K+ Efflux Agonists Induce NLRP3 Inflammasome Activation Independently of Ca2+ Signaling

Abstract

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+ concentration versus increased cytosolic Ca2+ concentration ([Ca2+]) remain disputed and incompletely defined. We investigated roles for elevated cytosolic [Ca2+] 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. The study provides three major findings relevant to this unresolved area of NLRP3 regulation. First, increased cytosolic [Ca2+] 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 Leu-Leu-O-methyl ester. Second, agonists for three Ca2+-mobilizing G protein–coupled receptors (formyl peptide receptor, P2Y2 purinergic receptor, and calcium-sensing receptor) expressed in murine dendritic cells were ineffective as activators of rapidly induced NLRP3 signaling when directly compared with the K+ efflux agonists. Third, the intracellular Ca2+ buffer, BAPTA, and the channel blocker, 2-aminoethoxydiphenyl borate, widely used reagents for disruption of Ca2+-dependent signaling pathways, strongly suppressed nigericin-induced NLRP3 inflammasome signaling via mechanisms dissociated from their canonical or expected effects on Ca2+ homeostasis. The results indicate that the ability of K+ efflux agonists to activate NLRP3 inflammasome signaling can be dissociated from changes in cytosolic [Ca2+] as a necessary or sufficient signal.