Regulating NLRP3 Expression NLRP3 Inflammasome Activation By

Home , NLRP3

Cutting Edge: NF-κB Activating Pattern Recognition and Cytokine Receptors License NLRP3 Inflammasome Activation by Regulating NLRP3 Expression This information is current as of October 2, 2021. Franz G. Bauernfeind, Gabor Horvath, Andrea Stutz, Emad S. Alnemri, Kelly MacDonald, David Speert, Teresa Fernandes-Alnemri, Jianghong Wu, Brian G. Monks, Katherine A. Fitzgerald, Veit Hornung and Eicke Latz

J Immunol 2009; 183:787-791; Prepublished online 1 July Downloaded from 2009; doi: 10.4049/jimmunol.0901363 http://www.jimmunol.org/content/183/2/787 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2009/07/02/jimmunol.090136 Material 3.DC1 References This article cites 18 articles, 3 of which you can access for free at: http://www.jimmunol.org/content/183/2/787.full#ref-list-1

Why The JI? Submit online. by guest on October 2, 2021

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Cutting Edge

Cutting Edge: NF-␬B Activating Pattern Recognition and Cytokine Receptors License NLRP3 Inflammasome Activation by Regulating NLRP3 Expression1 Franz G. Bauernfeind,*†¶ Gabor Horvath,* Andrea Stutz,* Emad S. Alnemri,‡ Kelly MacDonald,§ David Speert,§ Teresa Fernandes-Alnemri,‡ Jianghong Wu,‡ Brian G. Monks,* Katherine A. Fitzgerald,* Veit Hornung,2,3† and Eicke Latz2,3*

The IL-1 family cytokines are regulated on transcriptional tivators, and crystalline or aggregated materials can activate and posttranscriptional levels. Pattern recognition and cy- the Nod-like receptor protein NLRP3 (5). The molecular tokine receptors control pro-IL-1␤ transcription whereas mechanisms of how NLRP3 can recognize such a diverse inflammasomes regulate the proteolytic processing of array of activators and the role of transcriptionally active Downloaded from pro-IL-1␤. The NLRP3 inflammasome, however, as- signaling receptors for the activation of the NLRP3 inflam- sembles in response to extracellular ATP, pore-forming masome are controversial and mechanistically poorly under- toxins, or crystals only in the presence of proinflamma- stood (2, 5–8). Upon activation, NLRP3 forms a so-called tory stimuli. How the activation of gene transcription inflammasome complex with the adaptor molecule ASC, which controls the activation of caspase-1. Activated caspase-1,

by signaling receptors enables NLRP3 activation re- http://www.jimmunol.org/ in turn, cleaves pro-IL-1␤ and pro-IL-18 into the biologically mains elusive and controversial. In this study, we active, secreted forms (9). show that cell priming through multiple signaling re- In this study, we demonstrate that the expression of NLRP3 ceptors induces NLRP3 expression, which we identi- itself is tightly controlled by the activity of multiple signaling fied to be a critical checkpoint for NLRP3 activation. receptors. We reveal that enhanced expression of NLRP3 in re- ␬ Signals provided by NF- B activators are necessary sponse to NF-␬B is sufficient for NLRP3 inflammasome acti- but not sufficient for NLRP3 activation, and a second vation by ATP or pore-forming toxins or crystals. Thus, mac- stimulus such as ATP or crystal-induced damage is rophages need to acquire a licensing signal provided by a

required for NLRP3 activation. The Journal of Im- transcriptionally active signaling receptor that enables them to by guest on October 2, 2021 munology, 2009, 183: 787–791. respond to NLRP3 activators.

embers of the TLR and C-type lectin receptor fam- ilies signal in response to microbial or altered en- Materials and Methods Mice dogenous molecules when presented extracellularly M 4 or in endo-lysosomal compartments (1–3). In the cytoplasm, The following mice were provided as indicated: NLRP3-knockout (KO) and ASC-KO (Millenium Pharmaceuticals); TLR2-KO, TLR4-KO, TLR7-KO, IL- Nod-like receptors and Rig-I-like helicases respond to defined 1R-associated kinase 4 (IRAK4)-KO, MyD88 adaptor-like (MAL)-KO, Toll/ microbial components gaining access to the cytosol (3). IL-1R domain-containing adapter inducing IFN-␤ (TRIF)-KO, MyD88-KO, and Most innate signaling receptors respond to a relatively re- TRIF-related adapter molecule (TRAM)-KO (S. Akira, Osaka University, Suita, stricted ligand spectrum (4). In contrast, diverse molecular Japan); TLR3-KO (R. Flavell, Yale University, New Haven, CT); and MD-2-KO (K. Miyake, Tokyo University, Tokyo, Japan). C57BL/6 mice were purchased entities including bacteria, viruses, purified microbial prod- from Jackson Laboratories. All animal experiments were approved by the University ucts, components of dying cells, small molecule immune ac- of Massachussetts Animal Care and Use Committee (Worcester, MA).

