Retinoic acid inducible gene-I mediated detection of bacterial nucleic acids in human microglial cells.

BACKGROUND: Bacterial meningitis and meningoencephalitis are associated with devastating neuroinflammation. We and others have demonstrated the importance of glial cells in the initiation of immune responses to pathogens invading the central nervous system (CNS). These cells use a variety of pattern recognition receptors (PRRs) to identify common pathogen motifs and the cytosolic sensor retinoic acid inducible gene-1 (RIG-I) is known to serve as a viral PRR and initiator of interferon (IFN) responses. Intriguingly, recent evidence indicates that RIG-I also has an important role in the detection of bacterial nucleic acids, but such a role has not been investigated in glia. METHODS: In this study, we have assessed whether primary or immortalized human and murine glia express RIG-I either constitutively or following stimulation with bacteria or their products by immunoblot analysis. We have used capture ELISAs and immunoblot analysis to assess human microglial interferon regulatory factor 3 (IRF3) activation and IFN production elicited by bacterial nucleic acids and novel engineered nucleic acid nanoparticles. Furthermore, we have utilized a pharmacological inhibitor of RIG-I signaling and siRNA-mediated knockdown approaches to assess the relative importance of RIG-I in such responses. RESULTS: We demonstrate that RIG-I is constitutively expressed by human and murine microglia and astrocytes, and is elevated following bacterial infection in a pathogen and cell type-specific manner. Additionally, surface and cytosolic PRR ligands are also sufficient to enhance RIG-I expression. Importantly, our data demonstrate that bacterial RNA and DNA both trigger RIG-I-dependent IRF3 phosphorylation and subsequent type I IFN production in human microglia. This ability has been confirmed using our nucleic acid nanoparticles where we demonstrate that both RNA- and DNA-based nanoparticles can stimulate RIG-I-dependent IFN responses in these cells. CONCLUSIONS: The constitutive and bacteria-induced expression of RIG-I by human glia and its ability to mediate IFN responses to bacterial RNA and DNA and nucleic acid nanoparticles raises the intriguing possibility that RIG-I may be a potential target for therapeutic intervention during bacterial infections of the CNS, and that the use of engineered nucleic acid nanoparticles that engage this sensor might be a method to achieve this goal.

Herpes Simplex Virus 1 γ134.5 Protein Inhibits STING Activation That Restricts Viral Replication.

The γ134.5 gene of herpes simplex virus 1 (HSV-1) encodes a virulence factor that promotes viral pathogenesis. Although it perturbs TANK-binding kinase 1 (TBK1) in the complex network of innate immune pathways, the underlying mechanism is obscure. Here we report that HSV-1 γ134.5 targets stimulator of interferon genes (STING) in the intracellular DNA recognition pathway that regulates TBK1 activation. In virus-infected cells the γ134.5 protein associates with and inactivates STING, which leads to downregulation of interferon regulatory factor 3 (IRF3) and IFN responses. Importantly, HSV-1 γ134.5 disrupts translocation of STING from the endoplasmic reticulum to Golgi apparatus, a process necessary to prime cellular immunity. Deletion of γ134.5 or its amino-terminal domain from HSV-1 abolishes the observed inhibitory activities. Consistently, an HSV mutant that lacks functional γ134.5 replicated less efficiently in STING+/+ than in STING-/- mouse embryonic fibroblasts. Moreover, reconstituted expression of human STING in the STING-/- cells activated IRF3 and reduced viral growth. These results suggest that control of the DNA sensing pathway by γ134.5 is advantageous to HSV infection.IMPORTANCE Viral inhibition of innate immunity contributes to herpes simplex virus pathogenesis. Although this complex process involves multiple factors, the underlying events remain unclear. We demonstrate that an HSV virulence factor γ134.5 precludes the activation of STING, a central adaptor in the intracellular DNA sensing pathway. Upon HSV infection, this viral protein engages with and inactivates STING. Consequently, it compromises host immunity and facilitates HSV replication. These observations uncover an HSV mechanism that is likely to mediate viral virulence.

