- Email: [email protected]
P0465 : Persistent intrahepatic Vγ9Vδ2 T-cells impairment in HCV-infected persons
POSTERS Conclusions: Studies of genetic factors in mothers and newborns are necessary to understand the processes underlying VT and the risk of chronic infection in the newborn. It can be affirmed that type C1 and C2 HLA ligands and their binding to KIRs are related to VT, while HLA-Bw4 ligands are associated with viral chronicity P0464 KNOCKDOWN OF Gpbar1 (TGR5) RENDERS MICE HIGHLY SUSCEPTIBLE TOWARDS Listeria monocytogenes INFECTION 1 M. Reich1 , P. Lang1 , D. Haussinger ¨ , V. Keitel1 . 1 Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine University, Duesseldorf, Germany E-mail: [email protected]
Background and Aims: TGR5 (Gpbar1) is a membrane-bound bile acid receptor expressed in macrophages of several organs such as lung, liver and intestine as well as in peripheral blood mononuclear cells [1–3]. Stimulation of TGR5 inhibits the nuclear translocation of p65 and NF-úB transcriptional activation [3,4], thus reducing LPS-mediated production of pro-inflammatory cytokines [1,2]. Aim of the present study was to determine the role of TGR5 in an animal model for pathogen defense. Methods: Male, 8–12 week old TGR5 knockout (KO) and WT mice were injected intravenously with 8×104 CFU/ml L. monocytogenes and observed for 7 days. On the 3rd day after infection with 2.5×104 CFU/ml bacterial titers were determined in spleen and liver. Serum levels of AST, ALT and LDH were measured using Spotchemanalyzer. Cytokine levels were determined in serum using Luminex cytometric bead assay. HE staining was carried out for all liver samples. Results: Following L. monocytogenes infection TGR5 KO displayed a 50% mortality rate within a week of inoculation, while 90% of the TGR5 WT animals survived. In addition higher L. monocytogenes titers were detected in liver and spleen of TGR5 KO than in WT animals and HE staining revealed significantly more inflammatory infiltrates in livers from TGR5 KO mice. Accordingly, the levels of AST/ALT and LDH were elevated in serum from TGR5 KO animals as compared to the WT littermates. Furthermore, TGR5 KO mice had increased serum cytokine levels after L. monocytogenes infection as compared to WT mice. Conclusions: Compared to WT littermates, TGR5 knockout mice showed higher Listeria monocytogenes titers in liver and spleen despite elevated serum inflammatory cytokines, such as TNFalpha. Accordingly, TGR5 KO mice were unable to control bacterial infection and systemic inflammation and thus displayed a significantly increased mortality rate. Reference(s) [1] Kawamata Y et al. A G protein-coupled receptor responsive to bile acids. J Biol Chem 2003; 278: 9435–9440. [2] Keitel V et al. Expression and function of the bile acid receptor TGR5 in Kupffer cells. Biochem Biophys Res Commun 2008; 372: 78–84. [3] Wang YD et al. The G-Protein-coupled bile acid receptor, Gpbar1 (TGR5), negatively regulates hepatic inflammatory response through antagonizing nuclear factor kappa light-chain enhancer of activated B cells (NF-kappaB) in mice. Hepatology 2011; 54: 1421–1432. [4] Pols TW et al. TGR5 activation inhibits atherosclerosis by reducing macrophage inflammation and lipid loading. Cell Metab 2011; 14: 747– 757.
