Famotidine inhibits toll-like receptor 3-mediated inflammatory signaling in SARS-CoV-2 infection
2021; Elsevier BV; Volume: 297; Issue: 2 Linguagem: Inglês
10.1016/j.jbc.2021.100925
ISSN1083-351X
AutoresRukmini Mukherjee, Anshu Bhattacharya, Denisa Bojková, Ahmad Reza Mehdipour, Dong Hyuk Shin, Khadija Shahed Khan, Hayley Hei-Yin Cheung, Kam‐Bo Wong, Wai‐Lung Ng, Jindřich Činátl, Paul P. Geurink, Gerbrand J. van der Heden van Noort, Krishnaraj Rajalingam, Sandra Ciesek, Gerhard Hummer, Ivan Đikić,
Tópico(s)SARS-CoV-2 and COVID-19 Research
ResumoApart from prevention using vaccinations, the management options for COVID-19 remain limited. In retrospective cohort studies, use of famotidine, a specific oral H2 receptor antagonist (antihistamine), has been associated with reduced risk of intubation and death in patients hospitalized with COVID-19. In a case series, nonhospitalized patients with COVID-19 experienced rapid symptom resolution after taking famotidine, but the molecular basis of these observations remains elusive. Here we show using biochemical, cellular, and functional assays that famotidine has no effect on viral replication or viral protease activity. However, famotidine can affect histamine-induced signaling processes in infected Caco2 cells. Specifically, famotidine treatment inhibits histamine-induced expression of Toll-like receptor 3 (TLR3) in SARS-CoV-2 infected cells and can reduce TLR3-dependent signaling processes that culminate in activation of IRF3 and the NF-κB pathway, subsequently controlling antiviral and inflammatory responses. SARS-CoV-2-infected cells treated with famotidine demonstrate reduced expression levels of the inflammatory mediators CCL-2 and IL6, drivers of the cytokine release syndrome that precipitates poor outcome for patients with COVID-19. Given that pharmacokinetic studies indicate that famotidine can reach concentrations in blood that suffice to antagonize histamine H2 receptors expressed in mast cells, neutrophils, and eosinophils, these observations explain how famotidine may contribute to the reduced histamine-induced inflammation and cytokine release, thereby improving the outcome for patients with COVID-19. Apart from prevention using vaccinations, the management options for COVID-19 remain limited. In retrospective cohort studies, use of famotidine, a specific oral H2 receptor antagonist (antihistamine), has been associated with reduced risk of intubation and death in patients hospitalized with COVID-19. In a case series, nonhospitalized patients with COVID-19 experienced rapid symptom resolution after taking famotidine, but the molecular basis of these observations remains elusive. Here we show using biochemical, cellular, and functional assays that famotidine has no effect on viral replication or viral protease activity. However, famotidine can affect histamine-induced signaling processes in infected Caco2 cells. Specifically, famotidine treatment inhibits histamine-induced expression of Toll-like receptor 3 (TLR3) in SARS-CoV-2 infected cells and can reduce TLR3-dependent signaling processes that culminate in activation of IRF3 and the NF-κB pathway, subsequently controlling antiviral and inflammatory responses. SARS-CoV-2-infected cells treated with famotidine demonstrate reduced expression levels of the inflammatory mediators CCL-2 and IL6, drivers of the cytokine release syndrome that precipitates poor outcome for patients with COVID-19. Given that pharmacokinetic studies indicate that famotidine can reach concentrations in blood that suffice to antagonize histamine H2 receptors expressed in mast cells, neutrophils, and eosinophils, these observations explain how famotidine may contribute to the reduced histamine-induced inflammation and cytokine release, thereby improving the outcome for patients with COVID-19. The COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) affected more than 100 million people