Fecal filtrate transplantation attenuates liver and gut injury in experimental alcohol-associated liver disease

ABSTRACT Fecal microbiota transplantation (FMT) carries a risk of infection. Cell-free fecal filtrate transplantation (FFT) represents a potentially safer alternative. The benefit of FFT has not been explored in alcohol-associated liver disease (ALD). We evaluated the therapeutic potential of FFT in a murine model of ALD. C57BL/6N mice were pair-fed control or ethanol Lieber-DeCarli diets with thioacetamide for 12 weeks to induce ALD. FFT (0.22-µm-filtered stool slurry) from healthy mice was administered three times per week to ALD animals. Post-FFT day 7, hepatic and intestinal injury and inflammation were assessed. Fecal microbiota was assessed by 16S rRNA sequencing, and the hepatic/stool metabolome was assessed by mass spectrometry. FFT was significantly better than abstinence, with reduced hepatic pro-inflammatory markers at the protein level, including IL-6 (1.6 fold change [FC], P < 0.001), TNF-α (1.2 FC, P < 0.001), and also decreased collagen deposition (2.97 FC, P = 0.003) and TGF-β expression (2 FC, P < 0.001). FFT improved intestinal barrier integrity by upregulation of ZO-1 protein (1.5-fold, P = 0.002) and Muc2 mRNA (3.28 FC, P = 0.007), with concomitant reduction of plasma-endotoxin (2.5 FC, P < 0.001). FFT suppressed pathogenic taxa like Escherichia–Shigella (log2 FC = −9.5, padj = 3.42E-13), Proteus (log2 FC = −5.6, padj = 0.004), Desulfovibrio (log2 FC = −1.5, padj = 0.004), and increased Prevotellaceae NK3B31 group (log2 FC = 2.7, padj = 0.018), and Acinetobacter (log2 FC = 4.9, padj = 9.41E-16) in the gut. Metabolomic profiling of stool revealed significant increases ( P < 0.05) in butyric, valeric, isobutyric, and ursodeoxycholic acids. These metabolites enriched pathways like butyrate metabolism ( P = 0.04), mitochondrial β-oxidation ( P = 0.008), and PPARα signaling ( P = 0.01), indicating intestinal and hepatic repair. FFT confers hepatoprotection in ALD by attenuating inflammation and fibrosis, and restoring the intestinal barrier, gut microbiota composition, and beneficial SCFA and bile acid metabolism. These findings support FFT as a promising preclinical strategy for gut–liver axis modulation in ALD. IMPORTANCE Alcohol-associated liver disease (ALD) involves disrupted gut-liver communication, contributing to inflammation, impaired intestinal barrier function, and metabolic disturbances that exacerbate liver injury. This study demonstrates that fecal filtrate transplantation (FFT) attenuates alcohol-induced liver injury while modulating intestinal barrier-related markers, gut microbial communities, and microbial metabolites. These findings highlight the potential of cell-free microbial components as modulators of the gut–liver axis and provide a basis for developing alternative approaches to gut-based intervention in ALD.