of November 30, 2013.This information is current as Infection

Trypanosoma cruziHost Resistance to Response andβInflammasomes Mediate IL-1

Containing a Caspase Recruitment Domain like Protein−Apoptosis-Associated Speck

Santana SilvaRicardo Tostes Gazzinelli, Dario Simões Zamboni and JoãoGuedes, Warrison Athanásio Andrade, Mariana De Niz, Fredy Roberto Salazar Gutierrez, Paulo Marcos da MattaSilveira, Braulia Costa Caetano, Catarina Veltrini Horta, Grace Kelly Silva, Renata Sesti Costa, Tatiana Nunes

http://www.jimmunol.org/content/191/6/3373doi: 10.4049/jimmunol.1203293August 2013;

2013; 191:3373-3383; Prepublished online 21J Immunol 

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The Journal of Immunology

Apoptosis-Associated Speck–like Protein Containinga Caspase Recruitment Domain Inflammasomes MediateIL-1b Response and Host Resistance to Trypanosoma cruziInfection

Grace Kelly Silva,*,† Renata Sesti Costa,*,1 Tatiana Nunes Silveira,†,1

Braulia Costa Caetano,‡ Catarina Veltrini Horta,† Fredy Roberto Salazar Gutierrez,x

Paulo Marcos da Matta Guedes,{ Warrison Athanasio Andrade,‡,‖,# Mariana De Niz,**,††

Ricardo Tostes Gazzinelli,‡,‖,# Dario Simoes Zamboni,† and Joao Santana Silva*

The innate immune response to Trypanosoma cruzi infection comprises several pattern recognition receptors (PRRs), including

TLR-2, -4, -7, and -9, as well as the cytosolic receptor Nod1. However, there are additional PRRs that account for the host immune

responses to T. cruzi. In this context, the nucleotide-binding oligomerization domain–like receptors (NLRs) that activate the

inflammasomes are candidate receptors that deserve renewed investigation. Following pathogen infection, NLRs form large

molecular platforms, termed inflammasomes, which activate caspase-1 and induce the production of active IL-1b and IL-18.

In this study, we evaluated the involvement of inflammasomes in T. cruzi infection and demonstrated that apoptosis-associated

speck–like protein containing a caspase recruitment domain (ASC) inflammasomes, including NLR family, pyrin domain–con-

taining 3 (NLRP3), but not NLR family, caspase recruitment domain–containing 4 or NLR family, pyrin domain–containing 6, are

required for triggering the activation of caspase-1 and the secretion of IL-1b. The mechanism by which T. cruzi mediates the

activation of the ASC/NLRP3 pathway involves K+ efflux, lysosomal acidification, reactive oxygen species generation, and lyso-

somal damage. We also demonstrate that despite normal IFN-g production in the heart, ASC2/2 and caspase-12/2 infected mice

exhibit a higher incidence of mortality, cardiac parasitism, and heart inflammation. These data suggest that ASC inflammasomes

are critical determinants of host resistance to infection with T. cruzi. The Journal of Immunology, 2013, 191: 3373–3383.

The intracellular protozoan parasite Trypanosoma cruzi is theetiologic agent of Chagas disease, a chronic condition af-fecting more than 10 million people worldwide, mostly in

Latin America (1). The mechanisms involved in the pathogenesis ofChagas disease are unclear; however, the development and severityof American trypanosomiasis is known to depend on immune-mediated mechanisms. Because acquired immune responses aremodulated and shaped by innate immune responses, the efficient

innate recognition of the parasite by pattern recognition receptors(PRRs) is crucial for host resistance (2). This idea is supported byreports showing that T. cruzi is recognized by TLR-2, -4, -7, and -9(3‑6). The engagement of these receptors by parasite moleculestriggers the activation of an inflammatory cytokine response througha MyD88-dependent pathway. Mice that are deficient in MyD88 arepartially competent at producing cytokines, suggesting that in ad-dition to MyD88, other PRRs are required for the host immune

*Department of Biochemistry and Immunology, Ribeirao Preto Medical School, Uni-versity of Sao Paulo, 14049-900 Ribeirao Preto, Sao Paulo, Brazil; †Department ofCellular Biology, Ribeirao Preto Medical School, University of Sao Paulo, 14049-900Ribeirao Preto, Sao Paulo, Brazil; ‡Division of Infectious Diseases and Immunology,Department of Medicine, University of Massachusetts Medical School. Worcester,MA 01655; xSchool of Medicine, University Antonio Narino, 110231 Bogota,Colombia; {Departamento de Microbiologia e Parasitologia, Universidade Federal doRio Grande do Norte, 59072-970 Natal, Rio Grande do Norte, Brazil; ‖Departamento deBioquımica e Imunologia, Universidade Federal de Minas Gerais, 31270-901 BeloHorizonte, Minas Gerais, Brazil. #Centro de Pesquisas Rene Rachou, FundacaoOswaldo Cruz, 30190-002 Belo Horizonte, Minas Gerais, Brazil; **Institute of CellBiology, University of Bern, CH-3012 Bern, Switzerland; and ††Barcelona Center forInternational Health Research (Centre de Recerca en Salut Internacional de Barcelona,Hospital Clınic-Universitat de Barcelona), 4a 08036 Barcelona, Spain

1R.S.C. and T.N.S. contributed equally to the development of this work.

Received for publication November 30, 2012. Accepted for publication July 22,2013.

This work was supported by the Instituto Nacional de Ciencia e Tecnologia deVacinas/Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico and Fundacaode Amparo a Pesquisa do Estado de Sao Paulo Grants 2007/53940-0 (to J.S.S.)and 2010/50959-4 (to D.S.Z.). G.K.S., C.V.H., F.R.S.G., and T.N.S. were supportedby doctoral fellowships from Fundacao de Amparo a Pesquisa do Estado de SaoPaulo. B.C.C. is supported by a postdoctoral fellowship from the National Institutesof Health. M.D.N. is supported by an European Virtual Institute of Malaria ResearchPh.D. studentship. R.S.C. and P.M.d.M.G. are supported by postdoctoral fellowships

from Coordenacao de Aperfeicoamento de Pessoal de Nıvel Superior. W.A.A issupported by Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico. J.S.S.,D.S.Z., and R.T.G. are research fellows from Conselho Nacional de DesenvolvimentoCientıfico e Tecnologico.

The data in this study were deposited in the Gene Expression Omnibus database(http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE41089) under accession numberGSE41089.

Address correspondence and reprint requests to Dr. Joao Santana Silva, Departa-mento de Bioquımica e Imunologia, Ribeirao Preto Medical School, University ofSao Paulo, Avenue Bandeirantes 3900, 14049-900 Ribeirao Preto, SP, Brazil. E-mailaddress: jsdsilva@fmrp.usp.br

The online version of this article contains supplemental material.

