Necrotic cells trigger a sterile inflammatory responsethrough the Nlrp3 inflammasomeShankar S. Iyera,b, Wilco P. Pulskensc, Jeffrey J. Sadlera,b, Loes M. Butterc, Gwendoline J. Teskec, Tyler K. Ullanda,b,Stephanie C. Eisenbarthd,e, Sandrine Florquinc, Richard A. Flavelle,f, Jaklien C. Leemansc,1,2, and Fayyaz S. Sutterwalaa,b,1,2

aDivision of Infectious Diseases and the bInflammation Program, Department of Internal Medicine, University of Iowa, Iowa City, IA 52242; cDepartment ofPathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; and dDepartment of Laboratory Medicine, eDepartment ofImmunobiology, and the fHoward Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06520

Edited by Vincent T. Marchesi, Yale University School of Medicine, New Haven, CT, and approved October 14, 2009 (received for review July 31, 2009)

Dying cells are capable of activating the innate immune system andinducing a sterile inflammatory response. Here, we show that ne-crotic cells are sensed by the Nlrp3 inflammasome resulting in thesubsequent release of the proinflammatory cytokine IL-1�. Necroticcells produced by pressure disruption, hypoxic injury, or complement-mediated damage were capable of activating the Nlrp3 inflamma-some. Nlrp3 inflammasome activation was triggered in part throughATP produced by mitochondria released from damaged cells. Neu-trophilic influx into the peritoneum in response to necrotic cells invivo was also markedly diminished in the absence of Nlrp3. Nlrp3-deficiency moreover protected animals against mortality, renal dys-function, and neutrophil influx in an in vivo renal ischemic acutetubular necrosis model. These findings suggest that the inhibition ofNlrp3 inflammasome activity can diminish the acute inflammationand damage associated with tissue injury.

caspase-1 � interleukin-1� � necrosis

S terile inflammatory insults such as trauma or ischemia triggera robust influx of neutrophils and monocytes to the site of tissue

injury (1). Damaged cells are thought to release danger-associatedmolecular patterns (DAMPs) that can alert the innate immunesystem to the impending tissue damage. However, in the absence ofan infectious etiology, the collateral damage caused by the initiationof this inflammatory response can be extremely detrimental.Hence, it is important to understand the mechanisms initiating thesterile inflammatory response to devise strategies with which tocontrol it.

The innate immune system possesses germline-encoded patternrecognition receptors that are capable of recognizing highly con-served molecules. The best described class of these receptors areToll-like receptors (TLRs), which predominantly sense pathogen-associated molecular patterns (PAMPs) (2). The innate immunesystem serves to monitor for more than just the presence ofmicrobes; pattern recognition receptors also recognize DAMPsreleased by cells in response to cellular damage or stress. Althoughthere is evidence that some DAMPs, such as high-mobility groupbox 1 protein (HMGB1) and heat-shock proteins, can stimulateTLR2 and TLR4, the inflammatory response to necrotic cells invivo is predominantly independent of TLR signaling (3–6). How-ever, mice deficient in MyD88 [which in addition to TLR signalingis also essential for signaling through the IL-1 receptor I (IL-1R)]or IL-1R had markedly impaired recruitment of neutrophils inresponse to necrotic cells (6).

IL-1� and IL-1� signal through the IL-1R, and both requirecaspase-1 for secretion of their mature forms (7). Nlrp3 (also knownas Nalp3, cryopyrin, and CIAS1), a member of the NLR (nucle-otide-binding domain leucine-rich repeat containing) family, alongwith ASC and caspase-1, form a multiprotein complex called theNlrp3 inflammasome (8). The Nlrp3 inflammasome can activatecaspase-1 in response to a number of diverse stimuli including ATP,monosodium urate crystals (MSU), and bacterial pore formingtoxins resulting in the processing and secretion of the proinflam-matory cytokines IL-1�, IL-1�, and IL-18 (9–12).

Here, we investigate the mechanisms of necrotic cell-inducedinflammation and demonstrate that specific forms of cell death aresensed by the Nlrp3 inflammasome. We further found that viablemitochondria released from these necrotic cells were responsiblefor Nlrp3 inflammasome activation. Nlrp3-deficiency also had aprofound effect on mortality and renal function in an in vivo modelof renal ischemic acute tubular necrosis. Together our data indicatethat the Nlrp3 inflammasome plays a crucial role in initiating sterileinflammatory responses to tissue injury.

