Myeloid Dendritic Cells (DCs) of Mice Susceptible toParacoccidioidomycosis Suppress T Cell Responses whereas Myeloidand Plasmacytoid DCs from Resistant Mice Induce Effector andRegulatory T Cells

Adriana Pina, Eliseu Frank de Araujo, Maíra Felonato, Flávio V. Loures, Claudia Feriotti, Simone Bernardino,José Alexandre M. Barbuto, Vera L. G. Calich

Departamento de Imunologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brazil

The protective adaptive immune response in paracoccidioidomycosis, a mycosis endemic among humans, is mediated by T cellimmunity, whereas impaired T cell responses are associated with severe, progressive disease. The early host response to Paracoc-cidioides brasiliensis infection is not known since the disease is diagnosed at later phases of infection. Our laboratory establisheda murine model of infection where susceptible mice reproduce the severe disease, while resistant mice develop a mild infection.This work aimed to characterize the influence of dendritic cells in the innate and adaptive immunity of susceptible and resistantmice. We verified that P. brasiliensis infection induced in bone marrow-derived dendritic cells (DCs) of susceptible mice a preva-lent proinflammatory myeloid phenotype that secreted high levels of interleukin-12 (IL-12), tumor necrosis factor alpha, andIL-�, whereas in resistant mice, a mixed population of myeloid and plasmacytoid DCs secreting proinflammatory cytokines andexpressing elevated levels of secreted and membrane-bound transforming growth factor � was observed. In proliferation assays,the proinflammatory DCs from B10.A mice induced anergy of naïve T cells, whereas the mixed DC subsets from resistant miceinduced the concomitant proliferation of effector and regulatory T cells (Tregs). Equivalent results were observed during pulmo-nary infection. The susceptible mice displayed preferential expansion of proinflammatory myeloid DCs, resulting in impairedproliferation of effector T cells. Conversely, the resistant mice developed myeloid and plasmacytoid DCs that efficiently ex-panded gamma interferon-, IL-4-, and IL-17-positive effector T cells associated with increased development of Tregs. Our workhighlights the deleterious effect of excessive innate proinflammatory reactions and provides new evidence for the importance ofimmunomodulation during pulmonary paracoccidioidomycosis.

Dendritic cells (DCs), which continuously survey their envi-ronment for invading microorganisms, are considered pro-

fessional antigen-presenting cells (APCs) due to their unique abil-ity to activate T cells (1). During infection, the interaction ofpattern recognition receptors (PRRs) on immature DCs with con-served molecular patterns of microorganisms (PAMPs) results inincreased secretion of inflammatory mediators and enhanced ex-pression of major histocompatibility complex (MHC) and co-stimulatory molecules, characterizing their transition to the ma-ture phenotype and efficient APCs. DCs also downmodulate theirendocytic capacity and migrate to the T cell zone of draininglymph nodes, where they activate naïve antigen-specific T cells(2, 3). Three principal DC subsets have been identified in mice:conventional myeloid DCs (mDCs; CD11c� CD8�� CD11b�),plasmacytoid DCs (pDCs; CD11cinterm. B220� CD11b�), andlymphoid DCs (CD11c� CD8�). All DC subsets exhibit immu-nostimulatory (4, 5) and tolerogenic functions that reflect theirmaturation condition at the time of T cell interaction (6, 7). Thediverse DC subsets are characterized by a distinct pattern of PRRexpression, including Toll-like receptors (TLRs) and C-type lectinreceptors (CLRs), which recognize diverse conserved pathogenstructures (4, 8–10).

DCs sense different morphotypes of fungal pathogens in a spe-cific way, resulting in the expansion of distinct T-helper cells thatcan exert protective or deleterious effects on the host (10). T celldifferentiation to the Th1, Th2, or Th17 phenotype depends onthe innate receptor used by DCs to sense the fungal pathogen and

the predominant cytokine that is subsequently produced.Whereas interleukin-12 (IL-12) is associated with Th1 differenti-ation, IL-23 enhances the Th17 phenotype and is induced by theconcerted action of transforming growth factor � (TGF-�) andIL-6. The predominant synthesis of IL-10 or TGF-�, however,facilitates the differentiation and expansion of regulatory T cells(Tregs), which control autoimmunity and excessive inflammatoryreaction due to uncontrolled immune responses (9–13).

Paracoccidioides brasiliensis, a primary fungal pathogen fromLatin America, infects individuals from areas of endemicity via therespiratory route. The initial interaction with alveolar macro-phages appears to govern the subsequent disease outcome, whichcan evolve as a localized infection or overt disease (14, 15). Para-coccidioidomycosis (PCM) is usually asymptomatic and inducesprotective immunity, whereas the severity of disease is propor-

Received 21 July 2012 Returned for modification 13 August 2012Accepted 20 December 2012

Published ahead of print 22 January 2013

Editor: G. S. Deepe, Jr.

Address correspondence to Vera L. G. Calich, vlcalich@icb.usp.br.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.00736-12.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.00736-12

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tional to the impairment of the cellular immune response and theactivation of humoral immunity. A Th2/Th3-skewed immunityhas been correlated with severe forms of the disease, whereas mildforms are usually associated with Th1 immunity (16–18). Theregulatory mechanisms controlling protective or deleterious im-mune responses in PCM are not well-defined, but the apoptosis ofT cells, expression of CTLA-4 (a T cell-inhibitory molecule), andincreased presence of regulatory T cells have been described insevere forms of PCM (19–21)

Our laboratory has established a pulmonary model of paracoc-cidioidomycosis. In this model, susceptible B10.A mice, despitethe early proinflammatory response and control of fungal growth,develop T cell anergy and a fatal disseminated disease; in contrast,resistant A/J mice are initially permissive for fungal growth butlater develop positive delayed-type hypersensitivity (DTH) reac-tions and regressive disease (22–27). Studies of the main T cellsubsets involved in immunity to P. brasiliensis infection revealed apersistent CD4� T cell anergy but a preserved CD8� T cell re-sponse in susceptible B10.A mice. In contrast, the early unrespon-siveness of resistant mice was followed by a protective responsemediated by CD4� and CD8� T cells that secrete a mixed patternof cytokines with a prevalence of gamma interferon (IFN-�) (24,28, 29).

In human paracoccidioidomycosis, the innate phase of immu-nity has never been investigated because the disease is diagnosedduring late phases of infection. We have, however, investigatedsome aspects of innate immunity developed by both resistant andsusceptible mice (22) We verified that P. brasiliensis-infected alve-olar macrophages of susceptible mice have a proinflammatoryphenotype characterized by intense nitric oxide (NO) production,efficient fungal killing, and the prevalent synthesis of IL-12. Incontrast, alveolar macrophages from resistant mice show an anti-inflammatory behavior due to the prevalence of TGF-� secretionthat impairs their NO secretion and fungicidal properties (26).Importantly, the early administration of IL-12 does not reverse thesusceptibility of B10.A mice but induces an excessive pulmonaryinflammation (30). IL-4 depletion, instead of abolishing the sus-ceptibility of B10.A mice, induces a more severe disease (31).Moreover, early in infection, higher levels of IFN-� were found inthe lungs of B10.A but not A/J mice (32). Thus, several studiesusing our experimental model have demonstrated that the suscep-tibility of B10.A mice is most likely mediated by excessive proin-flammatory activity of innate immune cells and not by an earlyTh2-skewed response, whereas the resistance of A/J mice com-prises an early tolerance to fungal growth that subsequentlyevolves to a prevalent Th1 immunity (17, 22).