Abbreviations used in this article: ASC, apoptosis-associated speck–like proteincontaining a caspase recruitment domain; BMM, bone marrow macrophage; CFP,cyan fluorescent protein; DAMP, damage-associated molecular pattern; dpi, daypostinfection; FLICA, fluorescent-labeled inhibitor of caspases; HK, heat-killed; IR,irradiated; MOI, multiplicity of infection; NAC, N-acetylcysteine; NAIP, NLR family,apoptosis inhibitory protein; NLR, nucleotide-binding oligomerization domain–like re-ceptor; NLRC4, NLR family, caspase recruitment domain–containing 4; NLRP3, NLRfamily, pyrin domain–containing 3; NLRP6, NLR family, pyrin domain–containing 6;ROS, reactive oxygen species; TIRAP, TIR domain–containing adapter protein; WT,wild-type.

Copyright� 2013 by TheAmerican Association of Immunologists, Inc. 0022-1767/13/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1203293

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responses to T. cruzi through a MyD88-independent signaling path-way (2, 7). Recently, we demonstrated that the intracellular receptorNod1, a member of the nod-like receptor family, is essential for thehost resistance against T. cruzi (8). Our data also suggested thatother receptors in addition to Nod1 are necessary to control theT. cruzi infection because the Nod1-deficient mice produced arobust cytokine response despite their high susceptibility (8). Inthis context, the NLRs that activate the inflammasomes are can-didate receptors that deserve renewed investigation.Caspase-1, a cysteine protease that cleaves the mature secreted

bioactive forms of IL-1b and IL-18, is recruited and activated byinflammasomes in response to a wide variety of stimuli (9–11).Inflammasomes are large multiprotein complexes formed by thepolymerization of nucleotide-binding oligomerization domain–likereceptors (NLRs). The different types of inflammasomes are dis-tinguished by their NLRs, adaptors, and stimuli specificities. TheNLR pyrin domain–containing 3 (NLRP3) inflammasome, whichis possibly the most extensively studied platform, is activated bydifferent stimuli including prokaryotic RNA and different agentsthat trigger damage-associated molecular patterns (DAMPs), suchas urate crystals, pore-forming toxins, and silica, among others (11–15). It has also been demonstrated that the engagement of theNLRP3 inflammasome requires the generation of reactive oxygenspecies (ROS), potassium efflux, and lysosomal damage. In con-trast, the inflammasome composed of NLR family, apoptosisinhibitory protein (NAIP)/NLR caspase recruitment domain–con-taining 4 (NLRC4) is activated by bacterial proteins, such as fla-gellin, basal body rod proteins of the type III secretion systems andpilin (16–18). The central adaptor molecule, which is required forthe assembly of the NLRP3 inflammasome, is the apoptosis-as-sociated speck–like protein containing a caspase recruitment do-main (ASC). This pyrin-containing protein recruits procaspase-1and bridges its interaction with NLRP3 (19, 20). The inflamma-somes play an important role in the host defense against severalintracellular pathogens (21). Because there is a resistance againstT. cruzi that is independent of MyD88 and Nod1, we suggest thatinflammasomes are engaged in the activation of innate immuneresponses during T. cruzi infection.Our results indicate that T. cruzi induce caspase-1 activation and

IL-1b secretion in macrophages. This process is fully dependenton ASC and partially dependent on NLRP3. In addition, we de-termined a significant role for the ASC inflammasome and in thecontrol of inflammation, cardiac parasitism, and the survival ofT. cruzi–infected mice. Our findings demonstrate a critical rolefor the inflammasome in the host protection against T. cruzi.

Materials and MethodsMice

Mice aged 6–8 wk were used in the infection experiments and for the iso-lation of the bone marrow macrophages (BMMs). The C57BL/6, ASC2/2,NLRC42/2, caspase-12/2, IL-1R2/2, P2X7R2/2, NLRP32/2, and NLRfamily, pyrin domain–containing (NLRP)62/2 mice were bred at the animalfacilities at the Medical School of Ribeirao Preto, Universidade de SaoPaulo, or at the animal facilities at the University of Massachusetts MedicalSchool. The Institutional Ethics Committee for Animal Care and Researchat Faculdade de Medicina de Ribeirao Preto-Universidade de Sao Paulo(Comissao de Etica em Experimentacao Animal-Faculdade de Medicina deRibeirao Preto/Universidade de Sao Paulo, animal protocol 059/2008) andthe Institutional Animal Care and Use Committee at the University ofMassachusetts Medical School (protocol A-1817-09) approved the experi-mental protocols.

Parasites and infections

The T. cruzi Y strain was cultivated in the LLC-MK2 host cells as de-scribed elsewhere (22). For the in vitro experiments, the BMMs wereinfected at a multiplicity of infection (MOI) of five parasites per macro-

phage and were harvested at the indicated times. The infected macro-phages were cultured with IFN-g (1 ng/ml) or IL-1b (10–100 ng/ml) forthe evaluation of the parasite growth. After 5 d of infection, the amount ofreleased parasites was quantified using a Neubauer chamber. The NOproduction, 48 h postinfection, was estimated using the Griess reaction asdescribed previously (23). For the in vivo experiments, the mice wereinfected i.p with 103 blood-derived trypomastigote forms of T. cruzi, andthe mortality was monitored daily. The mice were sacrificed at 10 and18 d postinfection (dpi), and the serum and organ samples were collectedfor the detection of enzymatic markers of heart injury and the evaluation oftissue parasitism.

Evaluation of disease evolution and parasitism in infected mice

To determine heart injury, the serum samples obtained at 10 and 18 dpi wereevaluated using a commercially available creatine-kinase detection CK-MBassay kit (Labtest, Sao Paulo, Brazil). For the histological analysis of par-asitism, the tissue samples were fixed, stained with H&E, and examined bylight microscopy. The number of infected cells per area was estimated usingthe ImageTool 2.0 software (University of Texas). Alternatively, DNA sampleswere obtained from the non-fixed tissue using the QIAmp DNA kit (Qiagen).Quantitative PCR was performed using the SYBR reagents (Invitrogen)and OneStep Plus thermocycler (Applied Biosystems). The specific pri-mers and the PCR cycling conditions for the amplification of the T. cruzigenetic markers have been described elsewhere (24).

In vitro assays using BMMs

The BMMs were obtained as described previously (25). Briefly, the totalbone marrow cells were cultured for 7 d in RPMI 1640 medium (Sigma-Aldrich), supplemented with 20% FBS (Invitrogen) and 30% L-929 cellconditioned media, at 37˚C and 5% CO2. The cells were infected withtrypomastigotes of T. cruzi, as described above. Prior to infection, theBMMs were treated for 1 h with one of the following reagents: 130 mMpotassium chloride, 130 mM sodium chloride, 20 or 50 mM CA-074Me,20 mM cytochalasin D, 50 or 100 mM glibenclamide, 20 mM N-acetylcys-teine (NAC), or 0.01 or 0.1 mM chloroquine (all from Sigma-Aldrich).

