Critical role for calcium mobilization in activation ofthe NLRP3 inflammasomeTomohiko Murakamia,1, Johan Ockingera,1, Jiujiu Yua,2, Vanessa Bylesa,2, Aisleen McColla, Aldebaran M. Hoferb,and Tiffany Hornga,3

aDepartment of Genetics and Complex Diseases, Harvard School of Public Health, Boston, MA 02115; and bDepartment of Surgery, Brigham and Woman’sHospital and Harvard Medical School, and Veteran’s Administration Boston Healthcare System, West Roxbury, MA 02132

Edited by Jenny P. Y. Ting, University of North Carolina at Chapel Hill, Chapel Hill, NC, and accepted by the Editorial Board May 25, 2012 (received for reviewOctober 28, 2011)

The NLRP3 (nucleotide-binding domain, leucine-rich-repeat-containingfamily, pyrin domain-containing 3) inflammasome mediates pro-duction of inflammatory mediators, such as IL-1β and IL-18, and assuch is implicated in a variety of inflammatory processes, includinginfection, sepsis, autoinflammatory diseases, and metabolic dis-eases. The proximal steps in NLRP3 inflammasome activation arenot well understood. Here we elucidate a critical role for Ca2+

mobilization in activation of the NLRP3 inflammasome by multiplestimuli. We demonstrate that blocking Ca2+ mobilization inhibitsassembly and activation of the NLRP3 inflammasome complex, andthat during ATP stimulation Ca2+ signaling is pivotal in promotingmitochondrial damage. C/EPB homologous protein, a transcriptionfactor that can modulate Ca2+ release from the endoplasmic re-ticulum, amplifies NLRP3 inflammasome activation, thus linkingendoplasmic reticulum stress to activation of the NLRP3 inflamma-some. Our findings support a model for NLRP3 inflammasome acti-vation by Ca2+-mediated mitochondrial damage.

innate immunity | mitochondria

Inflammation must be tightly regulated to meet the demands ofhost defense and stress adaptation while limiting immunopa-

thology and the detrimental effects of chronic inflammation.Sensors of the innate immune system, such as pattern recogni-tion receptors, couple detection of proinflammatory triggers toinduction of inflammation in both adaptive and maladaptivesettings, so the regulation of this interaction is absolutely criticalin control of inflammation (1). A particularly intriguing “sensor”is the NLRP3 (nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3) inflammasomecomplex, which detects cellular stress in a variety of infectious and“sterile” settings and links this recognition to the induction ofa critical inflammatory pathway (2–5).The NLRP3 inflammasome is a cytoplasmic complex consist-

ing of the regulatory subunit NLRP3, the adaptor ASC, and theeffector subunit caspase-1. Stimulus dependent assembly of thecomplex activates the proteolytic activity of caspase-1, which isrequired for the processing and secretion of the inflammatorycytokines IL-1β and IL-18 (2–5). In recent years, many differentmolecules have been shown to activate the NLRP3 inflamma-some complex. These molecules include: extracellular ATP re-leased from dying cells (6); alum, the adjuvant activity in humanvaccines (7); nigericin, a bacterial toxin with potassium iono-phore activity (6); and cholesterol crystals (8), which contribute tothe development of atherosclerotic plaques. Such findings under-score the critical role of the NLRP3 inflammasome in inducinginflammation in a variety of diseases.How these diverse stimuli activate the NLRP3 inflammasome

is not clear, but the prevailing view is that they generate somesignal of cellular stress that is often referred to as signal 2. Al-though signal 1 is important for transcriptional up-regulation ofthe NLRP3 subunit and is typically provided by signaling throughToll-like receptors (9), three cellular processes have been sug-gested to provide signal 2 (2, 3, 10). The first is K+ efflux, whichhas been linked to most/all NLRP3 inflammasome activators, butjust how K+ efflux can activate the inflammasome in a specific

