Hemolysis-induced lethality involves inflammasomeactivation by hemeFabianno F. Dutraa, Letícia S. Alvesa, Danielle Rodriguesa, Patricia L. Fernandezb, Rosane B. de Oliveirac,Douglas T. Golenbockc, Dario S. Zambonid, and Marcelo T. Bozzaa,1

aLaboratório de Inflamação e Imunidade, Departamento de Imunologia, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, 21941-902,Rio de Janeiro, Brazil; bCentro de Biología Celular y Molecular de Enfermedades, Instituto de Investigaciones Científicas y Servicios de Alta Tecnología,0843-01103, Panama City, Panama; cDivision of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, MA 01605;and dDepartamento de Biologia Celular, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, 14049-900, São Paulo, Brazil

Edited* by Ruslan Medzhitov, Yale University School of Medicine, New Haven, CT, and approved August 11, 2014 (received for review March 21, 2014)

The increase of extracellular heme is a hallmark of hemolysisor extensive cell damage. Heme has prooxidant, cytotoxic, andinflammatory effects, playing a central role in the pathogenesis ofmalaria, sepsis, and sickle cell disease. However, the mechanismsby which heme is sensed by innate immune cells contributing tothese diseases are not fully characterized. We found that heme,but not porphyrins without iron, activated LPS-primed macrophagespromoting the processing of IL-1β dependent on nucleotide-bindingdomain and leucine rich repeat containing family, pyrin domain con-taining 3 (NLRP3). The activation of NLRP3 by heme required spleentyrosine kinase, NADPH oxidase-2, mitochondrial reactive oxygenspecies, and K+ efflux, whereas it was independent of heme in-ternalization, lysosomal damage, ATP release, the purinergic re-ceptor P2X7, and cell death. Importantly, our results indicatedthe participation of macrophages, NLRP3 inflammasome compo-nents, and IL-1R in the lethality caused by sterile hemolysis. Thus,understanding the molecular pathways affected by heme in innateimmune cells might prove useful to identify new therapeutic tar-gets for diseases that have heme release.

inflammation | mitochondria | ROS | NOX2 | Syk

Hemolysis, hemorrhage, and rhabdomyolysis cause the releaseof large amounts of hemoproteins to the extracellular space,

which, once oxidized, release the heme moiety, a potentiallyharmful molecule due to its prooxidant, cytotoxic, and inflam-matory effects (1, 2). Scavenging proteins such as haptoglobinand hemopexin bind hemoglobin and heme, respectively, pro-moting their clearance from the circulation and delivery to cellsinvolved with heme catabolism. Heme oxygenase cleaves hemeand generates equimolar amounts of biliverdin, carbon monox-ide (CO) and iron (2). Studies using mice deficient for hapto-globin (Hp), hemopexin (Hx), and heme oxygenase 1 (HO-1)demonstrate the importance of these proteins in controlling thedeleterious effects of heme. Both Hp−/− and Hx−/− mice haveincreased renal damage after acute hemolysis induced by phe-nyhydrazine (Phz) compared with wild-type mice (3, 4). Micelacking both proteins present splenomegaly and liver in-flammation composed of several foci with leukocyte infiltrationafter intravascular hemolysis (5). Hx protect mice against heme-induced endothelial damage improving liver and cardiovascularfunction (6–8). Lack of heme oxygenase 1 (Hmox1−/−) causesiron overload, increased cell death, and tissue inflammationunder basal conditions and upon inflammatory stimuli (9–15).This salutary effect of HO-1 has been attributed to its effect ofreducing heme amounts as well as generating the cytoprotectivemolecules, biliverdin and CO.Heme induces neutrophil migration in vivo and in vitro (16,

17), inhibits neutrophil apoptosis (18), triggers cytokine and lipidmediator production by macrophages (19, 20), and increases theexpression of adhesion molecules and tissue factor on endothe-lial cells (21–23). Heme cooperates with TNF, causing hepato-cyte apoptosis in a mechanism dependent on reactive oxygen

species (ROS) generation (12). Whereas heme-induced TNFproduction depends on functional toll-like receptor 4 (TLR4),ROS generation in response to heme is TLR4 independent (19).We recently observed that heme triggers receptor-interactingprotein (RIP)1/3-dependent macrophage-programmed necrosisthrough the induction of TNF and ROS (15). The highly un-stable nature of iron is considered critical for the ability of hemeto generate ROS and to cause inflammation. ROS generated byheme has been mainly attributed to the Fenton reaction. How-ever, recent studies suggest that heme can generate ROSthrough multiple sources, including NADPH oxidase and mito-chondria (22, 24–27).Heme causes inflammation in sterile and infectious con-

ditions, contributing to the pathogenesis of hemolytic diseases,subarachnoid hemorrhage, malaria, and sepsis (11, 13, 24, 28),but the mechanisms by which heme operates in different con-ditions are not completely understood. Blocking the prooxidanteffects of heme protects cells from death and prevents tissuedamage and lethality in models of malaria and sepsis (12, 13, 15).Importantly, two recent studies demonstrated the pathogenicrole of heme-induced TLR4 activation in a mouse model ofsickle cell disease (29, 30). These results highlight the greatpotential of understanding the molecular mechanisms ofheme-induced inflammation and cell death as a way to iden-tify new therapeutic targets.

Significance

Heme causes inflammation in sterile and infectious conditions,contributing to the pathogenesis of sickle cell disease, malaria,and sepsis, but the mechanisms by which heme operates arenot completely understood. Here we show that heme inducesIL-1β processing through the activation of the nucleotide-binding domain and leucine rich repeat containing family, pyrindomain containing 3 (NLRP3) inflammasome in macrophages.Our results suggest that among NLRP3 activators, heme hascommon as well as unique requirements to trigger inflamma-some activation. In vivo, hemolysis and heme cause inflam-masome activation. Importantly, macrophages, inflammasomecomponents, and IL-1R contribute to hemolysis-induced le-thality. These results highlight the potential of understandingthe molecular mechanisms by which heme is sensed by innateimmune receptors as away to identify new therapeutic strategiesto treat the pathological consequences of hemolytic diseases.