Detection of activated caspase-1 and IL-1b

The BMMs (106 cells) were infected with T. cruzi as described above. Thecell-free supernatants and cellular lysate were collected after 12, 24, and 48 hof incubation, and the caspase-1 and IL-1b production was detected byWestern blotting, as described previously (26). The BMMs incubated withATP (5 mM) plus 100 ng/ml LPS (both from Sigma-Aldrich) were used asthe positive control. The intracellular caspase-1 activity in the infectedBMMs was assessed using a caspase-1 fluorescent-labeled inhibitor of cas-pases (FLICA) kit (Immunochemistry Technologies) in accordance with themanufacturer’s instructions. The BMMs (1.53 106 cells) were infected withT. cruzi for 24 h, and the cells were stained with FLICA. The flow cytometricanalysis was performed in a BD LSRII machine (BD Biosciences), and thedata were analyzed using the FlowJo Software (Tree Star)

Cytokine and LDH detection

The BMMs were infected with T. cruzi (MOI 5), washed, and incubatedfor 24, 36, or 48 h. The cell culture supernatants were assayed for IL-1b,TNF, and IFN-g using ELISA kits (R&D Systems) in accordance with themanufacturer’s instructions. LDH was measured using CytoTox 96 (Promega).To normalize for spontaneous lysis, the percentage of LDH release wascalculated as follows: (LDH infected2 LDH uninfected/LDH total lysis2LDH uninfected 3 100).

Inflammasome assembly assays

The cyan fluorescent protein (CFP)-ASC–expressing macrophages weregenerated as described previously (27). Briefly, 1.5 3 106 cells were seededin 35-mm confocal dishes, infected with T. cruzi (MOI 5), and stained with1 mM of the cell tracker CPMTX (Invitrogen). At 3 d postinfection (dpi), thecells were analyzed by fluorescence microscopy (Leica SP2 AOBS), and thepercentage of specks in the infected cells were quantified.

Microarray analysis

The microarray was generated using the Affymetrix GeneChip MouseGenome 430A 2.0. The hearts from three uninfected (controls) and threeinfected C57BL/6micewere processed for RNA extraction, and the sampleswere hybridized individually. The extraction of the heart from the infectedmice was performed at 18 dpi with the Y strain of T. cruzi. The scannedimages were analyzed using the MAS 5.0 software for the determination ofthe chip quality, including intensity value background correction, deter-

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mining array outlier percentages, log2 transformation, and quantile nor-malization. The results of interest were confirmed by robust multi-arrayaverage using the Bioconductor package. Following this control step,12,344 probe sets were filtered for further analysis. The data for each geneprobe were expressed as the log2 ratio of the normalized fluorescenceintensity of the sample. The differentially expressed genes were identifiedusing the Significant Analysis of Microarrays 4.0; after fitting a linearmodel to the expression data, the cutoff significance level was chosen asa 2-fold change in gene expression at a significance level of 95%. Overall,1,471 probe sets (1,151 genes) were identified as demonstrating signifi-cantly different levels of expression. The hierarchical cluster analysis ofthe microarray data sets was performed using the Cluster and Treeviewsoftware. The functional annotation was performed using the DAVID Bio-informatics 6.7, which enabled the identification of enriched functionallyrelated gene groups, including those related to cytokine and chemokinesignaling, TLRs, cell death, and the inflammasome, as well as the visu-alization of such genes in KEGG pathway maps. To assign gene ontology–annotated terms to the sets of genes that were significantly differentiallyregulated, functional annotation clustering was based on p value and Ben-jamini scores set to a cutoff value of 0.05. The raw and normalized data weredeposited in the Gene Expression Omnibus database at the National Centerfor Biotechnology Information (accession number GSE41089). The link tothis Web site over the accession number: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE41089.

Isolation of inflammatory cells from cardiac tissues and flowcytometry

Hearts collected from 7 to 10 mice on day 10 p were minced, pooled, andincubated for 1 h at 37˚C with RPMI 1640 medium supplemented withNaHCO3, penicillin-streptomycin-gentamicin, and 0.05 g/ml liberaseblendzyme CI (Roche, Basel, Switzerland). After tissue digestion andwashes, cell viability was assessed by trypan blue exclusion. The cellswere counted in a hemocytometer and stained with a fluorescently labeledanti-CD11b Ab (BD Biosciences). The cell suspension was filtered through100-mm nylon mesh. Fluorocytometric analysis was performed with aFACSCanto II apparatus and FlowJo software.

Immunohistochemistry

For immunohistochemistry, hearts from three mice per experimental groupwere frozen in OCT compound (Sakura Finetek, Torrance, CA). Immu-nohistochemistry was performed with anti-F480 (eBioscience) followed byan avidin–biotin peroxidase reaction. The staining was developed with 3,3-

diaminobenzidine (Vector Laboratories, Burlingame, CA). Controls wereperformed by incubating slides with an IgG isotype control instead ofprimary Abs.

Statistical analysis

The data are expressed as the mean 6 SEM. For comparisons using morethan two experimental groups or time series, one-way ANOVA was usedfollowed by Tukey’s multiple comparison test. In the case of comparisonsusing two experimental groups, the unpaired t test was used. Comparisons ofcumulative survival curves were performed by log-rank (Mantel–Cox) test.All analyses were performed using the Prism 5.0 software (GraphPad).

ResultsT. cruzi upregulates expression of inflammasome genes in vivo

To identify additional receptors that are involved in the innate im-mune recognition of T. cruzi, we first performed a microarrayanalysis to assess the expression of innate immunity genes in theheart of the mice at 18 dpi. We determined that several genes relatedto chemokines, cytokines, and their receptors were upregulatedcompared with the uninfected control mice. In addition, variousgenes involved in the TLR signaling pathway were upregulated. Incontrast, the expression of IRAK4, RACl, TIR domain–containingadapter protein (TIRAP), and translocation-associated membraneprotein 1 was similar to the uninfected mice (Fig. 1). Curiously, theNLRP signaling was one of the top pathways upregulated (Supple-mental Table I). Specifically, we observed the increased expressionseveral genes in the inflammasome signaling pathway, includingNAIP5, NAIP2, Panx1, PYCARD/ASC, caspase-1, IL-18, and IL-1R(Fig. 1). The high expression of caspase-1 suggests that the inflam-masome is required during T. cruzi infection.

