title: Alcohol stimulates macrophage activation through caspase-dependent hepatocyte derived release of CD40L containing extracellular vesicles.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.affiliation: Gastroenterology Research Unit, Department of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA.
authors.forename: Vikas K
authors.initials: VK
authors.lastname: Verma
authors.affiliation: Gastroenterology Research Unit, Department of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA; Affiliated Hospital of Guiyang Medical College, Guiyang, Guizhou, China.
authors.forename: Haiyang
authors.initials: H
authors.lastname: Li
authors.affiliation: Gastroenterology Research Unit, Department of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA.
authors.forename: Ruisi
authors.initials: R
authors.lastname: Wang
authors.affiliation: Gastroenterology Research Unit, Department of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA.
authors.forename: Petra
authors.initials: P
authors.lastname: Hirsova
authors.affiliation: Gastroenterology Research Unit, Department of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA.
authors.forename: Malek
authors.initials: M
authors.lastname: Mushref
authors.affiliation: Gastroenterology Research Unit, Department of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA; First Hospital of Jilin University, China.
authors.forename: Yaming
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Gastroenterology Research Unit, Department of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA.
authors.forename: Sheng
authors.initials: S
authors.lastname: Cao
authors.affiliation: Conatus Pharmaceuticals, San Diego, CA, USA.
authors.forename: Patricia C
authors.initials: PC
authors.lastname: Contreras
authors.affiliation: Gastroenterology Research Unit, Department of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA.
authors.forename: Harmeet
authors.initials: H
authors.lastname: Malhi
authors.affiliation: Gastroenterology Research Unit, Department of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA.
authors.forename: Patrick S
authors.initials: PS
authors.lastname: Kamath
authors.affiliation: Gastroenterology Research Unit, Department of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA.
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Gores
authors.affiliation: Gastroenterology Research Unit, Department of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA. Electronic address: shah.vijay@mayo.edu.
authors.forename: Vijay H
authors.initials: VH
authors.lastname: Shah
mesh_terms: D000818:Animals; D017209:Apoptosis; D023201:CD40 Ligand / Q000502:physiology*; D020169:Caspases / Q000502:physiology*; D019392:Cytochrome P-450 CYP2E1 / Q000502:physiology; D000431:Ethanol / Q000378:metabolism / Q000633:toxicity*; D000067128:Extracellular Vesicles / Q000502:physiology*; D005260:Female; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D008108:Liver Diseases, Alcoholic / Q000209:etiology*; D008262:Macrophage Activation / Q000187:drug effects*; D051379:Mice; D008810:Mice, Inbred C57BL
chemical_list: D023201:CD40 Ligand; D000431:Ethanol; D019392:Cytochrome P-450 CYP2E1; D020169:Caspases
keywords: Alcohol; Alcoholic hepatitis; CD40L; Caspase inhibitor; Cyp2E1; Exosomes; Hepatocyte; Inflammation; Liver; Macrophage; TNF-α