Author contributions: F.F.D., D.T.G., D.S.Z., and M.T.B. designed research; F.F.D., L.S.A.,D.R., P.L.F., and R.B.d.O. performed research; F.F.D., L.S.A., D.R., P.L.F., R.B.d.O., D.S.Z., andM.T.B. analyzed data; and F.F.D. and M.T.B. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.1To whom correspondence should be addressed. Email: mbozza@micro.ufrj.br.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1405023111/-/DCSupplemental.

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Hemolysis and heme synergize with microbial molecules forthe induction of inflammatory cytokine production and inflam-mation in a mechanism dependent on ROS and Syk (24). Pro-cessing of pro–IL-1β is dependent on caspase-1 activity, requiringassembly of the inflammasome, a cytosolic multiprotein complexcomposed of a NOD-like receptor, the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), andcaspase-1 (31–33). The most extensively studied inflammasomeis the nucleotide-binding domain and leucine rich repeat con-taining family, pyrin domain containing 3 (NLRP3). NLRP3 andpro–IL-1β expression are increased in innate immune cellsprimed with NF-κB inducers such as TLR agonists and TNF (34,35). NLRP3 inflammasome is activated by several structurallynonrelated stimuli, such as endogenous and microbial molecules,pore-forming toxins, and particulate matter (34, 35). The acti-vation of NLRP3 involves K+ efflux, increase of ROS and Sykphosphorylation. Importantly, critical roles of NLRP3 havebeen demonstrated in a vast number of diseases (34, 36). Wehypothesize that heme causes the activation of the inflamma-some and secretion of IL-1β. Here we found that heme triggeredthe processing and secretion of IL-1β dependently on NLRP3inflammasome in vitro and in vivo. The activation of NLRP3 byheme was dependent on Syk, ROS, and K+ efflux, but inde-pendent of lysosomal leakage, ATP release, or cell death. Fi-nally, our results indicated the critical role of macrophages, theNLRP3 inflammasome, and IL-1R to the lethality caused bysterile hemolysis.

ResultsHeme Induces IL-1β Processing and Caspase-1 Activation. To examinethe ability of heme to act as a second signal, triggering inflam-masome activation, we pretreated bone-marrow–derived mac-rophages (BMMs) with LPS (signal 1) and stimulated with heme.Heme caused a dose-dependent secretion of IL-1β, including theprocessed form (Fig. 1A). In the absence of prestimulation withLPS, heme was unable to induce the secretion of IL-1β by BMMsor by thioglycollate-elicited peritoneal macrophages (Fig. S1 Aand B). We have previously shown that the synergistic effect ofheme on cytokine production triggered by microbial moleculesonly occurs in the presence of serum (24). Thus, we tested therequirement of serum on heme-induced IL-1β processing. UsingBMMs, we observed that heme caused a robust processing ofIL-1β and caspase-1 in the presence of serum, but only a modestprocessing in its absence, whereas ATP was highly effective inactivating the inflammasome in both conditions (Fig. 1B). Wemeasured lactate dehydrogenase (LDH) in the supernatants ofmacrophages stimulated with heme to quantify cell death. Asexpected, low amounts of LDH were present in the supernatantsof BMMs stimulated with heme in the presence of serum,whereas high concentrations of LDH were observed in macro-phages stimulated with heme without serum (Fig. 1C). Theseresults indicate that macrophage death is not involved ininflammasome activation by heme. Interestingly, heme inducedIL-1β processing by thioglycollate-elicited peritoneal macro-phages both in the presence and absence of serum (Fig. S1B).Next, we analyzed the effect of heme on in vivo production ofIL-1β. Injection of heme in the peritoneal cavity of mice alsocaused a significant increase in IL-1β (Fig. 1D). These resultsindicate that heme promotes the processing of caspase-1 andIL-1β in primed macrophages and the production of IL-1β in vivo.

Structural Motifs Involved in Inflammasome Activation by Heme. Todefine the structural requirement of heme to activate theinflammasome, we used several heme analogs. ProtoporphyrinIX (PPIX) is a direct precursor of heme that lacks the atom ofiron. CoPPIX (cobalt) and SnPPIX (tin) are heme analogs witha metal substitution. Treatment of BMMs or thioglycollate-eli-cited peritoneal macrophages with different porphyrins demon-strated that heme, but not its analogs lacking the atom of iron,caused IL-1β secretion (Fig. 2A and Fig. S1B). Incubation ofmacrophages with heme in the presence of the iron chelatordeferoxamine (DFO) abrogated heme-induced processing ofIL-1β and caspase-1 and IL-1β secretion, although it had no ef-fect on macrophages stimulated with ATP (Fig. 2 B and C).Treatment with DFO did not affect the amount of pro–IL-1β andprocaspase-1, as observed in cell extracts. Incubation of DFOwith heme inhibited inflammasome activation, although pre-treatment with DFO, followed by media change before stimu-lation with heme, was ineffective in blocking IL-1β processing(Fig. 2D). Stimulation of LPS-primed macrophages with iron(Fe2+ or Fe3+) did not cause IL-1β maturation (Fig. 2E). Ferritinis an endogenous iron chelator that binds intracellular iron andprotects cells from iron-induced oxidative damage (1, 37, 38).The stimulation of macrophages from ferritin heavy-chain–deficient (Fth−/−) mice with heme or ATP caused a similarprocessing of IL-1β compared with wild-type macrophages (Fig.2F). The presence of excess iron even in the absence of ferritinwas not capable of inducing IL-1β processing. The catabolism ofheme by HO-1 causes the intracellular release of the atom ofiron from the porphyrin ring (2). Thus, we tested the in-volvement of enzymatic release of intracellular iron from hemeby HO-1 on inflammasome activation. Treatment of wild-typeand Hmox1−/− macrophages with heme or ATP demonstratedthat HO-1 is dispensable for inflammasome activation by thesestimuli (Fig. 2G). Together, these results suggest that the iron

