RIG-I is a pivotal cytoplasmic sensor that recognizes different species of viral RNAs. This recognition leads to activation of the transcription factors NF-κB and IRF3, which collaborate to induce type I interferons (IFNs) and innate antiviral response. In this study, we identified the TRIM family protein TRIM4 as a positive regulator of RIG-I-mediated IFN induction. Overexpression of TRIM4 potentiated virus-triggered activation of IRF3 and NF-κB, as well as IFN-β induction, whereas knockdown of TRIM4 had opposite effects. Mechanistically, TRIM4 associates with RIG-I and targets it for K63-linked polyubiquitination. Our findings demonstrate that TRIM4 is an important regulator of the virus-induced IFN induction pathways by mediating RIG-I for K63-linked ubiquitination.

Toll/IL-1R domain-containing adaptor inducing IFN-β (TRIF) is an adaptor molecule that is recruited to TLR3 and -4 upon agonist stimulation and triggers activation of IFN regulatory factor 3 (IRF3) and expression of type 1 IFNs, which are critical for cellular antiviral responses. We show that Akt is a downstream molecule of TRIF/TANK-binding kinase 1 (TBK1) and plays an important role in the activation of IRF3 by TLR3 and -4 agonists. Blockade of Akt by a dominant-negative mutant or by short interfering RNA decreased IRF3 activation and IFN-β expression induced by polyinosinic:polycytidylic acid [poly(I:C)], LPS, TRIF, and TBK1. Association of endogenous TBK1 and Akt was observed in macrophages when stimulated with poly(I:C) and LPS. In vitro kinase assays combined with reversed-phase liquid chromatography mass spectrometry analysis showed that TBK1 enhanced phosphorylation of Akt on Ser(473), whereas knockdown of TBK1 expression by short interfering RNA in macrophages decreased poly(I:C)- and LPS-induced Akt phosphorylation. Embryonic fibroblasts derived from TBK1 knockout mice also showed impaired Akt phosphorylation in response to poly(I:C) and LPS. To our knowledge, our results demonstrate a new regulatory mechanism for Akt activation mediated by TBK1 and a novel role of Akt in TLR-mediated immune responses.

The BGLF4 protein kinase of Epstein-Barr virus (EBV) is a member of the conserved family of herpesvirus protein kinases which, to some extent, have a function similar to that of the cellular cyclin-dependent kinase in regulating multiple cellular and viral substrates. In a yeast two-hybrid screening assay, a splicing variant of interferon (IFN) regulatory factor 3 (IRF3) was found to interact with the BGLF4 protein. This interaction was defined further by coimmunoprecipitation in transfected cells and glutathione S-transferase (GST) pull-down in vitro. Using reporter assays, we show that BGLF4 effectively suppresses the activities of the poly(I:C)-stimulated IFN-beta promoter and IRF3-responsive element. Moreover, BGLF4 represses the poly(I:C)-stimulated expression of endogenous IFN-beta mRNA and the phosphorylation of STAT1 at Tyr701. In searching for a possible mechanism, BGLF4 was shown not to affect the dimerization, nuclear translocation, or CBP recruitment of IRF3 upon poly(I:C) treatment. Notably, BGLF4 reduces the amount of active IRF3 recruited to the IRF3-responsive element containing the IFN-beta promoter region in a chromatin immunoprecipitation assay. BGLF4 phosphorylates GST-IRF3 in vitro, but Ser339-Pro340 phosphorylation-dependent, Pin1-mediated downregulation is not responsible for the repression. Most importantly, we found that three proline-dependent phosphorylation sites at Ser123, Ser173, and Thr180, which cluster in a region between the DNA binding and IRF association domains of IRF3, contribute additively to the BGLF4-mediated repression of IRF3(5D) transactivation activity. IRF3 signaling is activated in reactivated EBV-positive NA cells, and the knockdown of BGLF4 further stimulates IRF3-responsive reporter activity. The data presented here thus suggest a novel mechanism by which herpesviral protein kinases suppress host innate immune responses and facilitate virus replication.

