Cutting Edge: Necrosis Activates the NLRP3 Hanfen Li, Aditya Ambade and Fabio Re This information is current as J Immunol 2009; 183:1528-1532; Prepublished online 13 of September 28, 2021. July 2009; doi: 10.4049/jimmunol.0901080 http://www.jimmunol.org/content/183/3/1528 Downloaded from

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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: Necrosis Activates the NLRP3 Inflammasome1 Hanfen Li, Aditya Ambade, and Fabio Re2

Cells undergoing necrosis release endogenous danger IL-1 family (IL-1␤, IL-18, and IL-33). The protease -1 signals that possess proinflammatory potential. In this and the adaptor molecule ASC are also part of the inflamma- study we show that mature IL-1␤ and IL-18 are released some. The function of NLR is to detect the presence of micro- by necrotic cells but not by apoptotic cells. We identify bial products, toxins, and danger signals in the cytoplasm lead- 7-bromoindirubin-3؅-oxime, an indirubin oxime deriv- ing to caspase-1 activation, a necessary step for the processing ␤ ative that induces necrosis, as a potent inducer of and secretion of the immature forms of IL-1 , IL-18, and IL- caspase-1 activation and release of mature IL-1␤ and IL- 33. One of the best-characterized NLRs is NLRP3 (also known as cryopyrin and Nalp3). This molecule mediates inflamma-

18. Inflammasome activation was triggered by other ne- Downloaded from some activation in response to several intracellular bacteria (3) crosis-inducing treatments but was not observed in re- and viruses (4, 5), danger signals such as extracellular ATP and sponse to -inducing stimuli. Necrosis-induced uric acid crystals (monosodium urate) (6), and particles of dif- inflammasome activation was mediated by the NLRP3 ␤ ferent origin such as asbestos fibers (7), silica particles (8, 9), and ASC molecules. Release of IL-18 and IL-1 in re- and alum crystals, as we and other groups have recently dem- sponse to necrosis-inducing stimuli was observed in onstrated (10–13). The differences in the structure and func- THP-1 macrophages and the MSTO-211H human me- tion of the factors reported to activate the NLRP3-inflamma- http://www.jimmunol.org/ sothelioma cell line independently of LPS priming. Us- some make it unlikely that they directly interact with the ing the in vivo model of naphthalene-induced airway ep- receptor. Rather, the emerging consensus is that NLRP3 may ithelial cell injury, we showed that necrosis activates the recognize a “messenger molecule” that is generated inside the ASC inflammasome in vivo. Our study identifies a new cell in response to the insult that these agonists may cause. Re- mechanism through which necrosis generates proin- cently published evidence suggests that lysosome destabiliza- flammatory molecules that contributes to the sterile in- tion and the protease cathepsin B are critically involved in flammatory response. The Journal of Immunology, NLRP3 activation (8). 2009, 183: 1528–1532. Considering the evolutionary advantage that detection of tis- by guest on September 28, 2021 sue damage confers to the organism, it is conceivable that, in addition to passive release of danger signals, necrotic cells may ecrotic cells release endogenous molecules, such as actively generate proinflammatory molecules as result of the ac- heath shock proteins, uric acid, ATP, and DNA tivation of endogenous signaling pathways. In this study we re- N that alert the innate immune system of the danger port that the NLRP3 inflammasome is activated in necrotic but associated with tissue damage and infection (1). These mol- not in apoptotic cells, leading to the release of IL-1␤ and IL-18. ecules act as endogenous “danger signals” by activating a va- Our study identifies a new mechanism through which necrosis riety of pattern recognition receptors, including TLR and generates proinflammatory molecules that contribute to the Nod-like receptors (NLR).3 As a result, necrotic cells and sterile inflammatory response. several danger signals directly trigger proinflammatory re- sponses, potentiate signaling by classical proinflammatory Materials and Methods stimuli, and possess adjuvant capacity. trig- Mice gered by tissue damage in the absence of infection is referred NLRP3Ϫ/Ϫ, ASCϪ/Ϫ, and NLRC4Ϫ/Ϫ mice on C57BL/6 background were to as sterile inflammatory response. kindly provided by Dr. V. Dixit, Genentech, South San Francisco, CA. Age Members of the NLR family are key components of the in- and sex-matched C57BL/6 were purchased from The Jackson Laboratory. All experiments using mice were approved by the University of Tennessee flammasome (2), a multiprotein cytoplasmic complex that reg- Health Science Center Institutional Animal Care and Use Committee ulates secretion and bioactivity of cytokines belonging to the (Memphis, TN).

