The role of gut proteases in determining intestinal permeability for patients with alcohol-related liver disease

Abstract

The precise mechanism of increased intestinal permeability and bacterial translocation in the pathogenesis of alcohol-related liver disease (ArLD) remains unclear. Recent data has implicated gut-derived virulence factors in the development of alcoholic hepatitis but it is not yet fully established how these gain access to the systemic circulation. This thesis aims to examine the effect that gut proteases may have on intestinal permeability and the link with alterations in the gut microbiome seen in ArLD. Stool samples were collected from patients with ArLD. Faecal water protease activity was measured and correlated with indirect markers of intestinal permeability and biochemical markers of liver disease. A monolayer model of the gut barrier using Madin Darby canine kidney (MDCK) cells enabled the effect of faecal water protease to be measured. Gelatin zymography differentiated stool proteases by weight followed by mass spectrometry. Protease-indicator agar allowed bacterial isolates showing a protease-producing phenotype to be characterised following DNA extraction. Stool protease activity on tight junction proteins was assessed using tight junction antibodies against MDCK monolayers and duodenal tissue samples taken from patients with ArLD. No correlation was seen between faecal protease activity and markers of intestinal permeability or biochemical markers of liver disease. A significant correlation was seen between faecal protease activity and the change in gut permeability as measured in the MDCK monolayer model. This correlation was abolished with the addition of a broad protease-inhibitor. Taxonomic characterisation of stool samples revealed reduced alpha diversity in samples from patients with ArLD but a higher proportion of Enterococcus. Tight junction protein staining was reduced in duodenal samples from patients with decompensated ArLD and in MDCK cells treated with ArLD faecal water compared to controls. These data suggest that faecal proteases may play a role in increasing intestinal permeability in ArLD through their effects on tight junction proteins.