Abstract
BACKGROUND & AIMS: Disruption of the intestinal barrier facilitates microbial translocation to the liver and contributes to chronic liver disease. We aimed to study the role of the fecal proteome for disease progression in patients with alcohol-associated hepatitis.
METHODS: We used fecal proteomics data from a multicenter cohort of patients with alcohol-associated hepatitis (n=80), alcohol use disorder (n=20), and controls (n=19) (InTeam), and a cathepsin B activity assay in an independent multicenter cohort of patients with alcohol-associated hepatitis (n=80), alcohol use disorder (n=20), and controls (n=18) (AlcHepNet). Mice lacking cathepsin B in myeloid cells and transgenic mice overexpressing occludin in intestinal epithelial cells, were subjected to the chronic-plus-binge ethanol feeding model (NIAAA).
RESULTS: Fecal proteomics and activity analysis revealed that the protease cathepsin B progressively increased with alcohol use disorder and alcohol-associated hepatitis compared to controls, and is associated with higher short-term mortality in patients with alcohol-associated hepatitis. Cathepsin B is predominantly expressed in intestinal macrophages and is upregulated by ethanol. Cathepsin B deficiency in myeloid cells or oral treatment with the gut-restricted cathepsin B inhibitor CA074 stabilized gut barrier by preserving the tight junction protein occludin, lowered serum LPS levels, and attenuated ethanol-induced steatohepatitis. Transgenic overexpression of occludin in intestinal epithelial cells sufficed to reduce steatohepatitis and blunted the effects of CA074 in ethanol-fed mice. Cathepsin B proteolytically cleaves occludin in enzymatic assays, and its inhibition prevented occludin degradation and barrier disruption in intestinal organoids and epithelial monolayers. Molecular modeling and peptide profiling reveal specific cathepsin B-induced cleavage sites in the extracellular region of occludin.
CONCLUSIONS: Intestinal cathepsin B is an essential mediator of gut barrier dysfunction and therapeutic target in alcohol-associated liver disease.
IMPACT AND IMPLICATIONS: Intestinal barrier disruption facilitates the microbial translocation to the liver, contributing to the progression of alcohol-associated hepatitis, however the molecular mechanisms driving barrier dysfunction remain incompletely understood. Our study identified the protease cathepsin B as a key contributor to the progression of alcohol-associated liver disease by degrading the extracellular region of tight junction protein occludin in the intestine, which in turn leads to barrier disruption. This work advances the field by addressing causality, uncovering the molecular target, and proposing cathepsin B as a promising therapeutic target in alcohol-associated hepatitis, a condition for which liver transplantation remains the only effective treatment in a limited subset of patients.