SARS-CoV-2 NSP7 inhibits type I and III IFN production by targeting the RIG-I/MDA5, TRIF, and STING signaling pathways.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a poor inducer of innate antiviral immunity, and the underlying mechanism still needs further investigation. Here, we reported that SARS-CoV-2 NSP7 inhibited the production of type I and III interferons (IFNs) by targeting the RIG-I/MDA5, Toll-like receptor (TLR3)-TRIF, and cGAS-STING signaling pathways. SARS-CoV-2 NSP7 suppressed the expression of IFNs and IFN-stimulated genes induced by poly (I:C) transfection and infection with Sendai virus or SARS-CoV-2 virus-like particles. NSP7 impaired type I and III IFN production activated by components of the cytosolic dsRNA-sensing pathway, including RIG-I, MDA5, and MAVS, but not TBK1, IKKε, and IRF3-5D, an active form of IRF3. In addition, NSP7 also suppressed TRIF- and STING-induced IFN responses. Mechanistically, NSP7 associated with RIG-I and MDA5 prevented the formation of the RIG-I/MDA5-MAVS signalosome and interacted with TRIF and STING to inhibit TRIF-TBK1 and STING-TBK1 complex formation, thus reducing the subsequent IRF3 phosphorylation and nuclear translocation that are essential for IFN induction. In addition, ectopic expression of NSP7 impeded innate immune activation and facilitated virus replication. Taken together, SARS-CoV-2 NSP7 dampens type I and III IFN responses via disruption of the signal transduction of the RIG-I/MDA5-MAVS, TLR3-TRIF, and cGAS-STING signaling pathways, thus providing novel insights into the interactions between SARS-CoV-2 and innate antiviral immunity.

COVID-19 plasma exosomes promote proinflammatory immune responses in peripheral blood mononuclear cells.

Elevated serum cytokine production in COVID-19 patients is associated with disease progression and severity. However, the stimuli that initiate cytokine production in patients remain to be fully revealed. Virus-infected cells release virus-associated exosomes, extracellular vesicles of endocytic origin, into the blood to deliver viral cargoes able to regulate immune responses. Here, we report that plasma exosomes of COVID-19 patients contain SARS-CoV-2 double stranded RNA (dsRNA) and stimulate robust production of interleukin-6 (IL-6), IL-8, tumor necrosis factor-α (TNF-α), and other inflammatory cytokines and chemokines by human peripheral mononuclear cells. Exosome depletion abolished these stimulated responses. COVID-19 plasma exosomes induced proinflammatory responses in CD4+ T cells, CD8+ T cells, and CD14+ monocytes but not significantly in regulatory T cells, Th17 T cells, or central memory T cells. COVID-19 plasma exosomes protect the SARS-CoV-2 dsRNA cargo from RNase and deliver the dsRNA into recipient cells. These exosomes significantly increase expression of endosomal toll-like receptor 3 (TLR3), TLR7, TLR8, and TLR9 in peripheral T cells and monocytes. A pharmacological inhibitor of TLR3 considerably reduced cytokine and chemokine production by CD4+ and CD8+ T cells but not by CD14+ monocytes, highlighting divergent signaling pathways of immune cells in response to COVID-19 plasma exosomes. Our results identify a novel model of intercellular crosstalk following SARS-CoV-2 infection that evoke immune responses positioned to contribute to elevated cytokine production associated with COVID-19 progression, severity, and long-haul symptoms.

Human Lung Fibroblasts Exhibit Induced Inflammation Memory via Increased IL6 Gene Expression and Release.

Fibroblasts of different origins are known to possess stromal memory after inflammatory episodes. However, there are no studies exploring human lung fibroblast memory which may predict a subsequent inflammatory response in chronic respiratory diseases and COVID-19. MRC-5 and HF19 human lung fibroblast cell lines were treated using different primary and secondary stimulus combinations: TNFα-WD-TNFα, Poly (I:C)-WD-TNFα, TNFα-WD-Poly (I:C), or LPS-WD-TNFα with a 24-h rest period (withdrawal period; WD) between the two 24-h stimulations. TLR3 and NF-κB inhibitors were used to determine pathways involved. The effect of SARS-Cov-2 spike protein to inflammatory response of lung fibroblasts was also investigated. mRNA expressions of genes and IL6 release were measured using qRT-PCR and ELISA, respectively. Statistical significance was determined by using one- or two-way ANOVA, followed by Bonferroni's post hoc analysis for comparison of multiple groups. Preexposure with Poly (I:C) significantly increased TNFα-induced IL6 gene expression and IL6 release in both cell lines, while it affected neither gene expressions of IL1B, IL2, IL8, and MMP8 nor fibrosis-related genes: ACTA2, COL1A1, POSTN, and TGFB1. Inhibition of TLR3 or NF-κB during primary stimulation significantly downregulated IL6 release. Simultaneous treatment of MRC-5 cells with SARS-CoV-2 spike protein further increased TNFα-induced IL6 release; however, preexposure to Poly (I:C) did not affect it. Human lung fibroblasts are capable of retaining inflammatory memory and showed an augmented response upon secondary exposure. These results may contribute to the possibility of training human lung fibroblasts to respond suitably on inflammatory episodes after viral infection.

mRNA expression of toll-like receptors 3, 7, 8, and 9 in the nasopharyngeal epithelial cells of coronavirus disease 2019 patients.

