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correlates with high cytokine levels in Ebolavirus infection. Currently we are investigating which cellular signaling pathways are involved in the observed differential cytokine induction in EBOV versus RESTV infection. http://dx.doi.org/10.1016/j.cyto.2013.06.192

190 Group-2 innate lymphoid cell/T cell interactions in helminth expulsion Christopher J. Oliphant a, See Heng Wong a,1, Jennifer A. Walker a, You Yi Hwang a, Jillian L. Barlow a, Emily Hams d, Padraic G. Fallon b,c,d, Andrew N.J. McKenzie a, a MRC Laboratory of Molecular Biology, Cambridge, UK, b Trinity Biomedical Sciences Institute, Trinity College Dublin, Ireland, c National Children’s Research Centre, Dublin, Ireland, d Institute of Molecular Medicine, Trinity College Dublin, Dublin 2, Ireland Group-2 innate lymphoid cells (ILC2) are a recently discovered subset of innate lymphocytes that are essential for both the protective and detrimental type-2 immune responses associated with helminth infections and allergens, respectively. ILC2 arise rapidly in response to the epithelial cytokines interleukin (IL)-25 and IL33, and are typically characterised by the release of the type-2 associated cytokines, IL-13 and IL-5. Previous data from our laboratory have demonstrated that IL-13 expression by ILC2 is essential for the successful clearance of the helminth parasite, Nippostrongylus brasiliensis. Interestingly, we also noted that an impaired ability of Rag2-deficient mice to expel N.brasiliensis correlated with a failure to maintain ILC2 beyond the first few days of infection. This finding suggested that proliferation, migration and/or prolonged survival of ILC2 are highly dependent on a dialogue between ILC2 and either with T or B cells. However, previous studies have shown that B cells, unlike T cells, are dispensable for helminth clearance leading us to examine the mediators associated with ILC2 and T cells in N. brasiliensis infections. Using in vitro co-culture systems we demonstrate that interactions with T cells are highly important for the expansion of ILC2, and their expression of IL-13 and IL-5. Furthermore, we show how these molecular interactions between ILC2 and T cells are important for the efficient expulsion of N. brasiliensis in vivo. Together our data demonstrate that ILC2/T cell interactions are a previously unappreciated mechanism for the initiation and maintenance of type-2 immunity. http://dx.doi.org/10.1016/j.cyto.2013.06.193

191 Difference in the activation of IFN gene expression in response to influenza A and B virus entry Pamela Österlund, Sanna Mäkelä, Veera Arilahti, Ilkka Julkunen, Virology Unit, National Institute for Health and Welfare, Helsinki, Finland Activation of interferon (IFN) system, which is triggered largely by the recognition of viral nucleic acids, is one of the most important host defense reactions against viral infection. Both influenza A and B viruses have a negative strand RNA genome and they possess significant structural similarities. However, influenza A and B viruses are evolutionally diverged with complete genetic incompatibility. We analysed the mechanisms of activation of innate immunity during the infection with type A and B influenza viruses in human dendritic cells. We have previously shown that IFN responses are induced significantly faster in cells infected with influenza B virus than type A virus. We demonstrated that influenza A virus infection activates IFN responses only after the viral RNA (vRNA) synthesis, whereas influenza B virus induces IFN responses even if the infectivity of the virus is destroyed by UV treatment. Thus, initial viral transcription, replication and viral protein synthesis are not required for influenza B virus-induced antiviral responses. Furthermore, we observed that the early induction of IFN gene expression by influenza B virus is mediated by multiple signalling pathways with strong dependency on NF-jB transcription factor in addition to IRF3. The disturbance of endosomal acidification prevented IFN induction suggesting cytosolic recognition of incoming influenza B virus structures to be important in regulating IFN gene expression. Moreover, influenza B virus NP located in the nuclei already at 15 min postinfection whereas the NP from incoming type A virus was found in nuclear fractions at later time points of infection and in significantly lesser extent. Collectively, our data provides new evidence of the mechanisms of influenza B virus recognition by the host cell providing new insights in the understanding of the pathogenesis of different influenza virus types. http://dx.doi.org/10.1016/j.cyto.2013.06.194

1

Present address: MedImmune, Milstein Building, Cambridge, UK.