*Department of Infectious Diseases and Immunology, University of Massachusetts Med- 2 V.H. and E.L. contributed equally to this study. ical School, Worcester, MA 01605; †Department of Clinical Chemistry and Pharmacol- 3 Address correspondence and reprint requests to Dr. Eicke Latz, University of Mas- ogy, University of Bonn, Bonn, Germany; ‡Department of Biochemistry and Molecular sachusetts Medical School, Department of Infectious Diseases and Immunology, 364 Biology, Center for Apoptosis Research, Kimmel Cancer Institute, Thomas Jefferson Uni- Plantation Street, Lazare Research Building 308, Worcester, MA 01605. E-mail ad- versity, Philadelphia, PA 19107; §Division of Infectious and Immunological Diseases, De- dress: [email protected] or Dr. Veit Hornung, University of Bonn, Department of partment of Pediatrics, University of British Columbia and British Columbia’s Children’s Clinical Chemistry and Pharmacology, Sigmund-Freud-Strasse 25, 53127 Bonn, Ger- Hospital, Vancouver, British Columbia, Canada; and ¶Division of Clinical Pharmacology, many. E-mail address: [email protected] Department of Medicine, University of Munich, Munich, Germany 4 Abbreviations used in this paper: KO, knockout; IRAK4, IL-1R-associated kinase 4; Received for publication May 1, 2009. Accepted for publication May 22, 2009. MDP, muramyl dipeptide; TRIF, Toll/IL-1R domain-containing adapter inducing The costs of publication of this article were defrayed in part by the payment of page charges. IFN-␤; YFP, yellow fluorescent protein. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 1 This work was supported by National Institutes of Health Grants AI-065483 (to E.L.), AI-067497 (to K.A.F.), and AG14357 and AR055398 (to E.S.A.), a grant from the Dana Foundation (to E.L.), German Research Foundation Grant Ho2783/2-1 (to V.H.), and a grant from the Canadian Institutes for Health Research (to D.P.S.).

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901363 788 CUTTING EDGE: NLRP3 INFLAMMASOME LICENSING BY PATTERN RECOGNITION RECEPTORS

Reagents ATP, poly(dA-dT), muramyl dipeptide (MDP), nigericin, cycloheximide, and Bay11-7082 were from Sigma-Aldrich. Pam2CysK4, polyinosinic:polycytidylic acid, ultra-pure LPS, R848, and ␥-D-glutamyl-meso-diaminopimelic acid (iE-DAP) were from InvivoGen. Anti-TLR4 Abs (UT18 and MTS510) were from eBioscience. The anti-NLRP3 polyclonal Ab was raised against the NLRP3 pyrin domain that was expressed in Escherichia coli. The mAb against mouse pro-IL-1␤ was from the National Cancer Institute and the mouse anti- actin mAb was from Sigma-Aldrich. Cell stimulation and analysis Human PBMCs were isolated by density-gradient centrifugation, stimulated in complete RPMI 1640, and the caspase-1 carboxyfluorescein-YVAD-fluorom- ethylketone FLICA (fluorescent labeled inhibitors of caspases) kit (Immuno- chemistry Technologies) was used to stain active caspase-1. CD14-positive cells were analyzed for FLICA positivity by flow cytometry. The respective local eth- ics committees approved experiments involving PBMCs. Immortalized macrophage cell lines were generated as described (10). Caspase-1 was detected in serum-free cell supernatants or cell lysates by SDS PAGE using caspase-1 polyclonal Ab (catalog no. sc-514; Santa Cruz Biotechnology). Human ASC and NLRP3 were cloned from cDNA into the lentiviral plasmid FugW and immortalized macrophages were transduced as described (11). Quantitative real-time PCR was performed as described (12). Primer sequences are available upon re- Downloaded from quest. The mouse NLRP3 promoter (Ϫ3000 bp to 0 bp upstream of the transcription start site 1) was cloned from cDNA into pGL3-basic. HEK293T cells were transfected with luciferase reporter plasmid and expression plasmids (100 ng each) using Lipofectamine (Invitrogen). Microscopy