P0465 PERSISTENT INTRAHEPATIC Vg9Vd2 T-CELLS IMPAIRMENT IN HCV-INFECTED PERSONS E. Cimini1 , V. Bordoni1 , U. Visco-Comandini2 , M. Montalbano2 , R. Lionetti2 , A. Sacchi1 , R. Casetti1 , N. Tumino1 , M.R. Capobianchi3 , F. Martini1 , C. Agrati1 . 1 Cellular Immunology Laboratory, 2 Clinical Department, 3 Virology Laboratory, National Institute for Infectious Diseases “Lazzaro Spallanzani”, Rome, Italy E-mail: [email protected] Background and Aims: dgHepatitis C virus (HCV) persistence in the host results from inefficiency of both innate and adaptive immune responses to eradicate the infection. Among innate immune cells, a functional impairment of peripheral Vg9Vd2 T-cells was described during chronic HCV infection; this functional defect can be partially restored by using IFN-a, opening the possibility to boost antiviral innate immune response. Unfortunately, no data are available on intrahepatic Vg9Vd2 T-cells that instead, may represent the key actors in the anti-HCV response. Aim of our work was to compare intrahepatic and peripheral Vg9Vd2 T-cells in chronic HCV-infected patients (HCV) and in healthy donors (HD) subjected to gut resection surgery. Methods: Phenotypic and functional features of intrahepatic and peripheral Vg9Vd2 T-cells were analyzed in 17 chronic HCV patients and in 14 HD by flow cytometry and ELISA assay. Results: Irrespective of HCV infection, intrahepatic compartment was characterized by a lower frequency of Vg9Vd2 T-cells than in the peripheral blood. Although expressing an effector/activated phenotype, intrahepatic Vg9Vd2 T-cells showed a drastic reduction of their ability to produce IFN-g after specific stimulation both in HCV and in HD subjects. Nevertheless, while intrahepatic Vg9Vd2 T-cells from HD can be functionally restored by using IFN-a costimulation, intrahepatic Vg9Vd2 T-cells from HCV-patients are refractory also to IFN-a co-stimulation, suggesting an exhaustion profile. Accordingly, Vg9Vd2 T-cells from HCV-patients presented a higher PD-1 expression than HD both in peripheral and intrahepatic compartments, probably due to persistent antigenic stimulation. Conclusions: Overall, intrahepatic Vg9Vd2 T-cells from HCV patients presented an exhaustion phenotype and are functional impaired also after IFN-a co-stimulation. New studies are mandatory in order to identify molecular mechanisms inducing this functional anergy and to discover pathway able to restore Vg9Vd2 T-cell functionality. P0466 IL-33 KNOCK OUT MICE ARE SENSITIZED TO SEVERE ConA LIVER INJURY BUT NOT CCl4 -MEDIATED LIVER INJURY M. Imran Arshad1 , G. Noel2 , A. Filliol2 , V. Genet2 , C. Lucas-Leclerc3 , J.-P. Girard4 , C. Piquet-Pellorce2 , M. Samson2 . 1 Universit´e de Rennes 1, U.1085 Inserm, 2 Universit´e de Rennes 1, IRSET – U.1085 Inserm, 3 Universit´e de Rennes 1, CHU Rennes, Rennes, 4 IPBS-CNRS, Universit´e de Toulouse, Toulouse, France E-mail: [email protected] Background and Aims: IL-33/ST2 axis play a protective role during acute hepatitis but little is known about the functional role of endogenous IL-33 in liver patho-physiology. We aimed to decipher the functional role of IL-33 by using IL-33 deficient mice during immune cell mediated and hepatotoxic driven liver injury. Methods: We used a genetic model of acute hepatitis by using IL-33 deficient mice in ConA (a T cell-mediated hepatitis) and CCl4 (a hepatotoxic agent-induced hepatitis) induced acute liver injury. The liver functions (AST/ALT), signature of cytokines and characterization of infiltrate cell in WT and IL-33−/− mice were carried out by biochemistry, qPCR and flow cytometry analyses. Results: Our results demonstrated that IL-33−/− mice exhibited more severe ConA liver injury than WT mice evidencing a protective effect of IL-33 in this hepatic model while no difference was
Journal of Hepatology 2015 vol. 62 | S263–S864
S487
Background and Aims: TGR5 (Gpbar1) is a membrane-bound bile acid receptor expressed in macrophages of several organs such as lung, liver and intestine as well as in peripheral blood mononuclear cells [1–3]. Stimulation of TGR5 inhibits the nuclear translocation of p65 and NF-úB transcriptional activation [3,4], thus reducing LPS-mediated production of pro-inflammatory cytokines [1,2]. Aim of the present study was to determine the role of TGR5 in an animal model for pathogen defense. Methods: Male, 8–12 week old TGR5 knockout (KO) and WT mice were injected intravenously with 8×104 CFU/ml L. monocytogenes and observed for 7 days. On the 3rd day after infection with 2.5×104 CFU/ml bacterial titers were determined in spleen and liver. Serum levels of AST, ALT and LDH were measured using Spotchemanalyzer. Cytokine levels were determined in serum using Luminex cytometric bead assay. HE staining was carried out for all liver samples. Results: Following L. monocytogenes infection TGR5 KO displayed a 50% mortality rate within a week of inoculation, while 90% of the TGR5 WT animals survived. In addition higher L. monocytogenes titers were detected in liver and spleen of TGR5 KO than in WT animals and HE staining revealed significantly more inflammatory infiltrates in livers from TGR5 KO mice. Accordingly, the levels of AST/ALT and LDH were elevated in serum from TGR5 KO animals as compared to the WT littermates. Furthermore, TGR5 KO mice had increased serum cytokine levels after L. monocytogenes infection as compared to WT mice. Conclusions: Compared to WT littermates, TGR5 knockout mice showed higher Listeria monocytogenes titers in liver and spleen despite elevated serum inflammatory cytokines, such as TNFalpha. Accordingly, TGR5 KO mice were unable to control bacterial infection and systemic inflammation and thus displayed a significantly increased mortality rate. Reference(s) [1] Kawamata Y et al. A G protein-coupled receptor responsive to bile acids. J Biol Chem 2003; 278: 9435–9440. [2] Keitel V et al. Expression and function of the bile acid receptor TGR5 in Kupffer cells. Biochem Biophys Res Commun 2008; 372: 78–84. [3] Wang YD et al. The G-Protein-coupled bile acid receptor, Gpbar1 (TGR5), negatively regulates hepatic inflammatory response through antagonizing nuclear factor kappa light-chain enhancer of activated B cells (NF-kappaB) in mice. Hepatology 2011; 54: 1421–1432. [4] Pols TW et al. TGR5 activation inhibits atherosclerosis by reducing macrophage inflammation and lipid loading. Cell Metab 2011; 14: 747– 757.