Previously we identified a DNA damage response-deficient (DDRD) molecular subtype within breast cancer. A 44-gene assay identifying this subtype was validated as predicting benefit from DNA-damaging chemotherapy. This subtype was defined by interferon signaling. In this study, we address the mechanism of this immune response and its possible clinical significance.

We used immunohistochemistry (IHC) to characterize immune infiltration in 184 breast cancer samples, of which 65 were within the DDRD subtype. Isogenic cell lines, which represent DDRD-positive and -negative, were used to study the effects of chemokine release on peripheral blood mononuclear cell (PBMC) migration and the mechanism of immune signaling activation. Finally, we studied the association between the DDRD subtype and expression of the immune-checkpoint protein PD-L1 as detected by IHC. All statistical tests were two-sided.

We found that DDRD breast tumors were associated with CD4+ and CD8+ lymphocytic infiltration (Fisher's exact test P < .001) and that DDRD cells expressed the chemokines CXCL10 and CCL5 3.5- to 11.9-fold more than DNA damage response-proficient cells (P < .01). Conditioned medium from DDRD cells statistically significantly attracted PBMCs when compared with medium from DNA damage response-proficient cells (P < .05), and this was dependent on CXCL10 and CCL5. DDRD cells demonstrated increased cytosolic DNA and constitutive activation of the viral response cGAS/STING/TBK1/IRF3 pathway. Importantly, this pathway was activated in a cell cycle-specific manner. Finally, we demonstrated that S-phase DNA damage activated expression of PD-L1 in a STING-dependent manner.

We propose a novel mechanism of immune infiltration in DDRD tumors, independent of neoantigen production. Activation of this pathway and associated PD-L1 expression may explain the paradoxical lack of T-cell-mediated cytotoxicity observed in DDRD tumors. We provide a rationale for exploration of DDRD in the stratification of patients for immune checkpoint-based therapies.

We have shown that activation of toll-like receptor 4 (TLR4) and its interferon regulatory factor 3 (IRF3)-dependent downstream signaling pathway are required for the development of liver ischemia/reperfusion injury (IRI). This study focused on the role of TLR4-IRF3 activation pathway products, in particular, chemokine (C-X-C motif) ligand 10 (CXCL10). The induction of CXCL10 by liver IR was rapid (1 hour postreperfusion), restricted (ischemic lobes), and specific (no CXCL9 and CXCL11 induction). Functionally, CXCL10 was critical for IR-induced liver inflammation and hepatocellular injury. CXCL10 knockout (KO) mice were protected from IRI, as evidenced by reduced serum alanine aminotransferase (sALT) levels and preserved liver histological detail. The induction of pro-inflammatory genes, such as tumor necrosis factor alpha (TNF-alpha), interleukin 1beta (IL-1beta), IL-6, and IL-12beta was diminished, whereas the induction of the IL-10 gene remained intact in CXCL10 KO mice, indicating an altered liver response against IR. This was accompanied by selective down-regulation of extracellular signal-regulated kinase (ERK), but intact Jun N-terminal kinase (JNK), activation in the KO IR livers. This altered liver inflammation response was (1) specific to IR, because lipopolysaccharide (LPS) induced a comparable pro-inflammatory response in CXCL10 KO and wild-type (WT) mice; and (2) responsible for liver cytoprotection from IR, because neutralization of IL-10 restored local inflammation and hepatocellular damage.

CONCLUSIONS

CXCL10 regulates liver inflammation response against IRI, and its deficiency protected livers from IRI by local IL-10-mediated cytoprotection. Targeting CXCL10 may provide a novel therapeutic means to ameliorate liver IRI in clinics.

