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. Primary mouse hepatocytes (PMHs) and Huh7 cells were treated with palmitate or lysophosphatidylcholine (LPC). Released EVs were isolated by differential ultracentrifugation. 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 colocalized with the red fluorescent protein (RFP)-tagged EV marker, CD63, after LPC treatment of cotransfected 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 wild-type mice. Conclusions: During hepatocyte lipotoxicity, activated MLK3 induces the release of CXCL10-bearing vesicles from hepatocytes, which are chemotactic for macrophages.
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