Background. Intestinal mucus layer disruption and gut microflora modification in conjunction with tight junction (TJ) changes can increase colonic permeability that allows bacterial dissemination and intestinal and systemic disease. Employing Citrobacter rodentium (CR) as a model system to mimic the pathogenic mechanisms of Enteropathogenic and Enterohaemorrhagic E. coli, we showed previously that a functional cross-talk between the Notch and Wnt/β-catenin pathways regulates hyperplasia and/or colitis following CR infection. Aim: To test the hypothesis that CR infection combined with chronic Notch inhibition will alter the composition of the colonic mucus to promote dysbiosis, tight junction disruption, loss of multipotent intestinal stem cells (ISCs) and colitis. Material/methods: Immune-competent NIH:Swiss outbred mice were infected with CR (108 CFUs) and treated with Notch blocker Dibenzazepine [DBZ, i.p. @ 10μmol/Kg body weight]. Mucus composition, fecal 16S rDNA analysis and components of TJ integrity were measured and correlated with changes in components of the Notch pathway. 6% Pectin diet served as a SCFA-delivery system to the colon. Results: In the colons of CR-infected/DBZ-treated mice, mucus analysis revealed dramatic alterations in the composition of trace O-glycans and complex type and hybrid N-glycans, compared to CR-infected alone that preceded alterations in 16S rDNA microbial community structure. Indeed, mucin degrading and colitogenic bacterium, Akkermansia muciniphila exhibited dramatic increases in the feces of CR-infected and DBZ-treated mice. At the same time, colons from CR-infected/DBZ-treated mice had decreased expression of antimicrobial peptides Angiogenin-4, Retnlβ, Intelectin-1 and Intelectin-2, respectively. Expression levels of both TJ and adherens junction proteins (e.g., Claudin-5, ZO-1, ZO-2, E-Cadherin, β-catenin) also decreased significantly in the colonic crypts of CR-infected/DBZ-treated mice that paralleled loss of Notch signaling. Both C3H/HeN mice that exhibit exaggerated response to CR infection and T- and B-cell deficient Rag-1 mice following CR-infection and DBZ-treatment, exhibited dramatic increases in paracellular permeability, goblet cell metaplasia and exacerbation of colitis. Chronic blockade of the Notch pathway depleted colonic epithelium of ISC markers Dclk1 and Lgr5 concomitant with exacerbation of colitis. 6% Pectin diet ameliorated colitis by restoring barrier integrity via recruitment of Bacteroides vulgatus, Streptococcus gordonii and Lactococcus lactis, bacteria with potential anti-inflammatory functions. Conclusions: 1. These studies help us delineate the mechanistic basis of colitis development in the aftermath of chronic Notch inhibition. 2. Our findings also caution against the use of Notch inhibitors for patients suffering from colitis-associated colon cancer.
Citation Format: Ishfaq Ahmed, Badal Roy, Rita-marie McFadden, Shrikant Anant, Seth Septer, Shahid Umar. Altered mucus composition and bacterial dysbiosis promote development of colitis following chronic Notch inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3297.