Because DCs are the most important antigen-presenting cellsand function as a bridge that links innate and adaptive immunity,we decided to analyze the behavior of these cells in our experimen-tal model. Thus, we characterized the influence of P. brasiliensisinfection on the phenotype and behavior of pulmonary and bonemarrow-derived dendritic cells (BMDCs) of resistant and suscep-tible mice. We also investigated the antigen-presenting ability ofDCs to naïve lymphocytes. We demonstrated that BMDCs fromB10.A mice, stimulated by P. brasiliensis yeasts, differentiated to apredominantly proinflammatory myeloid phenotype, whereasmyeloid and plasmacytoid subsets were detected with A/J mousecells. Both myeloid and plasmacytoid DCs underwent functionalmaturation in response to P. brasiliensis infection but activateddifferent programs of cytokine production.

While the proinflammatory behavior of B10.A DCs is associ-ated with T cell impairment, a common phenotype usually devel-oped by this susceptible strain, the concomitant production ofpro- and anti-inflammatory cytokines by A/J DCs results in theenhanced differentiation of IFN-�-positive (IFN-��), IL-4-posi-tive (IL-4�), and IL-17-positive (IL-17�) effector T cells associ-ated with increased Treg development. These data help, in part, toexplain the distinct immune responses of resistant and susceptiblemice to Paracoccidioides brasiliensis infection. Furthermore, ourwork demonstrates that excessive proinflammatory innate re-sponses are deleterious to the adaptive response against pulmo-nary paracoccidioidomycosis and immunoprotection is achievedwhen DCs induce well-balanced pro- and anti-inflammatory re-sponses.

MATERIALS AND METHODSMice. Mouse strains susceptible (B10.A) and resistant (A/J) to P. brasil-iensis infection were obtained from our Isogenic Breeding Unit (Departa-mento de Imunologia, Instituto de Ciências Biomédicas, Universidade deSão Paulo, São Paulo, Brazil) and used at 8 to 11 weeks of age. Specific-pathogen-free mice were fed sterilized laboratory chow and water ad libi-tum. The experiments were approved by the Ethics Committee on AnimalExperiments of the Institute of Biomedical Sciences of the University ofSão Paulo (process 76/04/CEEA).

Fungus. P. brasiliensis 18, a highly virulent isolate (33), was usedthroughout this investigation. P. brasiliensis 18 yeast cells were maintainedby weekly subcultivation in semisolid Fava Netto’s culture medium (34) at35°C and used on day 7 after culture. The yeast cells were washed inphosphate-buffered saline (pH 7.2) and adjusted to 5 � 104 cells/ml onthe basis of hemocytometer counts. Viability was determined with Janusgreen B vital dye (Merck, Darmstadt, Germany) and was always higherthan 80%. All solutions used to prepare yeast cell suspensions and DCs forcultivation were tested for the presence of lipopolysaccharide (LPS) usingthe Limulus amoebocyte lysate chromogenic assay (Sigma) and alwaysshowed LPS levels �0.015 endotoxin units/ml.

DC generation and maturation in vitro. Bone marrow-derived DCswere generated according to described methods (35) with some modifi-cations. Briefly, cells removed from the femurs were cultured with 20ng/ml of recombinant granulocyte-macrophage colony-stimulating fac-tor (rGM-CSF; BD Pharmingen) and 2 ng/ml recombinant interleukin-4(rIL-4; BD Pharmingen) in RPMI 1640 medium containing 10% fetal calfserum (FCS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 �g/mlstreptomycin. The medium with rGM-CSF and rIL-4 was renewed ondays 2 and 4 of culture. On day 5, the cells were matured with P. brasiliensisyeasts at a yeast/DC ratio of 1:20 for 48 h of incubation. On day 7, thenonadherent cells were removed and analyzed by flow cytometry, usingDC surface markers.

Dendritic cell phenotypes and intracellular cytokines. DCs were ad-justed to 1 � 106 viable cells/ml and incubated for 20 min at 4°C withmonoclonal antibodies (MAbs) to CD16/CD32 (Fc�R II/III, 2 · 4G2; Fcblock) and stained for 30 min at 4°C with fluorescein isothiocyanate(FITC)-, phycoerythrin (PE)-, or phycoerythrin-Cy5 (PE-Cy5)-labeledMAbs (1 �g/106 cells). The following antibodies (BD Biosciences, Phar-mingen) were used to characterize the expression of surface moleculesfrom DCs: anti-CD11c (HL3), MHC class II (MHC-II; I-Ak, 11-5.2),CD80 (B7-1, 16-10A1), CD86 (B7 · 2, Gl-1), CD40 (3/23), CD8� (Ly-2),CD45-B220 (RA3-6B2), and mouse plasmacytoid dendritic cell antigen-1(mPDCA-1; CD317). The stained cells were analyzed immediately onFACScan equipment using PC-Lysis software (Becton, Dickinson, SanJose, CA) with gating on macrophages, as judged from forward andside light scatter (FSC and SSC, respectively). Fifty thousand cells werecounted, and the data were expressed as the percentage of positive cellsor the median fluorescence intensity (MFI) obtained with specific an-tibodies.

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The expression of some intracellular cytokines (IL-12, tumor necrosisfactor alpha [TNF-�], IL-10, IL-1�, and TGF-�), as well as the presence ofmembrane-bound TGF-� (latency-associated peptide [LAP]), in the di-verse DC subsets was also assessed. Thus, the nonadherent cells were re-moved from cultures and incubated with 100 �g of MicroBeads (MiltenyiBiotec) conjugated to hamster anti-mouse CD11c MAbs. Positively se-lected DCs contained 85% CD11c� cells. For intracellular cytokinestaining, DCs (1 � 106 cells/ml) were stimulated for 6 h in the presence of50 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich), 500ng/ml ionomycin (Sigma-Aldrich), and brefeldin A (eBioscience). Cellswere incubated for 20 min at 4°C with MAbs to CD16/CD32 (Fc�R II/III,2 · 4G2; Fc block) and stained for 30 min at 4°C with PE-Cy7 anti-CD11c(clone RM4-5; eBioscience), Pacific blue anti-CD11b (M1-70; eBiosci-ence), allophycocyanin anti-CD45-B220 (RA3-6B2; eBioscience), AlexaFluor anti-CD8� (clone 53-6.7; BioLegend), or PERCY anti-human LAP–TGF-�1; FAB2463C; R&D Systems). Cells were then fixed, permeabilized,and stained by PE anti-human TGF-�1 (IC240P; R&D Systems), PE anti-IL-12 (554479; R&D Systems), PE anti-IL-10 (JeS5-16E3; BioLegend), orPE anti-TNF-� (554419; R&D Systems). Samples were washed twice withPerm buffer and immediately analyzed by flow cytometry.