Recognition of viral RNA structures by the intracytosolic RNA helicase RIG-I triggers induction of innate immunity. Efficient induction requires RIG-I ubiquitination by the E3 ligase TRIM25, its interaction with the mitochondria-bound MAVS protein, recruitment of TRAF3, IRF3- and NF-κB-kinases and transcription of Interferon (IFN). In addition, IRF3 alone induces some of the Interferon-Stimulated Genes (ISGs), referred to as early ISGs. Infection of hepatocytes with Hepatitis C virus (HCV) results in poor production of IFN despite recognition of the viral RNA by RIG-I but can lead to induction of early ISGs. HCV was shown to inhibit IFN production by cleaving MAVS through its NS3/4A protease and by controlling cellular translation through activation of PKR, an eIF2α-kinase containing dsRNA-binding domains (DRBD). Here, we have identified a third mode of control of IFN induction by HCV. Using HCVcc and the Huh7.25.CD81 cells, we found that HCV controls RIG-I ubiquitination through the di-ubiquitine-like protein ISG15, one of the early ISGs. A transcriptome analysis performed on Huh7.25.CD81 cells silenced or not for PKR and infected with JFH1 revealed that HCV infection leads to induction of 49 PKR-dependent genes, including ISG15 and several early ISGs. Silencing experiments revealed that this novel PKR-dependent pathway involves MAVS, TRAF3 and IRF3 but not RIG-I, and that it does not induce IFN. Use of PKR inhibitors showed that this pathway requires the DRBD but not the kinase activity of PKR. We then demonstrated that PKR interacts with HCV RNA and MAVS prior to RIG-I. In conclusion, HCV recruits PKR early in infection as a sensor to trigger induction of several IRF3-dependent genes. Among those, ISG15 acts to negatively control the RIG-I/MAVS pathway, at the level of RIG-I ubiquitination.These data give novel insights in the machinery involved in the early events of innate immune response.

Infection with Listeria monocytogenes strains that enter the host cell cytosol leads to a robust cytotoxic T cell response resulting in long-lived cell-mediated immunity (CMI). Upon entry into the cytosol, L. monocytogenes secretes cyclic diadenosine monophosphate (c-di-AMP) which activates the innate immune sensor STING leading to the expression of IFN-β and co-regulated genes. In this study, we examined the role of STING in the development of protective CMI to L. monocytogenes. Mice deficient for STING or its downstream effector IRF3 restricted a secondary lethal challenge with L. monocytogenes and exhibited enhanced immunity that was MyD88-independent. Conversely, enhancing STING activation during immunization by co-administration of c-di-AMP or by infection with a L. monocytogenes mutant that secretes elevated levels of c-di-AMP resulted in decreased protective immunity that was largely dependent on the type I interferon receptor. These data suggest that L. monocytogenes activation of STING downregulates CMI by induction of type I interferon.

Toll-like receptor 4 (TLR4) signals the induction of transcription factor IRF3-dependent genes from the early endosome via the adaptor TRAM. Here we report a splice variant of TRAM, TAG ('TRAM adaptor with GOLD domain'), which has a Golgi dynamics domain coupled to TRAM's Toll-interleukin 1 receptor domain. After stimulation with lipopolysaccharide, TRAM and TAG localized to late endosomes positive for the GTPase Rab7a. TAG inhibited activation of IRF3 by lipopolysaccharide. Knockdown of TAG with small interfering RNA enhanced induction of the chemokine CCL5 (RANTES), but not of interleukin 8, by lipopolysaccharide in human peripheral blood mononuclear cells. TAG displaced the adaptor TRIF from TRAM. TAG is therefore an example of a specific inhibitor of the adaptor MyD88-independent pathway activated by TLR4. Targeting TAG could be useful in the effort to boost the immunostimulatory effect of TLR4 without causing unwanted inflammation.

The sensing of pathogen infection and subsequent triggering of innate immunity are key to controlling zoonotic infections. Myxoma virus (MV) is a cytoplasmic DNA poxvirus that in nature infects only rabbits. Our previous studies have shown that MV infection of primary mouse cells is restricted by virus-induced type I interferon (IFN). However, little is known about the innate sensor(s) involved in activating signaling pathways leading to cellular defense responses in primary human immune cells. Here, we show that the complete restriction of MV infection in the primary human fibroblasts requires both tumor necrosis factor (TNF) and type I IFN. We also demonstrate that MV infection of primary human macrophages (pHMs) activates the cytoplasmic RNA sensor called retinoic acid inducible gene I (RIG-I), which coordinately induces the production of both TNF and type I IFN. Of note, RIG-I sensing of MV infection in pHMs initiates a sustained TNF induction through the sequential involvement of the downstream IFN-regulatory factors 3 and 7 (IRF3 and IRF7). Thus, RIG-I-mediated co-induction of TNF and type I IFN by virus-infected pHMs represents a novel innate defense mechanism to restrict viral infection in human cells. These results also reveal a new regulatory mechanism for TNF induction following viral infection.

