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Interferons (IFNs) are a group of cytokines with a well-established antiviral function. They can be induced by viral infection, are secreted and bind to specific receptors on the same or neighbouring cells to activate the expression of hundreds of IFN stimulated genes (ISGs) with antiviral function. Type I IFN has been known for more than half a century. However, more recently, type III IFN (IFNλ, IL-28/29) was shown to play a similar role and to be particularly important at epithelial surfaces. Here we show that airway epithelia, the primary target of influenza A virus, produce both IFN I and III upon infection, and that induction of both depends on the RIG-I/MAVS pathway. While IRF3 is generally regarded as the transcription factor required for initiation of IFN transcription and the so-called \"priming loop\", we find that IRF3 deficiency has little impact on IFN expression. In contrast, lack of IRF7 reduced IFN production significantly, and only IRF3(-/-)IRF7(-/-) double deficiency completely abolished it. The transcriptional response to influenza infection was largely dependent on IFNs, as it was reduced to a few upregulated genes in epithelia lacking receptors for both type I and III IFN (IFNAR1(-/-)IL-28Rα(-/-)). Wild-type epithelia and epithelia deficient in either the type I IFN receptor or the type III IFN receptor exhibit similar transcriptional profiles in response to virus, indicating that none of the induced genes depends selectively on only one IFN system. In chimeric mice, the lack of both IFN I and III signalling in the stromal compartment alone significantly increased the susceptibility to influenza infection. In conclusion, virus infection of airway epithelia induces, via a RIG-I/MAVS/IRF7 dependent pathway, both type I and III IFNs which drive two completely overlapping and redundant amplification loops to upregulate ISGs and protect from influenza infection.

Interferon (IFN) regulatory factor 3 (IRF3) is a transcription factor activated by phosphorylation in the cytoplasm of a virus-infected cell; by translocating to the nucleus, it induces transcription of IFN-β and other antiviral genes. We have previously reported IRF3 can also be activated, as a proapoptotic factor, by its linear polyubiquitination mediated by the RIG-I pathway. Both transcriptional and apoptotic functions of IRF3 contribute to its antiviral effect. Here, we report a nontranscriptional function of IRF3, namely, the repression of IRF3-mediated NF-κB activity (RIKA), which attenuated viral activation of NF-κB and the resultant inflammatory gene induction. In Irf3 −/− mice, consequently, Sendai virus infection caused enhanced inflammation in the lungs. Mechanistically, RIKA was mediated by the direct binding of IRF3 to the p65 subunit of NF-κB in the cytoplasm, which prevented its nuclear import. A mutant IRF3 defective in both the transcriptional and the apoptotic activities was active in RIKA and inhibited virus replication. Our results demonstrated IRF3 deployed a three-pronged attack on virus replication and the accompanying inflammation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a devastating global pandemic, infecting over 43 million people and claiming over 1 million lives, with these numbers increasing daily. Therefore, there is urgent need to understand the molecular mechanisms governing SARS-CoV-2 pathogenesis, immune evasion, and disease progression. Here, we show that SARS-CoV-2 can block IRF3 and NF-κB activation early during virus infection. We also identify that the SARS-CoV-2 viral proteins NSP1 and NSP13 can block interferon activation via distinct mechanisms. NSP1 antagonizes interferon signaling by suppressing host mRNA translation, while NSP13 downregulates interferon and NF-κB promoter signaling by limiting TBK1 and IRF3 activation, as phospho-TBK1 and phospho-IRF3 protein levels are reduced with increasing levels of NSP13 protein expression. NSP13 can also reduce NF-κB activation by both limiting NF-κB phosphorylation and nuclear translocation. Last, we also show that NSP13 binds to TBK1 and downregulates IFIT1 protein expression. Collectively, these data illustrate that SARS-CoV-2 bypasses multiple innate immune activation pathways through distinct mechanisms.