A Leica SP2 AOBS (acousto-optical beam splitter) confocal laser scanning mi- http://www.jimmunol.org/ croscope was used. ASC pyroptosomes were quantified by epifluorescence microscopy and ImageJ software. Results and Discussion Assembly of the NLRP3 inflammasome requires priming signals functioning upstream of ASC FIGURE 1. Caspase-1 activation and the formation of ASC pyropto- Fluorescent ASC forms speck-like structures, termed pyroptosomes require priming. A, ASC-YFP expressed by C57BL/6 macrophages somes, upon activation (13). We generated immortalized mouse were left untreated or stimulated as indicated, counterstained for mem- macrophages expressing ASC-yellow fluorescent protein (YFP) to branes (choleratoxin subunit B (red)) and nuclei (Hoechst dye (blue)), and by guest on October 2, 2021 test whether the NLRP3 inflammasome can assemble upstream of analyzed by confocal microscopy. B, ASC-CFP pyroptosome formation af- caspase-1 in the absence of priming. Resting cells or cells treated ter LPS priming and subsequent ATP stimulation was analyzed by epifluo- with ATP or LPS alone uniformly expressed ASC-YFP throughout rescence microscopy. C, Immunoblot (IB) for cleaved caspase-1 in supernatants from cells stimulated as in B. Data are representative of three the cells. However, when LPS-primed cells were treated with ATP, independent experiments (error bars, s.d. in B). ASC-YFP formed large, irregularly shaped pyroptosomes, indicating that LPS signaling was also required for ASC pyroptosome formation upon NLRP3 activation (Fig. 1A). Thus, mouse macrophages require two stimuli for NLRP3 inflammasome activation mechanisms. We made use of an activating anti- even at the level of ASC, similar to what is observed at the level of TLR4/MD-2 Ab (clone UT18) and tested whether the acti- caspase-1 (supplemental Fig. 1A and Ref. 6).5 Furthermore, py- vation of TLR4 in the absence of LPS was sufficient for roptosomes dose-responsively formed in response to LPS and ATP priming of the NLRP3 inflammasome. Preincubation with and caspase-1 cleavage closely correlated with the number of py- an activating Ab, but not with a blocking TLR4/MD-2 Ab roptosomes (Fig. 1, B and C, and supplemental Fig. 1B). Synthetic (clone MTS510), led to the cleavage of caspase-1 in wild- LPS or TLR2 and TLR7 activators also induced pyroptosomes to- type macrophages when stimulated with ATP. Furthermore, gether with ATP (supplemental Fig. 1, C and D) suggesting that activation by the stimulating Ab was dependent on TLR4 TLR activators and not an undefined contaminant were responsi- and MD-2, consistent with the fact that it fails to bind ble for pyroptosome formation in response to ATP. In line with and activate TLR4 or MD-2 alone (15) (Fig. 2A). Further- previous observations, pyroptosome formation in response to the more, activation of TLR2, TLR3, and TLR7 also induced AIM2 inflammasome activator, transfected dsDNA (poly(dA- priming of the NLRP3 inflammasome, and macrophages dT)), did not require LPS priming (supplemental Fig. 1B and Refs. lacking TLRs for the respective stimuli failed to activate 12 and 14). caspase-1 after activation via ATP (Fig. 2B). We next analyzed the requirement of TLR signaling for Different signaling receptor family members are able to license NLRP3 NLRP3 priming. Macrophages deficient in MyD88 or TRIF inflammasome activation responded normally to LPS and ATP with cleavage of We next aimed at dissecting the influence of TLR4 signaling on caspase-1, whereas macrophages doubly deficient in both NLRP3 inflammasome activation from TLR-independent MyD88 and TRIF or TLR4 failed to respond (supplemental Fig. 2A). These results show that both TRIF- and MyD88-de- 5 The online version of this article contains supplemental material. pendent signaling pathways can compensate for each other in The Journal of Immunology 789 Downloaded from FIGURE 2. Pattern recognition receptor activation is required for NLRP3 inflammasome activation. A, Immunoblot (IB) of caspase-1 in wild-type or KO macrophages primed for 4 h with LPS (200 ng/ml), Pam2CysK4 (50 ng/ml), TLR4 activating (UT18), or TLR4 blocking (MTS510) Abs and subsequently stimulated with ATP (1 h). B and C, Cells from wild-type or KO macrophages were left untreated or stimulated for 4 h with polyinosinic:polycytidylic acid (poly(I:C)) (1 ␮g/ml), Pam2CysK4 (50 ng/ml), or R848 (0.5 ␮g/ml) (B) or with Pam2CysK4 (50 ng/ml), MDP (10 ␮g/ml), LPS (200 ng/ml), or transfected with poly(dA-dT) (dAdT) (C). ATP was added for1hasindicated. D, Wild-type or TNFR1/2 double KO (dKO) macrophages were stimulated with TNF-␣ as indicated or with LPS as in A and analyzed for caspase-1 activation. Immunoblot analyses of cleaved caspase-1 from supernatants are show. Data are from one representative

