BACKGROUND & AIMS Hepatocyte cellular dysfunction and death induced by lipids, and macrophage-associated inflammation are characteristics of nonalcoholic steatohepatitis (NASH). The fatty acid palmitate can activate death receptor 5 (DR5) on hepatocytes, leading to their death, but little is known about how this process contributes to macrophage-associated inflammation. We investigated whether lipid-induced DR5 signaling results in release of extracellular vesicles (EV) from hepatocytes, and whether these can induce an inflammatory macrophage phenotype. METHODS Primary mouse and human hepatocytes and Huh7 cells were incubated with palmitate, its metabolite lysophosphatidylcholine, or diluent (control). The released EV were isolated, characterized, quantified, and applied to macrophages. C57BL/6 mice were placed on chow or a diet high in fat, fructose, and cholesterol to induce NASH. Some mice were also given the ROCK1 inhibitor fasudil; 2 weeks later, serum EVs were isolated and characterized by immunoblot and nanoparticle-tracking analyses. Livers were collected and analyzed by histology, immunohistochemistry, and quantitative PCR. RESULTS Incubation of primary hepatocytes and Huh7 cells with palmitate or lysophosphatidylcholine increased their release of EV, compared with control cells. This release was reduced by inactivating mediators of the DR5 signaling pathway or ROCK1 inhibition. Hepatocyte-derived EV contained TRAIL and induced expression of interleukin-1, beta (Il1b) and Il6 mRNAs in mouse bone marrow-derived macrophages. Activation of macrophages required DR5 and RIP1. Administration of the ROCK1 inhibitor fasudil to mice with NASH reduced serum levels of EV; this reduction was associated with decreased liver injury, inflammation, and fibrosis. CONCLUSIONS Lipids, which stimulate DR5, induce release of hepatocyte EV, which activate an inflammatory phenotype in macrophages. Strategies to inhibit ROCK1-dependent release of EV by hepatocytes might be developed for treatment of patients with NASH.

Background and aims Mixed lineage kinase 3 (MLK3) deficiency reduces macrophage associated-inflammation in a murine model of nonalcoholic steatohepatitis (NASH). However, the mechanistic links between MLK3 activation in hepatocytes and macrophage-driven inflammation in NASH are uncharted. Herein, we report that MLK3 mediates the release of (C-X-C motif) ligand 10 (CXCL10)-laden extracellular vesicles (EVs) from lipotoxic hepatocytes, which induce macrophage chemotaxis. Methods Primary mouse hepatocytes (PMH) and Huh7 cells were treated with palmitate or lysophosphatidylcholine (LPC). Released EVs were isolated by differential ultracentrifugation. Results LPC treatment of PMH or Huh7 cells induced release of EVs, which was prevented by either genetic or pharmacological inhibition of MLK3. Mass spectrometry identified the potent chemokine CXCL10 in the EVs, which was markedly enriched in EVs isolated from LPC-treated hepatocytes versus untreated cells. Green fluorescent protein (GFP)-tagged CXCL10 was present in vesicular structures and co-localized with the red fluorescent protein (RFP)-tagged EV marker cluster of differentiation (CD) 63 following LPC treatment of co-transfected Huh-7 cells. Either genetic deletion or pharmacological inhibition of MLK3 prevented CXCL10 enrichment in EVs. Treatment of mouse bone marrow-derived macrophages with lipotoxic hepatocyte-derived EVs induced macrophage chemotaxis, an effect blocked by incubation with CXCL10 neutralizing antisera. MLK3 deficient mice fed a NASH-inducing diet had reduced concentrations of total plasma EVs, and CXCL10 containing EVs compared to WT mice. In Conclusion during hepatocyte lipotoxicity, activated MLK3 induces the release of CXCL10-bearing vesicles from hepatocytes, which are chemotactic for macrophages.