Lymphocyte phenotypes and intracellular cytokines. After lympho-cyte proliferation assays, two- or three-color flow cytometry was used tomeasure the expression of surface molecules and intracellular FoxP3 orIFN-�. For surface molecules, PE-Cy7 anti-CD4 (clone RM4-5; eBiosci-ence), Alexa Fluor anti-CD8� (clone 53-6-7; BioLegend), FITC anti-CD3ε (17A2; BD Pharmingen), and PE anti-CD28 (37.51), CD40L(MR1), CD44 (IM7), CD25 (3C7), CTLA-4 (UC10-4F10-11), GITR(DTA-1), NKT/NK (U5A2-13), and � T cell receptor (TCR; GL3) fromBD Pharmingen were used. The stained cells were analyzed immediatelyon FACScan equipment with gating on lymphocytes, as judged from for-ward and side light scatter. For flow cytometric analysis of apoptotic andnecrotic lymphocytes, annexin V labeling and propidium iodide labeling,respectively, were used (36). For intracellular cytokine staining, DCs andT lymphocytes were cultivated by 3 days; cells were then stimulated for 6h in complete medium in the presence of 50 ng/ml PMA (Sigma-Aldrich),500 ng/ml ionomycin (Sigma-Aldrich), and brefeldin A (eBioscience).Treg cells were characterized by intracellular staining for FoxP3, using theTreg staining kit of BD Bioscience. Surface staining of CD25� and intra-cellular FoxP3 expression were back-gated on the CD4� T cell population.To measure the expression of intracellular IFN-�, after staining of surfacemolecules, cells were fixed, permeabilized, and stained by peridinin chlo-rophyll protein (PerCP)-Cy5 anti-IFN-� antibodies (eBioscience). Thecell surface expression of leukocyte markers and the intracellular expres-sion of FoxP3 and IFN-� in stimulated lymphocytes were analyzed in aFACScalibur flow cytometer (BD Pharmingen) using FlowJo software(Tree Star, Inc., Ashland, OR).

Detection of NO and cytokines in culture supernatants of DCs. Bonemarrow-derived DCs were challenged with P. brasiliensis yeasts, and cul-ture supernatants were harvested 48 h later. Supernatants were tested forNO by using the Griess reaction. Briefly, 50 �l of the supernatant wasincubated with 50 �l of Griess reagent for 5 min at room temperature, andthe NO2 concentration was determined by measuring the absorbance at550 nm in reference to a standard NaNO2 solution. The levels of IL-12,TNF-�, IFN-�, IL-10, IL-6, IL-1�, and TGF-� were measured by captureenzyme-linked immunosorbent assay (ELISA) with antibody pairs pur-chased from Pharmingen. Latent plus active TGF-� was measured usingcommercially available kits from R&D Systems. Some cytokines were alsomeasured in culture supernatants from lymphoproliferation assays. TheELISA procedure was performed according to the manufacturer’s proto-col, and absorbances were measured with a Versa Max microplate reader(Molecular Devices). The concentrations of cytokines were determinedwith reference to a standard curve for serial 2-fold dilutions of murinerecombinant cytokines.

Quantitative analysis of TGF-� mRNA expression. RNA was ex-tracted from P. brasiliensis-infected DCs from B10.A and A/J mice using

TRIzol reagent (Invitrogen). cDNA was synthesized from 2 �g RNA usinga High Capacity RNA-to-cDNA kit (Applied Biosystems) according to themanufacturer’s instructions. TGF-� mRNA expression was quantifiedrelative to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH)using assay-on-demand primers and probes, TaqMan universal mastermix, and an ABI Prism 7000 apparatus (Applied Biosystems).

Cell proliferation. DC preparations were used to study the stimula-tory activity of DCs on naïve lymphocytes previously stained with the vitaldye 5,6-carboxyfluorescein succinimidyl ester (CFSE; Molecular Probes,Eugene, OR), as described previously (37). To obtain lymphocytes, spleencells from healthy mice were collected, red blood cells were lysed, and thecells were washed and then plated onto 12-well plates for 2 h at 37°C.Nonadherent cells were collected, and T cells were obtained by positiveselection using anti-CD90 (Thy-1) magnetic microbeads (Miltenyi, Bio-tec, Surrey, United Kingdom). The cells were washed, labeled with 5 mMCFSE, and suspended in RPMI 1640 complete medium containing 10%FCS. P. brasiliensis-stimulated DCs (1 � 104) were cultured for 5 days withCFSE-stained naïve spleen lymphocytes (3 � 105) at a lymphocyte/DCratio of 30:1. Dead cells were discriminated by propidium iodide uptake,and cell division was determined by a decrease in the intensity of CFSEstaining by flow cytometry. As an additional positive control, CFSE-la-beled naïve lymphocytes were treated with 5.0 �g/ml of concanavalin A(Sigma). A minimum of 50,000 events were acquired on a FACScaliburflow cytometer using CellQuest software (BD Pharmingen). The prolifer-ation index (PI) was calculated as the MFI of unstimulated naïve T cells/Tcells cultured with mature DCs.

The proliferation assays were also performed with unlabeled naïvesplenic T lymphocytes (3 � 105) stimulated by P. brasiliensis-pulsed DCs(1 � 104) to characterize the phenotype of proliferating cells. Two- orthree-color flow cytometry was performed to measure the expression ofsurface and intracellular molecules, as described above.

In vivo infection. B10.A and A/J mice were anesthetized and submit-ted to intratracheal (i.t.) P. brasiliensis infection as previously described(23). Briefly, after intraperitoneal anesthesia, the mice were i.t. infectedwith 1 � 106 P. brasiliensis yeast cells, contained in 50 �l of fungus. Thenumber of CFU was determined using colony plate counts (32).

Isolation of pulmonary DCs and lymphocytes. B10.A and A/J micewere infected i.t. with 1 million yeast cells of P. brasiliensis; after 96 h,lungs were removed and digested enzymatically for 30 min with colla-genase (1 mg/ml) and DNase (30 �g/ml) in culture medium (Sigma).Large particulate matter was removed by passing the cell suspensionthrough a small, loose, nylon wool plug. DCs were purified by mag-netic cell sorting with microbeads (Miltenyi Biotec) conjugated tohamster anti-mouse CD11c monoclonal antibodies. Positively se-lected DCs contained more than 90% CD11c� cells. Cell surface mark-ers were characterized by flow cytometry as described for in vitro-derived DCs. Purified DCs were also cultured for 48 h at 37°C, andsupernatants were used to quantify some pro- and anti-inflammatorycytokines (IL-12, IL-1�, TNF-�, IL-6, IL-10, and TGF-�). In someexperiments, DCs were restimulated with PMA and ionomycin andsubjected to intracellular staining as described above.