Background Macrophage proinflammatory activation contributes to the pathology of severe acute pancreatitis (SAP) and, simultaneously, macrophage functional changes, and increased pyroptosis/necrosis can further exacerbate the cellular immune suppression during the process of SAP, where cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) plays an important role. However, the function and mechanism of cGAS–STING in SAP-induced lung injury (LI) remains unknown. Methods Lipopolysaccharide (LPS) was combined with caerulein-induced SAP in wild type, cGAS −/− and sting −/− mice . Primary macrophages were extracted via bronchoalveolar lavage and peritoneal lavage. Ana-1 cells were pretreated with LPS and stimulated with nigericin sodium salt to induce pyroptosis in vitro. Results SAP triggered NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome activation-mediated pyroptosis of alveolar and peritoneal macrophages in mouse model. Knockout of cGAS / STING could ameliorate NLRP3 activation and macrophage pyroptosis. In addition, mitochondrial (mt)DNA released from damaged mitochondria further induced macrophage STING activation in a cGAS- and dose-dependent manner. Upregulated STING signal can promote NLRP3 inflammasome-mediated macrophage pyroptosis and increase serum interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α levels and, thus, exacerbate SAP-associated LI (SAP-ALI). Downstream molecules of STING, IRF7, and IRF3 connect the mtDNA–cGAS–STING axis and the NLRP3–pyroptosis axis. Conclusions Negative regulation of any molecule in the mtDNA–cGAS–STING–IRF7/IRF3 pathway can affect the activation of NLRP3 inflammasomes, thereby reducing macrophage pyroptosis and improving SAP-ALI in mouse model.

Type I IFNs are key cytokines mediating innate antiviral immunity. cGMP-AMP synthase, ritinoic acid-inducible protein 1 (RIG-I)–like receptors, and Toll-like receptors recognize microbial double-stranded (ds)DNA, dsRNA, and LPS to induce the expression of type I IFNs. These signaling pathways converge at the recruitment and activation of the transcription factor IRF-3 (IFN regulatory factor 3). The adaptor proteins STING (stimulator of IFN genes), MAVS (mitochondrial antiviral signaling), and TRIF (TIR domain-containing adaptor inducing IFN-β) mediate the recruitment of IRF-3 through a conserved pLxIS motif. Here we show that the pLxIS motif of phosphorylated STING, MAVS, and TRIF binds to IRF-3 in a similar manner, whereas residues upstream of the motif confer specificity. The structure of the IRF-3 phosphomimetic mutant S386/396E bound to the cAMP response element binding protein (CREB)-binding protein reveals that the pLxIS motif also mediates IRF-3 dimerization and activation. Moreover, rotavirus NSP1 (nonstructural protein 1) employs a pLxIS motif to target IRF-3 for degradation, but phosphorylation of NSP1 is not required for its activity. These results suggest a concerted mechanism for the recruitment and activation of IRF-3 that can be subverted by viral proteins to evade innate immune responses.

SARS-CoV-2 is the pathogenic agent of COVID-19, which has evolved into a global pandemic. Compared with some other respiratory RNA viruses, SARS-CoV-2 is a poor inducer of type I interferon (IFN). Here, we report that SARS-CoV-2 nsp12, the viral RNA-dependent RNA polymerase (RdRp), suppresses host antiviral responses. SARS-CoV-2 nsp12 attenuated Sendai virus (SeV)- or poly(I:C)-induced IFN-β promoter activation in a dose-dependent manner. It also inhibited IFN promoter activation triggered by RIG-I, MDA5, MAVS, and IRF3 overexpression. Nsp12 did not impair IRF3 phosphorylation but suppressed the nuclear translocation of IRF3. Mutational analyses suggested that this suppression was not dependent on the polymerase activity of nsp12. Given these findings, our study reveals that SARS-CoV-2 RdRp can antagonize host antiviral innate immunity and thus provides insights into viral pathogenesis.

Cancer-associated fibroblasts (CAFs) perform diverse roles and can modulate therapy responses1. The inflammatory environment within tumours also influences responses to many therapies, including the efficacy of oncolytic viruses2; however, the role of CAFs in this context remains unclear. Furthermore, little is known about the cell signalling triggered by heterotypic cancer cell–fibroblast contacts and about what activates fibroblasts to express inflammatory mediators1,3. Here, we show that direct contact between cancer cells and CAFs triggers the expression of a wide range of inflammatory modulators by fibroblasts. This is initiated following transcytosis of cytoplasm from cancer cells into fibroblasts, leading to the activation of STING and IRF3-mediated expression of interferon-β1 and other cytokines. Interferon-β1 then drives interferon-stimulated transcriptional programs in both cancer cells and stromal fibroblasts and ultimately undermines the efficacy of oncolytic viruses, both in vitro and in vivo. Further, targeting IRF3 solely in stromal fibroblasts restores oncolytic herpes simplex virus function.Tumour fibroblasts influence oncolytic viral therapy. Arwert et al. show that transcytosis of cancer cells into fibroblasts activates STING and IRF3 to upregulate interferon-β1, eliciting a transcriptional program to reduce the effectiveness of oncolytic viral therapy.