T. cruzi triggers IL-1b production

To investigate whether T. cruzi induces caspase-1 activation, wefirst infected the BMMs from the wild-type (WT) mice with try-pomastigotes of the Y strain. The immunoblot analyses of theactivated caspase-1 in the cell culture supernatants demonstratedthat the infection with T. cruzi resulted in the cleavage of caspase-

FIGURE 1. T. cruzi upregulates

expression of inflammasome genes

in vivo. Gene expression profile of

myocardial tissue from WT mice in-

fected, 18 d after the i.p infection with

the T. cruzi Y strain parasite, as es-

tablished by the mouse Affymetrix

array (GeneChip Mouse Genome

430A 2.0). The results were normal-

ized, log2-transformed, and expressed

relative to the expression values of the

uninfected control mice. Each column

represents a cDNA from an infected

mice and each row represents one

probe set. Differentially expressed

genes were grouped according to their

functional roles. The color bar shows

the magnitude of gene expression

changes on a log scale: significantly

induced (red), unchanged (black), or

repressed (green).

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1 to produce its active p20 subunit at 24 h postinfection (Fig. 2A).As a positive control, we primed the BMMs using LPS followedby ATP stimulation. We also quantified the activated caspase-1 inthe T. cruzi–infected macrophages using the fluorescent markerFAM-YVAD-FMK (FLICACASP1), which specifically binds to theactive form of caspase-1 (28). The flow cytometry analysis re-vealed the robust activation of caspase-1 at 24 h postinfection(Fig. 2B). Active caspase-1 processes cytoplasmic pro–IL-1b togenerate the biologically active form of this cytokine, which issubsequently secreted from the cell to initiate inflammation andhost defense mechanisms (29). Because, T. cruzi induces caspase-1activation, we investigated the levels of IL-1b secretion in theinfected BMMs by analyzing the supernatants for IL-1b secretion(Fig. 2C). We observed that T. cruzi induced IL-1b release into thesupernatants at 24 h postinfection. Importantly, the experimentsthat were performed using the CL–Brener strain of T. cruzi yieldedsimilar results (Supplemental Fig. 1A), indicating that the acti-vation of the inflammasome in response to infection is a broadmechanism of host resistance and is not a feature that is restrictedto certain strains of the parasite. Because T. cruzi triggers caspase-1activation and IL-1b secretion, we next evaluated the mechanismsinvolved in IL-1b production in the T. cruzi–infected macrophages.We stimulated the WT BMMs with live, irradiated (IR), or heat-killed (HK) T. cruzi. Interestingly, live T. cruzi triggered more ef-ficient IL-1b release than IR or HK parasites at 24 h postinfection(Fig. 2C). However, HK and IR parasites stimulated a significantamount of IL-1b release compared with uninfected BMMs, sug-gesting that HK parasites are able to trigger pro–IL-1b or reduce theamount of the mature cytokine. To investigate this question, weperformed Western blotting to evaluate IL-1b production in BMMsinfected with live or HK trypomastigotes. We found that only liveT. cruzi induced efficient cleavage of pro–IL-1b at 24 and 48 hpostinfection (Fig. 2D), and this was concomitant with caspase-1activation, which started at 24 h and increased by 48 h postin-

fection with live T. cruzi (Fig. 2E). In contrast, HK parasites werenot able to induce cleavage of caspase-1 (Fig. 2E). In addition, thecytochalasin D treatment of the BMMs significantly reduced theirability to produce IL-1b in response to T. cruzi (Fig. 2F), suggestingthat parasite internalization is a prerequisite for inflammasome ac-tivation. In addition, we verified that T. cruzi induced IL-1b pro-duction in the myocardium, which is an important tissue affectedin trypanosomiasis (Fig. 2G). The results suggest that T. cruzipromotes caspase-1 activation and the invasion of the live parasiteis required for higher IL-1b production by macrophages in vitro.Moreover, T. cruzi induced IL-1b release in the cardiac tissue ofthe infected mice, indicating that inflammasome activation occursduring the in vivo infection with the protozoan parasite.

Caspase-1 activation and IL-1b release by T. cruzi–infectedmacrophages are dependent on ASC inflammasomes

Next, we investigated the inflammasome components involved inthe caspase-1 activation and subsequent IL-1b release in responseto T. cruzi infection. Because ASC is a common adaptor for a di-verse set of inflammasomes, we first infected the BMMs from WT,ASC2/2, and caspase-12/2 mice with trypomastigotes and as-sessed the cleaved caspase-1 using Western blots. We observedcaspase-1 activation in the infected BMMs from the WT mice butnot in the macrophages from the ASC2/2 or caspase-12/2 mice at24 h postinfection (Fig. 3A). To further investigate the require-ment for ASC in caspase-1 activation and inflammasome forma-tion, we used an immortalized macrophage cell line stably overex-pressing ASC tagged with CFP. In the absence of stimuli, thesecells exhibit a diffuse fluorescence in the cytoplasm, and the ac-tivation with certain stimuli causes a clustering of the ASC-CFPmolecules, which is visible under confocal microscopy as a singlebrightly fluorescent “speck” in the cytosol (27, 30). We infectedthe ASC-CFP–expressing macrophages with the Y strain trypo-mastigotes (MOI 5), previously stained with red fluorescent dye

FIGURE 2. T. cruzi triggers IL-1b production. BMMs from WT mice were infected for 24 h with live, IR, or HK trypomastigotes (MOI 5) or were

stimulated with LPS+ATP. The presence of the p20 cleavage product of active caspase-1 in the cell culture supernatants was analyzed by Western blot (A).

The arrowhead indicates the p20 subunit. BMMs (1 3 106) infected 24 h were probed with FAM-YVAD-FMK (FLICACASP1) and analyzed by flow

cytometry (B). The gray areas and the black lines in the histogram represent the noninfected and infected cells, respectively. BMMs were untreated (C) or

treated for 1 h with 20 mM cytochalasin (F) before infection with live, HK, or IR T. cruzi and the levels of IL-1b in the culture assayed by ELISA (C, F) and

Western blotting (D). In (E), active caspase-1 was quantified in WT BMMs infected with live or HK parasites. At 18 dpi, the levels of IL-1b in homogenates

of hearts tissues of T. cruzi–infected WT mice (five animals per group) were quantified (G). The results showed are representative of five experiments

performed in triplicate with similar results. *p , 0.05 as compared with not treated BMMs. NI, Noninfected; Tc, T. cruzi.