experiment of three (A and B) or two (C and D). http://www.jimmunol.org/

their ability to induce priming for NLRP3 activation. Of note, AIM2 inflammasome activation was independent of this the priming of macrophages was also a necessary step for type of priming (supplemental Fig. 3B). When primed with NLRP3 inflammasome activation by other established NLRP3 IRAK-4-independent ligands, monocytes from the IRAK4- activators such as the pore-forming toxin nigericin or crystalline mutant patient responded robustly with caspase-1 activation activators (supplemental Fig. 2, B and C), suggesting that prim- after ATP stimulation. In contrast and consistent with by guest on October 2, 2021 ing is generally required for NLRP3 inflammasome activation. IRAK4 deficiency in the murine system, we failed to observe MDP, which engages NOD2, also mediated priming for priming activity toward ATP after TLR2 or TLR7/8 stimu- ATP responsiveness, and the activity was dependent on RIP2, lation (supplemental Fig. 3B). Thus, these data indicate that the downstream signaling transducer of this signaling pathway NLRP3 activation is also critically dependent on priming ac- (Fig. 2C). We further observed that priming with a cytokine tivity by signaling receptors in human cells. stimulus (TNF-␣) was sufficient to induce caspase-1 activation by ATP. Notably, TNF-␣ failed to prime cells obtained from mice doubly deficient in TNFR1 and TNFR2, whereas LPS NF-␬B-dependent signals regulate NLRP3 expression was able to prime these cells (Fig. 2D). Collectively, these results Treatment of macrophages with the protein synthesis inhibitor suggest that multiple transcriptionally active signaling receptors cycloheximide dose-responsively led to reduced caspase-1 acti- can prime macrophages for subsequent NLRP3 inflammasome vation obtained by the combination of LPS and ATP, indicat- activation. ing that protein de novo synthesis was functionally limiting in Priming is required for NLRP3 activation in human monocytes mouse macrophages (Fig. 3A). In addition, priming of the NLRP3 inflammasome was dose-dependently reduced by a spe- IRAK4 is essentially required for signal transduction to ␬ cific inhibitor of NF-␬B (Bay11-7082), suggesting a key role NF- B downstream of MyD88. Indeed, ligands for TLRs ␬ that exclusively signal via the adapter MyD88 also failed to for NF- B in priming (Fig. 3B). prime IRAK4-deficient macrophages for NLRP3-mediated Overexpression of ASC is not sufficient to overcome the prim- ATP reactivity, whereas signaling cascades that operate in- ing requirement for NLRP3 activation (Fig. 1), suggesting that the ␬ dependently of MyD88 induced priming of IRAK4 inde- NF- B-induced activity was acting upstream of ASC. Consistent pendently (supplemental Fig. 3A). This differential require- with this idea, we found that LPS stimulation did not change Asc ␬ ment for priming allowed us to address the role of priming mRNA levels but led to strong, NF- B-dependent increases in for NLRP3 inflammasome activation in the human system. Nlrp3 mRNA in mouse macrophages (Fig. 3C). These studies We obtained monocytes from healthy volunteers and from a are in line with a report demonstrating NLRP3 induction by patient with a loss-of-function mutation (Q293X) in IRAK4 TNF and TLR ligands in human cells (16). To analyze the pu- and tested their ability to activate caspase-1 upon ATP stim- tative Nlrp3 promoter activity, we cloned the promoter region ulation after priming with IRAK-4-dependent and -inde- entailing Ϫ3,000 to 0 bp upstream of the Nlrp3 transcription pendent stimuli. Only primed monocytes displayed a robust start site and constructed a luciferase reporter gene construct. caspase-1 activation upon ATP stimulation, whereas the We made use of the fact that heterologous overexpression of 790 CUTTING EDGE: NLRP3 INFLAMMASOME LICENSING BY PATTERN RECOGNITION RECEPTORS

