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Abstract / Cytokine 70 (2014) 28–79

highly sensitive mass spectrometry analysis allows for the comprehensive analysis of the kinome [1]. In this study, we have used two kinase-capturing reagents to profile the kinome from various cellular sources including HEK293Ts, human G-CSF mobilized neutrophils and murine bone marrow-derived macrophages (BMDMs). Kinome enrichment was achieved using CTx-0294885 resin (KiNET-1 beads, Synkinase) and a pan-JAK1/2 inhibitor Cyt387 (Cytopia) coupled to NHS-Sepharose beads. In this way we have identified a number of novel kinases within specific cell types, some of which may play important roles in the context of disease. This kinome profiing strategy was performed alongside a phosphopeptide enrichment strategy for quantitative mass spectrometry analysis. Using this approach we identified a number of novel phosphopeptide sites within clinically-relevant kinases including the JAK protein tyrosine kinases. We identified 40 phosphorylation sites in murine JAK2 (23 novel and 17 known sites), while 43 phosphorylation sites were identified in murine JAK1 (32 novel and 11 known sites). Mutational studies are currently underway to screen a selection of these evolutionarily-conserved candidate phosphorylation sites for functional relevance. The authors would like to acknowledge Leanne Daly from Synkinase for supplying the KiNET-1 beads used in this study.

Reference [1] Zhang L et al.. Characterization of the novel broad-spectrum kinase inhibitor CTx0294885 as an affinity reagent for mass spectrometry-based kinome profiling. J Proteome Res 2013;12(7):3104–16. http://dx.doi.org/10.1016/j.cyto.2014.07.040

34 Expression and function of ADAR1 and PACT and their impact on PKR activation during HIV-1 infection Aïcha Daher 1, Samantha Burugu 1,2, Guerline Clerzius 1,3, Eileen Shaw 1,3, Roman Radetskyy 1,2, Jean-Pierre Routy 3,4, Anne Gatignol 1,2,3, 1 Lady Davis Institute for Medical Research, Montreal, QUE, Canada, 2 Department of Microbiology and Immunology, McGill University, Montreal, QUE, Canada, 3 Department of Medicine, Division of Experimental Medicine, McGill University, Montreal, QUE, Canada, 4 Division of Hematology, McGill University, Montreal, QUE, Canada Aims: A poor innate immune response contributes to the establishment of viral latency and persistence in human immunodeficiency virus (HIV)-1 infection. The protein kinase RNA-activated (PKR) is an Interferon-stimulated gene (ISG) that senses RNA viruses. PKR is a potent HIV-1 suppressor through the phosphorylation of the translation initiation factor eIF2a and consequent inhibition of protein synthesis. We showed that PKR is activated at the beginning of HIV-1 infection followed by a deactivation due, in part, to cellular proteins TRBP and ADAR1. Although PACT is a PKR activator, we showed that it becomes a PKR inhibitor in HIV-expressing cells [1]. Our hypothesis is that PKR inhibition contributes to HIV-1 replication and persistence through multiple mechanisms. Our objective is to elucidate how ADAR1 and PACT contribute to PKR inhibition during HIV-1 infection. Methods: We analysed the expression of PKR, ADAR1 and PACT in HIV-infected patients and in peripheral blood mononuclear cells (PBMCs) infected with HIV-1 by western blots and immunoprecipitations. We used short hairpin (sh)RNAs against ADAR1 and PACT to assess their impact on HIV-1 production. We assessed the interaction between ADAR1 and PACT and its impact on PACT function. Results: ADAR1 and PACT expression are increased in HIV-1 infected PBMCs and immunoprecipitate with PKR. This is correlated with PKR deactivation and increased viral expression. A decrease of either ADAR1 or PACT inhibits HIV-1 production. A direct interaction between ADAR1 and PACT suggests a functional significance on PACT and PKR functions during HIV replication. Conclusion: The increased expression of both ADAR1 and PACT contribute to PKR inhibition in HIV-1 infected cells. PACT changes its function for a PKR inhibitor in HIV-infected cells, which could be due to its interaction with ADAR1, thereby contributing to viral persistence in patients.

