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. 2010 May 21;5(5):e10757.
doi: 10.1371/journal.pone.0010757.

Immunological basis for the gender differences in murine Paracoccidioides brasiliensis infection

Camila Figueiredo Pinzan  1 , Luciana Pereira Ruas, Anália Sulamita Casabona-Fortunato, Fernanda Caroline Carvalho, Maria-Cristina Roque-Barreira
Affiliations

Immunological basis for the gender differences in murine Paracoccidioides brasiliensis infection

Camila Figueiredo Pinzan et al. PLoS One. .

Abstract

This study aimed to investigate the immunological mechanisms involved in the gender distinct incidence of paracoccidioidomycosis (pcm), an endemic systemic mycosis in Latin America, which is at least 10 times more frequent in men than in women. Then, we compared the immune response of male and female mice to Paracoccidioides brasiliensis infection, as well as the influence in the gender differences exerted by paracoccin, a P. brasiliensis component with carbohydrate recognition property. High production of Th1 cytokines and T-bet expression have been detected in the paracoccin stimulated cultures of spleen cells from infected female mice. In contrast, in similar experimental conditions, cells from infected males produced higher levels of the Th2 cytokines and expressed GATA-3. Macrophages from male and female mice when stimulated with paracoccin displayed similar phagocytic capability, while fungicidal activity was two times more efficiently performed by macrophages from female mice, a fact that was associated with 50% higher levels of nitric oxide production. In order to evaluate the role of sexual hormones in the observed gender distinction, we have utilized mice that have been submitted to gonadectomy followed by inverse hormonal reconstitution. Spleen cells derived from castrated males reconstituted with estradiol have produced higher levels of IFN-gamma (1291+/-15 pg/mL) and lower levels of IL-10 (494+/-38 pg/mL), than normal male in response to paracoccin stimulus. In contrast, spleen cells from castrated female mice that had been treated with testosterone produced more IL-10 (1284+/-36 pg/mL) and less IFN-gamma (587+/-14 pg/mL) than cells from normal female. In conclusion, our results reveal that the sexual hormones had a profound effect on the biology of immune cells, and estradiol favours protective responses to P. brasiliensis infection. In addition, fungal components, such as paracoccin, may provide additional support to the gender dimorphic immunity that marks P. brasiliensis infection.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Spleen cells from male and female infected mice secrete different levels of cytokines after paracoccin stimulus.
Spleen cells of mice of both sexes (n = 6) infected with P.brasiliensis yeast were collected after 7 and 30 days post infection and cultured for 48 hours under stimulation with paracoccin (0.7 µg/ml), LPS (1 µg/ml) or only medium. The concentrations of IL-12p40, TNF-α, IFN-γ and IL-10 in supernatants were measured by ELISA. Each bar represents the mean ± SD of duplicates and is representative of four experiments made in duplicate. * p<0.05 significant female versus male of the same group. All cytokine data were normalized against those of the non-infected male and female mice.
Figure 2
Figure 2. Expression of mRNA for IL-4, GATA-3 and T-bet from male and female infected mice.
Spleen cells of male and female mice infected with P.brasiliensis yeast were collected after 30 days post infection and cultured for 48 hours under paracoccin stimulation (0.7 µg/ml) (2A, B and C), LPS (1 µg/ml) (2C) or only medium (2C). The levels of mRNA relative expression for the IL-4, T-bet and GATA-3 were determined by real-time PCR, using the β-actin gene as control. All real-time PCR data were normalized against those of the non-infected male and female mice. The results represent the mean±SD of six mice per group, from a representative experiment of three assays. * p<0.05 significant when compared with opposite sex.
Figure 3
Figure 3. Quantification of P. brasiliensis through real-time PCR and Colony-forming units (CFU).
Evaluation of the fungal burdens in lungs and liver of male (grey bars) and female (open bars) mice 30 days after intraperitoneal infection with yeast cells. The bars represent the mean±SD obtained from duplicate samples in groups of six animals. * P<0.05, compared with female infected mice.
Figure 4
Figure 4. Phagocytosis of P. brasiliensis yeasts by macrophages from male and female mice.
Macrophages treated or not with paracoccin (50 µg/well) were challenged with Pb FITC- conjugated yeasts for 4 hs. The cells were stained with Evans Blue and counted in a fluorescence microscope (100x) to determine the yeast Phagocitic index (3A). Panel B illustrates the result and allow to observe that no extracellular attachment of yeasts after 4 hs of incubation. The results on panel A represent the mean±SD from a representative experiment of three assays.
Figure 5
Figure 5. NO production and viability of Pb yeasts after phagocytosis by macrophages from male and female mice.
Macrophages from male and female mice were incubated with paracoccin (PCN), P. brasiliensis yeasts (Pb), PCN plus Pb, PCN (pre-treated with GlcNac) plus Pb or only medium and nitrite levels were detected by the Griess method (4A). For the evaluation of viability of Pb cells macrophages were stimulated with PCN, PCN (pre-treated with GlcNac) or only medium and challenged with live Pb. The determination of the number of viable fungi was made by CFU counts (4B). The results represent the mean±SD from a representative experiment of three assays. * p<0.05 significant when compared with male of the same group.
Figure 6
Figure 6. IFN-γ (A) and IL-10 (B) production by spleen cells from male and female infected mice of different treated groups.
Spleen cells from male and female 30 days infected mice from different groups were collected and cultured for 48 hours under paracoccin stimulation (7 µg/ml). The concentration of IFN-γ and IL-10 in supernatants was measured by ELISA. The results represent the mean±SD of six mice per group, from a representative experiment of two assays. * p<0.05 significant when compared with opposite sex of the same group.
Figure 7
Figure 7. Possible role of hormones in the course of P. brasiliensis infection.
Male and female mice were infected with P. brasiliensis yeasts cells and several events of the immune responses mounted by both genders of mice are displayed in this Figure.
See this image and copyright information in PMC

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