manner is not obvious; moreover, modulating the ionic milieu inother ways also affects NLRP3 inflammasome activation (11, 12).Second, crystals trigger phagolysosomal membrane damage/rupture(13); but how this is linked to the proximal steps in NLRP3inflammasome activation is not clear, nor is its relevance forstimuli that are not phagocytosed. Finally, other studies impli-cate mitochondrial damage, including increased mitochondrialreactive oxygen species (mROS) production, loss of membranepotential (ΔΨ), and release of mtDNA into the cytosol (14–16).Importantly, how NLRP3 inflammasome activators trigger mito-chondrial damage has not been characterized.Ca2+ mobilization controls diverse cellular processes, including

proliferation and differentiation, transcription, cellular metabo-lism, and cell death (17). Such Ca2+ mobilization is made possibleby maintaining low levels of cytoplasmic Ca2+ at basal state, sothat signal-dependent influx of Ca2+ from the extracellular spaceand intracellular Ca2+ stores (e.g., endoplasmic reticulum, ER)leads to a rapid increase in cytoplasmic Ca2+ levels, activation ofCa2+ binding proteins, and induction of various cellular responses.A key player in Ca2+ signaling is the mitochondria, which take upCa2+ from the ER or the extracellular space to regulate spatio-temporal patterns of Ca2+ signaling (18–20). However, excessiveor sustained mitochondrial Ca2+ uptake can lead to mitochondrialdamage and cell death (21–23).In this article, we show that activation of the NLRP3 inflam-

masome requires Ca2+ signaling. Several NLRP3 inflammasomeactivators mobilize Ca2+, disruption of which inhibits NLRP3inflammasome activation. During ATP stimulation, the crucialrole of Ca2+ mobilization is in inducing mitochondrial damage.C/EPB homologous protein (CHOP), a protein known to regu-late Ca2+ release from the ER during ER stress, amplifiesNLRP3 inflammasome activation.

ResultsCa2+ Signaling Is Critical for NLRP3 Inflammasome Activation byExtracellular ATP. Extracellular ATP activates the NLRP3 in-flammasome through P2X7R (24), a ligand-gated ion channelof the purinergic receptor family. Using Ca2+-sensitive fluoro-metric probes and analysis by time-lapse microscopy to measurecytosolic Ca2+ levels, we confirmed previous findings (25) thatCa2+ is mobilized from both extracellular and intracellular poolsduring ATP stimulation (Fig. 1A). To determine which poolcontributes to NLRP3 inflammasome activation, we used exper-imental manipulations common in Ca2+ signaling research:

Author contributions: T.M. and T.H. designed research; T.M., J.O., J.Y., V.B., and A.M.performed research; A.M.H. contributed new reagents/analytic tools; T.M., J.O., A.M.H.,and T.H. analyzed data; and T.H. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. J.P.Y.T. is a guest editor invited by theEditorial Board.1T.M. and J.O. contributed equally to this work.2J.Y. and V.B. contributed equally to this work.3To whom correspondence should be addressed. E-mail: thorng@hsph.harvard.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1117765109/-/DCSupplemental.

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treatment with thapsigargin (Tg), an inhibitor of the SERCA(sarcoplasmic/ER Ca2+-ATPase) pump to deplete ER Ca2+

stores, or incubation in Ca2+ free media, to block extracellularCa2+ entry. We found that brief Tg pretreatment (Fig. 1B) andincubation in Ca2+-free media (Fig. 1C) both attenuate NLRP3inflammasome activation by ATP stimulation (as measured bycaspase-1 processing to the active p10 subunit), IL-1β processing,and release of processed caspase-1 and IL-1β into culturesupernatants. This finding suggests that ER Ca2+ release andinflux of extracellular Ca2+ are both required to activate theNLRP3 inflammasome.We sought to further characterize the mechanisms that regu-

