- Email: [email protected]
O037 : Macrophage autophagy mediated the beneficial effects of cannabinoid receptor 2 in alcoholic liver disease
ORAL PRESENTATIONS significant associated mortality and few treatment options: liver transplantation remains the only treatment option for those not responding to medical treatment. CCR9+ macrophages have recently been reported as playing a key role in a mouse model of acute liver injury. We investigated the CCR9/CCL25 receptor/ligand axis in acute liver injury. Methods: Serum concentration of CCL25, the ligand for CCR9, was measured by ELISA in patients with ALF admitted to Kings College Hospital, London. Blood and liver-infiltrating immune cells were analysed by FACS. In murine models of ALF, mice were administered carbon tetrachloride (CCl4 ) (1 ml/kg) or paracetamol (400 mg/kg) and sacrificed at specified time points. Liver injury was assessed by serum transaminase concentration. Results: In patients with ALF, serum CCL25 concentration was markedly higher than healthy controls (mean 3.96 ng/ml vs. 0.878, p < 0.001). This was an early phenomenon with levels highest on the first day after admission and then decreasing (p < 0.01). CCR9+ macrophages were identified in peripheral blood and explanted liver tissue of patients with ALF. In mice, liver-infiltrating macrophage numbers peaked within 24 hours of liver injury, which coincided with peak hepatic inflammation, measured by serum ALT levels. Expression of CCR9 on macrophages peaked between 6–12 hours and was greater in paracetamol than CCl4 induced liver injury (mean percentage CCR9 expression 25.8% vs 11.0%). Conclusions: The CCR9/CCL25 axis is upregulated early in both human and mouse acute liver failure. In mice, infiltration of liver tissue by CCR9+ macrophages coinceides with the peak of necroinflammation as assessed by ALT. These data suggest a proinflammatory role for CCR9+ macrophages in ALF. O036 OVEREXPRESSION OF C-MYC IN HEPATOCYTES PROMOTES INITIATION AND PROGRESSION OF ALCOHOLIC LIVER DISEASE Y.A. Nevzorova1 , W. Hu1 , F.J. Cubero1 , U. Haas1 , N. Gassler2 , P. Strnad1 , H.W. Zimmermann1 , C. Trautwein1 , C. Liedtke1 . 1 Department of Internal Medicine III, 2 Institute of Pathology, RWTH Aachen University, Aachen, Germany E-mail: [email protected] Background and Aims: Alcohol exposure may result in the overexpression of certain oncogenes in human cells thereby increasing the intracellular concentration of reactive oxygen species (ROS) and, thus, triggering initiation and progression of alcoholic liver disease (ALD). We previously showed that prolonged c-myc expression in hepatocytes leads to spontaneous fibrogenesis and end-stage high-latency tumor development. In the present study, we hypothesized that c-myc overexpression might exert a crucial role in the development of ALD. Methods: Expression of c-myc was measured in biopsies of patients with ALD by quantitative real-time PCR (qPCR) and by immunohistochemistry (IHC). ALD in mice carrying transgenic overexpression of c-myc in hepatocytes (alb-myctg) and wildtype (WT) controls was induced by administration of control or ethanol (EtOH) Lieber DeCarli diet for 4 weeks. Primary hepatocytes were isolated from WT and alb-myctg mice, subjected to EtOH treatment and investigated for markers of cell cycle progression and oxidative stress by Western blotting, immunofluorescence and electron microscopy. Results: Hepatic c-myc was strongly up-regulated in human patients with advanced ALD and in WT mice fed with EtOH-diet. Conversely, the over-expression of c-myc in hepatocytes led to early ballooning degeneration, increase of liver collagen deposition and ethanol-induced hepatic lipotoxicity, in conjunction with excessive CYP2E1-derived ROS after EtOH-diet. Unexpectedly, alb-myctg livers displayed impaired cell proliferation resulting in remarkable hepatic hypertrophy and hepatocyte enlargement in response to EtOH challenge. Moreover, EtOH-fed alb-myctg mice exhibited
profound dramatic changes in the mitochondrial morphology associated with mitochondrial dysfunction. Consistently, alb-myctg derived primary hepatocytes showed blockade of proliferation and dramatic increase of cellular ROS, in response to EtOH challenge. Therefore, we explored the underlying molecular mechanisms, which demonstrated that in our experimental model of ALD, c-myc overexpression leads to strong activation of AKT, and, consequently phosphorylation of Mdm2 and degradation of p53 function. Conclusions: Our findings show that the proto-oncogen c-myc accelerated the progression of ALD thereby increasing collagen deposition and intracellular concentrations of oxidants through a p53 pathway-depending mechanism. These results render c-myc as a plausible novel diagnostic and prognostic tool for early detection of ALD. O037 MACROPHAGE AUTOPHAGY MEDIATED THE BENEFICIAL EFFECTS OF CANNABINOID RECEPTOR 2 IN ALCOHOLIC LIVER DISEASE T. Denaes1,2 , J. Lodder1,2,3,4 , M.-N. Chobert1,2 , J.-M. Pawlotsky1,2 , S. Lotersztajn1,2,3,4 , F. Teixeira-Clerc1,2 . 