Fig. 1. Heme induces IL-1β maturation and secretion in vitro and in vivo. (A)LPS-primed BMMs (4 h) were stimulated for 3 h with various doses of hemeto analyze IL-1β secretion and maturation in cellular supernatants (SNs) byELISA and Western blot. (B) BMMs primed with LPS were stimulated withheme or ATP in the absence or presence of 1% serum to analyze IL-1β andcaspase-1 maturation in cellular SNs by Western blot. (C) BMMs primed ornot with LPS were stimulated with heme for 4 h in the absence or presenceof 1% serum to analyze LDH leakage in cellular SN. (D) WT mice wereinjected in the peritoneal cavity with heme (n = 4) or its vehicle (n = 4) toanalyze IL-1β production in the peritoneal cavity by ELISA. One representa-tive of two experiments. Data represent mean ± SE of two (C) or three (A)independent experiments. *P < 0.05.

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present in the heme molecule, but not the iron released by HO-1and the intracellular iron pool, is critical to the activationof inflammasome.

Heme Causes Activation of NLRP3 Inflammasome Dependent on K+

Efflux and ROS. Considering the essential role of NLRP3 inflam-masome on macrophage activation by damage-associated signals,we tested the role of NLRP3, ASC, and caspase-1 on heme-induced IL-1β processing. BMMs deficient in Nlrp3, Asc, andCaspase-1 were unable to process and secrete IL-1β whenstimulated with heme or ATP (Fig. 3A). The heme-inducedIL-1β processing by macrophages in the presence of serum wasindependent of ATP release or the purinergic receptor P2X7,while ATP-induced inflammasome activation requires P2X7receptor (39). This result was based on the following obser-vations: using BMMs from P2X7-deficient mice heme-inducedactivation of IL-1β secretion was preserved, whereas the effectof ATP was abrogated (Fig. 3B), the use of oxidized-ATP(o-ATP), an antagonist of P2X7 receptor, and the treatmentwith apyrase, that degrades ATP, inhibited the effect of ATPbut not of heme (Fig. 3C).Different models have been proposed to explain the activation

of NLRP3: K+ efflux, generation of ROS, mitochondrial dys-function, and lysosomal damage with cathepsin release (34, 35).Thus, we tested the role of these pathways on heme-induced

inflammasome activation in the presence of serum. High con-centration of K+ in the culture medium blocks the K+ efflux andis able to abrogate inflammasome assembly and caspase-1 pro-cessing induced by all known NLRP3 activators (40, 41). Simi-larly, processing of IL-1β triggered by heme was blocked by highconcentrations of K+ (Fig. 3 D and E). Treatment of LPS-primedmacrophages with N-acetyl cysteine, a precursor of the antioxi-dant glutathione, inhibited heme-induced secretion of IL-1β(Fig. 3F). As previously shown, antioxidants also inhibited ATP-induced IL-1β secretion (42, 43). Treatment with bafilomycin, aninhibitor of the vacuolar type H(+)-ATPase that prevents thephagosomal maturation and fusion to lysosome, had no effect onheme or ATP-induced inflammasome activation (Fig. S2A). Asexpected, bafilomycin blocked the inflammasome activation bysilica crystals (44). Similar results were obtained with the ca-thepsin inhibitor CA-074, able to block IL-1β secretion triggeredby silica but not by heme or ATP (Fig. S2B). Particulate matterhas been shown to activate the NLRP3 inflammasome ina mechanism that requires internalization and is inhibited bydrugs that cause cytoskeleton disturbance (43, 44). Becausehemozoin is a crystal formed by heme able to activate theNLRP3 inflammasome (45–47), we tested the necessity of en-docytosis for heme-induced inflammasome activation. Treat-ment with cytochalasin D, which blocks actin polymerization,abolished silica-induced inflammasome activation but had no

Fig. 2. The coordinated iron is essential for heme-induced IL-1β maturation. (A) BMMs primed with LPS were stimulated with heme or porphyrins withsubstitutions in place of the iron atom to analyze IL-1β secretion and maturation in cellular supernatants (SNs) by ELISA and Western blot. The porphyrinswere PPIX (a porphyrin ring without a metal atom), SnPPIX (tin), or CoPPIX (cobalt) and the stimulation was made with 50 μM for 3 h. (B and C) BMMs primedwith LPS were incubated with 2 mM of deferoxamine (DFO) for 2 h before stimulation with heme to analyze IL-1β secretion and maturation in cellularsupernatants by ELISA and Western blot. (D) DFO1 was incubated for 4 h and kept in culture all through the stimulation with heme. DFO2 was incubated for4 h and medium was changed to stimulate the cells with heme in the absence of DFO. (E) BMMs primed with LPS were stimulated with 100 μM of FeSO4 (Fe

+2)or FeCl3 (Fe

+3) for 16 h or with heme for 3 h to analyze IL-1β maturation in cellular supernatants by Western blot. The macrophages stimulated with FeSO4 orFeCl3 were cultured with nutridoma all through the experiment to maintain cellular viability. (F) WT and Ft−/− BMMs primed with LPS were stimulated withheme or ATP for 3 h or with 100 μM of FeSO4 for 16 h to analyze IL-1βmaturation in cellular supernatants by Western blot. BMMs stimulated with FeSO4 werecultured with nutridoma all through the experiment. (G) WT and Hmox−/− BMMs primed with LPS were stimulated with heme or ATP to analyze IL-1βmaturation in cellular supernatants by Western blot. Data represent mean ± SE of two (A) or three (B) independent experiments. *P < 0.05.

