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Front. Immunol. | doi: 10.3389/fimmu.2018.02980

Hepatocyte-derived Lipotoxic Extracellular Vesicle Sphingosine 1-phosphate Induces Macrophage Chemotaxis

 Chieh-Yu Liao1, Yandong Gao1, Amy S. Mauer1, Alexander Revzin1 and  Harmeet Malhi1*
  • 1Mayo Clinic, United States

BACKGROUND: The pathophysiology of nonalcoholic steatohepatitis involves hepatocyte lipotoxicity due to excess saturated free fatty acids and concomitant proinflammatory macrophage effector responses. These include the infiltration of macrophages into hepatic cords in response to incompletely understood stimuli. Stressed hepatocytes release an increased number of extracellular vesicles (EVs), which are known to participate in intercellular signaling and coordination of the behavior of immune cell populations via their cargo. We hypothesized that hepatocyte-derived lipotoxic EVs that are enriched in sphingosine 1-phosphate (S1P) are effectors of macrophage infiltration in the hepatic microenvironment. METHODS: Lipotoxic EVs were isolated from palmitate treated immortalized mouse hepatocytes and characterized by nanoparticle tracking analysis. Lipotoxic EV sphingolipids were quantified using tandem mass spectrometry. Wildtype and S1P1 receptor knockout bone marrow-derived macrophages were exposed to lipotoxic EV gradients in a microfluidic gradient generator. Macrophage migration toward EV gradients was captured by time-lapse microscopy and analyzed to determine directional migration. Fluorescence-activated cell sorting along quantitative PCR and immunohistochemistry were utilized to characterize the cell surface expression of S1P1 receptor on intrahepatic leukocytes and hepatic expression of S1P1 receptor, respectively. RESULTS: Palmitate treatment induced the release of EVs. These EVs were enriched in S1P. Palmitate-induced S1P enriched EVs were chemoattractive to macrophages. EV S1P enrichment depended on the activity of sphingosine kinases 1 and 2, such that, pharmacological inhibition of sphingosine kinases 1 and 2 resulted in a significant reduction in EV S1P cargo without affecting the number of EVs released. When exposed to EVs derived from cells treated with palmitate in the presence of a pharmacologic inhibitor of sphingosine kinases 1 and 2, macrophages displayed diminished chemotactic behavior. To determine receptor-ligand specificity, we tested the migration responses of macrophages genetically deleted in the S1P1 receptor toward lipotoxic EVs. S1P1 receptor knockout macrophages displayed a marked reduction in their chemotactic responses toward lipotoxic palmitate-induced EVs. CONCLUSIONS: Palmitate-induced lipotoxic EVs are enriched in S1P through sphingosine kinases 1 and 2. S1P-enriched EVs activate persistent and directional macrophage chemotaxis mediated by the S1P1 receptor, a potential signaling axis for macrophage infiltration during hepatic lipotoxicity, and a potential therapeutic target for nonalcoholic steatohepatitis.

Keywords: Nonalcholic Steatohepatitis, Palmitic Acid, sphingosine kinase, sphingolipid, ceramide

Received: 31 Aug 2018; Accepted: 04 Dec 2018.

Edited by:

Hannelie Korf, Faculty of Medicine, KU Leuven, Belgium

Reviewed by:

Myriam Aouadi, Karolinska Institute (KI), Sweden
Christoph Welsch, Goethe-Universität Frankfurt am Main, Germany  

Copyright: © 2018 Liao, Gao, Mauer, Revzin and Malhi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Dr. Harmeet Malhi, Mayo Clinic, Rochester, 55902, Minnesota, United States,