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(CMTPX), to monitor the infection and the speck formationsimultaneously. At 72 h, but not at 6, 12, 24, 36, and 48 h post-infection, we observed the formation of fluorescent clusters ofCFP-ASC in 31% of the infected cells, indicating that the ASCinflammasome is activated during T. cruzi infection (Fig. 3B, 3D).Similar results were obtained using the CL–Brener strain of T. cruzi(Supplemental Fig. 1B). To confirm the participation of the ASCand caspase-1 pathway in the T. cruzi experimental model, weevaluated the IL-1b secretion in the T. cruzi–infected macro-phages from WT, ASC2/2, and caspase-12/2 mice. We observeda time-dependent increase in IL-b release only in the infected WTBMMs (Fig. 3C). In contrast, the infection of the caspase-12/2

and ASC2/2 macrophages did not lead to IL-1b production (Fig.3C). The abrogation of IL-1b secretion observed in the infectedcaspase-12/2 and ASC2/2 BMMs was specifically related to theinflammasome pathway because the release of inflammasome-independent proinflammatory cytokines, such as TNF-a, was sim-ilar in all groups (Fig. 3C). Next, we investigated whether these

inflammasome components are involved in the control of parasiteproliferation. Therefore, we infected the BMMs from the WT,caspase-12/2 and ASC2/2 mice with T. cruzi in the presence orabsence of IFN-g and evaluated the trypomastigote growth. Weobserved that the non-primed macrophages did not kill the para-sites efficiently, whereas the IFN-g–primed macrophages releasedless T. cruzi. Notably, when primed with IFN-g, the ASC2/2 andcaspase-12/2 BMMs exhibited 51 and 56% parasite killing, re-spectively, compared with the primed WT BMMs, which eradicated80% of the trypomastigotes (Fig. 3E). These data suggest that ASCand caspase-1 are important for restricting the T. cruzi proliferation.Because NO is essential for the restriction of T. cruzi proliferation byBMMs (31), we investigated whether the impaired killing observedin the ASC2/2 and caspase-12/2 BMMs is related to the ability ofthe cells to produce NO. We observed that the IFN-g-primed BMMsfrom the ASC- and caspase-1–deficient mice produced less NO wheninfected with T. cruzi than the WT mice under the same conditions(Fig. 3F). Therefore, we suggest that the ASC inflammasome is

FIGURE 3. The ASC adaptor is

essential for T. cruzi–induced cas-

pase-1 activation and IL-1b secre-

tion. BMMs from the WT (white

bars), ASC2/2 (striped bars), and

caspase-12/2 (black bars) mice were

infected with trypomastigotes of T.

cruzi (MOI 5), and 24 h later, the

presence of active caspase-1 p20 sub-

unit and LDH release assayed in the

culture supernatant by Western blot-

ting and a nonradioactive cytotoxicity

assay, respectively (A, I). ASC-CFP–

expressing macrophages (green) were

infected for 3 d with trypomastigotes

previously stained with CMTPX (red)

and imaged (photomicrographs show

original magnification 31000) by

confocal microscopy (B). The num-

ber of specks in ASC-CFP–express-

ing macrophages was quantified for

infected cells (D). Similar to (A), the

BMMs were infected, and at the in-

dicated times, the IL-1b (top graph)

and TNF-a (bottom graph) were quan-

tified using ELISA (C). WT, ASC2/2,

and caspase 12/2 BMMs were treated

with IFN-g (1 ng/ml) or IL-1b (10–

100 ng/ml), followed by infection

with trypomastigotes. After 2 and 5

dpi, parasites (E, G) and NO (F, H)

were quantified in the culture super-

natants. The experiments in triplicate

were performed independently (four

times) and generated similar results.

The results of one representative ex-

periment are presented. *p , 0.05 as

compared with WT mice.

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important for NO production in IFN-g–activated BMMs following T.cruzi infection. Next, we investigated the mechanisms regulated byinflammasomes that are responsible for the control of T. cruzi in-fection and NO production. We treated WT-infected BMMs with IL-1b (10 or 100 ng/ml) and measured trypomastigote growth and NOproduction. We observed that IL-1b–treated macrophages releasedfewer trypomastigotes than untreated cells (Fig. 3G). We also foundthat IL-1b triggers NO release by infected BMMs in a dose-dependent manner (Fig. 3H). Interestingly, IL-1b was recentlydemonstrated to be critical for the restriction of Leishmania ama-zonensis infection (32). Because pyroptosis is required to control thereplication of certain intracellular pathogens (33), we quantified thistype of cell death in infected macrophages. We observed reducedLDH release in ASC2/2 and caspase-12/2 infected BMMs comparedwith WT-infected BMMs (Fig. 3I), suggesting that pyroptosis couldbe important in restricting the parasite burden in macrophages.Altogether, these results indicate that ASC and caspase-1 partic-ipate in the inflammasome response to T. cruzi infection in vitro.

Potassium efflux, ROS generation, and cathepsin B activity arenecessary for caspase-1 activation induced by T. cruzi

Next, we investigated the molecular mechanisms underlying theT. cruzi–induced caspase-1 activation. Cellular signals, includingpotassium efflux, lysosomal destabilization, extracellular ATP, andthe generation of ROS, have been shown to trigger inflammasomeassembly (34). Therefore, we investigated whether these processesare involved inflammasome activation in response to T. cruzi in-fection by evaluating IL-1b production as readout for caspase-1activation. First, we cultured the WT BMMs in a high concen-tration of KCl (130 mM) or in the presence of glibenclamide,compounds that impair the efflux of intracellular K+. Becauseglibenclamide can interact with ASC inflammasomes, impairingthe assembly of these molecules (35, 36), we used a high KClconcentration to confirm this phenomenon. We observed that theKCl and glibenclamide treatment inhibited the IL-1b release in theT. cruzi–infected BMMs (Fig. 4A). Moreover, the infected macro-phages exhibited a significant dose-dependent reduction in caspase-1 activation when treated with the glibenclamide (SupplementalFig. 2A). Under pathophysiological conditions, the efflux of K+ canresult from the activation of the P2X7R by extracellular ATP (37).We used P2X7R2/2 BMMs to determine whether the IL-1b se-cretion in our model was mediated through the P2X7R. However,we did not observe significant differences in the IL-1b secretion(Fig. 4A) or caspase-1 activation (Supplemental Fig. 2B) betweenthe WT and P2X7R2/2 T. cruzi-infected BMMs. To inhibit ROSgeneration, we used the antioxidant NAC. We observed that the T.cruzi–infected macrophages treated with NAC exhibited reducedIL-1b production and caspase-1 activation compared with thenontreated BMMs (Fig. 4B, Supplemental Fig. 2C). To investigatethe lysosomal destabilization in response to the T. cruzi infection,we treated the BMMs with chloroquine or CA-074-Me, which in-hibit endosomal–lysosomal acidification and cathepsin B activity,respectively. Both treatments resulted in a significant reduction inthe IL-1b release after T. cruzi infection (Fig. 4E, 4F). Collectively,our results indicated that potassium efflux, ROS generation, andlysosomal acidification are important mechanisms for inflamma-some activation by T. cruzi–infected macrophages.