BACKGROUND: The etiopathogenesis of coronavirus disease 2019 (COVID-19) stem partially from the abnormal activation of the innate and adaptive immune systems. Here in the current investigation, the mRNA expression levels of toll-like receptors (TLRs) were evaluated in the nasopharyngeal epithelial cells from COVID-19 patients. METHODS: Epithelial cells were obtained using nasopharyngeal swab samples from 90 COVID-19 patients and 50 controls. COVID-19 cases were classified into those without symptoms, with symptoms but not hospitalized, and with symptoms and hospitalized. To determine the mRNA expression levels of TLRs, first RNA was extracted and cDNA was synthesized, and finally Real-time PCR was exerted. RESULTS: It was seen that the transcript levels of TLR3, TLR7, TLR8, and TLR9 were overexpressed in the COVID-19 patients with clinical symptoms needing hospitalization as well as in those with clinical symptoms without needing for hospitalization compared to controls. Upregulation of TLRs was associated with clinical presentations of the patients. CONCLUSIONS: Modulation of TLR3, TLR7, TLR8, TLR9 in the epithelial cells of COVID-19 cases may estimate the disease severity and requirement for hospitalization.

TRIM56 overexpression restricts porcine epidemic diarrhoea virus replication in Marc-145 cells by enhancing TLR3-TRAF3-mediated IFN-ß antiviral response.

Infection with the porcine epidemic diarrhoea virus (PEDV) causes severe enteric disease in suckling piglets, causing massive economic losses in the swine industry worldwide. Tripartite motif-containing 56 (TRIM56) has been shown to augment type I IFN response, but whether it affects PEDV replication remains uncharacterized. Here we investigated the role of TRIM56 in Marc-145 cells during PEDV infection. We found that TRIM56 expression was upregulated in cells infected with PEDV. Overexpression of TRIM56 effectively reduced PEDV replication, while knockdown of TRIM56 resulted in increased viral replication. TRIM56 overexpression significantly increased the phosphorylation of IRF3 and NF-κB P65, and enhanced the IFN-ß antiviral response, while silencing TRIM56 did not affect IRF3 activation. TRIM56 overexpression increased the protein level of TRAF3, the component of the TLR3 pathway, thereby significantly activating downstream IRF3 and NF-κB signalling. We demonstrated that TRIM56 overexpression inhibited PEDV replication and upregulated expression of IFN-ß, IFN-stimulated genes (ISGs) and chemokines in a dose-dependent manner. Moreover, truncations of the RING domain, N-terminal domain or C-terminal portion on TRIM56 were unable to induce IFN-ß expression and failed to restrict PEDV replication. Together, our results suggested that TRIM56 was upregulated in Marc-145 cells in response to PEDV infection. Overexpression of TRIM56 inhibited PEDV replication by positively regulating the TLR3-mediated antiviral signalling pathway. These findings provide evidence that TRIM56 plays a positive role in the innate immune response during PEDV infection.

The Priming Potential of Interferon Lambda-1 for Antiviral Defense in the Oral Mucosa.

The oral mucosa is one of the first lines of the innate host defense system against microbial invasion. Interferon (IFN) lambda-1 (IFN-λ1), a type III IFN, exhibits type I IFN-like antiviral activity. In contrast to ubiquitously expressed type I IFN receptors, IFN-λ receptor 1 (IFN-λR1), which has higher affinity for type III IFNs than low-affinity interleukin (IL)-10 receptor 2, is mainly expressed on epithelial cells. Although IFN-λ1 has been shown to exert antiviral effects in the respiratory tract, gastrointestinal tract, and skin, the regulation of type III IFN receptor expression and its functions in the oral mucosa remain unclear. We herein showed the expression of IFN-λR1 in human gingival keratinocytes. The expression of IL-6, angiotensin-converting enzyme 2 (a critical molecule for severe acute respiratory syndrome coronavirus 2 infection), and IL-8 in human primary gingival keratinocytes (HGK) were significantly higher following treatments with either type I IFN (IFN-ß) or type II IFN (IFN-γ) than with IFN-λ1. However, the IFN-λ1 treatment strongly induced toll-like receptor (TLR) 3 and retinoic acid-inducible gene I (RIG-I), which mainly recognize viral nucleic acids, via the STAT1-mediated pathway. Furthermore, a stimulation with a RIG-I or TLR3 agonist promoted the production of IL-6, IL-8, and IFN-λ in HGK, which was significantly enhanced by a pretreatment with IFN-λ1. These results suggest that IFN-λ1 may contribute to the activation of innate immune responses to oral viral infections by up-regulating the expression of RIG-I and TLR3 and priming their functions in keratinocytes.

