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P091 Interferon-beta regulates Th17 and dendritic cell responses in EAE
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Abstract / Cytokine 59 (2012) 546–551
lem in this area is to understand how the formation of regulatory macrophages can be promoted at sites of inflammation to prevent and/or alleviate chronic inflammatory and autoimmune diseases. Methods. Bone-marrow derived macrophages were treated with protein kinase inhibitors prior to stimulation with Toll-like receptor (TLR) agonists. mRNA synthesis was measured by qPCR and cytokine secretion was measured using the BIOPLEX system. To identify the relevant substrate, the effects of the protein kinase inhibitors on the phosphoproteome were measured by LC-MS/MS. Subsequently, the repertoire of phosphorylation sites were mapped and their effects on protein function were analyzed using luciferase assays. Finally, the targets of the protein kinase inhibitors were validated in mouse knock-outs. Results. We have discovered a novel mechanism that restricts the formation of regulatory macrophages. This has led us to identify the first small-molecule inhibitors of protein kinases capable of inducing all the hallmarks of regulatory macrophages, including the production of high levels of IL-10 and IL-1ra. The molecular mechanism underlying the effects of these compounds will be presented. Conclusion. The remarkable effects of these protein kinase inhibitors on macrophage function suggest a novel approach for the development of anti-inflammatory drugs. Disclosure of interest: None declared.
of IL-6, yet decreased levels of IL-27, compared with DCs generated from IFN-b+/+mice. These data suggest that DCs from IFN-b / mice have a propensity to secrete cytokines perhaps associated with Th17 rather than Treg polarization. Moreover, the IFN-b / DCs produce lower levels of IL-1 and IL-10. The activation profiles of proteins, such as phospho-proteins that drive proliferation and activation signaling cascades, differ in disease states, both from a ‘‘normal” profile and among different pathologies. Using our mouse EAE model of MS and human MS patient peripheral blood, we are interrogating the phosphorylation status of signaling effectors, including STATs and MAPKs, by FACS-based multiparametric staining of cell surface and intracellular markers to determine the activation status of circulating discrete immune cell populations. Our staining panels allow us to discern the phosphorylation status of these signaling effectors in T cell populations and DCs. Data will be presented that describe the signature phospho-profiles during active disease/relapse and remission. Conclusion. Taken together, the data indicate that IFN-b plays a role in regulating the Th17 and DC responses during EAE, by limiting cell accumulation and trafficking and by regulating cytokine production. Disclosure of interest: None declared. http://dx.doi.org/10.1016/j.cyto.2012.06.179
http://dx.doi.org/10.1016/j.cyto.2012.06.177
P090 Role of TREM2 in the pathogenesis of DSS-induced IBD K.-H. Lee 1, H.-R. Choi 2, H.-S. Kang 1, 1 School of Biological Sciences and Technology, Chonnam National University, Republic of Korea, 2 Department of Food and Nutrition, Nambu University, Gwangju, Republic of Korea Introduction. Triggering receptor expressed on myeloid cells-2 (TREM-2) is a cell surface receptor that participates in diverse cellular functions including inflammation, bone homeostasis and neurological development. We examined the effects of TREM-2 on experimentally induced inflammatory bowel disease (IBD) in mice. Methods. Wild type (WT) and TREM2 transgenic (TREM2 TG) mice were administrated with dextran sulfate sodium (DSS) to induce IBD. The gene expression of proinflammatory cytokines were examined using RT-PCR and histopathological analysis was performed by H&E staining. The population of regulatory T cells was analyzed by flow cytometry. Results. We found that body weight and colon length were remarkably decreased by treatment of DSS in TREM2-TG mice compared with WT littermates. Gene expression of proinflammatory cytokines including TNF-a, IL-1b, IL-17F, and IL-6 was increased in colonic tissue of DSS-treated TREM2-TG mice. In addition, recruitment of immune cells into colon tissue was also increased by DSS treatment in TREM2TG mice, but population of regulatory T cells was decreased. Conclusion. Our data suggest that TREM2 may be involved in the pathogenesis of IBD by upregulating proinflammatory cytokines due to the reduction of regulatory T cells. Disclosure of interest: None declared. http://dx.doi.org/10.1016/j.cyto.2012.06.178
P091 Interferon-beta regulates Th17 and dendritic cell responses in EAE L.M. Pennell 1,2, C.L. Galligan 1,2, E.N. Fish 1,2, 1 Immunology, University of Toronto, Canada, 2 Cell and Molecular Biology, Toronto General Research Institute, Toronto, Canada Introduction: . Multiple sclerosis (MS) is an autoimmune disease that targets the central nervous system. Interferon (IFN)-b was the first approved therapy for relapsing-remitting MS more than 10 years ago, however, its mechanism of action remains ill-defined. Methods. Using a murine experimental autoimmune encephalomyelitis (EAE) model for MS, we show that mice null for IFN-b, IFN-b / , exhibit an earlier onset and a more rapid progression of disease compared to IFN-b+/+mice. Results. IFN-b / mice exhibit increased numbers of Th17 cells in their draining lymph nodes (DLN) as early as 4 days after disease induction. Th17 cells are implicated as critical effectors in different autoimmune diseases, including MS. Increased Th17 cell polarization may be driven by dendritic cells (DC), as DCs derived from IFN-b / mice induce greater proliferation of T cells derived from either IFN-b+/+ or IFN-b / mice, compared with DCs derived from IFN-b+/+mice. In addition, adoptive transfer of myelin oligodendrocyte glycoprotein (MOG) peptide-primed IFN-b / DC into IFN-b+/+ and IFN-b / mice with EAE resulted in their rapid migration into the CNS of recipient mice, visualized by high resolution fluorescence imaging. Cytokine expression analysis revealed that DCs generated from IFN-b / mice secrete increased levels