Cathepsin H deficiency in mice induces excess Th1 cell activation and early-onset of EAE though impairment of toll-like receptor 3 cascade.

OBJECTIVE: The objective of this study is to investigate the role of cathepsin H (CatH), a lysosomal cysteine protease, in the development of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. METHODS: EAE was induced in CatH-deficient mice (CatH-/-) and wild-type littermates (+/+) using myelin oligodendrocyte glycoprotein (MOG) 35-55. The effects of CatH deficiency were determined by clinical scoring, mRNA expression levels of Tbx21, Rorc and FoxP3, protein levels of poly(I:C)-induced toll-like receptor 3 (TLR3) and phosphorylation of IRF3, and secretion of interferon-ß (IFN-ß) by splenocytes. RESULTS AND CONCLUSIONS: CatH-/- showed a significantly earlier disease onset of EAE and increased Th1 cell differentiation in splenocytes. Splenocytes prepared from immunized CatH-/- showed a significant decrease in poly(I:C)-induced increased TLR3 expression, interferon regulatory factor 3 (IRF3) phospholylation and IFN-ß secretion. Therefore, CatH deficiency impaired TLR3-mediated activation of IRF3 and consequent secretion of IFN-ß from dendritic cells, leading to the enhancement of Th1 cell differentiation and consequent early disease onset of EAE.

Expression kinetics of ISG15, IRF3, IFNγ, IL10, IL2 and IL4 genes vis-a-vis virus shedding, tissue tropism and antibody dynamics in PPRV vaccinated, challenged, infected sheep and goats.

Here, we studied the in vivo expression of Th1 (IL2 and IFN gamma) and Th2 (IL4 and IL10) - cytokines and antiviral molecules - IRF3 and ISG15 in peripheral blood mononuclear cells in relation to antigen and antibody dynamics under Peste des petits ruminants virus (PPRV) vaccination, infection and challenge in both sheep and goats. Vaccinated goats were seropositive by 9 days post vaccination (dpv) while in sheep idiosyncratic response was observed between 9 and 14 dpv for different animals. Expression of PPRV N gene was not detected in PBMCs of vaccinated and vaccinated challenged groups of both species, but was detected in unvaccinated infected PBMCs at 9 and 14 days post infection. The higher viral load at 9 dpi coincided with the peak clinical signs of the disease. The peak in viral replication at 9 dpi correlated with significant expression of antiviral molecules IRF3, ISG15 and IFN gamma in both the species. With the progression of disease, the decrease in N gene expression also correlated with the decrease in expression of IRF3, ISG15 and IFN gamma. In the unvaccinated infected animals ISG15, IRF3, IFN gamma and IL10 expression was higher than vaccinated animals. The IFN gamma expression predominated over IL4 in both vaccinated and infected animals with the infected exhibiting a stronger Th1 response. The persistent upregulation of this antiviral molecular signature - ISG15 and IRF3 even after 2 weeks post vaccination, presumably reflects the ongoing stimulation of innate immune cells.

STING Is Involved in Antiviral Immune Response against VZV Infection via the Induction of Type I and III IFN Pathways.

Varicella zoster virus (VZV) is a human-restricted α-herpesvirus that exhibits tropism for the skin. The VZV host receptors and downstream signaling pathways responsible for the antiviral innate immune response in the skin are not completely understood. Here, we show that STING mediates an important host defense against VZV infection in dermal cells including human dermal fibroblasts and HaCaT keratinocytes. Inhibition of STING using small interfering-RNA or short hairpin RNA-mediated gene disruption resulted in enhanced viral replication but diminished IRF3 phosphorylation and induction of IFNs and proinflammatory cytokines. Pretreatment with STING agonists resulted in reduced VZV glycoprotein E expression and viral replication. Additionally, using RNA sequencing to analyze dual host and VZV transcriptomes, we identified several host immune genes significantly induced by VZV infection. Furthermore, significant up-regulation of IFN-λ secretion was observed after VZV infection, partly through a STING-dependent pathway; IFN-λ was shown to be crucial for antiviral defense against VZV in human dermal cells. In conclusion, our data provide an important insight into STING-mediated induction of type I and III IFNs and subsequent antiviral signaling pathways that regulate VZV replication in human dermal cells.