ResultsInflammatory Response to Necrotic Cells Is Mediated by the Nlrp3Inflammasome. To examine if the Nlrp3 inflammasome was involvedin the inflammatory response to cellular damage, we injectedpressure-disrupted B16 cells (a murine melanoma cell line) i.p. intomice deficient in IL-1R, caspase-1, Nlrp3, or ASC (Fig. 1 A–C).Sixteen hours after challenge with pressure disrupted B16 cells,wild-type (WT) mice displayed a marked influx of neutrophils intothe peritoneal cavity. This neutrophil influx was significantly di-minished in mice deficient in components of the Nlrp3 inflamma-some (Fig. 1 A–C). Neutrophil influx in Nlrp3- and ASC-deficientmice in response to thioglycollate remained intact demonstratingthat Nlrp3- and ASC-deficiency does not lead to a global defect inneutrophil migration (Fig. S1).

The cysteine protease caspase-1 is required for the processingand secretion of IL-1� and also partially for IL-1� (7, 13). Todetermine if necrotic cells were capable of inducing the secretion ofthese cytokines, LPS-primed macrophages (M�) were stimulatedwith necrotic B16 cells derived by UV-irradiation, freeze-thawing,or pressure disruption. Pressure-disrupted B16 cells induced arobust secretion of IL-1�, however, UV-irradiated or freeze-thawed B16 cells induced substantially less IL-1� (Fig. 2A). Thesecretion of IL-1� was not specific to B16 cells, as pressure-disrupted HEK293 cells similarly induced IL-1� secretion fromLPS-primed M� (Fig. S2A). In contrast to pressure-disrupted B16cells, which induce a robust neutrophilic influx in vivo following i.p.injection, necrotic B16 cells produced by freeze-thaw inducedsignificantly less i.p. inflammation, which was independent ofcaspase-1 (Fig. 2B). Together, these findings demonstrate thatspecific forms of necrotic cell death are capable of inducing thesecretion of IL-1� from LPS-primed M� and inflammatory re-sponses in vivo in a manner dependent on the Nlrp3 inflammasome.

Author contributions: S.S.I., W.P.P., S.F., J.C.L., and F.S.S. designed research; S.S.I., W.P.P.,J.J.S., L.M.B., G.J.T., and T.K.U. performed research; S.C.E. and R.A.F. contributed newreagents/analytic tools; S.S.I., W.P.P., J.J.S., L.M.B., G.J.T., S.F., J.C.L., and F.S.S. analyzeddata; and J.C.L. and F.S.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

1J.C.L. and F.S.S. contributed equally to this work.

2To whom correspondence may be addressed. E-mail: j.c.leemans@amc.uva.nl or fayyaz-sutterwala@uiowa.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0908698106/DCSupplemental.

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Nlrp3-, ASC-, and caspase-1-deficient LPS-primed M� displayeda marked defect in their ability to process and secrete IL-1� inresponse to pressure-disrupted B16 cells compared to WT M� (Fig.2 C–E). Caspase-1 activation in LPS-primed WT M� stimulatedwith pressure-disrupted B16 cells was detected by Western blot bythe appearance of the p10 cleavage product (Fig. 2E), whichoccurred by 3 h post-stimulation (Fig. S2B). We did not observecaspase-1 activation in response to pressure-disrupted B16 cells ineither Nlrp3- or ASC-deficient LPS-primed M� (Fig. 2E). Inaddition, there is a defect in IL-1� secretion in Nlrp3-deficientLPS-primed M� stimulated with pressure-disrupted B16 cells,although the absolute amounts of IL-1� secreted were substantiallylower compared to IL-1� (Fig. S2C). Thus, pressure-disruptednecrotic cells drive an inflammatory cell infiltrate in vivo and induce

the production of IL-1� and IL-1� in a Nlrp3 inflammasome-dependent manner.