late Ca2+ mobilization during ATP stimulation. PhospholipaseC (PLC) proteins are key players in Ca2+ signaling through theirproduction of the second messengers inositol triphosphate (IP3)and diacylglycerol (17). Using macrophages expressing a PLCreporter (consisting of the PLC pleckstrin homology domainfused to GFP) (26), we showed that the protein translocates fromthe plasma membrane to the cytosol during ATP stimulation,indicative of PLC activation (Fig. S1A). Importantly ATP-in-duced Ca2+ flux could be blocked by the PLC inhibitor U73122(Fig. 1D). PLC activation can be coupled to ER Ca2+ releasethrough the IP3 receptor (IP3R), and indeed treatment with theIP3R inhibitor XeC attenuates ATP-mediated Ca2+ signaling(Fig. 1D). Finally, ER Ca2+ release can trigger extracellular Ca2+

influx through store-operated Ca2+ entry (SOCE) (27). Thechemical 2-aminoethoxydiphenyl borate (2-APB), which blocksSOCE at multiple steps (including oligomerization and trans-location of the STIM proteins that sense ER Ca2+ depletion, aswell as the activity of the Ca2+ channels ORAI), also blocks Ca2+

mobilization (Fig. 1D). Inhibition of Ca2+ flux was strongest with2-APB and weaker with XeC and U73122, suggesting that otherCa2+-releasing second messengers or ER Ca2+ release mecha-nisms may be triggered by P2X7R.These findings indicate that during ATP stimulation, Ca2+

mobilization may be regulated by PLC proteins, IP3R-mediatedCa2+ release, and SOCE. Next, we addressed the role of suchCa2+ mobilization in activation of the NLRP3 inflammasome,using multiple read-outs of NLRP3 inflammasome activity. Wefound that XeC, U73122, and 2-APB blocked ATP-inducedcaspase-1 and IL-1β processing and release of processed caspase-1and IL-1β into culture supernatants (Fig. 1 E and F). As with

Ca2+ flux (Fig. 1D), inhibition was strongest with 2-APB andweakest with XeC. Finally, Ca2+ signaling inhibitors blockedassembly of the NLRP3 inflammasome complex as visualized byimmunofluorescence (Fig. S2). Although many ATP-stimulatedcells (>25%) accumulate foci corresponding to activated NLRP3inflammasome complexes, cotreatment with Ca2+ signalinginhibitors restricts formation of such foci (<5%). This findingdemonstrates that Ca2+ signaling regulates NLRP3 inflammasomeactivation at an upstream step, proximal to complex assembly.Of note, XeC, U73122, and 2-APB had no effect on the

transcriptional induction of NLRP3 (signal 1) or pro–IL-1β byLPS (Fig. S1B), indicating that Ca2+ mobilization does notregulate expression of the NLRP3 inflammasome or LPS sig-naling. We also rule out a role for the inhibitors in modulatingcell viability (Fig. S1C). In conclusion, Ca2+ signaling—probablyregulated by PLC, ER Ca2+ release through the IP3R, andSOCE—is important for NLRP3 inflammasome activation byextracellular ATP.

Ca2+ Signaling May Have a Central Role in NLRP3 InflammasomeActivation. Next, we asked if Ca2+ signaling plays a role inNLRP3 inflammasome activation by other stimuli. We found thatnigericin stimulation led to Ca2+ mobilization (Fig. 2A) (28), asdid monosodium uric acid crystals (MSU) (Fig. 2B). We wereinitially not successful in detecting alum-induced Ca2+ flux, butbecause this effort could be confounded by high rates of Ca2+

efflux from the cytosol, we took an alternative experimentalstrategy (29) and asked if alum pretreatment could block well-established mechanisms of ER Ca2+ release. Indeed, alum pre-treatment inhibited Ca2+ mobilization during P2Y receptor acti-vation, strongly suggesting that it triggered ER Ca2+ release anddepletion of ER Ca2+ stores (Fig. S3A). Thus, multiple stimulithat activate the NLRP3 inflammasome induce Ca2+ signaling.Next, we asked if such Ca2+ mobilization plays a role in

NLRP3 inflammasome activation. Indeed XeC, U73122, and2-APB blocked caspase-1 processing and IL-1β production dur-ing stimulation with nigericin, MSU, and alum (Fig. 2 C–F andFig. S3 B and C). Incubation in Ca2+-free media also attenuatedNLRP3 inflammasome activation by MSU and alum stimulation(Fig. S4). In contrast, activation of the NLRC4 inflammasome byFliC was attenuated by the PLC inhibitor U73122 but not by theIP3R or SOCE inhibitors (Fig. S5). This result may reflect