Activation of an ASC-dependent inflammasome is important forresistance against T. cruzi infection

To investigate the role of the ASC-dependent inflammasomein the host resistance to T. cruzi infection in vivo, we infectedcaspase-12/2, ASC2/2, and WT mice with the Y strain and eval-uated the parasitemia and mortality over a 40-d period. Strikingly,

100% of the ASC2/2 and 90% of caspase-12/2 mice succumbed tothe infection after 28 d, whereas 30% of the WT mice died duringthat period (Fig. 5A). Similar results were obtained in mice infectedwith the CL–Brener strain (Supplemental Fig. 1C). Although weobserved similar numbers of parasites in the blood from the ASC2/2

and caspase-12/2 mice, they exhibited higher parasitism in the heartat 18 dpi (Fig. 5B) and spleen (data not shown) than the WT mice.Interesting, at 10 dpi we observed reduced inflammation in thehearts of ASC2/2 and caspase-12/2-infected mice when comparedwith WT animals (Fig. 5C). In contrast, in the histopathologicalexamination of the heart muscle sections obtained at 18 dpi, both theASC2/2 and caspase-12/2 mice demonstrated more pronouncedmyocarditis compared with the WT mice (Fig. 5C, 5E). The mea-surement of the serum CK–MB levels confirmed a higher rate ofheart injury in the infected knockout mice (Fig. 5D). Finally, thequantification of cytokines in extracts of the infected heart tissuesamples demonstrated that the ASC2/2 and caspase-12/2 miceexhibited lower levels of IL-1b but similar levels of IFN-g per gramof tissue compared with the WTmice at 18 dpi (Fig. 5F). The resultsindicate that the ASC inflammasome–dependent response plays animportant role in the host resistance to acute T. cruzi infection.

NLRP3 inflammasome contributes to caspase-1 activation andIL-1b secretion during T. cruzi infection

The data presented in this study support that ASC is essential forcaspase-1 activation and IL-1b secretion following T. cruzi infec-tion. Because several NLRs, such as NLRC4, NLRP3, and NLRP6,recruit ASC to promote inflammasome formation, we investigatedthe participation of these receptors in the response to T. cruzi in-fection. To investigate the role of these NLRs in response to T. cruzi,

FIGURE 4. Potassium efflux, ROS generation and cathepsin B are

necessary for IL-1b production in BMMs infected with T. cruzi. The

BMMs (2 3 105) from the WT or P2X7R2/2 mice were infected with T.

cruzi (MOI 5 parasites) and, 24 h later, the supernatants were collected and

the IL-1b quantified. Prior to parasite infection (1 h), the BMMs were

treated or not with 130 mM KCl, 130 mM NaCl, 50 or 100 mM gliben-

clamide (A), 20 mM NAC (B), 0.1 or 0.01 mM chloroquine (C) or 20 or 50

mM CA-074-Me (D). The results showed are of one of three representative

experiments performed in triplicate with similar results. *p , 0.05 com-

paring treated infected and nontreated infected samples.

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we infected the BMMs from ASC2/2, NLRC42/2, NLRP32/2,NLRP62/2, and WT mice with T. cruzi and quantified the IL-1brelease. We observed that in addition to the ASC2/2 BMMs, onlythe NLRP32/2 BMMs exhibited significantly reduced IL-1b pro-duction and caspase-1 activation (Fig. 5G, 5H). Interestingly, thedecreased IL-1b production in the infected ASC2/2 BMMs cellswas still lower than in the NLRP32/2 BMMs (Fig. 5G). The partialcontribution of NLRP3 to the response following T. cruzi infectionwas confirmed in in vivo experiments in which the NLRP32/2,ASC2/2, caspase-12/2, and WT mice were infected with T. cruzi.The parasitemia was similar among the observed groups. Interest-ingly, whereas 100% of the ASC2/2mice and 87.5% of the caspase-1mice succumbed to the parasite infection, only 34% of the NLPR32/2

mice died (Fig. 5I). This result suggests that other inflammasomecomponents participated in the ASC-dependent response againstT. cruzi infection in vivo.

IL-1R is required for resistance to T. cruzi and the migration ofmacrophages to the infected heart

The experiments described thus far suggest that IL-1b plays a rolein the resistance to T. cruzi infection. To confirm the requirement

for this cytokine during infection, we evaluated cardiac parasitismand survival in IL-1R2/2 infected mice. We observed a highermortality and parasite load in the hearts of IL-1R2/2 mice com-pared with WT mice (Fig. 6 A‑C). These results suggest that IL-1R is crucial for resistance to T. cruzi infection and controls heartparasitism. Therefore, it is possible that the myocardia of ASC2/2

and caspase-12/2 infected mice are compromised because of theIL-1–dependent recruitment of innate immune effector cells in theearly phase of T. cruzi infection. Indeed, the histological exami-nation of heart muscle sections obtained at 10 dpi from ASC2/2,caspase-12/2, and IL-1R2/2 mice revealed a significant reduc-tion in myocarditis compared with WT mice (Fig. 6D, 6E).Using cytometry and immunohistochemistry techniques, we ob-served a significantly reduced amount of CD11b+ and F4/80+

cells in the hearts of ASC2/2, caspase-12/2, and IL-1R2/2 micewhen compared with infected WT mice (Fig. 6F, 6G), suggestingthat IL-1R is necessary for the recruitment of macrophages duringT. cruzi infection in an inflammasome-dependent manner. There-fore, we conclude that IL-1R is involved in the mechanism bywhich F4/80+ and CD11b+ cells migrate to the hearts of infectedmice during T. cruzi infection Our findings reveal an unrecognized

FIGURE 5. ASC inflammasome is necessary to control of T. cruzi infection in vivo. C57BL/6, ASC2/2, and caspase-12/2 mice (10 animals/group) were

infected with 103 blood trypomastigotes and the mortality evaluated (A). The amount of parasite DNA (B) and inflammatory infiltrate (C) was quantified in the

heart tissue of mice (four to five animals per group) on days 10 and 18 postinfection. The levels of creatine kinase in the serum of mice (six to seven animals per

group) on days 10 and 18 postinfection were evaluated as described in methods (D), and photomicrographs (original magnification3200) of heart tissues of mice

on day 18 postinfection are shown (E). IFN-g (left graph) and IL-1b (right graph) were quantified in the homogenates of heart tissues by ELISA (F). BMMs

from the WT, NLRC42/2, ASC2/2, NLRP32/2, and NLRP62/2mice were infected for 24 h with trypomastigotes (five parasites per cell) of T. cruzi and IL-1b in

the supernatants (G) and active caspase-1 (H) were measured. For caspase-1 activity assay, the infected cells were treated with FLICA and the green fluorescence

assayed using flow cytometer. The light gray, dark gray, and black lines represent the noninfected NLRP32/2 and infected WT cells, respectively. In (I) is shown

the mortality rate of WT, ASC2/2, caspase-12/2, and NLRP32/2 mice (8–10 animals/group) infected with 103 blood trypomastigotes. The graphs depict the

results from one representative experiment performed three times with similar results. *p, 0.05 indicates a significant difference among WTand knockout mice.

