Browsing by Author "Mody, Christopher Hugh"

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    NK cell killing of extracellular Pseudomonas aeruginosa
    (2020-06-26) Feehan, David; Mody, Christopher Hugh; Cobo, Eduardo R.; Harrison, Joe J.
    Pseudomonas aeruginosa is an opportunistic pathogen that commonly infects individuals with the respiratory illness, Cystic Fibrosis and contributes to airway blockage and loss of lung function. NK cells are cytotoxic, granular lymphocytes that are part of the innate immune system. NK cell secretory granules contain the cytolytic proteins granulysin, perforin and granzymes. In addition to their cytotoxic effects on cancer and virally infected cells, NK cells have been shown to play a role in an innate defense against microbes. The hypothesis of this project is that NK cells directly kill extracellular P. aeruginosa using NK effector molecules. Live cell imaging of a co¬-culture of YT cells, a human NK cell line, and GFP P. aeruginosa stained with the membrane viability dye propidium iodide, demonstrated that YT cell killing of P. aeruginosa is contact mediated. CRISPR knockout of granulysin or perforin in YT cells had no significant effect on YT cell killing of P. aeruginosa, as determined by CFU count. Pre-treatment of YT and NK cells with the serine protease inhibitor 3,4-dichloroisocoumarin (DCI) to inhibit all granzymes, resulted in an inhibition of killing. Although singular CRISPR knockout of granzyme B or H had no effect, knockout of both in YT cells completely abrogated killing of PAO1 in comparison to wild type controls. This demonstrates that granzymes are required for killing, but exhibit redundancy in their function. In summary, these results suggest that NK cell kill P. aeruginosa through a membrane damaging, contact-dependent process, requiring granzymes.
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    Subversion of dendritic cell immunity to Cryptococcus gattii by a novel phagosomal F-actin cage structure
    (2020-04-28) Jamil, Khusraw; Mody, Christopher Hugh; Ganguly, Anutosh; Amrein, Matthias W.; Yates, Robin Michael; Yong, Voon Wee
    The highly virulent fungus, Cryptococcus gattii, emerged as a novel respiratory pathogen on Vancouver Island (British Columbia, Canada) nearly two decades ago and has spread to the surrounding regions encompassing the Pacific Northwest of United States, where there is an ongoing outbreak. C. gattii is a major cause of life-threatening cryptococcosis in immunocompetent individuals and has a mortality rate of up to 33%. Host immune response is a key determining factor for the development of cryptococcal disease. It is now recognized that evasion of host immune recognition is a hallmark of C. gattii pathogenesis, but the mechanism of immune evasion remains unclear. There is increasing evidence that C. gattii subverts dendritic cell (DC) activation to evade the protective T helper cell-mediated immunity. This thesis demonstrates that primary human DC can phagocytose C. gattii yeasts but trafficking to the late phagolysosome is blocked by retention of a filamentous actin (F-actin) cage on the phagosomes. Structural studies by super resolution microscopy revealed a novel, highly branched F-actin cage that physically interfered with lysosomal fusion. C. gattii F-actin cage promoted immune evasion by silencing the canonical RelA signaling of the NF-κB pathway required for DC costimulation and T cell activation. Disruption of the F- actin cage through targeted inhibition or by TNF-α signaling reprogrammed quiescent DC to immunocompetent antigen-presenting cells (APCs). Furthermore, the presence of phagosomal F-actin cage corresponded with the presence of C. gattii polysaccharide capsule. Acapsular mutant strains did not retain phagosomal F-actin and were remarkable at inducing DC activation and T cell proliferation. Collectively, our results have uncovered a unique mechanism of DC immune subversion by intracellular pathogens such as hypervirulent C. gattii. Manipulations of this mechanism can potentially inform novel therapeutic interventions against C. gattii.
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    The Effect of COVID-19 on Natural Killer Cell Function
    (2024-06-26) Dagar, Arushi; Mody, Christopher Hugh; Corcoran, Jennifer A.; Jenne, Craig
    COVID-19 has caused more than 7 million deaths, and according to the World Health Organization, it continues to result in more than 1000 reported deaths per week at the time of this writing. Therefore, it is crucial to understand the immune response to COVID-19 since the virus has the potential to become endemic, like influenza A. Natural killer (NK) cells are essential for immune defence against viral infections and play a critical role in COVID-19. While it is well documented that infected patients have a reduction in lymphocytes and NK cells, gaps in knowledge exist regarding the function of NK cells. To study the function of NK cells in COVID-19 patients, peripheral blood was obtained from patients admitted to the medical (non-ICU) wards at the Foothills Medical Centre with a positive COVID-19 test. I demonstrated a decrease in the mature cytotoxic subset of NK cells within the peripheral blood of patients hospitalized with COVID-19. I also observed a notable decrease in the cytotoxic function of NK cells against tumour targets. I examined the sequence of events within NK cells that lead to killing in a stepwise manner. I found reductions in the intracellular levels of effector molecules, the degranulation of cytotoxic granules, and the extracellular concentrations of released effector molecules. I identified alterations in intracellular granule trafficking required to position the granules for release. I found alterations in the expression of multiple NK cell receptors, suggesting inhibitory signalling. Additionally, males with COVID-19 showed more pronounced NK cell defects than healthy males, which may partly be due to receptor expressions. My findings highlight defects in cytolytic effector molecules, granule trafficking and release, and increased expression of inhibitory receptors in hospitalized COVID-19 patients, in addition to a sex difference in cytolytic function, which contributes to defective NK cell function in COVID-19.