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Fig. 1. Extracellular ATP mobilizes Ca2+ to activate theNLRP3 inflammasome. (A) LPS-primed BMDMs were loadedwith Fura-2 followed by stimulation with 1 mM ATP andanalysis of Ca2+ flux by time-lapse microscopy. Ca2+ is re-leased from intracellular stores during the initial stimulationin Ca2+-free buffer, and exchange to 1 mM Ca2+-containingbuffer permits extracellular Ca2+ influx. (B and C) LPS-primedBMDMs were treated or not with Tg for 30 min to deplete ERCa2+ (B) or switched to Ca2+ free (−) or Ca2+-containing (+)media immediately before ATP stimulation (C). NLRP3 in-flammasome activation was assessed by Western blotting oflysates and supernatants (supes) as well as IL-1β ELISA. (D–F)BMDMs were pretreated with XeC, U73122, or 2-APB, fol-lowed by ATP stimulation and analysis of Ca2+ flux (D) andNLRP3 inflammasome activation (E and F).

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“noncanonical” functions of PLC (e.g., nuclear PLC) in settingswhere the roles of IP3R and SOCE are not well-defined (30, 31).U73122 is unlikely to have off-target effects during ATP stimu-lation because it blocked Ca2+ mobilization (Fig. 1D) concomi-tantly with NLRP3 inflammasome activity (Fig. 1E); moreover,the inactive analog U73343 did not have such effects (Fig. S1D).Collectively, these findings indicate that Ca2+ signaling may bea common step in NLRP3 inflammasome activation.

Ca2+ Signaling Promotes Mitochondrial Damage. Next, we askedhow the requirement for Ca2+ signaling could be integrated withcurrent models of NLRP3 inflammasome activation. K+ effluxhas been implicated by studies showing that high extracellularK+ blocks NLRP3 inflammasome activation by multiple stimuli(Fig. 3A) (10). K+ efflux is not directly linked to activation ofspecific signaling pathways but can regulate Ca2+ signaling throughits effects on plasma membrane polarization, and during extra-cellular Ca2+ entry K+ efflux counteracts membrane depolar-ization, thus promoting further Ca2+ influx. Indeed, we found thatATP-mediated Ca2+ flux is strongly reduced in the presence ofhigh extracellular K+ (Fig. 3B), suggesting that K+ efflux maypromote Ca2+ influx to activate the NLRP3 inflammasome.Phagolysosomal rupture has been posited to be crucial in NLRP3

inflammasome activation by crystals. In support of this, a lysoso-motropic peptide (Leu-Leu-OMe) that induces phagolysosomal

rupture through a completely distinct mechanism—osmotic stressof the lysosome—can activate the NLRP3 inflammasome (13).Importantly, the lysosomal compartment constitutes an intracellularCa2+ store that can be released in a signal-dependent way totrigger ER Ca2+ release, indicative of functional coupling be-tween these Ca2+ stores (32). Thus, we hypothesized thatphagolysosomal rupture may induce Ca2+ mobilization to acti-vate the NLRP3 inflammasome. Indeed, we demonstrated thatLeu-Leu-OMe stimulation induces ER Ca2+ release, because itblocks subsequent Ca2+ mobilization by P2YR activation (Fig.3C). Furthermore, Ca2+ signaling inhibitors block NLRP3inflammasome activation by Leu-Leu-OMe, indicating that suchCa2+ mobilization is critical (Fig. 3 D and E). Thus, phag-olysosomal rupture may induce Ca2+ mobilization to activate theNLRP3 inflammasome.Finally, mitochondrial damage has been linked to NLRP3

inflammasome activation. ATP stimulation leads to increasedmROS production, loss of membrane potential (ΔΨ), and re-lease of mtDNA into the cytosol (14–16), and manipulations thatblock any of these processes reduce inflammasome activation.Importantly, mitochondria play a key role in shaping the spa-tiotemporal dynamics of Ca2+ signaling by uptake and release ofcytosolic Ca2+ (18–20). However, excessive or sustained Ca2+

