IAI Accepts, published online ahead of print on 22 January 2013 Infect. Immun. doi:10.1128/IAI.00736-12 Copyright © 2013, American Society for Microbiology. All Rights Reserved.
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Myeloid dendritic cells (DCs) of susceptible mice to paracoccidioidomycosis
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suppress T cell responses whereas myeloid and plasmacytoid DCs from
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resistant mice induce effector and regulatory T cells
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Adriana Pina , Eliseu Frank de Araujo , Maíra Felonato , Flávio V. Loures , Claudia Feriotti
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Simone Bernardino , José Alexandre M. Barbuto and Vera L. G. Calich , #
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Paulo, São Paulo, SP, Brazil.
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Departamento de Imunologia, Instituto de Ciências Biomédicas, Universidade de São
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Keywords: Paracoccidioidomycosis, Dendritic cells, Innate immunity, Regulatory T cells
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Cytokines, T cell response
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Running Title: DCs and susceptibility to P.brasiliensis infection
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#- Corresponding author at: Departamento de Imunologia, Instituto de Ciências Biomédicas,
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Universidade de São Paulo, Av. Prof. Lineu Prestes 1730, CEP 05508-900, São Paulo, SP,
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Brazil. Tel.: +55 11 3091 7397; fax: + 55 11 3091 7224.
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E-mail address:
[email protected] (V.L.G. Calich)
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ABSTRACT
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The protective adaptive immune response in paracoccidioidomycosis, a human endemic
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mycosis, is mediated by T cell immunity whereas impaired T cell responses are associated
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with severe, progressive disease. The early host response to Paracoccidioides brasiliensis
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infection is not known since the disease is diagnosed at later phases of infection. Our
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laboratory established a murine model of infection where susceptible mice reproduce the
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severe disease while resistant mice develop a mild infection. This work aimed to characterize
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the influence of dendritic cells in the innate and adaptive immunity of susceptible and
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resistant mice. We verified that P. brasiliensis infection induced in bone marrow-derived DCs
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of susceptible mice a prevalent pro-inflammatory myeloid phenotype that secreted high levels
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of IL-12, TNF-α, and IL-β, whereas in resistant mice a mixed population of myeloid and
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plasmacytoid DCs, secreting pro-inflammatory cytokines and expressing elevated levels of
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secreted and membrane-bound TGF-β, was observed. In proliferation assays, the pro-
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inflammatory DCs from B10.A mice induced anergy of naïve T cells, whereas the mixed DC
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subsets from resistant mice induced the concomitant proliferation of effector and Treg cells.
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Equivalent results were observed during pulmonary infection. The susceptible mice displayed
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preferential expansion of pro-inflammatory myeloid DCs, resulting in impaired proliferation
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of effector T cells. Conversely, the resistant mice developed myeloid and plasmacytoid DCs
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that efficiently expanded IFN-γ+, IL-4+ and IL-17+ effector T cells associated with increased
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development of Tregs. Our work highlights the deleterious effect of excessive innate pro-
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inflammatory reactions and provides new evidence for the importance of immunomodulation
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during pulmonary paracoccidioidomycosis.
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INTRODUCTION
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Dendritic cells (DCs), which continuously survey their environment for invading
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microorganisms, are considered “professional APCs” due to their unique ability to activate T
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cells (5). During infection, the interaction of pattern recognition receptors (PRRs) on
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immature DCs with conserved molecular patterns of microorganisms (PAMPs) results in
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increased secretion of inflammatory mediators and enhanced expression of MHC and
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costimulatory molecules, characterizing their transition to the mature phenotype and efficient
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APCs. DCs also down-modulate their endocytic capacity and migrate to the T cell zone of
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draining lymph nodes, where they activate naïve antigen-specific T cells (4, 57). Three
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principal DCs subsets have been identified in mice: “conventional” myeloid DC (mDC;
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CD11c+ CD8α- CD11b+) plasmacytoid DC (pDC; CD11c interm. B220+ CD11b-), and lymphoid
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DC (CD11c+CD8+). All DC subsets exhibit immunostimulatory (21, 29) and tolerogenic
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functions that reflect their maturation condition at the time of T cell interaction (35, 63). The
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diverse DC subsets are characterized by a distinct pattern of PRR expression, including toll-
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like receptors (TLRs) and C-type lectin receptors (CLRs), which recognize diverse conserved
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pathogen structures (21, 38,47,54).
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DCs sense different morphotypes of fungal pathogens in a specific way, resulting in
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the expansion of distinct T-helper cells that can exert protective or deleterious effects on the
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host (54). T cell differentiation to Th1, Th2 or Th17 phenotypes depends on the innate
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receptor used by DCs to sense the fungal pathogen and the predominant cytokine that is
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subsequently produced. Whereas IL-12 is associated with Th1 differentiation, IL-23 enhances
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the Th17 phenotype and is induced by the concerted action of TGF-β and IL-6. The
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predominant synthesis of IL-10 or TGF-β, however, facilitates the differentiation and
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expansion of regulatory T cells (Treg), which control autoimmunity and excessive
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inflammatory reaction due to uncontrolled immune responses (7, 44, 47, 53,54).