Lungs of P. brasiliensis-infected B10.A and A/J mice were obtained at 2weeks of infection and digested enzymatically as described above. Lungcell suspensions were centrifuged in the presence of 20% Percoll (Sigma)to separate leukocytes from cell debris. Total lung leukocyte numberswere assessed in the presence of trypan blue using a hemocytometer; via-bility was 85%. The number and phenotype of CD4�, CD8�, and reg-ulatory T cells, as well as the presence of intracellular cytokines, weredetermined by flow cytometry, as described above.

Statistical analysis. Data were analyzed by the Student t test. Thesoftware program GraphPad Prism, version 4.0 (GraphPad, San Diego,CA), was used for all statistical tests. P values under 0.05 were consideredsignificant.

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RESULTSP. brasiliensis infection induces a prevalent myeloid phenotypein BMDCs of susceptible mice, while in resistant mice a mixedpattern of myeloid and plasmacytoid DCs was expanded. Bonemarrow-derived cells from B10.A and A/J mice were cultured withGM-CSF and IL-4 to induce DC differentiation, and at day 5 thecells were challenged in vitro with P. brasiliensis yeasts for 48 h.Cells were then analyzed according to their FSC and SSC charac-teristics by flow cytometry as well as for their expression of CD11c,CD11b, B220, and CD8�. Unstimulated DCs were analyzed, andno differences in CD11c� CD11b�, CD11c� B220�, and CD11c�

CD8� between immature B10.A and A/J mouse DCs were found.Activation by P. brasiliensis challenge, however, resulted in an in-crease in the frequency of myeloid CD11c� CD11b� DCs in B10.Amice, whereas a concomitant differentiation of plasmacytoid(CD11c� B220�) and myeloid (CD11c� CD11b�) DCs was ob-served in A/J mice (Fig. 1A). No differences in the frequency oflymphoid DCs (CD11c� CD8�) were observed between themouse strains. Importantly, a significantly increased expression ofmPDCA was observed in CD11c� B220�-gated cells from A/J

mice in comparison with that in cells from B10.A mice (MFIs,218 � 17 and 80 � 12, respectively; P � 0.05), supporting theincreased differentiation of pDCs by resistant mice. The frequency(percent) of cells and the intensity of expression (MFI) of someactivation markers and costimulatory molecules were also as-sessed in the three DC subsets to evaluate their maturation. Com-pared with A/J cells, B10.A DCs expressed higher levels of CD80 inthe myeloid and plasmacytoid DCs and CD86 in the lymphoidsubset of DCs. In contrast, A/J mice expressed increased levels ofCD86 and CD40 in their myeloid and lymphoid DC subsets, re-spectively (Fig. 1B to D). The frequency of positive cells for co-stimulatory molecules was similar to the MFI data presented here(data not shown). In conclusion, P. brasiliensis infection inducesin resistant and susceptible mice the expansion of different DCsubsets, showing small differences in their maturation degrees.

DCs from resistant and susceptible mice secrete differentpatterns of cytokines when activated by P. brasiliensis yeasts.Immature DCs from B10.A and A/J mice were in vitro challengedwith P. brasiliensis yeasts for 48 h, and the levels of cytokines andNO were determined in culture supernatants using ELISA and the

FIG 1 Subsets and activation profile of dendritic cells developed by resistant and susceptible mice. Bone marrow cells from B10.A and A/J mice were culturedwith rGM-CSF and rIL-4 for 5 days and in vitro challenged with P. brasiliensis yeasts (1:20 fungus/DC ratio) for 48 h, and DC subpopulations were characterizedby flow cytometry. (A to D) Frequency of myeloid (CD11c� CD11b�), plasmacytoid (CD11c� B220�), and lymphoid (CD11c� CD8�) DCs. The expression ofCD80, CD86, CD40, and MHC class II was measured in each DC subpopulation. Data are expressed as a percentage or the MFI and are representative of threeindependent experiments with similar results. *, P � 0.05 compared with DCs of B10.A mice; **, P � 0.01 compared with DCs of B10.A mice; ***, P � 0.001compared with DCs of B10.A mice.

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Griess reaction, respectively. P. brasiliensis-stimulated DCs fromsusceptible mice secreted increased levels of TNF-�, IL-12, IL-1�,and IL-10, whereas DCs from resistant mice produced high con-centrations of TGF-� and IL-6 (Fig. 2A and B). Although at levelslower than those secreted by B10.A cells, A/J DCs were also able toproduce TNF-�, IL-12, IL-1�, and IL-10. Because pDCs secretelarge amounts of type I IFN in response to viral infections (4, 38),we also assessed the presence of IFN-� in the supernatants of P.brasiliensis-stimulated DCs. However, no IFN-� was detected inDC supernatants of either mouse strain.

We further analyzed by quantitative PCR the expression ofTGF-� mRNA in B10.A and A/J mouse DCs. Confirming the ele-vated levels of protein observed (Fig. 2B), increased expression ofTGF-� mRNA was detected in A/J DCs (Fig. 2D). The more pro-inflammatory behavior of B10.A DCs was confirmed by the in-creased levels of NO that they secreted (Fig. 2C).

Resistant mice develop a high frequency of TGF-�-positive(TGF-��) and LAP-positive plasmacytoid DCs, while suscepti-ble mice develop a high frequency of IL-12� myeloid and plas-macytoid DCs. BMDCs from B10.A and A/J mice were in vitrochallenged for 48 h with P. brasiliensis, and nonadherent CD11c�

cells were isolated by magnetic microbeads. For intracellular cy-tokine staining, CD11c� DCs were restimulated with PMA andionomycin for 6 h and subjected to intracellular staining for IL-12,TNF-�, IL-10, TGF-�, and membrane-bound TGF-� (LAP). Themyeloid, plasmacytoid, and lymphoid DC subpopulations weregated, and the expression of intracellular cytokines and mem-brane LAP was determined from the MFI. Compared with A/Jcells, increased expression of IL-12 by mDCs and pDCs was de-tected in B10.A cells (Fig. 3A). Myeloid DCs of susceptible micealso presented increased expression of another proinflammatorycytokine, TNF-�, (Fig. 3A). Importantly, high levels of TGF-�were seen in A/J pDCs (Fig. 3B), while membrane-bound TGF-�(LAP) was expressed at a higher intensity by all three DC subsets ofresistant mice (Fig. 3A to C).

The poor proliferative response of naïve lymphocytes in-duced by B10.A DCs was associated with increased cell deathand IFN-� production. We further asked if the DCs from resis-tant and susceptible mice had the same immunogenic or tolero-genic activity when cocultivated with homologous naïve lympho-cytes. DCs from B10.A and A/J mice were stimulated by P.brasiliensis yeasts and further incubated for a period of 5 days at37°C in 5% CO2 with CFSE (5 mM)-labeled naïve T lymphocytes.After cocultivation, the cells were removed and analyzed by flowcytometry. The decrease of fluorescence corresponds to the pro-liferative activity of lymphocytes. P. brasiliensis-activated DCs ofA/J mice were able to induce an intense proliferative response ofnaïve A/J lymphocytes (PI � 4.0), but totally impaired lympho-cyte proliferation was detected when B10.A DCs were used (PI �1.1) (Fig. 4A).

