Insights into the Pathogenesis of Pediatric IBD: Epithelial Barrier, Immune Dysregulation, and the Gut Environment.

• Author / Creator

  Zaidi, Deenaz

• Introduction: Inflammatory bowel diseases (IBD), encompassing Crohn disease (CD) and ulcerative colitis (UC) are on a steadfast global increase, and in particular, are highly prevalent in children in Canada. Current treatment is targeted towards controlling inflammation and to date no certain etiology or cure have been found. Multiple factors play integral roles in disease pathogenesis, such as, dysfunctional epithelial barrier, altered microbial composition, and inappropriate immune responses, but the mechanism leading to disease is still unknown. In my PhD project, I investigated critical, interlinked factors associated with IBD pathogenesis, i.e., the gut barrier, microbial virulence, cytokines, and factors that control inflammation. The objectives of this thesis were to evaluate gap density in pediatric IBD patients, correlate it with changes in cytokines and inflammatory markers, and to establish its prognostic value; to investigate the role of A20, also known as tumor necrosis factor α-induced protein 3 (TNFAIP3), in IBD pathogenesis; and to study the relationship between microbial virulence, gut environment, and the gut barrier. Methods: The experimental approach to address my objectives was achieved through a cohort study of known IBD patients and non-IBD controls between the ages of 3-18 years. The control group included patients undergoing gastroscopy and colonoscopy for indications other than IBD. Epithelial gap density in the duodenum of recruited patients was evaluated using probe-based confocal laser endomicroscopy (pCLE) with fluorescein injected as contrast during endoscopy. Biopsy samples were obtained from the duodenum and terminal ileum and assessed for A20 and associated factors. Duodenal and terminal ileum aspirates were collected for analyses of bacteria and their function and virulence, using metabolomics and in vitro approaches, respectively. Results: Epithelial gaps assessed with pCLE were found to be increased in the duodenum of children with both CD and UC but unrelated to inflammation. This suggests that an altered epithelial barrier is an important systemic feature of pediatric IBD and is not only secondary to inflammation. I was also able to utilize pCLE to assess vascular flow in the duodenum of non-IBD and IBD patients and found capillary flow rates to be significantly higher in the duodenum of UC patients. Results from exploring the role of the TNF-α counteracting protein, A20 in IBD also showed important findings related to a possible mechanism for failure of A20 to down-regulate inflammation in pediatric CD. I found that despite an increase in A20 expression in TI biopsies of CD patients, the protein levels were low. The discrepancy between A20 expression and protein levels was possibly due to the concomitant lower expression of ABIN-1, an A20 accessory protein, and instability of the A20 protein due to lack of post-translational phosphorylation, possibly related to lower expression of IKKβ. Escherichia coli strain LF82 augmented A20 expression, but not A20 protein, suggesting that microbes can hinder the capacity of A20 to limit inflammation. Thus, factors affecting A20 and microbes could negatively impact the protein's anti-inflammatory action in pediatric CD, and contribute towards unremitting inflammation. Effects of intestinal aspirates on microbial invasion potential were evaluated using Gentamicin protection assays by infecting T-84 and Caco-2 cells with various E. coli strains. Invasion potential of some E. coli strains was greatly increased in the presence of intestinal aspirates. Metabolomic analysis of intestinal aspirates identified variation in the quantity of metabolites in the intestinal aspirates of non-IBD and IBD patients, which could possibly explain the invasion potential and virulence factors of bacteria. Conclusion: This thesis has revealed important information about key factors involved in causing and affecting the course of IBD. I found that epithelial gaps are increased in IBD, and that there is dysregulation in factors controlling inflammation. Findings from this work might help in improving treatment strategies, such as medications that enhance the gut barrier and reduce epithelial cell shedding and drugs that target specific proteins and reduce inflammation. Taken together, the results of this thesis show significant findings that are important contributors to our understanding of pediatric IBD pathogenesis, and provide a basis for additional scientific work targeted towards a therapeutic approach.

• Subjects / Keywords

  • IBD, pediatrics
  • INFLAMMATORY BOWEL DISEASES
  • PEDIATRICS

• Graduation date

  Spring 2017

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3FT8DX3Q

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.