Viral infection causes host cells to produce type I interferons (IFNs), which are critically involved in viral clearance. Previous studies have demonstrated that activation of the transcription factor interferon regulatory factor (IRF)3 is essential for virus-triggered induction of type I IFNs. Here we show that the E3 ubiquitin ligase RBCC protein interacting with PKC1 (RBCK1) catalyzes the ubiquitination and degradation of IRF3. Overexpression of RBCK1 negatively regulates Sendai virus-triggered induction of type I IFNs, while knockdown of RBCK1 has the opposite effect. Plaque assays consistently demonstrate that RBCK1 negatively regulates the cellular antiviral response. Furthermore, viral infection leads to induction of RBCK1 and subsequent degradation of IRF3. These findings suggest that the cellular antiviral response is controlled by a negative feedback regulatory mechanism involving RBCK1-mediated ubiquitination and degradation of IRF3.

Indoleamine 2,3-dioxygenase (IDO) is the first and rate-limiting enzyme in the kynurenine pathway of tryptophan catabolism and has been implicated in neurotoxicity and suppression of the antiviral T-cell response in HIV encephalitis (HIVE). Here we show that the Toll-like receptor 3 (TLR3) ligand poly(I:C) (PIC) induces the expression of IDO in human astrocytes. PIC was less potent than gamma interferon (IFN-gamma) but more potent than IFN-beta in inducing IDO. PIC induction of IDO was mediated in part by IFN-beta but not IFN-gamma, and both NF-kappaB and interferon regulatory factor 3 (IRF3) were required. PIC also upregulated TLR3, thereby augmenting the primary (IFN-beta) and secondary (IDO and viperin) response genes upon subsequent stimulation with PIC. In HIVE, the transcripts for TLR3, IFN-beta, IDO, and viperin were increased and IDO immunoreactivity was detected in reactive astrocytes as well as macrophages and microglia. PIC caused suppression of intracellular replication of human immunodeficiency virus pseudotyped with vesicular stomatitis virus G protein and human cytomegalovirus in a manner dependent on IRF3 and IDO. The involvement of IDO was demonstrated by partial but significant reversal of the PIC-mediated antiviral effect by IDO RNA interference and/or tryptophan supplementation. Importantly, the cytokine interleukin-1 abolished IFN-gamma-induced IDO enzyme activity in a nitric oxide-dependent manner without suppressing protein expression. Our results demonstrate that IDO is an innate antiviral protein induced by double-stranded RNA and suggest a therapeutic utility for PIC in human viral infections. They also show that IDO activity can be dissociated from protein expression, indicating that the local central nervous system cytokine and nitric oxide environment determines IDO function.

Dendritic cells (DCs) play a central role in innate immunity and antiviral responses. In this study, we investigated the production of alpha interferon (IFN-alpha) and inducible chemokines by human monocyte-derived dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) infected with West Nile virus (WNV), an emergent pathogen whose infection can lead to severe cases of encephalitis in the elderly, children, and immunocompromised individuals. Our experiments demonstrated that WNV grown in mammalian cells (WNV(Vero)) was a potent inducer of IFN-alpha secretion in pDCs and, to a lesser degree, in mDCs. The ability of WNV(Vero) to induce IFN-alpha in pDCs did not require viral replication and was prevented by the treatment of cells with bafilomycin A1 and chloroquine, suggesting that it was dependent on endosomal Toll-like receptor recognition. On the other hand, IFN-alpha production in mDCs required viral replication and was associated with the nuclear translocation of IRF3 and viral antigen expression. Strikingly, pDCs failed to produce IFN-alpha when stimulated with WNV grown in mosquito cells (WNV(C7/10)), while mDCs responded similarly to WNV(Vero) or WNV(C7/10). Moreover, the IFN-dependent chemokine IP-10 was produced in substantial amounts by pDCs in response to WNV(Vero) but not WNV(C7/10), while interleukin-8 was produced in greater amounts by mDCs infected with WNV(C7/10) than in those infected with WNV(Vero). These findings suggest that cell-specific mechanisms of WNV recognition leading to the production of type I IFN and inflammatory chemokines by DCs may contribute to both the innate immune response and disease pathogenesis in human infections.