1 INSERM, U955, 2 Universit´e Paris-Est, Facult´e de M´edecine, UMR-S955, Cr´eteil, 3 INSERM, U1149, 4 Universit´e Paris 7 Denis Diderot, Facult´e de M´edecine, Bichat, Paris, France E-mail: [email protected] Background and Aims: Kupffer cells play a major role in the pathogenesis of alcoholic liver disease. We have previously reported that during experimental ALD, the cannabinoid receptor 2 (CB2) limits Kupffer cells polarization towards a pro-inflammatory phenotype, thereby leading to decreased hepatic inflammation and steatosis. The aim of this study was to investigate the contribution of macrophagic CB2 receptor to these effects and the mechanisms involved. Methods: Female mice were fed a Lieber-DeCarli liquid diet containing 5% ethanol for 10 days then gavaged with a single dose of ethanol (5 g/kg body weight) and sacrificed 9 hours later (NIAAA model). Experiments were performed in mice invalidated for CB2 receptor in the myeloid lineage (CB2Mye−/− mice) or in mice invalidated for the autophagy gene ATG5 in the myeloid lineage (ATG5Mye−/− mice) and treated with the CB2 agonist JWH-133. In vitro studies were performed on peritoneal macrophages isolated from WT or ATG5Mye−/− mice. Results: As compared to WT littermates, CB2Mye−/− mice showed ennhanced alcohol-induced pro-inflammatory phenotype of Kupffer cells and hepatic steatosis. CB2 receptor activation by JWH-133 stimulated macrophage autophagy in the liver of alcoholfed mice, whereas macrophage autophagy was inhibited in the liver of alcohol-fed CB2Mye−/− mice. These findings were confirmed in studies with cultured peritoneal macrophages, showing that autophagy is induced in macrophages exposed to JWH-133 and inhibited in CB2-deficient macrophages. Finally, JWH-133 reduced the induction of inflammatory genes by LPS in WT peritoneal macrophages, but not in ATG5-deficient cells. The CB2 agonist also protected from alcohol-induced liver inflammation and steatosis in WT, but not in ATG5Mye−/− mice demonstrating that macrophage autophagy mediated the anti-inflammatory and anti-steatogenic effects of CB2 receptor. Conclusions: These results demonstrated that CB2 receptor activation in macrophages protects from alcohol-induced steatosis by inhibiting hepatic inflammation through an autophagydependent pathway in Kupffer cell.
Journal of Hepatology 2015 vol. 62 | S187–S212
S207
profound dramatic changes in the mitochondrial morphology associated with mitochondrial dysfunction. Consistently, alb-myctg derived primary hepatocytes showed blockade of proliferation and dramatic increase of cellular ROS, in response to EtOH challenge. Therefore, we explored the underlying molecular mechanisms, which demonstrated that in our experimental model of ALD, c-myc overexpression leads to strong activation of AKT, and, consequently phosphorylation of Mdm2 and degradation of p53 function. Conclusions: Our findings show that the proto-oncogen c-myc accelerated the progression of ALD thereby increasing collagen deposition and intracellular concentrations of oxidants through a p53 pathway-depending mechanism. These results render c-myc as a plausible novel diagnostic and prognostic tool for early detection of ALD. O037 MACROPHAGE AUTOPHAGY MEDIATED THE BENEFICIAL EFFECTS OF CANNABINOID RECEPTOR 2 IN ALCOHOLIC LIVER DISEASE T. Denaes1,2 , J. Lodder1,2,3,4 , M.-N. Chobert1,2 , J.-M. Pawlotsky1,2 , S. Lotersztajn1,2,3,4 , F. Teixeira-Clerc1,2 . 1 INSERM, U955, 2 Universit´e Paris-Est, Facult´e de M´edecine, UMR-S955, Cr´eteil, 3 INSERM, U1149, 4 Universit´e Paris 7 Denis Diderot, Facult´e de M´edecine, Bichat, Paris, France E-mail: [email protected] Background and Aims: Kupffer cells play a major role in the pathogenesis of alcoholic liver disease. We have previously reported that during experimental ALD, the cannabinoid receptor 2 (CB2) limits Kupffer cells polarization towards a pro-inflammatory phenotype, thereby leading to decreased hepatic inflammation and steatosis. The aim of this study was to investigate the contribution of macrophagic CB2 receptor to these effects and the mechanisms involved. Methods: Female mice were fed a Lieber-DeCarli liquid diet containing 5% ethanol for 10 days then gavaged with a single dose of ethanol (5 g/kg body weight) and sacrificed 9 hours later (NIAAA model). Experiments were performed in mice invalidated for CB2 receptor in the myeloid lineage (CB2Mye−/− mice) or in mice invalidated for the autophagy gene ATG5 in the myeloid lineage (ATG5Mye−/− mice) and treated with the CB2 agonist JWH-133. In vitro studies were performed on peritoneal macrophages isolated from WT or ATG5Mye−/− mice. Results: As compared to WT littermates, CB2Mye−/− mice showed ennhanced alcohol-induced pro-inflammatory phenotype of Kupffer cells and hepatic steatosis. CB2 receptor activation by JWH-133 stimulated macrophage autophagy in the liver of alcoholfed mice, whereas macrophage autophagy was inhibited in the liver of alcohol-fed CB2Mye−/− mice. These findings were confirmed in studies with cultured peritoneal macrophages, showing that autophagy is induced in macrophages exposed to JWH-133 and inhibited in CB2-deficient macrophages. Finally, JWH-133 reduced the induction of inflammatory genes by LPS in WT peritoneal macrophages, but not in ATG5-deficient cells. The CB2 agonist also protected from alcohol-induced liver inflammation and steatosis in WT, but not in ATG5Mye−/− mice demonstrating that macrophage autophagy mediated the anti-inflammatory and anti-steatogenic effects of CB2 receptor. Conclusions: These results demonstrated that CB2 receptor activation in macrophages protects from alcohol-induced steatosis by inhibiting hepatic inflammation through an autophagydependent pathway in Kupffer cell.
Journal of Hepatology 2015 vol. 62 | S187–S212
S207