129 NOD2 Mediates MDP-induced Inflammation Independent

Abstracts / Cytokine 39 (2007) 1–49 and Weil Medical College of Cornell University, New York, NY, USA Therapies for most autoimmune diseases rely on glucocorticoid drugs. Glucocorticoid receptor (GR) is a ligand-dependent transcription factor, which binds glucocorticoid response elements (GREs) and activates or represses gene expression. Glucocorticoid immunosuppression is believed to stem largely from GR interference with AP1 and NFjB activities. Although additional factors likely mediate dramatic effects of glucocorticoids on the immune system, their identity remains obscure. We previously identified a p160 family member GRIP1 as a GR corepressor at the AP1/NF-jB ‘tethering’ GREs. Its corepression domain (RD) did not resemble any known protein and functioned specifically at tethering GREs, suggesting its role as a determinant of immunosuppressive actions of glucocorticoids, perhaps beyond GR antagonism with AP1/NF-jB. A yeast 2-hybrid screen for RD-interacting proteins yielded Interferon (IFN) Regulatory Factor (IRF)3—an effector of innate immune responses downstream of TLRs 3 and 4 (recognizing dsRNA and LPS, respectively), which targets several IFN and chemokine genes. We refined the GRIP1:IRF3 interface and showed that GR and IRF3 competitively bind GRIP1 in vitro. Endogenous GRIP1 and IRF3 interacted in macrophages in a dsRNA-dependent glucocorticoid-sensitive manner. In functional studies, GRIP1 depletion by liganded GR or siGRIP1 inhibited, whereas GRIP1 overexpression rescued IRF3-dependent gene expression; IRF3 binding sites (ISREs) were responsible for both positive and negative regulation. Genetic approaches confirmed glucocorticoid disruption of MyD88-independent TLR3–TRIF–IRF3 pathway and not of autocrine IFN signaling or signaling by another TLR. Chromatin-IP survey of transcription complexes and histone modifications at IRF3-regulated genes in primary macrophages revealed glucocorticoid inhibition of transcription initiation. Conversely, activation of IRF3 lifted glucocorticoid repression of AP1/NF-jB, consistent with GRIP1 loss from GR repression complexes. These data identify GRIP1 as IRF3 coactivator and a potential regulator of innate immunity. Further, GRIP1 sequestration by liganded GR antagonizes IRF3-driven transcription establishing the TLR3–IRF3 pathway as a novel target for GR-mediated immunosuppression. doi:10.1016/j.cyto.2007.07.131

127 Type I Interferons as Virulence-determining Factors in Listeria Monocytogenes Infections Benjamin Reutterer 1, Silvia Stockinger 1, Andreas Pilz 1, Didier Soulat 1, Thomas Ru¨licke 2, Mathias Mu¨ller 3, Thomas Decker 1, 1 Max F. Perutz Laboratories, Department of Microbiology and Immunobiology, University of Vienna, Vienna, Austria, 2 Biomodels Austria GmbH, University of Veterinary Medicine, Vienna, Austria, 3 Institute of Animal Breeding and Genetics, University of Veterinary Medicine, Vienna, Austria Type I IFN (IFN-I) are known to initiate potent antimicrobial mechanisms against virus infections. Their role in non-viral infections is less defined. Following infection with the facultative intracellular, gram-positive bacteria Listeria monocytogenes, production of IFN-I sensitizes cells and mice to lethal outcome. Therefore, the amount of IFN-I produced during infection might be an important factor determining Listeria virulence. We were able to characterize two commonly used strains of L. monocytogenes, EGD and LO28, as, respectively, low and high inducers of IFN-I synthesis in infected macrophages. Increased IFN-I production resulted from the stronger ability of the LO28 strain to selectively trigger the IRF3 signaling pathway and correlated with an increased sensitization of macrophages to lethal infection. In contrast, stimulation of NF-jB, MAPK, or inflammasome signaling by the LO28 and EGD strains did not differ significantly. The LO28 strain was more virulent in wild-type (wt) C57Bl/6 mice than the EGD strain whereas both strains were similarly virulent in IFN-I receptor-deficient C57Bl/6 mice. Together our data stress the fact that isolates of wt L. monocytogenes differ in their ability to

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trigger the IRF3 signaling pathway and IFN-I production, and that the amount of IFN-I produced during infection is an important determinant of Listeria virulence. doi:10.1016/j.cyto.2007.07.132