Department Molecular Sciences, University of Tennessee Health Science Center, Mem- 3 Abbreviations used in this paper: NLR, Nod-like receptor; AEBSF, 4-(2-aminoethyl)- phis, TN 38163 benzenesulfonyl fluoride; BAL, bronchoalveolar lavage; 7BIO, 7-bromoindirubin-3Ј- oxime; BMM, bone marrow-derived mononuclear cell; HMGB1, high mobility group box Received for publication April 3, 2009. Accepted for publication June 10, 2009. 1; IO, indirubin oxime; LDH, lactate dehydrogenase; STS, staurosporine; Z-YVAD- The costs of publication of this article were defrayed in part by the payment of page charges. FMK, N-benzyloxycarbonyl-Tyr-Val-Ala-Asp-fluoromethyl ketone. 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 Grant R21AI076835 (to F.R.). 2 Address correspondence and reprint requests to Dr. Fabio Re, Department Molecular Sciences, University of Tennessee Health Science Center, 858 Madison Avenue, Memphis, TN 38163. E-mail address: [email protected] www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901080 The Journal of Immunology 1529

Reagents Indirubin derivatives (resuspended in DMSO at 25 mM) and the caspase-1 inhibitor N-benzyloxycarbonyl-Tyr-Val-Ala-Asp-fluoromethyl ketone (Z- YVAD-FMK) were purchased from Axxora. Alum (Alhydrogel) was from Sigma-Aldrich. The cathepsin B inhibitor CA-074-Me was from Biomol International. All other chemicals were from Sigma-Aldrich. The following Abs were used: rabbit anti-caspase-1 (Upstate Biotechnologies), goat anti- mIL-1␤ (R&D Systems), anti-human IL-1␤ 3ZD mAb (Preclinical Repos- itory, Biological Research Branch, National Cancer Institute), and rabbit anti-HMGB1 (where HMGB1 is high mobility group box 1; Santa Cruz Biotechnology). Human cell lines The THP-1 macrophages and MSTO-211H lung mesothelioma human cell lines were obtained from American Type Culture Collection and grown in RPMI 1640 with 10% FCS. PBMC and bone marrow-derived mononuclear cell (BMM) isolation Human PBMC were isolated from leukopacks by Ficoll-Histopaque density gradient centrifugation. Mouse BMM were generated by incubating bone mar- row in RPMI 1640 with FCS and supplemented with recombinant murine FIGURE 1. 7BIO activates the inflammasome. THP-1 (A) or PBMC Downloaded from GM-CSF (20 ng/ml) for 8 days. This procedure routinely results in 60–80% (B–D) were primed for 4 h with LPS (3 ng/ml) and stimulated for additional CD11cϩ dendritic cells. 6 h with the indicated indirubin derivatives (25 ␮MinA and 5, 25, and 125 ␮ ␤ ␤ Cytokine measurements MinB) and inhibitors. A, Human IL-1 (hIL-1 ) was measured in con- ditioned supernatants of cells stimulated with 5-iodine-indirubin-3Ј-oxime Cytokine levels in conditioned supernatants and bronchoalveolar lavage (BAL) (5IIO), 6-bromoindirubin-3Ј-oxime (6BIO), 7BIO, IO, indirubin (Indi), fluids were measured by ELISA using the following paired Abs kits: human 7BIO-Me, and alum crystals. B, Primed PBMC were also stimulated with IL-1␤ and human IL-18 (R&D Systems), murine IL-1␤ (eBioscience), and http://www.jimmunol.org/ 7BIO for 1, 3, and7horwith 7BIO, 6BIO, and indirubin at 125, 25, and murine IL-18 (MBL Nagoya, Japan). 5 ␮M. C, The presence of the immature (Pro-) and mature forms of Statistical analysis caspase-1 and IL-1␤ in cell culture supernatants was analyzed by immuno- blotting. C, CA-074-Me; Z, Z-YVAD-FMK. D, Inflammasome activation Ϯ All data were expressed as mean SEM. Student’s t test was used for statistical by 7BIO was blocked by treatment with Z-YVAD-FMK (ZYVAD) at 10 evaluation of the results. Significance was set at p Ͻ 0.05. ␮M) and CA-074-Me (CA094) but not by cytochalasin D (CytoD) or by uricase at 10 U/ml, apyrase at 2 U/ml, and oxidized ATP (oATP) at 0.25 ␮M. Results and Discussion Indirubin oxime (IO) derivative 7BIO activates the inflammasome