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Paracoccidioides brasiliensis, a primary fungal pathogen from Latin America, infects
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individuals from endemic areas via the respiratory route. The initial interaction with alveolar
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macrophages appears to govern the subsequent disease outcome that can evolve as localized
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infection or overt disease (10, 30). Paracoccidioidomycosis (PCM) infection is usually
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asymptomatic and induces protective immunity, whereas the severity of disease is
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proportional to the impairment of the cellular immune response and the activation of humoral
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immunity. A Th2/Th3-skewed immunity has been correlated with severe forms of the disease,
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whereas mild forms are usually associated with Th1 immunity (8, 13,43). The regulatory
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mechanisms controlling protective or deleterious immune responses in PCM are not well
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defined, but the apoptosis of T cells, expression of CTLA4 (a T cell inhibitory molecule), and
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increased presence of regulatory T cells have been described in severe forms of PCM (11, 12,
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18)
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Our laboratory has established a pulmonary model of paracoccidioidomycosis. In
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this model, susceptible B10.A mice, despite the early pro-inflammatory response and control
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of fungal growth, develop T cell anergy and a fatal disseminated disease; in contrast, resistant
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A/J mice are initially permissive for fungal growth, but later develop positive DTH reactions
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and regressive disease (14, 17, 39, 45, 49, 50). Studies of the main T cell subsets involved in
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immunity to P. brasiliensis infection revealed a persistent CD4+ T cell anergy but a preserved
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CD8+ T cell response in susceptible B10.A mice. In contrast, the early unresponsiveness of
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resistant mice was followed by a protective response mediated by CD4+ and CD8+ T cells that
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secrete a mixed pattern of cytokines with a prevalence of IFN-γ (15, 19, 39).
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In human paracoccidioidomycosis, the innate phase of immunity has never been
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investigated because the disease is diagnosed during late phases of infection. We have,
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however, investigated some aspects of innate immunity developed by both resistant and
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susceptible mice (14) We verified that P. brasiliensis-infected alveolar macrophages of
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susceptible mice have a pro-inflammatory phenotype characterized by intense nitric oxide
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(NO) production, efficient fungal killing, and prevalent synthesis of IL-12. In contrast,
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alveolar macrophages from resistant mice show an anti-inflammatory behavior due to the
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prevalence of TGF-β secretion that impairs their NO secretion and fungicidal properties (49).
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Importantly, the early administration of IL-12 does not reverse the susceptibility of B10.A
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mice but induces an excessive pulmonary inflammation (1). IL-4 depletion, instead of
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abolishing the susceptibility of B10.A mice, induces a more severe disease (3). Moreover,
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early in infection, higher levels of IFN-γ were found in the lungs of B10.A but not A/J mice
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(16). Thus, several studies using our experimental model have demonstrated that the
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susceptibility of B10.A mice is most likely mediated by excessive pro-inflammatory activity
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of innate immune cells and not by an early Th2-skewed response, whereas the resistance of
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A/J mice comprises an early tolerance to fungal growth that subsequently evolves to a
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prevalent Th1 immunity (13, 14).
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Because dendritic cells (DCs) are the most important antigen-presenting cells and
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function as a bridge that links innate and adaptive immunity, we decided to analyze the
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behavior of these cells in our experimental model. Thus, we characterized the influence of
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P.brasiliensis infection on the phenotype and behavior of pulmonary and bone marrow-
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derived dendritic cells (BMDCs) of resistant and susceptible mice. We also investigated the
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antigen-presenting ability of DCs to naïve lymphocytes. We demonstrated that BMDCs from
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B10.A mice, stimulated by P. brasiliensis yeasts, differentiated to a predominantly pro-
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inflammatory myeloid phenotype, whereas myeloid and plasmacytoid subsets were detected 5
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with A/J mice cells. Both, myeloid and plasmacytoid DCs underwent functional maturation in
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response to P. brasiliensis infection but activated different programs of cytokine production.
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While the pro-inflammatory behavior of B10.A dendritic cells (DCs) is associated with T cell
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impairment, a common phenotype usually developed by this susceptible strain, the
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concomitant production of pro- and anti-inflammatory cytokines by A/J DCs results in the
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enhanced differentiation of IFN-γ+, IL-4+ and IL-17+ effector T cells associated with increased
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Treg development. These data help, in part, to explain the distinct immune responses of
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resistant and susceptible mice to Paracoccidioides brasiliensis infection. Furthermore, our
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work demonstrates that excessive pro-inflammatory innate responses are deleterious to the
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adaptive response against pulmonary paracoccidioidomycosis, and immunoprotection is
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achieved when DCs induce well-balanced pro- and anti-inflammatory responses.
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MATERIALS AND METHODS
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Mice. Susceptible (B10.A) and resistant (A/J) mouse strains to P. brasiliensis infection were
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obtained from our Isogenic Breeding Unit (Departamento de Imunologia, Instituto de Ciências
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Biomédicas, Universidade de São Paulo, Brazil) and used at 8 to 11 weeks of age. SPF mice
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were fed sterilized laboratory chow and water ad libitum. The experiments were approved by
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the ethics committee on animal experiments of the Institute of Biomedical Sciences of
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University of São Paulo (Proc.76/04/CEEA).
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Fungus. P. brasiliensis 18, a highly virulent isolate (40) was used throughout this
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investigation. P. brasiliensis18 yeast cells were maintained by weekly subcultivation in
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semisolid Fava Netto’s culture medium (23) at 35°C and used on day 7 after culture. The
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yeast cells were washed in phosphate-buffered saline (pH 7.2) and adjusted to 5 x 104 cells/ml 6
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based on hemocytometer counts. Viability was determined with Janus Green B vital dye
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(Merck, Darmstadt, Germany) and was always higher than 80%. All solutions used to prepare
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yeast cell suspensions and DCs cultivation were tested for the presence of LPS using the
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Limulus amoebocyte lysate chromogenic assay (Sigma) and always showed LPS levels
85% CD11c+ cells. For intracellular cytokine staining, DCs (1x106 cells/mL)
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were stimulated for 6h in the presence of 50 ng/ml PMA (phorbol 12-myristate 13-acetate),
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500 ng/ml ionomycin (both from Sigma-Aldrich) and brefeldin-A (eBioscience). Cells were
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incubated for 20 min at 4°C with monoclonal antibodies (mAbs) to CD16/CD32 (FcγR II/III,
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2·4G2; Fc block) and stained for 30 min at 4° with PE-Cy7 anti-CD11c (clone RM4-5, e-
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Bioscience), Pacific blue anti-CD11b (M1-70, e-Bioscience), APC anti CD45-B220 (RA3-
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6B2, e-Bioscience), Alexa Fluor anti-CD8α (clone 53-6.7, BioLegend), PERCY anti-hLAP –
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TGF-β1, FAB2463C – R&D Systems) and cells were then fixed, permeabilized, and stained
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by PE anti-hTGF-β1 (IC240P, R&D Systems), PE anti-IL-12 (554479, R&D Systems), PE
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anti-IL-10 (JeS5-16E3, BioLegend ), PE anti-TNF-α (554419, R&D Systems). Samples were
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washed twice with Perm-buffer and analyzed immediately by flow cytometry.
