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+] versus increased cytosolic [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 (LLME). 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 LLME. Second, agonists for three Ca2+-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 Ca2+ buffer, BAPTA, and the channel blocker, 2-aminoethoxydiphenyl borate (2-APB), 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.

Ca2+ Signaling but Not Store-Operated Ca2+ Entry Is Required for the Function of Macrophages and Dendritic Cells

“…Because 2-APB non-selectively modulates many ion channels besides CRAC channels as discussed above, and BAPTA suppresses all intracellular Ca 2+ signaling, these data collectively suggest that Ca 2+ signals (potentially mediated by P2X7 receptors or Ca 2+ release from ER stores) but not SOCE are required for NLRP3 inflammasome activation. Given recent reports that K + efflux but not Ca 2+ influx is essential for NLRP3 activation (85, 86) further studies if and how Ca 2+ signaling exactly contributes to NLRP3 inflammasome activation are required.…”

Inflammasome activation in the liver: Focus on alcoholic and non-alcoholic steatohepatitis

“…ATP is released by damaged cells, triggering K+ efflux via stimulation of the ATP-gated P2X7 ion receptor [14,15]. High levels of ATP are detected in serum of chronic ethanolfed mice [10].…”

Caspase-8 as an Effector and Regulator of NLRP3 Inflammasome Signaling

“…We recently reported that the kinetics of nigericin-induced propidium 2ϩ influx tracks with accumulation of active caspase-1 as an early index of the change in plasma membrane permeability that defines pyroptosis (34). Intracellular accumulation of the normally impermeant propidum 2ϩ via increased plasma membrane permeability facilitates its intercalation with DNA resulting in increased 540 3 620 nm fluorescence.…”