Hepatocytes release ceramide-enriched pro-inflammatory extracellular vesicles in an IRE1α-dependent manner

Eiji Kakazu,Amy S Mauer,Meng Yin,Harmeet Malhi

doi:10.1194/jlr.m063412

Eiji Kakazu, Amy S Mauer + Show 2 more

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https://doi.org/10.1194/jlr.m063412

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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.

Highlights

Nonalcoholic steatohepatitis (NASH) is a lipotoxic disease wherein activation of endoplasmic reticulum (ER) stress response and macrophage-mediated hepatic inflammation are key pathogenic features
Ceramides are bioactive lipids that are the backbone for Abbreviations: ATF6␣, activating transcription factor 6␣; BMDM␾, bone marrow-derived macrophage; ER, endoplasmic reticulum; EV, extracellular vesicle; FFC, fat, fructose, and cholesterol; IMH, immortalized mouse hepatocyte; IRE1␣, inositol requiring enzyme 1␣; MVB, multivesicular body; NAFLD, nonalcoholic fatty liver disease; NAFLD activity score (NAS), nonalcoholic fatty liver disease activity score; NASH, nonalcoholic steatohepatitis; OA, oleate; PA, palmitate; PERK, protein kinase-like endoplasmic reticulum kinase; S1P, sphingosine-1-phosphate; S1P receptor 1 (S1P1), sphingosine-1-phosphate receptor 1; SphK, sphingosine kinase; SPT, serine palmitoyltransferase; Tg, thapsigargin; XBP-1, X-box binding protein-1
The principal findings of this study are: i) PAtreated hepatocytes release EVs in an IRE1␣-dependent manner; ii) lipotoxic EVs are enriched in C16:0 ceramide, and IRE1␣-regulated de novo ceramide biosynthesis is necessary for PA-stimulated EV release; iii) lipotoxic EVs stimulate macrophage chemotaxis via S1P generation; and iv) ceramide-enriched circulating EVs are increased in mouse and human NASH

Summary

Introduction

Nonalcoholic steatohepatitis (NASH) is a lipotoxic disease wherein activation of endoplasmic reticulum (ER) stress response and macrophage-mediated hepatic inflammation are key pathogenic features. Because PA activates ER stress, we used KO hepatocytes to demonstrate that PA-induced EV release was mediated by inositol requiring enzyme 1␣ (IRE1␣)/X-box binding protein-1. Hepatocytes release ceramide-enriched pro-inflammatory extracellular vesicles in an IRE1␣-dependent manner. Ceramides are bioactive lipids that are the backbone for Abbreviations: ATF6␣, activating transcription factor 6␣; BMDM␾, bone marrow-derived macrophage; ER, endoplasmic reticulum; EV, extracellular vesicle; FFC, fat, fructose, and cholesterol; IMH, immortalized mouse hepatocyte; IRE1␣, inositol requiring enzyme 1␣; MVB, multivesicular body; NAFLD, nonalcoholic fatty liver disease; NAS, nonalcoholic fatty liver disease activity score; NASH, nonalcoholic steatohepatitis; OA, oleate; PA, palmitate; PERK, protein kinase-like endoplasmic reticulum kinase; S1P, sphingosine-1-phosphate; S1P1, sphingosine-1-phosphate receptor 1; SphK, sphingosine kinase; SPT, serine palmitoyltransferase; Tg, thapsigargin; XBP-1, X-box binding protein-1. The exact pathways by which C16:0 ceramides mediate liver injury and inflammation remain incompletely defined

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