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Abstract / Cytokine 63 (2013) 243–314 Panel using LuminexÒ technology for simultaneous measurement of 13 Cytokines/ Chemokines (GM-CSF, IFNc, IL-1a, IL-1b, IL-1ra, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, and TNFa) in porcine serum, plasma and tissue culture samples. Our validation data showed that the assay is specific, sensitive, accurate and reproducible, and this panel detects serum and plasma samples in various porcine strains including Hanford, Yucatan, Gottingen, Sinclair, and Yorkshire. Using this new assay panel, we also measured Cytokine/Chemokine levels in porcine PBMC culture medium samples collected at different time intervals after treatment of LPS or Con A. Our results suggested that this robust multiplex assay panel is a very useful tool for porcine cytokine/chemokine measurement for various research areas and drug discovery.

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contrast to isotype-treated or untreated mice which cleared infection by day 4. This susceptibility was associated with reduced mucosal expression of specific IL-17-associated genes, such as lipocalin-2 and CXCL5. In contrast, anti-IL-17F treatment did not impair clearance. Additionally, IL-17A / mice fully cleared infection by day 4 akin to wild type mice, but IL-17RA / and Act1 / mice failed to clear infection by day 14 (end of study). Overall, these results highlight differences between cytokine blockade in vivo versus constitutive genetic ablation of individual IL-17 cytokines, receptors and key signaling components, and implicate a role for both IL-17A and alternative mechanisms in resistance to OPC. Funding from Novartis and NIH Grant DEO22550. http://dx.doi.org/10.1016/j.cyto.2013.06.282

http://dx.doi.org/10.1016/j.cyto.2013.06.280

278 Inflammatory disease and an impaired type I interferon response resulting from a de novo human NEMO hypomorphic mutation Alex W. Wessel a, Amy P. Hsu b, Jevgenia Zilberman-Rudenko a, Raphaela T. GoldbachMansky c, Richard M. Siegel a, Eric P. Hanson a, a Autoimmunity Branch, NIAMS, United States, b Laboratory of Clinical Infectious Diseases, United States, c NIAID, Pediatric Translational Research Branch, NIAMS, United States Toll-like receptor 3 and the RIG-I-like family of receptors (RLRs) mediate antiviral signaling through NEMO-dependent activation of transcription factors NF-jB and IRF3, which are recognized for their roles in inflammation and the induction of type I interferon. We identified an individual with Ectodermal Dysplasia, a history of in utero cytomegalovirus exposure, and persistent inflammatory disease. Sanger sequencing revealed a de novo synonymous mutation in IKBKG, which encodes NEMO, the NF-jB essential modulator. The mutation results in an mRNA splicing defect which leads to the production of a mutant protein containing an in-frame deletion of 51 residues, lacking a previously identified TANK (TRAF family member-associated NFKB activator) interaction domain. Co-immunoprecipitation studies revealed that the mutant form of NEMO from patient T cells is unable to recruit the IRF3 kinase TANK-binding kinase-1 (TBK1), due to impaired association with TANK. Patient fibroblasts stimulated with the RLR ligand poly (I:C) led to impaired induction of IRF3 response genes IFNB1, CCL5, CXCL10, IP10, OASL, and DDX58. Consequently, dermal fibroblasts infected with RSV or hPIV3 demonstrated increased virus propagation relative to healthy control cells. Impaired gene expression occurred despite enhanced nuclear translocation of IRF3, suggesting that the mechanism of impaired activation is not a simple defect in nuclear locof the transcription factor. In contrast to the impaired type I IFN response, canonical IKK activation and gene induction in dermal fibroblasts appeared relatively normal following TNF treatment. These results illustrate a novel mechanism of autoinflammatory disease due to aberrant expression of an alternately spliced NEMO and deficient IRF3-mediated response. http://dx.doi.org/10.1016/j.cyto.2013.06.281