SARS-CoV-2 causes senescence in human cells and exacerbates the senescence-associated secretory phenotype through TLR-3.

Senescent cells, which arise due to damage-associated signals, are apoptosis-resistant and can express a pro-inflammatory, tissue-destructive senescence-associated secretory phenotype (SASP). We recently reported that a component of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surface protein, S1, can amplify the SASP of senescent cultured human cells and that a related mouse ß-coronavirus, mouse hepatitis virus (MHV), increases SASP factors and senescent cell burden in infected mice. Here, we show that SARS-CoV-2 induces senescence in human non-senescent cells and exacerbates the SASP in human senescent cells through Toll-like receptor-3 (TLR-3). TLR-3, which senses viral RNA, was increased in human senescent compared to non-senescent cells. Notably, genetically or pharmacologically inhibiting TLR-3 prevented senescence induction and SASP amplification by SARS-CoV-2 or Spike pseudotyped virus. While an artificial TLR-3 agonist alone was not sufficient to induce senescence, it amplified the SASP in senescent human cells. Consistent with these findings, lung p16INK4a+ senescent cell burden was higher in patients who died from acute SARS-CoV-2 infection than other causes. Our results suggest that induction of cellular senescence and SASP amplification through TLR-3 contribute to SARS-CoV-2 morbidity, indicating that clinical trials of senolytics and/or SASP/TLR-3 inhibitors for alleviating acute and long-term SARS-CoV-2 sequelae are warranted.

Famotidine inhibits toll-like receptor 3-mediated inflammatory signaling in SARS-CoV-2 infection.

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.

Role of Host Immune and Inflammatory Responses in COVID-19 Cases with Underlying Primary Immunodeficiency: A Review.

Coronavirus disease 2019 (COVID-19) has spread rapidly and become a pandemic. Caused by a novel human coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe COVID-19 is characterized by cytokine storm syndromes due to innate immune activation. Primary immunodeficiency (PID) cases represent a special patient population whose impaired immune system might make them susceptible to severe infections, posing a higher risk to COVID-19, but this could also lead to suppressed inflammatory responses and cytokine storm. It remains an open question as to whether the impaired immune system constitutes a predisposing or protective factor for PID patients when facing SARS-CoV-2 infection. After literature review, it was found that, similar to other patient populations with different comorbidities, PID patients may be susceptible to SARS-CoV-2 infection. Their varied immune status, however, may lead to different disease severity and outcomes after SARS-CoV-2 infection. PID patients with deficiency in antiviral innate immune signaling [eg, Toll-like receptor (TLR)3, TLR7, or interferon regulatory factor 7 (IRF7)] or interferon signaling (IFNAR2) may be linked to severe COVID-19. Because of its anti-infection, anti-inflammatory, and immunomodulatory effects, routine intravenous immunoglobulin therapy may provide some protective effects to the PID patients.

IFIT Proteins Are Involved in CXCL10 Expression in Human Glomerular Endothelial Cells Treated with a Toll-Like Receptor 3 Agonist.

INTRODUCTION: Various viruses including a novel coronavirus (SARS-CoV-2) can infect the kidney. When viruses invade the glomeruli from the bloodstream, glomerular endothelial cells (GECs) initiate the innate immune reactions. We investigated the expression of interferon (IFN)-induced protein with tetratricopeptide repeats (IFIT) 1/2/3, antiviral molecules, in human GECs treated with a toll-like receptor (TLR) 3 agonist. Role of IFIT1/2/3 in the expression of C-X-C motif chemokine ligand 10 (CXCL10) was also examined. METHODS: Human GECs were cultured and stimulated with polyinosinic-polycytidylic acid (poly IC), a synthetic TLR3 agonist. Real-time qPCR, Western blotting, and ELISA were used to examine the expression of IFIT1/2/3, IFN-ß, and CXCL10. RNA interference against IFN-ß or IFIT1/2/3 was also performed. RESULTS: Expression of IFIT1/2/3 and CXCL10 was induced by poly IC in GECs. The inductions were inhibited by RNA interfering of IFN-ß. Knockdown of IFIT1/2/3 decreased the CXCL10 expression. Knockdown of IFIT3 decreased the expression of IFIT1 and IFIT2 proteins. CONCLUSION: IFIT1/2/3 and CXCL10 were induced by poly IC via IFN-ß in GECs. IFIT1/2/3 may increase the expression of CXCL10 which induces lymphocyte chemotaxis and may inhibit the replication of infected viruses. These molecules may play a role in GEC innate immune reactions in response to viruses.