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pathway of T. cruzi activation in the innate immune response(Fig. 7).

DiscussionThe innate immune response to T. cruzi infection is composedof diverse PRRs, including TLR-2, -4, -7, and -9, as well as thecytosolic receptor Nod1 (3–6, 8). However, cytokine productionin the mice deficient in the different TLRs, the adaptor proteinMyd88 and Nod1 was not completely abolished, thus suggestingthat additional PRRs account for the host immune responses to

T cruzi. To identify additional receptors that are involved in theinnate immune recognition of T. cruzi, we performed a microarrayanalysis of the infected mice and determined that the genes as-sociated with inflammasome activation were highly upregulatedin response to the infection. Further investigation established akey role for the ASC inflammasomes during T. cruzi infection inmacrophages and in vivo. In our microarray analysis, we detectedthe higher expression of inflammasome-related genes, includingASC, NAIP5, caspase-1, and IL-1R, in the hearts of the T. cruzi–infected mice compared with the uninfected C57BL/6 mice,

FIGURE 6. IL-1R is required to control T. cruzi infection in vivo. WT and IL-1R2/2 mice (10 per group) were infected with the Y (A) or CL–Brener

strain of T. cruzi (B), and mortality was evaluated. Hearts fromWT, ASC2/2, caspase-12/2, and IL-1R2/2 (five mice per group) infected mice (10 dpi) were

used to quantify parasitism by real-time PCR (C) and inflammation (D). The photomicrographs (original magnification3200) of heart tissues from infected

mice are shown (E). Leukocytes from the hearts were stained with anti-CD11b or anti-F4/80 Ab and analyzed by flow cytometry (F) and immunohisto-

chemistry (G). The photomicrographs (original magnification3200) of heart tissues from infected mice are shown (G). The results are representative of one

of two experiments performed independently and yielding similar results. *p , 0.05, a significant difference between WT and knockout mice.

FIGURE 7. Model of caspase-1 activation upon

T. cruzi infection. The trypomastigotes infect macro-

phages and at 8–16 h the parasites egress the vacuole

and differentiate into the amastigote form in the cyto-

plasm, where they begin to divide (41). The parasites

into the cytoplasm activate several DAMPs, including

lysosomal acidification, B-cathepsin, potassium efflux,

and ROS, which are important for inflammasome ac-

tivation by T. cruzi–infected macrophages (as in Fig. 4).

The DAMPs are sensed by the NLRP3/ASC/caspase-1

inflammasome resulting in caspase-1 activation and IL-

1b protein maturation. Moreover, possibly other re-

ceptors may participate in the caspase-1 activation us-

ing the ASC inflammasome. CARD, Caspase activation

and recruitment domain; NACHT, NACHT domain;

PYD, pyrin domain.

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suggesting that inflammasome-related genes operate during the invivo infection with T. cruzi in the tissues relevant to the diseasedevelopment. Further investigation of the role of the inflamma-some during the infection indicated that in T. cruzi–infected macro-phages, caspase-1 activation and IL-1b production are readily andstrongly induced in an ASC-dependent manner. These findingsrevealed a very important and yet unappreciated pathway that istriggered in response to the T. cruzi infection.To assess the mechanisms that are responsible for the IL-1b

secretion and inflammasome activation following T. cruzi infec-tion, we infected the BMMs from the WT mice with live, IR, orHK parasites. Notably, we observed that only live T. cruzi inducedcaspase-1 activation and efficient IL-b production. Moreover,caspase-1 and IL-1b maturation occurred concomitantly startingat 24 h postinfection. These results suggest that parasite virulencefactors are crucial for engaging inflammasomes to cleave caspase-1and trigger subsequent IL-1b production. In addition, the experi-ments performed using cytochalasin indicated that the internaliza-tion of the parasite by macrophages is required for inflammasomeactivation. Collectively, the data demonstrate that the invasion ofviable trypomastigotes is essential for triggering caspase-1 activa-tion, which is consistent with other studies showing that the in-vasion of the macrophages with viable pathogens is necessary tostimulate the caspase-1 pathway (38, 39). We speculate that theparasite escape from the vacuole to the cytoplasm is pivotal forinducing lysosomal damage and consequently the NLRP3-de-pendent activation of the inflammasome. A similar phenomenonoccurs with cholesterol and acid uric crystals, which promote thedestabilization of lysosomes and leakage into the cytoplasm,where they are detected by NLRP3 (10, 40).At 8–16 h after the cellular invasion by trypomastigote forms of

T. cruzi, the parasites egress the vacuole and differentiate into theamastigote form in the cytoplasm, where they begin to divide (41).Because our data demonstrate that the caspase-1 signaling inducedby T. cruzi occurs at a later stage (24 h postinfection) comparedwith the caspase-1 activation triggered by other pathogens, wespeculate that the parasites need to escape of the phagosome totrigger the inflammasome. Indeed, the inhibition of endosomal–lysosomal acidification by incubation of the cells with chlo-roquine, which inhibits the release of trypomastigotes into thecytosol (42), confirmed this hypothesis; in the presence of thechloroquine, the IL-1b secretion stimulated by T. cruzi was re-duced significantly. These data reinforce our previous results,which demonstrated that viable T. cruzi induced higher IL-1bproduction and caspase-1 activation than HK parasites. Interest-ing, we observed also that the formation of fluorescent clusters ofCFP-ASC in infected cells (ASC inflammasome activation) occursonly when the amastigotes (replicative form of T. cruzi) are in thecytoplasm, indicating again that parasites need to escape of thephagosome to trigger the inflammasome. Importantly, this time ofinflammasome activation is sufficient to kill the T. cruzi, becausethe amastigotes still are in the cellular cytoplasm.IFN-g is essential for the resistance against in vivo and in vitro

