type-I Interferon Induction Research Articles

SARS-CoV-2 ORF6 protein targets TRIM25 for proteasomal degradation to diminish K63-linked RIG-I ubiquitination and type-I interferon induction.

Evasion and antagonism of host cellular immunity upon SARS-CoV-2 infection provide replication advantage to the virus and contribute to COVID-19 pathogenesis. We explored the ability of different SARS-CoV-2 proteins to antagonize the host's innate immune system and found that the ORF6 protein mitigated type-I Interferon (IFN) induction and downstream IFN signaling. Our findings also corroborated previous reports that ORF6 blocks the nuclear import of IRF3 and STAT1 to inhibit IFN induction and signaling. Here we show that ORF6 directly interacts with RIG-I and blocks downstream type-I IFN induction and signaling by reducing the levels of K63-linked ubiquitinated RIG-I. This involves ORF6-mediated targeting of E3 ligase TRIM25 for proteasomal degradation, which was also observed during SARS-CoV-2 infection. The type-I IFN antagonistic activity of ORF6 was mapped to its C-terminal cytoplasmic tail, specifically to amino acid residues 52-61. Overall, we provide new insights into how SARS-CoV-2 inhibits type-I IFN induction and signaling through distinct actions of the viral ORF6 protein.

H1N1 Influenza A Virus Protein NS2 Inhibits Innate Immune Response by Targeting IRF7.

Influenza A virus (IAV) is a globally distributed zoonotic pathogen and causes a highly infectious respiratory disease with high morbidity and mortality in humans and animals. IAV has evolved various strategies to counteract the innate immune response, using different viral proteins. However, the mechanisms are not fully elucidated. In this study, we demonstrated that the nonstructural protein 2 (NS2) of H1N1 IAV negatively regulate the induction of type-I interferon. Co-immunoprecipitation experiments revealed that NS2 specifically interacts with interferon regulatory factor 7 (IRF7). NS2 blocks the nuclear translocation of IRF7 by inhibiting the formation of IRF7 dimers, thereby prevents the activation of IRF7 and inhibits the production of interferon-beta. Taken together, these findings revealed a novel mechanism by which the NS2 of H1N1 IAV inhibits IRF7-mediated type-I interferon production.

Cross-species analysis of viral nucleic acid interacting proteins identifies TAOKs as innate immune regulators

The cell intrinsic antiviral response of multicellular organisms developed over millions of years and critically relies on the ability to sense and eliminate viral nucleic acids. Here we use an affinity proteomics approach in evolutionary distant species (human, mouse and fly) to identify proteins that are conserved in their ability to associate with diverse viral nucleic acids. This approach shows a core of orthologous proteins targeting viral genetic material and species-specific interactions. Functional characterization of the influence of 181 candidates on replication of 6 distinct viruses in human cells and flies identifies 128 nucleic acid binding proteins with an impact on virus growth. We identify the family of TAO kinases (TAOK1, −2 and −3) as dsRNA-interacting antiviral proteins and show their requirement for type-I interferon induction. Depletion of TAO kinases in mammals or flies leads to an impaired response to virus infection characterized by a reduced induction of interferon stimulated genes in mammals and impaired expression of srg1 and diedel in flies. Overall, our study shows a larger set of proteins able to mediate the interaction between viral genetic material and host factors than anticipated so far, attesting to the ancestral roots of innate immunity and to the lineage-specific pressures exerted by viruses.

Synergy of HSP90 Inhibition and Radiation Therapy for cGAS/STING Pathway Activation

