Tolerogenic Plasmacytoid Dendritic Cells Control <i>Paracoccidioides brasiliensis</i> Infection by Inducting Regulatory T Cells in an IDO-Dependent Manner

<div><p>Plasmacytoid dendritic cells (pDCs), considered critical for immunity against viruses, were recently associated with defense mechanisms against fungal infections. However, the immunomodulatory function of pDCs in pulmonary paracoccidiodomycosis (PCM), an endemic fungal infection of Latin America, has been poorly defined. Here, we investigated the role of pDCs in the pathogenesis of PCM caused by the infection of 129Sv mice with 1 x 10⁶ <i>P</i>. <i>brasiliensis-</i>yeasts. <i>In vitro</i> experiments showed that <i>P</i>. <i>brasiliensis</i> infection induces the maturation of pDCs and elevated synthesis of TNF-α and IFN-β. The <i>in vivo</i> infection caused a significant influx of pDCs to the lungs and increased levels of pulmonary type I IFN. Depletion of pDCs by a specific monoclonal antibody resulted in a less severe infection, reduced tissue pathology and increased survival time of infected mice. An increased influx of macrophages and neutrophils and elevated presence of CD4⁺ and CD8⁺ T lymphocytes expressing IFN-γ and IL-17 in the lungs of pDC-depleted mice were also observed. These findings were concomitant with decreased frequency of Treg cells and reduced levels of immunoregulatory cytokines such as IL-10, TGF-β, IL-27 and IL-35. Importantly, <i>P</i>. <i>brasilienis</i> infection increased the numbers of pulmonary pDCs expressing indoleamine 2,3-dioxygenase-1 (IDO), an enzyme with immunoregulatory properties, that were reduced following pDC depletion. In agreement, an increased immunogenic activity of infected pDCs was observed when IDO-deficient or IDO-inhibited pDCs were employed in co-cultures with lymphocytes Altogether, our results suggest that in pulmonary PCM pDCs exert a tolerogenic function by an IDO-mediated mechanism that increases Treg activity.</p></div

pDC depletion determines increased presence of pulmonary macrophages, neutrophils and activated T lymphocytes.

<p>pDC-depleted and control groups were inoculated i.t. with 1 × 10⁶ <i>P</i>. <i>brasiliensis</i> yeasts and cell phenotypes determined at weeks 2 and 8 after infection. Lungs of both mouse groups (n = 4–5) were excised and digested enzymatically. Cell suspensions were obtained and stained as described in Materials and Methods. The stained cells were analyzed immediately on a FACSCanto II equipment with gating of lymphocytes or granulocytes, as judged from FSC and SSC scatters. Gated CD4⁺ and CD8⁺ cells were also analyzed for the expression of activation and deactivation markers. (A) Representative FACS plots demonstrating the gating strategy for lymphocytes, macrophages and neutrophils. (B) Number of total leukocytes. (C) Frequency and number of neutrophils CD11b⁺F4/80^-Gr1⁺, and (D) macrophages F4/80⁺CD11b⁺. (E) Total and activated CD4⁺ T and (F) CD8⁺ T cells. Markers of the suppressive activity of T cells (CTLA4, GITR, ICOS, PD-L1) were also measured by flow cytometry at weeks 2 (G) and 8 (H) after infection. One hundred thousand cells were counted and the data expressed as frequency and number of positive cells. Data are expressed as means ± SE of the mean and are representative of three independent experiments. *<i>p</i> < 0.05.</p

IDO-expressing pDCs and increased levels of kinurenines are observed in the lungs of <i>P</i>. <i>brasiliensis</i> infected mice.

<p>The presence of pDCs expressing intracellular IDO was determined by flow cytometry in the lungs of uninfected and <i>P</i>. <i>brasilienis</i>-infected mice (1×10⁶ yeasts cells). At 2 and 8 week post infection the lungs were removed, leukocytes obtained and the pDCs isolated by two rounds of positive selection as described in material and methods. The cells were then stained using the Cytofix/Cytoperm kit (BD Biosciences) and specific antibodies anti-IDO. (A) The pDCs were characterized as CD11c⁺B220⁺PDCA⁺ cells as indicated in the gate strategy. (B-C) Plots and bar graphics showing IDO-producing pDCs before and after <i>P</i>. <i>brasiliensis</i> infection. (D) The pDCs isolated from the lungs were also kept overnight in culture and the kynurenines were measured in the supernatants. Relative expression of IDO mRNA (E) and kynurenines measurements (F) in the lung homogenates of mice treated with anti-mPDCA or control IgG, after 96 h, 2 and 8 weeks of infection. Bars reflect mean ± SD of two independent experiments with five mice per group (* <i>p</i> < 0.05).</p

Depletion of pDC induces increased Th1/Th17-related transcription factors and reduced Treg-associated Foxp3.