In a search for small molecules capable of activating the inflam- activation by 7BIO is not mediated by the release of endoge- by guest on September 28, 2021 masome, we identified 7-bromoindirubin-3Ј-oxime (7BIO), an nous danger signals like monosodium urate or ATP. 4 IO derivative (see supplemental Fig. 1 for structure), as a po- Necrosis but not apoptosis activates the inflammasome tent inducer of the release of IL-1␤ and IL-18 from LPS-primed human THP-1 macrophages or human PBMC (Fig. 1, A and All of the indirubin derivatives tested have been demon- B). Surprisingly, indirubin and the other derivatives 6-bro- strated to inhibit several kinases and to induce apoptosis moindirubin-3Ј-oxime (6BIO), IO, 5-iodine-indirubin-3Ј- with the exception of 7BIO, which does not induce apopto- oxime (5IIO), and 7BIO-Me (an inactive compound with a sis or inhibit kinases but rather rapidly induces a form of methyl group on N1 of 7BIO) were poor activators of the caspase-independent necrosis (15–17). The unequal ability inflammasome. 7BIO induced processing of pro-IL-1␤ and of the indirubin derivatives to induce necrosis was confirmed procaspase-1 (Fig. 1C), a biochemical demonstration of inflam- by measuring release in the culture supernatant of the en- masome activation. Inflammasome activation by 7BIO was in- zyme lactate dehydrogenase (LDH) or the nuclear protein hibited by treatment with the caspase-1 inhibitor Z-YVAD- HMGB1, an endogenous danger signal that is released by FMK and the cathepsin B inhibitor CA-074-Me, but not by necrotic, but not apoptotic, cells or during infection (18). In cytochalasin D (Fig. 1D), which blocks inflammasome activa- agreement with previous results (15–17), 7BIO was a much tion by alum crystals (14) and other particles (7). Necrotic cells stronger inducer of necrosis than the other indirubin deriv- atives (Fig. 2A). The fact that only 7BIO, among the indiru- are known to release uric acid and ATP, both of which activate ␤ the inflammasome (3, 6). Inflammasome activation in response bin derivatives, induced IL-1 release suggested that infam- to 7BIO was not blocked by uricase, which inhibits inflamma- masome activation may be triggered in cells undergoing some activation by uric acid crystals. The ATP-hydrolyzing en- necrosis. To test this hypothesis, LPS-primed THP-1 cells or zyme apyrase or oxidized ATP, which blocks inflammasome ac- PBMC were rendered apoptotic or necrotic by additional tivation by extracellular ATP, did not inhibit IL-1␤ release in treatments and inflammasome activation was tested. response to 7BIO (Fig. 1D). Moreover, inflammasome activa- To induce apoptosis, cells were treated with the kinase in- tion by 7BIO was still observed in BMDC derived from mice hibitor staurosporine (STS) or the topoisomerase inhibitor etoposide or were UV irradiated. Cells were rendered ne- deficient in P2X7, the for extracellular ATP that is critically involved in inflammasome activation by ATP crotic by one cycle of freeze and thaw, by osmotic shock, or (supplemental Fig. 2). These results show that inflammasome by treatment with STS in presence of oligomycin (an inhib- itor of ATP synthase). It has been demonstrated that cells depleted of ATP through oligomycin treatment undergo ne- 4 The online version of this article contains supplemental material. crosis rather than apoptosis when cotreated with STS (19). 1530 CUTTING EDGE: NECROSIS ACTIVATES THE NLRP3 INFLAMMASOME Downloaded from FIGURE 2. Necrosis but not apoptosis activates the inflammasome. A, THP-1 cells were treated with the indicated indirubin derivatives (5-iodine- indirubin-3Ј-oxime (5IIO), 6-bromoindirubin-3Ј-oxime (6BIO), 7BIO, IO, indirubin (Indi), 7BIO-Me, and alum crystals) and the release of LDH FIGURE 3. Necrosis activates the NLRP3 inflammasome. BMM from the and HMGB1 in cell culture supernatants was measured. B, PBMC were indicated mouse strains were primed for 4 h with LPS and stimulated with the primed for 4 h with LPS (3 ng/ml) and stimulated for additional 6 h with indicated stimuli, including STS/oligomycin (STSϩOlig.) and one freeze-thaw the indicated stimuli (including UV irradiation, etoposide (Etop), STS, (F&T) cycle) at the concentrations given in the Fig. 2 legend. Mature murine http://www.jimmunol.org/ ϩ STS/oligomycin (STS Olig), one freeze-thaw cycle (F&T), and H2O. Hu- IL-␤ (mIL-1␤) and caspase-1 were measured by ELISA