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PS1-74 Interleukin 1β- modulates cardiac contractility through desensitization of the β1-adrenergic receptor
Abstracts / Cytokine 52 (2010) 17–34 events, the role of OSM-induced expression of Grp78 has to be further analyzed. However, OSM seems play an important role for hepatocytes and hepatoma cells and its potential role as target in hepatoma therapy should be further studied. doi:10.1016/j.cyto.2010.07.145
PS1-74 Interleukin 1b- modulates cardiac contractility through desensitization of the b1adrenergic receptor Benjamin W. Van Tassell, Stefano Toldo, Eleonora Mezzaroma, Neeru Goyal, Ignacio M. Seropian, Antonio Abbate, Virginia Commonwealth University, Richmond, VA Background: Heart failure (HF) is a complex clinical syndrome characterized by desensitization of the myocardial b1-adrenergic receptor (b1-AR). Interleukin-1 (IL1) is a prototypal inflammatory cytokine that induces contractile dysfunction in both cellular and animal models. We therefore sought to determine whether IL-1 induces contractile dysfunction in the heart through desensitization of the b1-AR Methods: Myocadial contractile reserve was determined by serial injection of escalating doses of a b-agonist, isoproterenol (ISOPT, 10 10 g to 10 5 g), in healthy mice with or without prior injection with a standardized dose of IL-1 (3 mcg/kg IP). LV function was measured by transthoracic echocardiography at baseline, 4 hours after IL-1 injection, and 10 minutes after each ISOPT injection. Relative b1-AR responsiveness was calculated as the ratio of ISO dose required to elicit a 25% increase (ED25) or 50% increase (ED50) in LV fractional shortening. Following ISOPT challenge, mice received injection with an adenylyl cyclase activator, forskolin (5 mg/kg IP) to determine whether inotropic signaling mechanisms were still intact downstream from the b1-AR. Results: IL1b produced a significant reduction in LV fractional shortening ( 33%, P < 0.001) at 4 hours after injection. This systolic dysfunction was associated with a blunted response to isoproterenol (+44% [ISOPT] vs +11% [ISOPT + IL-1], P < 0.05) that corresponded to a 5-fold increase in ED25 (4.8 10 9g [ISOPT] vs 24.3 10 9g [ISOPT + IL-1], P < 0.01) and 3-fold increase in ED50 (17.8 10 9g [ISOPT] vs 55.0 10 9g [ISOPT + IL-1], P < 0.05). Conversely, contractile response to forskolin challenge was not diminished by IL-1 administration (+46% [forskolin] vs +45% [forskolin + IL-1], P = NS). Conclusion: IL-1b modulates cardiac contractility through desensitization of the b1-adrenergic receptor and may represent a potential new mechanism modulating cardiac function in heart failure. doi:10.1016/j.cyto.2010.07.146
PS1-75 IL-17 stimulation results in a pro-inflammatory outcome during antiviral response in human cells Grigory Ryzhakov, Cheryl Lai, Katrina Blazek, Irina Udalova, Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College of Science, Technology and Medicine, London, United Kingdom IL-17 is a cytokine mostly produced by Th17 and cdT cells, which is best known for its protective properties during bacterial infections, but was also shown to contribute to pathogenesis of autoimmune disorders. IL-17 induces expression of proinflammatory mediators in stromal, epithelial and endothelial cells, enhancing leukocyte infiltration at sites of inflammation that can result in significant tissue damage. IL-17 is able to boost cytokine expression induced by TNF-a, and this synergy is believed to be responsible for pathogenic effects of IL-17. Recently IL-17 was shown to mediate hyper-inflammation during influenza infection in a TNF-a independent way. We hypothesized that IL-17 is able to enhance cytokine production during viral infections by directly enhancing the antiviral signalling. We used poly(I:C) to mimic viruses recognised by Toll-like receptor 3 (TLR3) expressed on the surface of host cells to explore the combined effect of the treatment with poly(I:C) and IL-17 on gene expression in human cells. Co-stimulation of primary human fibroblasts and smooth muscle cells with IL-17 greatly enhanced poly(I:C)-induced pro-inflammatory gene expression on mRNA and protein levels without affecting expression of the interferon stimulated genes. RNAi experiments revealed that this synergy depended on the IL-17 receptor signalling adaptor Act1. Interestingly, Act1 was able to induce IRF3 dependent gene expression. This induction required C-terminal phosphorylation of IRF3 as it was demonstrated by mutagenesis of the IRF3 C-terminal domain and the use of the dominant-negative IKKi that blocked Act1/IRF3 induced signalling. Overall, we have established that IL-17 may exert its physiological role during viral infections by super-inducing the pro-inflammatory branch of the antiviral signalling in virus infected cells.