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Lymphocyte phenotypes and intracellular cytokines. After lymphocyte proliferation
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assays, two- or three-color flow cytometry was used to measure the expression of surface
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molecules and intracellular FoxP3 or IFN-γ. For surface molecules, PE-Cy7 anti-CD4 (clone
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RM4-5, e Bioscience), Alexa Fluor anti-CD8α (clone 53-6-7, BioLegend), FITC anti-CD3ε
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(17A2, BD Pharmingen), and PE anti-CD28 (37.51), CD40L (MR1), CD44 (IM7), CD25
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(3C7), CTLA4 (UC10-4F10-11), GITR (DTA-1), NK-T/NK (U5A2-13), γδ TCR (GL3) from
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BD Pharmingen were used. The stained cells were analyzed immediately on a FACScan
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equipment, gating on lymphocytes as judged from forward and side light scatter. For flow
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cytometric analysis of apoptotic and necrotic lymphocytes, annexin V and propidium iodide
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labeling was used (62). For intracellular cytokine staining, DCs and T lymphocytes were
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cultivated by 3 days; cells were then stimulated for 6h in complete medium in the presence of
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50 ng/ml phorbol 12-myristate 13-acetate, 500 ng/ml ionomycin (both from Sigma-Aldrich)
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and brefeldin A (eBioscience). Treg cells were characterized by intracellular staining for
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FoxP3, using the Treg staining kit of BD Bioscience. Surface staining of CD25+ and
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intracellular FoxP3 expression were back-gated on the CD4+ T cell population. To measure
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the expression of intracellular IFN-γ, after staining of surface molecules, cells were fixed,
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permeabilized and stained by PerCP-Cy5 anti-IFN-γ antibodies (eBioscience). The cell
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surface expression of leukocyte markers as well as intracellular expression of FoxP3 and IFN-
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γ in stimulated lymphocytes were analyzed in a FACScalibur flow cytometer (BD
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Pharmingen) using the FlowJo software (Tree Star, Inc., Ashland,OR, USA).
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Detection of NO and cytokines in culture supernatants of DCs. Bone marrow DCs
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were challenged with P. brasiliensis yeasts and culture supernatants were harvested 48 h later.
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Supernatants were tested for NO by using the Griess reaction. Briefly, 50 µl of the
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supernatant were incubated with 50 µl of Griess reagent for 5 min at room temperature, and
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the NO2 concentration was determined by measuring the absorbance at 550 nm in reference to
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a standard NaNO2 solution. The levels of IL-12, TNF-α, IFN-γ, IL-10, IL-6, IL-1β, and TGF-
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β, were measured by capture enzyme-linked immunosorbent assay (ELISA) with antibody
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pairs purchased from PharMingen. Latent plus active TGF-β was measured using
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commercially available kits from R&D Systems. Some cytokines were also measured in
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culture supernatants from lymphoproliferation assays. The ELISA procedure was performed
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according to the manufacturer’s protocol, and absorbencies were measured with Versa Max
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Microplate Reader (Molecular Devices). The concentrations of cytokines were determined
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with reference to a standard curve for serial twofold dilutions of murine recombinant
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cytokines.
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Quantitative analysis of TGF-β mRNA expression. RNA was extracted from
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P.brasiliensis-infected DCs from B10.A and A/J mice using Trizol reagent (Invitrogen).
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cDNA was synthesized from 2μg RNA using High Capacity RNA-to-cDNA kit (Applied
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Biosystems) according to manufacturer’s instructions. TGF-β mRNA expression was
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quantified relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using assay-on-
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demand primers and probes, Taqman Universal Master Mix, and ABI Prism 7000 apparatus
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(Applied Biosystems).
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Cell proliferation. DC preparations were used to study their stimulatory activity to naïve
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lymphocytes previously stained with the vital dye 5,6-carboxy-succinimidyl-fluorescein-ester
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(CSFE; Molecular Probes, Eugene, OR), as described (42). To obtain lymphocytes, spleen
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cells from normal mice were collected, red blood cell lysed, cells washed and then plated onto
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12 wells plates for 2 hours at 37°C. Non adherent cells were collected, and T cells were
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obtained by positive selection using anti-CD90 (Thy-1) magnetic microbeads (Miltenyi,
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Biotec, Surry, UK). The cells were washed, labeled with 5 mM CSFE and suspended in
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RPMI-1640 complete medium containing 10% FCS. P. brasiliensis-stimulated DCs (1 x 104)
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were cultured during 5 days with CFSE-stained naïve spleen lymphocytes (3 x 105) at
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lymphocytes:DC ratio of 30:1. Dead cells were discriminated by propidium iodide uptake
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and cell division was determined by a decrease in the intensity of CFSE staining by flow
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cytometry. As additional positive control, CFSE-labeled naïve lymphocytes were treated with
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5.0 μg/ml of Concanavalin A (Sigma). A minimum of 50,000 events were acquired on a
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FACScalibur flow cytometer using Cell-Quest software (BD Pharmingen). The proliferation
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index (PI) was calculated as the mean fluorescence intensity (MFI) of unstimulated naïve T
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cells/ T cells cultured with mature DCs.