P092 A combined role for TNFR1 and IFNAR1 in psoriasis? L. Grine 1,2, R. Vandenbroucke 1,2, L. Dejager 1,2, C. Libert 1,2, 1 Department for Molecular Biomedical Research, Flanders Institute for Biotechnology, Zwijnaarde, Belgium, 2 Department of Biomedical Molecular Biology, University Ghent, Ghent, Belgium Introduction. Psoriasis is a chronic inflammatory skin disease affecting 2–3 percent of the world population. The disease is mainly characterized by epidermal hyperplasia, scaling and erythema. But it is also associated with a high degree of morbidity and patients suffer from the social impact of the disease. In addition, the existing longterm medications come with side effects. The disease forms therefore a major burden for health care systems and society. Until now, the underlying mechanism is extensively studied since the etiology of psoriasis is poorly understood and remains to be investigated. A prominent role for TNF in the disease development of psoriasis has been shown. So far, various types of TNF antagonists have been developed, e.g. infliximab and etanercept. Additionally, more recently, type I interferons have also gained interest as potential drug targets in psoriasis. In our research, we are interested in targeting the receptors of TNF and type I IFN, namely TNFR1 and IFNAR1. We believe that targeting the receptors of these cytokines is at least equally efficient as targeting the ligands themselves. We also believe that by this approach, the specificity of the therapy increases and subsequently less side effects are expected to occur. Methods. For this purpose, we have generated mice lacking either TNFR-receptor-1 (TNFR1-/-) or type I IFN-receptor-1 (IFNAR1-/-) on a C57BL/6 J background. Mice received a daily topical dose of 62.5 mg of imiquimod (IMQ) cream (Aldara) on the shaved back for 5 consecutive days. Wild type littermates received a similar treatment. Erythema and scaling were scored on a daily basis and on the last day, mice were euthanized and skin was isolated for histological analysis. Results. We found that TNFR1-/- mice suffered less from imiquimod-induced erythema and scaling. Histological analysis of skin epidermis showed less hyperplasia, characterized by a reduced epidermal thickness, compared to their wild type littermates. Similar results were obtained for the IFNAR1-/- mice. Conclusion. Mice lacking either the TNFR1 or IFNAR1 are protected against imiquimod-induced psoriasis. Both knockout mice show reduced erythema, scaling and epidermal thickness. Our data suggest that TNFR1 and IFNAR1 are potential drug targets in psoriasis and raise the question whether blocking both receptors might result in an even stronger protection. To investigate this, we have generated double knockout mice lacking both TNFR1 and IFNAR1. Our next goal is to test these mice in the imiquimod-induced psoriasis model and investigate whether we observe a synergistic protection against the insult. If we can prove that these mice have an increased resistance, then we can argue that the next step in treating psoriasis is combined inhibition therapy. Disclosure of interest: None declared. http://dx.doi.org/10.1016/j.cyto.2012.06.180
P093 Lymphotoxin-alpha produced by autoreactive CD4+ thymocytes controls formation and homeostasis of the thymic medulla M. Irla 1, L. Guerri 2, J. Guenot 1, A. Sergé 1, O. Lantz 2, B. Imhof 1, E. Palmer 3, W. Reith 1, 1 Department of Pathology and Immunology, University of Geneva Medical School, Geneva, Switzerland, 2 Institut Curie, Paris, France, 3 University Hospital-Basel, Basel, Switzerland
Abstract / Cytokine 59 (2012) 546–551
lem in this area is to understand how the formation of regulatory macrophages can be promoted at sites of inflammation to prevent and/or alleviate chronic inflammatory and autoimmune diseases. Methods. Bone-marrow derived macrophages were treated with protein kinase inhibitors prior to stimulation with Toll-like receptor (TLR) agonists. mRNA synthesis was measured by qPCR and cytokine secretion was measured using the BIOPLEX system. To identify the relevant substrate, the effects of the protein kinase inhibitors on the phosphoproteome were measured by LC-MS/MS. Subsequently, the repertoire of phosphorylation sites were mapped and their effects on protein function were analyzed using luciferase assays. Finally, the targets of the protein kinase inhibitors were validated in mouse knock-outs. Results. We have discovered a novel mechanism that restricts the formation of regulatory macrophages. This has led us to identify the first small-molecule inhibitors of protein kinases capable of inducing all the hallmarks of regulatory macrophages, including the production of high levels of IL-10 and IL-1ra. The molecular mechanism underlying the effects of these compounds will be presented. Conclusion. The remarkable effects of these protein kinase inhibitors on macrophage function suggest a novel approach for the development of anti-inflammatory drugs. Disclosure of interest: None declared.