192 The functional analysis of RIG-I-inducible microRNA Ryota Ouda a, Koji Onomoto b, Kiyohiro Takahasi a, Hiroki Kato a, Mitstoshi Yoneyama b, Takashi Fujita a, a Laboratory of Molecular Genetics, Institute for Virus Research, Kyoto University, Japan, b Medical Mycology Research Center, Chiba University, Japan In mammals, viral infections are detected by innate immune receptors, including Toll-like receptor (TLR) and retinoic acid inducible gene I (RIG-I)-like receptor (RLR), which activate the type I interferon (IFN) system. IFN essentially activates genes encoding antiviral proteins that inhibit various steps of viral replication as well as facilitate the subsequent activation of acquired immune responses. In this study, we investigated the expression of non-coding RNA upon viral infection or RLR activation. Using a microarray, we identified several microRNAs (miRNAs) specifically induced by the activation of RLR signaling. According to miRNA microarray, miR423-3p was one of the RIG-I-inducible miRNAs. We found that overexpression of miR-423-3p augments expression level of IFN-b upon viral infection. Conversely, introduction of anti-miR-423-3p suppressed expression level of IFN-b upon viral infection. To explore the possible target genes of miR-423-3p, we used three different databases (Target Scan, Micro Cosm, microRNA.org). Among these database, only one gene, Poly(A)-binding protein 1 (PABP1), was overlapped. PABP1 is known as one of the components of stress granules (SGs). Previously we have reported that SGs are important loci to mediate RIG-I signaling. Knockdown and overexpression experiment of PABP1 revealed enhanced and suppressed production of IFN-b respectively. These results suggest that PABP1 acts as a negative regulator of RIG-I mediate signaling and the function of PABP1 is finely regulated by miR-423-3p. http://dx.doi.org/10.1016/j.cyto.2013.06.195

193 Type I interferon treatment inhibits the formation of RNA virus replicationassociated membrane structures Diede Oudshoorn a, Corrine Beugeling a, Ronald W.A.L. Limpens b, Jochem Pronk a, Jessica Ruinard a, Eric J. Snijder a, Montserrat Bárcena b, Marjolein Kikkert a, a Department of Medical Microbiology, Section Molecular Virology, Leiden University Medical Center, Leiden, The Netherlands, b Department of Molecular Cell Biology, Section Electron Microscopy, Leiden University Medical Center, Leiden, The Netherlands The antiviral innate immune response is built around the prompt recognition of pathogen-associated molecular patterns (PAMPs) and the subsequent inhibition of the replication of the associated virus until the adaptive immune response is activated for ultimate clearance of the virus. All plus-stranded RNA viruses hijack intracellular host membranes to form elaborate virus-induced membrane modifications, such as the network of interconnected ER-derived double membrane vesicles formed by corona- and arteriviruses. These membrane structures not only accommodate the viral replication machinery, but also devastate the interior of the cell during infection. We hypothesized that the innate immune system may have ways to directly counteract the formation of these virus-induced structures and/or recognize the virally modified membranes as PAMPs. Ectopic expression of several arterivirus non-structural proteins (nsps) results in the formation of membrane structures similar to those observed during infection, in the absence of replicating virus. Using large scale 2D electron microscopy mosaic maps of cells expressing arterivirus nsp2–3, we found that type I interferon pre-treatment significantly reduced the formation of virus-associated membrane modifications, while the expression level and subcellular localization of the nsps was not affected. This suggests a direct inhibitory effect of type I interferon and/or interferon-stimulated genes on the formation of virus-induced membrane modifications. The underlying mechanism and the innate immune factors involved remain to be elucidated and future work will also aim to determine whether virus-induced membrane modifications function as PAMPs. http://dx.doi.org/10.1016/j.cyto.2013.06.196

194 IRF8 regulates autophagy and activates microglia to exacerbate neuroinflammation Keiko Ozato a, Ryusuke Yoshimi a, Yuko Yoshida a, Monica Gupta a, Hiroaki Yoshii a, Jeeva Munasinghe b, Olga Maximova c, Huabao Xiong d, Hongsheng Wang e, Herbert C. Morse e, a Program in Genomics of Differentiation, NICHD, National Institutes of Health, Bethesda, MD, USA, b Laboratory of Molecular Imaging, NINDS, National Institutes of Health, Bethesda, MD, USA, c Laboratory of Infectious Diseases, National Institutes of

