DIETARY LACTONES CAUSE INCREASED INTESTINAL PROTEOLYTIC ACTIVITY IN HUMANIZED MOUSE MODEL OF IRRITABLE BOWEL SYNDROME THROUGH A LOSS OF MICROBIAL β-GLURONIDASE ACTIVITY

Background: In irritable bowel syndrome (IBS), insufficient suppression of proteolytic activity (PA) leads to intestinal barrier disruption and visceral hypersensitivity. Our work shows host PA is suppressed by microbial β-glucuronidase (GUS) activity. Lactone sugars can inhibit GUS activity, and hence diets rich in lactone sugars may exacerbate protease mediated IBS symptomology. Aim: Determine the effect of diet on PA and microbiota using a humanized mouse model of IBS. Methods: High PA (>1078 BAEE Units/mg protein) and healthy human commensal microbiota were used to humanize germ-free mice (n=4 mice/group). Mice were fed chow supplemented with 1 g/kg apple, orange, carrot, and 200 mg/kg spinach to simulate high lactone consumption daily for 12 weeks. PA and GUS activity were measured pre and post diet intervention by FITC-casein and 4-Methylumbelliferyl-β-D-glucuronide cleavage assays respectively. Shotgun metagenomics with linear model and PERMANOVA were used to test relationships between microbiota diversity and PA. Results: In high PA humanized mice, lactones resulted in decreased α-diversity (Observed, Chao, Shannon and InvSimpson, p<0.01) and changes in β-diversity (Bray-Curtis, p<0.001) compared to controls. This associated with 3-fold increased fecal PA (3321±1322 vs 9875±2132 BAEE/mg of protein, p<0.01) and decreased fecal GUS activity (0.0048±0.0017 vs 0.0011±0.0003, GUS Units/mg protein, p<0.05). In contrast, dietary lactone supplementation in healthy humanized mice increased α-diversity (p<0.05) and mediated changes in β-diversity (p<0.001) compared to controls. Fecal PA (43.9±33.6 vs 69.6±63.9 BAEE/mg of protein) and GUS activity (0.034±0.004 vs 0.021±0.010, GUS Units/mg protein, p=0.06) remained unchanged. Cecal PA and GUS activity were unchanged in the high PA humanized mice after treatment; however, mice with human commensal microbiota had a 10-fold increase in cecal PA (147±31 vs 1420±560 BAEE/mg of protein, p<0.05) and 3-fold decrease in cecal GUS activity compared to controls (0.011±0.003 vs 0.031±0.008, GUS Units/mg protein, p<0.05). Critically, lactone removal increased cecal GUS activity (0.008±0.003 vs 0.015±0.001, Units/mg protein, p<0.05) and a reversal of fecal PA in in high PA mice (9875±2132 vs 3167±1191 BAEE/mg of protein, p<0.01). Conclusions: Dietary lactone sugars cause microbial taxa loss and further increase PA in high PA state. Lactones may directly inhibit PA; however, taxa loss suggests loss of microbial GUSes, leading to insufficient PA suppression. However, in healthy states, diversification of diet overcomes these effects, increasing overall microbial diversity. These data suggest dietary restrictions like low FODMAPs should be made in appropriate clinical context and indiscriminate elimination can result in depleted microbial diversity which can negatively influence host health. Supported by DK120745.