Pathogenesis mediated by extracellular vesicles in alcoholic steatohepatitis

Sandra Helinski

Eguchi A1 , Lazaro RG2 , Wang J2 , Kim J3 , Ohno-Machado L3 , Tsukamoto H2,4 and Feldstein AE1

1. Department of Pediatrics, University of California San Diego, La Jolla, California, USA 2. Southern California Research Center for ALPD and Cirrhosis and Departments of Pathology, Keck School of Medicine of the University of Southern California, Los Angeles, California, USA 3. Department of Biomedical Informatics, University of California San Diego, La Jolla, California, USA 4. Department of Veterans Affairs, Greater Los Angeles Healthcare System, Los Angeles, California, USA

Liver inflammation is key histological features associated with prognosis in patients with alcoholic steatohepatitis (ASH). Extracellular vesicles (EVs) are released during cell stress or demise, can contain a barcode of the cell of origin including specific microRNAs and are growingly recognized as key cell-to-cell communicators. Here we tested the hypothesis that during ASH development, hepatocyte damage release EVs with a microRNA signature that can fuse with hepatic macrophages (HM) to regulate their phenotype. Methods: C57/B6 mice were placed on intragastric feeding model of continuous ethanol infusion or control diet for 4 weeks to reproduce a physiologically relevant model of ASH. The extent of steatosis, inflammation, and fibrosis was assessed by histological and molecular analyses on liver specimens. Isolated hepatocytes (HC) or HM from ASH or control mice were incubated in medium-EV free serum and HC- or HM-derived EVs were isolated by ultracentrifugation from culture medium. A complete characterization of EVs was performed by FACS, electron microscopy, dynamic light scattering. HCEV biological function was investigated in isolated primary HMs derived from C57/B6 mice by qPCR. Comprehensive encapsulated miRNA in HC-EVs were assessed via transcriptome using illumina miRNA-seq. Results: We observed highly significant differences in the levels of HC- or HM-EVs between control group and ASH. HC represented the main source of EVs. The majority of HC-EV size was around 200 nm. Incubation of HM with HC-EVs from ASH mice resulted in a significant up-regulation of various macrophage activation markers including, IL-1b, IL-6, and TNF-a, compared to HC-EVs from control mice. Transcriptome analysis of HC-EVs from ASH detected various differentially expressed microRNAs, including 9 upregulated miRNAs and 4 down-regulated microRNAs compared to HC-EVs from control mice. Conclusions: Our results reveal that damaged hepatocytes from ASH mice are a key source of EVs with a specific microRNA cargo that may contribute liver injury via HM activation. These findings uncover EVs as potential novel therapeutic targets for ASH.