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The proliferation assays were also performed with unlabeled naïve splenic T
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lymphocytes (3 x 105 ) stimulated by P. brasiliensis-pulsed DCs (1 x 104) to characterize the
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phenotype of proliferating cells. Two or three color flow cytometry was performed to
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measure the expression of surface and intracellular molecules as above described.
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In vivo infection. B10.A and A/J mice were anesthetized and submitted to intratracheal
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(i.t.) P. brasiliensis infection as previously described (17). Briefly, after intraperitoneal
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anesthesia, the mice were i.t. infected with 1 x 106 P. brasiliensis yeasts cells, contained in 50
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µl of fungus. The number of CFUs was determined using colony plate counts (16).
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Isolation of pulmonary DCs and lymphocytes. B10.A and A/J mice were infected i.t.
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with one million yeast cells of P. brasiliensis; after 96 h, lungs were removed and digested
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enzymatically for 30 minutes with collagenase (1 mg/ml) and DNase (30 μg/ml) in culture
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medium (Sigma). Large particulate matter was removed by passing the cell suspension
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through a small, loose, nylon wool plug. DCs were purified by magnetic cell sorting with
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microbeads (Miltenyi Biotec) conjugated to hamster anti-mouse CD11c monoclonal
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antibodies. Positively selected DCs contained more than 90% CD11c+ cells. Cell-surface
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markers were characterized by flow cytometry as described for in vitro-derived DCs. Purified
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DCs were also cultured for 48 h at 37oC and supernatants used to quantify some pro- and anti-
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inflammatory cytokines (IL-12, IL-1β, TNF-α, IL-6, IL-10 and TGF-β). In some experiments,
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DCs were re-stimulated with PMA/ionomycin and subjected to intracellular staining as
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described above.
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Lungs of P. brasiliensis infected B10.A and A/J mice were obtained at 2 weeks of
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infection and digested enzymatically as previously described. Lung cell suspensions were
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centrifuged in presence of 20% percoll (Sigma) to separate leukocytes from cell debris. Total
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lung leukocyte numbers were assessed in the presence of trypan blue using a hemocytometer;
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viability was >85%. The number and phenotype of CD4+, CD8+ and regulatory T cells as well
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as the presence of intracellular cytokines were determined by flow cytometry as described
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above.
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Statistical analysis. Data were analyzed by the Student's t test. The software program
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GraphPad Prism version 4.0 (San Diego, CA, USA) was used for all statistical tests. P values
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under 0.05 were considered significant.
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RESULTS P. brasiliensis infection induces a prevalent myeloid phenotype in BMDCs of
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susceptible mice, while in resistant mice a mixed pattern of myeloid and plasmacytoid
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DCs were expanded. Bone-marrow derived cells from B10.A and A/J mice were cultured
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with GM-CSF and IL-4 to induce DC differentiation, and at day 5 the cells were challenged in
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vitro with P. brasiliensis yeasts during 48h. Cells were then analyzed according to their FSC
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and SSC characteristics by flow cytometry as well as for their expression of CD11c, CD11b,
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B220 and CD8α. Unstimulated DCs were analyzed and no differences of CD11c+ CD11b+,
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CD11c+B220+ and CD11c+CD8+ between immature B10.A and A/J DCs were found.
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Activation by P. brasiliensis challenge, however, resulted in increased in frequency of
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myeloid CD11c+CD11b+ DCs in B10.A mice, whereas a concomitant differentiation of
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plasmacytoid (CD11c+B220+) and myeloid (CD11c+CD11b+) DCs were observed in A/J
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mice (Fig. 1A). No differences in the frequency of lymphoid DCs (CD11c+ CD8+) were
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observed between the mouse strains. Importantly, a significantly increased expression of
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mPDCA was observed in CD11c+B220+ gated cells from A/J mice in comparison with B10.A
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mice (MFI 218 ± 17 X 80 ± 12; p< 0.05) supporting the increased differentiation of pDCs by
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resistant mice. The frequency (%) of cells and intensity of expression (MFI) of some
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activation markers and costimulatory molecules were also assessed in the three DC subsets to
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evaluate their maturation. Compared with A/J cells, B10.A DCs expressed higher levels of
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CD80 in the myeloid and plasmacytoid DCs, and CD86 in the lymphoid subset of DCs. In
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contrast, A/J mice expressed increased levels of CD86 and CD40 in their myeloid and
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lymphoid DCs subsets, respectively (Fig. 1B, C, D). The frequency of positive cells for
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costimulatory molecules was similar to the MFI data here presented (data not shown). In
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conclusion, P.brasiliensis infection induces in resistant and susceptible mice the expansion of
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different DC subsets showing small differences in their maturation degrees.
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DCs from resistant and susceptible mice secrete different patterns of cytokines when
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activated by P. brasiliensis yeasts. Immature DCs from B10.A and A/J mice were in vitro
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challenged with P. brasiliensis yeasts for 48 h and the levels of cytokines and nitric oxide
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(NO) were determined in culture supernatants using ELISA and Griess reaction, respectively.
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P. brasiliensis-stimulated DCs from susceptible mice secreted increased levels of TNF-α, IL-
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12, IL-1β and IL-10 whereas DCs from resistant mice produce high concentrations of TGF-β
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and IL-6 (Fig. 2A, B). Although in lower levels than those secreted by B10.A cells, A/J DCs
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were also able to produce TNF-α, IL-12, IL-1β, and IL-10. Because pDCs secrete large
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amounts of type I IFN in response to viral infections (21, 61), we have also assessed the
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presence of IFN-α in the supernatants of P.brasiliensis stimulated DCs. However, no IFN-
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α was detected in DCs supernatants of both mouse strains.
