TY - JOUR
T1 - Mixed lineage kinase 3 mediates release of C-X-C motif ligand 10-bearing chemotactic extracellular vesicles from lipotoxic hepatocytes
AU - Ibrahim, Samar H.
AU - Hirsova, Petra
AU - Tomita, Kyoko
AU - Bronk, Steven F.
AU - Werneburg, Nathan W.
AU - Harrison, Stephen A.
AU - Goodfellow, Val S.
AU - Malhi, Harmeet
AU - Gores, Gregory J.
N1 - Funding Information:
Received July 6, 2015; accepted September 22, 2015. Additional Supporting Information may be found at onlinelibrary.wiley.com/doi/10.1002/hep.28252/suppinfo. This work was supported by the Center for Clinical and Translational Science (CCaTS), KL2 program KL2TR000136-09 (to S.H.I.), a Pilot and Feasibility Award (to S.H.I.), and the optical microscopy core of the Mayo Clinic Center for Cell Signaling in Gastroenterology (P30DK084567), National Institutes of Health (NIH) DK97178 (to H.M.), NIH grant DK41876 (to G.J.G.), and the Mayo Clinic (Rochester, MN). Copyright VC 2015 by the American Association for the Study of Liver Diseases. View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.28252
Funding Information:
The authors thank Dr. B. Huang for his help in studies employing electron microscopy, Dr. A. Bamidele for his help in the study employing flow cytometry, and Eugene Krueger for his help in the study employing TIRF microcopy. The authors thank Dr. Cristine Charlesworth and the proteomics core at Mayo Clinic for performing the mass spectrometry analysis on the EVs. The authors thank B. Freeman and A. Mauer for their excellent technical assistance and C. Hoover for her excellent secretarial assistance. The authors thank Dr. Roger Davis, Howard Hughes Medical Institute Investigator at University of Mass Medical School, for providing the Mlk3-/- mice. This work was supported by the Center for Clinical and Translational Science (CCaTS), KL2 program KL2TR000136-09 (to S.H.I.), a Pilot and Feasibility Award (to S.H.I.), and the optical microscopy core of the Mayo Clinic Center for Cell Signaling in Gastroenterology (P30DK084567), National Institutes of Health (NIH) DK97178 (to H.M.), NIH grant DK41876 (to G.J.G.), and the Mayo Clinic (Rochester, MN).
Publisher Copyright:
© 2016 by the American Association for the Study of Liver Diseases.
PY - 2016/3/1
Y1 - 2016/3/1
N2 - 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.
AB - 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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U2 - 10.1002/hep.28252
DO - 10.1002/hep.28252
M3 - Article
C2 - 26406121
AN - SCOPUS:84949883269
SN - 0270-9139
VL - 63
SP - 731
EP - 744
JO - Hepatology
JF - Hepatology
IS - 3
ER -