with more than 2 million deaths as of February 18, 2021. Many drugs are being repurposed for COVID-19 therapy, often based on insufficient evidence from randomized trials. Famotidine is an approved drug for peptic ulcers and gastroesophageal reflux disease (GERD) (Fig. 1A). It acts as a competitive antagonist of histamine in gastric parietal cells. Histamine activates protein kinase A (PKA), which causes movement of H+/K+ transporters to the membrane resulting in more acid secretion. Famotidine counters this activity, thereby reducing acid secretion in GERD patients. Several independent case studies on COVID-19 patients have suggested the use of this H2-receptor antagonist in the treatment of disease. Famotidine reduces the risk of intubation and death in hospitalized COVID-19 patients (1Freedberg D.E. Conigliaro J. Wang T.C. Tracey K.J. Callahan M.V. Abrams J.A. Famotidine Research GroupFamotidine use is associated with improved clinical outcomes in hospitalized COVID-19 patients: A propensity score matched retrospective cohort study.Gastroenterology. 2020; 159: 1129-1131.e3Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 2Mather J.F. Seip R.L. McKay R.G. Impact of Famotidine use on clinical outcomes of hospitalized patients with COVID-19.Am. J. Gastroenterol. 2020; 115: 1617-1623Crossref PubMed Scopus (85) Google Scholar). It has also been suggested to reduce respiratory symptoms in nonhospitalized patients (3Janowitz T. Gablenz E. Pattinson D. Wang T.C. Conigliaro J. Tracey K. Tuveson D. Famotidine use and quantitative symptom tracking for COVID-19 in non-hospitalised patients: A case series.Gut. 2020; 69: 1592-1597Crossref PubMed Scopus (84) Google Scholar). In combination with the H1-receptor antagonist cetirizine, it has been shown to reduce pulmonary distress in hospitalized COVID-19 patients (4Hogan II, R.B. Hogan III, R.B. Cannon T. Rappai M. Studdard J. Paul D. Dooley T.P. Dual-histamine receptor blockade with cetirizine-famotidine reduces pulmonary symptoms in COVID-19 patients.Pulm. Pharmacol. Ther. 2020; 63: 101942Crossref PubMed Scopus (52) Google Scholar). These studies indicated that famotidine has a beneficial role in managing COVID-19 disease symptoms, but the molecular basis of these observations remained elusive. The SARS-CoV-2 main protease (CoV-2 3CLpro) and Papain-like protease (SARS-CoV-2 PLpro) have been suggested to be the targets of famotidine based on several virtual screening studies (5Wu C. Liu Y. Yang Y. Zhang P. Zhong W. Wang Y. Wang Q. Xu Y. Li M. Li X. Zheng M. Chen L. Li H. Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods.Acta Pharm. Sin. 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The histamine H2 receptor targeted by famotidine is not limited to the stomach, but is also found in the brain, the endocrine and exocrine glands, the pulmonary system, and the cardiovascular system. H2 receptors are also present on mast cells (MCs), which are deregulated in viral infections including those caused by coronaviruses (8Kritas S.K. Ronconi G. Caraffa A. Gallenga C.E. Ross R. Conti P. Mast cells contribute to coronavirus-induced inflammation: New anti-inflammatory strategy.J. Biol. Regul. Homeost Agents. 2020; 34: 9-14PubMed Google Scholar, 9Malone R.W. Tisdall P. Fremont-Smith P. Liu Y. Huang X.P. White K.M. Miorin L. Moreno E. Assaf A. Delaforge E. Hennecker C.D. Wang G. Pottel J. Blair R.V. Roy C.J. et al.COVID-19: Famotidine, Histamine, Mast Cells, and Mechanisms. Front. Pharm., 2021Google Scholar, 10Theoharides T.C. Potential association of mast cells with COVID-19.Ann. Allergy Asthma Immunol. 