Complement- and Hypoxia-Mediated Cellular Damage Is Sensed by theNlrp3 Inflammasome. Although the complement system plays animportant role in protecting the host against infectious diseases andaiding in the repair of damaged tissue, unregulated or inappropriateactivation of the complement cascade can in itself lead to tissueinjury (14). To determine if complement-mediated damage in-duced an inflammatory response similar to pressure-disruptednecrotic cells, we challenged M� with complement-lysed spleno-cytes. Complement-lysed splenocytes did induce the secretion ofIL-1� from LPS-primed WT M� (Fig. 3A). Heat inactivation ofserum used for complement lysis abrogated the ability of thesplenocytes to induce IL-1� secretion (Fig. 3B), suggesting thatcellular damage was required for this process. Nlrp3-, ASC-, andcaspase-1-deficient M� failed to secrete IL-1� in response tocomplement-lysed splenocytes (Fig. 3A). In contrast, M� deficientin Nlrc4, which is required for caspase-1 activation in response toinfection with type III and type IV secretion system carryingGram-negative bacteria (15, 16), were capable of secreting IL-1� inresponse to complement-lysed splenocytes (Fig. 3A). Similar toprimary splenocytes, complement-mediated lysis of a hybridomacell line was also capable of inducing the activation of caspase-1 andthe secretion of IL-1� from LPS-primed M� in a Nlrp3-dependentmanner (Fig. 3 C–D and Fig. S2D). Therefore, complement-mediated damage to host cells can be sensed by the Nlrp3 inflam-masome. These findings also demonstrate a communication be-tween these two evolutionary ancient components of the innateimmune system.

To assess if hypoxia-mediated cellular damage was capable ofactivating the Nlrp3 inflammasome, M� were exposed to B16 cellsgrown under hypoxic conditions. LPS-primed M� exposed tohypoxic B16 cells, but not B16 cells grown under normoxic condi-tions, secreted IL-1� in a Nlrp3-dependent manner (Fig. 3E). Theseresults suggest that in addition to pressure-disruption and comple-ment-mediated damage, the Nlrp3 inflammasome is capable ofsensing cellular damage induced by hypoxic conditions.

Extracellular Matrix Molecules Can Prime the Nlrp3 Inflammasome forActivation. Activation of the Nlrp3 inflammasome is a two-stepprocess requiring a priming step (signal 1) followed by an activationstep (signal 2). Signal 1 serves two functions: in addition tostimulating the production of pro-IL-1� it is also a prerequisite forinflammasome activation. LPS priming of M� allowed for pressure-

Fig. 1. Inflammation induced in vivo by pressure-disrupted necrotic cells isdependent on the Nlrp3 inflammasome. (A–C) Neutrophil influx into theperitoneum of WT, IL-1R-, caspase-1-, ASC-, and Nlrp3-deficient mice 16 h afteri.p. challenge with pressure-disrupted B16 cells (B16). Control WT mice werechallenged i.p. with PBS. *, P � 0.0017; **, P � 0.0021; ***, P � 0.0159; ****,P � 0.0240.

Fig. 2. Cellular injury induced by pressure-disruption is sensed by the Nlrp3 inflammasome. (A) LPS-primed WT M� were stimulated with either UV-treated,freeze-thawed, or pressure-disrupted B16 cells at a ratio of 10 necrotic cells per M�; culture supernatants were collected 12 h later, and IL-1� release was measured byELISA. (B) Neutrophil influx into the peritoneum of WT and caspase-1-deficient mice 16 h after i.p. challenge with either 1 � 107 pressure-disrupted B16 cells (B16) orfreeze-thawed B16 cells. *, P � 0.0041. (C and D) LPS-primed WT, Nlrp3-, caspase-1-, or ASC-deficient M� were stimulated with pressure-disrupted B16 cells andsupernatants collected 12 h later or at the indicated time. IL-1� release was measured by ELISA. (E) Lysates from LPS-primed WT, Nlrp3-, or ASC-deficient M� stimulatedwith pressure-disrupted B16 cell for 12 h were immunoblotted with antibodies against the p10 subunit of caspase-1, IL-1�, and GAPDH. (A, C, and D) Determinationswere performed in triplicate and expressed as the mean � SEM. Results are representative of two (A) and three (C and D) separate experiments.