uptake can lead to mitochondrial damage characterized by in-creased production of mROS, mitochondrial permeability tran-sition, and eventually rupture of the mitochondria (21–23). Thus,we hypothesized that the requirement for Ca2+ mobilization inactivation of the NLRP3 inflammasome may reflect the ability ofCa2+ signaling to trigger mitochondrial damage. Consistent withthis idea, Ca2+ signaling inhibitors block mROS productionduring ATP stimulation as measured by MitoSOX, a specificprobe of mROS (Fig. 3F). Ca2+ signaling inhibitors also rescueATP-mediated loss of membrane potential as measured bystaining with Mitotracker Deep Red, a dye that accumulates ina ΔΨ-dependent manner (Fig. 3G). Moreover, Ca2+ signalinginhibitors block release of mtDNA into the cytosol during ATPstimulation (Fig. S6). Thus, we propose that Ca2+ mobilization isrequired for inducing mitochondrial damage. Together with re-cent studies, our results support the model whereby Ca2+ sig-naling critically regulates NLRP3 inflammasome activation bytriggering mitochondrial damage.

CHOP Amplifies Activation of the NLRP3 Inflammasome. Our resultsimplicate a critical role for Ca2+ mobilization in NLRP3 inflam-masome activation, thus we hypothesized that regulators of thisprocess may modulate NLRP3 inflammasome activity. CHOP isa transcription factor that can regulate ER Ca2+ release throughthe IP3R, and during ER stress CHOP deficiency leads to atten-uated ER Ca2+ release concomitant with reduced oxidative stressand improved cellular survival (33–35). To test if CHOP plays anyrole in modulating NLRP3 inflammasome activity, we examinedNLRP3 inflammasome activation in WT and Chop−/− bone mar-row-derived macrophages (BMDMs). We observed a consistentdecrease in NLRP3 inflammasome activation in Chop−/− BMDMsin response to ATP stimulation (Fig. 4A). In contrast, LPS-mediated IL-6 and TNF-α production (Fig. S7B); NLRP3 andpro–IL-1β up-regulation (Fig. S7C); and NLRC4 inflammactionactivation were normal (Fig. S7D), suggesting no general defect ininflammatory responses. Expression of Asc, P2X7R, and cathepsinB was also normal (Fig. 4 and Fig. S7A).These findings suggested a specific involvement of CHOP in

modifying NLRP3 inflammasome activity, and we asked whethersuch a role could be related to control of ER Ca2+ release via theIP3R. First, we showed that ATP-mediated Ca2+ mobilization isreduced in Chop−/− BMDMs (Fig. 4B). Second, we reasoned thatif CHOP and IP3R function in the same pathway, XeC shouldblock NLRP3 inflammasome activity in WT but not Chop−/−

BMDMs. Indeed, although XeC treatment and CHOP deficiencyboth inhibited ATP-induced IL-1β production by ∼30–40% in WTBMDMs, no further block of IL-1β production was observed uponXeC addition to Chop−/− BMDMs (Fig. 4C). Thus, CHOP may

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Fig. 2. Ca2+ signaling may be a common mechanism for NLRP3 inflamma-some 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-oper-ated Ca2+ entry to Ca2+ flux. (C–F) BMDMs stimulated as indicated wereexamined for NLRP3 inflammasome activation.

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amplify NLRP3 inflammasome activity by regulating IP3R-medi-ated Ca2+ release.To demonstrate physiological relevance, we turned to a mouse

model of sepsis in which IL-1β production is dependent on theNLRP3 inflammasome (6, 36). WT and Chop−/− mice wereinjected with high dose LPS (40 mg/kg), followed by analysis ofserum IL-1β levels. Significantly reduced IL-1β secretion wasfound in Chop−/− mice compared with WT mice, but levels ofTNF-α and IL-6 were similar (Fig. 4D). Thus, CHOP has a specificrole in regulation of the NLRP3 inflammasome in vivo.