or immunoblotting in ␤ ␤ man IL-1 (hIL-1 ), caspase-1, and HMGB-1 were measured in condi- culture supernatants. Asterisks indicate statistically significant difference com- .p Ͻ 0.01 ,ءء ;tioned supernatants by ELISA and immunoblotting. C, THP-1 or MSTO- pared with the wild-type (WT) cells 211H cell lines were stimulated, in absence of LPS, with 7BIO or STS/ oligomycin for6hinpresence of the indicated inhibitors, including Z-YVAD-FM (Z) and CA-074-Me (C), and AEBSF (A), IL-1␤ and IL-18 were measured in conditioned supernatants. The inset in the MSTO-211H markably, secretion of IL-18, which is constitutively ex- ␤ graph shows detection by immunoblotting of mature IL-1 in the concen- pressed by several cell types, was induced by 7BIO or STS/ trated supernatants of cells stimulated with 7BIO (A, AEBSF). Concentra- tions used in these experiments were 25 mM for etoposide, 50 mJ/cm2 for oligomycin in the absence of LPS priming in THP-1 cells by guest on September 28, 2021 UV, 0.5 ␮M for STS, 10 ␮M for oligomycin, 25 ␮M for indirubin deriv- (Fig. 2C). Similarly, these necrotic stimuli induced the re- atives, 10 ␮M for Z-YVAD-FM (ZYVAD), 25 ␮M for CA-074-Me, and lease of mature IL-1␤ independently of LPS in MSTO- 300 ␮M AEBSF. 211H, a mesothelioma cell line that constitutively expresses pro-IL-1␤ (our unpublished observation). In these cells, in- flammasome activation by 7BIO was inhibited by 4-(2-ami- As shown in Fig. 2B, the necrotic stimuli were strong induc- noethyl)-benzenesulfonyl fluoride (AEBSF), a serine pro- ers of inflammasome activation as measured by the level of tease inhibitor that was reported to block the induction of IL-1␤ released in culture supernatants and by the processing necrosis by 7BIO (17), demonstrating that induction of ne- of pro-IL-1␤ and procaspase-1. In contrast, inflammasome crosis is necessary for inflammasome activation by 7BIO. activation in apoptotic cells was much weaker. It should be noted, however, that during prolonged stimulation with ap- Necrosis activates the NLRP3 inflammasome optotic agents we observed IL-1␤ release as previously shown The fact that the cathepsin B inhibitor CA-074-Me suppressed (20). This is a likely reflection of the induction of secondary IL-1␤ release in response to necrotic stimuli suggested the in- necrosis of the apoptotic cells and, in fact, the release of volvement of the NLRP3 inflammasome (8). To test this hy- LDH and HMGB1 was greatly increased by overnight incu- pothesis, BMM from wild-type, NLRP3-, ASC-, or NLRC4 bation of the apoptotic cells (not shown). It is also interest- (Ipaf)-deficient mice were pretreated with LPS and then stim- ing that among the indirubin derivatives, after 7BIO the ulated with the necrotic stimuli 7BIO, STS/oligomycin, or one most potent inducer of inflammasome activation was 5IIO, cycle of freeze-thaw. As shown in Fig. 3, caspase-1 activation which has been shown to induce a form of with and secretion of mature IL-1␤ in response to these treatments Ϫ Ϫ Ϫ Ϫ both necrotic and apoptotic hallmarks (16). Inflammasome was abolished in NALP3 / and ASC / cells, whereas it was Ϫ Ϫ activation by the necrotic treatments was blocked by the induced at a comparable level in wild-type and NLRC4 / caspase-1 inhibitor Z-YVAD-FMK. Attesting to the degree cells. These results demonstrate that the NLRP3 inflammasome of necrosis induction, the danger molecule HMGB-1 was is activated in cells undergoing necrosis. detected in the supernatants of cells stimulated with the ne- crosis-inducing, but not the apoptosis-inducing, treatments Necrosis activates the inflammasome in vivo (Fig. 2B, right panel). In contrast, cleaved poly-ADP-ribose To test whether necrosis can activate the inflammasome in vivo, polymerase, a hallmark of apoptosis, was strongly detected in we used the naphthalene-induced lung injury model. In this cells treated with apoptosis-inducing agents but to greatly model, i.p. injection of naphthalene results in selective necrosis reduced levels in necrotic cells (supplemental Fig. 3). Re- of the epithelial cell lining the airways. As shown in Fig. 4, the The Journal of Immunology 1531