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disrupted B16 cells to activate caspase-1 and induce the secretionof IL-1� as unprimed M� failed to activate caspase-1 and secreteIL-1� (Fig. S3 A and B). Importantly, endogenous molecules canalso serve a priming role for Nlrp3 inflammasome activation.Biglycan and hyaluronic acid, components of the extracellularmatrix, were capable of priming M� for Nlrp3 inflammasomeactivation in response to pressure-disrupted B16 cells (Fig. 3F).Hence, extracellular matrix components that accumulate in non-physiological sites or amounts can function as signal 1, suggestingthat Nlrp3 inflammasome activation can occur in vivo in sterilesettings without microbes being present to provide signal 1.

Mitochondria Activate the Nlrp3 Inflammasome. To determine whatcellular component from necrotic cells was capable of inducing the

activation of the Nlrp3 inflammasome, we performed subcellularfractionation of the pressure-disrupted B16 cells. Cytosolic, nu-clear, and plasma membrane fractions all failed to induce thesecretion of IL-1� from LPS-primed M� (Fig. 4A). Surprisingly,mitochondria isolated from pressure-disrupted B16 cells stimulatedthe secretion of IL-1� from LPS-primed WT M� (Fig. 4A). Thiswas not unique to B16 cells, as mitochondria isolated from HEK293cells were similarly capable of inducing the secretion of IL-1� fromLPS-primed M� (Fig. S4A). Purity of the mitochondrial fractionwas confirmed by Western blot analysis using antibodies againstCox4 (Fig. S4B). Unprimed M� failed to secrete IL-1� in responseto mitochondria, demonstrating that a separate priming step wasrequired for mitochondria to activate caspase-1 (Fig. S3 A and B).Similar to results obtained using pressure-disrupted cells, we ob-served that LPS-primed M� from Nlrp3-, ASC-, and caspase-1-deficient mice failed to secrete IL-1� in response to mitochondria(Fig. 4B). LPS-primed M� from Nlrc4-deficient mice had an intactresponse to mitochondria and secreted IL-1� (Fig. 4B), suggestingthat M� that encounter extracellular mitochondria specificallyactivate the Nlrp3 inflammasome. Stimulation of LPS-primed WTM�, but not Nlrp3- or ASC-deficient M�, with mitochondriaresulted in activation of caspase-1 as detected by Western blot (Fig.4C). Mitochondria injected i.p. into WT mice resulted in a markedinflammatory response as determined by neutrophilic influx; how-ever, Nlrp3-deficient mice had a significantly diminished inflam-matory response in vivo compared to WT mice (Fig. 4D).

Pretreatment of mitochondria with rotenone or myxothiazol,inhibitors of mitochondrial complex I and III, respectively, apyrase,an ATPase, or heat treatment resulted in the loss of ATP contentof the mitochondria (Fig. 4E) as well as diminished their ability toinduce M� secretion of IL-1� (Fig. 4F), suggesting that mitochon-dria must be actively respiring to induce the activation of the Nlrp3inflammasome. To confirm that residual rotenone, myxothiazol,and apyrase were not affecting M� function, silica, an activator ofthe Nlrp3 inflammasome, was added in combination with untreatedand treated mitochondria; no inhibition in silica-induced IL-1� wasobserved (Fig. S4C). Consistent with these findings, pretreatmentof pressure-disrupted necrotic B16 cells with rotenone, myxothia-zol, apyrase, or heat treatment also resulted in a diminished abilityto induce M� secretion of IL-1� (Fig. S4D). Thus, mitochondriaactivate the Nlrp3 inflammasome, and this activation can beblocked by inhibitors of cellular respiration.

Mitochondrial Activation of the Nlrp3 Inflammasome in Vitro PartiallyRequires P2X7R. Given its association with cellular damage, it waspossible that liberated uric acid (monosodium urate; MSU) frompressure-disrupted B16 cells was responsible for Nlrp3 inflamma-some activation (11, 17). We thus challenged LPS-primed M� withpressure-disrupted B16 cells or mitochondria that had been pre-treated with uricase. As expected, uricase treatment of MSUabolished its ability to induce the secretion of IL-1� (Fig. S5A) (18).However, uricase treatment of pressure-disrupted B16 cells ormitochondria did not inhibit their ability to induce IL-1� secretion(Fig. S5A), suggesting that pressure-disrupted necrotic cells andmitochondria activate the Nlrp3 inflammasome in a manner that isindependent of MSU.