280 Essential role of the phosphatase PP1 in the RIG-I and MDA5 mediated antiviral interferon response Effi Wies a, May K. Wang a,b, Natalya P. Maharaj a, Kan Chen a, Shenghua Zhou c, Robert W. Finberg c, Michaela U. Gack a,b, a New England Primate Research Center, Harvard Medical School, Southborough, MA, USA, b Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, USA, c Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA Innate sensing of viral infection and the subsequent activation of signaling pathways are essential for an effective antiviral immune response. Retinoic acid-inducible gene-I (RIG-I; also called DDX58) and melanoma differentiation-associated gene 5 (MDA5; also known as IFIH1) have emerged as key cytosolic sensors for the detection of RNA viruses. Upon viral RNA binding, the N-terminal caspase recruitment domains (CARDs) of RIG-I and MDA5 initiate downstream signaling, resulting in the production of type-I interferon (IFN) and proinflammatory cytokines. Recent studies highlighted the important role of multisite posttranslational modification for controlling RIG-I antiviral activity; in contrast, the regulation of MDA5 signaling activity remains largely undetermined. Here, we show that MDA5 signal-transducing activity is regulated by a dynamic balance between phosphorylation and dephosphorylation of its N-terminal CARDs, and further identify the phosphoprotein phosphatase 1 a (PP1a) and PP1c as essential activators of both MDA5 and RIG-I antiviral signal transduction. Using mass spectrometry and a phosphorylation state-specific MDA5 antibody, we identified that MDA5 is robustly phosphorylated at S88 under normal conditions; upon viral infection, however, MDA5 is rapidly dephosphorylated, allowing CARDmediated downstream signaling. Employing a phosphatome RNA interference (RNAi) screen, we identified PP1a and PP1c as primary phosphatases responsible for the MDA5 and RIG-I CARD dephosphorylation, which leads to their activation. Depletion of endogenous PP1a and PP1c markedly enhanced the CARD phosphorylation of the sensors MDA5 and RIG-I, and strongly reduced antiviral IFN-b production. Furthermore, cells in which PP1a and PP1c had been silenced using specific siRNAs were impaired in their ability to induce the expression of various IFN-stimulated genes, which correlated with an enhanced RNA virus replication in these cells. Collectively, this work identifies PP1a and PP1c as critical regulators of antiviral innate immune responses to a wide range of RNA viruses, including paramyxoviruses, orthomyxoviruses, dengue virus and picornaviruses. http://dx.doi.org/10.1016/j.cyto.2013.06.283

279 Experimental analysis of acute murine oral candidiasis after in vivo Anti-IL-17A/ IL-17F treatment and in knockout mice Natasha Whibley a, Anna J. Mamo a, Elisabetta Traggiai b, Frank Kolbinger b, Beate Vogel b, Michael Kammüller b, Ulrich Siebenlist c, Sarah L. Gaffen a, a Division of Rheumatology and Clinical Immunology, University of Pittsburgh, United States, b Novartis Institutes for Biomedical Research, Basel, Switzerland, c NIAID, NIH, Bethesda, MD, United States Inhibitors targeting IL-17A or one of its receptor chains (IL-17RA) are now in clinical trials for the treatment of autoimmune diseases, with promising results. However, potential side effects associated with anti-IL-17 pathway therapy, in particular the potential for increased susceptibility to infections, are poorly understood. A number of genetic diseases with increased chronic mucocutaneous candidiasis (CMC) in humans have implicated the Th17/ IL-17 pathway in immune protection against Candida albicans infections. However, the individual roles of IL-17A or IL-17F regarding mucosal candidiasis have not been well defined. To further examine the potential role of the IL-17 pathway in fighting mucosal candidiasis, we took advantage of a murine model of acute oropharyngeal candidiasis (OPC) in which wild type mice clear infection by day 4. Following oral C. albicans exposure, C57BL/6 (wild type) mice were treated with isotype control Abs or neutralizing anti-IL-17A or anti-IL-17F Abs and disease symptoms, fungal burden in the tongue and body weight change were assessed. IL-17A blockade resulted in delayed clearance of OPC with anti-IL-17A-treated mice failing to clear infection by day 4, but with clearance by day 7 onwards in

