Alcohol stimulates macrophage activation through caspase-dependent hepatocyte derived release of CD40L containing extracellular vesicles

Vikas K. Verma, Haiyang Li, Ruisi Wang, Petra Hirsova, Malek Mushref, Yaming Liu, Sheng Cao, Patricia C. Contreras, Harmeet Malhi, Patrick S. Kamath, Gregory J. Gores, Vijay H. Shah

Research output: Contribution to journalArticlepeer-review

105 Scopus citations


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 (HepG2ADH) or cytochrome P450 2E1 (HepG2Cyp2E1) 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 HepG2Cyp2E1 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.

Original languageEnglish (US)
Pages (from-to)651-660
Number of pages10
JournalJournal of hepatology
Issue number3
StatePublished - Mar 1 2016


  • Alcohol
  • Alcoholic hepatitis
  • CD40L
  • Caspase inhibitor
  • Cyp2E1
  • Exosomes
  • Hepatocyte
  • Inflammation
  • Liver
  • Macrophage
  • TNF-α

ASJC Scopus subject areas

  • Hepatology


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