<p>mRNA relative expression of Tbet, Gata3, Rorc and Foxp3 in the lungs of mice treated with anti-mPDCA or control isotype after 96 h, 2 and 8 weeks of infection. Bars show mean ± SD from at least four mice per group and are representative of three independent experiments (*<i>p</i>< 0.05).</p

Depletion of pDCs reduces fungal loads, tissue injury and mortality rates.

<p>Groups of anti-CD317 (anti-PDCA; clone BX44) or control rat IgG (clone HRPN) treated mice were infected i.t. with 1×10⁶ yeasts cells of <i>P</i>.<i>brasiliensis</i>. At 96 h, 2 and 8 weeks post-infection lungs were removed, leukocytes obtained and the numbers of pDC analyzed by flow cytometry (A). Colony-forming unit (CFU) counts from lungs (B) and liver (C) were determined 96 h, 2 and 8 weeks after <i>P</i>. <i>brasiliensis</i> infection. The bars represent means ± standard errors of the mean (SEM) of log₁₀ CFU counts obtained from groups of 4–5 mice. (D–G) Photomicrographs of lung lesions of control (D and F) and pDC-depleted mice (E and G) at weeks 2 (D And E) and 8 (F and G) of infection. Lesions were stained with hematoxylin-eosin (left panels) and Grocott (right panels). (See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006115#ppat.1006115.s001" target="_blank">S1 Fig</a> for liver lesions). (H) Total area of lesions in the lungs at week 2 and 8 of infection. (I) Survival curves of pDC-depleted and control infected mice were determined in a period of 110 days. Data represent the means ± SEM of at least 4 mice/group and are representative of two independent experiments with equivalent results (*<i>p</i> < 0.05).</p

pDC response to <i>P</i>. <i>brasilienis</i> infection The influx of pDCs to the lungs of <i>P</i>. <i>brasiliensis</i>-infected mice (1×10⁶ yeasts cells) was determined by flow cytometry at weeks 2 and 8 post-infection.

<p>Lungs were removed, leukocytes obtained and the number of pDCs evaluated. The pDCs were characterized as CD11c⁺B220⁺PDCA⁺ cells as indicated in the gate strategy used (A) and the activation measured by the expression of CD40, CD80, C86 and MHC-II molecules on their surface (B-C). The number of pDCs that migrated to the lungs was also determined by flow cytometric analysis (D). The levels of TNF-α and IFN-β were measured by ELISA in pDC supernatants obtained after18 hr of cell culture (E). Data represent the means ± SEM of at least 8 mice and are representative of two independent experiments with equivalent results (*<i>p</i> < 0.05).</p

Absence of type I IFN signaling during <i>P</i>. <i>brasiliensis</i> infection increases mortality rates associated with increased fungal loads and tissue pathology.

<p>Colony-forming unit (CFU) counts from organs were determined at 2 (A) and 8 weeks (B) after <i>P</i>. <i>brasiliensis</i> infection of 129Sv WT, IFNαβR^−/−, IFNγR^-/- and IRF1^-/- mice. The bars represent means ± standard errors of the mean (SEM) of log₁₀ CFU counts obtained from groups of 4–5 mice. Photomicrographs of lesions of WT mice (C), IFNαβR^−/− (D), IFNγR^-/- (E) and IRF1^-/- (F) mice at week 8 of infection with 1 × 10⁶ <i>P</i>. <i>brasiliensis</i> yeasts. Staining of lesions was performed with hematoxylin-eosin (left panels) and Grocott (right panels). (G) Total area of pulmonary lesions at week 8 after infection. (H) Survival times of the four studied mouse strains infected i.t. with 1 × 10⁶ <i>P</i>. <i>brasiliensis</i> yeast cells were determined in a period of 110 days. Data represent the means ± SEM of at least 10 mice/group and are representative of two independent experiments with equivalent results. (*<i>p</i> < 0.05).</p

pDC depletion increases the differentiation of type 1 and type 17 CD4⁺ and CD8⁺ T cells.