281 Assembly of IFNAR1/IFNAR2 is regulated by ubiquitin specific protease 18 Stephan Wilmes a, Zhi Li b, Katharina Hötte a, Oliver Beutel a, Cindy Kroll a, Friedrich Roder a, Patrizia Hanhart a, Dennis Janning a, Changjiang You a, Christian Paolo Richter a, Véronique François-Newton b, Gilles Uzé c, Sandra Pellegrini b, Jacob Piehler a, a University of Osnabrück, FB Biology, Department of Biophysics, Osnabrück, Germany, b Institut Pasteur, Départment Immunologie, Unité de Signalisation de Cytokines, Paris, France, c CNRS UMR 5235, University Montpellier II, Place Eugène Bataillon, 34095 Montpellier cedex 5, France Type I Interferons (IFN) are key cytokines in the regulation of the innate immune response. All IFNs bind to a shared cell surface receptor comprised of two subunits, IFNAR1 and IFNAR2. Together with IFN the receptors assemble to hetero-trimeric ‘‘ternary” complexes which initiate phosphorylation cascades through the intracellularly associated Janus-Kinases Jak1 and Tyk2. Detailed structure–function analysis of IFNs has established that the dynamics and affinities of IFN interactions with the receptor subunits play a critical role for regulating signalling specificities. Here we have explored the IFN-induced assembly of the ternary signalling complex at physiological expression level by dual colour single molecule imaging. To this end, we employed highly specific and efficient orthogonal posttranslational labelling approaches with photostable organic fluorophores combined with TIRF-microscopy.

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By single molecule co-localization and co-tracking analysis we have mapped lateral distribution and diffusion of the two receptor subunits with very high spatial and temporal resolution in living cells. Thus, we were able to monitor IFN-induced assembly, co-diffusion and dissociation of individual ternary signalling complexes in the plasma membrane. Systematic modulation of the binding affinities towards the receptor subunits revealed that binding affinity of IFNa2 towards IFNAR1 is optimized for efficient ternary complex formation at physiological receptor surface concentrations. Interestingly, the negative feedback regulator ubiquitin-specific protease 18 (USP18) reduces receptor dimerization by IFNa2, but not IFNb, which binds to IFNAR1 with higher affinity. Strikingly, this USP18-dependent loss of 2D affinity could be mimicked by truncating the receptor’s intracellular domains. These observations suggest that ternary complexes are stabilized by intracellular interactions of Jak1 and Tyk2 and that these interactions are interfered by USP18, thus selectively abrogating signalling by IFNs with low affinity towards IFNAR1.

