Rabbit Anti-Ire1P (C-Terminal) Polyclonal antibody

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 (IRE1 alpha(Delta hep)) 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 4 mu 8C. 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 hepatocyte-derived EVs, and also from cultured primary hepatocytes. Mice given intravenous injections of IRE1A-stimulated, hepatocyte-derived 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 serine palmitoyltransferase genes, which encode the rate-limiting enzyme for ceramide biosynthesis. Administration of a pharmacologic inhibitor of serine palmitoyltransferase to mice reduced the release of EVs. Levels of XBP1 and serine palmitoyltransferase were increased in liver tissues, and numbers of EVs were increased in plasma, from patients with NASH compared with control samples and correlated with the histologic features of inflammation. CONCLUSIONS: In mouse hepatocytes, activated IRE1A promotes transcription of serine palmitoyltransferase genes via XBP1, resulting in ceramide biosynthesis and release of EVs. The EVs recruit monocyte-derived macrophages to the liver, resulting in inflammation and injury in mice with diet-induced steatohepatitis. Levels of XBP1, serine palmitoyltransferase, and EVs are all increased in liver tissues from patients with NASH. Strategies to block this pathway might be developed to reduce liver inflammation in patients with NASH.

Drug-induced hepatotoxicity (DIH) is a common adverse event that is associated with both antiretroviral (ARV) and anti-tuberculosis drugs (ATD). Moreover, the genetic variations predisposing ARV- and ARV-ATD-induced liver toxicity in African populations are not well investigated, despite the two diseases being the major global health problems in sub-Saharan Africa. We performed a genome-wide association study (GWAS) and replication study to identify the genetic variants linked to the risk of developing DIH due to ARV drugs alone, and ARV-ATD co-treatment in Ethiopian HIV-positive patients. Treatment-naive newly diagnosed HIV patients (n=719) with or without tuberculosis (TB) co-infection were enrolled prospectively and received efavirenz-based ARV therapy with or without rifampicin-based short course ATD, respectively. Whole-genome genotyping was performed by using the Illumina Omni Express Exome Bead Chip genotyping array with 951,117 single nucleotide polymorphisms (SNPs) on a total of 41 cases of DIH, and 452 people without DIH (treatment tolerants). The replication study was carried out for 100 SNPs with the lowest p-values (top SNPs) by using an independent cohort consisting of 18 DIH cases and 208 treatment tolerants. We identified a missense SNP rs199650082 (2756GA, R919Q, p=1.4x10(-6), odds ratio [OR]=18.2, 95% confidence interval [CI]=7.1-46.9) in an endoplasmic reticulum to the nucleus signaling-1 (ERN1) gene on chromosome 17 to be associated with DIH in the ARV-only cohort. In the ARV-ATD co-treatment groups, rs4842407, a long intergenic noncoding RNAs (lincRNAs) transcript variant on chromosome 12, was associated with DIH (p=5.3x10(-7), OR=5.4, 95% CI=2.8-10.3). These genetic variants that are putatively associated with DIH due to ARV drugs alone and ARV-ATD co-treatment establish a foundation for future personalized medicine in people with HIV and TB and call for larger studies in independent populations.