At late stages of infection, IL-10 deficiency leads to reduced production of pro- and anti-inflammatory cytokines in the lungs of <i>P. brasiliensis</i>-infected mice.

<p>Levels of cytokines in lung homogenates of IL-10^−/− and WT control mice were measured after i.t. infection with 10⁶ yeast cells. Lungs were disrupted at weeks 8 and 16 after infection, and supernatants were analyzed for cytokine content by using the BD CBA mouse Th1/Th2/Th17 cytokine kit. The bars depict means ± SEM of cytokine levels (6 to 8 animals per group). * <i>p</i><0.05; ** <i>p</i><0.01, compared with WT control.</p

IL-10 inhibits the phagocytic and fungicidal abilities of macrophages.

<p>(A) Macrophages from WT and IL-10^−/− mice either treated with IFN-γ or left untreated were infected for 2 h with <i>P. brasiliensis</i> yeasts labeled with propidium iodide (1∶1, fungus∶macrophage ratio). Co-cultures were gently washed and macrophages were analyzed by flow cytometry. (B) For fungicidal assay, macrophages were infected with <i>P. brasiliensis</i> yeasts (1∶12.5, fungus∶macrophage ratio) during 2 h, washed, and further cultivated for 48 h at 37°C in 5% CO₂. Supernatants were removed, the monolayers were washed with distilled water to lyse macrophages, and 100 µl of cell homogenates were assayed for the presence of viable yeasts by a CFU assay (C) NO production was measured in culture supernatants by Griess reagent. Data are means ± SEM of three independent experiments with similar results (* <i>p</i><0.05, ** <i>p</i><0.01 and *** <i>p</i><0.001).</p

IL-10^−/− mice develop a less severe PCM associated with increased cellular immunity that results in decreased mortality rates.

<p>(A) IL-10^−/− and WT mice were inoculated with 1×10⁶<i>P. brasiliensis</i> yeast cells by the i.t. route, and severity of infection was evaluated by determining fungal loads in the lungs, livers and spleens at five post-infection periods (2, 4, 8, 16 and 23 weeks). The bars depict means ± SEM of the numbers of log₁₀ CFU obtained from groups of 6 to 8 mice. The results are representative of 3 experiments. ** <i>p</i><0.01, and *** <i>p</i><0.001, compared with WT controls. (B) Survival of IL-10^−/− and WT control mice after i.t. infection with 1×10⁶<i>P. brasiliensis</i> yeast cells was determined for a period of 220 days. Results are representative of two independent experiments (n = 12; * <i>p</i><0.05). (C) DTH responses mounted by <i>P. brasiliensis</i> infected mice. Control and infected WT and IL-10^−/− mice were injected intrafootpad with soluble <i>P. brasiliensis</i> yeast antigen (5 µg in 25 µl PBS) 24 hours before measurement of the footpad response at weeks 4 and 8 after fungal infection. Results are representative of two independent experiments. The bars depict means ± SEM of footpad swelling (* <i>p</i><0.05; ** <i>p</i><0.01; n = 6–8 mice).</p

IL-10 deficiency determines increased humoral immunity in early <i>P. brasiliensis</i> infection.

<p>Levels of total specific Ig (A) and fungus-specific isotypes (B) in the sera of IL-10^−/− and WT mice at weeks 2, 4, 8, and 16 after i.t. infection with 10⁶ yeast cells. Sera were assayed for total Ig, IgM, IgA, IgG1, IgG2a, IgG2b and IgG3 by using an isotype-specific ELISA as detailed in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002512#s2" target="_blank"><i>Materials and Methods</i></a>. The bars depict means ± serum titers (6 to 8 mice per group). The results are representative of 3 experiments. ** <i>p</i><0.01; *** <i>p</i><0.001, compared with WT controls.</p

IL-10^−/− mice clear <i>P. brasiliensis</i> infection and develop mild pulmonary inflammatory reactions.

<p>Photomicrographs of lungs, livers and spleens from WT (A to F) and IL-10^−/− (G to L) mice at week 8 after infection with 1×10⁶<i>P. brasiliensis</i> yeast cells. Compared with IL-10^−/− mice, increased number of inflammatory cells and more severe lesions were detected in WT mice. For panels: A, C, E, G, I and K, HE-stained, magnification ×10; for B, D, F, H, J and L, Groccot-stained, magnification ×10. Morphometrical analysis (M) confirmed the more extensive areas occupied by the lung and liver lesions of WT mice (*** <i>p</i><0.001).</p

In pulmonary paracoccidioidomycosis, 1MT treatment increases fungal loads and pulmonary NO but reduces kynurenines levels and IDO mRNA production.