Even though immunotherapy is a common part in cancer treatment, only a small subset of patients profits long term. Since radiation therapy (RT) has been shown to improve tumor immunogenicity, treatment strategies combining RT and immunotherapy are an emerging field in cancer research. The inhibition of DNA damage repair (DDR) mechanisms is a potential target, because its impairment enhances, like RT alone, type-I interferon induction by activating the cGAS/STING pathway. Type-I interferons mediate the immunomodulatory effects following RT. Therefore, combination of RT and DDR inhibition appears to be a promising approach to enhance tumor immunogenicity. To induce broad DDR impairment, we here employ TAS-116 (Taiho Pharmaceutical Co., Ltd.), an inhibitor of Heat Shock Protein 90 (HSP90). First, we determined the tumor type-dependent toxicity by titering TAS-116 in six murine tumor cell lines and measured cell death via flow cytometry after 24h. Treatment with subtoxic concentrations of TAS-116 one hour prior to RT (up to 8Gy) resulted in increased γH2AX foci, indicating DNA damage, both in RP157.8 SCLC and B16F10 melanoma cells. Declining γH2AX foci were seen upon 12Gy in vitro. Our data suggest that irreparable DNA damage may occur which leads to cell death. The induction of DNA damage following TAS-116 treatment in combination with 8Gy RT was confirmed in vivo using a transplantable mouse tumor model. To confirm our hypothesis that the observed DNA damage mediates STING pathway activation in vitro, we performed Western blot analysis for pSTING at timepoints up to 72h after RT. While TAS-116 alone led to an increase up to 72h, highest pSTING levels were observed after 3h in combination with 8Gy RT, demonstrating synergistic effects for STING activation by accelerating and enhancing STING phosphorylation. While our results display an enhanced specific CD8+ T-cell-mediated killing in vitro using the transfected lymphoma cell line E.G7 after either treatment alone, combination of TAS-116 and RT up to 8Gy led to a significant reduction of this effect. We hypothesize that an observed upregulation of PD-L1 on the tumor cell surface, a known adverse effect of STING activation and suppressor of CD8+ T-cell-mediated killing, causes this phenomenon. Here, we demonstrate a synergy of HSP90 inhibition and RT to activate the immunogenic cGAS/STING pathway. However, to harness this effect therapeutically, anti-PD-1 therapy seems to be required.

N6-methyladenosine modification of HIV-1 RNA suppresses type-I interferon induction in differentiated monocytic cells and primary macrophages.

N6-methyladenosine (m6A) is a prevalent RNA modification that plays a key role in regulating eukaryotic cellular mRNA functions. RNA m6A modification is regulated by two groups of cellular proteins, writers and erasers that add or remove m6A, respectively. HIV-1 RNA contains m6A modifications that modulate viral infection and gene expression in CD4+ T cells. However, it remains unclear whether m6A modifications of HIV-1 RNA modulate innate immune responses in myeloid cells that are important for antiviral immunity. Here we show that m6A modification of HIV-1 RNA suppresses the expression of antiviral cytokine type-I interferon (IFN-I) in differentiated human monocytic cells and primary monocyte-derived macrophages. Transfection of differentiated monocytic U937 cells with HIV-1 RNA fragments containing a single m6A-modification significantly reduced IFN-I mRNA expression relative to their unmodified RNA counterparts. We generated HIV-1 with altered m6A levels of RNA by manipulating the expression of the m6A erasers (FTO and ALKBH5) or pharmacological inhibition of m6A addition in virus-producing cells, or by treating HIV-1 RNA with recombinant FTO in vitro. HIV-1 RNA transfection or viral infection of differentiated U937 cells and primary macrophages demonstrated that HIV-1 RNA with decreased m6A levels enhanced IFN-I expression, whereas HIV-1 RNA with increased m6A modifications had opposite effects. Our mechanistic studies indicated that m6A of HIV-1 RNA escaped retinoic acid-induced gene I (RIG-I)-mediated RNA sensing and activation of the transcription factors IRF3 and IRF7 that drive IFN-I gene expression. Together, these findings suggest that m6A modifications of HIV-1 RNA evade innate immune sensing in myeloid cells.

Therapeutic potential of tricarboxylic acid cycle metabolite itaconate in cardiovascular diseases.

Therapeutic potential of tricarboxylic acid cycle metabolite itaconate in cardiovascular diseases.