IB: - LPS + ATP (kDa) IB: - LPS + ATP (kDa) cleaved cleaved -10 -10 caspase-1 caspase-1 -- 200408 --3.1 6.3 12.5 Cycloheximide Bay11-7082 (ng/ml) (µM) )

ASC 6 0.12 0.5 NLRP3 80 0.1 0.4 60 0.08 0.3 0.06 40 0.2 0.04 20 0.02 0.1 relative mRNA level mRNA relative relative mRNA level mRNA relative 0 0 units (x10Luciferase 0 S LPS - +++ None LP Bay11-7082 --6.3 12.5(µM) pcDNA3MyD88

C57BL/6 C57BL/6 NLRP3-KONLRP3-KO(kDa) (kDa) + NLRP3 IB: -225 IB: -225 NLRP3 NLRP3 IB: IB: pro-IL-1β pro-IL-1β Downloaded from -31 -31 IB: IB: β-actin β-actin -38 -38 LPS: - + +++ -- FIGURE 4. Stable NLRP3 expression is sufficient for priming-independent 1 5 10 50 100 200 13612 None 1000 NLRP3 activation. Macrophages were stimulated for 4 h and ATP or nigericin was added for an additional1hasindicated. A, Confocal microscopy of caspase- FIGURE 3. NLRP3 induction involves NF-␬B activity. A and B, Immuno- 1-KO macrophages expressing ASC-cyan fluorescent protein (CFP) and NLRP3-YFP. B, Expression of ASC-CFP and NLRP3-YFP assessed by GFP blotting (IB) of caspase-1 from supernatants of wild-type macrophages pre- http://www.jimmunol.org/ treated with cycloheximide (A) or Bay11-7082 (B) as indicated for 1 h followed immunoblotting after GFP immunoprecipitation. C, ASC pyroptosome quan- by LPS (200 ng/ml, 4h) and stimulated with ATP (1 h). C, Messenger RNA tification in caspase-1-KO macrophages expressing ASC-CFP and NLRP3- expression of ASC or NLRP3 in LPS-primed or untreated macrophages. Cells YFP or ASC-CFP and ASC-YFP. D, Immortalized C57BL/6 (left)or were pretreated with Bay11-7082 for 1 h where indicated. D, HEK293T cells NLRP3-KO (right) macrophages with or without stable expression of NLRP3- were transfected with pcDNA3-MyD88 or control (pcDNA3) together with a mCitrine were left untreated or primed with LPS (200 ng/ml) for 4 h and stim- NLRP3 promoter reporter and assessed for luciferase activity after 20 h (error ulated with nigericin for1hasindicated. Caspase-1 immunoblots of combined bars, SD). E and F, Immunoblots for NLRP3, pro-IL-1␤, and ␤-actin in lysates supernatant and cell lysates are shown. NLRP3 expression was verified by im- from C57BL/6 macrophages treated with LPS for6hasindicated, or treated munoblotting for GFP and NLRP3. One representative experiment of three (A, with LPS (200 ng/ml) (F) for the indicated periods of time. Controls are lysates C, and D) or two (B) is shown. from NLRP3-KO macrophages with and without heterologous NLRP3 expres- by guest on October 2, 2021 sion. Data are from one representative experiment of three (A–D)oroftwo(E and F) experiments. NLRP3 expression is a limiting factor for NLRP3 inflammasome activation To determine whether enhanced expression of NLRP3 was not only required but also sufficient for NLRP3 inflammasome ac- MyD88 activates downstream signaling and therefore ex- tivation by a “bona fide” NLRP3 stimulus, we next generated pressed the Nlrp3 promoter-luciferase construct alone or to- macrophage cell lines expressing NLRP3 controlled by a con- gether with MyD88. Indeed, coexpression of the Nlrp3 pro- stitutively active viral promoter (Fig. 4). In contrast to cell lines moter-luciferase construct with MyD88 led to a ϳ10-fold overexpressing ASC alone, cells that overexpressed fluorescent induction of luciferase activity, indicating that MyD88-medi- NLRP3 together with fluorescent ASC responded to NLRP3 ated signaling can activate the promoter of Nlrp3. Notably, stimuli in the absence of a prior priming step with the forma- ϳ40% of patients with autoinflammatory diseases present with tion of NLRP3/ASC pyroptosomes (Fig. 4, A, and C). We next classical clinical symptoms without carrying any mutations in assessed caspase-1 activity in wild-type and NLRP3-deficient the coding region of NLRP3 (17). Recently, unique NLRP3 macrophages with and without overexpressed NLRP3. Consis- promoter sequence variants leading to enhanced NLRP3 pro- tent with earlier results, LPS priming led to significant induc- moter activity were identified in patients with autoinflamma- tion of NLRP3 protein in wild-type cells, and LPS was required