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inhibitory effect on IL-1β processing induced by heme or ATP(Fig. S2C). A caveat in these experiments was the increased celldeath observed on macrophages treated with heme and cyto-chalasin D. Together these results suggest that heme-inducedNLRP3 inflammasome activation involves K+ efflux and ROSgeneration but not lysosomal disruption, cathepsin release, hemeinternalization, ATP release, or P2X7 activation.

Mitochondrial ROS and NOX2 Are Involved in Heme-InducedInflammasome Activation. Heme is a potent inducer of ROS pro-duction, activating different sources of ROS generation in macro-phages (19, 24). Recent studies indicate an essential role ofmitochondrial ROS (mtROS) on NLRP3 activation inducedby several stimuli (48, 49). We observed that heme caused thegeneration of mtROS by macrophages using a probe for mito-chondrial superoxide, MitoSOX (Fig. 4A). The selectivity of thisresponse was confirmed using MitoTEMPO, a specific scavengerof mitochondrial superoxide (Fig. 4A). Treatment of LPS-stim-ulated macrophages with MitoTEMPO caused the abrogation ofheme- and ATP-induced IL-1β and caspase-1 processing (Fig.4B). The NADPH oxidase inhibitor apocynin blocked heme-induced IL-1β processing/secretion but was ineffective on ATP-induced inflammasome activation (Fig. 4 C and D). Part of thecontroversy related to the role of ROS on NLRP3 activation isdue to results obtained with nonselective drugs. Thus, we testedthe role of NADPH oxidase 2 (NOX2) using macrophages deficientin the membrane subunit gp91phox (gp91phox−/−). Heme-inducedIL-1β and caspase-1 processing was abrogated in gp91phox−/−

macrophages (Fig. 4 E and F). As previously demonstrated, ATP-

induced secretion of IL-1β was not affected by gp91phox deficiency(44). These results demonstrated a selective role of NOX2 onheme-induced NLRP3 activation, an effect that is not observed withATP and that occurred despite similar amounts of pro–IL-1β andprocaspase-1. To evaluate whether NOX2 affects heme-inducedmtROS generation, we stimulated wild-type and gp91phox−/− mac-rophages with heme and analyzed mtROS. Heme caused mtROSgeneration in wild-type but not in gp91phox−/− macrophages (Fig.4G). The role of NOX2 on mtROS was selective to heme be-cause LPS +ATP caused a similar mtROS induction on wild typeand gp91phox−/− (Fig. S3A). Treatment of macrophages withhigh concentrations of K+ had no effect on heme-inducedmtROS generation (Fig. S3B).Heme-induced ROS is largely dependent on Syk activation

(24). Thus, we analyzed the involvement of Syk on heme-inducedinflammasome activation. Treatment with piceatannol, an in-hibitor of Syk, abrogated the processing of IL-1β induced byheme but did not affect ATP-induced inflammasome activation(Fig. 4H). Treatment with piceatannol was effective in blockingthe generation of mtROS by macrophages stimulated with heme(Fig. 4I). The inhibitory effect of DFO on heme-induced inflam-masome activation prompted us to characterize the mechanism.The simultaneous incubation of macrophages with DFO andheme caused the inhibition of heme-induced Syk phosphoryla-tion and mtROS generation (Fig. S3 C and D). Together theseresults indicate that DFO blocked the early events of macro-phage activation by heme and that heme caused mtROS-dependentNLRP3 inflammasome activation, an event that required Syk.

Fig. 3. Heme activates the NLRP3 inflammasome through the canonical pathway. (A) WT, Nlrp3−/−, Asc−/−, and Caspase-1−/− BMMs primed with LPS werestimulated with heme or ATP to analyze IL-1β maturation in cellular supernatants by Western blot. (B) WT and P2X7−/− BMMs primed with LPS were stim-ulated with heme or ATP to analyze IL-1β secretion in cellular supernatants by ELISA. (C) BMMs primed with LPS were incubated with apyrase (10 un/ml) oro-ATP (100 μM) for 30 min before stimulation with heme or ATP to analyze IL-1β secretion in cellular supernatants by ELISA. (D and E) BMMs primed with LPSwere incubated with various concentrations of KCl for 15 min before stimulation with heme or ATP to analyze IL-1β maturation in cellular supernatants byELISA and Western blot. (F) BMMs primed with LPS were incubated with 10 mM of N-acetyl cysteine (NAC) for 30 min before stimulation with heme or ATP toanalyze IL-1β secretion in cellular supernatants by ELISA. Data are from one representative of two independent experiments (B, C, and E) and data representmean ± SE of four (F) independent experiments. *P < 0.05.

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Inflammasome Components Are Essential to Heme-Induced Inflammationand Lethality Caused by Hemolysis. The in vitro activation of theinflammasome by heme suggested a possible participation of thispathway causing inflammation on hemolytic conditions. In fact,instillation of heme in the peritoneal cavity of wild-type micecaused the recruitment of neutrophils, which was significantlyreduced in Asc−/− mice (Fig. 5A). Next, we analyzed if lyzed redblood cells also cause neutrophil recruitment dependent oninflammasome. The injection of lyzed erythrocytes in the peri-toneal cavity promoted neutrophil recruitment in wild-type mice,an effect significantly reduced in Asc−/− mice (Fig. 5A). Severalstudies documented that during the hemolytic process or rhab-domyolysis, the high amounts of hemoglobin, myoglobin, andsubsequently free heme promote inflammation and tissue dam-age (3–5, 37). We used Phz to characterize the involvement ofmacrophages and inflammasome components on the pathogen-esis of hemolytic diseases. The challenge with a high dose of Phzcaused hemolysis in mice evident by the dark brown coloration ofurine as early as 4 h after challenge. This dose of Phz caused 70–100% lethality within 9 d (Fig. 5 B–E). Because macrophages areconsidered an important target of the inflammatory effects ofheme and the main source of IL-1β production upon inflam-