Immunoinformatic design of a COVID-19 subunit vaccine using entire structural immunogenic epitopes of SARS-CoV-2.

Coronavirus disease 2019 (COVID-19) is an acute pneumonic disease, with no prophylactic or specific therapeutical solution. Effective and rapid countermeasure against the spread of the disease's associated virus, SARS-CoV-2, requires to incorporate the computational approach. In this study, we employed various immunoinformatics tools to design a multi-epitope vaccine polypeptide with the highest potential for activating the human immune system against SARS-CoV-2. The initial epitope set was extracted from the whole set of viral structural proteins. Potential non-toxic and non-allergenic T-cell and B-cell binding and cytokine inducing epitopes were then identified through a priori prediction. Selected epitopes were bound to each other with appropriate linkers, followed by appending a suitable adjuvant to increase the immunogenicity of the vaccine polypeptide. Molecular modelling of the 3D structure of the vaccine construct, docking, molecular dynamics simulations and free energy calculations confirmed that the vaccine peptide had high affinity for Toll-like receptor 3 binding, and that the vaccine-receptor complex was highly stable. As our vaccine polypeptide design captures the advantages of structural epitopes and simultaneously integrates precautions to avoid relevant side effects, it is suggested to be promising for elicitation of an effective and safe immune response against SARS-CoV-2 in vivo.

A survey of genetic variants in SARS-CoV-2 interacting domains of ACE2, TMPRSS2 and TLR3/7/8 across populations.

The COVID-19 pandemic highlighted healthcare disparities in multiple countries. As such morbidity and mortality vary significantly around the globe between populations and ethnic groups. Underlying medical conditions and environmental factors contribute higher incidence in some populations and a genetic predisposition may play a role for severe cases with respiratory failure. Here we investigated whether genetic variation in the key genes for viral entry to host cells-ACE2 and TMPRSS2-and sensing of viral genomic RNAs (i.e., TLR3/7/8) could explain the variation in incidence across diverse ethnic groups. Overall, these genes are under strong selection pressure and have very few nonsynonymous variants in all populations. Genetic determinant for the binding affinity between SARS-CoV-2 and ACE2 does not show significant difference between populations. Non-genetic factors are likely to contribute differential population characteristics affected by COVID-19. Nonetheless, a systematic mutagenesis study on the receptor binding domain of ACE2 is required to understand the difference in host-viral interaction across populations.

Type III interferons disrupt the lung epithelial barrier upon viral recognition.

Viral infections of the lower respiratory tract are a leading cause of mortality. Mounting evidence indicates that most severe cases are characterized by aberrant immune responses and do not depend on viral burden. In this study, we assessed how type III interferons (IFN-λ) contribute to the pathogenesis induced by RNA viruses. We report that IFN-λ is present in the lower, but not upper, airways of patients with coronavirus disease 2019 (COVID-19). In mice, we demonstrate that IFN-λ produced by lung dendritic cells in response to a synthetic viral RNA induces barrier damage, causing susceptibility to lethal bacterial superinfections. These findings provide a strong rationale for rethinking the pathophysiological role of IFN-λ and its possible use in clinical practice against endemic viruses, such as influenza virus as well as the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

Irisin modulates genes associated with severe coronavirus disease (COVID-19) outcome in human subcutaneous adipocytes cell culture.

Obesity patients are more susceptible to develop COVID-19 severe outcome due to the role of angiotensin-converting enzyme 2 (ACE2) in the viral infection. ACE2 is regulated in the human cells by different genes associated with increased (TLR3, HAT1, HDAC2, KDM5B, SIRT1, RAB1A, FURIN and ADAM10) or decreased (TRIB3) virus replication. RNA-seq data revealed 14857 genes expressed in human subcutaneous adipocytes, including genes mentioned above. Irisin treatment increased by 3-fold the levels of TRIB3 transcript and decreased the levels of other genes. The decrease in FURIN and ADAM10 expression enriched diverse biological processes, including extracellular structure organization. Our results, in human subcutaneous adipocytes cell culture, indicate a positive effect of irisin on the expression of multiple genes related to viral infection by SARS-CoV-2; furthermore, translatable for other tissues and organs targeted by the novel coronavirus and present, thus, promising approaches for the treatment of COVID-19 infection as therapeutic strategy to decrease ACE2 regulatory genes.