Lipoxin Inhibits Fungal Uptake by Macrophages and Reduces the Severity of Acute Pulmonary Infection Caused by Paracoccidioides brasiliensis

Cysteinyl leukotrienes (CysLTs) and lipoxins (LXs) are lipid mediators that control inflammation, with the former inducing and the latter inhibiting this process. Because the role played by these mediators in paracoccidioidomycosis was not investigated, we aimed to characterize the role of CysLT in the pulmonary infection developed by resistant (A/J) and susceptible (B10.A) mice. 48 h after infection, elevated levels of pulmonary LTC 4 and LXA 4 were produced by both mouse strains, but higher levels were found in the lungs of susceptible mice. Blocking the CysLTs receptor by MTL reduced fungal loads in B10.A, but not in A/J mice. In susceptible mice, MLT treatment led to reduced influx of PMN leukocytes, increased recruitment of monocytes, predominant synthesis of anti-inflammatory cytokines, and augmented expression of 5-and 15-lipoxygenase mRNA, suggesting a prevalent LXA 4 activity. In agreement, MTL-treated macrophages showed reduced fungal burdens associated with decreased ingestion of fungal cells. Furthermore, the addition of exogenous LX reduced, and the specific blockade of the LX receptor increased the fungal loads of B10.A macrophages. This study showed for the first time that inhibition of CysLTs signaling results in less severe pulmonary paracoccidioidomycosis that occurs in parallel with elevated LX activity and reduced infection of macrophages

Lipoxin Inhibits Fungal Uptake by Macrophages and Reduces the Severity of Acute Pulmonary Infection Caused by Paracoccidioides brasiliensis

Cysteinyl leukotrienes (CysLTs) and lipoxins (LXs) are lipid mediators that control inflammation, with the former inducing and the latter inhibiting this process. Because the role played by these mediators in paracoccidioidomycosis was not investigated, we aimed to characterize the role of CysLT in the pulmonary infection developed by resistant (A/J) and susceptible (B10.A) mice. 48 h after infection, elevated levels of pulmonary LTC4 and LXA4 were produced by both mouse strains, but higher levels were found in the lungs of susceptible mice. Blocking the CysLTs receptor by MTL reduced fungal loads in B10.A, but not in A/J mice. In susceptible mice, MLT treatment led to reduced influx of PMN leukocytes, increased recruitment of monocytes, predominant synthesis of anti-inflammatory cytokines, and augmented expression of 5- and 15-lipoxygenase mRNA, suggesting a prevalent LXA4 activity. In agreement, MTL-treated macrophages showed reduced fungal burdens associated with decreased ingestion of fungal cells. Furthermore, the addition of exogenous LX reduced, and the specific blockade of the LX receptor increased the fungal loads of B10.A macrophages. This study showed for the first time that inhibition of CysLTs signaling results in less severe pulmonary paracoccidioidomycosis that occurs in parallel with elevated LX activity and reduced infection of macrophages

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

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

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

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 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