http://dx.doi.org/10.1016/j.cyto.2013.06.284

282 TSLP-elicited basophil responses mediate the pathogenesis of eosinophilic esophagitis Elia D. Tait Wojno a,b,1, Mario Noti a,b,1, Brian S. Kim a,b, Mark C. Siracusa a,b, Paul R. Giacomin a,b,d, Meera G. Nair a,b, Alain J. Benitez e, Kathryn R. Ruymann f, Amanda B. Muir e, David A. Hill a,b, Kudakwashe R. Chikwava g, Amin E. Moghaddam h, Quentin J. Sattentau h, Aneesh Alex i,j,k, Chao Zhou i,j,k, Paul Menard-Katcher l, Masato Kubo m,n, Kazushige Obata-Ninomiya o,p, Hajime Karasuyama o,p, Michael R. Comeau q, Terri Brown-Whitehorn f, Patrick M. Sleiman r,s,t, Hakon Hakonarson r,s,t, Antonella Cianferoni f, Gary W. Falk l, Mei-Lun Wang e, Jonathan M. Spergel f, David Artis a,b,c, a Department of Microbiology, University of Pennsylvania, Philadelphia, PA, USA, b Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA, c Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA, d Queensland Tropical Health Alliance and School of Public Health and Tropical Medicine, James Cook University, Cairns, Australia, e Division of Gastroenterology, Hepatology, and Nutrition, Children’s Hospital of Philadelphia, Philadelphia, PA, USA, f Department of Pediatrics, Division of Allergy and Immunology, Children’s Hospital of Philadelphia, Philadelphia, PA, USA, g Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA, USA, h The Sir William Dunn School of Pathology, The University of Oxford, Oxford, UK, i Department of Electrical and Computer Engineering, Lehigh University, Bethlehem, PA, USA, j Center for Photonics and Nanoelectronics, Lehigh University, Bethlehem,PA, USA, k Bioengineering Program, Lehigh University, Bethlehem, PA, USA, l Division of Gastroenterology, Hospital of the University of Pennsylvania, Philadelphia, PA, USA, m Laboratory for Signal Network, Research Center for Allergy and Immunology, RIKEN Yokohama Institute, Yokohama, Japan, n Division of Molecular Pathology, Research Institute for Biological Science, Tokyo University of Science, Chiba, Japan, o Department of Immune Regulation, Tokyo Medical and Dental University Graduate School, Tokyo, Japan, p JST, CREST, Tokyo Medical and Dental University Graduate School, Tokyo, Japan, q Inflammation Research, Amgen Inc., Seattle, WA, USA, r Center for Applied Genomics, University of Pennsylvania, Philadelphia, PA, USA, s Division of Human Genetics, Abramson Research Center, The Children’s Hospital of Philadelphia, University of Pennsylvania, Philadelphia, PA, USA, t Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Eosinophilic esophagitis (EoE) is a food allergy-associated disease characterized by esophageal eosinophilia and associated inflammation. EoE has become increasingly common in industrialized countries. However, current management strategies, such as treatment with swallowed steroids and dietary restrictions, are nonspecific or negatively impact the quality of life of EoE patients. In addition, use of more specific therapies that target immunoglobulin E (IgE) and interleukin (IL)-5 have been largely unsuccessful in ameliorating EoE. Thus, there is an urgent need to identify novel, specific immunological pathways that underlie the pathogenesis of EoE that could be targeted to treat this disease. Recently, a genome-wide association study identified that EoE is associated with a gain-of-function polymorphism in the gene that encodes thymic stromal lymphopoietin (TSLP). TSLP is an IL-7 family member cytokine that promotes allergic inflammation by eliciting T helper type 2 cytokine responses, IgE production, and the expansion of a distinct population of basophils. However, whether TSLP directly promotes allergic inflammation in the esophagus and the mechanisms by which TSLP might contribute to the pathogenesis of EoE remain unknown. Here, we describe a new murine model of experimental EoE-like disease that was used to investigate the role of TSLP in allergic inflammation of the esophagus. Murine experimental EoE-like disease developed independently of IgE but was dependent on TSLP-elicited basophils. Critically, therapeutic antibody-mediated neutralization of TSLP or depletion of basophil populations ameliorated established EoElike disease in mice. Finally, elevated TSLP levels and exaggerated basophil responses 1

These authors contributed equally to this work.

were observed in esophageal biopsies from EoE patients, and a gain-of-function polymorphism in TSLP correlated with increased basophil responses in patients with EoE. Together, these data indicate that TSLP-elicited basophil responses may play a key role in mediating the pathogenesis of EoE, suggesting that targeting the TSLP-basophil axis could represent a new and promising therapeutic target in the treatment of EoE. http://dx.doi.org/10.1016/j.cyto.2013.06.285