Abstract / Cytokine 63 (2013) 243–314 Health, Bethesda, MD, USA, d Immunology Institute, Mt. Sinai School of Medicine, New York, NY, USA, e Laboratory of Immunogenetics, NIAID, National Institutes of Health, Bethesda, MD, USA IRF8 is a transcription factor of the IRF family expressed in macrophages and dendritic cells (DCs). It enhances type I interferon induction in pDCs and IL12p40 in CD8+DCs and macrophages. We have observed that IRF8 regulates transcription of a series of genes in the autophagy pathway in macrophages and DCs in response to IFNg and toll-like receptor signaling, and plays a critical role in the formation of autophagosomes and their fusion with lysosomes. Recently, IRF8 was identified as a risk factor for several autoimmune diseases including multiple sclerosis (MS) in SNP based epidemiological studies. MS is a neuroinflammatory disease that involves activation and expansion of Th17, Th1 T cells as well as microglia, the antigen presenting cells of the central nervous system. Since the SNPs for MS lie in the 30 noncoding regions of IRF8, regulated expression of IRF8 likely accounts for its role in MS neuroinflammation. Because the mechanism by which IRF8 contributes to MS pathogenesis has not been clarified, we have undertaken in depth analysis of IRF8 activity in the mouse model of MS. Our results indicate that IRF8 plays a critical role at multiple steps of the disease, from the onset to progression in the periphery and the CNS, including activation and expansion of microglia during neuroinflammation. These observations are discussed in the context of its role in regulating autophagy. http://dx.doi.org/10.1016/j.cyto.2013.06.197

195 Type I interferon signaling distinguishes commensal from virulent Staphylococcus aureus Dane Parker, Grace Soong, Paul Planet, Alice Prince, Department of Pediatrics, Columbia University, New York, NY, USA Staphylococcus aureus is a leading cause of pneumonia with much of the disease caused by the host response. We hypothesized the host would differentially respond to a strain of S. aureus that is a well characterized commensal (502A) compared to USA300, a major cause of respiratory, skin and soft tissue infections that is currently epidemic in the United States. As predicted, 502A was less invasive than USA300 in invasion of several skin and airway epithelial cell lines. We observed a significant increase in the type I interferon (IFN) response to 502A compared to USA300 in epithelial (107-fold, P < 0.05), macrophage (66-fold, P < 0.05) and dendritic cells (8.7fold, P < 0.05). In dendritic and macrophage cells only type I IFN signaling was increased. The increased 502A response required live organism (89% decrease, P < 0.05), cellular uptake (inhibited by cytochalasin D and dynasore) and lysosomal fusion (inhibited by bafilomycin). Signaling did not require TRIF, STING, TLRs 2, 7 or 9 but involved MyD88 (93% decrease, P < 0.05) along with NOD2 (83% decrease, P < 0.05) and IRF5 (79% decrease, P < 0.05). Potential ligands for this response, cell lysate, DNA, RNA and peptidoglycan were equivalent however, 502A exhibited increased autolysis. The biological consequences of enhanced signaling by 502A, when the mucosal barrier is bypassed, led to increased mortality in both sepsis (40% vs 0%, P < 0.05) and pneumonia (100% vs 0%, 502A vs USA300, P < 0.05) models. In our model of acute pneumonia, mice infected with 502A displayed increased bacterial load, lung pathology, cytokines (KC, IL-6) and immune cell influx compared to USA300, which was ameliorated in Ifnar/ mice. These results indicate that multiple mechanisms exist to sense bacteria within a given species and highlight a difference in host signaling between virulent and commensal strains. http://dx.doi.org/10.1016/j.cyto.2013.06.198

196 Interleukin-7-mediated NFAT activation sets the threshold for T cell development Amiya K. Patra a, Andris Avots a, René P. Zahedi b, Thomas Schüler c, Albert Sickmann b,d, Ursula Bommhardt c, Edgar Serfling a, a Department of Molecular Pathology, Institute of Pathology, University of Würzburg, Josef Schneider-Str. 2, D97080 Würzburg, Germany, b Department of Bioanalytics, Leibniz-Institute for Analytical Sciences – ISAS, Otto-Hahn-Str. 6b, D-44227 Dortmund, Germany, c Institute of Molecular and Clinical Immunology, Otto-von-Guericke University Magdeburg, Leipziger-Str. 44, D39120 Magdeburg, Germany, d Medical Proteom-Center (MPC), Ruhr-University Bochum, D-44801 Bochum, Germany Interleukin-7 (IL-7), a member of the common cytokine gamma-chain family, plays critical role in T cell biology. Studies using gene knock out mice for IL-7 or