tory diseases that lack NLRP3 coding sequence mutations. This for caspase-1 cleavage in combination with an NLRP3 activa- suggests that dysregulated NLRP3 expression could evoke au- tor. NLRP3-mCitrine-overexpressing wild-type cells, however, toinflammatory symptoms (18). responded to nigericin (or ATP; data not shown) in the absence We further found that LPS stimulation led to both NLRP3 of prior priming (Fig. 4D, left). Moreover, heterologous expres- and pro-IL-1␤ protein induction in a dose- and time-depen- sion of NLRP3 joined to mCitrine (a monomeric YFP) func- dent manner (Fig. 3, E and F). In addition, LPS failed to induce tionally reconstituted NLRP3-KO macrophages, which also NLRP3 or pro-IL-1␤ in cells lacking TLR4 or doubly deficient did not require priming to induce a functionally active NLRP3 in MyD88 and TRIF, and NF-␬B inhibition led to a dose-de- inflammasome (Fig. 4D, right). Notably, overexpression of pendent reduction of NLRP3 protein induction by LPS (sup- NLRP3 per se had no influence on caspase-1 cleavage in the plemental Fig. 4, A and B). Collectively, these data indicate that absence of nigericin stimulation, demonstrating the need for an NLRP3 expression is tightly controlled by signals culminating NLRP3 activator at this level of expression. Altogether, these in the activation of NF-␬B. results establish that the requirement for priming of the NLRP3 The Journal of Immunology 791 inflammasome can be explained by the restricted expression of 2. Meylan, E., J. Tschopp, and M. Karin. 2006. Intracellular pattern recognition receptors in the host response. Nature 442: 39–44. NLRP3 itself and that the NLRP3 expression level is a limiting 3. Takeuchi, O., and S. Akira. 2008. MDA5/RIG-I and virus recognition. Curr. Opin. step for the NLRP3 inflammasome activation in macrophages Immunol. 20: 17–22. 4. Akira, S., S. Uematsu, and O. Takeuchi. 2006. Pathogen recognition and innate im- (2, 5). We revealed that the NLRP3 inflammasome is, in fact, munity. Cell 124: 783–801. rather restricted in its ability to directly recognize microbial-de- 5. Martinon, F., A. Mayor, and J. Tschopp. 2009. The inflammasomes: guardians of the rived substances. We found that the activation of the NLRP3 body. Annu. Rev. Immunol. 27: 229–265. 6. Kahlenberg, J. M., K. C. Lundberg, S. B. Kertesy, Y. Qu, and G. R. Dubyak. 2005. inflammasome requires two steps that are controlled by differ- Potentiation of caspase-1 activation by the P2X7 receptor is dependent on TLR signals ent mechanisms. First, NLRP3 expression itself needs to be and requires NF-␬B-driven protein synthesis. J. Immunol. 175: 7611–7622. 7. Kanneganti, T. D., M. Lamkanfi, Y. G. Kim, G. Chen, J. H. Park, L. Franchi, transcriptionally induced, and a second, posttranscriptional P. Vandenabeele, and G. Nunez. 2007. Pannexin-1-mediated recognition of bacterial step, leads to the activation of NLRP3, allowing for NLRP3 molecules activates the cryopyrin inflammasome independent of Toll-like receptor signaling. Immunity 26: 433–443. inflammasome assembly. The key mediator of immunity, NF- 8. Hornung, V., F. Bauernfeind, A. Halle, E. O. Samstad, H. Kono, K. L. Rock, ␬B, plays a critical role for the priming of the NLRP3 inflam- K. A. Fitzgerald, and E. Latz. 2008. Silica crystals and aluminum salts activate the masome. We thus speculate that other NF-␬B activators such as NALP3 inflammasome through phagosomal destabilization. Nat. Immunol. 9: 847–856. UV light or reactive oxygen species can also induce NLRP3 9. Dinarello, C. A. 2009. Immunological and inflammatory functions of the interleu- priming. kin-1 family. Annu. Rev. Immunol. 27: 519–550. 10. Roberson, S. M., and W. S. Walker. 1988. Immortalization of cloned mouse splenic The fact that priming is a necessary step for NLRP3 inflam- macrophages with a retrovirus containing the v-raf/mil and v-myc oncogenes. Cell. masome assembly suggests that macrophages need to acquire a Immunol. 116: 341–351. signal that indicates either the presence of infection (via activa- 11. Halle, A., V. Hornung, G. C. Petzold, C. R. Stewart, B. G. Monks, T. Reinheckel,