<p>The presence of IL-17⁺, IL-4 and IFN-γ⁺ in CD4⁺ and CD8⁺ T cells in the LILs was assessed by intracellular cytokine staining by flow cytometry at weeks 2 and 8 after infection. Lung cells were stimulated <i>in vitro</i> with PMA/ionomycin for 6 hours and subjected to intracellular staining for IL-17 and IFN-γ. The lung infiltrating lymphocytes were gated by FSC/SSC analysis. Lymphocytes were gated for CD4 or CD8 expression and then for IFN-γ, IL-4 and IL-17 expression. Results are expressed in frequency and absolute number of cells and are representative of 3 independent experiments. Data are expressed as means ± SE of the mean. *<i>p</i> < 0.05.</p

Data_Sheet_2.PDF

<p>Plasmacytoid dendritic cells (pDCs), which have been extensively studied in the context of the immune response to viruses, have recently been implicated in host defense mechanisms against fungal infections. Nevertheless, the involvement of human pDCs during paracoccidioidomycosis (PCM), a fungal infection endemic to Latin America, has been scarcely studied. However, pDCs were found in the cutaneous lesions of PCM patients, and in pulmonary model of murine PCM these cells were shown to control disease severity. These findings led us to investigate the role of human pDCs in the innate phase of PCM. Moreover, considering our previous data on the engagement of diverse Toll-like receptors and C-type lectin receptors receptors in Paracoccidioides brasiliensis recognition, we decided to characterize the innate immune receptors involved in the interaction between human pDCs and yeast cells. Purified pDCs were obtained from peripheral blood mononuclear cells from healthy donors and they were stimulated with P. brasiliensis with or without blocking antibodies to innate immune receptors. Here we demonstrated that P. brasiliensis stimulation activates human pDCs that inhibit fungal growth and secrete pro-inflammatory cytokines and type I IFNs. Surprisingly, P. brasiliensis-stimulated pDCs produce mature IL-1β and activate caspase 1, possibly via inflammasome activation, which is a phenomenon not yet described during pDC engagement by microorganisms. Importantly, we also demonstrate that dectin-2 and dectin-3 are expressed on pDCs and appear to be involved (via Syk signaling) in the pDC-P. brasiliensis interaction. Moreover, P. brasiliensis-stimulated pDCs exhibited an efficient antigen presentation and were able to effectively activate CD4⁺ and CD8⁺ T cells. In conclusion, our study demonstrated for the first time that human pDCs are involved in P. brasiliensis recognition and may play an important role in the innate and adaptive immunity against this fungal pathogen.</p

Data_Sheet_1.PDF

<p>Plasmacytoid dendritic cells (pDCs), which have been extensively studied in the context of the immune response to viruses, have recently been implicated in host defense mechanisms against fungal infections. Nevertheless, the involvement of human pDCs during paracoccidioidomycosis (PCM), a fungal infection endemic to Latin America, has been scarcely studied. However, pDCs were found in the cutaneous lesions of PCM patients, and in pulmonary model of murine PCM these cells were shown to control disease severity. These findings led us to investigate the role of human pDCs in the innate phase of PCM. Moreover, considering our previous data on the engagement of diverse Toll-like receptors and C-type lectin receptors receptors in Paracoccidioides brasiliensis recognition, we decided to characterize the innate immune receptors involved in the interaction between human pDCs and yeast cells. Purified pDCs were obtained from peripheral blood mononuclear cells from healthy donors and they were stimulated with P. brasiliensis with or without blocking antibodies to innate immune receptors. Here we demonstrated that P. brasiliensis stimulation activates human pDCs that inhibit fungal growth and secrete pro-inflammatory cytokines and type I IFNs. Surprisingly, P. brasiliensis-stimulated pDCs produce mature IL-1β and activate caspase 1, possibly via inflammasome activation, which is a phenomenon not yet described during pDC engagement by microorganisms. Importantly, we also demonstrate that dectin-2 and dectin-3 are expressed on pDCs and appear to be involved (via Syk signaling) in the pDC-P. brasiliensis interaction. Moreover, P. brasiliensis-stimulated pDCs exhibited an efficient antigen presentation and were able to effectively activate CD4⁺ and CD8⁺ T cells. In conclusion, our study demonstrated for the first time that human pDCs are involved in P. brasiliensis recognition and may play an important role in the innate and adaptive immunity against this fungal pathogen.</p