Intestinal proteases in alcohol-associated liver disease

University Of California, San Diego, La Jolla CA

Investigators

Abstract

Project Summary Alcohol-associated health problems are a major medical burden in industrialized countries. Patients with alcohol-associated liver disease (ALD) show intestinal dysbiosis and gut barrier dysfunction. Increased intestinal permeability allows microbe-associated molecular patterns (MAMPs) such as lipopolysaccharides (LPS) to translocate to the liver and cause progression of ALD. The molecular mechanisms how alcohol mediates disruption of tight junctions are incompletely understood. Using multiplexed quantitative proteomics as an unbiased approach, our preliminary results demonstrate that fecal samples from patients with alcohol-associated hepatitis (AH) contain an increased abundance of host-derived proteases as compared with healthy controls and patients with alcohol use disorder (AUD). Fecal Cathepsin B (Ctsb) levels were associated with increased mortality in patients with AH. Our laboratories further demonstrate that Ctsb is predominantly expressed in macrophages in the intestine and its secretion is upregulated after chronic ethanol feeding in mice. Fecal supernatant from patients with AH induces barrier disruption in a Caco2 cell monolayer, which is blocked with the specific Ctsb inhibitor CA-074. This is supported by data demonstrating that oral administration of the gut-restricted Ctsb inhibitor CA-074 stabilizes the gut barrier and reduces ethanol-induced liver disease in mice. The testable central hypothesis of this proposed collaborative and multidisciplinary research application implicates that the ethanol-mediated induction of Ctsb in intestinal macrophages contributes to gut barrier dysfunction by directly degrading intestinal tight junction proteins and promoting ALD. Through the proposed studies we will characterize host gut proteases and the immune response in a human cohort and a mouse model of ethanol-induced liver disease. Towards this goal, we will characterize human proteases in fecal samples and intestinal biopsies of patients with ALD. We predict that increased fecal Ctsb correlates with clinical severity and outcome in patients with ALD (Aim 1). We will mechanistically determine the role of Ctsb in contributing to intestinal tight junction disruption. We will characterize the source, regulation and substrates of Ctsb using mouse intestinal macrophages and human intestinal organoids. We will use mice with a myeloid specific deletion of Ctsb and subject them to chronic-plus binge ethanol feeding (Aim 2). We will use a pharmacological intervention with an intestine restricted and specific Ctsb inhibitor in a mouse model of ethanol feeding (Aim 3). We believe these studies will provide important insights into alcohol-mediated changes of intestinal proteases that result in gut barrier dysfunction and promote ALD. Eventually this approach will lead to new therapeutic targets for patients with ALD.