Objectives: Fecal microbiota transplantation (FMT) is a novel therapeutic strategy for alcohol-associated liver disease (ALD). However, adverse effects have been reported due to possible pathogenic bacteria colonization. Extracellular vesicles (EVs) derived from the feces have been suggested as an important mediator in disease pathogenesis. We aimed to evaluate whether oral administration of fecal EVs (fEVs) from mice fed alcohol and patients with alcohol-associated hepatitis (AH) to mice exacerbates experimental ALD.
Methods: fEVs from the feces of mice fed alcohol or pair-fed and patients with moderate alcoholic hepatitis (AHm) patients and healthy controls (HC) were isolated and analyzed by proteomic analysis. Male C57BL/6J mice were subjected to NIAAA alcohol exposure model (10-day Lieber DeCarli diet containing 5% ethanol, and a binge ethanol gavage at 5g/kg). fEVs (50 µg/mouse/day) or PBS were given to alcohol-fed mice orally for the last 3 days before the end of the experiments. Serum, feces, and liver tissues were collected for analysis.
Results: First, we showed that the fEVs from control mice suppressed, while fEVs from alcohol-fed mice exacerbated or had no effect, on pro-inflammatory cytokine expression in macrophage stimulated by endotoxin. Second, mice received fEVs from mice fed alcohol had increased levels of serum AST and AST compared with the mice received fEVs from pair-fed mice. Third, three-day AHm-fEVs treatment significantly increased serum ALT levels and fatty liver in mice compared to HC-fEVs controls. Interestingly, HC-fEVs decreased alcohol-induced fatty liver compared to the PBS controls. RNA-seq analysis of liver tissues revealed that genes involved in cytokine production were upregulated, while those involved in cholesterol metabolism and lipophagy regulation were downregulated, when comparing AF-AHm vs AF-HC. Notably, the hepatic expression of long non-coding RNA nuclear paraspeckle assembly transcript 1 (NEAT1), which has been shown to promote steatosis, was increased significantly in AF-AHm group, compared to AF-HC group. Proteomics analysis of donors’ fEVs identified proteins from both bacterial- and human-origin. Of note, AHm-fEVs had a dramatic decrease in proteins of butyrate-producing bacteria and flagellin domain proteins, which are critical of intestinal IL-22 and antimicrobial peptide expression through toll like receptor 5 activation. In contrast, murein-lipoprotein, one of the most abundant membrane proteins in Gram-negative bacterial cell walls and a virulence factor, was abundantly present in AHm-fEVs but almost absent in HC-fEVs.
Conclusions: Fecal EVs from AH subjects (mice and humans) and healthy controls contain distinct protein cargos from both gut microbiota and host intestinal cells that have distinct effects on alcohol-induced liver steatosis and injury in mice. (Supported by NIAAA).

Lihua Zhang

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