Reference [1] Clerzius et al.. Retrovirology 2013;10:96. http://dx.doi.org/10.1016/j.cyto.2014.07.041

35 Oral rapamycin prevents carcinogen and inflammation-induced skin carcinogenesis through an IFN-c-dependent mechanism Vinh Dao 1,2, Sri Lakshmi Pandeswara 1, Kim Cardenas 1, Vincent Hurez 1, Sherry Dodds 3, Yang Liu 1, Aijie Liu 1, Z. Dave Sharp 3, Paul Hasty 3, Tyler J. Curiel 1,2,4, 1 Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA, 2 Microbiology & Immunology, Graduate School of Biomedical Sciences, San Antonio, TX, USA, 3 Molecular Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA, 4 Cancer Therapy & Research Center, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA Background: Cancer prevention is a cost-effective alternative to treatment. Oral rapamycin (eRapa) prevents distinct cancers and extends life in mice[1], making it a candidate broad-spectrum cancer prevention agent. Rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), has significant immune effects that are unstudied in cancer. We hypothesize that eRapa prevents cancer through improved cancer immune surveillance, which we tested in a carcinogen-induced, inflammation-driven skin cancer model. Methods: eRapa was used at 14 ppm microencapsulated rapamycin. Skin cancer. Wild-type C57BL/6 (WT) or syngeneic bd knockout (KO) and interferon (IFN)-c KO mice (lacking all T cells or IFN-c, respectively) got eRapa or control for 1 month, followed once with the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA), then twice weekly applications with the inflammatory agent 12-O-tetradecanoylphorbol (TPA) for 24 weeks. Flow cytometry assessed cells and IFN-c. Statistics. Tumors (unpaired t test and 2-way ANOVA) and malignant degeneration (Fischer exact test). Results: eRapa reduced benign (p = .004) and malignant (p = .03) tumors in WT mice. T cells and IFN-c mediate cancer immune surveillance so their effects on eRapa cancer prevention were studied. eRapa reduced skin tumors in bd KO mice lacking all T cells (p = .04), but not in IFN-c KO mice (p = .13), consistent with loss of beneficial eRapa-induced, non-T cell IFN-c. In support, WT or IFN-c KO T cell transfer into IFN-c KO mice did not alter eRapa cancer prevention in this model. In WT mice on DMBA/ TPA, eRapa increased IFN-c producing dermal natural killer (NK) cells (p < .0001) that can mediate skin cancer immune surveillance. There was a loss of this eRapa-mediated dermal NK cell increase in IFN-c KO mice, suggesting that eRapa-mediated IFN-c facilitated local NK cell accumulation. Conclusions: eRapa prevents carcinogen and inflammation-induced skin cancer, which involves a non-T cell that could be an NK cell, and is IFN-c dependent. We also show that eRapa prevents cancer and extends life in distinct mouse cancer models, which paves the way for use of mTOR inhibitors as broad spectrum human cancer prevention agents.

Reference [1] Miller RA, Harrison DE, Astle CM, Baur JA, Boyd AR, de Cabo R, et al.. Rapamycin, but not resveratrol or simvastatin, extends life span of genetically heterogeneous mice. J Gerontol A Biol Sci Med Sci 2011;66:191–201. http://dx.doi.org/10.1016/j.cyto.2014.07.042

36 Modulation of innate immune responses by ATF3 Dominic De Nardo 1, Larisa I. Labzin 1, Susanne Schmidt 2, Seth L. Masters 3, Rainer Stahl 1, Joachim Schultze 2, Eicke Latz 1,4,5, 1 Institute of Innate Immunity, Bonn, Germany, 2 Life and Medical Sciences Institute (LIMES), University of Bonn, Bonn, Germany, 3 The Walter and Eliza Hall Institute (WEHI) for Medical Research, Parkville, VIC, Australia, 4 Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, USA, 5 German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany Our body is continuously exposed to a diverse array of infectious agents, foreign antigens, and host-derived danger signals. The innate immune system plays a vital role as the first line of defence against these insults by protecting the host from infection and maintaining tissue homeostasis. Families of signalling receptors (e.g. TLRs) recognize invading microbes and co-ordinate an appropriate inflammatory response via the production of pro-inflammatory cytokines and type I interferons (IFNs) to clear infection. However, inappropriate activation of innate immune receptors and unregulated production of inflammatory mediators is thought to be the basis for many acute and chronic inflammatory conditions. Indeed, excessive IFN production is implicated in the pathogenesis of lupus and atherosclerosis. Maintaining a balance between the beneficial and detrimental effects of IFNs is therefore crucial and their production must be tightly regulated. Yet these precise regulatory mechanisms remain poorly understood. We have discovered that macrophages deficient in the transcriptional modulator, ATF3, display significantly greater IFNb production follow-

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