DiscussionIn this study, we elucidate a critical role for Ca2+ signaling inactivation of the NLRP3 inflammasome. Previous studies relyingmainly on 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraaceticacid (BAPTA)-AM treatment or incubation in Ca2+-free mediahave made somewhat inconsistent conclusions regarding the roleof Ca2+ in NLRP3 inflammasome activation (28, 37–39). Giventhe pleitropic effects of BAPTA-AM on many cellular processesincluding LPS signaling (40), we wished to more carefully examinethis issue by defining specific regulators of Ca2+ mobilization.Using XeC, U73122, and 2-APB, we implicate PLC proteins,IP3R-mediated Ca2+ release, and SOCE as critical regulatorsduring ATP stimulation (Fig. 1D). Although not well-character-ized, induction of such a pathway by stimuli that induce Ca2+

influx across the plasma membrane is not without precedence (41,42). Definitive demonstration of the role of this pathway requiresgenetic models, but pharmacological inhibitors are commonlyused in the field of Ca2+ signaling because of redundancy inregulation (i.e., multiple PLCs) or, conversely, lethality of cells andmice lacking critical regulators (i.e., IP3R1). Most importantly, wecorrelate block of Ca2+ mobilization (Fig. 1D) with NLRP3

inflammasome activity (Fig. 1 E and F), and use independentmethods to implicate ER Ca2+ release and extracellular Ca2+

entry (Fig. 1 B and C). Ca2+ signaling inhibitors do not regulatesignal 1 (Fig. S1B) or affect cell viability (Fig. S1C), but blockNLRP3 inflammasome activation at every step examined, in-cluding complex assembly (Fig. S2), caspase-1 processing, IL-1βprocessing, and IL-1β release to culture supernatants (Fig. 1 Eand F). Collectively, these data indicate a role for Ca2+ mo-bilization in regulating the upstream, proximal steps in NLRP3inflammasome activation (although not ruling out additionaleffects more distally).How can our findings be integrated into existing models for

activation of the NLRP3 inflammasome? K+ efflux and phag-olysomal rupture have been implicated in NLRP3 inflammasomeactivation but the critical intermediate events have not beendefined. Our results are consistent with the idea that high K+

buffers perturb Ca2+ mobilization and that K+ efflux may reg-ulate Ca2+ signaling (Fig. 3B), while rupture of phagolysosomesleads to ER Ca2+ release (Fig. 3 C–E), likely as a result of Ca2+

mobilization from the lysosomal compartment. Thus, we proposethat K+ efflux and phagolysosomal rupture promote Ca2+ mo-bilization to activate the NLRP3 inflammasome. Conversely, wereport that Ca2+ signaling inhibitors block ATP-mediated mi-tochondrial Ca2+ damage, as measured by mROS production(Fig. 3F), loss of ΔΨ (Fig. 3G), and release of mtDNA into thecytosol (Fig. S6). Because these processes are required forNLRP3 inflammasome activation, this finding suggests that thecritical role of Ca2+ signaling, at least during ATP stimulation, isto mediate mitochondrial damage. Consistent with this theory,activation of the NLRP3 inflammasome by transfection of oxi-dized mitochondrial DNA (14) is not blocked by Ca2+ signalinginhibitors (Fig. S8).

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Fig. 3. Ca2+ signaling promotes mitochondrial damageduring ATP stimulation. (A) BMDMs were stimulated withATP 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 pres-ence of hK+ or hNa+ buffers. Fold-increase represents theratio of maximal fluorescence to baseline fluorescence.Ionomycin stimulation was included as a control for Fura-2 loading. (C) BMDMs were pretreated with Leu-Leu-OMeor not (control), followed 1 h later by stimulation with100 μM ATP in Ca2+-free HBSS (white arrow) to induce ERCa2+ release through the P2Y receptor. Stimulation withthe Ca2+ ionophore Ionomycin (black arrow) mobilizedCa2+ from distinct Ca2+ stores resistant to Leu-Leu-OMeand P2YR activation and served as a positive control forFura-2 loading. (D and E) BMDMs, stimulated as indicated,were examined for NLRP3 inflammasome activation. (F andG) BMDMs stimulated as indicated were stained withMitoSOX (F) or Mitotracker Green and Deep Red (G) fol-lowed by flow cytometry analysis.

