Investigating the role of calcium ion mobilization in NLRP3 inflammasome activation of J774A.1 macrophages and optimizing cell morphology and IL-1β quantification protocols

Abstract

The nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3 (NLRP3) inflammasome is responsible for sensing and regulating the inflammatory response. When activated, NLRP3 inflammasomes can upregulate inflammatory cytokines such as IL-1β and IL-18, which are often associated with changes in cell morphology and can lead to pyroptosis. NLRP3 activation is also regulated by several upstream events such as calcium mobilization, but explicit mechanisms of activation have yet to be resolved. To investigate the effect of calcium mobilization on NLRP3 activation, we quantified changes in cell morphology and IL-1β release in response to LPS and ATP. LPS-primed J774A.1 cells were stimulated with 5mM or 2mM ATP for various lengths of time, and we used phase-contrast microscopy to visualize cell morphology changes as well as protein-based assays to quantify IL-1β release. We observed cell morphology changes in response to stimulation, and areas for troubleshooting and further optimization were identified for these methods of quantification. However, we were not able to detect the release of cleaved IL-1β. Future studies could explore more suitable conditions for LPS and ATP stimulation and formally investigate the role of calcium in inflammasome activation.