K+ efflux agonists induce NLRP3 inflammasome activation independently of Ca2+ signaling1

Perturbation of intracellular ion homeostasis is a major cellular stress signal for activation of NLRP3 inflammasome signaling that results in caspase-1 mediated production of IL-1β and pyroptosis. However, the relative contributions of decreased cytosolic [K + ] versus increased cytosolic [Ca 2+ ] remain disputed and incompletely defined. We investigated roles for elevated cytosolic [Ca 2+ ] in NLRP3 activation and downstream inflammasome signaling responses in primary murine dendritic cells and macrophages in response to two canonical NLRP3 agonists (ATP and nigericin) that facilitate primary K + efflux by mechanistically distinct pathways or the lysosome-destabilizing agonist Leu-Leu-O-methyl ester (LLME). The study provides three major findings relevant to this unresolved area of NLRP3 regulation. First, increased cytosolic [Ca 2+ ] was neither a necessary nor sufficient signal for the NLRP3 inflammasome cascade during activation by endogenous ATP-gated P2X7 receptor channels, the exogenous bacterial ionophore nigericin, or the lysosomotropic agent LLME. Second, agonists for three Ca 2+ -mobilizing G protein-coupled receptors (formyl peptide receptor/FPR; P2Y2 purinergic receptor/P2Y2R; calcium-sensing receptor/CaSR) expressed in murine dendritic cells were ineffective as activators of rapidly induced NLRP3 signaling when directly compared to the K + efflux agonists. Third, the intracellular Ca 2+ buffer, BAPTA, and the channel blocker, 2-aminoethoxydiphenyl borate (2-APB), widely used reagents for disruption of Ca 2+ -dependent signaling pathways, strongly suppressed nigericin-induced NLRP3 inflammasome signaling via mechanisms dissociated from their canonical or expected effects on Ca 2+ homeostasis. The results indicate that the ability of K + efflux agonists to activate NLRP3 inflammasome signaling can be dissociated from changes in cytosolic [Ca 2+ ] as a necessary or sufficient signal.