Mannan treatment induced increased fungicidal ability and NO production by macrophages from resistant (A/J) and susceptible (B10.A) mice.

<p>(A, B) CFU assays were performed to determine the recovery of viable fungi in cell homogenates. Macrophages were primed or unprimed with IFN-γ (20 ng/mL) overnight, treated by mannan (2.5, 0.5 and 0.1 mg/mL) and then challenged with viable <i>P. brasiliensis</i> yeasts (1∶25, fungus:macrophages ratio). Two hours later the cultures were gently washed, cultivated for an additional 48 h period, and the number of recovered viable yeasts measured by a CFU assay. (C, D) Nitric oxide (NO) production was measured in culture supernatants by a Griess reagent. Data are means ± SEM of triplicate samples from two experiment determinations. (*P<0.05, **P<0.01 and ***P<0.001).</p

TLR4 and CR3 blockade by anti-TLR4 and anti-CD11b antibodies modulated the cytokines production of <i>P. brasiliensis</i> infected A/J and B10.A macrophages.

<p>Macrophages from A/J and B10.A mice were untreated or treated with anti-TLR4 and anti-CD11b antibodies (10 µg/ml) for 30 min., infected or not with <i>P. brasiliensis</i> yeasts cells, and then cultivated for 48 h. The levels of cytokines were assessed by ELISA in the cell supernatants. Data are means ± SEM of triplicate samples from two experiment determinations. (*P<0.05, **P<0.01 and ***P<0.001).</p

TLR4 and CR3 control the recognition of <i>P.brasiliensis</i> yeasts by A/J than B10.A macrophages.

<p>Macrophages from A/J and B10.A mice were untreated or treated with anti-TLR4 and anti-CD11b antibodies (10 µg/ml) for 30 min. and then infected or not with <i>P. brasiliensis</i> yeasts cells. (A, B) Adherence/ingestion activity, (C, D) fungicidal activity, and, (E, F) NO production were assessed as described before. Data are means ± SEM of triplicate samples from two experiment determinations. (*P<0.05, **P<0.01 and ***P<0.001).</p

MR signaling controls the different patterns of cytokines produced by A/J and B10.A macrophages.

<p>Macrophages from A/J and B10.A mice were treated by anti-MR antibodies (20 µg/mL) for 30 min. After, some cultures were challenged with viable <i>P. brasiliensis</i> yeasts (1∶25, fungus:macrophages ratio) for 48 h. Supernatants were removed and used for cytokines measurements by ELISA. Data are means ± SEM of triplicate samples from two experiment determinations. (*P<0.05, **P<0.01 and ***P<0.001).</p

<i>P. brasiliensis-</i> and mannan-activated macrophages from susceptible mice preferentially upregulate SOCS3 whereas macrophages from resistant mice upregulate SOCS1 expression.

<p>Quantitative PCR analysis of (A) suppressor of cytokine signaling-3 (SOCS3), (B) SOCS1 mRNA expression. Graph (C) represents the SOCS1/SOCS3 ratio. Macrophages from A/J and B10.A mice were untreated or treated by mannan (2.5 mg/mL) for 30 min and cultivated for 12 h. Some cultures were only infected by viable <i>P. brasiliensis</i> yeasts (1∶25, fungus:macrophages ratio) for 12 h. Total RNA from macrophages cultures was obtained, reverse transcribed, and cDNA amplified. Real-time PCR was performed using TaqMan universal master mix. Amplified products were normalized to the amount of GAPDH products from in vitro cultivated macrophages. Data represent the means ± SEM of at least 5 mice/group and are representative of two independent experiments. (**P<0.01 and ***P<0.001).</p

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

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

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

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

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