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

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

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

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

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

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

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

Treg cells from resistant mice have a higher suppressive potency than those of susceptible mice.

<p>CFSE-labeled responder CD4⁺CD25⁻ T cells from naïve mice were stimulated by irradiated naïve APCs plus anti-CD3 antibodies and cultured in the presence or absence of several ratios of CD25⁺ T cells obtained from lungs of resistant and susceptible mice at weeks 2 (A) and 10 (B) after infection with 1×10⁶<i>P.brasiliensis</i> yeasts. Cells were cultured for 5 days and the proliferative response of CFSE-labeled cells was measured by flow cytometry. The proliferation index (PI) was calculated as describe in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051071#s2" target="_blank">materials and methods</a> and the percentage of inhibition considered as 100% the PI of APC-stimulated CD4⁺CD25⁻ responder cells in the absence of CD4⁺CD25⁺ cells. The data represent the mean ± SEM of the results from 6 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 A/J mice.</p