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

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

<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

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

Depletion of Treg cells induces balanced Th1/Th2/Th17 immunity in both stages of infection.

<p>(A) Quantification of activated/memory and naïve CD4⁺ and CD8⁺ T lymphocytes in lungs from mice treated with anti-CD25 or control IgG and analyzed after 2 (left) and 10 (right) weeks of infection. (B) mRNA relative expression of Tbet, GATA3, RORγC, Foxp3, PD-L1, CCR5, CCR6 and Granzyme B in lung cells of mice treated with anti-CD25 or control IgG, after 2 (top) and 10 (bottom) weeks of infection. <b>(C)</b> Cytokine quantitation by ELISA in lung homogenates from mice treated with anti-CD25 or control IgG. Bars show mean ± SD from at least five mice per group and are representative of three independent experiments (*<i>p</i>< 0.05, **<i>p</i> < 0.005, ***<i>p</i> <0.001).</p

Adoptive transfer of Treg cells improves the protective effect of CD4⁺Foxp3^- T cells.

<p>(A) Survival curves of Rag1^-/- mice infected with Pb18 after adoptive transfer of 2×10⁵ Treg cells, 2×10⁶ CD4⁺Foxp3^- T cells, 2×10⁵ Treg cells + 2×10⁶ CD4⁺Foxp3^- T cells, or vehicle only (<i>p</i> < 0.05 between PBS and Treg; <i>p</i> < 0.001 between PBS and CD4⁺Foxp3^- T cells; <i>p</i> <0.001 between PBS and CD4⁺Foxp3^- + Treg; <i>p</i> < 0.05 between Treg and CD4⁺Foxp3^-; <i>p</i> < 0.001 between Treg and CD4⁺Foxp3^- + Treg; n ≥ 13). (B and C) Analysis of disease severity through recovery of CFU from lungs, livers and spleens from Rag1^-/- mice infected with Pb18 after adoptive transfer of 2×10⁵ Treg cells, 2×10⁶ CD4⁺Foxp3^- T cells, 2×10⁵ Treg cells + 2×10⁶ CD4⁺Foxp3^- T cells, or vehicle only. Horizontal bars indicate the mean value in each group (* = <i>p</i> < 0.05; ** = <i>p</i> < 0.005; *** = <i>p</i> < 0.001; n ≥ 10).</p

Treg cells display a natural-like phenotype and express typical surface and intracellular markers during infection.

<p>(A) Expression of surface and intracellular markers suggest a natural Treg-associated phenotype, as demonstrated by increased expression of Neuropilin-1, CD39, CD73 and Helios. Solid lines represent infected mice, whereas dashed lines indicate uninfected mice. (B) Treg cells in the lungs exhibit augmented expression of the chemokine receptor CCR5 and the suppressive cytokine IL-10 after infection with Pb18. Cells were stained for surface CCR5 or permeabilized and stained for intracellular IL-10. Histograms were gated on Foxp3^GFP+ cells and show infected (shaded area) and uninfected (black line) mice. Dot plots display the frequency of Foxp3^GFP+ cells expressing IL-10. Histograms and dot plots are representative of three independent experiments with at least five mice per group. (C) IL-10 mRNA levels in Treg cells increase after infection, as analyzed by RT-PCR. Treg cells from lungs of uninfected and infected mice (weeks 2 and 10 after infection) were isolated and total RNA was extracted. After cDNA synthesis, RT-PCR was performed using primers for IL-10. Bars represent mean ± SD from at least 5 mice per group. One representative experiment is shown (* <i>p</i> < 0.05; **<i>p</i> < 0.005).</p