MDA5 cleavage by the Leader protease of foot-and-mouth disease virus reveals its pleiotropic effect against the host antiviral response

The RIG-I-like receptor (RLR) melanoma differentiation-associated gene 5 (MDA5) plays a key role in triggering innate antiviral response during infection by RNA viruses. MDA5 activation leads to transcription induction of type-I interferon (IFN) and proinflammatory cytokines. MDA5 has also been associated with autoimmune and autoinflammatory diseases by dysfunctional activation of innate immune response in the absence of infection. Here, we show how foot-and-mouth disease virus (FMDV) counteracts the specific antiviral effect exerted by MDA5 targeting the protein for cleavage by the viral Leader protease (Lpro). MDA5 overexpression had an inhibitory effect on FMDV infection in IFN-competent cells. Remarkably, immunostimulatory viral RNA co-immunoprecipitated with MDA5 in infected cells. Moreover, specific cleavage of MDA5 by Lpro was detected in co-transfected cells, as well as during the course of FMDV infection. A significant reduction in IFN induction associated with MDA5 cleavage was detected by comparison with a non-cleavable MDA5 mutant protein with preserved antiviral activity. The Lpro cleavage site in MDA5 was identified as the RGRAR sequence in the conserved helicase motif VI, coinciding with that recently reported for Lpro in LGP2, another member of the RLRs family involved in antiviral defenses. Interestingly, specific mutations within the MDA5 Lpro target sequence have been associated with immune disease in mice and humans. Our results reveal a pleiotropic strategy for immune evasion based on a viral protease targeting phylogenetically conserved domains of immune sensors. Identification of viral strategies aimed to disrupt MDA5 functionality may also contribute to develop new treatment tools for MDA5-related disorders.

Effect of porcine epidemic diarrhea virus nsp1 on type Ⅰ interferon response

Porcine epidemic diarrhea virus (PEDV) inhibits the host typeⅠinterferon and cellular antiviral response, but its inhibition mechanism is unclear, and the roles of PEDV nonstructural proteins in regulating typeⅠinterferon responses have been seldom studied. To study the effect of nsp1 on typeⅠinterferon response, nsp1 gene was cloned into a eukaryotic expression vector pCAGGS. The expression of nsp1 in transfected cells was determined by Western blot and indirect immunofluorescence assay. The effects of nsp1 on the induction of typeⅠinterferon were evaluated by dual luciferase reporter gene assay, ELISA and VSV bioassay. Western blot and indirect immunofluorescence assay showed that nsp1 was highly expressed in transfected cells and PEDV-infected cells. Dual luciferase reporter gene assay results indicated that nsp1 strongly inhibited the IFN-β promoter activity, and the inhibitory effect was nsp1 dose-dependent. ELISA results showed that nsp1 significantly inhibited the expression of IFN-β in protein level. And VSV replication-inhibition bioassay revealed that nsp1 significantly inhibited typeⅠIFN antiviral activities induced by poly(I:C). Our results implied that nsp1 was a highly conserved protein of PEDV and exhibited antagonistic function on interferon promoter activity. The results have laid a foundation for further understanding the immune evasion mechanism of PEDV and for developing new effective vaccine against PEDV.

Microbial-Derived Metabolites Drive Protective Type-I Interferon Responses in Models of Gut Epithelial Damage and Limit Graft-Versus-Host Disease