K. A. Fitzgerald, E. Latz, K. J. Moore, and D. T. Golenbock. 2008. The NALP3 Downloaded from tion of pattern recognition receptors by microbial products) or inflammasome is involved in the innate immune response to amyloid-␤. Nat. Immu- the activation of other cells (via the presence of proinflamma- nol. 9: 857–865. 12. Hornung, V., A. Ablasser, M. Charrel-Dennis, F. Bauernfeind, G. Horvath, tory cytokines) to commit to sense danger signals in their im- D. R. Caffrey, E. Latz, and K. A. Fitzgerald. 2009. AIM2 recognizes cytosolic dsDNA mediate environment via the activation of the NLRP3 inflam- and forms a caspase-1-activating inflammasome with ASC. Nature 458: 514–518. 13. Fernandes-Alnemri, T., J. Wu, J. W. Yu, P. Datta, B. Miller, W. Jankowski, masome. This dual stimulation requirement may operate to S. Rosenberg, J. Zhang, and E. S. Alnemri. 2007. The pyroptosome: a supramolecular prevent accidental or uncontrolled NLRP3 activation, which assembly of ASC dimers mediating inflammatory cell death via caspase-1 activation. Cell Death Differ. 14: 1590–1604. http://www.jimmunol.org/ can have devastating consequences for the host as exemplified 14. Fernandes-Alnemri, T., J. W. Yu, P. Datta, J. Wu, and E. S. Alnemri. 2009. AIM2 by the clinical presentation of patients with autoinflammatory activates the inflammasome and cell death in response to cytoplasmic DNA. Nature diseases (5, 17). 458: 509–513. 15. Ohta, S., U. Bahrun, R. Shimazu, H. Matsushita, K. Fukudome, and M. Kimoto. 2006. Induction of long-term lipopolysaccharide tolerance by an agonistic monoclo- Acknowledgments nal antibody to the Toll-like receptor 4/MD-2 complex. Clin. Vaccine Immunol. 13: We acknowledge P. Vandenabeele (University of Ghent, Ghent, Belgium) for 1131–1136. supplying a caspase-1 p20 antibody. 16. O’Connor, W., Jr., J. A. Harton, X. Zhu, M. W. Linhoff, and J. P. Ting. 2003. Cut- ting edge: CIAS1/cryopyrin/PYPAF1/NALP3/CATERPILLER 1.1 is an inducible inflammatory mediator with NF-␬B suppressive properties. J. Immunol. 171: 6329–6333.

Disclosures by guest on October 2, 2021 The authors have no financial conflict of interest. 17. Masters, S. L., A. Simon, I. Aksentijevich, and D. L. Kastner. 2009. Horror autoin- flammaticus: the molecular pathophysiology of autoinflammatory disease. Annu. Rev. Immunol. 27: 621–668. References 18. Anderson, J. P., J. L. Mueller, A. Misaghi, S. Anderson, M. Sivagnanam, 1. Takeda, K., T. Kaisho, and S. Akira. 2003. Toll-like receptors. Annu. Rev. Immunol. R. D. Kolodner, and H. M. Hoffman. 2008. Initial description of the human NLRP3 21: 335–376. promoter. Genes Immun. 9: 721–726. Supplementary Figure 1: Assembly of the ASC pyroptosomes requires TLR priming. (A) Caspase-1 immunoblot of cell lysates (pro-caspase-1) and cell free supernatants (cleaved caspase-1) of C57BL/6, NLRP3-KO, and ASC-KO macrophages. (B) Immortalized wild type macrophages stably expressing ASC-YFP were primed as indicated with crude LPS from E. coli, re-purified LPS from E. coli or synthetic LPS, Pam2CysK4 (50 ng/ml), Pam3CysK4 (50 ng/ml) or R848 (0.5 μg/ml) for 4h. ATP was added for an additional 1h as indicated. Epifluorescence images of ASC-YFP wild type macrophages (a and b left) and calculated number of ASC pyroptosomes per visual field (C right and D). One representative experiment out of three (A) or four (B-D) is shown.