<p>(<b>A, B</b>) Determination of fungal loads by CFU assays in the lungs of A/J and B10.A mice (n = 5–6), treated or not with 1MT, at weeks 2 and 8 after infection with 1×10⁶ yeasts. (<b>C, D</b>) Groups (n = 6–8) of B10.A and A/J mice received subcutaneous implants of 1MT releasing or control pellets (5 mg/ml/day) and disease severity assessed by CFU assays at weeks 4 and 8 after infection with 1×10⁶ yeast cells of <i>P. brasiliensis</i>. The bars depict means ± SEM of log10 CFU. The results are representative of 2 experiments with equivalent results (**<i>p</i><0.01, and ***<i>p</i><0.001). Levels of (<b>E, F</b>) nitric oxide and (<b>G, H</b>) kynurenines were measured in lung homogenates. NO production was measured by Griess reagent, and kynurenines were evaluated using Ehrlich's reagent. (<b>I, J</b>) IDO mRNA was measured using TaqMan real-time PCR assay. Data are means ± SEM of three independent experiments at weeks 2 and 8 after infection with similar results (*<i>p</i><0.05, **<i>p</i><0.01 and ***<i>p</i><0.001).</p

1MT treatment increases fungal loads, tissue pathology and mortality rates of susceptible but not resistant mice to <i>P. brasiliensis</i> infection.

<p>Photomicrographs of lungs from 1MT treated and untreated A/J (<b>A</b>) and B10.A (<b>B</b>) mice at week 8 after infection with 1×10⁶<i>P. brasiliensis</i> yeast cells obtained from groups of 5–6 mice. For panels: <b>A</b> and <b>B</b>, HE-stained, magnification ×10, Grocott-stained, magnification ×10. (<b>C</b>) Morphometric analysis of lung lesions of 1MT treated and untreated B10.A and A/J mice at week 8 post-infection (n = 5–6). (<b>D</b>) At weeks 2, 8 and 26 after infection, the presence of viable yeasts was determined in the liver of 1MT treated and untreated B10.A and A/J mice (n = 5–6). (<b>E, F</b>) Survival times of 1MT treated or untreated A/J and B10.A mice (9–10 mice per group) infected with 1×10⁶<i>P. brasiliensis</i> yeasts were determined for a period of 250 days. The results are representative of two independent experiments (**<i>p</i><0.01; ***<i>p</i><0.001).</p

IDO inhibition increases the migration of naïve and effector/memory CD4⁺ and CD8⁺ T cells to the lungs of A/J and B10.A mice, but reduces the number of regulatory T cells.

<p>(<b>A, B, C, D</b>) Characterization of CD4⁺, CD8⁺ T cells by flow cytometry in the lung infiltrating leucocytes (LIL) from 1MT treated and untreated A/J and B10.A mice inoculated i.t. with 1×10⁶<i>P. brasiliensis</i> yeast cells. At weeks 2 and 8 after infection, lung cell suspensions were obtained and stained as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003330#s2" target="_blank">Materials and Methods</a>. The acquisition and analysis gates were restricted to lymphocytes. The total numbers of effector (CD44^highCD62L^low) and naïve (CD44^low CD62L^high) CD4⁺ and CD8⁺ T cells at weeks 2 and 8 after infection were determined. (<b>E, F</b>) To characterize the number of Treg cells in LIL at weeks 2 and 8 weeks of infection, cells positive for surface staining of CD25 and intracellular Foxp3 expression were back-gated on the CD4⁺ T cell population. The data represent the means ± SEM of 6–8 mice per group and are representative of three independent experiments (*<i>p</i><0.05 and **<i>p</i><0.01).</p

1MT treatment reduces IL-12 and increases IL-6 and TGF-β production by A/J and B10.A macrophages.

<p>(<b>A, B</b>) IFN-γ-primed and unprimed macrophages were treated with 1MT or left untreated and then cultivated for 48 h. Some cultures were infected with viable <i>P. brasiliensis</i> yeasts (1∶25, fungus∶macrophages ratio) for 48 h. Supernatants were removed and used for cytokine measurements by means of ELISA. Data are means ± SEM of triplicate samples from three independent determinations (*<i>p</i><0.05, **<i>p</i><0.01 and ***<i>p</i><0.001).</p

IDO blockade increases the presence of IL-17⁺, but reduces the number of IFN-γ⁺ T cells in the lungs of A/J and B10.A mice.

<p>Lymphocytes from lung infiltrating leukocytes (LIL) obtained from groups of 5–6 mice were gated based on forward/side scatters analysis. Gated cells were assessed for CD4⁺, CD8⁺, γδ⁺ and NKT⁺ markers using labeled antibodies. The presence of IL-17⁺, IFN-γ⁺ and IL-4⁺ T cells in the LIL was determined by intracellular cytokine staining at week 2 after infection. Lung cells were stimulated <i>in vitro</i> with PMA/ionomycin for 6 h, the last 4 h in the presence of brefeldin A, and subjected to intracellular staining for IL-17, IL-4 and IFN-γ. The results are representative of three experiments with equivalent results (*<i>p</i><0.05, **<i>p</i><0.01 and ***<i>p</i><0.001).</p