2021; 126: 217-218Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Studies show that famotidine (unlike cimetidine) reaches systemic concentrations that are sufficient to antagonize H2 receptors on other cell types such as those on MCs and neutrophils (9Malone R.W. Tisdall P. Fremont-Smith P. Liu Y. Huang X.P. White K.M. Miorin L. Moreno E. Assaf A. Delaforge E. Hennecker C.D. Wang G. Pottel J. Blair R.V. Roy C.J. et al.COVID-19: Famotidine, Histamine, Mast Cells, and Mechanisms. Front. Pharm., 2021Google Scholar). Deregulation of MCs contributes to a robust inflammatory response leading to a pulmonary cytokine storm that is seen in severe COVID-19 infections (11Afrin L.B. Weinstock L.B. Molderings G.J. Covid-19 hyperinflammation and post-Covid-19 illness may be rooted in mast cell activation syndrome.Int. J. Infect. Dis. 2020; 100: 327-332Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). 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Toll-like receptor 3 signaling via TRIF contributes to a protective innate immune response to severe acute respiratory syndrome coronavirus infection.MBio. 2015; 6: e00638Crossref PubMed Scopus (329) Google Scholar). In humans, the TLR family comprises ten members (TLR1–TLR10), which are expressed in innate immune cells including macrophages, epithelial cells, and MCs. TLRs are pattern recognition receptors that recognize several pathogen-associated molecular patterns (PAMPs) present in bacteria, viruses, and other pathogens. TLRs on activation produce inflammatory cytokines, type I IFN, and other mediators. TLRs can be localized either on the cell surface (TLR-1, -2, -4, -5, -6, -10) or in endosomes (TLR-3, -7, -8, -9). SARS-CoV-2 enters the cell through the endosomal pathway and hence activates the endosomal TLRs. TLR activation via MyD88-dependent and TRIF-dependent pathways causes nuclear translocation of the transcription factors NF-κB, IRF-3, and IRF-7, with production of innate proinflammatory cytokines (IL-1, IL- 6, TNF-α) and type I IFN-α/β, which are essential for antiviral responses. TLR3-dependent signaling is an important innate immune response to coronaviral infections (16Iwasaki A. Yang Y. The potential danger of suboptimal antibody responses in COVID-19.Nat. Rev. Immunol. 2020; 20: 339-341Crossref PubMed Scopus (346) Google Scholar). Though TLR3 is beneficial in the initial viral clearance, hyperactivation may contribute to hyperinflammation and cause cytokine storms characteristic of severe cases of the disease (17Onofrio L. Caraglia M. Facchini G. Margherita V. Placido S.D. Buonerba C. Toll-like receptors and COVID-19: a two-faced story with an exciting ending.Future Sci. OA. 2020; 6: FSO6O5Crossref Scopus (82) Google Scholar). In this study, we assess the effect of famotidine on viral proteases (SARS-CoV-2 3CLPro and PLpro) and host cells using computational docking, in vitro and cell biological assays and infection studies. Our studies show that on infection, famotidine does not affect the viral life cycle and replication, but affects host cells by histamine-induced signaling processes. In cells treated with poly(I:C), which mimics viral RNA, histamine increases TLR3 expression leading to an increase in the downstream IRF3 and NF-κB-dependent signaling. SARS-CoV-2-infected Caco2 cells pretreated with famotidine show reduced activation of IRF3/NF-κB and had lower mRNA levels of the interferon gene ISG15 and inflammatory mediator CCL2. The inhibitory effect of famotidine on TBK1/IRF3 signaling can be restored by pretreating cells with the TLR3 inhibitor CU CPT4a. These observations indicate a molecular basis of how on-target effects of famotidine may help in management of histamine-induced inflammation in severe COVID-19 patients. Since several studies suggested that SARS-CoV-2 PLpro may be a target of famotidine (5Wu C. Liu Y. Yang Y. Zhang P. Zhong W. Wang Y. Wang Q. Xu Y. Li M. Li X. Zheng M. Chen L. Li H. Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods.Acta Pharm. Sin. B. 2020; 10: 766-788Crossref PubMed Scopus (1505) Google Scholar, 6Gupta A. Zhou H.X. Profiling SARS-CoV-2 main protease (MPRO) binding to repurposed drugs using molecular dynamics simulations in classical and neural network trained force fields.ACS Comb. Sci. 2020; 22: 826-832Crossref PubMed Scopus (25) Google Scholar), we checked for the possibility of famotidine binding to SARS-CoV-2 PLpro by docking famotidine to the SARS-CoV-2 PLpro protein structure (PDB ID: 7CJM). The results of docking showed two putative binding sites. At the catalytic site of SARS-CoV-2 PLpro, where naphthalene inhibitors such as GRL-0617 bind (Fig. 1B) (18Ratia K. Pegan S. Takayama J. Sleeman K. Coughlin M. Baliji S. Chaudhuri R. Fu W. Prabhakar B.S. Johnson M.E. Baker S.C. Ghosh A.K. Mesecar A.D. A noncovalent class of papain-like protease/deubiquitinase inhibitors blocks SARS virus replication.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 16119-16124Crossref PubMed Scopus (337) Google Scholar, 19Shin D. Mukherjee R. Grewe D. Bojkova D. Baek K. Bhattacharya A. Schulz L. Widera M. Mehdipour A.R. Tascher G. Geurink P.P. Wilhelm A. van der Heden van Noort G.J. Ovaa H. Müller S. et al.Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity.Nature. 2020; 587: 657-662Crossref PubMed Scopus (598) Google Scholar), the famotidine-binding mode is mainly electrostatic, dominated by three ionic interactions with R154, E165, and D162. This binding mode also shows three hydrogen bonds with the side chains of Y268, Q269, and T301, but lacks key interactions with P247 and P248 known from the GRL-0617-binding mode. The hydrogen bond between the sulfur of famotidine and the backbone of Q269 (S…NH) was far weaker than the CO…NH hydrogen bond in GRL-0617 binding. Interestingly, docking found another potential binding site near the S2 helix, which overlaps with the binding site for the N-lobes of ISG15 and K48-Ub2 (near F69 and T75) (Fig. 1C) (19Shin D. Mukherjee R. Grewe D. Bojkova D. Baek K. Bhattacharya A. Schulz L. Widera M. Mehdipour A.R. Tascher G. Geurink P.P. Wilhelm A. van der Heden van Noort G.J. Ovaa H. Müller S. et al.Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity.Nature. 2020; 587: 657-662Crossref PubMed Scopus (598) Google Scholar, 20Békés M. van der Heden van Noort G.J. Ekkebus R. Ovaa H. Huang T.T. Lima C.D. Recognition of Lys48linked di-ubiquitin and deubiquitinating activities of the SARS coronavirus papain-like protease.Mol. Cell. 2016; 62: 572-585Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Binding of famotidine in this site may thus interfere with the binding of ISG15. However, this binding mode is less promising because the only remarkable interactions were an ionic interaction with R65 and a suboptimal cation-π interaction with F69. The interactions with P59 and P75 were too weak to contribute significantly to binding affinity. To test whether famotidine binds to the SARS-CoV-2 PLpro in vitro, we employed a thermal shift assay. In this assay inhibitor binding is assessed by the shift in the melting temperature of the target protein, which correlates with the strength of interaction between the protein and the putative drug. We found that the previously confirmed SARS-CoV-2 PLpro inhibitor GRL-0617 increased the Tm of SARS-CoV-2 PLpro. By contrast, famotidine showed no effect on the denaturation curve of the protein (Fig. 1D). Similarly, famotidine did not affect the catalytic activity of either SARS-CoV-2 PLpro or SARS-CoV PLpro, measured by the velocity of AMC release from ubiquitin-AMC (ubiquitin–7-amido-4-methylcoumarin) in the presence of different concentrations of famotidine. On the other hand, GRL-0617 reduced the catalytic activity of both SARS-CoV-2 PLpro and SARS-CoV PLpro in the same assay (Fig. 1E). To examine whether famotidine can alter the binding of ISG15 to SARS-CoV-2 PLpro, we followed the modification of SARS-CoV-2 PLpro with ISG15-activity-based probes carrying a propargyl warhead. Whereas the level of modified SARS-CoV-2 PLpro decreased with GRL-0617, there were no such differences with famotidine (Fig. 1F). This result indicates that famotidine does not interact with PLpro or affect its