- Email: [email protected]
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Abstract / Cytokine 63 (2013) 243–314
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
Abstract / Cytokine 63 (2013) 243–314
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