IFNs lambda1, lambda2, and lambda3, or type III IFNs, are recently identified cytokines distantly related to type I IFNs. Despite an early evolutionary divergence, the 2 types of IFNs display similar antiviral activities, and both are produced primarily in dendritic cells. Although virus induction of the type I IFN-beta gene had served as a paradigm of gene regulation, relatively little is known about the regulation of IFN-lambda gene expression. Studies of virus induction of IFN-lambda1 identified an essential role of IFN regulatory factors (IRF) 3 and 7, which bind to a regulatory DNA sequence near the start site of transcription. Here, we report that the proximal promoter region of the IFN-lambda1 regulatory region is not sufficient for maximal gene induction in response to bacterial LPS, and we identify an essential cluster of homotypic NF-kappaB binding sites. Remarkably, these sites, which bind efficiently to NF-kappaB and function independently of the IRF3/7 binding sites, originate as transposable elements of the Alu and LTR families. We also show that depletion of the NF-kappaB RelA protein significantly reduces the level of the IFN-lambda1 gene expression. We conclude that IFN-lambda1 gene expression requires NF-kappaB, and we propose a model for IFN-lambda1 gene regulation, in which IRF and NF-kappaB activate gene expression independently via spatially separated promoter elements. These observations provide insights into the independent evolution of the IFN-lambda1 and IFN-beta promoters and directly implicate transposable elements in the regulation of the IFN-lambda1 gene by NF-kappaB.

TANK-binding kinase 1 (TBK1/NAK/T2K) and I-kappaB Kinase (IKK-i/IKK-epsilon) play important roles in the regulation of interferon (IFN)-inducible genes during the immune response to bacterial and viral infections. Cell stimulation with ssRNA virus, dsDNA virus or gram-negative bacteria leads to activation of TBK1 or IKK-i, which in turn phosphorylates the transcription factors, IFN-regulatory factor (IRF) 3 and IRF7, promoting their translocation in the nucleus. To understand the molecular basis of activation of TBK1, we analyzed the sequence of TBK1 and IKK-i and identified a ubiquitin-like domain (ULD) adjacent to their kinase domains. Deletion or mutations of the ULD in TBK1 or IKK-i impaired activation of respective kinases, failed to induce IRF3 phosphorylation and nuclear localization and to activate IFN-beta or RANTES promoters. The importance of the ULD of TBK1 in LPS- or poly(I:C)-stimulated IFN-beta production was demonstrated by reconstitution experiments in TBK1-IKK-i-deficient cells. We propose that the ULD is a regulatory component of the TBK1/IKK-i kinases involved in the control of the kinase activation, substrate presentation and downstream signaling pathways.

Toll-like receptor 3 (TLR3) mediates antiviral response by recognizing double-stranded RNA. Its cytoplasmic domain is tyrosine phosphorylated upon ligand binding and initiates downstream signaling via the adapter TIR-containing adaptor inducing interferon-β (TRIF). However, the kinase responsible for TLR3 phosphorylation remains unknown. We show here that Bruton's tyrosine kinase (BTK)-deficient macrophages failed to secrete inflammatory cytokines and IFN-β upon TLR3 stimulation and were impaired in clearing intracellular dengue virus infection. Mutant mice were also less susceptible to d-galactosamine/p(I:C)-induced sepsis. In the absence of BTK, TLR3-induced phosphoinositide 3-kinase (PI3K), AKT and MAPK signaling and activation of NFκB, IRF3, and AP-1 transcription factors were all defective. We demonstrate that BTK directly phosphorylates TLR3 and in particular the critical Tyr759 residue. BTK point mutations that abrogate or led to constitutive kinase activity have opposite effects on TLR3 phosphorylation. Loss of BTK also compromises the formation of the downstream TRIF/receptor-interacting protein 1 (RIP1)/TBK1 complex. Thus, BTK plays a critical role in initiating TLR3 signaling.