necrosis, however, was not dependent on inflammasome ac- tivation (as judged by LDH or HMGB1 release; data not shown), as it was induced to the same extent in wild-type or NLRP3- or ASC-deficient cells and was not blocked by caspase-1 inhibitors, cathepsin B inhibitors, or necrostatin, a inhibitor (23). It is also interesting to note that other necrosis-inducing conditions such as treatment with hydrogen peroxide or paclitaxel, which induces caspase-in- dependent necrosis, failed to induce inflammasome activa- tion. Similarly, induction of necroptosis through TNF-␣ FIGURE 4. Necrosis activates the NLRP3 inflammasome in vivo. Wild- stimulation in the presence of caspase-3 and caspase-9 inhib- type (WT), NLRP3Ϫ/Ϫ,orASCϪ/Ϫ mice were injected i.p. with corn oil itors (23) did not result in inflammasome activation. Finally, (Oil; n ϭ 3) or naphthalene in oil (Oil-Napht.; 250 mg/kg, n ϭ 9 for WT if necrosis was induced too rapidly by repeated cycles of and NLRP3Ϫ/Ϫ and n ϭ 8 for ASCϪ/ Ϫ). BAL fluids were collected 20 h freeze-thaw or excessive osmotic shock, inflammasome acti- later and murine IL-1␤ (mIL-1␤) and IL-18 (mIL-18) were measured by vation was greatly reduced. Based on these observations, we ELISA. speculate that the dissolution of the cellular architecture that occurs during some specific forms of necrosis may lead to

lysosome destabilization, resulting in activation of the Downloaded from BAL fluid collected from wild-type mice 20 h following naph- NLRP3 inflammasome (8). Recently, it has been shown that thalene injection contained elevated levels of IL-18. In contrast, necrotic cells release IL-1␣ (24), although inflammasome ac- Ϫ Ϫ Ϫ Ϫ the BAL fluid from NLRP3 / and ASC / mice contained tivation was not reported. Interestingly, in contrast to IL- significantly lower levels of this cytokine. Similarly, IL-1␤ was 1␤, the biological activity of which depends on caspase-1- Ϫ Ϫ detected at a lower level in the BAL fluid of ASC / mice (al- mediated processing, the bioactivity of IL-1␣ does not Ϫ Ϫ though the reduction in NLRP3 / mice was not statistically require processing of the precursor. Our results identified http://www.jimmunol.org/ significant). The fact that the levels of IL-18 and IL-1␤ were activation of the NLRP3 inflammasome during necrosis as a Ϫ Ϫ more prominently decreased in ASC / mice than in novel mechanism through which necrotic cells release proin- Ϫ Ϫ NLRP3 / mice may suggest that other yet uncharacterized flammatory molecules that contribute to the sterile inflam- ASC-dependent may be also activated during matory response. necrosis in vivo. In conclusion, our results show for the first time that the Acknowledgments Ϫ/Ϫ Ϫ/Ϫ NLRP3 inflammasome is activated in cells that undergo ne- We are grateful to Vishva Dixit, Genentech, for NLRP3 , ASC , and NLRC4Ϫ/Ϫ mice and to Fayyaz Sutterwala, University of Iowa, for a gift of crosis, resulting in the production of mature IL-1␤ and by guest on September 28, 2021 P2X bone marrow cells. IL-18 under sterile conditions. It is remarkable that we could 7 observe inflammasome activation and the release of IL-1␤ Disclosures and IL-18 in vitro or in vivo in the absence of LPS priming The authors have no financial conflict of interest. or other proinflammatory stimulation, conditions under which the sterile inflammatory response occurs. Mechanis- References tically, it is still unclear how pro-IL-1␤ is induced in a sterile 1. Rock, K. L., and H. Kono. 2008. The inflammatory response to cell death. Annu. Rev. environment. Several endogenous danger signals released by Pathol. 3: 99–126. 2. Franchi, L., T. Eigenbrod, R. Munoz-Planillo, and G. Nunez. 2009. The inflamma- necrotic cells have been shown to stimulate TLR and other some: a caspase-1-activation platform that regulates immune responses and disease pattern recognition receptors and therefore have the poten- pathogenesis. Nat. Immunol. 10: 241–247. 3. Mariathasan, S., D. S. Weiss, K. Newton, J. McBride, K. O’Rourke, M. 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