We also assessed the number of apoptotic and necrotic cells inthe lymphoproliferation assays. As depicted in Fig. 4B, increasednumbers of apoptotic and necrotic cells were observed whenB10.A lymphocytes were stimulated by their P. brasiliensis-acti-vated DCs.

Supernatants of the lymphoproliferation assay mixtures werecollected, and the presence of cytokines was measured by ELISA.The supernatants of B10.A cells presented increased levels ofIFN-�, whereas A/J cell supernatants showed increased levels ofIL-2 and IL-6. No differences in IL-4, TGF-�, and IL-10 produc-tion were observed (Fig. 4C).

Susceptible mice show an increased frequency of IFN-�� in-nate lymphocytes. To further define the phenotype of cells in-volved in IFN-� secretion in lymphoproliferation assays, P. brasil-iensis-activated DCs were cocultured with naïve lymphocytes andthe presence of intracellular IFN-� in several lymphocyte sub-populations was assessed by flow cytometry. Table 1 shows thatnaïve, nonstimulated B10.A cells presented increased frequenciesof NKT and � T cells expressing intracellular IFN-�. When stim-ulated by P. brasiliensis-pulsed DCs, the same lymphocyte subsets

FIG 2 Cytokine and nitric oxide production by DCs from resistant and sus-ceptible mice. Bone marrow-derived DCs from B10.A and A/J mice were invitro challenged for 48 h with P. brasiliensis yeasts, production of NO wasmeasured by the Griess reagent, and cytokines were determined by ELISA orquantitative PCR. (A and B) DCs from susceptible mice secrete high levels ofTNF-�, IL-12, IL-1-�, and IL-10, whereas DCs from resistant mice producelarge amounts of IL-6 and TGF-�. Data are representative of three experimentswith equivalent results. (C) DC supernatants were used to determine the levelsof nitrites. Data are means � SEMs of quintuplicate samples from one exper-iment representative of 3 independent determinations. (D) Quantitative PCRanalysis of TGB-� expression in DCs from A/J and B10.A mice stimulated withP. brasiliensis cells for 48 h. Amplified products were normalized to the amountof GAPDH products. Data are representative of two independent experimentsusing 3 mice per group and are expressed as means � SEMs. *, P � 0.05; **,P � 0.01; ***, P � 0.001.

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increased their expression of intracellular IFN-�. In contrast,IFN-�� CD4� and CD8� T cells were seen when A/J lymphocyteswere stimulated by A/J DCs. As a whole, innate immune cells(NKT and � ) appear to be the major IFN-� source in B10.Amice, while in A/J mice this activity was possibly dependent onCD4� and CD8� T cells.

DCs from A/J mice induce increased proliferation of effectorand regulatory T cells. P. brasiliensis-activated DCs from B10.Aand A/J mice were cultivated with homologous T lymphocytes fora period of 5 days. After cocultivation, the cells were removed andlabeled with specifics antibodies. The CD3� lymphocyte popula-tion was gated, and the expression of CD28, CD40L, CD25,CTLA-4, GITR, and CD44 on CD4� cells was evaluated. Whenactivated by P. brasiliensis-stimulated DCs, A/J lymphocytes pre-sented an increased frequency of CD4� T cells expressing activa-tion markers (CD4� CD28�, CD4� CD40L�, CD4� CD44�).Interestingly, the expression of other activation molecules which

are also used as Treg cell markers (CD4� CD25�, CD4� GITR�)was also seen at an increased frequency (Fig. 5A). In addition, A/Jcell cocultures expressed an increased percentage of CD8� CD28�

lymphocytes. With B10.A cells, a small amount of all T cell subsetswas seen, reflecting their poor lymphoproliferative activity andenhanced cell death (Fig. 5A). We also assessed the expression ofFoxP3, a transcription factor which determines the phenotype andactivity of Treg cells. As shown in Fig. 5B and C, DCs from resis-tant mice induced in A/J lymphocytes a higher frequency of CD4�

CD25� FoxP3� Treg cells than B10.A DCs. These data demon-strate that DCs of resistant mice are efficient inducers of lym-phoproliferative responses but also stimulate an increased prolif-eration of Treg cells.

In vivo, susceptible mice preferentially develop mDCs,whereas resistant mice develop increased numbers of pDCs. Wehave further asked if the main features detected in our in vitrostudies were also present during the in vivo infection of resistant

FIG 3 Intracellular cytokines and membrane TGF-� expressed by DCs. Bone marrow-derived DCs from B10.A and A/J mice were in vitro challenged for 48 hwith P. brasiliensis, and nonadherent CD11c� cells were isolated by magnetic microbeads. For intracellular cytokine staining, CD11c� DCs were restimulatedwith PMA and ionomycin for 6 h and subjected to intracellular staining for IL-12, TNF-�, IL-10, TGF-�, and membrane-bound TGF-� (LAP). The myeloid (A),plasmacytoid (B), and lymphoid (C) DC subpopulations were gated, and the expression of intracellular cytokines and LAP was determined from the MFI. Resultsare from one experiment and are representative of two independent experiments. The bars depict means � SEMs. *, P � 0.05 between strains.

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and susceptible mice. Thus, B10.A and A/J mice were infected i.t.with 1 million P. brasiliensis yeast cells, and at 96 h after infection,pulmonary DCs were purified from digested lungs using CD11c�

magnetic beads. DCs were then stained, and the expression ofsurface molecules was assessed by flow cytometry. Consistent withour in vitro findings, a high number of B10.A DCs showed aCD11c� CD11b� myeloid phenotype, while A/J cells were pre-dominantly CD11c� B220� or CD11c� PDCA positive(PDCA�), characterizing the plasmacytoid phenotype. DCs ex-pressing the CD8 marker were also seen in higher numbers in thelungs of A/J mice (Fig. 6A). In addition, higher numbers of pul-monary DCs from resistant mice were CD11c� I-Ak positive, in-dicating an efficient APC ability.

