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Abstract / Cytokine 70 (2014) 28–79 ing activation of TLRs and other innate immune receptors, as well as reduced LCMV viral infection. In contrast, overexpression of ATF3 in macrophages reduced the induction of IFNb and increased viral replication. Utilizing a range of molecular biology, biochemical and immunological techniques, integrated with powerful bioinformatics approaches we have investigated the molecular mechanisms responsible for ATF3mediated regulation of IFNb. Our ATF3 ChIP-seq data has revealed that ATF3 regulates IFNb by binding directly to its promoter. Furthermore, we have found that ATF3 itself is regulated by IFNs and thus forms part of a negative feedback loop during the induction of IFN signalling. Together our findings indicate ATF3 is a critical regulator of the IFN innate immune response. A better understanding of how the innate immune response is regulated is imperative for the development of future therapeutic strategies against autoimmune and inflammatory diseases.

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ISGF3 activity and ISG expression by LPS is critically dependent on intermediate production of IFN-b and autocrine signaling through type I IFN receptors. http://dx.doi.org/10.1016/j.cyto.2014.07.045

39 The effect of Mal D96N SNP in TLR signalling Jennifer K. Dowling 1, Michelle D. Tate 1, Douglas T. Golenbock 2, Ashley Mansell 1, MIMR-PHI Institute of Medical Research, Clayton, VIC, Australia, 2 Division of Infectious Disease and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA 1

http://dx.doi.org/10.1016/j.cyto.2014.07.043

37 Indigofera cordifolia seeds attenuate the oxidative stress and inflammation associated with Streptozotocin induced Type 2 Diabetes mellitus Padmini Deshpande, Jayesh Dhodi, Nitin Gawali, Pankaj Kothavade, Amrita Chowdhury, Archana Juvekar, Department of Pharmaceutical Science and Technology, Institute of Chemical Technology, Mumbai, Maharashtra, India Aim: The key pathogenic events involved in Type 2 Diabetes mellitus are impaired glucose regulation and insufficient carbohydrate utilization caused either by the lack of sufficient Insulin or resistance to it. The present study was hypothesized to investigate the beneficial effects of Indigofera cordifolia on hyperglycemia-induced oxidative damage in Streptozotocin (STZ) induced diabetic rats. Methods: Diabetes was induced by an intraperitoneal injection of STZ (45 mg/kg body weight). The seeds of I. Cordifolia were extracted with 70% methanol and fractionated with n-butanol (ICMB fraction). An oral dose of 500 mg/kg of ICMB was given once a day for 14 days following diabetes induction. At the end of the experimental period, blood was obtained from retro-orbital route. Liver and pancreas were used for estimations of antioxidant parameters while biochemical parameters were estimated in serum. Results: In the diabetic control group, levels of glucose, SGOT, SGPT, Total cholesterol, TNF-a and IL-6 were significantly increased, while levels of SOD and GSH were decreased. Administration of ICMB fraction significantly reversed these alterations. In-vitro antioxidant assays demonstrated the radical scavenging property of ICMB against DPPH and ABTS radicals. Moreover, ICMB caused significant inhibition of the carbohydrate hydrolyzing enzymes a-glucosidase and a-amylase. Histopathological examinations of pancreas demonstrated increase in the number and size of the islets in the ICMB treated group as compared to the diabetic control group. Conclusion: Thus, it can be safely concluded that Indigofera cordifolia exerts a protective action against STZ induced hyperglycaemia and inflammation. http://dx.doi.org/10.1016/j.cyto.2014.07.044

