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Abstract
Simple SummaryConsumption of alcohol increases liver damage in HIV people; however, the mechanisms remain elusive. By utilizing alcohol (ethanol) exposed HIV-infected human hepatocytes liver cells and ethanol-fed liver humanized mouse model (immunodeficient mice livers harbor human hepatocytes), we show that the combination of alcohol and HIV enhances oxidative stress and impairs lysosome activity, which in turn stimulates pathogenic nanosized extracellular vesicles. These vesicles are of great importance, as manipulating their numbers and contents will eliminate the spread of infection in other cell types.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 RLWXP-GFP (Huh 7.5 cells stably transfected with CYP2E1 and XPack-GFP) cells and validated on ethanol-fed liver-humanized fumarylacetoacetate hydrolase (Fah)-/-, Rag2-/-, common cytokine receptor gamma chain knockout (FRG-KO) mice. Cells and mice were infected with HIV-1ADA 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 hepatocyte-transplanted 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-metabolism-induced EVs release is tightly controlled by lysosome status in hepatocytes and participates in the development of double-insult-induced liver injury.
Highlights
Despite significant improvement in the survival of people living with HIV-1 (PLWH) in the era of highly efficient antiretroviral therapy, liver disease is emerging as a major cause of morbidity and mortality [1]
Hepatocytes were plated on polyelectrolyte multilayer (PEM) film coating on top of the polydimethylsiloxane surface (two-dimensional (2D) culture) to support a long-term cell functionality since cells plated on collagen undergo fast de-differentiation and lose expression of ethanol metabolizing enzymes, cytochrome P4502E1 (CYP2E1), and alcohol dehydrogenase (ADH) in 24 h [14,15]
Given that hepatocytes represent the majority of cells in the liver and are the primary site of ethanol metabolism, we sought to investigate the effects of alcohol and HIV on extracellular vesicles (EVs) release from hepatocytes
Summary
Despite significant improvement in the survival of people living with HIV-1 (PLWH) in the era of highly efficient antiretroviral therapy, liver disease is emerging as a major cause of morbidity and mortality [1]. Alcohol potentiates HIV-mediated liver damage, and alcohol consumption in PLWH exacerbates liver injury, leading to hepatic fibrosis or cirrhosis It enhances pathological infection features by increasing viremia, suppressing the immune response, and promoting non-adherence to treatment, resulting in poor HIV treatment outcomes [3,4]. 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. 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
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