128 Oncostatin M-receptor Deficient Phenotype Provides Evidence that Oncostatin M is Critical for IL-4-induced Eosinophil Accumulation in Lungs In Vivo Carl D. Richards 1, Zhou Xing 1, Christine Kerr 1, Dominik Fritz 1, David Smyth 1, Atsushi Miyajima 2, Carrie M. Langdon 1, 1 Centre for Gene Therapeutics, Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ont., Canada, 2 University of Tokyo, Tokyo, Japan Cytokines play important roles in inflammation, and regulate responses in context of the inflammatory milieu that may vary depending on tissue site and stage of immunological/pathological processes. Oncostatin (OSM), member of the Interleukin (IL)-6/gp130 cytokine family, synergises with IL-1, TNF or IL-17 in regulation of IL-6 and metalloproteinases in chondrocytes, but can also synergise with IL-4 or IL-13 in the regulation of different genes such as eotaxin-1 in lung fibroblasts. Over-expression of IL-4 in mouse lungs using Adenovirus (Ad IL-4) induces eosinophil accumulation in lungs in vivo. To determine the role of OSM in eosinophil accumulation, we examined the responses of OSMReceptor / deficient cells in vitro and of / mice in vivo. Mouse lung fibroblasts (MLF) cultures were generated from +/+ or / mice and treated for response to OSM, IL-4 and IL-13 by analysis of 24 h supernantant content. Results show that OSM, IL-4 or IL-13 could induce eotaxin-1 in MLF+/+ whereas IL-4/IL-13 but not OSM could induce eotaxin-1 in OSMR / MLF. Typical responses to IL-1 or TNF were equivalent in the two cell types, suggesting that OSMR / MLF are able to respond normally to cytokines other than OSM. Treatment of +/+ mice with AdIL-4 resulted in accumulation of eosinophils in the broncho-alveolar (BAL) fluid at seven days after intranasal administration. In parallel experiments, treatment of OSMR / mice (same background strain) resulted in a marked decrease in eosinophil accumulation in the BAL. Analysis of mRNA levels taken at day 7 from whole lung extracts, showed that while eotaxin mRNA was increased in +/+ lungs, levels were reduced in OSMR / lungs. Thus, the effects of AdIL-4 in mouse lungs is dependent on the presence of a functional OSMReceptor. Since OSM synergises with IL-4, we suggest that lack of synergy in vivo can determine characteristics of inflammation. Acknowledgment Studies supported by the Canadian Institutes for Health Research. doi:10.1016/j.cyto.2007.07.133

129 NOD2 Mediates MDP-induced Inflammation Independent H.L. Rosenzweig 1, T.M. Martin 1, M. Jann 2, K. Goodwin 1, S.R. Planck 1, M.P. Davey 2, J.T. Rosenbaum 1, 1 Casey Eye Institute, Oregon Health & Science University, Portland, OR, USA, 2 Veterans Affairs Medical Center, Portland, OR, USA NOD2/CARD15 belongs to the NOD-like receptor (NLR) family, which plays an important role in the recognition of intracellular bacteria. NOD2 specifically senses a component of peptidoglycan, muramyl dipeptide (MDP). Mutations in NOD2 are associated with inflammatory diseases such as Crohn’s disease and early-onset sacroidosis. Specific mutations in NOD2 have been identified that cause Blau syndrome, an autosomal dominant form of uveitis, arthritis and dermatitis. Intriguingly, mutations in other NLR genes such as

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Abstracts / Cytokine 39 (2007) 1–49

NALP3/CIAS1 are associated with other autoinflammatory diseases: Muckle–Wells syndrome, familial cold urticaria and chronic infantile neurological cutaneous and articular syndrome (CINCA/NOMID). These share some clinical characteristics with Blau syndrome. NALP3 regulates IL-1b production which in turn mediates NALP3-associated autoinflammatory diseases. We recently described a mouse model of MDP-induced uveitis, in which mice treated with an intravitreous injection of MDP develop ocular inflammation. MDP-induced uveitis is dependent on NOD2, as NOD2 knockout mice fail to show an ocular inflammatory response to MDP. IL-1b production as assessed by ELISA was significantly increased in the eye in response to MDP in a NOD2-dependent manner. MDP-induced IL-1b production in the eye was impaired in caspase-1 knockout mice. This indicates that MDP-induced IL-1b production requires both NOD2 as well as caspase-1, a component of the NALP3 inflammasome. Unexpectedly, IL-1b does not appear to be a predominant mediator of MDP-induced uveitis, as deletion of either IL-1-receptor-1 or caspase-1 had no impact on the intravascular inflammatory response to MDP as measured by intravitral microscopic quantification of leukocyte rolling and sticking. In contrast, deletion of interferon gamma does reduce the ocular inflammatory response induced by MDP. Our studies shed light onto the role of NOD2 as a key regulator of IL-1b production. Despite clinical similarities of NLR-associated autoinflammatory diseases, NOD2 may contribute to inflammation in an IL-1b-independent mechanism. doi:10.1016/j.cyto.2007.07.134