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PS1-76 Deregulated STAT-independent cytokine signaling by IL-6 promotes pulmonary emphysema associated with apoptosis in mice Saleela M. Ruwanpura a, Louise McLeod a, Alistair Miller a, Jessica Jones b, Philip Bardin a, Gary Anderson b, Brendan J. Jenkins a, a Monash Institute of Medical Research, Clayton, Victoria, Australia, b The University of Melbourne, Parkville, Victoria, Australia The interleukin (IL)-6 cytokine family is linked with the pathogenesis of pulmonary emphysema, the major component of the lethal Chronic Obstructive Pulmonary Disease (COPD). However, studies into the definitive mechanisms by which these cytokines cause emphysema have been hampered by the absence of informative animal disease models that allow for the identification of specific emphysema-causing intracellular cytokine signaling pathways. To address this issue, we have employed a sophisticated animal model (gp130F/F mice) with a subtle mutation in the IL-6 cytokine family co-receptor gp130 which promotes signal transducers and activator of transcription (STAT)3 and STAT1 hyperactivation and impaired Src-homology phosphatase (SHP)2-mediated mitogen activated protein kinase (MAPK) and phosphoinositol 3’ kinase (PI3K) activation. These mice spontaneously develop pulmonary inflammation and emphysema by 6 months of age characterized by increased lung compliance. Among the IL-6 cytokine family, IL-6 expression was significantly up-regulated in the lungs of gp130F/F mice, and genetic ablation of IL-6 in gp130F/F mice prevented lung disease. Despite the reduced levels of STAT3 and STAT1 activity in the lungs of gp130F/F: IL-6 / mice, emphysema persisted in the absence or presence of pulmonary inflammation upon genetic reduction of either STAT3 or STAT1 activity, respectively, in gp130F/F mice. Detailed molecular and cellular characterization of emphysematous gp130F/F mouse lungs revealed elevated apoptosis of alveolar cells that correlated with increased activation of caspase 8, thus implicating molecular alterations to the extrinsic apoptotic pathway. Finally, the synergy between aberrant gp130 signaling pathways and cigarette smoke-induced molecular and cellular events in gp130F/F mice subjected to acute and chronic smoke exposure will be discussed. Collectively, these observations provide new mechanistic insights linking endogenous deregulated IL-6 signaling independent of STAT activity with aberrant apoptotic events in the lung which drive the pathogenesis of emphysema. doi:10.1016/j.cyto.2010.07.148
PS1-77 Interleukin-6 signal transduction and gene regulation in astroglia versus microglia: The impact of defective gp130 signaling Ricardo F. Frausto 1, Jürgen Scheller 2, Stefan Rose-John 2, Brendan J. Jenkins 3, Matthias Ernst 4, Gareth Denyer 1, Iain L. Campbell 1, 1 School of Molecular Bioscience, University of Sydney, Sydney, NSW, Australia, 2 Department of Biochemistry, Christian-AlbrechtsUniversity, Kiel, Germany, 3 Monash Institute of Medical Research, Clayton, Vic, Australia, 4 Ludwig Institute for Cancer Research, Melbourne, Vic, Australia IL-6 induces marked activation of astroglia and microglia. This response is mediated by gp130-STAT and gp130-SHP2 pathways, which are negatively regulated by SOCS3. We investigated the role of IL-6 signaling in the glial response to Hyper-IL6 or IL-6 using astroglia and microglia from Y757F and DSTAT mice that were defective in gp130-SHP2 and gp130-STAT signaling, respectively. Compared with WT astroglia, Y757F astroglia had higher and more sustained activation of gp130-STAT signaling, while the gp130-SHP2 pathway remained unchanged. This was also observed in microglia. In DSTAT astroglia there was weak activation of the gp130-STAT pathway, while gp130-SHP2 activation was higher and prolonged compared with WT astroglia. Compared with DSTAT astroglia no gp130STAT activation was observed in DSTAT microglia but there was higher and prolonged gp130-SHP2 activation. In WT astroglia and microglia SOCS3 production was strongly inversely correlated with the level of pY-STAT3 but not with pYSHP2, pERK or pS-Akt and directly correlated with SOCS3 mRNA. This is in contrast with the increased production of SOCS3 from 2hrs in Y757F astrocytes and microglia, even in the presence of decreased pY-STAT3 from peak levels. In contrast with astroglia, SOCS3 production in microglia showed a strong inverse correlation with all the phospho-proteins investigated. Analysis of downstream gene regulation in WT, Y757F and DSTAT astroglia showed a multitude of upregulated gp130-STATdependent genes at 2hrs; however, in Y757F astroglia many of these genes were further increased at 12hrs compared with WT astrocytes. Similar results were obtained for WT versus Y757F microglia while the DSTAT microglia showed only a weak upregulation of a small set of genes. Few genes showed dependence on the gp130-SHP2 pathway – the primary function of which appears to be modulation of gp130-STAT signaling. Taken together, these results identify glial-specific differences in the execution of IL-6 signaling and gene regulation in astroglia versus microglia.
