At week 2 after infection lungs from anti-CD25 treated mice presented decreased levels of IL-10, TGF-ß and GM-CSF, but at week 10 increased levels of Th1-, Th2-, and Th17-associated cytokines.

<p>At weeks 2 and 10 after i.t. infection with 1×10⁶ yeast cells of <i>P. brasiliensis</i>, lungs from anti-CD25 treated and untreated A/J and B10.A mice were collected, disrupted in 5.0 ml of PBS and supernatants analyzed for cytokines content by capture ELISA. (A, B, and C) Th1, Th2 and Th17 cytokines at week 2 of infection, respectively. (D, E, and F) Th1, Th2 and Th17 cytokines at week 10 of infection, respectively. The bars depict means ± SEM of cytokine levels (6–8 per group). The results are representative of two independent experiments. * (<i>P</i><0.05), ** (<i>P</i><0.01), and *** (<i>P</i><0.001) compared with IgG treated controls or the susceptible strain.</p

Anti-CD25 treatment increases the influx of inflammatory cells to the lungs of resistant but not susceptible mice to <i>P. brasiliensis</i> infection.

<p>Anti-CD25-treated and untreated A/J and B10.A mice were inoculated i.t. with 1×10⁶<i>P. brasiliensis</i> yeast cells. At weeks 2 and 10 of infection lungs of both mouse strains (n = 6) were excised, minced, and digested enzymatically. Lung cells suspensions were obtained, counted, stained for CD3 (T cells), CD19 (B cells) and GR1 (myeloid cells, including neutrophils and monocytes) by flow cytometry. Anti-CD25 treatment significantly alters the number (A) but not the frequency of inflammatory cells in the lungs (B) of infected mice. At week 2, anti-CD25-treated A/J mice showed increased influx of T cells, B cells and myeloid cells, whereas at week 10 these populations appeared in decreased numbers (C, D). In B10.A mice only GR1⁺ cells appeared in decreased numbers at week 10 of infection (C, D). The data represent the mean ± SEM of the results from 6–8 mice per group and are representative of two independent experiments. * (<i>P</i><0.05), ** (<i>P</i><0.01), and *** (<i>P</i><0.001) compared with IgG-treated mice.</p

Depletion of CD25⁺ cells diminishes the inflammatory reactions in the liver of A/J mice and abolishes the hepatic fungal lesions of B10.A mice.

<p>Characterization of leukocyte subsets and activation profile of cells by flow cytometry in the liver infiltrating leucocytes (LIL) from anti-CD25-treated and untreated A/J and B10.A mice inoculated i.t. with 1×10⁶<i>P. brasiliensis</i> yeast cells. At week 10 after infection liver cell suspensions were obtained and stained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051071#s2" target="_blank">Materials and Methods</a>. The acquisition and analysis gates were restricted to macrophages or lymphocytes. A, CD4⁺ T cells; B, CD8⁺ T cells; C- Activated/Treg CD4⁺ T cells. D- Liver macrophages. E- CD4⁺CD25⁺Foxp3⁺ Treg cells. The data represent the mean ± SEM of the results from 5–6 mice per group and are representative of two experiments. * (<i>P</i><0.05), ** (<i>P</i><0.01), and *** (<i>P</i><0.001) compared with IgG controls or B10.A strain. F- Histopathology of liver. Anti-CD25 treated and untreated A/J mice did not show hepatic lesions at week 10 of infection (data not shown). In contrast, control B10.A mice presented extensive hepatic lesions containing large numbers of fungal cells (F, a,b). Anti-CD25 treatment practically abolished the inflammatory lesions (F, c,d) and the fungal loads of B10.A mice. a, c (HE, X 100); b, d (Groccot X 100).</p

Anti-CD25 causes intense alterations in the migration of macrophages and dendritic cells to the lungs of A/J mice, but has a minor effect in B10.A mice.

<p>Characterization of macrophages and dendritic cells by flow cytometry in the lung infiltrating leucocytes (LIL) from anti-CD25-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 10 after infection lung cell suspensions were obtained and stained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051071#s2" target="_blank">Materials and Methods</a>. The acquisition and analysis gates were restricted to macrophages. Macrophages (A), and dendritic cells (B) at weeks 2 and 10 of infection. The data represent the mean ± SEM of the results from 6–8 mice per group and are representative of two independent experiments. * (<i>P</i><0.05), ** (<i>P</i><0.01), and *** (<i>P</i><0.001) compared with IgG treated controls or the susceptible strain.</p

Depletion of CD25⁺ cells abolishes the increased mortality of susceptible mice but does not induce sterile immunity.