After in vitro cultivation, DCs were disrupted and the numberof viable P. brasiliensis yeasts was evaluated by CFU counts. Asseen in Fig. 6B, decreased numbers of yeast cells were measured inB10.A DCs, possibly reflecting their prevalent secretion of NO

FIG 4 Lymphoproliferative responses, cell death, and cytokines induced by DCs from resistant and susceptible mice. A/J and B10.A mouse naïve spleenlymphocytes were previously stained with the vital dye CFSE and then cultivated for 5 days with homologous DCs previously activated by P. brasiliensis yeasts (A).Cell division was determined by the decreased intensity of CFSE staining by flow cytometry. The gray peak represents the MFI of control lymphocytes. PI is theMFI of naïve T cells divided by the MFI of T cells cultured with mature DCs. (B) Cells from lymphoproliferation assays were removed and labeled withannexin-FITC and propidium iodide to determine by flow cytometry the frequency of apoptotic and necrotic cells. Fluorescent-activated cell sorter analyses wereperformed using gates for lymphocytes defined by FSC and SSC. Data are representative of two experiments with similar results. (C) DCs from A/J and B10.Amice previously activated with P. brasiliensis yeasts were cultured for 5 days with naïve homologous or heterologous splenic lymphocytes (lymphocyte/DC ratio,30:1). After 5 days, the culture supernatants were removed and levels of cytokines were measured by ELISA (3 to 5 wells per group). Data are representative of twoexperiments with similar results and are expressed as means � SEMs. *, P � 0.05; **, P � 0.01; ***, P � 0.001.

TABLE 1 Phenotypic characterization of IFN-�-producinglymphocytesa

Mouse group PI

Mean frequency (%) of positive cells � SEM

� NKTb T CD4 T CD8

Control A/J Ly 0.3 � 0.0 1.5 � 0.1 2.0 � 0.2 1.0 � 0.4Control B10.A Ly 2.2 � 0.4* 6.9 � 1.4* 1.7 � 0.1 0.9 � 0.2DC A/J � Ly A/J 4.2 1.3 � 0.5 3.5 � 0.0 7.5 � 0.3* 7.6 � 1.4*DC B10.A � Ly B10.A 1.1 7.0 � 3.2* 12.3 � 0.4*** 3.8 � 0.5 4.0 � 0.8a B10.A and A/J lymphocytes were cocultivated for 3 days with P. brasiliensis-activatedDCs. Intracellular IFN-� and the phenotype of IFN-�-positive cells were assessed byflow cytometry. For intracellular cytokine staining, DCs and splenocytes were cultivatedby 3 days; cells were then stimulated for 6 h in the presence of PMA, ionomycin, andbrefeldin A (eBioscience). Cells were incubated for 20 min at 4°C with MAbs to CD16/CD32 (Fc block) and stained for 30 min at 4°C with PE-Cy7 anti-CD4, Alexa Fluoranti-CD8�, FITC anti-CD3ε, PE NKT/NK, and PE � TCR. Cells were then fixed,permeabilized, and stained by PerCP-Cy5 anti-IFN-� antibodies (eBioscience). Data arerepresentative of two independent experiments with similar results. *, P � 0.05,compared with equivalent group of A/J mice; ***, P � 0.001.b NKT, IFN-�� cells in the double-positive NK1.1� CD3ε� gate.

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(Fig. 6C). As a whole, our ex vivo studies with pulmonary DCsrecapitulated the main findings detected in vitro with BMDCs.

Pulmonary DCs from resistant mice express high levels ofTNF-� and TGF-�, whereas B10.A DCs express elevated levelsof IL-12. Isolated DCs were cultivated for 48 h, and cytokines incell supernatants were measured by ELISA. Higher levels of IL-12were detected in the supernatants of B10.A DCs, whereas A/J DCssecreted higher levels of TGF-� and TNF-�. Equivalent concen-trations of IL-6, IL-1�, and IL-10 were observed in the superna-tants of both mouse strains (Fig. 7A). Isolated CD11c� cells wererestimulated in vitro with PMA and ionomycin, and the presenceof intracellular cytokines was characterized by flow cytometry.Increased numbers of IL-1��, TNF-�-positive (TNF-��), andTGF-�� DCs were detected in the lung cell preparations of A/Jmice. In contrast, B10.A mice presented increased numbers ofIL-10� and IL-12� DCs (Fig. 7B).

Resistant mice develop an increased influx of effector andregulatory T cells in the lungs. B10.A and A/J mice were infectedwith 1 � 106 P. brasiliensis yeast cells, and 2 weeks later their lungswere removed and the lymphocytes were isolated and phenotypedby flow cytometry. Compared with B10.A mice, increased num-bers of CD4� and CD8� naïve (CD4� CD44low CD62Lhigh andCD8� CD44low CD62Lhigh, respectively) as well as CD4� andCD8� effector (CD4� CD44high CD62Llow and CD8� CD44high

CD62Llow, respectively) T cells were detected in A/J mice (Fig. 8).When other activation markers of Treg/effector T cells were stud-ied, enhanced numbers of CD4� CD25�, CD4� CTLA-4�, andCD4� GITR� T cells were seen in the lungs of A/J mice. We fur-ther characterized Treg cells, determining the number of CD4�

CD25� T cells expressing FoxP3. Consistent with in vitro data, A/Jmice displayed increased numbers of Treg cells in their lungs(Fig. 8).

Resistant mice develop increased numbers of CD4� andCD8� T cells expressing IFN-�, IL-4, and IL-17, whereas suscep-tible mice show elevated numbers of IL-17� and IFN-�� �� Tcells. B10.A and A/J mice were infected with 1 � 106 P. brasiliensisyeast cells, and 2 weeks later their lungs were removed, the lym-phocytes were isolated, and the presence of intracellular cytokineswas characterized by flow cytometry. Compared with B10.A mice,A/J mice developed increased numbers of CD4� and CD8� T cellsexpressing IFN-�, IL-4, and IL-17, whereas susceptible mice showelevated numbers of IL-17� and IFN-�� � T cells. These datademonstrate that resistant mice develop mixed Th1/Th2/Th17adaptive immunity responses, while in B10.A mice, innate im-mune cells are mainly involved in IL-17 and IFN-� secretion(Fig. 9).

DISCUSSION

The present work was undertaken to better understand some as-pects of the innate and adaptive immunity of mice geneticallyresistant and susceptible to P. brasiliensis infection.

We confirmed our previous findings that early proinflamma-tory innate immunity leads to susceptibility, whereas an anti-in-flammatory (TGF-�-mediated) reaction counterbalanced by aproinflammatory response results in host resistance. Indeed, ourlab has previously demonstrated that the typical Th1/Th2 re-sponses do not explain the immunological mechanisms that con-fer resistance or susceptibility to P. brasiliensis infection.

