IRE1A Stimulates Hepatocyte-Derived Extracellular Vesicles That Promote Inflammation in Mice With Steatohepatitis

Background & Aims: Endoplasmic reticulum to nucleus signaling 1 (ERN1, also known as IRE1A) serves as a key sensor in the unfolded protein response and is activated in the livers of patients with nonalcoholic steatohepatitis (NASH). Upon IRE1A activation, hepatocytes release inflammatory extracellular vesicles (EVs) enriched in ceramide. This study investigates the effects of IRE1A inhibition on the release of these inflammatory EVs in mice with diet-induced steatohepatitis.

Methods: C57BL/6J mice and mice with hepatocyte-specific deletion of Ire1a (IRE1αΔhep) were fed a high-fat, fructose, and cholesterol diet to induce steatohepatitis, while control mice received a standard chow diet. Some mice were administered the IRE1A inhibitor 4μ8C via intraperitoneal injection. Liver samples and primary hepatocytes were transduced with adenovirus or adeno-associated virus expressing IRE1A. Liver tissues were analyzed using quantitative polymerase chain reaction and chromatin immunoprecipitation assays; plasma samples underwent enzyme-linked immunosorbent assay. EVs from hepatocytes were injected intravenously into mice. Plasma EVs were characterized through nanoparticle-tracking analysis, electron microscopy, immunoblots, and nanoscale flow cytometry, with a membrane-tagged reporter mouse used to identify hepatocyte-derived EVs. Plasma and liver tissues from patients with and without NASH were assessed for EV concentration and analyzed using RNAscope and gene expression analyses.

Results: Disruption of Ire1a in hepatocytes or IRE1A inhibition resulted in reduced EV release, liver injury, inflammation, and macrophage accumulation in mice on the high-fat diet. Activation of IRE1A in the livers stimulated the release of hepatocyte-derived EVs, as observed in cultured primary hepatocytes. Mice receiving intravenous injections of IRE1A-stimulated EVs showed increased monocyte-derived macrophage accumulation in the liver. These EVs were rich in ceramides. Chromatin immunoprecipitation revealed that IRE1A activated X-box binding protein 1 (XBP1), enhancing transcription of serine palmitoyltransferase genes, which are crucial for ceramide biosynthesis. Administration of a pharmacological inhibitor of serine palmitoyltransferase to mice led to decreased EV release. Elevated levels of XBP1 and serine palmitoyltransferase, along with increased EV concentrations, were observed in liver tissues and plasma from patients with NASH compared to controls, correlating with histological signs of inflammation.

Conclusions: In mouse hepatocytes, activated IRE1A enhances transcription of serine palmitoyltransferase genes through XBP1, leading to ceramide production and EV release. These EVs subsequently recruit monocyte-derived macrophages to the liver, contributing to inflammation and injury in diet-induced steatohepatitis. Increased levels of XBP1, serine palmitoyltransferase, and EVs in the liver of NASH patients suggest potential therapeutic strategies targeting this pathway to mitigate liver inflammation in affected individuals.