doi:10.1016/j.cyto.2010.07.147 doi:10.1016/j.cyto.2010.07.149
PS1-74 Interleukin 1b- modulates cardiac contractility through desensitization of the b1adrenergic receptor Benjamin W. Van Tassell, Stefano Toldo, Eleonora Mezzaroma, Neeru Goyal, Ignacio M. Seropian, Antonio Abbate, Virginia Commonwealth University, Richmond, VA Background: Heart failure (HF) is a complex clinical syndrome characterized by desensitization of the myocardial b1-adrenergic receptor (b1-AR). Interleukin-1 (IL1) is a prototypal inflammatory cytokine that induces contractile dysfunction in both cellular and animal models. We therefore sought to determine whether IL-1 induces contractile dysfunction in the heart through desensitization of the b1-AR Methods: Myocadial contractile reserve was determined by serial injection of escalating doses of a b-agonist, isoproterenol (ISOPT, 10 10 g to 10 5 g), in healthy mice with or without prior injection with a standardized dose of IL-1 (3 mcg/kg IP). LV function was measured by transthoracic echocardiography at baseline, 4 hours after IL-1 injection, and 10 minutes after each ISOPT injection. Relative b1-AR responsiveness was calculated as the ratio of ISO dose required to elicit a 25% increase (ED25) or 50% increase (ED50) in LV fractional shortening. Following ISOPT challenge, mice received injection with an adenylyl cyclase activator, forskolin (5 mg/kg IP) to determine whether inotropic signaling mechanisms were still intact downstream from the b1-AR. Results: IL1b produced a significant reduction in LV fractional shortening ( 33%, P < 0.001) at 4 hours after injection. This systolic dysfunction was associated with a blunted response to isoproterenol (+44% [ISOPT] vs +11% [ISOPT + IL-1], P < 0.05) that corresponded to a 5-fold increase in ED25 (4.8 10 9g [ISOPT] vs 24.3 10 9g [ISOPT + IL-1], P < 0.01) and 3-fold increase in ED50 (17.8 10 9g [ISOPT] vs 55.0 10 9g [ISOPT + IL-1], P < 0.05). Conversely, contractile response to forskolin challenge was not diminished by IL-1 administration (+46% [forskolin] vs +45% [forskolin + IL-1], P = NS). Conclusion: IL-1b modulates cardiac contractility through desensitization of the b1-adrenergic receptor and may represent a potential new mechanism modulating cardiac function in heart failure. doi:10.1016/j.cyto.2010.07.146
PS1-75 IL-17 stimulation results in a pro-inflammatory outcome during antiviral response in human cells Grigory Ryzhakov, Cheryl Lai, Katrina Blazek, Irina Udalova, Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College of Science, Technology and Medicine, London, United Kingdom IL-17 is a cytokine mostly produced by Th17 and cdT cells, which is best known for its protective properties during bacterial infections, but was also shown to contribute to pathogenesis of autoimmune disorders. IL-17 induces expression of proinflammatory mediators in stromal, epithelial and endothelial cells, enhancing leukocyte infiltration at sites of inflammation that can result in significant tissue damage. IL-17 is able to boost cytokine expression induced by TNF-a, and this synergy is believed to be responsible for pathogenic effects of IL-17. Recently IL-17 was shown to mediate hyper-inflammation during influenza infection in a TNF-a independent way. We hypothesized that IL-17 is able to enhance cytokine production during viral infections by directly enhancing the antiviral signalling. We used poly(I:C) to mimic viruses recognised by Toll-like receptor 3 (TLR3) expressed on the surface of host cells to explore the combined effect of the treatment with poly(I:C) and IL-17 on gene expression in human cells. Co-stimulation of primary human fibroblasts and smooth muscle cells with IL-17 greatly enhanced poly(I:C)-induced pro-inflammatory gene expression on mRNA and protein levels without affecting expression of the interferon stimulated genes. RNAi experiments revealed that this synergy depended on the IL-17 receptor signalling adaptor Act1. Interestingly, Act1 was able to induce IRF3 dependent gene expression. This induction required C-terminal phosphorylation of IRF3 as it was demonstrated by mutagenesis of the IRF3 C-terminal domain and the use of the dominant-negative IKKi that blocked Act1/IRF3 induced signalling. Overall, we have established that IL-17 may exert its physiological role during viral infections by super-inducing the pro-inflammatory branch of the antiviral signalling in virus infected cells.