Interferon regulatory factor 3 (IRF3) is a key transcription factor involved in the induction of interferon (IFN) in response to viral infection. Rotavirus non-structural protein NSP1 binds to and targets IRF3 for proteasome degradation early post-infection. Mutational analysis of cysteine and histidine residues within the conserved N-terminal zinc-binding domain in NSP1 of bovine rotavirus strain B641 abolished IRF3 degradation in transfected cells. Thus, the integrity of the zinc-binding domain in NSP1 is important for degradation of IRF3. In contrast to bovine strain B641, IRF3 was stable in cells infected with porcine rotavirus strain OSU and OSU NSP1 bound only weakly to IRF3. Both B641 NSP1 and OSU NSP1 were stabilized in cells or cell-free extracts in the presence of the proteasome inhibitor MG132 and when the zinc-binding domain was disrupted by site-directed mutagenesis. Data from the B641 analyses that show IRF3 degradation is dependent on the presence of NSP1 and the integrity of the N-terminal zinc-binding domain, coupled with the regulated stability of IRF3 and NSP1 by the proteasome, collectively support the hypothesis that NSP1 is an E3 ubiquitin ligase.

Ro52 is a common target of circulating autoantibodies in autoimmune disease. Data indicate that anti-Ro52 antibodies are associated with distinct clinical manifestations. It is therefore of major interest to understand how it becomes an antigenic target and what cells express this protein under what conditions and what cellular function it has. Ro52 contains a RING and a B-box motif, followed by a coiled-coil domain and a B30.2 (or PRYSPRY) region in the C-terminal end. This molecular structure places Ro52 within the family of tripartite motif proteins (TRIM), and it is also denoted TRIM21. Like several other TRIM proteins, Ro52 has E3 ligase activity and functions in the process of ubiquitination. Ro52 is expressed in the immune system as a predominantly cytoplasmic protein that can be upregulated and translocate to the nucleus in a proinflammatory environment. Reported substrates for Ro52-mediated ubiquitination include IRF3, IRF5, IRF7 and IRF8, and via these transcription factors Ro52 regulates type 1 interferon and cytokine production. Ro52 is upregulated at the site of autoimmune inflammation, such as cutaneous lupus lesions. This implies that Ro52 may have an important role in the pathogenesis of autoimmunity, and this paper will review the available data on the role of Ro52 in immune responses and autoimmune pathogenesis.

Toll-like receptor (TLR) ligands that signal via TIR-domain-containing adapter-inducing IFNβ (TRIF) activate the IκB kinase (IKK)-related kinases, TRAF associated NFκB activator (TANK)-binding kinase-1 (TBK1) and IKKε, which then phosphorylate IRF3 and induce the production of IFNβ. Here we show that TBK1 and IKKε are also activated by TLR ligands that signal via MyD88. Notably, the activation of IKKε is rapid, transient, and it precedes a more prolonged activation of TBK1. The MyD88- and TRIF-dependent signaling pathways activate the IKK-related kinases by two signaling pathways. One is mediated by the canonical IKKs, whereas the other culminates in the autoactivation of the IKK-related kinases. Once activated, TBK1/IKKε then phosphorylate and inhibit the canonical IKKs. The negative regulation of the canonical IKKs by the IKK-related kinases occurs in both the TRIF- and MyD88-dependent TLR pathways, whereas IRF3 phosphorylation is restricted to the TRIF-dependent signaling pathway. We have discovered that the activation of IKKε is abolished, the activation of TBK1 is reduced, and the interaction between the IKK-related kinases and the canonical IKKs is suppressed in TANK(-/-) macrophages, preventing the IKK-related kinases from negatively regulating the canonical IKKs. In contrast, IRF3 phosphorylation and IFNβ production was normal in TANK(-/-) macrophages. Our results demonstrate a key role for TANK in enabling the canonical IKKs and the IKK-related kinases to regulate each other, which is required to limit the strength of TLR signaling and ultimately, prevent autoimmunity.

