Multi-Omics Identifies Associations of Gut Roseburia Abundance and Butyrate Levels with Antiviral Responses to Entecavir Plus Interferon-α in HBV Mouse Models

Background: Individuals with chronic hepatitis B virus (HBV) infection present with gut dysbiosis. Evidence suggests that gut microbiota can affect the efficacy of anti-HBV therapy. However, limited research has comprehensively explored how gut microbiota and its related metabolites interact with HBV treatment response after antiviral treatment. Our study aims to identify an association between the dominant microbial cluster and HBV treatment response during anti-viral therapies. Methods: HBV carrier mice were utilized to assess the antiviral efficacy and peripheral and intrahepatic immune responses of entecavir (ETV) and ETV plus interferon-α (EI) therapies. We analyzed the gut microbiome, metabolome, and non-parenchymal hepatic cells transcriptome to determine the role of gut microbes and related metabolites in virological and intrahepatic immune responses to ETV and EI. Mice receiving no therapy (HBV control), ETV monotherapy, and EI therapy were used for comparison. Furthermore, we used immune cells to evaluate the effects of metabolites produced by the identified bacteria. Results: EI therapy showed a greater trend toward reducing serum HBsAg and HBeAg levels compared with ETV monotherapy, and significantly decreased intrahepatic HBcAg expression. Principal coordinates analysis revealed that gut microbes and metabolites were significantly different in the EI treatment group and the ETV and HBV groups. Notably, the relative abundance of short-chain fatty acid-producing bacteria was higher in the EI group, particularly in gut Roseburia abundance and butyrate levels. Butyrate levels were also elevated in the liver. However, whether this reflects intestinal absorption or hepatic synthesis remains undetermined. Butyrate is a potent inhibitor of histone deacetylases (HDACs), which can reduce the expression of hepatic HDAC and also suppress its activity. Hepatic HDAC1 protein levels were lower in the EI group than ETV group, consistent with the possibility that butyrate may modulate HDAC1 expression. Furthermore, EI treatment increased hepatic NKG2D+ NK cells and upregulated the NK cell-mediated cytotoxicity pathway. In vitro studies revealed that 0.5 mM butyrate enhanced the cytokine response of CD8+ T cells to HBV surface-specific antigens. Conclusions: Our multi-omics analysis reveals an association of gut Roseburia abundance and butyrate levels with improved virological responses to EI therapy. In vitro, butyrate enhanced the cytokine response of HBV-specific CD8+ T cells, suggesting a hypothesis that butyrate (but does not verify mediation of the virological responses to EI) may contribute to the antiviral response. While our multi-omics and in vitro data support a testable working hypothesis that butyrate may contribute to intrahepatic immune modulation, we acknowledge that causality is not established by this study. These results provide potential possibilities for future functional investigations of butyrate-producing microbiota as an adjunct to anti-HBV therapy.