41 Angiotensin I (Ang I) is an inactive peptide that gets activated to Ang II by ACE activity. Ang II a potent vasoconstrictor, has an important role in maintaining blood pressure and in cell proliferation by mediating the release of proinflammatory cytokines. Ang II gets converted to Ang (1-7) by ACE2. 4 Under normal circumstances, Ang II directly binds to the NF-κB and expresses inflammatory cytokines; whereas Ang (1-7) maintains the anti-inflammatory response by upregulation of P13K/ Akt and ERK signaling. But due to the binding of SARS-COV-2 with ACE2 receptors, the free concentration of ACE2 decreases, and as a result, Ang II will not get converted into Ang (1-7) leading to the accumulation of Ang II. This may further augment inflammation and pulmonary injury. 43, 45 Endothelial dysfunction is a result of cytokine storm, which activates the complement cascade, increasing vascular permeability and vasculitis. Due to the wide expression of ACE2 receptors on the endothelial cell surface, the virus entry into the host becomes easier.

The complement activation is a defensive mechanism and will take place via the lectin pathway and followed by the classical pathway, resulting in the accumulation of complement 3b (C3b), which triggers the formation of C5 and its product C5a to C5b-9. 46 Apart from inducing vascular inflammation, C5a and C5b-9 promote thrombomodulin loss, P-selectin exocytosis and von-Willebrand factor triggering coagulation cascade. This may further result in vascular injury and platelet aggregation. 46 

Severe cases of COVID-19 may cause cardiovascular disorders like myocardial injury, acute coronary syndrome, thromboembolism, cardiac failure. 33 The cardiac symptoms could be a clinical symptom for the patients who are not showing the symptoms of SARS-COV-2 infection like dry cough and fever. Cardiac pericytes have a higher expression of ACE2 than cardiomyocytes. 35 Cardiac pericytes are responsible for endothelial stability, binding of SARS-COV-2 causes endothelial dysfunction and results in microcirculatory disorders.

Hence, despite low expression of ACE2, the patient suffers cardiac injury. Patients with a history of cardiac failure have highly expressed ACE2, hence are more susceptible to cardiac injury. 33, 48, 50 TLR4 is expressed on cardiomyocytes, fibroblasts, and macrophages. Among all the TLRs, TLR4 is the most widely expressed on cardiomyocytes. 35 An association between TLR4 and Ang II is known to exist. Ang II is found to mediate vascular dysfunction through TLR4. SARS-COV-2 downregulates the ACE2 receptor, which decreases the conversion of ACE2 into Ang (1-7). Ang (1-7) has efficacy in opposing pro-inflammatory, profibrotic, vasoconstriction activity, and its decreased level results in exaggeration of the cytokine storm. 51 TLR4 is also associated with high blood pressure in several models depicting hypertension. The pro-inflammatory response to Ang II has been mediated by TLR7/8/9 in response to the exogenous ligand in spontaneously hypertensive rats. TLR9 is considered as a negative regulator for cardiac vagal tone as well as for baroreflex function, activation of TLR9 results in the increment in blood pressure and vascular dysfunction in a normotensive rat. 18 Hence, TLR9 activation through SARS-COV-2 may result in vascular MANIK AND SINGH | 5 dysfunction. TLR activation was also found to mediate calcium homeostasis in cells. 24 of viral entry causing acute kidney injury (AKI) has not been evidenced yet. AKI has been seen widely in patients with ARDS suggesting lung-kidney crosstalk postinfection. 20, 52 The possible mechanism was described as hemodynamic effect, hypoxemia, and hyperinflammation associated with ARDS. AKI may increase the levels of inflammatory cytokines production, especially IL-6, and decrease their clearance. IL-6 causes infiltration of neutrophils and macrophages, which increases lung permeability. 37, 53 The viral entry showed the activation of the complement system in the kidney. The complement C5a is responsible for neutrophil attraction to the sites, platelet aggregation, and endothelial injury. Altogether these damaging effects may contribute to the worsening of AKI. In the kidney biopsy studies, platelets adhesion along with fibrin were seen in peritubular capillaries and venules. TLR4 mRNA expression has been found on endothelial cells as well as tubular cells of the kidney, activation of which by SARS-COV-2 could contribute to tubular necrosis, ischemia, inflammation, and injury. 20,53 However, a direct effect of cytokine storm has shown its effect on renal epithelial cells along with coagulopathy and hemodynamic changes. 12, 46 Not only these vital organs are susceptible to SARS-COV-2, but also it has an impact on the metabolism of the host, it may also affect the gastric system causing diarrhea and vomiting postinfection. It also affects patients with diabetes mellitus, as it messes with the metabolism of the host, causing glucotoxicity and lipotoxicity.

The cytokine storm refers to the abnormal increment in the level of from the lung to other organs such as the kidney, heart, pancreas, and so forth, it may stimulate an exaggerated inflammation and cause damage to that organ. 29, 57 Hence, it can be said that the cytokine storms are a crucial cause of ARDS, systemic inflammatory response, and multiple organ failure in SARS-COV-2. Based on these consequences, the cytokine storm has been categorized into two stages: the temporary immune-deficient condition is described as the first stage, whereas the hyperactive immune state resulting due to the compensatory mechanism to eliminate the virus, failure of which spike up the cytokine levels, is considered as the second stage.

The cytokine storm can also be mediated either by ACE2mediated inflammatory response, by cell pyroptosis or by the delayed release of Type I IFNs. Cell pyroptosis is caused due to programmed cell deaths in SARS-COV-2, which occurs by activation of NLRP3 inflammasomes by viroporin 3, a protein from coronavirus having a role in IL-1β production. 10, 54, 58 Delayed release of type-I IFN is due to the activation of the JAK/STAT pathway initiating the transcription of the ISG and driving the production of cytokines like IL-1β, IL-6, and so forth, which are mainly pro-inflammatory in nature. 59 This IL-1β is highly responsible for the induction of bronchiolar and alveolar inflammation, whereas both the IL-1B and IL-6 activate the complement cascade that increases the vascular permeability. have shown a correlation (R = 0.902) between serum viral load (RNAaemia) and the occurrence of cytokine storm, which is directly related to the level of IL-6. Apart from highly elevated IL-6, a low count of lymphocytes and thrombocytes may also predict disease severity in COVID-19. 18 The TLR induced organ damage has also been related to ACE2 polymorphism and its expression across patient gender and age group. Recently WHO has reported MIS-C in children, hence the reason behind this exception also needs to be explored.

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How to cite this article: Manik M, Singh RK. Role of toll-like receptors in modulation of cytokine storm signaling in SARS-CoV-2-induced COVID-19

The authors declare that there are no conflict of interests.

Moumita Manik has prepared the first draft. Rakesh K. Singh has conceptualized, read, and modified the final draft of the manuscript for submission. Both the authors have read and finalized the submitted version of the manuscript.

Data are available from the corresponding author upon reasonable request.

http://orcid.org/0000-0002-7834-6162