305 306
We further analyzed by quantitative PCR the expression of TGF-β mRNA in B10.A and A/J DCs. Confirming the elevated levels of protein observed (Fig. 2B), increased expression
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of TGF-β mRNA was detected in A/J DCs (Figure 2D). The more pro-inflammatory behavior
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of B10.A DCs was confirmed by the increased levels of NO they secreted (Fig. 2C).
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Resistant mice develop a high frequency of TGF-β+ and LAP+ plasmacytoid DCs
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while susceptible mice develop a high frequency of IL-12+ myeloid and plasmacytoid
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DCs.
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BMDCs from B10.A and A/J mice were in vitro challenged during 48 h with P.brasiliensis,
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and non-adherent CD11c+ cells isolated by magnetic microbeads. For intracellular cytokine
314
staining, CD11c+ DCs were restimulated with PMA/ionomycin for 6h and subjected to
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intracellular staining for IL-12, TNF-α, IL-10, TGF-β and membrane-bound TGF-β (LAP,
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latency associated peptide). The myeloid, plasmacytoid and lymphoid DC subpopulations
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were gated, and the expression of intracellular cytokines and membrane LAP determined by
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median fluorescence intensity (MFI). Compared with A/J cells, increased expression of IL-12
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by mDCs and pDCs was detected in B10.A cells (Figure 3A). Myeloid DCs of susceptible
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mice also presented increased expression of another proinflammatory cytokine, TNF-α, (Fig.
321
3A). Importantly, high levels of TGF-β was seen in A/J pDCs (Fig. 3B), while membrane
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TGF-β (LAP) was expressed in higher intensity by all three DC subsets of resistant mice (Fig.
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3A-C).
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The poor proliferative response of naïve lymphocytes induced by B10.A DCs was
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associated with increased cell death and IFN-γ production. We further asked if the DCs of
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resistant and susceptible had the same immunogenic or tolerogenic activity when co-
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cultivated with homologous naïve lymphocytes. DCs from B10.A and A/J mice were
328
stimulated by P. brasiliensis yeasts, and further incubated for a period of 5 days at 37°C in
329
5% CO2 with CFSE (5mM)-labeled naïve T lymphocytes. After co-cultivation, the cells were
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removed and analyzed by flow cytometry. The decrease of fluorescence corresponds to the
331
proliferative activity of lymphocytes. P.brasiliensis-activated DCs of A/J were able to induce
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332
intense proliferative response of naïve A/J lymphocytes (Proliferation Index, PI = 4.0) but
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totally impaired lymphocyte proliferation was detected when B10.A DCs were used (PI = 1.1)
334
(Fig. 4A).
335
We have also assessed the number of apoptotic and necrotic cells in the
336
lymphoproliferation assays. As depicted in Figure 4B, increased numbers of apoptotic and
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necrotic cells were observed when B10.A lymphocytes were stimulated by their
338
P.brasiliensis-activated DCs.
339
Supernatants of lymphoproliferation assays were collected and the presence of cytokines
340
was measured by ELISA. The supernatants of B10.A cells presented increased levels of IFN-γ
341
whereas A/J supernatants showed increased levels of IL-2 and IL-6. No differences were
342
observed in IL-4, TGF−β and IL-10 production (Fig. 4C).
343
Susceptible mice show an increased frequency of IFN-γ+ innate lymphocytes. To
344
further define the phenotype of cells involved in IFN-γ secretion in lymphoproliferation
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assays, P.brasiliensis-activated DCs were co-cultured with naïve lymphocytes and the
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presence of intracellular IFN-γ in several lymphocyte subpopulations was assessed by flow
347
cytometry. Table 1 shows that naïve, non-stimulated B10.A cells presented increased
348
frequency of NKT and γδ T cells expressing intracellular IFN-γ. When stimulated by
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P.brasiliensis pulsed DCs, the same lymphocyte subsets increased their expression of
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intracellular IFN-γ. In contrast, IFN-γ+ CD4+ and CD8+ T cells were seen when A/J
351
lymphocytes were stimulated by their DCs. As a whole, innate immune cells (NKT and γδ Τ)
352
appear to be the major IFN-γ source in B10.A mice while in A/J mice this activity was
353
possibly dependent on CD4+ and CD8+ T cells.
354
DCs from A/J mice induce increased proliferation of effector and regulatory T cells.
355
P.brasiliensis-activated DCs from B10.A and A/J mice were cultivated with homologous T
356
lymphocytes for a period of 5 days. After co-cultivation, the cells were removed and labeled 15
357
with specifics antibodies. The CD3+ lymphocyte population was gated and the expression
358
CD28, CD40L, CD25, CTLA-4, GITR and CD44 on CD4+ cells was evaluated. When
359
activated by P. brasiliensis-stimulated DCs, A/J lymphocytes presented increased frequency
360
of CD4+ T cells expressing activation markers (CD4+CD28+, CD4+CD40L+, CD4+CD44+).
361
Interestingly, the expression of other activation molecules which are also used as Treg cells
362
markers (CD4+CD25+, CD4+GITR+) were also seen in increased frequency (Fig. 5A). In
363
addition, A/J co-cultures expressed an increased percentage of CD8+CD28+ lymphocytes.
364
With B10.A cells a small frequency of all T cell subsets was seen, reflecting their poor
365
lymphoproliferative activity and enhanced cell death (Fig. 5A). We have also assessed the
366
expression of FoxP3, a transcription factor which determines the phenotype and activity of
367
Treg cells. As shown in Figure 5 B-C, DCs from resistant mice induced in A/J lymphocytes a
368
higher frequency of CD4+CD25+FoxP3+ Treg cells than B10.A DCs. These data demonstrated
369
that DCs of resistant mice are efficient inducers of lymphoproliferative responses but also
370
stimulate an increased proliferation of Treg cells.
