- Email: [email protected]
GP130 SIGNALING PATHWAY IN MYELOID CELLS MODULATES THE INFLAMMATORY RESPONSE DURING SEPSIS
POSTERS synthetase-1) are best served low-affinity enzymes (Gls-2, Cps-1) to salvage glutamine if amino acids are scarce. Although ammonia detoxification is a crucial function of GS, GS in any organ outside the brain is dispensable due to extensive redundancy of its expression among organs. 586 CARDIAC THICK-FILAMENT STRUCTURAL AND FUNCTIONAL CHANGES CONTRIBUTE TO CIRRHOTIC CARDIOMYOPATHY IN RATS H. Liu, H. Honar, T. Glenn, H.E.D.J. ter Keurs, S.S. Lee. University of Calgary, Calgary, AB, Canada E-mail: [email protected] Background and Aims: Cirrhotic cardiomyopathy is one of the complications in cirrhosis, but the pathogenic mechanisms, in particular the cellular contractile machinery at the myofilament level, remain to be fully clarified. Cardiomyocyte contraction is the result of thick and thin myofilament sliding and thus contraction/relaxation. Myosin heavy chain is the main structure in the thick filament. The present study aimed to elucidate the role of myosin heavy chain (MHC), T-tubular integrity and Ca2+ transients in cardiac contractile abnormalities in cirrhosis. Methods: Cirrhosis was induced in male Lewis Brown-Norway rats by bile duct-ligation (BDL). Contractile force of trabeculae was measured using a He-Ne laser beam; cell shortening was quantified using a video sarcomere detector (Ionoptix, Milton, MA); and Ca2+ transients were studied at varied frequency and extracellular calcium concentrations [Ca2+ ]. T-tubular integrity was assessed by power spectrum analysis of images of myocytes stained with di-8ANEPPS and myosin heavy chain (MHC) isoform distribution by gel electrophoresis. Results: BDL rats showed a mild loss of transverse tubular integrity; a reduced maximum and a reduced rate of rise of the Ca2+ transient (Max F/Fo) without a change in the rate of relaxation of the Ca2+ transient; a reduction of both the rate of rise and fall of contraction; a decreased maximal force-generating capacity; a loss of the inotropic effect of increased stimulus frequency; a increased spontaneous diastolic sarcomere length fluctuation. Myosin heavy chain isoforms were shifted from V1 to V3 in BDL heart. In other words, the predominant alpha-MHC of normal rat hearts that produces stronger contractility was shifted to a dominance of betaMHC that contracts less vigorously but requires less ATP energy per contraction than the former. Conclusion: Cardiomyocyte contractility in cirrhotic rats showed features typical of heart failure including systolic and diastolic prolongation, impaired force-frequency relation, and decreased force-generating capacity. At least part of the mechanism for the above is likely due to a myosin heavy chain isoform shift and abnormal calcium kinetics in the cirrhotic heart. 587 MACROPHAGE INFILTRATION AND CARDIAC DYSFUNCTION IN CIRRHOTIC CARDIOMYOPATHY IN RATS H. Liu, C. D’Mello, S.S. Lee. University of Calgary, Calgary, AB, Canada E-mail: [email protected] Background and Aims: Cirrhosis is associated with blunted cardiovascular response to stimuli such as hemorrhage, but the mechanism remains unclear. In this study, we aimed to clarify the role of macrophagein blunted cardiovascular response to hemorrhage in cirrhotic rats. Methods: Cirrhosis was induced by bile duct-ligation (BDL). Ventricular and systemic hemodynamics were measured. The resident monocytes and macrophages were quantified by immunohistochemical staining. Animals were also treated with vehicle or Gadolinium Chloride (GdCl3) to deplete monocytes and macrophages and finally, isolated monocytes were directly applied S240
