Aditya Ambade

The end-organ effects of alcohol span throughout the entire body, from the gastrointestinal tract to the central nervous system (CNS). In the intestine, alcohol use changes the microbiome composition and increases gut permeability allowing translocation of microbial components into the circulation. Gut-derived pathogen-associated signals initiate inflammatory responses in the liver and possibly elsewhere in the body. Because previous studies showed that the gut microbiome contributes to alcohol-induced liver disease, we hypothesized that antibiotic administration to reduce the gut microbiome would attenuate alcohol-induced inflammation in the brain and small intestine (SI). Six- to 8-week-old C57BL/6J female mice were fed alcohol in a liquid diet or a calorie-matched control diet for 10 days with an acute alcohol binge or sugar on the final day (acute-on-chronic alcohol administration). Some mice were treated with oral antibiotics daily to diminish the gut microbiome. We compared serum levels of TNFα, IL-6, and IL-1β by ELISA; expression of cytokines Tnfα, Mcp1, Hmgb1, Il-17, Il-23, Il-6, and Cox2; and inflammasome components Il-1β, Il-18, Casp1, Asc, and Nlrp3 in the CNS and SI by qRT-PCR. Microglial morphology was analyzed using immunohistochemical IBA1 staining in the cortex and hippocampus. Antibiotics dramatically reduced the gut microbiome load in both alcohol- and pair-fed mice. Alcohol-induced neuroinflammation and increase in SI cytokine expression were attenuated in mice with antibiotic treatment. Acute-on-chronic alcohol did not induce serum TNFα, IL-6, and IL-1β. Alcohol feeding significantly increased the expression of proinflammatory cytokines such as Tnfα, Mcp1, Hmgb1, Il-17, and Il-23 in the brain and intestine. Reduction in the gut bacterial load, as a result of antibiotic treatment, attenuated the expression of all of these alcohol-induced proinflammatory cytokines in both the brain and SI. Alcohol feeding resulted in microglia activation and morphologic changes in the cortex and hippocampus characterized by a reactive phenotype. These alcohol-induced changes were abrogated following an antibiotic-induced reduction in the gut microbiome. Unexpectedly, antibiotic treatment increased the mRNA expression of some inflammasome components in both the brain and intestine. Our data show for the first time that the acute-on-chronic alcohol administration in mice induces both neuroinflammation and intestinal inflammation and that reduction in the intestinal bacterial load can attenuate alcohol-associated CNS and gut inflammation. Gut microbiome-derived signals contribute to neuroinflammation in acute-on-chronic alcohol exposure.

Inflammatory cell activation drives diverse cellular programming during hepatic diseases. Hypoxia‐inducible factors (HIFs) have recently been identified as important regulators of immunity and inflammation. In nonalcoholic steatohepatitis (NASH), HIF‐1α is upregulated in hepatocytes, where it induces steatosis; however, the role of HIF‐1α in macrophages under metabolic stress has not been explored. In this study, we found increased HIF‐1α levels in hepatic macrophages in methionine‐choline‐deficient (MCD) diet‐fed mice and in macrophages of patients with NASH compared with controls. The HIF‐1α increase was concomitant with elevated levels of autophagy markers BNIP3, Beclin‐1, LC3‐II, and p62 in both mouse and human macrophages. LysM^Cre HIF^dPAfl/fl mice, which have HIF‐1α levels stabilized in macrophages, showed higher steatosis and liver inflammation compared with HIF^dPAfl/fl mice on MCD diet. In vitro and ex vivo experiments reveal that saturated fatty acid, palmitic acid (PA), both induces HIF‐1α and impairs autophagic flux in macrophages. Using small interfering RNA–mediated knock‐down and overexpression of HIF‐1α in macrophages, we demonstrated that PA impairs autophagy via HIF‐1α. We found that HIF‐1α mediates NF‐κB activation and MCP‐1 production and that HIF‐1α—mediated impairment of macrophage autophagy increases IL‐1β production, contributing to MCD diet‐induced NASH. Conclusion: Palmitic acid impairs autophagy via HIF‐1α activation in macrophages. HIF‐1α and impaired autophagy are present in NASH in vivo in mouse macrophages and in human blood monocytes. We identified that HIF‐1α activation and decreased autophagic flux stimulate inflammation in macrophages through upregulation of NF‐κB activation. These results suggest that macrophage activation in NASH involves a complex interplay between HIF‐1α and autophagy as these pathways promote proinflammatory overactivation in MCD diet‐induced NASH.