Towards optimizing ATP-induced activation of the NLRP3 inflammasome in glycolysis-inhibited J774A.1 murine macrophages

Abstract

Persistent inflammatory reactions underlie many of the physiological changes observed during aging. During the aging process, levels of self-antigens such as extracellular ATP increase and induce homeostatic changes as well as the activation of the NLRP3 inflammasome, a multi-protein complex containing the NLRP3 sensor protein which becomes activated in response to foreign pathogens and tissue damage. Aberrant inflammasome activation contributes to the aging process and has been linked to metabolic disturbances, though the exact mechanism for this remains unclear. An exploration of the link between cellular metabolic changes and inflammatory responses to age-associated molecules would provide further insight into the interplay between chronic inflammation and aging. In this study, we attempted to describe this interaction by inhibiting glycolysis in J774A.1 murine macrophages with 2-deoxyglucose (2-DG) and glucose 6-phosphate (G6P), and quantifying NLRP3 inflammasome activation using the production of cleaved IL-1β. Through Western blot, ELISA, and cell morphology analysis, we observed pro-IL-1β upregulation following LPS priming of J774A.1 cells, but were unable to detect cleaved IL-1β and observe distinctive cell morphology changes following ATP treatment. Glycolysis inhibition using 2-DG and G6P was also unsuccessful, as confirmed via a glycolysis assay.