To determine if necrotic cells and mitochondria require inter-nalization to activate the Nlrp3 inflammasome in a manner similarto MSU, silica, and alum (11, 18–23), LPS-primed M� werepretreated with cytochalasin B or D. As expected, cytochalasin Band D inhibited silica-induced IL-1� secretion (Fig. S5B) (23, 24);however, cytochalasin B or D did not markedly inhibit pressure-disrupted B16 cells or mitochondria from inducing IL-1� secretionfrom LPS-primed WT M� (Fig. S5B). Hence, endocytosis ofpressure-disrupted necrotic cells or mitochondria is not required forNlrp3 inflammasome activation.

ATP, acting via the purinergic P2X7 receptor, can activate theNlrp3 inflammasome without a requirement for endocytosis (10,

Fig. 3. Complement- and hypoxia-induced cellular injury is sensed by the Nlrp3inflammasome. (A) LPS-primed WT, caspase-1-, ASC-, Nlrp3-, or Nlrc4-deficientM� were stimulated with complement-lysed splenocytes at a ratio of 25 spleno-cytes per M�; supernatants were collected 12 h later, and IL-1� release assessedby ELISA. (B–D) Splenocytes or the hybridoma cell line 6F10 were either leftuntreated or opsonized with anti-MHC class II A�(b) IgG2a antibody followed byincubation with human serum (serum) or heat-inactivated human serum (HI-serum). LPS-primed WT or Nlrp3�/� M� were stimulated with complement-damaged cells at a ratio of 25 splenocytes per M�, 20 6F10 cells per M�, or asindicated; supernatants were collected 12 h later, and IL-1� release assessed byELISA.*,P�0.0003. (E)LPS-primedWTorNlrp3�/�M�werestimulatedwithB16cells grown under normoxic (control) or hypoxic conditions at a ratio of five cellsper M�; supernatants were collected 12 h later, and IL-1� release assessed byELISA. **, P � 0.0013. (F) WT M� were primed with either biglycan (8 �g/mL) orhyaluronic acid (25 �g/mL) for 12 h followed by stimulation with pressure-disrupted B16 cell at a ratio of 10 necrotic cells per M�; supernatants werecollected 12 h later, and IL-1� release measured by ELISA. (A–F) Determinationswere performed in triplicate and expressed as the mean � SEM. Results arerepresentative of two (C–F) and three (A and B) separate experiments.

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12, 25). To determine if signaling through the P2X7 receptor wasrequired for the inflammatory response to injured cells, we usedM� from P2X7R-deficient mice. As expected P2X7R-deficientLPS-primed M� failed to secrete IL-1� in response to ATP,however, IL-1� secretion in response to silica remained intact (Fig.4G). P2X7R-deficient LPS-primed M� had a marked defect intheir ability to secrete IL-1� in response to either pressure-disrupted B16 cells, mitochondria, or complement-lysed 6F10 cells(Fig. 4G and Fig. S5C); suggesting that ATP released from dis-rupted cells or generated by actively respiring mitochondria iscapable of activating the Nlrp3 inflammasome. However, whileIL-1� secretion from P2X7R-deficient LPS-primed M� was dimin-ished in response to necrotic cells or mitochondria, it was notabrogated (Fig. 4G and Fig. S5C), suggesting that additionalpathways leading to Nlrp3 inflammasome activation and IL-1�secretion exist. Consistent with these in vitro findings, in vivo i.p.challenge of P2X7R�/� mice with pressure-disrupted B16 cells didnot result in a significant decrease in neutrophilic influx into theperitoneal cavity in comparison to WT mice (Fig. S5D).

Nlrp3- and ASC-Deficient Mice Are Protected from Renal Ischemia-Reperfusion Injury. The events that occur during ischemia-reperfusion (I/R) injury are complex and involve altered renalmorphology and hemodynamics, death of epithelial and endothelialcells, and exuberant inflammation (26). It is known that micedeficient in the components of the membrane attack complex areprotected from renal I/R injury, suggesting cell lysis by complementis a key initiator of the inflammatory response (27). To explore themechanism by which necrotic injury is translated in vivo into