A herbal formula comprising Rosae Multiflorae Fructus and Lonicerae Japonicae Flos inhibits the production of inflammatory mediators and the IRAK-1/TAK1 and TBK1/IRF3 pathways in RAW 264.7 and THP-1 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: As documented in the Chinese Materia Medica Grand Dictionary (), a herbal formula (RL) consisting of Rosae Multiflorae Fructus (multiflora rose hips) and Lonicerae Japonicae Flos (Japanese honeysuckle flowers) has traditionally been used in treating inflammatory disorders. RL was previously reported to inhibit the expression of various inflammatory mediators regulated by NF-κB and MAPKs that are components of the TLR4 signalling pathways. AIM OF THE STUDY: This study aims to provide further justification for clinical application of RL in treating inflammatory disorders by further delineating the involvement of the TLR4 signalling cascades in the effects of RL on inflammatory mediators. MATERIALS AND METHODS: RL consisting of Rosae Multiflorae Fructus and Lonicerae Japonicae Flos (in 5:3 ratio) was extracted using absolute ethanol. We investigated the effect of RL on the production of cytokines and chemokines that are regulated by three key transcription factors of the TLR4 signalling pathways AP-1, NF-κB and IRF3 in LPS-stimulated RAW264.7 cells using the multiplex biometric immunoassay. Phosphorylation of AP-1, NF-κB, IRF3, IκB-α, IKKα/ß, Akt, TAK1, TBK1, IRAK-1 and IRAK-4 were examined in LPS-stimulated RAW264.7 cells and THP-1 cells using Western blotting. Nuclear localizations of AP-1, NF-κB and IRF3 were also examined using Western blotting. RESULTS: RL reduced the secretion of various pro-inflammatory cytokines and chemokines regulated by transcription factors AP-1, NF-κB and IRF3. Phosphorylation and nuclear protein levels of these transcription factors were decreased by RL treatment. Moreover, RL inhibited the activation/phosphorylation of IκB-α, IKKα/ß, TAK1, TBK1 and IRAK-1. CONCLUSIONS: Suppression of the IRAK-1/TAK1 and TBK1/IRF3 signalling pathways was associated with the effect of RL on inflammatory mediators in LPS-stimulated RAW264.7 and THP-1 cells. This provides further pharmacological basis for the clinical application of RL in the treatment of inflammatory disorders.

Suppression of the TRIF-dependent signaling pathway of Toll-like receptor by CDr10b in RAW264.7 macrophages.

Toll-like receptors (TLRs) recognize distinct pathogen-associated molecular patterns and play a critical role in innate immune responses. TLR signaling pathways can be largely classified as either myeloid differential factor 88 (MyD88)- or toll-interleukin-1 receptor domain-containing adapter inducing interferon-ß (TRIF)-dependent pathways. Compound of Designation red 10 binding (CDr10b) was synthesized to investigate its role in neuroinflammatory diseases. This study was conducted to determine whether CDr10b can affect TLR signaling pathways. CDr10b suppressed NF-κB activation as well as COX-2 and iNOS expression induced by TLR3 or TLR4 agonists. CDr10b also suppressed the activation of interferon regulatory factor 3 (IRF3) and the expression of interferon inducible protein-10 (IP-10) induced by TLR3 or TLR4 agonists. These results indicate that CDr10b can modulate the TRIF-dependent pathway of TLRs and has the potential to become a new therapeutic drug for chronic inflammatory diseases.

Phosphatidylinositol-3-kinase and Akt are required for RIG-I-mediated anti-viral signalling through cross-talk with IPS-1.