T. cruzi infection (43). This cytokine induces the cytoplasmicguanosine 59-triphosphatases, which have been associated withantimicrobial defenses (44). The human GBP5 and its mouseortholog are required for the NLRP3-dependent IL-1b secretion(45); therefore, we suggest that T. cruzi triggers GBP5, whichinduces the assembly of the NLRP3/ASC inflammasome that isimportant for killing the parasite. Furthermore, we demonstratedthat the ASC2/2 and caspase-12/2 macrophages primed with IFN-gand infected with T. cruzi exhibited increased numbers of parasitesand reduced levels of NO compared with the WT macrophages,thus reinforcing our hypothesis that the caspase-1 pathway is some-

how connected to the production of NO, as reported previously (46).Notably, we showed that IL-1b triggered robust NO production bymacrophages and controlled T. cruzi growth in BMMs. Therefore,because IFN-g (47) and IL-1b are produced soon postinfection,they likely cooperate to increase macrophage activation. We alsoshowed that T. cruzi triggered pyroptosis, which was dependent onthe ASC inflammasome. Therefore, in addition to cytokine controlof parasite replication, we suggest that pyroptosis can also be animportant mechanism to restrict the parasite burden in macro-phages. To our knowledge, this is the first description of the in-volvement of pyroptosis in a parasitic infection.To elucidate the mechanisms that are required to induce the

activation of the ASC inflammasomes, we performed experimentsblocking the DAMPs that trigger the NLRP3/ASC inflammasome.The caspase-1 activation and IL-1b release after T. cruzi infectionwere dependent of the potassium efflux, ROS generation, lyso-somal acidification, and cathepsin B release. Stimulation of theP2X7R, an ATP-gated ion channel, triggers rapid activation ofcaspase-1 (37, 48). However, the P2X7R was dispensable forcaspase-1 activation in our studies. Similar results were observedfollowing infection with Streptococcus pyogenes, which triggersNLRP3 independent of P2X7R (11). Importantly, because gli-benclamide can act directly on ASC inflammasome assembly in-dependently of K+ channel inhibition (35, 36), we used a high KClconcentration to show that potassium efflux is a significant mecha-nism required to caspase-1 activation induced by T. cruzi.Importantly, the absence of ASC and caspase-1 resulted in

a significant increase in the susceptibility to T. cruzi infectionin vivo, revealing a novel role for ASC inflammasomes in medi-ating the host resistance to the parasite infection. The mechanisminvolved in the susceptibility of the ASC2/2 mice is likely de-pendent on IL-1 because the IL-1R2/2 infected mice are moresusceptible to the infection compared with WT mice (Fig. 6). Thedownstream effects of IL-1 signaling in host resistance are likelymediated through cell recruitment, because a lack of IL-1 sig-naling significantly reduced macrophage infiltration into the car-diac tissue. Strikingly, IL-1R–infected mice were more susceptible(started died at 10 dpi) to the infection than were the ASC2/2 andcaspase-12/2 animals (started died at 17 dpi), suggesting that IL-1R is more relevant than ASC2/2 and caspase-1 in resistance to T.cruzi. Other study has shown an important function of IL-1 sig-naling, but limited role of inflammasome activation (49). Then, wespeculated that IL-1R regulates other mechanisms that are inde-pendent of the inflammasomes activation to control the T. cruziinfection.Interestingly, at 18 dpi, the ASC2/2 and caspase-12/2 infected

mice exhibited increased inflammation in the heart compared withthe WT animals, possibly because of the increased parasite load inthe cardiac tissue. We also observed reduced levels of IL-1b in thehearts of the ASC2/2 and caspase-12/2 mice, whereas the IFN-grelease was not affected. It is likely that the IFN-g productionobserved in the ASC2/2 and caspase-12/2 infected mice are de-pendent on IL-12, because IL-18 could be reduced in ASC2/2 andcaspase-12/2 infected mice. In addition, IL-182/2 animals in-fected with T. cruzi presented similar mortality and parasitemiacompared with WT-infected mice (data not shown), thus rein-forcing data that have previously been published (50). Thus, thesusceptibility of ASC and caspase-1–deficient mice is likely to bedependent on IL-1.Therefore, ASC and caspase-1 are importantfor the induction of IL-1b production after T. cruzi infection. IL-1b, in turn, may be required to trigger NO production and theconsequent control of T. cruzi growth in the cardiac myocytes, ashas been demonstrated previously in isolated cultures of car-diomyocytes (51, 52). In this study, we showed the results of IL-

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1b in heart using ELISA, which detect both forms of this cytokine.Therefore, more studies are necessary to determine whether IL-1bin the hearts of infected mice is cleaved or in an immature form.Moreover, it is required to define whether IL-1b in the heart tissuetakes effect immediately or plays a biological role in other affectedtissues upon T. cruzi infection. We show that T. cruzi inducesa significant production of total IL-1b (protein) in infected heartsby ELISA, and we believe this cytokine has an important bio-logical role.The role of the inflammasome has been investigated in the malaria

cerebral model. In this study, the absence of NLRP3 reduced thefever in the infected mice and increased the survival rate of theseanimals, suggesting that the ASC/NLRP3 inflammasome is notfavorable for resistance to this parasite (53, 54). Our data, however,demonstrated that the ASC signaling was essential for the control ofthe T. cruzi infection in vivo, whereas NLRP3 participation was notcrucial for the resistance to the parasite. Therefore, an adequateinnate immune response to produce a specific immunity against thedistinct parasites is necessary to overcome the different infections.Several NLRs, including NLRP6, NLRC4 and NLRP3, associate

with the ASC inflammasome to trigger caspase-1 activation andIL-1b secretion following stimulation with different pathogens(55). We observed that NLRC4 and NLRP6 were dispensable forthe caspase-1 activation after T. cruzi infection, whereas in theabsence of NLRP3, the IL-1b production and caspase-1 activationwere significantly reduced. However, the decrease in IL-1b andcaspase-1 activation in the NLRP32/2 BMMs was partial becauseit did not exhibit the same drastic IL-1b production phenotypeexhibited by the ASC2/2 infected macrophages. Consistent withthese results, we observed that the NLRP32/2 infected mice dem-onstrated a similar survival rate as the WT mice. In addition, theNLRP3 expression in the hearts from the WT mice was not alteredsignificantly postinfection, as demonstrated by the microarray(Fig. 1). Other NLRs are potentially engaged by the ASC inflam-masome to induce an adequate immune response against T. cruzi.Nonetheless, further investigation will be required to identify thespecific NLRs that are involved in the activation of the ASCinflammasomes in response to T. cruzi infection. As demonstratedin this study, NLRP3 is involved in the response; however, be-cause of the possible functional redundancy of NLRP3 with otherNLRs, the NLRP32/2 phenotype is not as susceptible to infectionas the mice deficient in ASC and caspase-1.Our findings reveal an unrecognized pathway of T. cruzi activation

in the innate immune response (Fig. 7). T. cruzi activates the ASCinflammasome using NLRP3-dependent and -independent mecha-nisms, and this process leads to the potent induction of active IL-1b,which is essential for regulating the parasite burdens in the hearts ofthe infected mice. The data add to our understanding of the hostresistance mechanisms against T. cruzi infection and may aid thedevelopment of future therapeutics against Chagas disease.

AcknowledgmentsWe thank Cristiane Maria Milanezi for technical assistance. We also thank

Prof. Douglas Golenbock, University of Massachusetts, for several sugges-

tions about experiments performed in this study.

DisclosuresThe authors have no financial conflicts of interest.

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