masome activation, we initially analyzed the participation of mac-rophages in the pathogenesis of hemolysis. We used clodronateliposomes to deplete macrophages and after 2 d of treatment wechallenged mice with Phz. Mice depleted of macrophages weresignificantly more resistant to lethality induced by hemolysis com-pared with mice that received control liposomes (Fig. 5B). Therole of inflammasome components was also characterized in thismodel. The lack of Nlrp3, Asc, or Caspase-1 strikingly preventedhemolysis-induced lethality (Fig. 5C). These results indicate anessential role of the NLRP3 inflammasome on heme-inducedinflammation and to the lethal effects of hemolysis. To definethe importance of IL-1 in the lethality caused by hemolysis, wecompared the effect of Phz in wild-type and Il1r−/− mice. Ourresults demonstrated that IL-1R receptor was essential to thehemolysis-induced lethality (Fig. 5D). These results suggest thatheme promotes inflammasome activation and IL-1β secretion bymacrophages that participate in the inflammatory response, tis-sue damage, and lethality induced by hemolysis. In the presenceof serum, inflammasome activation was independent of celldeath, ATP, and functional P2X7, whereas in the absence ofserum, it correlated with cell death. Thus, we tested the role ofP2X7 on hemolysis pathogenesis. The challenge of wild-type and

Fig. 4. Heme activates the inflammasome through ROS generation induced by mitochondria and NOX2. (A and B) BMMs were incubated with 500 μM ofMitoTEMPO (MT) for 1 h before stimulation with heme or ATP. (A) BMMs were stimulated with heme for 1 h and then loaded with MitoSOX to analyze mtROSby flow cytometry. (B) BMMs incubated or not with MitoTEMPO were stimulation with heme or ATP to analyze IL-1β and caspase-1 maturation in cellularsupernatants by Western blot. (C and D) BMMs primed with LPS were incubated with 100 μM of apocynin (Apo) for 1 h before stimulation with heme or ATP toanalyze IL-1β secretion and maturation in cellular supernatants by ELISA and Western blot. (E and F) WT and gp91phox−/− BMMs primed with LPS werestimulated with heme or ATP to analyze in cellular supernatants IL-1β secretion by ELISA, and IL-1β and caspase-1 maturation by Western blot. A line wasincluded to identify that the Western blot was cut. (G) WT and gp91phox−/− BMMs were stimulated with heme for 1 h to analyze mitochondrial ROS by flowcytometry by loading the cells with MitoSOX. (H) BMMs primed with LPS were incubated with 10 μM of piceatanol (PIC) for 1 h before stimulation with heme orATP to analyze IL-1β maturation in cellular supernatants by Western blot. (I) BMMs were incubated with 10 μM of PIC for 1 h before stimulation with heme for1 h to analyze mitochondrial ROS generation by flow cytometry. Data represent mean ± SE of two (C) or five (E) independent experiments. *P < 0.05.

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P2X7−/− mice with Phz demonstrated a similar susceptibility(Fig. 5E). These results indicate that hemolysis-induced lethalityinvolves NLRP3 activation by a mechanism independent ofP2X7 activation.

DiscussionIn this study, we show that heme is an inducer of IL-1β pro-cessing through the activation of the NLRP3 inflammasome inmacrophages. The molecular mechanism by which heme pro-motes the NLRP3 activation in the presence of serum requiresSyk phosphorylation, ROS, and K+ efflux, although it is in-dependent of lysosomal damage, cathepsin activity, heme in-ternalization, heme catabolism by HO-1, cell death, release ofATP, or a functional P2X7 receptor (Fig. 6). In vivo, hemolysiscauses inflammasome activation and IL-1β secretion, both ofwhich participate in the inflammatory response and contribute tothe lethality. Together, these data confirm and extend the notionthat heme is sensed by innate immune receptors affecting theinflammatory response in sterile and infectious conditions (50).Iron was involved in the activation of NLRP3 induced by

heme. The following observations suggested that the coordinatediron of the prophyrin ring, not free iron, was involved in thisresponse: (i) Heme, but not CoPPIX, SnPPIX, or PPIX, pro-moted the secretion of mature IL-1β. (ii) Stimulation of LPS-treated macrophages with free iron (Fe2+ or Fe3+) did not causesecretion of mature IL-1β. (iii) The iron chelator DFO inhibitedheme-induced inflammasome activation only when incubatedtogether with heme in the culture media. (iv) The lack of HO-1,which cleaves the porphyrin ring releasing the iron inside thecells, or the lack of ferritin H, which stores labile iron, did notaffect the heme-induced inflammasome activation. However,we cannot formally exclude the possibility that iron releasedfrom heme, through oxidative assault inside macrophages, is

involved in Syk phosphorylation, mtROS generation, and in-flammasome activation.The importance of ROS in the activation of NLRP3 inflam-