371
In vivo, susceptible mice preferentially develop mDCs whereas resistant mice
372
develop increased numbers of pDCs. We have further asked if the main features detected in
373
our in vitro studies, were also present during the in vivo infection of resistant and susceptible
374
mice. Thus, B10.A and A/J mice were infected i.t. with one million P.brasiliensis yeasts, and
375
96 hs after infection pulmonary DCs were purified from digested lungs using CD11c+
376
magnetic beads. DCs were then stained and the expression of surface molecules was assessed
377
by flow cytometry. Consistent with our in vitro findings, a high number of B10.A DCs
378
showed a CD11c+CD11b+ myeloid phenotype while A/J cells were predominantly
379
CD11c+B220+ or CD11c+PDCA+, characterizing the plasmacytoid phenotype DCs expressing
380
the CD8 marker were also seen in higher number in the lungs of A/J mice (Fig. 6A). In
16
381
addition, a higher number of pulmonary DCs from resistant mice was CD11c+ IAK+,
382
indicating an efficient APC ability.
383
After in vitro cultivation, DCs were disrupted and the number of viable P.brasiliensis
384
yeasts evaluated by CFU counts. As seen in Figure 6B, a decreased number of yeast cells was
385
measured in B10.A DCs, possibly reflecting their prevalent secretion of NO (Fig. 6C). As a
386
whole, our ex-vivo studies with pulmonary DCs recapitulated the main findings detected in
387
vitro with BMDCs.
388
Pulmonary DCs from resistant mice express high levels of TNF-α and TGF-β whereas
389
B10.A DCs express elevated levels of IL-12.
390
Isolated DCs were cultivated for 48 h and cytokines in cell supernatants measured by
391
ELISA. Higher levels of IL-12 were detected in the supernatants of B10.A DCs, whereas A/J
392
DCs secreted higher levels of TGF-β and TNFα. Equivalent concentrations of IL-6, IL-1β
393
and IL-10 were observed in the supernatants of both mouse strains (Fig. 7A). Isolated CD11c+
394
cells were re-stimulated in vitro with PMA/ionomycin and the presence of intracellular
395
cytokines characterized by flow cytometry. Increased numbers of IL-1β+, TNF-α+ and TGF-
396
β+ DCs were detected in the lung cell preparations of A/J mice. In contrast, B10.A mice
397
presented increased numbers of IL-10+ and IL-12+ DCs (Fig. 7B).
398
Resistant mice develop an increased influx of effector and regulatory T cells in the
399
lungs. B10.A and A/J mice were infected with 1 x 106 P.brasiliensis yeasts and 2 weeks later
400
their lungs were removed, the lymphocytes isolated and phenotyped by flow cytometry.
401
Compared with B10.A mice, increased numbers of CD4+ and CD8+ naïve
402
(CD4+CD44lowCD62Lhigh and CD8+CD44lowCD62Lhigh, respectively) as well as CD4+ and
403
CD8+ effector (CD4+CD44highCD62Llow and CD8+CD44highCD62Llow) T cells were detected
404
in A/J mice (Fig. 8). When other activation markers of Treg/effector T cells were studied,
405
enhanced numbers of CD4+CD25+, CD4+ CTLA4+ and CD4+GITR+ T cells were seen in the
17
406
lungs of A/J mice. We further characterized Treg cells, determining the number of
407
CD4+CD25+ T cells expressing FoxP3. Consistent with in vitro data, A/J mice displayed
408
increased numbers of Treg cells in their lungs (Fig. 8).
409
Resistant mice develop increased numbers of CD4+ and CD8+ T cells expressing
410
IFN-γ, IL-4 and IL-17, whereas susceptible mice show elevated numbers of IL-17+ and
411
IFN-γ+ γδ T cells. B10.A and A/J mice were infected with 1 x 106 P.brasiliensis yeasts and 2
412
weeks later their lungs were removed, the lymphocytes isolated and the presence of
413
intracellular cytokines characterized by flow cytometry. Compared with B10.A mice, A/J
414
mice developed increased numbers of CD4+ and CD8+ T cells expressing IFN-γ, IL-4 and IL-
415
17, whereas susceptible mice show elevated numbers of IL-17+ and IFN-γ+ γδ T cells. These
416
data demonstrate that resistant mice develop mixed Th1/Th2/Th17 adaptive immunity
417
responses, while in B10.A mice innate immune cells are mainly involved in IL-17 and IFN-γ+
418
secretion (Fig. 9).
419 420 421
DISCUSSION The present work was undertaken to better understand some aspects of the innate and
422
adaptive immunity of mice genetically resistant and susceptible to P.brasiliensis infection.
423
We confirmed our previous findings that early pro-inflammatory innate immunity leads to
424
susceptibility, whereas an anti-inflammatory (TGF-β-mediated) reaction counterbalanced by a
425
pro-inflammatory response results in host resistance. Indeed, our lab has previously
426
demonstrated that the typical Th1/Th2 responses do not explain the immunological
427
mechanisms that confer resistance or susceptibility to P. brasiliensis infection.
428
Our previous data suggest that alternative mechanisms of innate immunity underlie the
429
suppressed T cell immunity of B10.A and the delayed but protective immunity of A/J mice (1,
430
2, 15, 16, 19, 24, 39, 49, 50). These data are in contrast with some studies suggesting that
18
431
susceptibility of B10.A mice correlates with a Th2-stimulatory activity of DCs whereas
432
resistance of A/J mice depends on the Th1-inducing ability of DCs (26-28)
433
The present investigation demonstrates that P.brasiliensis induces a preferential
434
myeloid phenotype in DCs precursors of susceptible mice, whereas in resistant mice a high
435
frequency of plasmacytoid and myeloid DCs differentiate concomitantly. TGF-β was the
436
main cytokine produced by A/J DCs, whereas elevated levels of pro-inflammatory cytokines
437
(TNF-α, IL-12, IL-1β) and NO were preferentially secreted by mDCs from susceptible mice.