Retinoic acid-inducible gene I (RIG-I) is a cytosolic pattern-recognition receptor that recognizes viruses and triggers anti-viral immune responses. Activation of intracellular RIG-I signalling is mediated through interferon-ß (IFN-ß) promoter stimulator-1 (IPS-1), an adaptor of RIG-I, which induces IFN regulatory factor (IRF) 3 activation and type I IFN expression. The phosphatidylinositol-3-kinase (PI3K) and Akt pathway is activated in host immune cells upon viral infection. However, the mechanism as to how they work in RIG-I signalling has not been fully elucidated. Therefore, we investigated the role of PI3K and Akt in the regulation of RIG-I-mediated IRF3 activation and type I IFN expression in macrophages. Our results show that Sendai virus infection, which is recognized by RIG-I, led to IRF3 activation and IFN-ß expression and these responses were attenuated by the PI3K inhibitor (LY294002) and an Akt dominant-negative mutant in the macrophage cell line(RAW264.7). IRF3 phosphorylation and dimerization as well as IFN-ß expression induced by a synthetic RIG-I agonist, short poly(I:C), were suppressed by LY294002 or siRNA-Akt in bone marrow-derived macrophages. Suppression of PI3K and Akt using a dominant-negative mutant and siRNA knockdown resulted in attenuation of IRF3 activation and IFN-ß expression induced by RIG-I itself or its adaptor, IPS-1. Association of Akt with IPS-1 increased with short poly(I:C) stimulation and required the pleckstrin homology domain of Akt and caspase-recruitment domain in IPS-1. Collectively, our results show that PI3K and Akt are required downstream of IPS-1 for RIG-I-mediated anti-viral immune responses. The results describe a novel, interactive relationship between RIG-I downstream signalling molecules resulting in efficient anti-viral immunity.

IFIT5 potentiates anti-viral response through enhancing innate immune signaling pathways.

Humans have a distinct combination of IFIT (IFN-induced protein with tetratricopeptide repeats) family orthologs, including IFIT1 (ISG56), IFIT2 (ISG54), IFIT3 (ISG60), and IFIT5 (ISG58). The function of IFIT1/IFIT2/IFIT3 has been intensively investigated. However, little is known about the role of IFIT5 in any cellular processes. In this study, we reported that both the mRNA and protein levels of IFIT5 are up-regulated in response to RNA virus infection or polyinosinic-cytidylic acid stimulation. Ectopic expression of IFIT5 could synergize IRF3- and NF-κB-mediated gene expression, whereas knockdown of IFIT5 impairs the transcription of these genes. Consistently, anti-viral responses of host cells are significantly increased or decreased in the presence or absence of IFIT5. Mechanistically, IFIT5 co-localizes partly with mitochondria and interacts with RIG-I and MAVS. Our study identified that IFIT5 is an important enhancer in innate immune response.

Virus-induced expression of retinoic acid inducible gene-I and melanoma differentiation-associated gene 5 in the cochlear sensory epithelium.

The inner ear has been regarded as an immunoprivileged site because of isolation by the blood-labyrinthine barrier. Several reports have indicated the existence of immune cells in the inner ear, but there are no reports showing immunocompetence of the cochlear tissue. In this report, we examined the potential involvement of retinoic acid inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5), which are critical for initiating antiviral innate immune responses. We found that RIG-I and MDA5 are expressed in the mouse cochlear sensory epithelium, including Hensen's and Claudius' cells. Ex vivo viral infection using Theiler's murine encephalomyelitis virus revealed that the virus replicates in these cells and that protein levels of RIG-I and MDA5 are up-regulated. Furthermore, the critical antiviral transcription factor, interferon (IFN) regulatory factor-3, is activated in the infected cells as judged by its nuclear translocation and the accumulation of type I IFN transcripts. These results strongly suggest that RIG-I and MDA5 participate in innate antiviral responses in cochlear tissue.

Protein extract of ultraviolet-irradiated human skin keratinocytes promote the expression of mitogen-activated protein kinases, nuclear factor-κB and interferon regulatory factor-3 in Langerhans cells via Toll-like receptor 2 and 4.