Our previous data suggest that alternative mechanisms of in-nate immunity underlie the suppressed T cell immunity of B10.Amice and the delayed but protective immunity of A/J mice (24,26–30, 32, 39, 40). These data are in contrast to those from somestudies suggesting that the susceptibility of B10.A mice correlateswith a Th2-stimulatory activity of DCs, whereas the resistance ofA/J mice depends on the Th1-inducing ability of DCs (41–43)

The present investigation demonstrates that P. brasiliensis in-duces a preferential myeloid phenotype in DC precursors of sus-ceptible mice, whereas in resistant mice, high frequencies of plas-macytoid and myeloid DCs differentiate concomitantly. TGF-�was the main cytokine produced by A/J DCs, whereas elevatedlevels of proinflammatory cytokines (TNF-�, IL-12, IL-1�) andNO were preferentially secreted by mDCs from susceptible mice.The expression of costimulatory and activation molecules (CD80,CD86, CD40, and MHC class II) indicates that DCs from bothmouse strains undergo functional maturation. Despite the ele-

FIG 5 Phenotypic characterization of DC-activated lymphocytes. P. brasilien-sis-activated DCs from B10.A and A/J mice were cocultivated with homolo-gous lymphocytes for a period of 5 days. After this period, the cells were re-moved and labeled with specific antibodies, as described in Materials andMethods. (A) The CD4� or CD8� populations were separated by an electronicgate and analyzed for the expression of CD28, CD40L, CD25, CTLA-4, GITR,and CD44. The data represent the means � SEMs and are representative of twoindependent experiments. (B) Representative flow cytometric analysis of DC-induced Treg cells. For Treg (CD4� CD25� FoxP3�) cell characterization,cells were labeled with specific antibodies anti-CD4 (PE-Cy7) and anti-CD25(Alexa Fluor 488) and the intracellular marker Foxp3 (PE). The CD4� CD25�

population was separated by an electronic gate, and the frequency of theFoxp3� marker was determined. (C) Frequency of regulatory T cells inducedin vitro by B10.A and A/J DCs. Data represent the means � SEMs and arerepresentative of two independent experiments. *, P � 0.05; **, P � 0.01; ***,P � 0.001.

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vated levels of certain costimulatory molecules (CD80 and CD86),B10.A DCs did show efficient APC capabilities.

Characterization of intracellular cytokines expressed by differ-ent DC subsets confirmed the prevalent production of IL-12 bymDCs and pDCs of B10.A mice and the increased expression ofTGF-� by pDCs of A/J mice. Importantly, the elevated expressionof TGF-� mRNA and the increased presence of membrane-boundTGF-� on all DC subpopulations of A/J mice firmly suggest theinvolvement of this cytokine in the tolerogenic activity of A/J cells.Although the GM-CSF- and IL-4-differentiated DCs are not sim-ilar to the Fms-related tyrosine kinase 3 ligand (Flt3L)-inducedcells that resemble steady-state DCs of secondary lymphoid or-gans and other tissues (44), our in vitro-generated DCs can beviewed as monocyte-derived inflammatory DCs that respond totheir environment (e.g., PRR activation, cytokines) by differenti-ating into a variety of DC-like cells (45).

Consistent with our in vitro results, the phenotypic character-ization of DCs at an early phase of in vivo infection confirmed theprevalent myeloid phenotype of B10.A mouse DCs and the in-creased presence of plasmacytoid cells in the lungs of A/J mice.Moreover, pulmonary DCs from susceptible mice secreted highlevels of IL-12, whereas DCs from resistant mice produced ele-vated levels of TGF-� and TNF-�. Thus, these ex vivo data supportour main in vitro findings.

The behavior of B10.A mouse DCs characterized here is similarto that previously described for B10.A mouse alveolar macro-

phages (26). Thus, phagocytes from different tissues, at diversestages of maturation, exhibit similar behaviors. The secretion ofIL-12 indicates that B10.A DCs would induce a prevalent Th1pattern in naïve lymphocytes. However, this was not the case inour in vitro studies or in our in vivo model of infection. Indeed,during pulmonary infection, an early anergy in DTH responses(Th1, CD4� T lymphocytes) concomitant with an unexpectedelevated level of IFN-� in the lungs of infected B10.A mice waspreviously characterized (23, 32). These in vivo findings are con-sistent with the T cell anergy and elevated levels of IFN-� observedwhen naïve B10.A lymphocytes were stimulated in vitro by P.brasiliensis-activated DCs. Importantly, in vitro and in vivo studiesof intracellular IFN-� expression indicated that innate T lympho-cytes (primarily � T cells) from B10.A mice were the mainsources of this cytokine. The low levels of IL-2 and the increasedcell death of B10.A lymphocytes reported here could also contrib-ute to the suppressed T cell immunity observed in vitro and in vivo.This behavior led us to suppose that the increased synthesis ofIFN-� by innate immune cells could increase the expression ofNO by B10.A mDCs, enhancing their suppressive activity on Tlymphocytes (25, 26). Taken together, our findings suggest thatthe susceptibility of B10.A mice is likely due to the excessive pro-inflammatory activity of DCs and innate immune lymphocytesthat contribute to the control of initial fungal loads but suppress Tcell immunity, resulting in progressive disease and a fatal outcomefor infected mice.

FIG 6 Phenotype, fungicidal ability, and NO production by pulmonary DCs from infected resistant and susceptible mice. B10.A and A/J mice were infected i.t.with 1 � 106 yeast cells of P. brasiliensis, and at 96 h after infection, lung CD11c� DCs were purified using magnetic beads. DCs were labeled with specificantibodies, and cell phenotypes were analyzed by flow cytometry as described in Materials and Methods. (A) Myeloid (CD11c� CD11b�) DCs are prevalent inthe lungs of susceptible mice, whereas plasmacytoid (CD11c� B220� or CD11c� PDCA�) and lymphoid (CD11c� CD8�) DCs are prevalent in the lungs ofresistant mice. (B) After in vitro cultivation, A/J and B10.A DCs were ruptured by osmotic shock, the pellet was obtained, and the number of viable yeasts wasdetermined by a CFU assay. (C) NO levels were measured in DC supernatants. The data represent the means � SEMs of 5 to 7 mice per group and arerepresentative of two independent experiments. *, P � 0.05; **, P � 0.01; ***, P � 0.001.

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Plasmacytoid DCs are major players in host defense againstseveral types of pathogens. These cells are involved in Th cell dif-ferentiation, and depending on the cytokine milieu that they de-velop, they control immunity or tolerance (4, 38, 46). Interest-ingly, our in vitro model demonstrated that P. brasiliensisstimulation of immature DCs from resistant mice resulted in ahigh frequency of a plasmacytoid phenotype, as demonstrated bythe increased expression of B220 and PDCA-1 molecules on theirmembranes. These pDCs expressed high levels of intracellularTGF-� and membrane LAP, the inactive form of membrane-bound TGF-�, possibly playing an important role in the expan-sion of Treg cells and tolerance to fungal growth exhibited by A/Jmouse DCs. The decreased production of IL-12 could also be at-tributed to the presence of TGF-�, which was shown to inhibitIL-12 and reduce the stability of IL-12p40 mRNA (47). Interest-

ingly, pDCs were shown to play a substantial role in immunopro-tection against Aspergillus fumigatus infection, exerting an un-usual fungicidal activity following interaction with fungal hyphae(48). These cells secreted high levels of type I IFN, although an-other study using pDCs stimulated by Aspergillus fumigatus rest-ing conidia failed to detect this cytokine (49).