Background: Graft-versus-host disease (GVHD) is a dreaded complication after stem-cell transplantation (SCT). Standard treatment relies on immunosuppressants but is associated with an increased risk of infection and relapse. Up 50% of patients develop steroid-refractory GVHD, with dismal impact on SCT outcomes. New studies in mice suggest that induction of type-I interferon (IFN-I) signalling or activation of IFN-I inducing pathways such as cGAS/STING or RIG-I/MAVS can promote gut barrier integrity and limit GVHD. However, the endogenous ligands that drive this "protective" IFN-I response are poorly defined. Recent data in mice and humans suggest that microbial-derived metabolites such as small-chain fatty acids or indoles can decrease GHVD mortality. Here, we describe IFN-I inducing metabolites that improve outcomes in mouse models of gut epithelial damage and acute GVHD via a mechanism dependent on STING signalling. Methods: To investigate which cell types mediate protection and how, we generated intestinal organoids and antigen presenting cells (APC) from bone marrow of WT or genetically deficient mice (MAVS-/-, STING-/-, IFNAR-/-) under steady state conditions versus chemotherapy, total body irradiation and after allogeneic SCT in the presence or absence of microbial metabolites. Analysis was performed by microscopy, immunoblotting, qPCR, ELISA and flow cytometry. Outcomes of gut-injured mice were assessed by clinical scoring, histopathology, flow cytometry and organoid recovery. Results: Metabolite treatment promoted regeneration of intestinal organoids as assayed by organoid numbers as well as proliferation. These effects were dependent on IFN-I and STING signalling. In addition, we found activated pro-inflammatory NFkB signalling and decreased apoptosis as evidenced by reduced caspase-3 cleavage. In APCs, IFN-I responses were enhanced in the presence of metabolites including increased IFN-β production and upregulation of IFN stimulated genes. Metabolite-treated mice showed improved recovery of intestinal stem cells following gut injury by chemotherapy and irradiation and increased survival in acute GVHD. Conclusions: Our findings uncover a mechanism by which microbial metabolites amplify IFN-I signals, promote tissue regeneration and thereby prevent allo-activation and GVHD. We show for the first time that these IFN-I inducing metabolites are protective in diverse models of gut damage -- from chemotherapy and radiation to immune-mediated damage -- via a STING-dependent mechanism. Perhaps the poor prognosis of GVHD patients exhibiting a loss of microbiota diversity can be explained in part by an absence of "protective" metabolites able to amplify critical IFN signals. We are currently studying whether metabolite levels correlate with severity and outcome of GVHD in humans. Figure Disclosures No relevant conflicts of interest to declare.

Predictive value of TLR7 polymorphism for cetuximab-based chemotherapy in patients with metastatic colorectal cancer

The TLR7 and TLR9 signalings are implicated in the regulation of the immune system through type-I interferon induction. Preclinical studies have demonstrated the immunomodulatory and antitumor effects of TLR7 and TLR9 agonists in combination with cetuximab. We tested the hypothesis that genetic variations in TLR7 and TLR9 and their downstream molecules IRF5 and IRF7 were associated with outcomes in metastatic colorectal cancer (mCRC) patients receiving cetuximab-based chemotherapy. Six single nucleotide polymorphisms (SNPs) in TLR7, TLR9, IRF5 and IRF7 were tested for the association with RR, PFS, and OS in KRAS-wild type mCRC patients. Patients treated with FOLFIRI + cetuximab or FOLFIRI + bevacizumab in the FIRE-3 trial served as a discovery set (FIRE3-Cet, n = 244) or a control set (FIRE3-Bev, n = 246), respectively. Patients treated with FOLFOX or SOX + cetuximab in the JACCRO-CC05/06 trial served as a validation set (JACCRO, n = 76). Genomic DNA isolated from tumor tissue samples was analyzed by PCR-based direct sequencing. In the discovery cohort, patients with the TLR7 rs3853839 G/G variant showed a trend toward longer PFS than those with any C variants (median 10.0 vs. 11.8 months, HR 1.39, p = 0.092). This preliminary association was confirmed in the validation cohort, and those with the G/G genotype showed a PFS benefit compared with others (univariate: 9.1 vs. 11.6 months, HR 2.04, p = 0.005, multivariate: HR 2.02, 95% CI: 1.14-3.55, p = 0.015). This association was not observed in the control cohort. Our findings suggest that TLR7 rs3853839 predicts the outcome of cetuximab-based chemotherapy in mCRC patients.

The mitochodnrial E3 ligase MARCH5 resolves RIG-I and MAVS aggregates in innate immunity

Abstract Ubiquitin proteasome system (UPS) on the mitochondrial is one of quality control systems that works as a first line of defense barrier against aggregated or misfolded proteins. In innate immunity formation of the MAVS(Mitochondrial anti-viral signaling protein) oligomers elicits robust type-I interferon induction upon viral infection and however, persistent RIG-I and MAVS complex rather leads to host immunopathology. We recently reported that mitochondria-resident E3 ligase, MARCH5, recognizes the oligomeric form of RIG-I and MAVS complex. MARCH5+/− mice and MARCH5 deficient immune cells exhibited low viral replication and elevated type-I interferon response to viral infection. MARCH5 bound RIG-I and MAVS complex only during viral infection when MAVS forms oligomer. MARCH5 mediates Lys-48-linked poly-ubiquitination chain addition to RIG-I and MAVS oligomer and induces the RIG-I/MAVS monomer. Next, we studies have suggested that a novel function of MARCH5 in inflammation signaling. Together, these data demonstrates that MARCH5 regulates oligomeric RIG-I and MAVS complex.

