Critical role for calcium mobilization in activation of the NLRP3 inflammasome

Abstract

The NLRP3 (nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3) inflammasome mediates production of inflammatory mediators, such as IL-1β and IL-18, and as such is implicated in a variety of inflammatory processes, including infection, sepsis, autoinflammatory diseases, and metabolic diseases. The proximal steps in NLRP3 inflammasome activation are not well understood. Here we elucidate a critical role for Ca(2+) mobilization in activation of the NLRP3 inflammasome by multiple stimuli. We demonstrate that blocking Ca(2+) mobilization inhibits assembly and activation of the NLRP3 inflammasome complex, and that during ATP stimulation Ca(2+) signaling is pivotal in promoting mitochondrial damage. C/EPB homologous protein, a transcription factor that can modulate Ca(2+) release from the endoplasmic reticulum, amplifies NLRP3 inflammasome activation, thus linking endoplasmic reticulum stress to activation of the NLRP3 inflammasome. Our findings support a model for NLRP3 inflammasome activation by Ca(2+)-mediated mitochondrial damage.

Fig. 1.

Extracellular ATP mobilizes Ca²⁺ to activate the NLRP3 inflammasome. (A) LPS-primed BMDMs were loaded with Fura-2 followed by stimulation with 1 mM ATP and analysis of Ca²⁺ flux by time-lapse microscopy. Ca²⁺ is released from intracellular stores during the initial stimulation in Ca²⁺-free buffer, and exchange to 1 mM Ca²⁺-containing buffer permits extracellular Ca²⁺ influx. (B and C) LPS-primed BMDMs were treated or not with Tg for 30 min to deplete ER Ca²⁺ (B) or switched to Ca²⁺ free (−) or Ca²⁺-containing (+) media immediately before ATP stimulation (C). NLRP3 inflammasome activation was assessed by Western blotting of lysates and supernatants (supes) as well as IL-1β ELISA. (D–F) BMDMs were pretreated with XeC, U73122, or 2-APB, followed by ATP stimulation and analysis of Ca²⁺ flux (D) and NLRP3 inflammasome activation (E and F).

Fig. 2.

Ca²⁺ signaling may be a common mechanism for NLRP3 inflammasome activation. (A and B) LPS-primed BMDMs stimulated with nigericin (A) or MSU (B) were analyzed by Ca²⁺ imaging. In B, buffer exchange to remove (0 Ca²⁺) and replace Ca²⁺ (1 mM Ca²⁺) indicates contribution of store-operated Ca²⁺ entry to Ca²⁺ flux. (C–F) BMDMs stimulated as indicated were examined for NLRP3 inflammasome activation.

Fig. 3.

Ca²⁺ signaling promotes mitochondrial damage during ATP stimulation. (A) BMDMs were stimulated with ATP in the presence of 40 mM K⁺ (hK⁺) or Na⁺ (hNa⁺), followed by analysis of NLRP3 inflammasome activation. (B) Ca²⁺ imaging of ATP-stimulated BMDMs in the presence of hK⁺ or hNa⁺ buffers. Fold-increase represents the ratio of maximal fluorescence to baseline fluorescence. Ionomycin stimulation was included as a control for Fura-2 loading. (C) BMDMs were pretreated with Leu-Leu-OMe or not (control), followed 1 h later by stimulation with 100 μM ATP in Ca²⁺-free HBSS (white arrow) to induce ER Ca²⁺ release through the P2Y receptor. Stimulation with the Ca²⁺ ionophore Ionomycin (black arrow) mobilized Ca²⁺ from distinct Ca²⁺ stores resistant to Leu-Leu-OMe and P2YR activation and served as a positive control for Fura-2 loading. (D and E) BMDMs, stimulated as indicated, were examined for NLRP3 inflammasome activation. (F and G) BMDMs stimulated as indicated were stained with MitoSOX (F) or Mitotracker Green and Deep Red (G) followed by flow cytometry analysis.

Fig. 4.

CHOP amplifies NLRP3 inflammasome activation. (A) WT and Chop^−/− BMDMs were examined for inflammasome activation. (B) Ca²⁺ flux in ATP-stimulated WT and Chop^−/− BMDMs was examined by Ca²⁺ imaging. (C) WT and Chop^−/− BMDMs were pretreated with XeC or not followed by ATP stimulation. (D) WT and Chop^−/− mice were injected with LPS to induce sepsis, followed by analysis of serum cytokine levels. ns, not significant.

Fig. 5.

Model. Our results support a model in which Ca²⁺ signaling is critical for NLRP3 inflammasome activation. In response to ATP and perhaps other stimuli, Ca²⁺ mobilization from ER stores and the extracellular space triggers mitochondrial damage, including increased mROS production, loss of membrane potential, and release of mtDNA into the cytosol.