Mixed lineage kinase 3 mediates release of C‐X‐C motif ligand 10–bearing chemotactic extracellular vesicles from lipotoxic hepatocytes

Abstract

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.

Mixed-lineage kinase 3 pharmacological inhibition attenuates murine nonalcoholic steatohepatitis

“…These findings are consistent with our previous in vitro report that hepatocyte lipotoxicity triggers the activation of a MAPK signaling cascade consisting of the MAP3K MLK3, which activates the MAP2Ks MMK3/MKK6, and its downstream MAPK p38, resulting in the subsequent induction of the chemokine CXCL10. Likewise, URMC099 treatment in vitro inhibits this signaling cascade in hepatocytes under lipotoxic stress (11,23).…”

“…Based on our previous observation that MLK3 activation in hepatocytes under lipotoxic conditions induces the upregulation of the potent macrophage chemotactic ligand CXCL10 (11,23), we first assessed the expression of CXCL10 in whole liver by quantitative real-time PCR to assess whether systemic administration of URMC099 suppressed CXCL10 induction in the liver. Interestingly, URMC099 treatment significantly reduced CXCL10 hepatic mRNA expression in FFC-fed mice ( Figure 2A).…”

“…We have reported previously that MLK3 -/-mice are protected against liver injury, fibrosis, and inflammation in a murine model of NASH-inducing diet (22,23). We also demonstrated that hepatocytes under lipotoxic stress release chemotactic extracellular vesicles (EVs) by an MLK3-dependent mechanism (23); these vesicles are enriched with the potent macrophage chemotactic ligand (C-X-C motif) ligand 10 (CXCL10), expression of which is regulated by MLK3 in hepatocytes (11).…”

“…We also demonstrated that hepatocytes under lipotoxic stress release chemotactic extracellular vesicles (EVs) by an MLK3-dependent mechanism (23); these vesicles are enriched with the potent macrophage chemotactic ligand (C-X-C motif) ligand 10 (CXCL10), expression of which is regulated by MLK3 in hepatocytes (11). Furthermore, mice fed the NASH-inducing diet have increased CXCL10 in plasma EVs in an MLK3-dependent fashion (23), and CXCL10 -/-mice are protected against diet-induced NASH (24). Likewise, MLK3 proinflammatory properties have been recently recognized in different disease models (25)(26)(27).…”

“…Bolstered by our data supporting MLK3 as a crucial proinflammatory and proapoptotic mediator in our preclinical in vitro and in vivo model of NASH (22,23), we elected to test the protective effect of the MLK3 pharmacological inhibitor URMC099 in our murine model of diet-induced NASH and obesity. URMC099 is a selective small-molecule MLK3 pharmacological inhibitor that was developed for human use.…”