130 MAGUK Family Member Dlgh1 Coordinates TCR-dependent p38 Activation, Specifically Linking TCR Engagement to Activation of NFAT but not NF-jB J.L. Round 1, L.A. Humphries 1, T. Tommasian 2, P. Mittelstadt 3, M. Zhang 1, M.C. Miceli1 1,2,3, 1 Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90066, USA, 2 Molecular Biology Institute, University of California, Los Angeles, CA 90066, USA, 3 Laboratory of Immune Cell Biology, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA Proximal kinase activation couples TCR engagement to discrete signaling cascades capable of differentially contributing to T cell function. However, mechanisms of channeling TCR signals toward specific targets remain undefined. Previously, we established a role for Dlgh1 in TCR/CD28 NFAT activation and lymphokine production. Here, we show that alternatively activated p38 associates with the MAGUK family member Dlgh1 and identify a novel Dlgh1/p38/Zap70 signaling complex. TCR stimulation utilizes a unique ‘‘alternative’’ p38 activation whereby Lck and ZAP70 cooperate to directly phosphorylate and activate p38. Our data suggest that Dlgh1 may act to coordinate T cell-specific p38 activation and begin to define downstream effectors of this pathway. By manipulating Dlgh1 levels in T cells we demonstrate that Dlgh1 facilitates alternative p38 and NFAT, but not JNK or NF-jB, activation. Further, expression of a p38-binding-deficient-Dlgh1 inhibits TCR-induced p38 and NFAT activation and pharmacologic inhibition of p38 ablates Dlgh1-mediated NFAT upregulation. Taken together, our findings elucidate a novel signaling mechanism whereby TCR proximal kinase activity is selectively channeled toward p38-mediated NFAT activation through Dlgh1. In combination with reports that the MAGUK family member CARMA1 regulates JNK and NF-jB, but not p38 or NFAT activity, these data suggest independent roles for MAGUKs in orchestrating TCR signal specificity. Lisa A Humphries is a recipient of the Arthritis Foundation Postdoctoral Fellowship. E-mail address: [email protected] (M.C. Miceli1). doi:10.1016/j.cyto.2007.07.135

131 Ultra Small Doses of Antibodies to Human Gamma Interferon in the Treatment of Patients with Allergy and Viral Infections M. Rudenko 1, N. Markin 1, A. Zabelev 1, I. Rudenko 2, A. Shemshura 3, 1 Clinical Center EuroDon, Rostov-on-Don, Russian Federation, 2 629 Outpatients’ Clinic, Rostov-on-Don, Russian Federation, 3 Rostov Research Institute of Microbiology and Parasitology, Rostov-on-Don, Russian Federation Recent studies showed that immune changes caused by viral infections can lead to decrease of the number of T cells with the prevalence of Th2 response. [Luss L, Russian Immunology Journal 2006;1:17; Fedoskova T, Allergology 2007;2:265; Markova T, Cytokines and Inflammation 2005;3:129]. The aim of our study was to detect the peculiar changes in immune system and to try to influence on them. Twenty-six allergic patients 25–32 years with persistent HSV, EBV, CMV infections were carefully examined. We used following methods: microscopy of nasal smears, PCR, ELISA, blood and urine tests, immunologic examination and tests (CD3, CD4, CD8, CD16, CD20, CD3; CD95, CD3; HLADR, NST, IgA, IgM IgG IgE, Th1 and Th2). The patients were divided randomly into two groups. In the first one we used ultra small doses of antibodies to human gamma interferon (C12, C30 and C200) 3 times per day during 20 days, in the second-placebo. After the treatment we compared results in two groups and revealed improvement of allergy symptoms, deceased quantity of eosinophils in nasal smears in the first group. The immune parameters also improved Th1 was increased (first group 8.07 ± 0.09%; second group 6.06 ± 1.01%; p < 0.05) Th2 was decreased (first group 0.8 ± 0.19%; second group 3.56 ± 0.67%; p < 0,05), the number of T cells NK cells normalized. We can conclude that stimulation of gamma interferon production leads to positive changes in immune system and though that to clinical improvement. doi:10.1016/j.cyto.2007.07.136

132 Toll-like Receptors (TLRS) Contribute in the Inflammation and Pathogenesis of RA Sandra Sacre, Alexandra Lo, Mino Medghalchi, Bernard Gregory, Lynn Williams, Fionula Brennan, Richard Williams, Brian Foxwell, Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College of Science, Technology and Medicine, London, UK TLRs are a family of innate immune receptors that have been shown to respond to both exogenous as well as endogenous ligands. They are attractive candidates for a more targeted therapy in autoimmune diseases where many endogenous TLR ligands could be present. Recently, we demonstrated that the TLR signaling molecules MyD88 and Mal both have a role in pro-inflammatory cytokine production from human rheumatoid arthritis (RA) synovial membrane cell cultures. RA is one of the most common autoimmune diseases affecting 1:100 people and causes inflammation and damage to the joints. Further studies have identified inhibitors of TLR induced cytokine production. These inhibitors were able to significantly inhibit disease progression in the murine collagen induced arthritis (CIA) model as measured by an improvement in clinical score, decreased cell infiltration and reduced histology scoring. We also examined the effects of the inhibitors in human RA synovial membrane cultures. These cultures spontaneously release inflammatory cytokines, three of which, TNF, IL-6 and IL-1 are targets for biological therapies in human RA. We observed that the spontaneous release of these cytokines from human RA cell cultures was significantly decreased in the presence of the TLR inhibitors, suggesting that inhibition of the relevant TLRs may be beneficial in RA. doi:10.1016/j.cyto.2007.07.137