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

Abstract

BACKGROUND & AIMS: Endoplasmic reticulum to nucleus signaling 1 (ERN1, also called IRE1A) is a sensor of the unfolded protein response that is activated in the livers of patients with nonalcoholic steatohepatitis (NASH). Hepatocytes release ceramide-enriched inflammatory extracellular vesicles (EVs) after activation of IRE1A. We studied the effects of inhibiting IRE1A on release of inflammatory EVs in mice with diet-induced steatohepatitis. METHODS: C57BL/6J mice and mice with hepatocyte-specific disruption of Ire1a (IRE1a Dhep) were fed a diet high in fat, fructose, and cholesterol to induce development of steatohepatitis or a standard chow diet (controls). Some mice were given intraperitoneal injections of the IRE1A inhibitor 4m8C. Mouse liver and primary hepatocytes were transduced with adenovirus or adeno-associated virus that expressed IRE1A. Livers were collected from mice and analyzed by quantitative polymerase chain reaction and chromatin immunoprecipitation assays; plasma samples were analyzed by enzyme-linked immunosorbent assay. EVs were derived from hepatocytes and injected intravenously into mice. Plasma EVs were characterized by nanoparticle-tracking analysis, electron microscopy, immunoblots, and nanoscale flow cytometry; we used a membrane-tagged reporter mouse to detect hepatocyte-derived EVs. Plasma and liver tissues from patients with NASH and without NASH (controls) were analyzed for EV concentration and by RNAscope and gene expression analyses. RESULTS: Disruption of Ire1a in hepatocytes or inhibition of IRE1A reduced the release of EVs and liver injury, inflammation, and accumulation of macrophages in mice on the diet high in fat, fructose, and cholesterol. Activation of IRE1A, in the livers of mice, stimulated release of hepatocytederived EVs, and also from cultured primary hepatocytes. Mice given intravenous injections of IRE1A-stimulated, hepatocytederived EVs accumulated monocyte-derived macrophages in the liver. IRE1A-stimulated EVs were enriched in ceramides. Chromatin immunoprecipitation showed that IRE1A activated X-box binding protein 1 (XBP1) to increase transcription of

Immunological mechanisms and therapeutic targets of fatty liver diseases

“…278 Hepatocytes release ceramide-enriched inflammatory EVs by activating IRE1A, and EVs recruit monocyte-derived macrophages to the liver, resulting in inflammation in mice with steatohepatitis. 279 Thus, lipotoxic injury of hepatocytes boosts the release of EVs and activates macrophages to promote hepatic inflammation, which plays an important role in triggering NAFLD. These findings provide strong support for the development of EVs as biomarkers, and EVs are also potential therapeutic targets and tools.…”

Inhibiting IRE1α-endonuclease activity decreases tumor burden in a mouse model for hepatocellular carcinoma

“…On the other hand, tumor cells may hijack the ER-stress pathways to provide survival signals required for uncontrolled growth and eventually avoid apoptosis ( Kim et al, 2015 ). Activation of the UPR has also been shown to affect different fibrotic diseases ( Heindryckx and Li, 2018 ), including non-alcoholic fatty liver disease ( Bandla et al, 2018 ; Kwanten et al, 2016 ; Dasgupta et al, 2020 ), hepatitis-B-induced carcinogenesis ( Li et al, 2017a ), and biliary cirrhosis ( Sasaki et al, 2015 ). We have previously shown that inhibiting the IRE1α-branch of the UPR-pathway using 4μ8C, blocks TGFβ-induced activation of fibroblasts and stellate cells in vitro and reduces liver fibrosis in vivo ( Heindryckx et al, 2016 ).…”

Extracellular Vesicles as Drivers of Non-Alcoholic Fatty Liver Disease: Small Particles with Big Impact

“…In humans, the ceramide content in EVs was reported to be higher in patients with steatosis or NASH compared to in obese controls, and ceramide concentrations were nominally higher in NASH patients compared to in patients with only steatosis [ 23 ]. IRE1α activation also increased ceramide synthesis, resulting in augmented EV production and increased hepatic macrophage accumulation in mice with NAFLD [ 25 ]. Moreover, the intravenous injection of these EVs enhanced hepatic macrophage accumulation in otherwise healthy mice [ 25 ].…”

“…IRE1α activation also increased ceramide synthesis, resulting in augmented EV production and increased hepatic macrophage accumulation in mice with NAFLD [ 25 ]. Moreover, the intravenous injection of these EVs enhanced hepatic macrophage accumulation in otherwise healthy mice [ 25 ]. Collectively, this suggests a mechanistic link between EVs released via IRE1α activation and subsequent ceramide synthesis and the hepatic infiltration of macrophages in NAFLD/NASH [ 25 ].…”

“…Moreover, the intravenous injection of these EVs enhanced hepatic macrophage accumulation in otherwise healthy mice [ 25 ]. Collectively, this suggests a mechanistic link between EVs released via IRE1α activation and subsequent ceramide synthesis and the hepatic infiltration of macrophages in NAFLD/NASH [ 25 ].…”