Alcohol-and-hiv-induced lysosomal dysfunction regulates extracellular vesicles secretion in vitro and in liver-humanized mice

Background: Alcohol abuse is common in people living with HIV-1 and dramatically enhances the severity of HIV-induced liver damage by inducing oxidative stress and lysosomal dysfunction in the liver cells. We hypothesize that the increased release of extracellular vesicles (EVs) in hepatocytes and liver humanized mouse model is linked to lysosome dysfunction. Methods: The study was performed on primary human hepatocytes and human hepatoma RLW_XP-GFP (Huh 7.5 cells stably transfected with CYP2E1 and XPack-GFP) cells and validated on ethanol-fed liverhumanized fumarylacetoacetate hydrolase (Fah)^-/-, Rag2^-/-, common cytokine receptor gamma chain knockout (FRG-KO) mice. Cells and mice were infected with HIV-1_ADA virus. Results: We observed an increase in the secretion of EVs associated with a decrease in lysosomal activity and expression of lysosomal-associated membrane protein 1. Next-generation RNA sequencing of primary human hepatocytes revealed 63 differentially expressed genes, with 13 downregulated and 50 upregulated genes in the alcohol–HIV-treated group. Upstream regulator analysis of differentially expressed genes through Ingenuity Pathway Analysis identified transcriptional regulators affecting downstream genes associated with increased oxidative stress, lysosomal associated disease, and function and EVs biogenesis. Our in vitro findings were corroborated by in vivo studies on human hepatocytetransplanted humanized mice, indicating that intensive EVs’ generation by human hepatocytes and their secretion to serum was associated with increased oxidative stress and reduction in lysosomal activities triggered by HIV infection and ethanol diet. Conclusion: HIV-and-ethanol-metabolisminduced EVs release is tightly controlled by lysosome status in hepatocytes and participates in the development of double-insult-induced liver injury.