The calcium-sensing receptor regulates the NLRP3 inflammasome through Ca2+ and cAMP

Abstract

Mutations in the gene encoding NLRP3 cause a spectrum of autoinflammatory diseases known as cryopyrin-associated periodic syndromes (CAPS). NLRP3 is a key component of one of several distinct cytoplasmic multiprotein complexes (inflammasomes) that mediate the maturation of the proinflammatory cytokine interleukin-1β (IL-1β) by activating caspase-1. Although several models for inflammasome activation, such as K(+) efflux, generation of reactive oxygen species and lysosomal destabilization, have been proposed, the precise molecular mechanism of NLRP3 inflammasome activation, as well as the mechanism by which CAPS-associated mutations activate NLRP3, remain to be elucidated. Here we show that the murine calcium-sensing receptor (CASR) activates the NLRP3 inflammasome, mediated by increased intracellular Ca(2+) and decreased cellular cyclic AMP (cAMP). Ca(2+) or other CASR agonists activate the NLRP3 inflammasome in the absence of exogenous ATP, whereas knockdown of CASR reduces inflammasome activation in response to known NLRP3 activators. CASR activates the NLRP3 inflammasome through phospholipase C, which catalyses inositol-1,4,5-trisphosphate production and thereby induces release of Ca(2+) from endoplasmic reticulum stores. The increased cytoplasmic Ca(2+) promotes the assembly of inflammasome components, and intracellular Ca(2+) is required for spontaneous inflammasome activity in cells from patients with CAPS. CASR stimulation also results in reduced intracellular cAMP, which independently activates the NLRP3 inflammasome. cAMP binds to NLRP3 directly to inhibit inflammasome assembly, and downregulation of cAMP relieves this inhibition. The binding affinity of cAMP for CAPS-associated mutant NLRP3 is substantially lower than for wild-type NLRP3, and the uncontrolled mature IL-1β production from CAPS patients' peripheral blood mononuclear cells is attenuated by increasing cAMP. Taken together, these findings indicate that Ca(2+) and cAMP are two key molecular regulators of the NLRP3 inflammasome that have critical roles in the molecular pathogenesis of CAPS.

Figure 1. Extracellular calcium and ATP activate the NLRP3 inflammasome through CASR

a, b, LPS-primed BMDMs from wild-type (WT) or designated knockout mice were stimulated in RPMI medium supplemented with the indicated reagents. c, BMDMs were transiently transfected with scrambled (N.C.), Nlrp3, Asc, or Casr siRNA, and then stimulated with 2 mM ATP or 1 mM CaCl₂ after priming with LPS. Cell culture supernatants (Sup), cell lysates (Lys), and crosslinked pellets from whole-cell lysates were analysed by immunoblotting as indicated (a–c). All immunoblot data shown are representative of at least three independent experiments.

Figure 2. PLC-InsP₃-mediated calcium release from the ER triggers NLRP3 inflammasome activation

a–d, f, LPS-primed BMDMs were treated as indicated. Intracellular IP-one, [Author: Please give IP-one in full. inositol monophosphate]a surrogate for InsP₃ levels, was measured (c). Data represent the mean ± s.e.m. from three independent experiments. e, BMDMs transiently transfected with scrambled (N.C.) or Ip3r1 siRNA were primed with LPS and stimulated with 5 mM ATP or 1 mM CaCl₂. g, LPS-primed BMDMs were immunoprecipitated with anti-ASC antibody in buffer containing no additives (Non), 1 mM CaCl₂, or 1 mM EGTA. Supernatants and lysates were analysed by immunoblotting. Data are representative of three independent experiments.

Figure 3. cAMP binds to NLRP3 and suppresses inflammasome activation

a–e, LPS-primed BMDMs were treated as indicated. IL-1β secretion (b–d), ASC–NLRP3 interaction (e), or intracellular cAMP levels (a–c) were analysed. Data represent the mean ± s.e.m. from five experiments. f, BMDMs transfected with scrambled or Adcy3 + Adcy6 + Adcy7 + Adcy9 siRNAs were analysed for ASC oligomerization after LPS priming. g, LPS-primed BMDMs were pulled down with indicated beads and analysed by immunoblot for NLRP3. h, Indicated NLRP3 proteins were expressed in 293T cells and pulled down with cAMP beads. LRR, leucine-rich repeats; NBD, nucleotide-binding domain; PYD, PYRIN domain. Data are representative of three independent experiments.

Figure 4. The role of cAMP and calcium in the pathogenesis of CAPS

a, Wild-type or CAPS-associated mutant NLRP3 proteins were expressed in 293T cells and pulled down with cAMP beads. Densitometric analysis of cAMP-associated NLRP3 bands, normalized to the NLRP3 in lysates, is shown in the histogram. Data represent the mean ± s.e.m. of the seven experiments. b–e, PBMCs from CAPS patients with the designated mutations in NLRP3 were primed with LPS and treated with the indicated dose of forskolin (b), Ro-20-1724 or (R)-(-)-rolipram (c), 2-APB (d), or BAPTA-AM (e). Lysates and supernatants were analysed for IL-1β secretion by immunoblot.