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Characterizing the Role of IL-1β in Macrophage Clearance of Citrobacter rodentium Infection

  • Author / Creator
    Michael Bording-Jorgensen
  • Inflammatory Bowel Diseases (IBD) are a group of conditions characterized by chronic inflammation and a dysregulated immune response of the gastrointestinal tract. The etiology is unknown; however, research has indicated a complex relationship between environmental stimuli, genetic predisposition, and changes in the microbiome. The nod-like receptor protein complex 3 (NLRP3) is part of a cytosolic microbial sensor complex called the inflammasome, involved in the maturation and secretion of the proinflammatory cytokine IL-1β. Some individuals with IBD have a mutation in the NLRP3 gene that causes a hypoproduction of IL-1β, contrasting many other immune-mediated diseases involving the inflammasome. Citrobacter rodentium is a mouse pathogen used as a model for IBD as it causes IBD-like colitis in mice. In previous work, we showed that extraneous IL-1β improved the ability of macrophages to phagocytose C. rodentium in Nlrp3-/- mice and that clearance of infection is dependent on the NLRP3 inflammasome. In my PhD, I investigated the involvement of the NLRP3 inflammasome in the ability of macrophages to clear C. rodentium infection. Furthermore, I investigated how macrophages interact with epithelial cells during infection and how anaerobic bacteria isolated from IBD patients can activate this complex. My hypothesis was that inflammasome activation is required for macrophages to phagocytose and kill Citrobacter rodentium.
    My experimental approach was to infect the mouse macrophage cell lines, J774A.1 and RAW 264.7, with C. rodentium in vitro. Gentamicin protection assays were used to determine intracellular bacterial survival. Nigericin and extracellular ATP were used to stimulate the inflammasome, with YVAD and KCl used as inhibitors. Reactive oxygen species (ROS) expression was analyzed using the fluorescent dye DCFDA with ROS inhibition by DPI, NaC, or Acetovanillone. Gene expression was analyzed using qPCR and proteins were analyzed using Western blot, ELISA, and a Proteome profiler. Immunofluorescence was done using spinning disk confocal microscopy. Epithelial cell line CMT-93 was used in transwells to understand epithelial interaction with macrophages during infection. Tight junction permeability was measured using trans-epithelial electrical resistance with a volt-o-meter. Anaerobic bacteria were collected from patient ileal washes and cultured in an anaerobic chamber using brain heart infusion media. Human monocyte cell line THP-1 was differentiated using phorbol 12-myristate 13-acetate into macrophages, which were then infected with the anaerobic bacteria.
    Nigericin decreased intracellular bacterial survival in RAW 264.7 macrophages; however, these macrophages do not express the ASC protein and thus cannot activate the NLRP3 inflammasome. Therefore, I proposed a model for alternative pathway activated by Nigericin for killing intracellular pathogens. In addition, extracellular ATP decreased intracellular bacterial survival in J774A.1 macrophages through ROS generation. ATP activation caused an increase in cytosolic maturation of IL-1β but no change in secretion. ROS generation induced by ATP-activated inflammasomes was found to be mediated by the mitochondria. Furthermore, supernatant from ATP-activated macrophages was able to induce inflammasome activation and ROS production in naïve macrophages. ATP also caused a cytokine shift towards an anti-inflammatory state after the infection was cleared. In the presence of epithelial cells, these macrophages facilitated increased tight junction recovery. Macrophages were recruited to the epithelial apical membrane during infection and showed expression and production of ZO-1 and claudin 1 tight junction proteins. NLRP3 gene expression was increased in epithelial cells after exposure to extracellular ATP during infection but there was no change in secretion of IL-1β.Anaerobic bacteria isolated from patients with Crohn disease increased secretion of IL-1β and induced ROS generation compared to those isolated from individuals that did not have IBD.
    This thesis describes the complex nature of the NLRP3 inflammasome within macrophages and its interaction with epithelial cells, in the context of the gastrointestinal tract. My in vitro studies reinforce the importance of understanding pathways involved in enteric pathogen clearance by macrophages. Improving our understanding of the molecular pathways within macrophages will aid in regulating and clearing enteric pathogens and possibly explain the unique role of inflammasomes in the gut, in the context of IBD, and why this is different from other settings. This understanding has the potential for development of new therapies targeting the uncontrolled immune activation due to defective bacterial clearance, as seen in IBD.

  • Subjects / Keywords
  • Graduation date
    Fall 2018
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/R3TD9NQ61
  • License
    Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.