to cardiomyocytes to test the direct effects of monocytes on cardiac contractility. Results: Sham hearts showed virtually no monocytes or macrophages before or after hemorrhage, whereas BDL hearts had significantly more baseline white cells which further increased after hemorrhage. GdCl3 treatment significantly reduced cardiac endocannabinoid levels both at baseline and after hemorrhage. This treatment also restored cardiovascular response to hemorrhage in BDL rats but did not affect sham-controls. Monocytes isolated from BDL rats inhibited cardiomyocyte contractility significantly more than those from sham rats. Conclusions: The cirrhotic heart showed increased monocyte recruitment. Monocytes isolated from BDL rats inhibited cardiac contractility. GdCl3 treatment blocked monocyte infiltration and restored blunted cardiovascular response to hemorrhage. Inflammatory signals mediated by mononuclear cells may contribute to the pathogenesis of cirrhotic cardiomyopathy. 588 THE IL-6/GP130 SIGNALING PATHWAY IN MYELOID CELLS MODULATES THE INFLAMMATORY RESPONSE DURING SEPSIS A. Mohs1 , S. Dutton Sackett1 , L.E. Sander2 , F.J. Cubero1 , S. Strauch1 , D. Kroy1 , K. Streetz1 , C. Trautwein1 . 1 Department of Medicine III, University Hospital Aachen, Aachen, 2 Department of Infectious Disease and Pulmonary Medicine, Charit´e University Hospital Berlin, Berlin, Germany E-mail: [email protected] Background and Aim: Ascites and its bacterial infection (SBP) is a frequent and life-threatening complication in patients suffering from liver cirrhosis. After bacterial peritonitis, the mobilization of the regulatory myeloid cells is a major requirement to prevent sepsis. Current research on inflammatory pathways has shown that GP130 in hepatocytes is essential in the modulation of inflammation. Therefore, we investigated the contribution of the GP130 pathway for the skewing of macrophage towards the regulatory M2 phenotype in a model of peritonitis. Methods: Depletion of GP130 in hematopoietic cell-deficient mice was achieved by bone marrow transplantation (BMC) using MxCre GP130loxp/loxp GFP (cre− BMC-WT; cre+ BMC-KO) as donors into WT as recipients. Subsequently, the recipients underwent coecal ligation and puncture (CLP) – a model of bacterial peritonitis. Additionally, bone marrow derived macrophages (BMDM) generated from BMC-WT and BMC-KO mice were stimulated with LPS, LPS/IFNg, IL-4, IL-10/IL-4 or medium. Results: Clinical signs of sepsis such as lethargy, piloerection, tremors and respiratory distress associated with a significant decrease in survival and increased organ damage – liver, kidney and spleen – were more severely manifested in BMC-KO mice after CLP. Moreover, BMC-KO mice exhibited a pronounced pro-inflammatory response assessed by the expression of iNOS and IL-12. Concomitantly, TNFa, IL-12 and IL-6 levels in the peritoneal lavage fluid were significantly elevated in the BMC-KO mice. Next, we sought to determine the general role of GP130 in macrophage polarization. Thus, we generated BMDM from BMC-WT and BMC-KO mice and stimulated them with LPS, LPS/IFNg, IL-4 or medium. Our data clearly showed (a) reduced expression of Arginase I and Macrophage mannose receptor (MMR) after IL-4 stimulation and; (b) defective IL-6 response after stimulation with LPS/IFNg. The antiinflammatory phenotype, associated with a reduction of IL-4Ra, was rescued by the exogenous addition of IL-10 in vivo and in vitro. Conclusion: These data identify IL-6/GP130 signaling pathway as a critical component in myeloid lineage of immune cells for host survival and control of inflammation and infection during bacterial peritonitis. The IL-6/GP130 signaling cascade is required for the maintenance of the cytokine responses by regulating the activation of M1 macrophages during bacterial peritonitis and sepsis.