283 Suppression of SMAD-1 signaling pathway in mouse lung tissue remodelling by oncostatin M Steven Wong, Rebecca Rodrigues, Christine Kerr, Carl D. Richards, McMaster Immunology Research Centre, Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada Introduction: Abnormal remodeling of the extracellular matrix (ECM) in lung tissue marks the pathology and compromises lung function in diseases such as pulmonary fibrosis and severe asthma. Various cytokine networks, including TGF-beta and gp130 cytokines, have been shown to modulate the ECM. More recent work has suggested a protective role for bone morphogenetic proteins (BMP) in pulmonary fibrosis. We have previously shown that over-expression of Oncostatin M (OSM), one member of the gp130 cytokine family, induces ECM remodeling in mouse lung that remains evident in SMAD3 / and IL-6 / mice. Here we examine the regulation of BMP/SMAD1 signaling pathways upon transient pulmonary over-expression of OSM. Methods: C57Bl/6 mice were intubated with adenovirus vector encoding OSM (AdOSM) to induce transient pulmonary over-expression for 7 days. Lungs were processed for histology, immunohistochemistry, RNA analysis for ECM proteins (collagen) and BMP-associated proteins (BMP-2, BMP-4, BMP-7, gremlin), or for protein analysis by immunoblot for (activated) pSTAT3, pSMAD1/5/8 and BMPReceptor2. Results: AdOSM induced ECM remodeling as indicated by increased parenchymal collagen (PSR stain), coll1a1 and coll1a2 mRNA levels. Immunoblots of signaling responses in total lung homogenates showed increased levels of pSTAT3, but decreased levels of pSMAD1/5/8 and BMPR2 in AdOSM-treated mice. Immunohistochemistry showed a marked reduction of pSMAD1/5/8 detected in airway epithelial cells due to AdOSM, whereas pSTAT3 was up-regulated in airway epithelial and other cells. Levels of mRNA of BMP-2 and BMP-4 were reduced whereas gremlin mRNA was increased in total lungs of AdOSM-treated compared to control (AdDl70) mice. Conclusions: Transient over-expression of OSM induces rapid ECM remodeling in mouse lungs and caused marked suppression of BMP-2/4 mRNA, BMPR2 and pSMAD1 signaling. Such effects would decrease the putative protective function of BMPs in lung fibrosis. Thus OSM-induced suppression of the SMAD1 signaling, in context of STAT3 activation, may reflect a novel pathway that contributes to lung ECM remodeling. This work was supported by the Canadian Institutes for Health Research. http://dx.doi.org/10.1016/j.cyto.2013.06.286

284 Caspase-8-independent role of c-FLIP in Inflammasome Activation Yung-Hsuan Wu a, Wen-Chi Kuo a, Yu-Jong Wu a,b, Kai-Ting Yang a, Shui-Tein Chen c, Si-Tse Jiang d, Ming-Zong Lai a,b, a Institute of Molecular Biology, National Taiwan University, Taipei, Taiwan, b Institute of Immunology, National Taiwan University, Taipei, Taiwan, c Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, d National Laboratory Animal Center, National Applied Research Laboratories, Tainan, Taiwan Cellular FLICE inhibitory protein (c-FLIP) is an inhibitor of caspase-8 and is required for macrophage development. Recent studies reveal a selective role of caspase-8 in non-canonical IL-1b production that is independent of caspase-1 or inflammasome. Here we demonstrated that c-FLIP is an unexpected contributor to canonical inflammasome activation for the generation of caspase-1 and active IL-1b. Lipopolysaccharides (LPS)-, R848-, and heat-killed Listeria monocytogenes (HKLM)-induced IL1b production in macrophages was attenuated in the absence of c-FLIP. Decreased IL1b expression was attributed to a reduced activation of caspase-1 in c-FLIP-deficient cells. In contrast, the production of TNF-a was not affected by deficiency in c-FLIP. cFLIP interacted with NLRP3 or procaspase-1. Proximity ligation assay revealed that cFLIP is required for the full NLRP3 inflammasome assembly, and c-FLIP associates with NLRP3 inflammasome components in situ. c-FLIP-deficiency also reduced AIM2 inflammasome activation. In contrast, c-FLIP inhibited SMAC mimetic- or FasLinduced IL-1b generation that is caspase-8-mediated. Our results demonstrate a caspase-8-independent role of c-FLIP in the optimal activation of the NLRP3 and AIM2