Deciphering the role of Notch1 in ulcerative colitis

Abstract

BackgroundUlcerative colitis (UC) is an inflammatory bowel disease, that causes ofchronic inflammation and ulcers affecting the innermost layer of the colon. UC patients have an increased risk of colitis associated cancer. Notch1 signaling is a conserved intracellular signaling pathway. It regulates cell fate, cell proliferation, differentiation, and cell death. However, Notch1 can act as a mediator or suppressor depending on the cellular context and disease stage.AimAim of this study is to understand the mechanistic role of Notch1 in colitis.MethodsFor our in vivo model, epithelial cell‐specific deletion of Notch1 was done using the Cre/Lox system. Villin‐cre+/−/Notch1flox/flox (Ncre‐C57/B6) mice were generated, which cannot express Notch1 in epithelium. We used 2% DSS as inflammation inducer. Biopsy tissues‐ 3 normal and 6 UC patients (Emory University bio‐repository).ResultsAcute colitis was induced among 8 weeks old both male and female wild type (WT‐ C57/B6) and Ncre mice by giving 2%DSS for 5 days. After the colitis phase mice were allowed to recover for 10 days and were sacrificed. Ncre mice had significant loss of body weight compared to WT mice at the end of the recovery phase . Colonoscopy indicated severe inflammation and mucosal thickening among Ncre mice. Histology exhibited significant loss of crypt architecture and infiltration of neutrophils among Ncre mice compared to WTs. Active Notch1 (NICD/Notch1 inter cellular domain) was overexpressed in HCT116 cells by transient transfection. Wound healing assay was performed by scratching with a pipet tip. HCT116 cells overexpressing NICD exhibited faster wound closure at 24 and 48 hours . Quantification of wound closure was done by calculating the wound area using the ImageJ program. To assess the role of Notch1 in chronic inflammation, 10 weeks old both male and female Ncre and WT mice were given 2 cycles of 2% DSS followed by a 21 days recovery cycle. Mice were sacrificed on day 56. Ncre mice were more susceptible to chronic inflammation as indicated by colonoscopy, significant body weight loss, shorter colon length, and lower histological score compared to WT mice in chronic colitis. Barrier function was assessed by using 4kD FITC‐dextran tracer showing leaky epithelium among Ncre mice compared to WT mice. QPCR analysis showed a significant decrease in the mRNA levels of 16SrRNA (universal RNA for microbial population) among Ncre mice . QPCR analysis of human biopsies indicated a significant decrease in mRNA levels of TGFβ and MMP7 (wound healing markers) among UC biopsy tissues compared to healthy biopsy tissues.ConclusionThese results together suggest a novel protective role of Notch1 during the recovery (after acute colitis) as well as in the chronic colitis. Notch1 mediates preferential differentiation of epithelial cells, therefore is necessary to maintain epithelial integrity. It mediates its wound healing role by promoting the expression of wound healing markers such as TGFβ and MMP7. It maintains the microbial population by recruiting healthy epithelial cells to maintain epithelial homeostasis in chronic colitis. Therefore, as long‐term cure the beneficial role of Notch1 in wound healing and in chronic colitis could be translated as an efficient targeted therapy for UC patients.