inflammation, mice were subjected to bilateral renal artery occlu-sion and killed one day after reperfusion. This revealed thatnonlethal renal I/R injury resulted in a significant upregulation ofNlrp3 gene expression (Fig. 5A), which was accompanied bypronounced acute tubular necrosis (Fig. S6) that was similarbetween WT and Nlrp3-deficient animals (Fig. 5B). There was alsoincreased expression of the extracellular matrix molecules biglycanand hyaluronic acid following renal I/R injury (Fig. S7 A and B),consistent with previous findings (28). Importantly, Nlrp3 defi-ciency protected animals from lethal renal ischemic injury; Nlrp3-deficient mice had a 86% survival following renal acute tubularnecrosis at 5.5 days post-ischemia compared with 8% survival inWT mice (P � 0.0001) (Fig. 5C). Nlrp3-deficient mice weremoreover functionally protected against renal dysfunction as re-flected by significantly lower plasma urea and creatinine levels ascompared to WT animals 24 h after nonlethal renal ischemic injury(Fig. 5 D and E). This protection was associated with reducednumbers of neutrophils infiltrating the renal interstitium on day 1compared to WT mice (Fig. 5 F and G). Consistent with this, thelevel of neutrophil chemoattractant KC and total IL-1� in thekidneys of Nlrp3-deficient mice after ischemic acute tubular ne-crosis was reduced compared to WT mice (Fig. 5 H and I).

ASC gene expression was significantly upregulated 5 days afternonlethal renal I/R injury, while there was no difference after 1 day(Fig. S8A). The amount of acute tubular necrosis was similarbetween WT and ASC-deficient animals at both time points (Fig.S8B). ASC deficiency protected animals from lethal renal ischemicinjury, although the difference was less pronounced than in Nlrp3-deficient mice. ASC-deficient mice had a 38% survival following

Fig. 4. Mitochondria released from necrotic cells stimulate Nlrp3 inflammasome activation. (A) LPS-primed WT M� were stimulated with pressure-disrupted B16 cellsor nuclei, plasma membrane, cytosol, and mitochondrial fractions derived from B16 cells at a ratio of five cell equivalents per M�; supernatants were collected 12 h later,and IL-1� release was measured by ELISA. (B) LPS-primed WT, caspase-1-, ASC-, Nlrp3-, or Nlrc4-deficient M� were left untreated or stimulated with mitochondria (100�g/mL); supernatants were collected 12 h later, and IL-1� release was assessed by ELISA. (C) Lysates from LPS-primed WT, Nlrp3-, or ASC-deficient M� stimulated withmitochondria for 12 h were immunoblotted with antibodies against the p10 subunit of caspase-1 and GAPDH. (D) Neutrophil influx into the peritoneum of WT andNlrp3-deficient mice 16 h after i.p. challenge with mitochondria isolated from 1 � 107 B16 cells. Control WT mice were challenged i.p. with homogenization buffer.

*, P � 0.0317. (E) Mitochondria were treated with rotenone (10 �M), myxothiazol (10 �M), apyrase (1 U) for 20 min or heat-treated at 65 °C for 20 min, washed, andATP content quantified. (F) LPS-primed WT M� were stimulated with mitochondria (100 �g/mL) that had been treated as described; supernatants were collected 12 hlater, and IL-1� release was measured by ELISA. (G) LPS-primed WT or P2X7R-deficient M� were stimulated with ATP (5 mM), silica (50 �g/cm2), pressure-disrupted B16cells,ormitochondria; supernatantswerecollected12h later, and IL-1� releasewasmeasuredbyELISA.**,P�0.0041;***,P�0.0009.Determinationswereperformedin triplicate and expressed as the mean � SEM. Results are representative of two (A, E, and F) and three (B and G) separate experiments.

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renal acute tubular necrosis at 5.5 days post-ischemia, while noneof the WT mice survived (P � 0.001) (Fig. S8C). ASC-deficientmice also displayed a more preserved renal function 5 days afterrenal I/R injury as compared to WT (Fig. S8 D and E). In line withthe Nlrp3-deficient mice, protection of the ASC-deficient micecorrelated with reduced neutrophil influx and renal KC and IL-1�levels (Fig. S8 F–H). The difference seen between Nlrp3-deficientand ASC-deficient mice in survival and early renal dysfunctionfollowing renal ischemic injury suggests that Nlrp3 may play addi-tional roles independently of ASC and, by extension, caspase-1.Together these data demonstrate that the Nlrp3 inflammasomeinduces an exaggerated acute inflammatory response during isch-emic acute tubular necrosis and as a consequence contributes toI/R-induced renal dysfunction and lethality.