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Additionally, we find that Ca2+ is mobilized during stimulationwith alum, MSU, and nigericin, and that such Ca2+ signaling isrequired for NLRP3 inflammasome activation (Fig. 2, and Figs. S3and S4). This finding suggests that Ca2+ mobilization is a com-mon, proximal step in activation of the NLRP3 inflammasome.We propose that the multitude of stimuli that activate the NLRP3inflammasome can be rationalized in light of the many mecha-nisms for mobilizing Ca2+, including G protein-coupled receptors,receptor tyrosine kinases, immunoreceptor tyrosine-based acti-vation motif-coupled receptors, and Ca2+ entry channels (17).However, we note that Ca2+ signaling cannot be sufficient forNLRP3 inflammasome activation, as indicated by the inability ofthe Ca2+ ionophore ionomycin to induce IL-1β production (Fig.S9), despite triggering Ca2+ mobilization and mitochondrial Ca2+uptake (43). Presumably, many other stimuli that induce “physi-ological” Ca2+ signaling would also not activate the NLRP3inflammasome. Importantly, Ca2+ signaling is well-established tobe necessary but not sufficient for mitochondrial damage (21–23),and we propose that only stimuli that mobilize Ca2+ in a mannerleading to mitochondrial damage would activate the NLRP3inflammasome (Fig. 5).Finally, we wondered about a potential role for the NLRP3

inflammasome in “sensing” dysregulated Ca2+ homeostasis andinducing inflammation in chronic diseases, such as neuro-degeneration, atherosclerosis, and obesity-associated metabolicdiseases (44–46). In considering this possibility, we focused on

CHOP, an effector of the ER-stress pathway (35). ER stress isclosely linked to dysregulated Ca2+ homeostasis and CHOP canregulate ER Ca+ levels (33); moreover, a recent report links ERstress to NLRP3 inflammasome activation (47). Indeed, CHOPdeficiency attenuates ATP-mediated NLRP3 inflammasome acti-vation (Fig. 4A) and IL-1β production during sepsis (Fig. 4D), butother inflammatory responses were not affected (Fig. 4D and Fig.S7). The underlying mechanism is likely to be attenuated Ca2+

release from the IP3R, as supported by Ca2+ imaging studies (Fig.4B) and the inability of IP3R inhibition to further block inflam-masome activation (Fig. 4C). Somewhat surprisingly, alum stimu-lation of Chop−/− BMDMs leads to normal NLRP3 inflammasomeactivation (Fig. S10 A and B), but it also depletes ER Ca2+ storescomparably in WT and Chop−/− BMDMs (Fig. S10C). Less ROSis generated by alum stimulation compared with ATP (Fig. S10 Dand E), and CHOP has been proposed to regulate ER Ca2+

release in a manner dependent on ER redox status, so one pos-sible interpretation of these results is that CHOP amplifies NLRP3inflammasome activation in a manner dependent on ROS levels.Our results reveal CHOP as a modulator of the NLRP3 inflam-masome pathway and support the idea that in many chronic dis-eases, CHOP and ER stress may amplify NLRP3 inflammasomeactivity to augment inflammation.

Materials and MethodsMice. B6 and Chop−/− mice were from the Jackson Laboratories and Nlrp3−/−,Asc−/−, and Caspase-1−/− mice were gifts of Visha Dixit (Genentech, SanFrancisco, CA) and Ruslan Medzhitov (Yale University, New Haven, CT). Allmice are on the B6 background. Animals were bred and maintained atHarvard Medical School and all animal experiments were done with ap-proval by and in accordance with regulatory guidelines and standardsset by the Institutional Animal Care and Use committee of Harvard Med-ical School.