of IL-6, yet decreased levels of IL-27, compared with DCs generated from IFN-b+/+mice. These data suggest that DCs from IFN-b / mice have a propensity to secrete cytokines perhaps associated with Th17 rather than Treg polarization. Moreover, the IFN-b / DCs produce lower levels of IL-1 and IL-10. The activation profiles of proteins, such as phospho-proteins that drive proliferation and activation signaling cascades, differ in disease states, both from a ‘‘normal” profile and among different pathologies. Using our mouse EAE model of MS and human MS patient peripheral blood, we are interrogating the phosphorylation status of signaling effectors, including STATs and MAPKs, by FACS-based multiparametric staining of cell surface and intracellular markers to determine the activation status of circulating discrete immune cell populations. Our staining panels allow us to discern the phosphorylation status of these signaling effectors in T cell populations and DCs. Data will be presented that describe the signature phospho-profiles during active disease/relapse and remission. Conclusion. Taken together, the data indicate that IFN-b plays a role in regulating the Th17 and DC responses during EAE, by limiting cell accumulation and trafficking and by regulating cytokine production. Disclosure of interest: None declared. http://dx.doi.org/10.1016/j.cyto.2012.06.179
http://dx.doi.org/10.1016/j.cyto.2012.06.177
P090 Role of TREM2 in the pathogenesis of DSS-induced IBD K.-H. Lee 1, H.-R. Choi 2, H.-S. Kang 1, 1 School of Biological Sciences and Technology, Chonnam National University, Republic of Korea, 2 Department of Food and Nutrition, Nambu University, Gwangju, Republic of Korea Introduction. Triggering receptor expressed on myeloid cells-2 (TREM-2) is a cell surface receptor that participates in diverse cellular functions including inflammation, bone homeostasis and neurological development. We examined the effects of TREM-2 on experimentally induced inflammatory bowel disease (IBD) in mice. Methods. Wild type (WT) and TREM2 transgenic (TREM2 TG) mice were administrated with dextran sulfate sodium (DSS) to induce IBD. The gene expression of proinflammatory cytokines were examined using RT-PCR and histopathological analysis was performed by H&E staining. The population of regulatory T cells was analyzed by flow cytometry. Results. We found that body weight and colon length were remarkably decreased by treatment of DSS in TREM2-TG mice compared with WT littermates. Gene expression of proinflammatory cytokines including TNF-a, IL-1b, IL-17F, and IL-6 was increased in colonic tissue of DSS-treated TREM2-TG mice. In addition, recruitment of immune cells into colon tissue was also increased by DSS treatment in TREM2TG mice, but population of regulatory T cells was decreased. Conclusion. Our data suggest that TREM2 may be involved in the pathogenesis of IBD by upregulating proinflammatory cytokines due to the reduction of regulatory T cells. Disclosure of interest: None declared. http://dx.doi.org/10.1016/j.cyto.2012.06.178
P091 Interferon-beta regulates Th17 and dendritic cell responses in EAE L.M. Pennell 1,2, C.L. Galligan 1,2, E.N. Fish 1,2, 1 Immunology, University of Toronto, Canada, 2 Cell and Molecular Biology, Toronto General Research Institute, Toronto, Canada Introduction: . Multiple sclerosis (MS) is an autoimmune disease that targets the central nervous system. Interferon (IFN)-b was the first approved therapy for relapsing-remitting MS more than 10 years ago, however, its mechanism of action remains ill-defined. Methods. Using a murine experimental autoimmune encephalomyelitis (EAE) model for MS, we show that mice null for IFN-b, IFN-b / , exhibit an earlier onset and a more rapid progression of disease compared to IFN-b+/+mice. Results. IFN-b / mice exhibit increased numbers of Th17 cells in their draining lymph nodes (DLN) as early as 4 days after disease induction. Th17 cells are implicated as critical effectors in different autoimmune diseases, including MS. Increased Th17 cell polarization may be driven by dendritic cells (DC), as DCs derived from IFN-b / mice induce greater proliferation of T cells derived from either IFN-b+/+ or IFN-b / mice, compared with DCs derived from IFN-b+/+mice. In addition, adoptive transfer of myelin oligodendrocyte glycoprotein (MOG) peptide-primed IFN-b / DC into IFN-b+/+ and IFN-b / mice with EAE resulted in their rapid migration into the CNS of recipient mice, visualized by high resolution fluorescence imaging. Cytokine expression analysis revealed that DCs generated from IFN-b / mice secrete increased levels