Journal of Hepatology 2013 vol. 58 | S229–S407
to cardiomyocytes to test the direct effects of monocytes on cardiac contractility. Results: Sham hearts showed virtually no monocytes or macrophages before or after hemorrhage, whereas BDL hearts had significantly more baseline white cells which further increased after hemorrhage. GdCl3 treatment significantly reduced cardiac endocannabinoid levels both at baseline and after hemorrhage. This treatment also restored cardiovascular response to hemorrhage in BDL rats but did not affect sham-controls. Monocytes isolated from BDL rats inhibited cardiomyocyte contractility significantly more than those from sham rats. Conclusions: The cirrhotic heart showed increased monocyte recruitment. Monocytes isolated from BDL rats inhibited cardiac contractility. GdCl3 treatment blocked monocyte infiltration and restored blunted cardiovascular response to hemorrhage. Inflammatory signals mediated by mononuclear cells may contribute to the pathogenesis of cirrhotic cardiomyopathy. 588 THE IL-6/GP130 SIGNALING PATHWAY IN MYELOID CELLS MODULATES THE INFLAMMATORY RESPONSE DURING SEPSIS A. Mohs1 , S. Dutton Sackett1 , L.E. Sander2 , F.J. Cubero1 , S. Strauch1 , D. Kroy1 , K. Streetz1 , C. Trautwein1 . 1 Department of Medicine III, University Hospital Aachen, Aachen, 2 Department of Infectious Disease and Pulmonary Medicine, Charit´e University Hospital Berlin, Berlin, Germany E-mail: [email protected] Background and Aim: Ascites and its bacterial infection (SBP) is a frequent and life-threatening complication in patients suffering from liver cirrhosis. After bacterial peritonitis, the mobilization of the regulatory myeloid cells is a major requirement to prevent sepsis. Current research on inflammatory pathways has shown that GP130 in hepatocytes is essential in the modulation of inflammation. Therefore, we investigated the contribution of the GP130 pathway for the skewing of macrophage towards the regulatory M2 phenotype in a model of peritonitis. Methods: Depletion of GP130 in hematopoietic cell-deficient mice was achieved by bone marrow transplantation (BMC) using MxCre GP130loxp/loxp GFP (cre− BMC-WT; cre+ BMC-KO) as donors into WT as recipients. Subsequently, the recipients underwent coecal ligation and puncture (CLP) – a model of bacterial peritonitis. Additionally, bone marrow derived macrophages (BMDM) generated from BMC-WT and BMC-KO mice were stimulated with LPS, LPS/IFNg, IL-4, IL-10/IL-4 or medium. Results: Clinical signs of sepsis such as lethargy, piloerection, tremors and respiratory distress associated with a significant decrease in survival and increased organ damage – liver, kidney and spleen – were more severely manifested in BMC-KO mice after CLP. Moreover, BMC-KO mice exhibited a pronounced pro-inflammatory response assessed by the expression of iNOS and IL-12. Concomitantly, TNFa, IL-12 and IL-6 levels in the peritoneal lavage fluid were significantly elevated in the BMC-KO mice. Next, we sought to determine the general role of GP130 in macrophage polarization. Thus, we generated BMDM from BMC-WT and BMC-KO mice and stimulated them with LPS, LPS/IFNg, IL-4 or medium. Our data clearly showed (a) reduced expression of Arginase I and Macrophage mannose receptor (MMR) after IL-4 stimulation and; (b) defective IL-6 response after stimulation with LPS/IFNg. The antiinflammatory phenotype, associated with a reduction of IL-4Ra, was rescued by the exogenous addition of IL-10 in vivo and in vitro. Conclusion: These data identify IL-6/GP130 signaling pathway as a critical component in myeloid lineage of immune cells for host survival and control of inflammation and infection during bacterial peritonitis. The IL-6/GP130 signaling cascade is required for the maintenance of the cytokine responses by regulating the activation of M1 macrophages during bacterial peritonitis and sepsis.
Journal of Hepatology 2013 vol. 58 | S229–S407