Abstract 1629: An integrated view of Notch signaling that regulates tissue renewal in response to enteric infection

Abstract

Abstract Background: Microbial dysbiosis and the associated gut inflammation unbalances epithelial renewal, potentially leading to cancer which is increasingly being recognized as a stem cell disease. Notch signaling is active in multipotent intestinal stem cells (ISCs); yet, how Notch signaling orchestrates communication between the gut microbes and ISC-dependent tissue renewal following a pathogenic insult, is poorly understood. Aim: To investigate how Notch signaling contributes towards ISC regeneration and pathogenesis of infection. Methodology: Rag-1-/- mice and wild type littermates were infected with Citrobacter rodentium (CR; 108CFUs) and treated with Notch blocker Dibenzazepine [(DBZ), ip at 10 µmol/kg body weight]. Whole distal colon or purified crypts were isolated for analyses. Fecal 16S rDNA analysis was performed. Transgenic mice expressing MHC-II either in IEC (EpithTg) or in dendritic cells (CD11cTg) were crossed to Rag2-/- mice and received Helicobacter bilis (Hb) to induce colitis. De-identified sections from control or Crohn’s Disease (CD) and Ulcerative Colitis (UC) patients were stained for markers of ISCs and immune cells, respectively. Results: Rag-1-/- mice but not WT littermates, exhibited dramatic increases in Dclk1 (Doublecortin-like kinase 1; an ISC marker) expression in the colonic crypts, measured via flow cytometry and IHC at 12-days post CR-infection that co-localized with Notch Intracellular Domain (NICD). CR-infected mice when treated with DBZ for 10 days exhibited: i) significant dysbiosis with Proteobacteria dominating (48% compared to 27% after CR infection), ii) increases in paracellular permeability concomitant with almost complete attenuation of Dclk1 expression and, iii) exacerbation of inflammation/colitis. Intriguingly, Dclk1 immunoreactivity shifted towards the stroma wherein, Dclk1 co-localized with NICD and with CD11c+ dendritic cells, CD11b+;F4/80+ macrophages and MHCII. Both EpithTg/Rag2-/- and CD11cTg/Rag2-/- mice when infected with Hb compared to uninfected mice, exhibited loss of crypt Dclk1 and its co-localization with NICD that coincided with severity of colitis. Sections prepared from the colons of Crohn’s Disease (CD) or Ulcerative Colitis (UC) patients paralleled loss of crypt Dclk1 seen in mice while Dclk1 continued to co-localize with NICD and with markers of immune cells within the stroma. When CR infected and DBZ-treated Rag-1-/- mice were given a cocktail of antibiotics (500mg/l Vancomycin, 1g/l metronidazole and 0.2 g/l ciprofloxacin) for 7 days, we observed increased survival and decreases in colon myeloperoxidase activity that coincided with an elevated Dclk1 levels in the crypt. Conclusions: 1. Bacterial dysbiosis following chronic Notch inhibition coupled with loss of crypt Dclk1 impairs crypt regeneration. 2. Co-localization of stromal Dclk1 with markers of immune cells and with MHCII, suggests a novel role for Dclk1 in antigen presentation. Citation Format: Badal C. Roy, Ishfaq Ahmed, Audrey Seamons, Shrikant Anant, Lillian Maggio-Price, Seth Septer, Shahid Umar. An integrated view of Notch signaling that regulates tissue renewal in response to enteric infection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1629. doi:10.1158/1538-7445.AM2017-1629