P092 A combined role for TNFR1 and IFNAR1 in psoriasis? L. Grine 1,2, R. Vandenbroucke 1,2, L. Dejager 1,2, C. Libert 1,2, 1 Department for Molecular Biomedical Research, Flanders Institute for Biotechnology, Zwijnaarde, Belgium, 2 Department of Biomedical Molecular Biology, University Ghent, Ghent, Belgium Introduction. Psoriasis is a chronic inflammatory skin disease affecting 2–3 percent of the world population. The disease is mainly characterized by epidermal hyperplasia, scaling and erythema. But it is also associated with a high degree of morbidity and patients suffer from the social impact of the disease. In addition, the existing longterm medications come with side effects. The disease forms therefore a major burden for health care systems and society. Until now, the underlying mechanism is extensively studied since the etiology of psoriasis is poorly understood and remains to be investigated. A prominent role for TNF in the disease development of psoriasis has been shown. So far, various types of TNF antagonists have been developed, e.g. infliximab and etanercept. Additionally, more recently, type I interferons have also gained interest as potential drug targets in psoriasis. In our research, we are interested in targeting the receptors of TNF and type I IFN, namely TNFR1 and IFNAR1. We believe that targeting the receptors of these cytokines is at least equally efficient as targeting the ligands themselves. We also believe that by this approach, the specificity of the therapy increases and subsequently less side effects are expected to occur. Methods. For this purpose, we have generated mice lacking either TNFR-receptor-1 (TNFR1-/-) or type I IFN-receptor-1 (IFNAR1-/-) on a C57BL/6 J background. Mice received a daily topical dose of 62.5 mg of imiquimod (IMQ) cream (Aldara) on the shaved back for 5 consecutive days. Wild type littermates received a similar treatment. Erythema and scaling were scored on a daily basis and on the last day, mice were euthanized and skin was isolated for histological analysis. Results. We found that TNFR1-/- mice suffered less from imiquimod-induced erythema and scaling. Histological analysis of skin epidermis showed less hyperplasia, characterized by a reduced epidermal thickness, compared to their wild type littermates. Similar results were obtained for the IFNAR1-/- mice. Conclusion. Mice lacking either the TNFR1 or IFNAR1 are protected against imiquimod-induced psoriasis. Both knockout mice show reduced erythema, scaling and epidermal thickness. Our data suggest that TNFR1 and IFNAR1 are potential drug targets in psoriasis and raise the question whether blocking both receptors might result in an even stronger protection. To investigate this, we have generated double knockout mice lacking both TNFR1 and IFNAR1. Our next goal is to test these mice in the imiquimod-induced psoriasis model and investigate whether we observe a synergistic protection against the insult. If we can prove that these mice have an increased resistance, then we can argue that the next step in treating psoriasis is combined inhibition therapy. Disclosure of interest: None declared. http://dx.doi.org/10.1016/j.cyto.2012.06.180
P093 Lymphotoxin-alpha produced by autoreactive CD4+ thymocytes controls formation and homeostasis of the thymic medulla M. Irla 1, L. Guerri 2, J. Guenot 1, A. Sergé 1, O. Lantz 2, B. Imhof 1, E. Palmer 3, W. Reith 1, 1 Department of Pathology and Immunology, University of Geneva Medical School, Geneva, Switzerland, 2 Institut Curie, Paris, France, 3 University Hospital-Basel, Basel, Switzerland