Date of Award

2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Immunology

First Advisor

De'Broski R. Herbert

Abstract

The gastrointestinal (GI) epithelium is a wide array of cell lineages that

form a dynamic barrier to the external environment that regulates nutrient absorption, immunity and a physical barrier against organisms entering the intestinal lamina propria. However, the specific proteins expressed by epithelia to maintain GI function during homeostasis and inflammation and to promote tissue repair following injury are incompletely understood. This body of work demonstrates that one member of the Leucine rich repeat Ig domain containing nogo interacting protein family (LINGO) called LINGO3 serves a critical and previously unappreciated role in maintaining the intestinal barrier under homeostasis and in promoting tissue regeneration following acute tissue injury in the GI tract. Under steady-state conditions in co-housed animals, Lingo3 gene deficient (Lingo3 KO) mice have a marked dysregulation in the architecture of the adherens junction complex between small and large intestinal epithelial cells, which correlated with increased intestinal permeability and serum endotoxin levels. This mucosal barrier defect was also associated with a significant increase in Type 1 cytokines and pro-inflammatory myeloid lineage antigen presenting cells. Interestingly, Lingo3 deficiency did not confer resistance or susceptibility to the enteropathogenic bacterium Citrobacter rodentium. However, when Lingo3 KO mice were exposed to the dextran sodium sulfate (DSS) model of colitis, they developed significantly worse disease than co-housed wild-type (WT) controls as defined by overall morbidity, clinical score, reduced colon length, and histological changes, particularly during the recovery phase of DSS. Curiously, this basal defect and DSS recovery phase defect was remarkably similar to the phenotype of mice genetically deficient in Trefoil factor 2 (Tff2), a goblet cell-derived cytokine known to promote tissue repair. This dissertation also demonstrates that treatment of DSS-treated WT mice with a long-acting agonist of TFF2 (TFF2-Fc) promotes faster tissue recovery than Lingo3 KO mice. This enhanced tissue recovery mediated by TFF2-Fc was associated with a greater number of intestinal epithelial cells showing phosphorylation of ERK, which is a key transcription factor in the MAPK pathway that promotes cellular proliferation. To directly test whether LINGO3 and TFF2 functioned to drive epithelial cell regeneration distinct from their impact upon inflammation, we generated small and large intestinal organoids. Results show that both Lingo3 deficient and Tff2 deficient organoids had defective growth kinetics and impaired architectural structure. Strikingly, both Lingo3 deficient and Tff2 deficient small intestinal organoids had decreased mRNA transcript levels of the intestinal stem cell marker, Lgr5, implying that defects in stem cell abundance and/or function. In conclusion, the body of work comprising this dissertation demonstrates that LINGO3 serves a previously unappreciated role in mucosal epithelial cell barrier function under homeostasis and during tissue repair following colitic injury. This role partially functions through the reparative cytokine TFF2, which collectively warrants further investigation into how this putative TFF2-LINGO3 axis regulates enterocyte and intestinal stem cell function during health and disease.

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