<p>A- Survival times of anti-CD25-treated and untreated B10.A and A/J mice (n = 6–7) after i.t. infection with 1×10⁶<i>P. brasiliensis</i> yeast cells were determined in a period of 190 days. The results are representative of two independent experiments. **<i>P</i><0.01. Recovery of fungal loads (CFU) from lungs (B), and liver (C) of survivor mice at day 190 after infection. The bars represent means ± SEM of log₁₀ CFU obtained from groups of 6–7 mice. The results are representative of two experiments with equivalent results. **<i>P</i><0.01.</p

Administration of anti-CD25 antibody decreases the early and late organ fungal burdens of resistant and susceptible mice.

<p>Anti-CD25 mAb (PC61) or control IgG (500 µg) were administered at days −3 and +3 of <i>P. brasiliensis</i> infection. CFU counts were determined in the lungs (A), liver (B) and spleen (C) of resistant and susceptible mice at weeks 2 and 10 after infection. The points represent means ± SEM of log₁₀ CFU obtained from groups of six mice. The results are representative of 3 experiments * (<i>P</i><0.05), ** (<i>P</i><0.01), and *** (<i>P</i><0.001) compared with IgG inoculated controls.</p

Anti-CD25 treatment induces reduced expression of IDO mRNA and diminished production of kynurenine and NO.

<p>IDO mRNA expression (A) in the lungs of IgG or anti-CD25 treated normal and infected B10.A and A/J mice was monitored by quantitative RT-PCR. The data are reported as a ratio of IDO/GAPDH. Lung homogenates were obtained from anti-CD25 treated and untreated B10.A and A/J mice infected with 1×10⁶<i>P. brasiliensis</i> yeasts. The concentration of kynurenine (B) and nitrite (C) was measured in lung supernatants obtained at weeks 1 and 2 after infection. Concentrations of NO and kynurenine were measured using colorimetric assays. The bars represent means ± SEM of data obtained from groups of 6–7 mice. The results are representative of two independent experiments. * (<i>P</i><0.05), ** (<i>P</i><0.01), and *** (<i>P</i><0.001) compared with IgG treated controls or the susceptible strain.</p

Anti-CD25 causes intense alterations in the migration of inflammatory CD4⁺ and CD8⁺ T cells to the lungs of A/J mice, but does not affect B10.A mice.

<p>Characterization of CD4⁺, CD8⁺ T cells and activation profile of cells by flow cytometry in the lung infiltrating leucocytes (LIL) from anti-CD25-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 10 after infection lung cell suspensions were obtained and stained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051071#s2" target="_blank">Materials and Methods</a>. The acquisition and analysis gates were restricted to lymphocytes. CD4⁺ T cells (A), CD8⁺ T cells (B) and activated/Treg (C) cells at week 2 of infection. CD4⁺ T cells (D), CD8⁺ T cells (E) and activated/Treg (F) cells at week 10 of infection. To characterize the number of Treg cells in LIL, surface staining of CD25⁺ and intracellular Foxp3 expression were back-gated on the CD4⁺ T cell population (G). The data represent the mean ± SEM of the results from 6–8 mice per group and are representative of two independent experiments. * (<i>P</i><0.05), ** (<i>P</i><0.01), and *** (<i>P</i><0.001) compared with IgG treated controls or the susceptible strain.</p

Histopathology of pulmonary lesions of anti-CD25-treated and untreated A/J and B10.A mice at week 10 post-infection with 1×10⁶<i>P. brasiliensis</i> yeasts.

<p>IgG-treated (A, B) and anti-CD25-treated (C, D) A/J mice showed equivalent diffuse inflammatory reactions characterized by a small number of yeasts in the presence of elevated number of macrophages, lymphocytes and plasma cells; small portions of lung tissue were preserved, with limited signs of inflammatory cell recruitment. IgG-treated B10.A mice (E, F) presented an elevated number of well-defined, confluent, necrotic, granulomas of various sizes (E) containing an elevated number of fungal cells (arrows in F); these lesions occupy a large area of lung tissue (E, F). Compared with control B10.A mice, anti-CD25-treated B10.A mice showed significantly smaller lesions (G) containing a few number of yeasts (H). A, C, E, G, (HE, X 100); B, D, F, H (Groccot X 100). I- Total area of lung lesions of mice (n = 6) at week 10 after infection. ** (<i>P</i><0.01), and *** (<i>P</i><0.001) compared with IgG-treated controls or the susceptible strain.</p