Investigation of the importance of intestinal epithelial cell specific nuclear factor-κB subunits in regulating the response to intestinal damage-inducing stimuli



James, S
(2017) Investigation of the importance of intestinal epithelial cell specific nuclear factor-κB subunits in regulating the response to intestinal damage-inducing stimuli. PhD thesis, University of Liverpool.

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Abstract

The epithelium of the gastrointestinal (GI) tract is highly specialised and tightly regulated to ensure efficient absorption of dietary nutrients whilst preventing invasion by luminal microorganisms that may cause local and systemic infection and inflammation. Dysregulation of this single layer of columnar epithelium is a contributing factor to several intestinal diseases and toxicities. However, mechanisms that govern the susceptibility of intestinal epithelia to damage-inducing stimuli are not currently fully understood. NFĸB signalling is known to modulate the immune system and regulate homeostasis and repair in the GI tract. The NFĸB family of transcription factors consists of 5 main subunits (NFĸB1, RelA, c-Rel, NFĸB2 and RelB) that function as homo or hetero-dimers and is broadly categorised into the classical and alternative signalling pathways. Previous studies have indicated that the alternative (NFĸB2 and RelB) NFĸB signalling pathway regulates susceptibility to the onset of inflammatory bowel disease (IBD) and GI apoptosis. However, the contribution of intestinal epithelial cells and immune cells in regulating the GI mucosal response following the induction of experimental colitis was not clear from these studies. This thesis demonstrates using bone marrow reconstituted wild-type C57BL/6J and NFĸB2 null mice that the alternative NFĸB signalling pathway is important in both the haematopoietic cell and epithelial cell compartments in regulating GI damage following the induction of dextran sulphate sodium (DSS)-induced colitis. NFĸB2 deletion in the hematopoietic compartment caused wild-type mice to experience reduced colitis severity compared with non-reconstituted wild-type mice, whereas the presence of wild-type bone marrow caused NFĸB2 null mice to experience more severe colitis than that observed in NFĸB2 null mice. Therefore, the susceptibility to DSS-induced colitis is mediated by both the immune and epithelial cell compartments. As GI damage-inducing stimuli act on the epithelium by different mechanisms, this thesis explores whether NFĸB signalling pathways specifically in intestinal epithelial cells are important in regulating the epithelial response to further stimuli including mucositis-inducing chemotherapy drugs (5-fluorouracil (5-FU), etoposide and irinotecan) and tyrosine kinase inhibitors (TKIs; imatinib, nilotinib, dasatinib, bosutinib erlotinib and gefitinib) that are known to cause GI toxicity. Intestinal organoid (enteroid) cultures consisting of solely epithelial cells were established from NFĸB1 null, NFĸB2 null, c-Rel null and RelB null mice. c-Rel deletion protected enteroids from damage induced by 5-FU and etoposide, but not against irinotecan. To validate this in vitro enteroid model, mice were injected intraperitoneally with 5-FU (40mg/kg) for 24 hours and c-Rel null small intestine demonstrated persistent mitosis and reduced apoptosis compared with wild-type C57BL/6J mice. Therefore, c-Rel plays a role in regulating the response to the DNA damage caused by 5-FU and the in vivo data supports the in vitro model and analysis methods established in this study. The TKIs erlotinib, gefitinib and dasatinib caused significant increases in circularity (a measure of cell death validated against active-caspase-3 immunohistochemistry) of enteroids, while bosutinib, imatinib and nilotinib did not. This indicates that the GI side effects in erlotinib, gefitinib and dasatinib treated patients may be due to direct damage to GI epithelia. Deletion of NFĸB2 protected enteroids from erlotinib-induced cell death, indicating that the NFĸB2 signalling pathway may have a role in regulating the response of the intestinal mucosa to erlotinib. Deletion of NFĸB2 from intestinal epithelia therefore appears to confer a protective effect against GI damage following some damage-inducing stimuli. Further investigation of this pathway and the potential development of NFĸB2 inhibitors for targeted use in the clinic is therefore warranted.

Item Type: Thesis (PhD)
Divisions: Fac of Health & Life Sciences > Faculty of Health and Life Sciences
Depositing User: Symplectic Admin
Date Deposited: 29 Aug 2018 07:22
Last Modified: 29 Sep 2020 08:16
DOI: 10.17638/03019242
Supervisors:
URI: http://livrepository.liverpool.ac.uk/id/eprint/3019242