AIM: In this study, we investigated whether the protein extract of ultraviolet-irradiated human skin keratinocytes can activate Toll-like receptor 2 and Toll-like receptor 4 of Langerhans cells and induce the downstream gene expression of mitogen-activated protein kinases, nuclear factor-κB and interferon regulatory factor-3. METHODS: The protein expression of mitogen-activated protein kinases, nuclear factor-κB and interferon regulatory factor-3 in Langerhans cells and the protein expression of HSP60, HSP70 and ß-defensin 2 in keratinocytes were examined using Western blot analysis. Langerhans cells were pretreated with or without Toll-like receptor 2 and Toll-like receptor 4 siRNA. RESULTS: We found that the protein extract of ultraviolet-irradiated keratinocytes upregulated the expression of mitogen-activated protein kinases, nuclear factor-κB and interferon regulatory factor-3 in Langerhans cells via Toll-like receptor 2 and Toll-like receptor 4. We also found that ultraviolet radiation upregulated the expression HSP60, HSP70 and ß-defensin 2 in keratinocytes. CONCLUSIONS: Our previous study demonstrated that ultraviolet radiation upregulated Toll-like receptor 2 and Toll-like receptor 4 expression in Langerhans cells. Ultraviolet radiation also upregulated mitogen-activated protein kinases and nuclear factor-κB/p65 expression via Toll-like receptor 2 and Toll-like receptor 4, and upregulated interferon regulatory factor-3 expression partially via Toll-like receptor 4. So we conclude that ultraviolet radiation can directly or indirectly activate keratinocytes to induce endogenous ligands which stimulate Toll-like receptor 2- or Toll-like receptor 4-dependent signaling cascade in Langerhans cells, sequentially influence innate and adaptive immune responses.

Mechanism of inhibition of lipopolysaccharide-induced interferon-ß production by 2-aminopurine.

2-Aminopurine (2-AP) is widely used as an inhibitor for double stranded RNA-dependent protein kinase (PKR). Previously, we reported that 2-AP inhibits Toll-like receptor (TLR) ligand-induced nitric oxide production through the prevention of interferon (IFN)-ß production. In this study, we investigated the mechanisms for 2-AP inhibition of lipopolysaccharide (LPS)-induced IFN-ß production. A reporter gene assay showed that LPS-induced IFN-ß promoter, but not nuclear factor (NF)-κB, activation was significantly inhibited by 2-AP. IFN-ß promoter activation induced by the overexpression of Toll/interleukin-1 receptor domain-containing adaptor inducing IFN-ß (TRIF) was significantly inhibited by 2-AP in a dose-dependent manner, while TRIF- or myeloid differentiation primary response gene 88-dependent NF-κB activation was not inhibited. IFN-ß promoter activation induced by expression of the downstream signaling molecules, tumor necrosis factor receptor-associated factor family member-associated NF-κB activator-binding kinase 1, inhibitor of NF-κB kinase i and a constitutively active mutant of interferon regulatory factor (IRF)-3, was also inhibited by 2-AP. Another PKR inhibitor harboring the imidazolo-oxindole structure, however, did not affect TRIF signaling molecules-induced IFN-ß promoter activation, suggesting that the inhibition of IFN-ß transcription by 2-AP is independent of PKR inhibition. Further, we examined the effect of 2-AP on LPS-induced IRF-3 activation by immunoblotting. While 2-AP did not affect LPS-induced phosphorylation of IRF-3, nuclear translocation of IRF-3 was inhibited. Moreover, we revealed that LPS-induced phosphorylation of Akt, another key molecule involved in IRF-3 activation, was inhibited by 2-AP. These results suggest that 2-AP inhibits nuclear translocation of phosphorylated-IRF-3 by inhibiting Akt activation.

Mutations within the human parainfluenza virus type 3 (HPIV 3) C protein affect viral replication and host interferon induction.

