Abstract 654: Dietary interventions ameliorate infectious colitis through differential regulation of Lgr5

Abstract

Abstract Background: During inflammatory bowel disease (IBD), the disruption of the epithelial barrier and translocation of bacteria drive inappropriate immune responses and unresolved inflammation. Rapid wound healing responses orchestrated by intestinal stem cells (ISCs) are central to inflammatory resolution and normalization of the mucosal barrier. Commensal microbiota ferment fibers to produce short-chain-fatty-acids (SCFAs) such as butyrate and its decrease has been linked to IBD. However, the mechanism through which SCFAs promote wound healing is poorly understood. Aims: The present study was designed to test the hypothesis that dietary fibers/butyrate ameliorate infectious colitis by differentially regulating Lgr5-dependent crypt regeneration and wound healing. Methods: Lgr5CreERT2/+; Rosa26LacZ reporter (Lgr5-R) mice and wild-type littermates were infected with Citrobacter rodentium (CR; 108 CFUs) and fed with either 6% Pectin (Pec) or 6% Tributyrin (Tbt) diets followed by euthanasia at 12 days post-infection. To partially deplete microbiota, mice were also given a cocktail of vancomycin (500mg/L), metronidazole (1g/L) and Ciprofloxacin (0.2g/L) for 10 days starting 3-days post-CR infection. Fresh feces pre- and post-infection/treatments were taken for 16S rDNA sequencing. Lineage tracing post-tamoxifen and Lgr5 promoter reporter activity assays were performed. Results: Both Pec and Tbt reduced the severity of CR-induced colitis as was evidenced by increased body weight and colon length, reduced immune cell infiltration and increased mucus production compared with CR-infected but untreated mice. 16s rDNA sequencing revealed significant dysbiosis during CR infection with the dominance of Proteobacteria and loss of Firmicutes and Bacteroidetes. Both Pec and Tbt diets reduced the levels of Proteobacteria and restored Firmicutes and Bacteroidetes phyla to pre-infection levels. X-gal staining revealed that there was an expansion of LacZ-labeled Lgr5(+) stem cells in the colons of CR infected Lgr5-R mice when subjected to dietary intervention via Pectin and Tributyrin as sources of butyrate compared with controls. Interestingly, Pec-induced Lgr5 regulation was dependent upon the presence of gut microbiota as antibiotics treatment reduced Pec-induced Lgr5 expansion and the extent of crypt regeneration. Tbt-treatment, on the other hand, regulated Lgr5 independently of the microbiota. Butyrate, in a dose-dependent (1-10mM) manner, increased Lgr5 promoter reporter activity. Docking studies further revealed butyrate’s ability to efficiently bind Lgr5 with a -4.0 Kcal/mol binding energy. The cellular thermal shift assay showed that butyrate was indeed able to bind Lgr5. Conclusions: Thus, dietary interventions, by altering the gut microbiota, can differentially regulate Lgr5’s ability to orchestrate crypt regeneration and wound healing to ameliorate colitis. Citation Format: Ishfaq Ahmed, Badal C. Roy, Rita-Marie T. Raach, Sharon J. Manley, Pugazhendhi Srinivasan, Prasad Dandawate, Afreen Sayed, Danny R. Welch, Shrikant Anant, Venkatesh Sampath, Shahid Umar. Dietary interventions ameliorate infectious colitis through differential regulation of Lgr5 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 654.