Although the transcription factors IRF-3 and IRF-7 are considered master regulators of type I interferon (IFN) induction and IFN stimulated gene (ISG) expression, Irf3(-/-)×Irf7(-/-) double knockout (DKO) myeloid dendritic cells (mDC) produce relatively normal levels of IFN-β after viral infection. We generated Irf3(-/-)×Irf5(-/-)×Irf7(-/-) triple knockout (TKO) mice to test whether IRF-5 was the source of the residual induction of IFN-β and ISGs in mDCs. In pathogenesis studies with two unrelated positive-sense RNA viruses (West Nile virus (WNV) and murine norovirus), TKO mice succumbed at rates greater than DKO mice and equal to or approaching those of mice lacking the type I IFN receptor (Ifnar(-/-)). In ex vivo studies, after WNV infection or exposure to Toll-like receptor agonists, TKO mDCs failed to produce IFN-β or express ISGs. In contrast, this response was sustained in TKO macrophages following WNV infection. To define IRF-regulated gene signatures, we performed microarray analysis on WNV-infected mDC from wild type (WT), DKO, TKO, or Ifnar(-/-) mice, as well as from mice lacking the RIG-I like receptor adaptor protein MAVS. Whereas the gene induction pattern in DKO mDC was similar to WT cells, remarkably, almost no ISG induction was detected in TKO or Mavs(-/-) mDC. The relative equivalence of TKO and Mavs(-/-) responses suggested that MAVS dominantly regulates ISG induction in mDC. Moreover, we showed that MAVS-dependent induction of ISGs can occur through an IRF-5-dependent yet IRF-3 and IRF-7-independent pathway. Our results establish IRF-3, -5, and -7 as the key transcription factors responsible for mediating the type I IFN and ISG response in mDC during WNV infection and suggest a novel signaling link between MAVS and IRF-5.

The initial response to viral infection is anticipatory, with host antiviral restriction factors and pathogen sensors constantly surveying the cell to rapidly mount an antiviral response through the synthesis and downstream activity of interferons. After pathogen clearance, the host’s ability to resolve this antiviral response and return to homeostasis is critical. Here, we found that isoforms of the RNA-binding protein ZAP functioned as both a direct antiviral restriction factor and an interferon-resolution factor. The short isoform of ZAP bound to and mediated the degradation of several host interferon messenger RNAs, and thus acted as a negative feedback regulator of the interferon response. In contrast, the long isoform of ZAP had antiviral functions and did not regulate interferon. The two isoforms contained identical RNA-targeting domains, but differences in their intracellular localization modulated specificity for host versus viral RNA, which resulted in disparate effects on viral replication during the innate immune response. Savan and colleagues show that distinct isoforms of the RNA-binding protein ZAP function as an antiviral restriction factor or as an interferon-resolution factor, based on differences in their intracellular localization.

The AIM2 inflammasome induces maturation of the proinflammatory cytokines IL-1β and IL-18. Using AIM2-deficient mice, Fitzgerald and colleagues and Alnemri and colleagues show that the AIM2 inflammasome is essential for host defense against cytosolic bacteria and DNA viruses. Francisella tularensis , the causative agent of tularemia, infects host macrophages, which triggers production of the proinflammatory cytokines interleukin 1β (IL-1β) and IL-18. We elucidate here how host macrophages recognize F. tularensis and elicit this proinflammatory response. Using mice deficient in the DNA-sensing inflammasome component AIM2, we demonstrate here that AIM2 is required for sensing F. tularensis . AIM2-deficient mice were extremely susceptible to F. tularensis infection, with greater mortality and bacterial burden than that of wild-type mice. Caspase-1 activation, IL-1β secretion and cell death were absent in Aim2 −/− macrophages in response to F. tularensis infection or the presence of cytoplasmic DNA. Our study identifies AIM2 as a crucial sensor of F. tularensis infection and provides genetic proof of its critical role in host innate immunity to intracellular pathogens.