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PS1-76 Deregulated STAT-independent cytokine signaling by IL-6 promotes pulmonary emphysema associated with apoptosis in mice Saleela M. Ruwanpura a, Louise McLeod a, Alistair Miller a, Jessica Jones b, Philip Bardin a, Gary Anderson b, Brendan J. Jenkins a, a Monash Institute of Medical Research, Clayton, Victoria, Australia, b The University of Melbourne, Parkville, Victoria, Australia The interleukin (IL)-6 cytokine family is linked with the pathogenesis of pulmonary emphysema, the major component of the lethal Chronic Obstructive Pulmonary Disease (COPD). However, studies into the definitive mechanisms by which these cytokines cause emphysema have been hampered by the absence of informative animal disease models that allow for the identification of specific emphysema-causing intracellular cytokine signaling pathways. To address this issue, we have employed a sophisticated animal model (gp130F/F mice) with a subtle mutation in the IL-6 cytokine family co-receptor gp130 which promotes signal transducers and activator of transcription (STAT)3 and STAT1 hyperactivation and impaired Src-homology phosphatase (SHP)2-mediated mitogen activated protein kinase (MAPK) and phosphoinositol 3’ kinase (PI3K) activation. These mice spontaneously develop pulmonary inflammation and emphysema by 6 months of age characterized by increased lung compliance. Among the IL-6 cytokine family, IL-6 expression was significantly up-regulated in the lungs of gp130F/F mice, and genetic ablation of IL-6 in gp130F/F mice prevented lung disease. Despite the reduced levels of STAT3 and STAT1 activity in the lungs of gp130F/F: IL-6 / mice, emphysema persisted in the absence or presence of pulmonary inflammation upon genetic reduction of either STAT3 or STAT1 activity, respectively, in gp130F/F mice. Detailed molecular and cellular characterization of emphysematous gp130F/F mouse lungs revealed elevated apoptosis of alveolar cells that correlated with increased activation of caspase 8, thus implicating molecular alterations to the extrinsic apoptotic pathway. Finally, the synergy between aberrant gp130 signaling pathways and cigarette smoke-induced molecular and cellular events in gp130F/F mice subjected to acute and chronic smoke exposure will be discussed. Collectively, these observations provide new mechanistic insights linking endogenous deregulated IL-6 signaling independent of STAT activity with aberrant apoptotic events in the lung which drive the pathogenesis of emphysema. doi:10.1016/j.cyto.2010.07.148
PS1-77 Interleukin-6 signal transduction and gene regulation in astroglia versus microglia: The impact of defective gp130 signaling Ricardo F. Frausto 1, Jürgen Scheller 2, Stefan Rose-John 2, Brendan J. Jenkins 3, Matthias Ernst 4, Gareth Denyer 1, Iain L. Campbell 1, 1 School of Molecular Bioscience, University of Sydney, Sydney, NSW, Australia, 2 Department of Biochemistry, Christian-AlbrechtsUniversity, Kiel, Germany, 3 Monash Institute of Medical Research, Clayton, Vic, Australia, 4 Ludwig Institute for Cancer Research, Melbourne, Vic, Australia IL-6 induces marked activation of astroglia and microglia. This response is mediated by gp130-STAT and gp130-SHP2 pathways, which are negatively regulated by SOCS3. We investigated the role of IL-6 signaling in the glial response to Hyper-IL6 or IL-6 using astroglia and microglia from Y757F and DSTAT mice that were defective in gp130-SHP2 and gp130-STAT signaling, respectively. Compared with WT astroglia, Y757F astroglia had higher and more sustained activation of gp130-STAT signaling, while the gp130-SHP2 pathway remained unchanged. This was also observed in microglia. In DSTAT astroglia there was weak activation of the gp130-STAT pathway, while gp130-SHP2 activation was higher and prolonged compared with WT astroglia. Compared with DSTAT astroglia no gp130STAT activation was observed in DSTAT microglia but there was higher and prolonged gp130-SHP2 activation. In WT astroglia and microglia SOCS3 production was strongly inversely correlated with the level of pY-STAT3 but not with pYSHP2, pERK or pS-Akt and directly correlated with SOCS3 mRNA. This is in contrast with the increased production of SOCS3 from 2hrs in Y757F astrocytes and microglia, even in the presence of decreased pY-STAT3 from peak levels. In contrast with astroglia, SOCS3 production in microglia showed a strong inverse correlation with all the phospho-proteins investigated. Analysis of downstream gene regulation in WT, Y757F and DSTAT astroglia showed a multitude of upregulated gp130-STATdependent genes at 2hrs; however, in Y757F astroglia many of these genes were further increased at 12hrs compared with WT astrocytes. Similar results were obtained for WT versus Y757F microglia while the DSTAT microglia showed only a weak upregulation of a small set of genes. Few genes showed dependence on the gp130-SHP2 pathway – the primary function of which appears to be modulation of gp130-STAT signaling. Taken together, these results identify glial-specific differences in the execution of IL-6 signaling and gene regulation in astroglia versus microglia.
doi:10.1016/j.cyto.2010.07.147 doi:10.1016/j.cyto.2010.07.149