Nod-like receptors (NLRs) are intracellular sensors that respond to a variety of pathogen and intracellular danger signals to induce innate immune responses. NLRC5 has recently been identified to be an important regulator of NF-κB, type I interferon (IFN) and inflammasome signaling pathways, but the in vivo function and mechanisms of NLRC5 remain to be defined. Here, we describe the generation and characterization of NLRC5 knockout mice. We show that induction of NLRC5 expression by Toll-like receptor (TLR) ligand or cytokine stimulation requires the signal transducers and activators of transcription (Stat)1-mediated signaling pathway. NLRC5 ablation reduces MHC class I expression, and enhances IKK and IRF3 phosphorylation in response to TLR stimulation or viral infection. Consistent with these observations, we found that NLRC5 deficiency enhanced IL-6 and IFN-β production in mouse embryonic fibroblasts (MEFs), peritoneal macrophages and bone marrow-derived macrophages (BMMs), but not bone marrow-derived dendritic cells (BMDCs) after LPS stimulation or vesicular stomatitis virus (VSV) infection. Furthermore, we found that NLRC5-deficient mice produced higher amounts of IL-6 and IFN-β in the sera when they were challenged with LPS or infected with VSV. Taken together, these results provide in vivo evidence that NLRC5 plays critical roles in MHC class I expression, innate immune signaling and antiviral innate immune responses, thus serving as an important target for modulating innate immune signaling and regulation.

The vaccinia virus double-stranded RNA binding protein E3 has been demonstrated to inhibit the expression of cytokines, including beta interferon (IFN-beta) and tumor necrosis factor alpha (TNF-alpha). However, few details regarding the molecular mechanisms of this inhibition have been described. Using real-time PCR arrays, we found that E3 suppressed the induction of a diverse array of cytokines representing members of the IFN, interleukin (IL), TNF, and transforming growth factor cytokine families. We discovered that the factor(s) responsible for the induction of IL-6, TNF-alpha, and inhibin beta A (INHBA) was associated with the early and late phases of virus infection. In contrast, the factor(s) which regulates IFN-beta induction was associated with the late phase of replication. We have found that expression of these cytokines can be induced by transfection of cells with RNA isolated from vaccinia virus-infected cells. Moreover, we provide evidence that E3 antagonizes both PKR-dependent and PKR-independent pathways to regulate cytokine expression. PKR-dependent activation of p38 and NF-kappaB was required for vaccinia virus-induced INHBA expression, whereas induction of TNF-alpha required only PKR-dependent NF-kappaB activation. In contrast, induction of IL-6 and IFN-beta was largely PKR independent. IL-6 induction is regulated by NF-kappaB, while IFN-beta induction is mediated by IFN-beta promoter stimulator 1 and IFN regulatory factor 3/NF-kappaB. Collectively, these results indicate that E3 suppresses distinct but interlinked host signaling pathways to inhibit the expression of a diverse array of cytokines.

Protection against mucosally transmitted infections probably requires immunity at the site of pathogen entry, yet there are no mucosal adjuvant formulations licensed for human use. Polyethyleneimine (PEI) represents a family of organic polycations used as nucleic acid transfection reagents in vitro and DNA vaccine delivery vehicles in vivo. Here we show that diverse PEI forms have potent mucosal adjuvant activity for viral subunit glycoprotein antigens. A single intranasal administration of influenza hemagglutinin or herpes simplex virus type-2 (HSV-2) glycoprotein D with PEI elicited robust antibody-mediated protection from an otherwise lethal infection, and was superior to existing experimental mucosal adjuvants. PEI formed nanoscale complexes with antigen, which were taken up by antigen-presenting cells in vitro and in vivo, promoted dendritic cell trafficking to draining lymph nodes and induced non-proinflammatory cytokine responses. PEI adjuvanticity required release of host double-stranded DNA that triggered Irf3-dependent signaling. PEI therefore merits further investigation as a mucosal adjuvant for human use.