Cells and Stimulations. To activate the NLRP3 inflammasome, BMDMs (48)were primed with LPS before stimulation with ATP (1–5 mM, 30 min), alum(200 μg/mL, 4 h), nigericin (20 μM, 1 h), MSU (100 μg/mL, 4 h), and Leu-Leu-OMe (1 mM, 5 h). To activate the NLRC4 inflammasome, LPS-primed BMDMswere transfected with FliC (80 ng/0.7 × 106 cells) using the Lipofectamine2000 reagent (8 μL) 5 h before harvest. Transfection of oxidized mtDNAwas done as previously described (14). U73122 and U73343 (10 μM), 2-APB(100 μM), XeC (5 μM), and YVAD (40 μM) were added following LPS priming,30 min before addition of the NLRP3 inflammasome activators. Ca2+ freemedia (Invitrogen) was added 10 min before addition of NLRP3 inflamma-some activators, as was KCl or NaCl (40 mM final concentration). To tran-siently express PLC-PH-GFP, BMDMs were transfected with DNA using

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Fig. 5. Model. Our results support a model in which Ca2+ signaling is criticalfor NLRP3 inflammasome activation. In response to ATP and perhaps otherstimuli, Ca2+ mobilization from ER stores and the extracellular space triggersmitochondrial damage, including increased mROS production, loss ofmembrane potential, and release of mtDNA into the cytosol.

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Nucleofector technology (Lonza) and rested for 4 h before stimulation; 100μM ATP was used to activate the P2YR.

Antibodies. Antibodies were obtained from Santa Cruz (caspase-1), Alexis(NLRP3), Sigma (Tubulin), R&D Systems (IL-1β), and National Cancer Institute-Frederick Biological Resources Branch Repository (IL-1β).

Western Blotting and ELISA. In some Western blotting experiments, super-natants were concentrated by addition of TCA (20% final volume, 10 min at4 °C), followed by centrifugation (17,000 × g, 5 min at 4 °C) and twowashes of the protein pellet with ice-cold acetone. IL-1β ELISA kit was fromeBioscience, and IL-6 and TNF-α ELISA kits were from Biolegend.

Ca2+ Imaging. BMDMs were loaded with the Ca2+-sensitive indicator dye Fura-2-AM (1.5 μM, 30 min, 37 °C). Ca2+ flux was measured by time-lapse mi-croscopy, and fluorescence images were collected using either a YokogawaSpinning Disk microscope equipped with a Sutter DG-4 illuminator and a 40×objective or a Nikon TE200 inverted microscope with Nomarski optics. The340/380 ratio was calculated using ImageJ (National Institutes of Health).

Flow Cytometry. MitoSOX, MitoTracker Green and Red, and DCFDA stainingwere done according to manufacturer’s instructions (Invitrogen). Data were

acquired with a FACSCalibur flow cytometer (BD Biosciences) and analyzedwith FlowJo analytical software (TreeStar).

Measuring Cytosolic mtDNA. Measurements of cytosolic mtDNA were donefollowing an established protocol (15).

Sepsis. Mice age 6–10 wk were injected intraperitoneally with 40 mg/kg LPS.After 3 h, mice were killed and blood was obtained by cardiac puncture.Serum levels of IL-1β, TNF-α, and IL-6 were analyzed by ELISA.

Statistical Analysis. Statistics were calculated using GraphPad Prism 5. Com-parisons of two groups were analyzed using two-tailed t test, and compar-isons of multiple groups were analyzed using one-way or two-way ANOVAas indicated. P values <0.05 were considered significant.

ACKNOWLEDGMENTS. We thank K. Fitzgerald, V. Dixit, and R. Medzhitovfor sharing Nlrp3−/−, Asc−/−, Caspase-1−/− mice, and femurs. This work wassupported in part by the Uehara Memorial Foundation, the Kanae Founda-tion for the Promotion of Medical Sciences, and a Japan Society for thePromotion of Science Fellowship (T.M.); a Postdoctoral Fellowship from theSwedish Research Council (to J.O.); and a Harvard School of Public HealthCareer Incubator Fund and a Pilot and Feasibility Grant from the HarvardDigestive Diseases Center (to T.H.).

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