438
The expression of costimulatory and activation molecules (CD80, CD86, CD40 and MHC
439
class II) indicates that DCs from both mouse strains undergo functional maturation. Despite
440
the elevated levels of certain costimulatory molecules (CD80 and CD86), B10.A DCs did
441
show efficient APC capabilities.
442
Characterization of intracellular cytokines expressed by different DCs subsets
443
confirmed the prevalent production of IL-12 by mDCs and pDCs of B10.A mice and the
444
increased expression of TGF-β by pDCs of A/J mice. Importantly, the elevated expression of
445
TGF-β mRNA and the increased presence of membrane-bound TGF-β on all DC
446
subpopulations of A/J mice firmly suggest the involvement of this cytokine in the tolerogenic
447
activity of A/J cells. Although the GM-CSF- and IL-4-differentiated DCs are not similar the
448
Flt3L (Fms-related tyrosine kinase 3 ligand)-induced cells that resemble steady state DCs of
449
secondary lymphoid organs and other tissues (31), our in vitro generated DCs can be viewed
450
as monocyte-derived inflammatory DCs that respond to their environment (e.g.: PRRs
451
activation, cytokines, and etc) by differentiating into a variety of DCs-like cells (59).
452
Consistent with our in vitro results, the phenotypic characterization of DCs at an early
453
phase of in vivo infection confirmed the prevalent myeloid phenotype of B10.A DCs and the
454
increased presence of plasmacytoid cells in the lungs of A/J mice. Moreover, pulmonary DCs
455
from susceptible mice secreted high levels of IL-12, whereas DCs from resistant mice
19
456
produced elevated levels of TGF-β and TNF-α. Thus, these ex vivo data support our main in
457
vitro findings.
458
The behavior of B10.A DCs here characterized is similar to that previously described
459
for their alveolar macrophages (49). Thus, phagocytes from different tissues, at diverse stages
460
of maturation, exhibit similar behaviors. The secretion of IL-12 indicates that B10.A DCs
461
would induce a prevalent Th1 pattern in naïve lymphocytes. However, this was not the case in
462
our in vitro studies or in our in vivo model of infection. Indeed, during pulmonary infection,
463
an early anergy in DTH responses (Th1, CD4+ T lymphocytes), concomitant with an
464
unexpected elevated level of IFN-γ in the lungs of infected B10.A mice was previously
465
characterized (16, 17). These in vivo findings are consistent with the T cell anergy and
466
elevated levels IFN-γ observed when naïve B10.A lymphocytes were stimulated in vitro by
467
P.brasiliensis activated DCs. Importantly, in vitro and in vivo studies of intracellular IFN-γ
468
expression indicated that innate T lymphocytes (primarily γδ T cells) from B10.mice were the
469
main sources of this cytokine. The low levels of IL-2 and the increased cell death of B10.A
470
lymphocytes here reported could also contribute to the suppressed T cell immunity observed
471
in vitro and vivo. This behavior led us to suppose that the increased synthesis of IFN-γ by
472
innate immune cells could increase the expression of NO by B10.A mDCs, enhancing their
473
suppressive activity on T lymphocytes (45, 49). Taken together, our findings suggest that the
474
susceptibility of B10.A mice is likely due to the excessive pro-inflammatory activity of DCs
475
and innate immunity lymphocytes that contribute to the control of initial fungal loads but
476
suppress T cell immunity, resulting in progressive disease and a fatal outcome of infected
477
mice.
478
Plasmacytoid DCs are major players in host defense against several types of
479
pathogens. These cells are involved in Th cells differentiation and, depending on the cytokine
480
milieu they develop, they control immunity or tolerance (21, 33, 61). Interestingly, our in
20
481
vitro model demonstrated that P.brasiliensis stimulation of immature DCs from resistant mice
482
resulted in a high frequency of a plasmacytoid phenotype, as demonstrated by the increased
483
expression of B220 and PDCA-1 molecules on their membranes. These pDCs expressed high
484
levels of intracellular TGF-β and membrane LAP, the inactive form of membrane-bound
485
TGF-β, possibly playing an important role in the expansion of Treg cells and tolerance to
486
fungal growth exhibited by A/J DCs. The decreased production of IL-12 could also be
487
attributed to the presence of TGF-β, which was shown to inhibit IL-12 and reduce the stability
488
of IL-12p40 mRNA (22). Interestingly, pDCs were shown to play a substantial
489
immunoprotection against Aspergillus fumigatus infection, exerting an unusual fungicidal
490
activity following interaction with fungal hyphae (52). These cells secreted high levels of type
491
I IFN, although another study using pDCs stimulated by A. fumigatus resting conidia failed to
492
detect this cytokine (55).
493
Despite the high level of TGF-β produced by A/J DCs and its known inhibitory effect
494
on T cell activation (32), a significant proliferation was observed when A/J DCs were
495
cultured with homologous naive lymphocytes (PI= 4.0). This effect was most likely due to the
496
presence of immunogenic DC subsets, secretion of activating cytokines such as TNF-α, IL-6
497
and IL-1β and high expression of MHC class II and costimulatory molecules by A/J DCs,
498
providing efficient secondary signals for T cell activation. This APC activity resulted in an
499
increased proliferation of CD4+ and CD8+ T lymphocytes displaying activation/deactivation
500
markers on their membranes. Interestingly, the increased frequency of lymphocytes
501
expressing activation molecules such as CD28, CD44, and CD40L was concomitant with
502
increased expression of CTLA4, CD25, and GITR, late markers of T cell activation that
503
control excessive T cell activation, and also markers of Treg cells (6, 48). Indeed, compared
504
with B10.A DCs, A/J DCs induced a greater proliferation of CD4+CD25+Foxp3+ Treg cells
505
when co-cultured with naïve lymphocytes.