masome is a subject of debate. It was suggested that inhibitors ofROS block the priming of macrophages, impairing the pro–IL-1βand NLRP3 expression instead of affecting the NLRP3 activa-tion (51). Thus, we treated macrophages with antioxidants after4 h of stimulation with LPS to avoid any effect on signal 1, andsaw that this treatment did not affected the amount of pro–IL-1βproduced. Several reports indicate the participation of mito-chondrial dysfunction on NLRP3 activation by stimuli, includingATP, pore forming toxins, and particulate matter (48, 49, 52,53). We observed that macrophages stimulated with heme hadincreased mtROS, and the selective blockage with MitoTEMPOabrogated mtROS and inflammasome activation by heme. ROSgenerated by NOX was initially considered important to NLRP3activation especially by crystals and other particles (43). How-ever, monocytes from patients with chronic granulomatous disease(CGD) due to a genetic deficiency of p22phox, a component re-quired for the function of NOX1–4, produced equivalentamounts of IL-1β, compared with control subjects, whenstimulated with uric acid or silica (54). Similar results wereobtained with cells from patients with CGD carrying mutationsin the CYBB (the NOX2 gene) or with macrophages fromgp91phox−/− (44, 54). A recent study, however, demonstrated therequirement of NOX2 for activation of NLRP3 by homo-cysteine in podocytes, both in vitro and in vivo, contributing tokidney inflammation and tissue damage (55). Inhibition ofNADPH oxidase activity by apocynin and the genetic deficiencyof gp91phox−/− on primary BMMs abrogated the activation ofinflammasome by heme. The mechanism involves the regulationof mtROS, which indicates that NOX2 is upstream of mtROSgeneration. In fact, a recent study demonstrated that angiotensin

Fig. 5. Heme and hemolysis induce in vivo biological effects dependent on inflammasome. (A) WT and Asc−/− mice were injected with heme, lyzed red bloodcells (RBCs) or its vehicle in the peritoneal, and neutrophil numbers were analyzed after 4 h of instillation. Data are the sum of two independent experiments.(B) Phenylhydrazine (Phz) was injected in mice treated with clodronate (n = 16) or with the liposome control suspension (n = 16), and survival was analyzed for9 d. (C) WT (n = 40), Nlrp3−/− (n = 16), Caspase-1−/− (n = 22), and Asc−/− (n = 17) mice were injected with Phz and survival was analyzed for 9 d. (D) WT (n = 17)and Il1r−/− (n = 17) were injected with Phz and survival was analyzed for 9 d. (E) WT (n = 8) and P2X7−/− (n = 8) were injected with Phz and survival wasanalyzed for 9 d.

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II induces superoxide production by the mitochondria dependenton NOX2 (56).The role of Syk in activation of caspase-1 and secretion of

IL-1β and IL-18 by macrophages was demonstrated in studiesusing activators of NLRP3 and AIM2 (45, 57, 58). The phos-phorylation of Syk in response to Candida albicans participatesin signal 1 and signal 2 required for inflammasome activation,and this role of Syk was attributed to its effect on CARD9 andROS generation (57). Syk has been shown to interact with ASCpresent in the NLRP3 inflammasome (45). Nigericin-inducedNLRP3 activation in macrophages, but not in dendritic cells, alsorequires Syk (58). This effect is related to the phosphorylation ofASC with subsequent formation of ASC specks, but is unrelatedto CARD9 or mtROS generation. In the case of heme, wepreviously observed the pivotal role of Syk phosphorylation inthe production of ROS by macrophages (24). Here, we foundthat Syk was essential to heme-induced mtROS and IL-1β pro-cessing, whereas activation of NLRP3 by ATP was not affectedby Syk inhibition. Syk is activated by receptors and adaptorproteins that contain immunoreceptor tyrosine-based activationmotifs (ITAMs), affecting several signaling pathways (59). Thenature of a putative receptor involved in recognition of hemeand triggering of Syk phosphorylation is unknown. Interestingly,it has been shown that molecules that intercalate the cellmembrane interacting with lipid rafts, such as monosodium uratecrystals, can activate Syk in a mechanism independent of specificreceptors (60). DFO abrogated heme-induced Syk phosphory-lation and mtROS generation, events that are upstream of

NLRP3 activation by heme. These results suggest that the in-hibitory effect of DFO might be related to its ability to physicallyinteract with heme (61, 62), interfering with the association ofheme with a receptor or with the cell membrane, but not relatedto its free iron chelating property. Future studies will define themolecular mechanism by which heme causes phosphorylation ofSyk and if heme-induced Syk activation participates exclusivelyin ROS generation or also in ASC phosphorylation.A recent study has described heme as an activator of NLRP3

(63), showing, however, important distinctions from our presentwork. Some of the discrepancies between the two studies aredifficult to reconcile. We followed the protocols used in theirstudy, including the use of thioglycollate-elicited peritoneal macro-phages, but we were unable to observe processing or secretion ofIL-1β by PPIX with or without prestimulation with LPS. We alsodid not confirm heme-induced inflammasome activation in theabsence of signal 1. We observed that BMMs or thioglycollate-elicited peritoneal macrophages primed with LPS and treatedwith heme secreted mature IL-1β in the presence of serum, inwhich condition heme induced negligible amounts of TNF andcaused few cell deaths in the 4 h of stimulation. Moreover, heme-induced inflammasome activation in the presence of serum wasindependent of macrophage death, ATP release, and functionalP2X7 receptor. In the absence of serum, thioglycollate-elicitedperitoneal macrophages stimulated with heme processed IL-1βand this involved required P2X receptor (63). We observed thatin the absence of serum, inflammasome activation correlatedwith macrophage cell death determined by LDH release. It iswell established that upon necrosis, macrophages release ATPthat in turn activates the NLRP3 inflammasome (53, 64). Thus,heme-induced inflammasome activation has different mecha-nisms in the presence or absence of serum.Injection of heme or lyzed red blood cells into the peritoneal