38 An essential role for Interferon-b in the induction of interferon-stimulated gene expression by lipopolysaccharide in macrophages Raymond P. Donnelly 1, Faruk Sheikh 1, Harold Dickensheets 1, Ana M. Gamero 2, Stefanie N. Vogel 3, 1 Division of Therapeutic Proteins, U.S. Food and Drug Administration, Bethesda, MD, USA, 2 Department of Biochemistry, Temple University School of Medicine, Philadelphia, PA, USA, 3 Dept. of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD, USA TLR agonists such as lipopolysaccharide (LPS) and poly(I:C) induce expression of type I interferons such as IFN-alpha and IFN-beta by macrophages. To examine the role of IFN-b in the induction of IFN-stimulated genes (ISGs) by LPS, we compared the ability of LPS to induce ISGF3 activity and ISG expression in bone marrow-derived macrophages from wild-type (WT) and IFN-b gene knockout (Ifnb1/) mice. We found that LPS treatment activated ISGF3 and induced expression of ISGs such as Oas1, Mx1, Ddx58 (RIG-I) and Ifih1 (MDA5) in WT macrophages, but not in macrophages derived from Ifnb1/ mice or IFN-a/b receptor gene knockout (Ifnar1/) mice. The inability of LPS to induce activation of ISGF3 and ISG expression in Ifnb1/ macrophages correlated with the failure of LPS to induce activation of STAT1 and STAT2 in these cells. Consistent with these findings, LPS treatment also failed to induce ISG expression in bone marrow-derived macrophages from Stat2 knockout mice. Although activation of ISGF3 and induction of ISG expression by LPS was abrogated in Ifnb1/ and Ifnar1/ macrophages, activation of NF-kB and induction of NFkB responsive genes such as Tnf (TNF-a) and Il1b (IL-1bÞ were not affected by deletion of either the IFN-b or IFN-aR1 genes. These findings demonstrate that induction of

The adaptor Mal is crucially involved in TLR4 and TLR2 signal transduction via the recruitment of MyD88 to the plasma membrane. Mal is known to have eight non-synonymous single nucleotide polymorphisms (SNPs) in its coding region. Previous studies using overexpression systems suggest the Mal D96N SNP is associated with reduced NF-jB activation and cytokine production via the inability of Mal to recruit MyD88. Here with the use of the recently generated D96N mouse we aim to characterise the effect of Mal D96N on TLR signalling and the biological outcomes of this during bacterial and viral infection. Bone marrow derived macrophages (BMDMs) from D96N mice display reduced inflammatory responses to the TLR4 ligand lipopolysaccharide (LPS). Macrophages from Mal D96N mice produced significantly less IL-6 and TNFa with no effect on LPS-induced IFNb. Delayed MAP kinase activation and phosphorylation of NF-jB subunit p65 was also observed in D96N macrophages in response to both LPS and the TLR2 ligand Pam3 Cys. Furthermore, Mal D96N mice are protected from LPS-induced lethality. Data suggests reduced levels of IL-6 in the serum of D96N mice at early time points following administration of LPS (i.p). Future work will focus on examining the interaction of Mal D96N with MyD88 and its recruitment to the membrane following TLR4 and TLR2 activation. While Mal knock-out mice display ablated inflammation, studies with the D96N mouse will provide insight as to the non-bridging effects of Mal in TLR signalling during inflammation. http://dx.doi.org/10.1016/j.cyto.2014.07.046

40 Inhibition of euchromatic histone methyltransferase 1 and 2 sensitizes chronic myeloid leukemia cells to interferon treatment Chee-kwee Ea, Shengwei Loh, Weilun Ng, Koksiong Yeo, University of Malaysia, Kuala Lumpur, Malaysia Background: H3K9 methylation is one of the essential histone post-translational modifications for heterochromatin formation and transcriptional repression. Recently, several studies have demonstrated that H3K9 methylation negatively regulates the type I interferon response. Results: We report the application of EHTM1 and EHMT2 specific chemical inhibitors to sensitize CML cell lines to interferon and imatinib treatments. Inhibition of EHMT1 and EHMT2 with BIX01294 enhances the cytotoxicity of IFNa2a in all four CML cell lines tested but not three non-CML cell lines. Additionally, BIX01294 augments IFNa2a- and imatinib-mediated apoptosis in CML cells. Moreover, our data suggest that the expression level of EHMT1 and EHMT2 inversely correlates with the type I interferon response in CML cell lines. Conclusions: Our study sheds light on the role of EHMT1 and EHMT2 as potential targets in improving the efficacy of standard treatments of CML. http://dx.doi.org/10.1016/j.cyto.2014.07.047

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cb T cells, a novel T cell subset with a pathogenic role in IL-17-mediated CNS autoimmunity Sarah C. Edwards, Caroline E. Sutton, Kingston H.G. Mills, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland Vc4+ T cells have been identified as the main IL-17-producing cd T cell in the CNS of mice with experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. The findings of this study demonstrate that Vc4+ T cells are present in T cell receptor (TCR)d= mice, and furthermore these Vc4+ T cells co-express TCRb. The data reveals that Vc4b T cells respond to IL-1b and IL-23 stimulation in the