Date of Award

2017

Document Type

Open Access Dissertation

Department

Biological Sciences

Sub-Department

College of Arts and Sciences

First Advisor

Lydia E. Matesic

Abstract

The mucosal barrier of the small intestine is highly dynamic, enabling the passage of nutrients that are necessary for the body’s function while simultaneously preventing a breach by harmful microorganisms that are damaging to the host. The effectiveness of the mucosal barrier is dependent on the cohesive relationship established between the luminal mucosal epithelium and the underlying immune compartment in the small intestine. The epithelium provides the first line of defense against pathogens by establishing a physical barrier separating the external environment from the body’s internal milieu, while the immune system secondarily responds to clear bacteria that have breached the epithelial barrier. The HECT E3 ubiquitin ligase ITCH is known to regulate immune responses, and loss of function of ITCH has been associated with gastrointestinal inflammatory disorders. However, the high level of ITCH expression within the intestinal epithelium suggests that it may have an important function(s) in that tissue for maintaining gut homeostasis. Indeed, we identified that global loss of ITCH (Itcha18H/a18H) in young adult animals influenced intestinal architecture characterized by increases in both crypt and villus area that were more prominent in the distal part of the small intestine. Increased crypt area was found to result from expansion of both the proliferating transit amplifying progenitor population and terminally differentiated Paneth cells. Lack of ITCH also resulted in changes in numbers of goblet cells on the villus. Epithelial cell turnover was also accelerated in Itcha18h/a18H animals, as evidenced by increases in both proliferation and apoptosis within the crypt, as well a more rapid cell migration of bromodeoxyuridine-labeled epithelial cells along the crypt-villus axis. Consistent with the observed enhancement of cellular migration, Itcha18H/a18H mice carrying the Min mutation (Itcha18H/a18H; ApcMin/+) displayed a 76% reduction in tumor burden as compared to ApcMin/+ littermates with normal levels of ITCH. To identify which aspects of these changes were cell autonomous, intestinal organoids were generated from the crypts of ITCH sufficient and ITCH deficient animals. Interestingly, epithelial cell proliferation and differentiation were not perturbed in ITCH deficient organoids, in contrast to the in vivo phenotype of the Itcha18H/a18H small intestines. However, increased cell death was observed in organoids lacking ITCH, which was also consistent with increased cleavedcaspase 3 staining in the intestines of mice lacking ITCH exclusively in the intestinal epithelium. The failure to recapitulate the Itcha18H/a18H epithelial phenotype prompted us to investigate how loss of ITCH in immune cells impacts the intestinal epithelium. Animals lacking ITCH within the myeloid cell lineage have similar defects in crypt area, as well as increases goblet and Paneth cell numbers, as compared to the Itcha18H/a18H animals, albeit delayed. These finding highlight a cell autonomous as well as non-cell autonomous function for ITCH in mediating epithelial homeostasis, and emphasize the importance of ITCH in small intestinal barrier function.

Rights

© 2017, Heather L. Mentrup

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