cavity caused neutrophil recruitment in a mechanism dependenton inflammasome components. In a model of unilateral uretralobstruction, heme and the active forms of caspase-1 and IL-1βare increased (63). It has been demonstrated that inflammation,renal dysfunction, and death triggered by transient renal arteryocclusion, a model of tubular necrosis induced by ischemia–reperfusion, was dependent on NLRP3 (53), and intracerebralhemorrhage also activates the NLRP3 inflammasome in a mecha-nism dependent on mtROS (65). We observed that the pathogen-esis of hemolysis was dependent on inflammasome components,demonstrated by the high resistance of mice lacking Nlrp3, Asc,or Caspase-1. In this model, the activation of the NLRP3 andlethality was likely unrelated to ATP, because wild-type andP2X7−/− mice displayed similar susceptibility. The increased re-sistance of Il1r−/− mice suggested that upon hemolysis, hemeactivates the inflammasome on macrophages, promoting thesecretion of processed IL-1β that participates in inflammationand tissue damage. These results add a component of the innateimmune system to the recently demonstrated essential partici-pation of heme-induced TLR4 activation in sickle cell disease(29, 30). Moreover, inflammasome activation is likely importantin malaria pathogenesis (45, 66). Thus, understanding the mo-lecular signaling pathways affected by heme might prove usefulto the identification of new options for treating pathologicalconditions that have increased extracellular heme.

MethodsReagents and Materials. Heme, PPIX, SnPPIX, and CoPPIX were purchasedfrom Frontier Scientific. They were dissolved in NaOH (0.1 M) diluted in RPMI,and filtered just before. Stock solutions of porphyrins were prepared in thedark to avoid free radical generation. LPS 0111:B4 from Escherichia coliwas obtained from InvivoGen. Apocynin, N-acetyl cysteine, deferoxamine,piceatanol, ATP, FeSO4, FeCl3, KCl, oxidized ATP, apyrase, cytochalasin D, anti–IL-1β, and Phz were obtained from Sigma-Aldrich. Clodronate liposomes andcontrol liposomes were obtained from www.ClodronateLiposomes.com.

Fig. 6. Mechanisms involved in heme-induced NLRP3 inflammasome acti-vation in macrophages (1). Pathogen-associated molecular patterns (PAMPs),such as LPS or TNF, prime macrophages to activate NF-κB and induce theexpression of NLRP3, caspase-1, and IL-1β (2). Heme induces Syk activationthrough its coordinated iron. An unknown receptor or direct alterations inlipid rafts might be involved in Syk activation. Syk is involved in heme-induced mtROS generation and NLRP3 activation (3). Heme induces NOX2activation essential for mtROS generation and NLRP3 activation (4). mtROSgeneration is essential for heme-induced NLRP3 activation. (?) The Sykpathway might be involved in NOX2 activation (5). K+ efflux is also essentialfor heme-induced NLRP3 activation (6). Heme depends on mtROS, NOX2,and K+ efflux to activate NLRP3 (7). Active caspase-1 cleaves pro–IL-1β andthe mature form is secreted.

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Anti–caspase-1 was obtained from Genentech. Protease inhibitors andluminol were obtained from Santa Cruz Biotechnology. X-ray films werefrom Kodak. ELISA for IL-1β detection was obtained from Peprotech.MitoSOX was obtained from Molecular Probes. MitoTEMPO CA-074, andbafilomycin were obtained from Enzo Life Sciences. RPMI medium 1640and penicillin–streptomycin were obtained from LGC Biotechnology, FCSwas from Invitrogen, and nutridoma was obtained from Roche.

Mice. C57BL/6 (WT) mice were obtained from the animal facility at theUniversidade Federal do Rio de Janeiro (Rio de Janeiro, Brazil). P2X7−/− micewere obtained from the animal facility of the Instituto de Biofísica, Uni-versidade Federal do Rio de Janeiro. Nlrp3−/−, Asc−/−, Caspase-1−/−, and Il1r−/−

were obtained from the animal facility at the Universidade de São Paulo(Ribeirao Preto, SP, Brazil). gp91phox−/− mice were obtained from the animalfacility at the Universidade Federal de Minas Gerais (Belo Horizonte, MG,Brazil). Tibias and femurs of WT, Hmox1−/−, and Fth−/− mice were obtainedfrom the animal facility at the Instituto Gulbenkian de Ciência (Oeiras, Por-tugal). All experiments followed guidelines of the institutional ethical com-mittee and underwent approval [approval ID: CEUA/CCS/UFRJ/IMPPG 011(Comissão de Ética no Uso de Animais do Centro de Ciências da Saúde daUniversidade Federal do Rio de Janeiro RJ, Brazil)].

Macrophages. BMMs were prepared using tibia and femur from 6- to 12-wk-old mice. Bone marrow was obtained flushing bones with cold sterile RPMI.The differentiationmediumwas RPMI supplementedwith 20% (vol/vol) heat-inactivated FCS and 30% (vol/vol) L929 cell supernatant. Initially, 5 × 106

bone marrow cells were suspended in 10 mL of differentiation medium, thenseeded in 100-mm Petri dishes (BD Biosciences) at 37 °C in humidified 5%CO2. After 3 d, 10 mL of differentiation medium was added. Finally, after 3more days, cells were washed with cold RPMI and suspended and seeded atthe required density for all experiments. Before any experiment, cells restedfor at least 12 h. Peritoneal exudate cells were harvested by lavage fromC57BL/6 mice (4–6 wk) 4 d after i.p. injection of 2 mL of 3% (wt/vol) thio-glycollate (VETEC). Briefly, cells were collected by lavage with 5 mL of coldRPMI, washed twice by centrifugation at 400 × g for 10 min at 4 °C, andsuspended in RPMI with 10% (vol/vol) FCS. Cell viability was consistently 95%in Trypan blue exclusion test. Peritoneal macrophages were plated in 96-welltissue culture plates at 2 × 105 cells per well in medium [RPMI plus 10%(vol/vol) FCS]. Cells were kept at a 2-h incubation time at 37 °C 5% CO2.Nonadherent cells were removed by washing with RPMI and 98% of theremaining adherent cells were macrophages as determined by FACS stain-ing. Macrophages were stimulated with 50 μM of heme for 3 h and 2 mM ofATP for 1 h. The conditions of the stimulation with porphyrins, FeSO4, FeCl3,and silica are described in the figure legends. All of the incubations with theinhibitors (N-acetyl cysteine, apocynin, MitoTEMPO, deferoxamine, piceata-nol, bafilomycin, CA-074, cytochalasin D, apyrase, oxidized ATP, and KCl)were made between the priming and the stimulation to avoid interferencewith the expression of pro–IL-1β and procaspase-1. The time and the dose ofthe inhibitors used in the experiments are described in the figure legends.

