A190 ATP-INDUCED INFLAMMASOME ACTIVATION GENERATES MITOCHONDRIAL ROS PRODUCTION IN MACROPHAGES. (4th March 2021)

• Record Type: Journal Article
• Authors: Bording-Jorgensen, M; Armstrong, H; Wine, E
• Is Part Of: Journal of the Canadian Association of Gastroenterology. Volume 4(2021)Supplement 1
• Page Start: 206
• Page End: 207
• Publication Date: 2021-03-04
• Subjects: Gastroenterology -- Periodicals; 616.33005
• DOI: 10.1093/jcag/gwab002.188
• Languages: English
• ISSNs: 2515-2084

Abstract

Abstract: Background: The etiology of Inflammatory Bowel Diseases is unknown; however, a dysfunctional immune response has been well characterized. The role of the NLRP3 inflammasome in IBD is controversial with both beneficial and detrimental results. This pathway is required for the secretion of the proinflammatory cytokine IL-1β. Extracellular ATP is a well characterized inflammasome activator, which we have previously shown can increase the ability of J774A.1 macrophages to clear the mouse pathogen Citrobacter rodentium in an in vitro environment through the generation of reactive oxygen species (ROS). Aims: Our objectives were to determine: 1) if extracellular ATP was inducing mitochondrial stress, causing the production of ROS, leading to microbial death; and 2) what effects these activated macropahges have on naïve macrophages. Methods: Murine macrophage J774A.1 cells were infected with C. rodentium ; extracellular ATP was added as an inflammasome activator and YVAD as an inhibitor. Lysotracker red and MitoSOX were used to determine cellular location of bacteria and quantify mitochondrial ROS, respectively. Secreted cytokines were measured using ELISA and a proteome profiler, ROS was measured using DCFDA, Gasdermin D and Caspase 11 activities were determined by Western Blot. Supernatants taken from infected macrophages were filtered and then added to naïve macrophages during infection with C. rodentium . Results: Activation of mitochondrial ROS by ATP was found to be independent of infection. Secreted cytokines sICAM-1, MIP-1α, and MCP-2 were all increased by ATP but not inhibited by YVAD. Cleavage of Gasdermin D was increased with the addition of ATP but not inhibited by YVAD whereas Caspase 11 was unchanged between treatments. Supernatants from ATP-induced macrophages were able to induce IL-1β secretion in naïve macrophages and increase bacterial killing. Conclusions: Mitochondrial ROS production in response to extracellular ATP may be involved in the decrease of bacterial survival. ATP induces the secretion of cytokines, chemokines, and other factors that affect newly infected macrophages. Gasdermin D cleavage, independent of caspase 11, suggests that a noncannonical pathway is activated; this may explain the lack of pyroptotic cells in our study. In addition, we have shown that these macrophages are able to illicit the same behavior in naive macrophages, suggesting that a corrected dysfunctional pathway in macrophages can have beneficial effects downstream. Understanding how the NLRP3 inflammasome is activated and what the downstream pathways are may lead to potential therapies for inflammatory conditions, including IBD. Funding Agencies: CCC, CIHR