Systems-based analysis of RIG-I-dependent signalling identifies KHSRP as an inhibitor of RIG-I receptor activation.

Retinoic acid inducible gene-I (RIG-I) receptor recognizes 5′-triphosphorylated RNA and triggers a signaling cascade that results in the induction of type-I IFN-dependent responses. Its precise regulation represents a pivotal balance between antiviral defenses and autoimmunity. To elucidate cellular cofactors that regulate RIG-I signaling, we performed two global RNAi analyses to identify both positive and negative regulatory nodes operating on the signaling pathway during virus infection. These factors were integrated with experimentally and computationally derived interactome data to build a RIG-I protein interaction network. Our analysis revealed diverse cellular processes, including the unfolded protein response, WNT signaling, and RNA metabolism, as critical cellular components governing innate responses to non-self RNA species. Importantly, we identified K-Homology Splicing Regulatory Protein (KHSRP) as a negative regulator of this pathway. We find that KHSRP associates with the regulatory domain of RIG-I to maintain the receptor in an inactive state and attenuate it’s sensing of viral RNA (vRNA). Consistent with increased RIG-I antiviral signaling in the absence of KHSRP, viral replication is reduced when KHSRP expression is knocked down both in vitro and in vivo. Taken together, these data indicate that KHSRP functions as a checkpoint regulator of the innate immune response to pathogen challenge.

Expression of interferon regulatory factor 5 is regulated by the Sp1 transcription factor.

The transcription factor, interferon regulatory factor5 (IRF5), is important in the induction of typeI interferon, proinflammatory cytokines and chemokines, and is involved in autoimmune diseases and tumourigenesis. However, the mechanisms underlying the transcriptional regulation of wild‑type IRF5 remain to be fully elucidated. The present study was primarily designed to clarify whether specificity protein 1 (Sp1) was involved in the regulation of IRF5. Initially, the IRF5 promoter region was cloned and its promoter activity was examined using Hela and HEK293 cells. Deletion analyses revealed that the region spanning‑179to +62 was the minimal promoter of IRF5. Bioinformatics analyses showed that this region contained three putative Sp1 binding sites, and mutational analyses revealed that all the Sp1 sites contributed to transcriptional activity. Secondly, the overexpression of Sp1 was found to increase the activity of the IRF5 promoter and the mRNA level of IRF5, determined using reporter gene assays and polymerase chain reaction analysis, respectively. By contrast, treatment with mithramycin and Sp1 small interfering RNA significantly reduced the activity of the IRF5 promoter and the mRNA level of IRF5. Finally, the results of an electrophoretic mobility shift assay and a chromatin immunoprecipitation assay demonstrated that Sp1 bound to the promoter region of IRF5 invitro and invivo. These results suggested that the Sp1 transcription factor is the primary determinant for activating the basal transcription of the IRF5.

Circulating nano-particulate TLR9 agonist scouts out tumor microenvironment to release immunogenic dead tumor cells.

Recent evidence suggest that a β-glucan derived from mushroom Schizophyllan(SPG) complexed with a humanized TLR9 agonistic CpG DNA, K3 (K3-SPG) is a promising vaccine adjuvant that induces robust CD8 T cell responses to co-administered antigen. However, it has not been investigated whether K3-SPG alone can act as an anti-cancer immunotherapeutic agent or not. Here, we demonstrate that intravenous injection of K3-SPG, but not CpG alone, is accumulated in the tumor microenvironment and triggered immunogenic cell death (ICD) of tumor cells by local induction of type-I interferon (IFN) as well as IL-12. Resultant innate immune activation as well as subsequent tumor-specific CD8 T cell responses were contributed the tumor growth suppression. This anti-tumor effect of K3-SPG monotherapy was also confirmed by using various tumor models including pancreatic cancer peritoneal dissemination model. Taken together, nano-particulate TLR9 agonist injected intravenously can scout out tumor microenvironment to provoke local innate immune activation and release dead tumor cells into circulation that may induce broader and protective tumor antigen-specific CD8 T cells.

