Abstract

Background: Alcohol-associated liver disease (ALD) is a major public health problem worldwide, and gut microbial dysbiosis is an important contributor to ALD pathogenesis. Our previous study showed that patients with alcohol-associated hepatitis (AH) have increased proportions of mammalian viruses including retroviruses in the fecal virome. However, the role of the gut virome and in particular of endogenous retroviruses (ERVs) for ALD development is unknown. The aim of this study was to evaluate the contributions of ERVs to ALD onset and progression.

Methods: We used transcriptomics for human duodenal biopsies. Gnotobiotic and genetically modified C57BL/6 mice were subjected to the chronic plus binge ethanol feeding model (NIAAA).

Results: Three specific ERVs were upregulated in duodenal biopsies of patients with alcohol use disorder and ALD (n=45) as compared with non-alcoholic controls (n=15) as analyzed by RNAseq. Using the NIAAA model, we confirmed that chronic ethanol feeding of mice resulted in an increased abundance of ERVs in intestinal epithelial cells (IECs) of the small intestine and colon, but not in the liver. Ethanol treatment (50mM) induced the expression of ERVs in cultured small intestinal mouse organoids, which was blocked with the histone acetylation inhibitor A485, indicating that ethanol-mediated histone acetylation is involved in upregulation of ERVs. To further define the importance of ERVs in ethanol-induced liver disease, we colonized germ-free mice with stool from fecal retrovirus-positive and -negative AH patients. All patients were HIV negative. Germ-free mice transplanted with stool from fecal retrovirus-positive patients showed exacerbated ethanol-induced liver injury, steatosis, and inflammation. Oral treatment with antiretroviral medications (Emtricitabine, Tenofovir, Nevirapine) reduced ethanol-induced liver disease and intestinal ERV abundance in gnotobiotic mice colonized with stool from retrovirus-positive patients. Z- nucleic -binding protein 1 (Zbp1) is a sensor of ERVs and is known to trigger cell death in IECs and to induce gut barrier dysfunction. Zbp1 was upregulated in the intestine of ethanol-fed mice and reduced following antiretroviral medications. To demonstrate that Zbp1 mediates the effect of ERVs in the intestine, we generated mice with an IEC specific deletion of Zbp1 (Zbp1^ΔIEC). Zbp1^ΔIEC showed reduced ethanol-induced steatohepatitis as compared with floxed wildtype littermates. Deletion of Zbp1 in hepatocytes or Kupffer cells did not affect ethanol-induced liver disease.

Conclusion: Our results indicate that alcohol upregulates endogenous retroviral elements in the intestine of mice and humans. Reducing gut ERVs or deletion of intestinal Zbp1 in mice protects against ethanol-induced liver disease, indicating that the ERV-Zbp1 axis could be a potential therapeutic strategy for ALD.