P0465 PERSISTENT INTRAHEPATIC Vg9Vd2 T-CELLS IMPAIRMENT IN HCV-INFECTED PERSONS E. Cimini1 , V. Bordoni1 , U. Visco-Comandini2 , M. Montalbano2 , R. Lionetti2 , A. Sacchi1 , R. Casetti1 , N. Tumino1 , M.R. Capobianchi3 , F. Martini1 , C. Agrati1 . 1 Cellular Immunology Laboratory, 2 Clinical Department, 3 Virology Laboratory, National Institute for Infectious Diseases “Lazzaro Spallanzani”, Rome, Italy E-mail: [email protected] Background and Aims: dgHepatitis C virus (HCV) persistence in the host results from inefficiency of both innate and adaptive immune responses to eradicate the infection. Among innate immune cells, a functional impairment of peripheral Vg9Vd2 T-cells was described during chronic HCV infection; this functional defect can be partially restored by using IFN-a, opening the possibility to boost antiviral innate immune response. Unfortunately, no data are available on intrahepatic Vg9Vd2 T-cells that instead, may represent the key actors in the anti-HCV response. Aim of our work was to compare intrahepatic and peripheral Vg9Vd2 T-cells in chronic HCV-infected patients (HCV) and in healthy donors (HD) subjected to gut resection surgery. Methods: Phenotypic and functional features of intrahepatic and peripheral Vg9Vd2 T-cells were analyzed in 17 chronic HCV patients and in 14 HD by flow cytometry and ELISA assay. Results: Irrespective of HCV infection, intrahepatic compartment was characterized by a lower frequency of Vg9Vd2 T-cells than in the peripheral blood. Although expressing an effector/activated phenotype, intrahepatic Vg9Vd2 T-cells showed a drastic reduction of their ability to produce IFN-g after specific stimulation both in HCV and in HD subjects. Nevertheless, while intrahepatic Vg9Vd2 T-cells from HD can be functionally restored by using IFN-a costimulation, intrahepatic Vg9Vd2 T-cells from HCV-patients are refractory also to IFN-a co-stimulation, suggesting an exhaustion profile. Accordingly, Vg9Vd2 T-cells from HCV-patients presented a higher PD-1 expression than HD both in peripheral and intrahepatic compartments, probably due to persistent antigenic stimulation. Conclusions: Overall, intrahepatic Vg9Vd2 T-cells from HCV patients presented an exhaustion phenotype and are functional impaired also after IFN-a co-stimulation. New studies are mandatory in order to identify molecular mechanisms inducing this functional anergy and to discover pathway able to restore Vg9Vd2 T-cell functionality. P0466 IL-33 KNOCK OUT MICE ARE SENSITIZED TO SEVERE ConA LIVER INJURY BUT NOT CCl4 -MEDIATED LIVER INJURY M. Imran Arshad1 , G. Noel2 , A. Filliol2 , V. Genet2 , C. Lucas-Leclerc3 , J.-P. Girard4 , C. Piquet-Pellorce2 , M. Samson2 . 1 Universit´e de Rennes 1, U.1085 Inserm, 2 Universit´e de Rennes 1, IRSET – U.1085 Inserm, 3 Universit´e de Rennes 1, CHU Rennes, Rennes, 4 IPBS-CNRS, Universit´e de Toulouse, Toulouse, France E-mail: [email protected] Background and Aims: IL-33/ST2 axis play a protective role during acute hepatitis but little is known about the functional role of endogenous IL-33 in liver patho-physiology. We aimed to decipher the functional role of IL-33 by using IL-33 deficient mice during immune cell mediated and hepatotoxic driven liver injury. Methods: We used a genetic model of acute hepatitis by using IL-33 deficient mice in ConA (a T cell-mediated hepatitis) and CCl4 (a hepatotoxic agent-induced hepatitis) induced acute liver injury. The liver functions (AST/ALT), signature of cytokines and characterization of infiltrate cell in WT and IL-33−/− mice were carried out by biochemistry, qPCR and flow cytometry analyses. Results: Our results demonstrated that IL-33−/− mice exhibited more severe ConA liver injury than WT mice evidencing a protective effect of IL-33 in this hepatic model while no difference was
Journal of Hepatology 2015 vol. 62 | S263–S864
S487