21
506
Importantly, our in vitro findings were validated, at least partially, by our in vivo
507
studies. Indeed, 96 h after infection, the lungs of A/J mice showed increased numbers of
508
plasmacytoid DCs, and there was a consistent presence of TGF-β, as assessed by both
509
intracellular staining and supernatant assays. In addition to the plasmacytoid DCs, a relevant
510
number of myeloid and lymphoid DCs was also detected at the site of infection, and this
511
number was associated with elevated levels of pro-inflammatory (TNF-α, IL-6 and IL-1β)
512
cytokines. This complex behavior of pulmonary DCs appears to explain the low NO
513
production, the poor control of fungal growth, the increased presence of Foxp3+ Tregs and the
514
elevated numbers of IFN-γ-, IL-4- and IL-17- secreting CD4+ and CD8+ T cells at the site of
515
infection. Therefore, P. brasiliensis infection appears to induce the concomitant expansion of
516
immunogenic and tolerogenic DCs in A/J mice, which promote the equilibrated expansion of
517
effector Th1/Th2/Th17 cells. These effector cells are then possibly controlled by increased
518
numbers of FoxP3+ Treg cells. It is possible that the synergistic suppressive effects of TGF-β
519
secreted by alveolar macrophages (49), tolerogenic DCs and Treg cells (25) contribute to the
520
poor NO secretion and pathogen clearance of resistant mice at the initial phase of P.
521
brasiliensis infection (2, 10, 19). At the chronic phase, tightly controlled CD4+ and CD8+ T
522
cell responses appear sufficient to restrain fungal growth without excessive inflammation and
523
tissue damage. This interpretation is consistent with the histopathology of the lungs and livers
524
of A/J mice 10 weeks post-infection (Fig. S1). Intense, non-organized lesions containing high
525
numbers of fungal cells were observed in the lungs and liver of susceptible mice, whereas
526
discrete inflammatory reactions presenting low number of yeast cells and preserved organ
527
parenchyma were seen in resistant mice.
528
The immune response against fungal pathogens depends on the cooperation between
529
different DC subsets, which develop different activation programs induced by diverse
530
intracellular signaling following PRR activation. Inflammatory DCs usually induce Th2 and
22
531
Th17 immunity, whereas tolerogenic DCs activate Th1 and Treg cell differentiation (9). In
532
our experimental model, DCs from resistant mice showed simultaneous tolerogenic and
533
immunogenic behavior. This resulted in protective Th1/Th17 immunity that was tightly
534
regulated by Th2 and Treg cells. Importantly, we showed for the first time that genetic
535
resistance to P. brasiliensis infection was associated with Th17 and Tc17 immunity, and this
536
finding is consistent with the increased production of IL-6 and TGF-β by A/J DCs.
537
Our murine model demonstrates that susceptibility to a pulmonary fungal pathogen
538
can paradoxically be associated with efficient mechanisms of innate immunity, whereas
539
resistance can be based on immune mechanisms that are initially inefficient in the control of
540
pathogen growth, but evolve into complex effector T cell responses that are tightly regulated
541
by Treg cells. The unusual mechanism described in this and other studies (1, 2, 15, 16, 19, 24,
542
39, 49, 50) demonstrates that conviviality with a slow growing pathogen can be less dangerous
543
to the host than an initial aggressive response that results in parasite killing but aggregates
544
pernicious inflammation, tissue damage and eventually suppressed adaptive immunity.
545
Indeed, with other important pulmonary pathogens such as Streptococcus pneumonia and
546
Mycobacterium tuberculosis, the early recruitment of TGF-β+ anti-inflammatory DCs and
547
regulatory T cells to the infected lungs was associated with host resistance to pneumococcal
548
pneumonia and tuberculosis, respectively (41, 46). This concept of host resistance, mediated
549
by reduction or avoidance of damage caused by an infectious agent, has been described by
550
plant ecologists (20). However, despite its importance to a comprehensive view of host-
551
pathogen interactions, this concept has rarely been applied to mammalian models of infection
552
(51, 58). Finally, our model describes a resistance mechanism that can protect hosts without
553
damaging the lung, an organ whose main physiological function is substantially impaired by
554
inflammatory processes. Our data also contribute to the understanding of severe cases of
555
fungal infections, such as those caused by Pneumocystis carinii or even P. brasiliensis, where
23
556
successful therapy can be achieved only when an antibiotic is administered in combination
557
with an anti-inflammatory drug such as a corticosteroid (34, 36).
558 559
ACKNOWLEDGMENTS
560
This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São
561
Paulo (FAPESP) e Conselho Nacional de Pesquisas (CNPq). We are grateful to Márcio Y.
562
Tomiyoshi and Tania A. Costa for invaluable technical assistance.
563 564
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565
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32
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FIGURE LEGENDS
749
FIG 1 Subsets and activation profile of dendritic cells developed by resistant and
750
susceptible mice. Bone-marrow cells from B10.A and A/J mice were cultured with rGM-
751
CSF and rIL-4 for 5 days, in vitro challenged with P.brasiliensis yeasts (1:20 fungus/DC
752
ratio) for 48 h and DCs subpopulations characterized by flow cytometry. (A) Frequency of
753
myeloid (CD11c+CD11b+), plasmacytoid (CD11c+ B220+) and lymphoid (CD11c+ CD8+)
754
DCs. The expression of CD80, CD86, CD40 and MHC Class II was measured in each DC
755
subpopulation. Data are expressed in percentage or MFI (median fluorescence intensity)
756
and are representative of three independent experiments with similar results. (*) P< 0.05;
757
** P