activity in vitro. To test whether famotidine can affect PLpro in transfected cells, we checked the interaction between ISG15 and the catalytically inactive SARS-CoV-2 PLpro in interferon-α-treated A549 cells. ISG15 was coimmunoprecipitated with the mutant SARS-CoV-2 PLpro in interferon-α-treated cells. GRL-0617 inhibited this interaction, but famotidine had no effect (Fig. S1a). Previous studies have shown an inhibitory effect of coronaviral PLproteases on the type I interferon response and on NF-κB signaling (19Shin D. Mukherjee R. Grewe D. Bojkova D. Baek K. Bhattacharya A. Schulz L. Widera M. Mehdipour A.R. Tascher G. Geurink P.P. Wilhelm A. van der Heden van Noort G.J. Ovaa H. Müller S. et al.Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity.Nature. 2020; 587: 657-662Crossref PubMed Scopus (598) Google Scholar, 20Békés M. van der Heden van Noort G.J. Ekkebus R. Ovaa H. Huang T.T. Lima C.D. Recognition of Lys48linked di-ubiquitin and deubiquitinating activities of the SARS coronavirus papain-like protease.Mol. Cell. 2016; 62: 572-585Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). SARS-CoV-2 PLpro and SARS-CoV PLpro inhibit interferon signaling by inhibiting IRF3-dependent gene expression (19Shin D. Mukherjee R. Grewe D. Bojkova D. Baek K. Bhattacharya A. Schulz L. Widera M. Mehdipour A.R. Tascher G. Geurink P.P. Wilhelm A. van der Heden van Noort G.J. Ovaa H. Müller S. et al.Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity.Nature. 2020; 587: 657-662Crossref PubMed Scopus (598) Google Scholar, 21Ortega J.T. Serrano M.L. Jastrzebska B. Class AG protein-coupled receptor antagonist famotidine as a therapeutic alternative against SARS-CoV2: An in silico analysis.Biomolecules. 2020; 10: 954Crossref Scopus (38) Google Scholar, 22Chen X. Yang X. Zheng Y. Yang Y. Xing Y. Chen Z. SARS coronavirus papain-like protease inhibits the type I interferon signaling pathway through interaction with the STING-TRAF3-TBK1 complex.Protein Cell. 2014; 5: 369-381Crossref PubMed Scopus (190) Google Scholar). Phosphorylation of TBK1 and IRF3 was inhibited in SARS-CoV-2 PLpro expressing A549 cells treated with interferon-α. GRL-0617 causes partial rescue of this inhibition, but famotidine has no effect (Fig. S1b). From existing literature and previous studies, it is known that SARS-CoV PLpro inhibits NF-κB signaling while SARS-CoV-2 PLpro exerts a stronger inhibitory effect on interferon signaling (18Ratia K. Pegan S. Takayama J. Sleeman K. Coughlin M. Baliji S. Chaudhuri R. Fu W. Prabhakar B.S. Johnson M.E. Baker S.C. Ghosh A.K. Mesecar A.D. A noncovalent class of papain-like protease/deubiquitinase inhibitors blocks SARS virus replication.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 16119-16124Crossref PubMed Scopus (337) Google Scholar, 19Shin D. Mukherjee R. Grewe D. Bojkova D. Baek K. Bhattacharya A. Schulz L. Widera M. Mehdipour A.R. Tascher G. Geurink P.P. Wilhelm A. van der Heden van Noort G.J. Ovaa H. Müller S. et al.Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity.Nature. 2020; 587: 657-662Crossref PubMed Scopus (598) Google Scholar). Famotidine treatment (4 h, 50 μM) had no significant effect on interferon-β and NF-κB promoter activation in either SARS-CoV-2 PLpro or SARS-CoV PLpro transfected cells (Fig. S1, c and d). Also, famotidine did not affect the expression of cytokines, which are downstream of the type I interferon pathway (ISG15) or NF-κB-dependent inflammatory signaling (IL-6, IL-8) in SARS-CoV-2 PLpro expressing cells (Fig. S1f). Therefore, these experiments corroborate that famotidine does not show any effect on the interferon and NF-κB pathways in SARS-CoV-2 PLpro transfected cells. Several reports suggest that the antiviral effect of famotidine on patients is due to inhibition of the coronavirus main protease (23Matthews K. Schäfer A. Pham A. Frieman M. The SARS coronavirus papain like protease can inhibit IRF3 at a post