Polymorphisms in toll-like receptor (TLR) genes in the prediction of outcome for cetuximab-based treatment in patients with metastatic colorectal cancer (mCRC).

3588Background: TLR7 and TLR9 signaling pathways are implicated in the regulation of immune system through type-I interferon induction. Immune responses within the tumor microenvironment may influe...

Roles of innate immune regulator TAPE in RIG-I signaling and antiviral defenses

Abstract Pattern-recognition receptors detect pathogen-associated molecular patterns to trigger downstream pathways leading to type I IFN and inflammatory cytokines production to defend pathogen infection. RIG-I-like receptors (RLRs) are key cytosolic sensors for recognizing viral RNA to trigger antiviral immunity. The underlying mechanisms linking RLR-mediated viral recognition to antiviral immunity remain to be further explored. Through our previous work, we uncovered an innate immune regulator termed TAPE (TBK1-Associated Protein in Endolysosomes), also known as CC2D1A, which is implicated in the viral RNA senor TLR3 and RLR pathways. Yet, the in vivo role of TAPE in antiviral defenses and the mechanistic mechanisms of how TAPE regulates cytosolic RIG-I signaling still remain to be established. TAPE conditional knockout mice were generated for our study. Results from in vivo studies showed that TAPEf/f CD11c-Cre mice exhibited a more severe mortality than WT mice upon influenza A virus (IAV) infection. Ex vivo studies also showed that TAPE-deficient mouse embryonic fibroblasts and macrophages were defective in type-I interferon induction upon RLR ligand stimulation. In addition, our biochemical analyses showed that the N-terminal region of TAPE was critical for interacting with the CARD domain of RIG-I while the C-terminal region of TAPE contributed most to the interaction with MAVS/IPS-1, a RLR downstream mediator. Together, our results suggest a crucial role for TAPE in linking RIG-I to type I IFN-mediated antiviral responses. Future work will further determine the in vivo role of TAPE in IAV and other RNA virus infection, and explore the mechanistic mechanisms of how TAPE regulates RIG-I signaling.

The mitochondrial E3 ubiquitin ligase MARCH5 resolves MAVS aggregates during antiviral signaling.

Abstract Mitochondria serve as platforms for innate immunity. The mitochondrial antiviral signalling (MAVS) protein forms aggregates that elicit robust type-I interferon induction on viral infection, but persistent MAVS signalling leads to host immunopathology; it remains unknown how these signalling aggregates are resolved. Here we identify the mitochondria-resident E3 ligase, MARCH5, as a negative regulator of MAVS aggregates. March5(+/−) mice and MARCH5-deficient immune cells exhibit low viral replication and elevated type-I interferon responses to RNA viruses. MARCH5 binds MAVS only during viral stimulation when MAVS forms aggregates, and these interactions require the RING domain of MARCH5 and the CARD domain of MAVS. MARCH5, but not its RING mutant (MARCH5(H43W)), reduces the level of MAVS aggregates. MARCH5 transfers ubiquitin to Lys7 and Lys500 of MAVS and promotes its proteasome-mediated degradation. Our results indicate that MARCH5 modulates MAVS-mediated antiviral signalling, preventing excessive immune reactions.

The mitochondrial ubiquitin ligase MARCH5 resolves MAVS aggregates during antiviral signalling.