Human parainfluenza virus type 3 (HPIV 3) encodes a multifunctional C protein that is capable of inhibiting viral replication and counteracting the host interferon (IFN) signaling pathway. We recently demonstrated that the C protein is phosphorylated both in vitro and in vivo and mutations within the phosphorylation sites exhibit differential inhibitory activities in vitro. In this study, we report for the first time the successful recovery of mutant HPIV 3 viruses containing mutations within the C protein. Three mutant viruses, Cm-1, Cm-3 and Cm-4, harboring individual mutations of S7, S47T48 and S81 residues, respectively, were examined for their replication profiles and their ability to abrogate host IFN induction. Viral transcription was similar for all viruses; however Cm-3 displayed a relatively higher replication. Infection of cells with Cm-1 and Cm-3 led to the activation of IFN regulatory transcription factor 3 (IRF-3) and subsequent increase in IFN-ß mRNA levels as determined by immunofluorescence assay and RT-PCR analyses, respectively. Moreover, Cm-3 was able to partially resist the interferon induced antiviral state in Vero cells. Taken together, these results suggest that mutations within the C protein differentially affect viral replication and host interferon induction.

Isoflavone agonists of IRF-3 dependent signaling have antiviral activity against RNA viruses.

There is a growing need for novel antiviral therapies that are broad spectrum, effective, and not subject to resistance due to viral mutations. Using high-throughput screening methods, including computational docking studies and an interferon-stimulated gene 54 (ISG54)-luciferase reporter assay, we identified a class of isoflavone compounds that act as specific agonists of innate immune signaling pathways and cause activation of the interferon regulatory factor (IRF-3) transcription factor. The isoflavone compounds activated the ISG54 promoter, mediated nuclear translocation of IRF-3, and displayed highly potent activity against hepatitis C virus (HCV) and influenza virus. Additionally, these agonists efficiently activated IRF-3 in the presence of the HCV protease NS3-4A, which is known to blunt the host immune response. Furthermore, genomic studies showed that discrete innate immune pathways centered on IRF signaling were regulated following agonist treatment without causing global changes in host gene expression. Following treatment, the expression of only 64 cellular genes was significantly induced. This report provides the first evidence that innate immune pathways dependent on IRF-3 can be successfully targeted by small-molecule drugs for the development of novel broad-spectrum antiviral compounds.

Transcription factors Sp1 and Sp3 regulate basal transcription of the human IRF-3 gene.

Interferon regulatory factor 3 (IRF-3) plays a crucial role in initiation and development of the IFN antiviral response. The expression level of human IRF-3 is thought to be closely related to antiviral state of cells. However, the mechanisms of the transcription regulation of IRF-3 have remained largely unknown. We previously reported that transcription factor E2F1 negatively regulates the basal transcriptional activity of IRF-3. Here we demonstrate that transcription factors Sp1 and Sp3 up-regulate the basal transcriptional activity of IRF-3 and increase IRF-3 expression at mRNA level. By transient transfection analysis we revealed that mutation of Sp1/NRF-1 binding site resulted in a profound reduction of IRF-3 promoter activity. Overexpression of Sp1 and Sp3, but not NRF-1, transactivated the IRF-3 promoter activity in reporter gene assays while knocking-down of endogenous Sp1 and Sp3 by a shRNA strategy markedly inhibited IRF-3 promoter activity. Chromatin immunoprecipitation (ChIP) assays showed that Sp1 and Sp3 interact with the IRF-3 promoter in vivo. These results suggest that basal expression level of IRF-3 is regulated by transcription factors Sp1 and Sp3.

The adaptor protein FHL2 enhances the cellular innate immune response to influenza A virus infection.

The innate immune response of influenza A virus-infected cells is predominantly mediated by type I interferon-induced proteins. Expression of the interferon ß (IFNß) itself is initiated by accumulating viral RNA and is transmitted by different signalling cascades that feed into activation of the three transcriptional elements located in the IFNß promoter, AP-1, IRF-3 and NF-κB. FHL2 (four-and-a-half LIM domain protein 2) is an adaptor molecule that shuttles between membrane and nucleus regulating signalling cascades and gene transcription. Here we describe FHL2 as a novel regulator of influenza A virus propagation. Using mouse FHL2 wild-type, knockout and rescued cells and human epithelial cells with different expression levels of FHL2 we showed that FHL2 decreases influenza A virus propagation by regulating the intrinsic cellular antiviral immune response. On virus infection FHL2 translocates into the nucleus, potentiating the IRF-3-dependent transcription of the IFNß gene.

