title: Hepatocytes release ceramide-enriched pro-inflammatory extracellular vesicles in an IRE1α-dependent manner.
abstract: Nonalcoholic steatohepatitis (NASH) is a lipotoxic disease wherein activation of endoplasmic reticulum (ER) stress response and macrophage-mediated hepatic inflammation are key pathogenic features. However, the lipid mediators linking these two observations remain elusive. We postulated that ER stress-regulated release of pro-inflammatory extracellular vesicles (EVs) from lipotoxic hepatocytes may be this link. EVs were isolated from cell culture supernatants of hepatocytes treated with palmitate (PA) to induce lipotoxic ER stress, characterized by immunofluorescence, Western blotting, electron microscopy, and nanoparticle tracking analysis. Sphingolipids were measured by tandem mass spectrometry. EVs were employed in macrophage chemotaxis assays. PA induced significant EV release. Because PA activates ER stress, we used KO hepatocytes to demonstrate that PA-induced EV release was mediated by inositol requiring enzyme 1α (IRE1α)/X-box binding protein-1. PA-induced EVs were enriched in C16:0 ceramide in an IRE1α-dependent manner, and activated macrophage chemotaxis via formation of sphingosine-1-phosphate (S1P) from C16:0 ceramide. This chemotaxis was blocked by sphingosine kinase inhibitors and S1P receptor inhibitors. Lastly, elevated circulating EVs in experimental and human NASH demonstrated increased C16:0 ceramide. PA induces C16:0 ceramide-enriched EV release in an IRE1α-dependent manner. The ceramide metabolite, S1P, activates macrophage chemotaxis, a potential mechanism for the recruitment of macrophages to the liver under lipotoxic conditions.
authors.affiliation: Division of Gastroenterology and Hepatology Mayo Clinic, Rochester, MN 55905 Department of Community Medical Supports, Tohoku Medical Megabank Organization, Tohoku University, Aobaku, Sendai, 980-8573 Japan.
authors.forename: Eiji
authors.initials: E
authors.lastname: Kakazu
authors.affiliation: Division of Gastroenterology and Hepatology Mayo Clinic, Rochester, MN 55905.
authors.forename: Amy S
authors.initials: AS
authors.lastname: Mauer
authors.affiliation: Department of Radiology, Mayo Clinic, Rochester, MN 55905.
authors.forename: Meng
authors.initials: M
authors.lastname: Yin
authors.affiliation: Division of Gastroenterology and Hepatology Mayo Clinic, Rochester, MN 55905 Malhi.harmeet@mayo.edu.
authors.forename: Harmeet
authors.initials: H
authors.lastname: Malhi
mesh_terms: D002478:Cells, Cultured; D002518:Ceramides / Q000378:metabolism; D059865:Endoplasmic Reticulum Stress / Q000235:genetics; D004722:Endoribonucleases / Q000235:genetics* / Q000378:metabolism; D000067128:Extracellular Vesicles / Q000378:metabolism* / Q000473:pathology; D022781:Hepatocytes / Q000378:metabolism / Q000473:pathology; D006801:Humans; D007249:Inflammation / Q000139:chemically induced / Q000235:genetics / Q000378:metabolism*; D007294:Inositol / Q000378:metabolism; D008246:Lysophospholipids / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism / Q000473:pathology; D065626:Non-alcoholic Fatty Liver Disease / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D010168:Palmitates / Q000378:metabolism / Q000633:toxicity; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D002518:Ceramides; D008246:Lysophospholipids; D010168:Palmitates; C060506:sphingosine 1-phosphate; D007294:Inositol; C528229:ERN1 protein, human; D017346:Protein Serine-Threonine Kinases; D004722:Endoribonucleases; D013110:Sphingosine
keywords: endoplasmic reticulum stress; exosome; inositol requiring enzyme 1α; lipoinflammation; microvesicle; nonalcoholic steatohepatitis