DiscussionWe postulate that specific forms of cellular injury result in therelease of viable mitochondria into the extracellular space, trigger-ing the activation of the Nlrp3 inflammasome, in part through therelease of ATP. Extracellular ATP is quickly degraded by exo-nucleases, therefore release of intact ATP-producing mitochondriaprovides an effective mechanism to alert the immune system tocellular damage. Triggering of the Nlrp3 inflammasome results inactivation of caspase-1 and the processing and secretion of IL-1�,which can recruit neutrophils to sites of infection and injury, andalso possibly lead to host tissue damage (Fig. S9).

It is interesting that specific modes of cell death, such as pressuredisruption, complement lysis and hypoxia, were sensed by the Nlrp3inflammasome, whereas cell death induced by freeze-thaw and UV

irradiation failed to induce a robust secretion of IL-1� (Fig. 2A).We speculate that freeze-thaw or UV irradiation may either resultin damaged nonviable mitochondria or that mitochondria remainwithin the killed cell, inaccessible to the extracellular space andhence fail to activate the Nlrp3 inflammasome. A recent study byLi and colleagues also demonstrated that necrosis induced by7-bromoindirubin-3�-oxime was capable of activating the Nlrp3inflammasome (29). These findings highlight the heterogeneity ofcell death even within the necrotic subset.

I/R injury occurs as blood flow is restored to ischemic tissueresulting in a profound inflammatory response. It is a significantcause of the pathology associated with clinical conditions such asmyocardial infarction, cerebral ischemia, and in the operativemanagement of trauma. Renal I/R injury is a major cause of acuteand end-stage renal failure and is associated with increased acuterenal transplant rejection and delayed allograft function (30–34).Although the pathophysiology of renal I/R injury is complex, it isclear that necrotic cellular injury mediated by I/R and complementactivation play a major role in initiating subsequent inflammatoryresponses. Our studies show that although the initial necrotic insultfollowing I/R injury is the same in WT, Nlrp3-, and ASC-deficientmice, the ensuing inflammatory response to the necrotic cells ismarkedly reduced in mice deficient in components of the Nlrp3inflammasome. Limiting this Nlrp3 inflammasome-driven inflam-matory response is crucial to preventing further organ damage andin preserving renal function. An important role for IL-1� in thepathogenesis of renal I/R injury is also supported by in vivo studiesdemonstrating that the lack of functional IL-1R, IL-1�/�, or thetreatment with IL-1 receptor antagonist (IL-1Ra) impairs the

Fig. 5. Nlrp3-deficiency protects animals against mortality, renal dysfunction, and impairs an inflammatory response during renal ischemic acute tubular necrosis.(A) Relative Nlrp3 mRNA levels were measured in kidneys of WT mice 1 day after nonlethal renal I/R or sham operation. (B) Tubular necrosis score of WT mice (n � 8)and Nlrp3-deficient mice (n � 8) after nonlethal renal I/R injury using PAS-D-stained renal tissue sections. (C) Survival of Nlrp3-deficient mice (n � 14) compared to WTmice (n � 13) after lethal renal ischemia. (D and E) Renal dysfunction of Nlrp3-deficient mice (n � 8) compared to WT mice (n � 8) as reflected by increased levels ofurea (D) and creatinine (E) in plasma after nonlethal renal acute tubular necrosis. (F and G) Neutrophil influx in kidneys from WT and Nlrp3-deficient mice 1 day afterrenal I/R or sham operation as assessed by immunohistochemistry (F) and counted in at least 10 randomly selected high-power fields in the outer medulla (G). Originalmagnification of pictures, �400. (H and I) Total KC and IL-1� levels in kidneys from Nlrp3-deficient and WT mice subjected for 1 day to renal I/R injury. *, P � 0.05.

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inflammatory response and subsequently accelerates renal recov-ery (35) or reduces renal tissue destruction (36, 37) after renal I/Rinjury. We have also recently shown that Nlrp3-deficient mice areprotected from acetaminophen-induced hepatotoxicity (38). Ourcurrent findings provide mechanistic insight into this process,suggesting that necrotic hepatocytes themselves may be responsiblefor triggering the inflammatory response through the Nlrp3 in-flammasome.

