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

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 (HepG2ADH) or cytochrome P450 2E1 (HepG2Cyp2E1) were treated with ethanol and EV release was quantified with nanoparticle tracking analysis (NTA). EV mediated macrophage activation was monitored by analyzing inflammatory cytokines and macrophage associated mRNA expression, immunohistochemistry, biochemical serum ALT 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 (WT) 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 (AH) 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.

Mitochondrial DNA–enriched microparticles promote acute-on-chronic alcoholic neutrophilia and hepatotoxicity

“…Whether this mechanism also contributes to caspase-1-induced MP release in our E10d + 1B model required further studies. In addition, activation of the caspase apoptotic pathway (caspase-3 and caspase-8) promotes MP release in several types of cells, including neutrophils (37), adipocytes (38), and hepatocytes (21). It will be interesting to investigate whether caspase-1 activation of pyroptosis, a type of programed cell death that is different from apoptosis (39), also contributes to MP formation and release.…”

“…Blockage of the CXCL1 receptor CXCR1/2 ameliorated alcoholic steatohepatitis in mice (14). In addition to chemokines, extracellular vesicles (EVs) are new players in cellto-cell communication and play an important role in promoting inflammation and neutrophil infiltration in a variety of diseases (15,16), including liver diseases (17)(18)(19)(20)(21)(22)(23)(24)(25). EVs are nanometer-sized, membrane-bound extracellular milieu vesicles derived from cells (17).…”

“…The functions of EVs are mediated by delivering their contents from one cell to another. For example, stressed hepatocytes can activate macrophages via the release of EVs that contain lipids, proteins, chemokines, and nucleic acids (e.g., mitochondrial DNA [mtDNA]) (17)(18)(19)(20)(21)(22)(23)(24)(25). However, whether EVs also contribute to the elevation of circulating and hepatic neutrophils induced by acute-on-chronic alcoholic liver injury in human and mice remains unknown.…”

Hepatic sinusoids in liver injury, inflammation, and fibrosis: new pathophysiological insights

“…In a very recent study, MV have now been identified as an important pathomechanism in early ethanol-induced damage and as a novel form of crosstalk between hepatocytes and liver-resident macrophages. Verma et al [92] could show that ethanol results in the release of MV from hepatocytes via activation of the pro-apoptotic protein caspase 3. These MV have further been shown to stimulate macrophage activation by CD40 ligand-a member of the TNF family-and to induce inflammatory cytokines leading to liver inflammation [92].…”

Therapeutic opportunities for alcoholic steatohepatitis and nonalcoholic steatohepatitis: exploiting similarities and differences in pathogenesis

“…In NASH, EV cargoes include the proinflammatory chemokine CXCL10, which recruits macrophages, and TRAIL, which activates macrophages through death receptor signaling and leads to increases in IL-1β and IL-6 (65, 100, 101). In ALD, CD40 ligand-containing (CD40L-containing) EVs are released by hepatocytes and, in turn, activate CD40 on macrophages, which induces proinflammatory signaling (102). Sphingolipid cargoes, specifically C16:0 ceramide and sphingosine 1-phosphate (S1P), on EVs are implicated in the recruitment of macrophages to the steatotic liver in NASH (103).…”