Mitochondria serve as platforms for innate immunity. The mitochondrial antiviral signalling (MAVS) protein forms aggregates that elicit robust type-I interferon induction on viral infection, but persistent MAVS signalling leads to host immunopathology; it remains unknown how these signalling aggregates are resolved. Here we identify the mitochondria-resident E3 ligase, MARCH5, as a negative regulator of MAVS aggregates. March5+/− mice and MARCH5-deficient immune cells exhibit low viral replication and elevated type-I interferon responses to RNA viruses. MARCH5 binds MAVS only during viral stimulation when MAVS forms aggregates, and these interactions require the RING domain of MARCH5 and the CARD domain of MAVS. MARCH5, but not its RING mutant (MARCH5H43W), reduces the level of MAVS aggregates. MARCH5 transfers ubiquitin to Lys7 and Lys500 of MAVS and promotes its proteasome-mediated degradation. Our results indicate that MARCH5 modulates MAVS-mediated antiviral signalling, preventing excessive immune reactions.

C1q-based tolerance mechanism in HMGB1-mediated activation of human monocytes (IRM5P.649)

Abstract C1q, a component of the classical complement pathway, is required for clearance of apoptotic cells. C1q also regulates activation of innate immune cells through engagement of C1q receptors present on the immune cell surface. C1q deficiency is highly associated with the development of systemic lupus erythematosus (SLE). High Mobility Group Box 1 (HMGB1), a pro-inflammatory mediator, is secreted by inflammatory cells and passively released from necrotic cells. HMGB1 interacts with TLR4 and receptor for advanced glycation end products (RAGE), to augment the production of type 1 interferon and proinflammatory cytokines. Here we show that C1q inhibits HMGB1-mediated monocyte activation through cross-linking RAGE and Leukocyte-associated Ig-like receptor-1 (LAIR-1), which is an inhibitory receptor for collagen and C1q. This crosslinking causes LAIR-1 to enter lipid rafts following HMGB1 and C1q stimulation. C1q suppress HMGB-1 mediated induction of type I-interferon and pro-inflammatory cytokines including IL-12 and TNF-α. Moreover, exposure of monocytes to HMGB1 and C1q together leads to upregulation of a number of anti-inflammatory molecules that might function to induce phagocytosis and to maintain monocyte quiescence. Our results suggest that C1q regulates the activation of innate immunity by HMGB1 and maintains immune homeostasis. These data are consistent with the established requirement for C1q in the prevention of autoimmunity.

P101 The role of MDA5 and the effect of rotavirus in type 1 diabetes

Introduction The melanoma differentiation associated factor 5 (MDA5) is a member of the RIG- like helicase family that function in the antiviral response. MDA5 is a cytoplasmic sensor of viral RNAs that regulates the activity of the interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NFκB) transcription factors [1] . It has been reported that polymorphisms in the gene that encodes MDA5 (ifih1) correlate with the risk for developing Type 1 Diabetes (T1D) [2] . Although the triggers for this autoimmune disease are unknown, it is believed that both genetic and environmental factors are involved. Viral infection has been proposed as an important environmental trigger for T1D. We test the role of MDA5 in the antiviral response to rotavirus, which has been proposed as a likely environmental trigger, and test the different responsiveness of protein polymorphs that have been associated with T1D [3] . Methods (1) Induction of type-I interferon (IFN) and cytokines and replications of rotavirus were investigated in Mda5 wild type and knock out MEFs infected with rotavirus. (2) Point mutations of Mda5 polymorphisms associated with the risk of developing T1D were cloned. IRF3 and NFkB pathways were investigated in Hela cells transfected with Mda5 polymorphisms in response to double stranded RNA. (3) Glucose tolerant tests and levels of insulitis are investigated in Mda5 wild type and knock out mice inoculated with rotavirus. Results (1) We confirm that MDA5 is important in the immune response to rotavirus infection. The type-I IFN and cytokine responses are diminished in MDA5-ablated cells. And rotavirus replication is increased in the absence of MDA5. (2) Point mutations in MDA5 associated with the risk of developing T1D respond differentially to double stranded RNA. MDA5 polymorphisms show diminished capacity to induce IRF3 and NFkB. (3) We are currently investigating the role of MDA5 in insulitis initiated by rotavirus infection in vivo. Conclusion MDA5 is shown to be important for the immune response to a virus infection that has been proposed to be causal in T1D. Furthermore, polymorphisms associated with the risk of developing T1D are shown to have reduced antiviral function. Hence, our findings are consistent with a direct role for MDA5 in the development of the autosomal conditions that cause T1D.