TANK-binding kinase 1 (TBK1) plays an essential role in Toll-like receptor (TLR)- and retinoic acid-inducible gene I (RIG-I)-mediated induction of type I interferon (IFN; IFN-α/β) and host antiviral responses. How TBK1 activity is negatively regulated remains largely unknown. We report that TNF receptor-associated factor (TRAF)-interacting protein (TRIP) promotes proteasomal degradation of TBK1 and inhibits TLR3/4- and RIG-I-induced IFN-β signaling. TRIP knockdown resulted in augmented activation of IFN regulatory factor 3 (IRF3) and enhanced expression of IFN-β in TLR3/4- and RIG-I-activated primary peritoneal macrophages, whereas overexpression of TRIP had opposite effects. Consistently, TRIP impaired Sendai virus (SeV) infection-induced IRF3 activation and IFN-β production and promoted vesicular stomatitis virus (VSV) replication. As an E3 ubiquitin ligase, TRIP negatively regulated the cellular levels of TBK1 by directly binding to and promoting K48-linked polyubiquitination of TBK1. Therefore, we identified TRIP as a negative regulator in TLR3/4- and RIG-I-triggered antiviral responses and suggested TRIP as a potential target for the intervention of diseases with uncontrolled IFN-β production.

Astrocytes, together with microglia and macrophages, participate in innate inflammatory responses in the CNS. Although inflammatory mediators such as interferons generated by astrocytes may be critical in the defense of the CNS, sustained unopposed cytokine signaling could result in harmful consequences. Interferon regulatory factor 3 (IRF3) is a transcription factor required for IFNβ production and antiviral immunity. Most cells express low levels of IRF3 protein, and the transcriptional mechanism that upregulates IRF3 expression is not known. In this study, we explored the consequence of adenovirus-mediated IRF3 gene transfer (Ad-IRF3) in primary human astrocytes. We show that IRF3 transgene expression suppresses proinflammatory cytokine gene expression upon challenge with IL-1/IFNγ and alters astrocyte activation phenotype from a proinflammatory to an anti-inflammatory one, akin to an M1-M2 switch in macrophages. This was accompanied by the rescue of neurons from cytokine-induced death in glial-neuronal co-cultures. Furthermore, Ad-IRF3 suppressed the expression of microRNA-155 and its star-form partner miR-155*, immunoregulatory miRNAs highly expressed in multiple sclerosis lesions. Astrocyte miR-155/miR155* were induced by cytokines and TLR ligands with a distinct hierarchy and involved in proinflammatory cytokine gene induction by targeting suppressor of cytokine signaling 1, a negative regulator of cytokine signaling and potentially other factors. Our results demonstrate a novel proinflammatory role for miR-155/miR-155* in human astrocytes and suggest that IRF3 can suppress neuroinflammation through regulating immunomodulatory miRNA expression. © 2011 Wiley-Liss, Inc.

Interferon regulatory factor 3 (IRF3) plays a crucial role in mediating cellular responses to virus intrusion. The protein kinase TBK1 is a key regulator inducing phosphorylation of IRF3. The regulatory mechanisms during IRF3 activation remain poorly characterized. In the present study, we have identified by yeast two-hybrid approach a specific interaction between IRF3 and chaperone heat-shock protein of 90 kDa (Hsp90). The C-terminal truncation mutant of Hsp90 is a strong dominant-negative inhibitor of IRF3 activation. Knockdown of endogenous Hsp90 by RNA interference attenuates IRF3 activation and its target gene expressions. Alternatively, Hsp90-specific inhibitor geldanamycin (GA) dramatically reduces expression of IRF3-regulated interferon-stimulated genes and abolishes the cytoplasm-to-nucleus translocation and DNA binding activity of IRF3 in Sendai virus-infected cells. Significantly, virus-induced IRF3 phosphorylation is blocked by GA, whereas GA does not affect the protein level of IRF3. In addition, TBK1 is found to be a client protein of Hsp90 in vivo. Treatment of 293 cells with GA interferes with the interaction of TBK1 and Hsp90, resulting in TBK1 destabilization and its subsequent proteasome-mediated degradation. Besides maintaining stability of TBK1, Hsp90 also forms a novel complex with TBK1 and IRF3, which brings TBK1 and IRF3 dynamically into proximity and facilitates signal transduction from TBK1 to IRF3. Our study uncovers an essential role of Hsp90 in the virus-induced activation of IRF3.