activation step that requires deubiquitination activity.Virol. J. 2014; 11: 209Crossref PubMed Scopus (51) Google Scholar). The main protease of SARS-CoV-2 is a cysteine protease that shares ~95% homology to the 3CLpro of SARS-CoV. It cleaves the viral polyproteins and is hence essential for viral replication. Boceprevir has been found to be one of the strongest inhibitors of SARS-CoV-2 3CLPro, inhibiting ~60% activity of the protease at a concentration of 20 μM in a fluorescence resonance energy transfer (FRET)-based protease activity assay (24Ma C. Sacco M.D. Hurst B. Townsend J.A. Hu Y. Szeto T. Zhang X. Tarbet B. Marty M.T. Chen Y. Wang J. Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease.Cell Res. 2020; 30: 678-692Crossref PubMed Scopus (501) Google Scholar). We observed a robust decrease in 3CL-pro activity upon boceprevir treatment with an IC50 of 2.3 ± 0.2 μM. On the other hand, famotidine did not inhibit the activity of CoV-2 3CLpro (Fig. 2A). We further checked viral replication in cells treated with famotidine or remdesivir. Remdesivir inhibits the RNA-dependent RNA polymerase of SARSCoV-2 (25Williamson B.N. Feldmann F. Schwarz B. Meade-White K. Porter D.P. Schulz J. van Doremalen N. Leighton I. Kwe Yinda C. Pérez-Pérez L. Okumura A. Lovaglio J. Hanley P.W. Saturday G. Bosio C.M. et al.Clinical benefit of remdesivir in rhesus macaques infected with SARS-CoV-2.Nature. 2020; 585: 273-276Crossref PubMed Scopus (432) Google Scholar, 26Kokic G. Hillen H.S. Tegunov D. Dienemann C. Seitz F. Schmitzova J. Farnung L. Siewert A. Höbartner C. Cramer P. Mechanism of SARS-CoV-2 polymerase stalling by remdesivir.Nat. Commun. 2021; 12: 279Crossref PubMed Scopus (306) Google Scholar). There was no significant change in viral replication or any cytopathic effect (CPE) (measured by spike positive area) in famotidine-treated cells (Fig. 2B). Remdesivir caused a gradual dose-dependent inhibition of SARS-CoV-2-induced CPE with 1.25 μM remdesivir almost completely inhibiting CPE (Fig. 2B). Since famotidine is an H2 receptor antagonist, we assumed that the beneficial effects of famotidine in patients may be due to its on-target effect on H2 receptors. To see a global effect of famotidine in SARS-CoV-2 infected cells, we analyzed the proteome of cells infected with SARSCoV-2 in the presence of histamine with or without famotidine treatment. Famotidine treatment led to a decrease in several proteins associated with the interferon pathway (IFIT1, IFIT2, ISG20, Herc5, Ube2L6), NF-κB pathway (NKAP, NFKBIB, RIPK1), and those associated to TLR signaling (TRAF6, MyD88) (Fig. S2, Table S1). Gene ontology and network analysis of the proteomic data using the Metascape software identified pathways associated with interferon response, cytokine production, viral infection and NF-KB signaling to be significantly altered by famotidine treatment (Fig. S3, a and b). Some proteins were also upregulated in famotidine-treated cells, though from the GO analysis these appear to be unrelated to viral infection (Fig. S3c). These results suggested that though famotidine does not affect viral replication, it may affect the antiviral response in infected cells and production of cytokines. PCR amplification of histamine receptors from cDNA prepared from A549 cells showed that they expressed histamine H1 and H2 receptors and therefore may be used to monitor the on-target effect of histamine and famotidine on H2 receptors (Fig. S4a). Since toll-like receptors are an important arm of innate immune signaling, we decided to check the expression of TLRs in SARS-CoV-2-infected cells. Caco2 cells infected with SARS-CoV-2 for 24 h showed an upregulation of TLR3, TLR4, and TLR7 (Fig. S4b). Treatment of different cell types with histamine has been reported to upregulate expression of toll-like-receptor 3 (TLR3) (27Hou Y.F. Zhou Y.C. Zheng X.X. Wang H.Y. Fu Y.L. Fang Z.M. He S.H. Modulation of expression and function of Toll-like receptor 3 in A549 and H292 cells by histamine.Mol. Immunol. 