Despite the high level of TGF-� produced by A/J DCs and itsknown inhibitory effect on T cell activation (50), significant pro-liferation was observed when A/J DCs were cultured with homol-ogous naïve lymphocytes (PI � 4.0). This effect was most likelydue to the presence of immunogenic DC subsets, secretion ofactivating cytokines such as TNF-�, IL-6, and IL-1�, and the highlevel of expression of MHC class II and costimulatory moleculesby A/J DCs, providing efficient secondary signals for T cell activa-tion. This APC activity resulted in the increased proliferation of

FIG 7 Cytokines produced by pulmonary DCs from infected resistant and susceptible mice. B10.A and A/J mice were infected i.t. with 1 � 106 yeast cells of P.brasiliensis, and at 96 h after infection, lung CD11c� DCs were purified using magnetic beads. (A) DCs were cultured for 48 h at 37°C, and supernatants wereanalyzed for the presence of cytokines. (B) For intracellular cytokine staining, CD11c� DCs were restimulated with PMA and ionomycin for 6 h and subjectedto intracellular staining for cytokines. Results are from one experiment and are representative of two independent experiments. The bars depict means � SEMs.*, P � 0.05; **, P � 0.01; ***, P � 0.001.

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CD4� and CD8� T lymphocytes displaying activation/deactiva-tion markers on their membranes. Interestingly, the increased fre-quency of lymphocytes expressing activation molecules such asCD28, CD44, and CD40L was concomitant with the increasedexpression of CTLA-4, CD25, and GITR, late markers of T cellactivation that control excessive T cell activation and also markersof Treg cells (51, 52). Indeed, compared with B10.A DCs, A/J DCsinduced a greater proliferation of CD4� CD25� Foxp3-positive(Foxp3�) Treg cells when cocultured with naïve lymphocytes.

Importantly, our in vitro findings were validated, at least par-tially, by our in vivo studies. Indeed, at 96 h after infection, thelungs of A/J mice showed increased numbers of plasmacytoidDCs, and there was a consistent presence of TGF-�, as assessed byboth intracellular staining and supernatant assays. In addition tothe plasmacytoid DCs, a relevant number of myeloid and lym-phoid DCs was also detected at the site of infection, and this num-ber was associated with elevated levels of proinflammatory cyto-kines (TNF-�, IL-6, and IL-1�). This complex behavior ofpulmonary DCs appears to explain the low NO production, thepoor control of fungal growth, the increased presence of Foxp3�

Tregs, and the elevated numbers of IFN-�-, IL-4-, and IL-17-se-creting CD4� and CD8� T cells at the site of infection. Therefore,P. brasiliensis infection appears to induce the concomitant expan-sion of immunogenic and tolerogenic DCs in A/J mice, whichpromote the equilibrated expansion of effector Th1/Th2/Th17cells. These effector cells are then possibly controlled by increasednumbers of FoxP3� Treg cells. It is possible that the synergisticsuppressive effects of TGF-� secreted by alveolar macrophages(26), tolerogenic DCs, and Treg cells (53) contribute to the poorNO secretion and pathogen clearance of resistant mice at the ini-tial phase of P. brasiliensis infection (14, 29, 39). At the chronicphase, tightly controlled CD4� and CD8� T cell responses appearto be sufficient to restrain fungal growth without excessive inflam-mation and tissue damage. This interpretation is consistent withthe histopathology of the lungs and livers of A/J mice at 10 weekspostinfection (see Fig. S1 in the supplemental material). Intense,

nonorganized lesions containing high numbers of fungal cellswere observed in the lungs and liver of susceptible mice, whereasdiscrete inflammatory reactions presenting low numbers of yeastcells and preserved organ parenchyma were seen in resistant mice.

The immune response against fungal pathogens depends onthe cooperation between different DC subsets, which develop dif-ferent activation programs induced by diverse intracellular signal-ing following PRR activation. Inflammatory DCs usually induceTh2 and Th17 immunity, whereas tolerogenic DCs activate Th1and Treg cell differentiation (54). In our experimental model, DCsfrom resistant mice showed simultaneous tolerogenic and immu-nogenic behavior. This resulted in protective Th1/Th17 immunitythat was tightly regulated by Th2 and Treg cells. Importantly, weshow for the first time that genetic resistance to P. brasiliensisinfection is associated with Th17 and Tc17 immunity, and thisfinding is consistent with the increased production of IL-6 andTGF-� by A/J mouse DCs.

Our murine model demonstrates that susceptibility to a pul-monary fungal pathogen can, paradoxically, be associated withefficient mechanisms of innate immunity, whereas resistance canbe based on immune mechanisms that are initially inefficient inthe control of pathogen growth but evolve into complex effector Tcell responses that are tightly regulated by Treg cells. The unusualmechanism described in this and other studies (24, 26–30, 32, 39,40) demonstrates that conviviality with a slow-growing pathogencan be less dangerous to the host than an initial aggressive re-sponse that results in parasite killing but aggregates perniciousinflammation, tissue damage, and eventually, suppressed adaptiveimmunity. Indeed, with other important pulmonary pathogenssuch as Streptococcus pneumoniae and Mycobacterium tuberculosis,the early recruitment of TGF-�� anti-inflammatory DCs and reg-ulatory T cells to the infected lungs was associated with host resis-tance to pneumococcal pneumonia and tuberculosis, respectively(55, 56). This concept of host resistance mediated by a reductionor avoidance of damage caused by an infectious agent has beendescribed by plant ecologists (57). However, despite its impor-

FIG 8 Characterization of effector and regulatory T cells in the lungs of A/J and B10.A mice. Characterization of T cells by flow cytometry in the lung-infiltratingleukocytes (LIL) from A/J and B10.A mice inoculated i.t. with 1 � 106 P. brasiliensis yeast cells. At week 2 after infection, lung cell suspensions were obtained andstained as described in Materials and Methods. The acquisition and analysis gates were restricted to lymphocytes. The total numbers of effector (CD44high

CD62Llow) and naïve (CD44low CD62Lhigh) CD4� and CD8� T cells, as well as CD4� T cells expressing CD25, CTLA-4, and GITR, in the lungs of A/J and B10.Amice are presented. For characterization of Treg cells (CD4� CD25� Foxp3�), surface staining of CD25� and intracellular FoxP3 expression were back-gated onthe CD4� T cell population. The data represent the means � SEMs of the results from 5 to 7 mice per group and are representative of two independentexperiments. *, P � 0.05; **, P � 0.01.

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tance to a comprehensive view of host-pathogen interactions, thisconcept has rarely been applied to mammalian models of infec-tion (58, 59). Finally, our model describes a resistance mechanismthat can protect hosts without damaging the lung, an organ whosemain physiological function is substantially impaired by inflam-matory processes. Our data also contribute to the understandingof severe cases of fungal infections, such as those caused by Pneu-mocystis jirovecii or even P. brasiliensis, where successful therapycan be achieved only when an antibiotic is administered in com-bination with an anti-inflammatory drug such as a corticosteroid(60, 61).

ACKNOWLEDGMENTS

This work was supported by grants from the Fundação de Amparo àPesquisa do Estado de São Paulo (FAPESP) e Conselho Nacional de Pes-quisas (CNPq).

We are grateful to Márcio Y. Tomiyoshi and Tania A. Costa for invalu-able technical assistance.

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