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the components of IL-7 signaling have unequivocally established that IL-7-Jak3STAT5 signaling is an absolute necessity for development of a normal T cell repertoire. However, subtle differences in the phenotype of STAT5 deficient mice to that of IL-7 or IL-7R deficient mice implicate that transcription factors other than STAT5 might be activated upon IL-7 signaling. Transcription factors of NFAT family play prominant role in T cell development and function. However, the role of NFAT family proteins in the early cytokine-dependent stages of thymocyte development is unclear. Additionally, whether the activity of individual NFAT family members during thymocyte development are complementary, antagonistic or redundant is unknown. Differentiation of DN thymocytes from the early thymic progenitor (ETP) cells is critically dependent on IL-7 signaling. We have elucidated a novel aspect of IL-7 signaling, whereby IL-7 signals activate NFATc1 in the DN1-DN3 thymocytes by Jak3-mediated tyrosine phosphorylation, which occurs parallel to that of STAT5 activation. Conventionally, in T cells NFAT proteins are activated via calcineurinmediated multiple serine/threonine dephosphorylation in its regulatory domain upon TCR signaling. However the alternative NFAT activation pathway we have elucidated is distinct from that of the calcineurin-mediated NFAT activation pathway. First, instead of ser/thr dephosphorylation, IL-7 activates NFAT via tyrosine (tyr) phosphorylation. Second, JAK3 activated downstream to IL-7 signals is the kinase responsible for NFAT activation. Third, IL-7 induced NFAT activation is insensitive to cyclosporin A treatment and finally, this alternative NFAT activation pathway is vital for the survival of the early DN thymocytes which have not yet acquired the cell surface pre-TCR. We show that NFATc1 is the pre-dominant NFAT protein over NFATc2 or NFATc3 in regulating early thymocyte development and synergistically with STAT5, it controls the gene expression programs associated with early T cell survival. Accordingly, ablation of NFATc1, and not NFATc2 or NFATc3, severely impaired the T cell development resulting in T-cell lymphopenia similar to that observed in mice deficient for STAT5 or IL-7 signaling. Thus by elucidating a previously unknown aspect of IL-7 signaling, we demonstrate that in addition to STAT5, NFATc1 activation by IL-7/JAK3 sets the threshold between development of lymphopenia or that of a normal functional T cell repertoire during the cytokine-dependent phases of thymocyte development.

http://dx.doi.org/10.1016/j.cyto.2013.06.199

197 The tyrosine-based motif of UNCc93B1 differentially regulates the activity of human endosomal toll-like receptors Karin Pelka a, Larisa Labzin a, Rainer Stahl a, Jana Zimmermann b, Stefan Hoenig b, Eicke Latz a,c,d, a Institute of Innate Immunity, University Hospitals Bonn, Bonn, Germany, b Biochemistry-Center, University Hospitals Cologne, Cologne, Germany, c UMass Medical School, Department of Infectious Diseases and Immunology, Worcester, MA, USA, d German Center for Neurodegenerative Diseases, Bonn, Germany Endosomal toll-like receptors, namely human TLR3, TLR7, TLR8, and TLR9 sense nucleic acids (NAs) derived from viruses and bacteria. Yet, these TLRs can also contribute to autoimmunity by recognizing host NAs. NA-sensing TLR activity is highly regulated by the correct subcellular localization. Experimental mislocalization of endosomal TLRs to the cell surface leads to insufficient microbial recognition and inflammatory disease due to the recognition of endogenous NAs. Under physiological conditions, efficient activation of murine TLR7 and TLR9 is restricted to acidic compartments due to the need for proteolytic processing. TLR trafficking within the endolysosomal system further determines the nature of signaling events, as type I IFN and proinflammatory responses are initiated from different compartments. UNC93B1 is a key-regulator of NA-sensing TLRs by controlling the translocation of TLRs from the endoplasmic reticulum (ER) into endolysosomes. Without functional UNC93B1, NA-sensing TLRs are retained in the ER, incapable of ligand recognition. Patients lacking UNC93B1 display defective signaling of endosomal TLRs, and are at increased risk of developing herpes simplex virus encephalitis. The precise mechanism of how UNC93B1 regulates TLR trafficking remains unknown. Recently, a highly conserved tyrosine-based motif of UNC93B1 was identified. This motif is essential for the correct localization and function of murine TLR9, but dispensable for murine TLR7, TLR11, TLR12, and TLR13. TLR11, TLR12, and TLR13 are not expressed in humans. In contrast, human TLR8 plays a crucial role in the initiation of immune responses to certain pathogens. To date, molecular insights into UNC93B1-mediated trafficking of human endosomal TLRs are missing. Our study reveals that the tyrosine-based motif of UNC93B1 differentially regulates human TLR7, TLR8, and TLR9 responses and thereby contributes to the understanding of human TLR trafficking. http://dx.doi.org/10.1016/j.cyto.2013.06.200