It has been unclear what the initial priming step for Nlrp3inflammasome activation is in vivo, but our findings suggest it maybe through stimulation by endogenous DAMPs that are releasedconcomitantly with cellular injury. Both biglycan and hyaluronicacid were capable of priming M� for Nlrp3 inflammasome activa-tion in response to pressure-disrupted necrotic cells in vitro. Bigly-can and hyaluronic acid were also found to be expressed at higherlevels in tissue following renal I/R injury, suggesting that they maybe responsible for priming the Nlrp3 inflammasome for activationin vivo.

The sterile inflammatory response to injured cells is thought tocontribute to the pathogenesis of not only ischemic diseases, suchas renal-, myocardial-, and cerebral-ischemia, but also to inflam-mation associated with traumatic injury and chemotherapeutic-induced tumor death. It is clear this inflammatory response is anindependent source of profound morbidity and mortality. Blockadeof an excessive and prolonged sterile inflammatory response withspecific Nlrp3 inflammasome antagonists could represent an ap-proach to treat or prevent the adverse outcomes associated withtissue injury.

Materials and MethodsMice. The generation of Nlrp3-, ASC-, caspase-1-, and Nlrc4-deficient mice hasbeen described (7, 12, 39). IL-1R-deficient and P2X7R-deficient mice (25) werepurchased from Jackson Laboratories. Caspase-1-, IL-1R-, and P2X7R-deficient

mice were backcrossed onto the C57BL/6 genetic background for 10, five, andseven generations, respectively. ASC- and Nlrp3-deficient mice were backcrossedonto the C57BL/6 genetic background for nine generations. Nlrc4-deficient micewere backcrossed onto the C57BL/6 genetic background for six generations. Age-and sex-matched C57BL/6 mice purchased from NCI were used as WT controls. Allprotocols used in this study were approved by the Institutional Animal Care andUse Committee at the University of Iowa and the University of Amsterdam.

In Vivo Peritonitis. Mice were injected i.p. with either 1 � 107 pressure-disruptedB16 cells or freeze-thaw-treated B16 cells in 500 �L PBS, 1 mL 3% thioglycollate,or PBS alone. Sixteen hours later, animals were euthanized and peritoneal lavagewas performed. The number of neutrophils (Ly-6G � 7/4�) in the lavage wasassessed by flow cytometry as described in ref. 6.

Ischemic Acute Tubular Necrosis. Ischemic acute tubular necrosis was induced asdescribed in ref. 40. Briefly, renal arteries of mice were clamped for 30 min(nonlethal) or 45 min (lethal) using microaneurysm clamps through a midlineabdominal incision under general anesthesia. After surgery, all mice received as.c. injection of 50 �g/kg buprenorphin (Temgesic; Shering-Plough) for analgesicpurposes and were allowed to recover from surgery for 12 h at 28 °C in aventilated stove and were killed 1 day after surgery. Sham-operated mice (n � 6per group) underwent the same procedure without clamping. At the time ofsacrifice, blood was collected by heart puncture in heparin-containing tubes andstored at �80 °C, and kidneys were harvested for further analysis. Total RNA wasextracted from renal tissue sections with TRIzol reagent (Invitrogen) and con-verted to cDNA.

Detailed information about materials and methods is in the SI Text.

ACKNOWLEDGMENTS. We thank Suzanne Cassel and William Nauseef for crit-ical review of the manuscript and Anthony Coyle, Ethan Grant, and John Bertinfor providing Nlrp3�/�, ASC�/�, and Nlrc4�/� mice. We thank Stephen Wrz-esinski for providing B16 cells. This work was supported by grant number IRG-77–004-31 from the American Cancer Society, administered through The HoldenComprehensive Cancer Center at the University of Iowa (to F.S.S.), NationalInstitutesofHealthK08AI065517(toF.S.S.),bygrantnumber916.56.168fromtheNetherlands Organization for Scientific Research (to W.P.P. and J.C.L.) and bygrant number C06.6023 from the Dutch Kidney foundation (to L.M.B. and J.C.L.).R.A.F. is an investigator of the Howard Hughes Medical Institute.

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