Investigating the role of ADORA2B receptors in pyroptosis of inflammatory macrophages through gene knockdown and overexpression

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Deuna, Fatima Almira
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Adenosine receptors (ARs) regulate the immune response by activating pro- and anti- inflammatory cascades through mechanisms that are not completely defined. Macrophages also play a central role in the regulation of inflammation from initiation to resolution. ARs on macrophages have been shown to direct major functions including macrophage cell death. Pyroptosis is a highly immunogenic cell death induced by inflammasomes that cause cleavage of caspases and gasdermins. This process leads to the release of inflammatory cytokines Interleukin 18 (IL-18) and Interleukin -1β (IL-1β). The canonical pathway that leads to pyroptosis involves the NLRP3 (nucleotide-binding domain, leucine-rich–containing family, pyrin domain–containing-3) inflammasome, caspase-1 and gasdermin-D (GSDMD), though other pathways exist. Recently, our lab found that knocking down the Adenosine 2B (A2B) receptor renders macrophages susceptible to pyroptosis. In this thesis, we seek to confirm the role of the NLRP3 inflammasome specifically in pyroptosis observed after A2B receptor knockdown, and whether lost receptor-protein interactions promote this pathway. Results show that in A2B receptor deficient macrophages, NLRP3 inhibition was able to prevent IL-1β secretion and GSDMD cleavage. However, addition of adenosine to macrophages induced GSDMD cleavage that was not sensitive to NLRP3 inhibition. Furthermore, preliminary experiments showed that caspase-4 was activated by adenosine. Proteomic analysis was used to study receptor-protein interactions and pyroptotic proteins were identified in immunoprecipitation assays. A2B coimmunoprecipitation in epithelial cells and macrophages show that A2B receptors enrich and interact with cellular communication, calcium signaling and gap-junction proteins as well as Toll-like receptor signaling, and serine proteases. Results from proteomics and gene ontology analysis have identified possible alternative pathways through which the A2B receptor might influence macrophage pyroptosis.
Adenosine receptors, Immune response, Pyroptosis, Macrophages