IL-1β ELISA. Experiments to analyze IL-1β secretion were performed with2 × 105 cells and the cellular supernatants were collected for ELISA anal-ysis (Peprotech). All of the measurements were performed according tothe manufacturer’s protocol.

IL-1β, Caspase-1, and p-Syk Western Blots. Experiments to analyze the mat-uration of IL-1β (p17) and caspase-1 (p20) were performed with 2 × 106 cellsand the cellular supernatants were collected and concentrated for Westernblot analysis. Cellular extracts were collected in experiments involvinginhibitors and knockout cells to analyze the amounts of pro–IL-1β (p37) andprocaspase-1 (p45). Also, cellular extracts were used to analyze Syk phos-phorylation. Cellular supernatants were concentrated with trichloroaceticacid (TCA). Briefly, TCA was incubated with cellular supernatants for 1 h in

wet ice and centrifuged to obtain a pellet that was washed with acetone[90% (vol/vol)] and then suspended in laemmli buffer and boiled. To preparecellular extracts, the cells were lysed in RIPA buffer (150 mM NaCl, 20 mMTris-Cl pH 8.0, 0.1% SDS, 0.5% sodium deoxycholate, and 1.0% Nonidet P-40supplemented with complete protease inhibitors) for 15 min in wet ice. Thesamples were then centrifuged and the supernatants were mixed withlaemmli buffer and boiled. All samples (supernatants and cellular extracts)were subjected to SDS/PAGE and the proteins were transferred to nitrocel-lulose membranes that were blocked with TBS-T, containing 5% (wt/vol) fat-free milk. Primary antibodies (anti–IL-1β, anti–caspase-1, anti–p-Syk, anti-ERKand anti–β-actin) were incubated overnight at 4 °C in blocking buffer.Detection of the primary antibodies was performed with appropriate HRP-conjugated antibodies, followed by incubation with luminol and expositionof the membranes to X-ray films. The experiments presented are represen-tative of two or three experiments.

Measurement of Mitochondrial ROS. Mitochondrial ROS was measured inBMMs (5 × 105 cells) loaded with 5 μM of MitoSOX for 30 min at 37 °C with5% CO2 after the stimulation with 50 μM of heme for 1 h. Cells were leftuntreated or were pretreated with 2 mM of DFO for 2 h or 10 μM ofpiceatanol for 1 h or 500 μM of MitoTEMPO for 1 h before the stimulationwith heme. Mitochondrial ROS was measured by flow cytometry usingFACScan flow cytometer (BD Biosciences). The experiments presented arerepresentative of three to five experiments.

IL-1β and Neutrophil Migration in Peritoneal Fluid. Neutrophil migration andIL-1β was induced by an i.p. injection of heme (100 μg/cavity) or lyzed RBC ina volume of 200 μL. The control group received an i.p. injection of endo-toxin-free PBS solution in a same volume. After 5 h of injection, the animalswere killed, and their peritoneal cavities were rinsed with 3 mL of cold PBS.Total leukocytes in the peritoneal fluid were determined on Neubauerchambers after dilution in Turk solution. Differential counting of leukocyteswas carried out on DiffQuik (Baxter Travenol Laboratories)-stained slices.Also, the peritoneal fluid was centrifuged and the amount of IL-1β in thesupernatants was quantified by ELISA.

Hemolysis.Hemolysis was induced by Phz injection. Phzwas dissolved in sterilePBS and the pH was adjusted to 7.4 with NaOH. Age-matched adult males ofall genotypes under investigation were injected intraperitoneally with freshlyprepared Phz. To analyze the survival in hemolytic conditions, mice wereinjected twice with Phz. In the first step, mice were injected with 0.1 mg/g ofPhz. Sixteen hours later, mice were injected with 0.05 mg/g of Phz. Survivalwas thenmonitored for 9 d. To analyze the involvment ofmacrophages in theleyhality induced by PHZ, 10 μl/g of clodronate liposomes were instilled in theperitoneal cavity of mice 48 h before PHZ instillation. The control group wasinstilled with 10 μl/g of PBS liposomes.

Statistical Analysis. Differences between the groups were analyzed usinga Student test or one-way ANOVA with Newman–Keuls multiple comparisontest (Prism 5.0). A significant difference between groups was considered *P <0.05. Comparisons of survival curves were analyzed by log-rank (Mantel–Cox test).

ACKNOWLEDGMENTS. We thank Dr. Miguel Soares for providing tibias andfemurs of WT, Hmox1−/−, and Fth−/− mice; Dr. Leda Vieira for providing tibiasand femurs of WT and gp91phox−/−; Dr. Pedro Persechini for providing P2X7mice; and Dr. Miriam Werneck for critical reading of the manuscript. M.T.B.received financial support from Conselho Nacional de Pesquisa, Fundação deAmparo à Pesquisa do Rio de Janeiro (FAPERJ), and Instituto Nacional deCiência e Tecnologia em Dengue (Brazil). F.F.D. has a postdoctoral fellowshipfrom Programa de Apoio ao Pós-Doutorado no Rio de Janeiro, Coordenaçãode Aperfeiçoamento de Pessoal de Nível Superior/FAPERJ.

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