A20 (Tnfaip3) deficiency in myeloid cells protects against influenza A virus infection.

The innate immune response provides the first line of defense against viruses and other pathogens by responding to specific microbial molecules. Influenza A virus (IAV) produces double-stranded RNA as an intermediate during the replication life cycle, which activates the intracellular pathogen recognition receptor RIG-I and induces the production of proinflammatory cytokines and antiviral interferon. Understanding the mechanisms that regulate innate immune responses to IAV and other viruses is of key importance to develop novel therapeutic strategies. Here we used myeloid cell specific A20 knockout mice to examine the role of the ubiquitin-editing protein A20 in the response of myeloid cells to IAV infection. A20 deficient macrophages were hyperresponsive to double stranded RNA and IAV infection, as illustrated by enhanced NF-κB and IRF3 activation, concomitant with increased production of proinflammatory cytokines, chemokines and type I interferon. In vivo this was associated with an increased number of alveolar macrophages and neutrophils in the lungs of IAV infected mice. Surprisingly, myeloid cell specific A20 knockout mice are protected against lethal IAV infection. These results challenge the general belief that an excessive host proinflammatory response is associated with IAV-induced lethality, and suggest that under certain conditions inhibition of A20 might be of interest in the management of IAV infections.

Synoviocyte innate immune responses: TANK-binding kinase-1 as a potential therapeutic target in rheumatoid arthritis.

OBJECTIVES: Innate immune responses in the rheumatoid synovium contribute to inflammation and joint destruction in RA. Two IκB kinase (IKK)-related kinases, TNF receptor associated factor (TRAF) family member-associated nuclear factor κ-light-chain enhancer of activated B cells (NF-κB) activator (TANK)-binding kinase 1 (TBK1) and IKKε, potentially regulate synovitis by activating IFN response genes. These kinases induce the expression of inflammatory mediators such as C-X-C motif ligand 10 (CXCL10)/IFN-γ-induced protein 10 kDa (IP-10) in fibroblast-like synoviocytes (FLS). Since IP-10 is a promising therapeutic target in RA, we evaluated whether blocking TBK1 might be an effective way to modulate IP-10 expression. METHODS: Wild-type (WT) and IKKε(-/-) FLS were transfected with TBK1 or control small interfering RNA (siRNA) and stimulated with polyinosinic acid : polycytidylic acid [poly(I:C)]. Gene expression was assayed using quantitative PCR. Cytokine production in culture supernatants was measured by Luminex multiplex analysis. IFN-regulatory factor (IRF3) dimerization was determined by native PAGE. IFN-ß and IP-10 promoter activity was measured using luciferase reporter constructs. RESULTS: Initial studies showed that siRNA markedly decreased TBK1 expression in cultured FLS. Poly(I:C)-induced IRF7 gene expression was inhibited in the absence of TBK1, but not IKKε. IRF3 gene expression was similar to WT cells in TBK1 or IKKε-deficient FLS. IRF3 dimerization required both TBK1 and IKKε. Surprisingly, IRF3-mediated gene and protein expression of IFN-ß and IP-10 was dependent on TBK1, not IKKε. Promoter constructs showed that TBK1 decreased IP-10 gene transcription and IP-10 mRNA stability was unaffected by TBK1 deficiency. CONCLUSION: Based on the selective regulation of IP-10 in FLS, TBK1 appears to be the optimal IKK-related kinase to target in RA.

Interferon regulatory factor 3 inhibits astrocyte inflammatory gene expression through suppression of the proinflammatory miR-155 and miR-155*.

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 IFNß 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.

Hepatitis C virus reveals a novel early control in acute immune response.

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.