- Email: [email protected]
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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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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.