• Information Technology
  • Human Resources
  • Careers
  • Giving
  • Library
  • Bookstore
  • Active Living
  • Continuing Education
  • Go Dinos
  • UCalgary Maps
  • UCalgary Directory
  • Academic Calendar
My UCalgary
Webmail
D2L
ARCHIBUS
IRISS
  • Faculty of Arts
  • Cumming School of Medicine
  • Faculty of Environmental Design
  • Faculty of Graduate Studies
  • Haskayne School of Business
  • Faculty of Kinesiology
  • Faculty of Law
  • Faculty of Nursing
  • Faculty of Nursing (Qatar)
  • Schulich School of Engineering
  • Faculty of Science
  • Faculty of Social Work
  • Faculty of Veterinary Medicine
  • Werklund School of Education
  • Information TechnologiesIT
  • Human ResourcesHR
  • Careers
  • Giving
  • Library
  • Bookstore
  • Active Living
  • Continuing Education
  • Go Dinos
  • UCalgary Maps
  • UCalgary Directory
  • Academic Calendar
  • Libraries and Cultural Resources
View Item 
  •   PRISM Home
  • Graduate Studies
  • The Vault: Electronic Theses and Dissertations
  • View Item
  •   PRISM Home
  • Graduate Studies
  • The Vault: Electronic Theses and Dissertations
  • View Item
JavaScript is disabled for your browser. Some features of this site may not work without it.

Defining the Impact of Metabolic Stress and the loss of NOD2 in Epithelial-microbial Interaction

Thumbnail
Download
PhD Thesis (6.363Mb)
Advisor
McKay, Derek Mark
Author
Saxena, Alpana
Committee Member
Armstrong, Glen Douglas
MacNaughton, Wallace Keith
Devinney, Rebekah P
Gibson, Deanna
Other
Barrier
NOD2
Commensal bacteria
epithelium
mitochondria
Subject
Anthropology--Medical and Forensic
Microbiology
Type
Thesis
Metadata
Show full item record

Abstract
Reduced ATP levels and irregular mitochondrial structures have been found in gut epithelia in some IBD patients, suggesting that metabolic stress could be an etiologic factor. At the same time, Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) depletion is an important susceptibility trait for IBD. As a result, in our study, we assessed if loss of NOD2 further reduces epithelial barrier integrity under conditions of metabolic stress, instigated by the addition of the oxidative phosphorylation un-coupler, dinitrophenol (DNP). T84 (human colon cell line) cells treated with non-invasive E. coli + DNP (16 hrs) showed a significant increase in NOD2 protein. The increase in intracellular bacteria in both wild-type (WT) and NOD2 knock-down cells (NOD2 KD) was dependent on reactive oxygen species (ROS) and resulted in MAPK ERK 1/2 pathway activation; this was determined by adding an antioxidant (mitoTEMPO) or an ERK (U0126) inhibitor. While there was an increase in internalized E. coli in cells, no change was seen in the internalization of bead levels or dead E. coli suggesting that reduced killing of the bacteria was the primary cause for the increased numbers of bacteria in the NOD2 KD epithelia. This conclusion is reinforced by evidence of attenuated autophagy in NOD2 KD T84 cells. Neutrophils are potent contributors to IBD. IL-8 is a major neutrophil chemoattractant and signaling molecule; IL-8 levels have been shown to be elevated in IBD patients. As such, we assessed whether metabolic stress induced heightened IL-8 secretion. T84 cells exposed to DNP yielded heightened IL-8 secretion after 16 hrs compared to cells treated with E. coli alone in a time-dependent manner. Secretion significantly declined after mitoTEMPO treatment. Bacteria- derived products are necessary for IL-8 secretion; no response was seen to bead co-treatment, but persisted after T84 cells were treated with dead E. coli. IL-8 secretion is dependent on endosome maturation and cytoskeletal rearrangement; administration of chloroquine or cytochalsin D both significantly reduced IL-8 levels. Finally, IL-8 secretion is MyD88 dependent, implicating TLR signaling in the epithelial cell response to metabolic stress in a commensal rich environment. Taken together, we offer a three-hit hypothesis: lowered barrier function, as a result of metabolic dysfunction is amplified in the absence of NOD2, resulting in increased numbers of live intracellular bacteria. Bacterial presence in the intestinal epithelial cells provokes the release of the neutrophil chemotactic chemokine, IL-8, resulting in inflammation. In NOD2 depleted patients, greater levels of internalized bacteria yield a potent inflammatory effect, given that the antimicrobial effects of both macrophages and Paneth cells are compromised.
Corporate
University of Calgary
Faculty
Graduate Studies
Doi
http://dx.doi.org/10.5072/PRISM/25538
Uri
http://hdl.handle.net/11023/4168
Collections
  • The Vault: Electronic Theses and Dissertations

Browse

All of PRISMCommunities & CollectionsBy Issue DateAuthorsTitlesSubjectsThis CollectionBy Issue DateAuthorsTitlesSubjects

My Account

LoginRegister

Statistics

Most Popular ItemsStatistics by CountryMost Popular Authors

  • Email
  • SMS
  • 403.220.8895
  • Live Chat

Energize: The Campaign for Eyes High

Privacy Policy
Website feedback

University of Calgary
2500 University Drive NW
Calgary, AB T2N 1N4
CANADA

Copyright © 2017