A Primary Epithelial-Immune Cell Co-Culture Model to Investigate the Interactions between the Intestinal Epithelium and Innate Immune Cells in Response to Clostridioides difficile toxins
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2024-01-04
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Abstract
Clostridioides difficile is a leading cause of healthcare-associated diarrhea in the United States, with symptoms ranging from mild diarrhea to toxic megacolon. Upon disruption of the colonic epithelium by C. difficile toxins A and B (TcdAB), epithelial cells are thought to signal to underlying immune cells including group-3 innate lymphoid cells (ILC3s) and macrophages to mount an effective immune response. ILC3s and macrophages both modulate pathogen defense (including C. difficile), tissue repair, and inflammation. ILC3s do so largely through interleukin- 22 (IL-22) secretion, and macrophages do so through phagocytosis and the secretion of a wide array of cytokines and chemokines. However, pathways by which the intestinal epithelium responds to C. difficile TcdAB to affect underlying immune cells, such as ILC3s and macrophages, remain poorly understood. We therefore hypothesized that the response to C. difficile toxins throughout the colonic epithelium and underlying tissue is modulated by interactions between the intestinal epithelium, ILC3s and IL-22, and macrophages. To test this hypothesis and address the limitations of cancer cell lines and single lineage models, we developed two novel primary epithelial cell-innate immune cell co-culture models to investigate epithelial-immune interactions in response to C. difficile toxins in vitro. Co-culture of monolayers with either MNK-3 (ILC3-like) cells or bone marrow derived macrophages did not influence monolayer permeability in response to TcdAB. However, in the process of characterizing these models, we established that MNK-3 cells and macrophages remained viable in co-culture and released relevant mediators with respective stimulation. While MNK-3 cells were not activated in co-culture, macrophages were activated by both monolayer media and epithelial presence in co-culture. With these novel experiments, we have provided groundwork for the further development of multi-lineage models.
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Smith, L. (2024). A primary epithelial-immune cell co-culture model to investigate the interactions between the intestinal epithelium and innate immune cells in response to Clostridioides difficile toxins (Master's thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.