Supplementary Figure 2: Priming requirement for NLRP3 inflammasome activation for different NLRP3 inflammasome activators. (A-C) LPS primed or resting macrophages were stimulated with nigericin (10 μM), ATP (5 mM) or monosodium uric acid crystals (MSU, 250 μg/ml) as indicated. Caspase-1 immunoblots of stimulated wild type macrophages or knock-out macrophages are shown. One representative experiment out of three independent experiments is depicted.

Supplementary Figure 3: IRAK4 deficiency impairs MyD88 dependent inflammasome formation. (A) Wild type, MyD88-KO or IRAK4-KO macrophages were treated with Pam2CysK4 (50 ng/ml), R848 (2 μg/ml), poly(I:C) (1 μg/ml), MDP (10 μg/ml), LPS (200 ng/ml), or transfected with poly(dA-dT) for 4h or left untreated. ATP was added for an additional 1h as indicated. Immunoblot analysis of cleaved caspase-1 from supernatants is shown. (B) PBMCs of an IRAK4 mutant patient and a healthy volunteer were stimulated with Pam2CysK4 (5 ng/ml), R848 (100 ng/ml), iE-DAP (1 μg/ml), MDP (1 μg/ml), LPS (30 pg/ml) or transfected with poly(dA-dT) followed by addition of ATP (5 mM) for 1h. Caspase-1 activity in CD14 positive cells was assessed by flow cytometric analysis using the caspase-1 fluorescent inhibitor FAM-YVAD-FMK (FLICA). Percentage of FLICA peptide positive CD14+ cells is shown. Data are from one representative experiment of three (A and B left). Data involving PBMCs from the IRAK4 mutant patient are from one single experiment.

1 Supplementary Figure 4: NALP3 induction by LPS is dependent on LPS signaling and NF-B (A) Immunoblots for NLRP3, pro-IL-1 and -actin in lysates from untreated or LPS- treated (200 ng/ml, 6 h) TLR4-KO, MyD88/TRIF-dKO or C57BL/6 macrophages. (B) Immunoblots for NLRP3, pro-IL-1 and -actin from lysates of C57BL/6 macrophages treated as in A in the absence or presence of Bay11-7082. One representative experiment out of two independent experiments is depicted.

IB: C57BL/6 (kD) MyD88-KO (kD) IB: MyD88/Trif-dKO (kD) pro- pro- pro- - 45 - 45 - 45 caspase-1 caspase-1 caspase-1 cleaved cleaved - 10 - 10 cleaved - 10 caspase-1 caspase-1 caspase-1 Trif-KO TLR4-KO LPS ATP LPS pro- - 45 pro- control + ATP - 45 caspase-1 caspase-1 cleaved - 10 cleaved - 10 caspase-1 caspase-1

ATP LPS LPS LPS ATP + ATP control control LPS + ATP B IB: C (kD) IB: (kD) pro- pro- caspase-1 -45 caspase-1 -45

cleaved cleaved -10 caspase-1 caspase-1 -10 LPS − + + − + + LPS −+++ −+++ ATP − + − − + − −−+− −−+− ATP Nigericin − −+− −+ −+−− −+−− MSU crystals

MyD88/ C57BL/6 MyD88/ TRIF-dKO C57BL/6 TRIF-dKO

Supplementary Figure 2 A IB: C57BL/6 (kDa) cleaved -10 caspase-1 MyD88-KO cleaved -10 caspase-1 IRAK4-KO cleaved -10 caspase-1

p:IC LPS None R848 MDP dAdT

Pam2CysK4 +ATP B 30 Control + ATP 25

5 % FLICA positive CD14+ cells % FLICA 0

None R848 MDP LPS dAdT None R848 MDP LPS dAdT iE-DAP iE-DAP

Pam2CysK4 Pam2CysK4

Healthy control IRAK4 mutation

Supplementory Figure 3: AB TLR4- MyD88/TRIF- KO dKO C57BL/6 C57BL/6(kDa) (kDa) IB: -225 IB: -225 NLRP3 NLRP3 -120 -120 IB: IB: pro-IL-1β pro-IL-1β -31 -31 IB: IB: β-actin β-actin -38 -38 LPS: - + - ++ LPS: - +++ μ Bay11-7082: --6.3 12.5 ( M)

Supplementary Figure 4