2006; 43: 1982-1992Crossref PubMed Scopus (28) Google Scholar, 28Xie G. Wang F. Peng X. Liang Y. Yang H. Li L. Modulation of mast cell toll-like receptor 3 expression and cytokines release by histamine.Cell Physiol. Biochem. 2018; 46: 2401-2411Crossref PubMed Scopus (13) Google Scholar). In A549 cells, treatment with histamine for 12 h caused a modest upregulation of TLR3 and TLR7 mRNA levels (Fig. 3A). Pretreatment of these cells with famotidine reduced TLR3 mRNA levels, but did not affect TLR7 expression (Fig. 3B). To mimic the effect of viral RNA, cells were treated with poly(I:C) for 12 h and expression of TLRs was measured by RT-PCR. Treatment with poly(I:C) upregulated the expression of endosomal TLRs including TLR3, TLR7, TLR8, and TLR9 (Fig. S4c). To mimic a scenario where the virus is present in high concentration of systemic histamine, we pretreated A549 cells with histamine and famotidine and then transfected them with poly(I:C). Pretreatment with histamine led to a significant increase in TLR3 levels, which was greater than that caused by poly(I:C) alone. This cumulative effect of histamine and poly(I:C) could be reversed when famotidine was present in the medium (Fig. 3C). Cell viability was not significantly affected by poly(I:C) treatment or by treatment with 100 μM histamine or 50 μM famotidine when measured with an MTT assay (Fig. S4d). The effect of histamine and poly(I:C) on TLR3 expression was also evident when endogenous TLR3 was immunostained with TLR3 antibody and in cell lysates immunoblotted for TLR3. Cells pretreated with histamine for 12 h prior to poly(I:C) treatment had the highest levels of TLR3, which was again rescued by famotidine treatment (Fig. 3, D and E). The effect of histamine on TLR3 expression is absent in TLR3 siRNA-treated cells (Fig. S4e). These results collectively show that poly(I:C) and histamine increase TLR3 levels, which can be reversed by famotidine. Next we measured the effect of famotidine on TLR3 signaling. Cells were pretreated with histamine alone or in combination with famotidine and then treated with the TLR3 agonist poly(I:C) to activate TLR3-dependent signaling (Fig. 4A). Histamine alone was not enough to trigger TLR3 signaling though it can affect TLR3 expression. Poly(I:C) treatment activated TLR3 signaling causing phosphorylation of TANK binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3) and a subsequent increase in interferon-stimulated gene 15 (ISG15). Pretreatment with histamine further increases the activation of the TBK1/IRF3 pathway. Famotidine counters this effect leading to a decrease in IRF3 activation and lower ISG15 levels (Fig. 4B). Histamine and poly(I:C) treatment for 12 h increased IFN-β promotor activation, which could be reversed by famotidine (Fig. 4C). A similar effect was also seen on NF-κB(p65) promoter activation after 2 h of poly(I:C) treatment (Fig. 4D). NF-κB activation was also measured in histamine and poly(I:C)-treated cells by measuring nuclear NF-κB(p65) levels by immunofluorescence. Histamine and poly(I:C) caused an increase in the fraction of nuclear p65, which is reduced on famotidine treatment. RT-PCR to detect gene expression of ISG15 and IL8, which are downstream of IRF3 and NF-κB, showed that famotidine could reduce levels of these markers in poly(I:C)-treated cells (Fig. 4F). Taken together, these results indicate that famotidine may affect interferon and inflammatory pathways, which are activated by viral dsRNA downstream of TLR3. Next we evaluated the effect of histamine on TLR3 expression in cells infected with SARS-CoV-2 infected Caco2 cells 24 h postinfection. Expression of TLR2, TLR3, and TLR7 was upregulated while TLR5 was downregulated in virus-infected cells compared with uninfected control (Fig. 5A). Famotidine treatment redu
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