Vikas K Verma1, Haiyang Li2, Ruisi Wang1, Petra Hirsova1, Malek Mushref1, Yaming Liu3, Sheng Cao1, Patricia C Contreras4, Harmeet Malhi1, Patrick S Kamath1, Gregory J Gores1, Vijay H Shah5. 1. Gastroenterology Research Unit, Department of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA. 2. Gastroenterology Research Unit, Department of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA; Affiliated Hospital of Guiyang Medical College, Guiyang, Guizhou, China. 3. Gastroenterology Research Unit, Department of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA; First Hospital of Jilin University, China. 4. Conatus Pharmaceuticals, San Diego, CA, USA. 5. Gastroenterology Research Unit, Department of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA. Electronic address: shah.vijay@mayo.edu.
Abstract
BACKGROUND & AIMS: The mechanisms by which hepatocyte exposure to alcohol activates inflammatory cells such as macrophages in alcoholic liver disease (ALD) are unclear. The role of released nano-sized membrane vesicles, termed extracellular vesicles (EV), in cell-to-cell communication has become increasingly recognized. We tested the hypothesis that hepatocytes exposed to alcohol may increase EV release to elicit macrophage activation. METHODS: Primary hepatocytes or HepG2 hepatocyte cell lines overexpressing ethanol-metabolizing enzymes alcohol dehydrogenase (HepG2(ADH)) or cytochrome P450 2E1 (HepG2(Cyp2E1)) were treated with ethanol and EV release was quantified with nanoparticle tracking analysis. EV mediated macrophage activation was monitored by analysing inflammatory cytokines and macrophage associated mRNA expression, immunohistochemistry, biochemical serum alanine aminotransferase and triglycerides analysis in our in vitro macrophage activation and in vivo murine ethanol feeding studies. RESULTS: Ethanol significantly increased EV release by 3.3-fold from HepG2(Cyp2E1) cells and was associated with activation of caspase-3. Blockade of caspase activation with pharmacological or genetic approaches abrogated alcohol-induced EV release. EV stimulated macrophage activation and inflammatory cytokine induction. An unbiased microarray-based approach and antibody neutralization experiments demonstrated a critical role of CD40 ligand (CD40L) in EV mediated macrophage activation. In vivo, wild-type mice receiving a pan-caspase, Rho kinase inhibitor or with genetic deletion of CD40 (CD40(-/-)) or the caspase-activating TRAIL receptor (TR(-/-)), were protected from alcohol-induced injury and associated macrophage infiltration. Moreover, serum from patients with alcoholic hepatitis showed increased levels of CD40L enriched EV. CONCLUSION: In conclusion, hepatocytes release CD40L containing EV in a caspase-dependent manner in response to alcohol exposure which promotes macrophage activation, contributing to inflammation in ALD.
BACKGROUND & AIMS: The mechanisms by which hepatocyte exposure to alcohol activates inflammatory cells such as macrophages in alcoholic liver disease (ALD) are unclear. The role of released nano-sized membrane vesicles, termed extracellular vesicles (EV), in cell-to-cell communication has become increasingly recognized. We tested the hypothesis that hepatocytes exposed to alcohol may increase EV release to elicit macrophage activation. METHODS: Primary hepatocytes or HepG2 hepatocyte cell lines overexpressing ethanol-metabolizing enzymes alcohol dehydrogenase (HepG2(ADH)) or cytochrome P450 2E1 (HepG2(Cyp2E1)) were treated with ethanol and EV release was quantified with nanoparticle tracking analysis. EV mediated macrophage activation was monitored by analysing inflammatory cytokines and macrophage associated mRNA expression, immunohistochemistry, biochemical serum alanine aminotransferase and triglycerides analysis in our in vitro macrophage activation and in vivo murineethanol feeding studies. RESULTS:Ethanol significantly increased EV release by 3.3-fold from HepG2(Cyp2E1) cells and was associated with activation of caspase-3. Blockade of caspase activation with pharmacological or genetic approaches abrogated alcohol-induced EV release. EV stimulated macrophage activation and inflammatory cytokine induction. An unbiased microarray-based approach and antibody neutralization experiments demonstrated a critical role of CD40 ligand (CD40L) in EV mediated macrophage activation. In vivo, wild-type mice receiving a pan-caspase, Rho kinase inhibitor or with genetic deletion of CD40 (CD40(-/-)) or the caspase-activating TRAIL receptor (TR(-/-)), were protected from alcohol-induced injury and associated macrophage infiltration. Moreover, serum from patients with alcoholic hepatitis showed increased levels of CD40L enriched EV. CONCLUSION: In conclusion, hepatocytes release CD40L containing EV in a caspase-dependent manner in response to alcohol exposure which promotes macrophage activation, contributing to inflammation in ALD.
Authors: Yaming Liu; Vikas K Verma; Harmeet Malhi; Greg J Gores; Patrick S Kamath; Arun Sanyal; Naga Chalasani; Bin Gao; Vijay H Shah Journal: Am J Physiol Cell Physiol Date: 2017-06-21 Impact factor: 4.249
Authors: Yan Cai; Ming-Jiang Xu; Erik H Koritzinsky; Zhou Zhou; Wei Wang; Haixia Cao; Peter St Yuen; Ruth A Ross; Robert A Star; Suthat Liangpunsakul; Bin Gao Journal: JCI Insight Date: 2017-07-20
Authors: Petra Hirsova; Samar H Ibrahim; Vikas K Verma; Leslie A Morton; Vijay H Shah; Nicholas F LaRusso; Gregory J Gores; Harmeet Malhi Journal: Hepatology Date: 2016-10-20 Impact factor: 17.425