BALB/c mice (6-to 8-week-old, males) used for infection and bone marrow harvesting were bred at the School of Medicine – University of São Paulo in pathogen-free conditions. All animal experiments were performed in strict accordance with the Brazilian Federal Law 11,794 establishing the Animal Protection Code for the State of São Paulo. This study was approved by the Ethics Committee on Animal Experiments at the School of Medicine, University of São Paulo (189/14).

The virulent isolate P. brasiliensis Pb18 was cultivated on solid Sabouraud medium at 37°C for 7 days. The fungus was collected and washed three times in phosphate buffered saline (PBS, pH 7.2). After decanting large and agglutinated cells, small isolated yeast cells were counted in a Neubauer chamber. The yeast cells used for experimentation displayed >95% viability as determined by staining with Trypan blue.

The animals were immunosuppressed as previously described (Muñoz et al., 2014). Dexamethasone phosphate (Sigma, St Louis, MO, United States) was administrated daily in drinking water and the dose was calculated as 0.15 mg/kg considering an average water intake of 5 ml per day. Dexamethasone phosphate treatment was initiated 20 days before intratracheal (i.t.) infection and continued until the end of experiment. Immunosuppression was confirmed by the blood leukocyte levels in comparison with untreated mice. Animals were maintained in isolator cages. Cages were exchanged twice a week in a laminar flow hood and all bedding, food and water was autoclaved prior to use. Total leukocyte counts were determined using ∼200 μl of blood obtained via the ocular plexus. The blood was mixed with 20 μL of ACD (citric acid, sodium citrate, dextrose) to prevent clot formation and then Turk solution was used to hemolyze erythrocytes and stain leukocytes. Leukocyte counting was performed using blood smears on glass slides. Blood samples were also counted in a Neubauer chamber.

BALB/c mice were infected intratracheally (i.t.) with P. brasiliensis Pb18 20 days after immunosuppression was initiated. For the procedure, animals were anesthetized intraperitoneally using ∼200 μl of a solution containing 80 mg/kg ketamine and 10 mg/kg xylazine (both from União Química Farmacêutica, Brazil). Five minutes after the injection of the anesthetics, a small longitudinal skin incision was made in the neck to expose the trachea and 3 × 10⁵ yeast cells in 50 μl of PBS was injected. The incision was sutured with 5-0 silk.

P10 peptide with amidated C-terminal was purchased from Peptide 2.0 (Chantilly, VA, United States). HPLC and MS analyses performed by the manufacturer showed that the synthetic P10 was 98% pure.

Dendritic cells were obtained from bone marrow as described previously (Inaba et al., 1992). Briefly, femurs and tibias were collected from male BALB/c mice and flushed with RPMI (Vitrocell, Campinas, Brazil). Samples containing 10⁷ cells were cultivated in RPMI supplemented with 10% fetal calf serum (FCS; Vitrocell), 20 μg/ml gentamicin (Gibco BRL Life Technologies, Grand Island, NY, United States) and recombinant cytokines GM-CSF (30 ng/ml) and IL-4 (15 ng/ml) (both from Peprotech, Rocky Hill, NJ, United States) in 25 cm² cell culture flasks. On the second day, 80% of medium was removed and the same amount of fresh medium with growth factors was added. On the fourth, sixth, and eighth day, cell supernatants were centrifuged and pellets were plated in fresh medium containing growth factor. On the ninth day, cells were harvested and used in the experiments.

Dendritic cells were distributed into 24-well plates at a 10⁶/ml cell density and cultivated in medium alone or with P10 (2.55 × 10^-1mM) at 5% CO₂ and 37°C. After 2 h, cells were removed, washed in FACS buffer and incubated with antibodies to CD11c (BV711, Clone N418), MHC-II (APC-Cy7, Clone M5/114.15.2), CD86 (APC, Clone 16-10A1) and CD-80 (PE, GL1) for 30 min at 4°C. Cells were analyzed by flow cytometry (BD FACSCanto) to assess the expression of surface molecules. In addition, culture supernatants of DCs pulsed with or without P10 or IFN-γ (20 ng/ml) for 2, 12, and 24 h were assessed for IL-10 and IL-12 production using enzyme-linked immunosorbent assay (ELISA) kits (BD OpTeia, San Diego, CA, United States).

Dendritic cells (10⁶/ml) were distributed into 24-well plates and cultivated in medium alone or with P10. After 2 h, cells were harvested and a total of 3 × 10⁵ cells were injected subcutaneously in immunosuppressed mice 30 days post-infection. Mice were also randomized to receive either trimethoprim-sulfamethoxazole (TMP-SMX) 3–15 mg/kg every day for 15 days. Controls groups were treated with PBS.

Mice were euthanized 45 days after infection (day 65 of experiment) and lungs were removed. Portions of lung were weighed, homogenized in PBS and aliquots were plated on agar brain heart infusion (BHI) supplemented with 4% fetal bovine serum (Gibco, Grand Island, NY, United States), 5% supernatant of spent culture of P. brasiliensis Pb192, 10 IU/ml streptomycin-penicillin (Cultilab, Brazil) and 500 mg/ml cycloheximide (Sigma, St. Louis, MO, United States) (Moscardi-Bacchi et al., 1994). Plates were incubated at 37°C for 20 days, and the resulting Colony Forming Units (CFUs) were enumerated.

Lung sections were homogenized in PBS with protease inhibitors: benzamidine HCl (4 mM), EDTA disodium salt (1 mM), N-ethylmaleimide (1 mM), and pepstatin (1.5 mM) (Sigma, St. Louis, MO, United States). Supernatants were assayed for IL-4, IL-10, IL-12, and IFN-γ using ELISA kits (BD OpTeia, San Diego, CA, United States). The detection limits of the assays were as follow: 7.8 pg/ml for IL-4, 31.3 pg/ml for IFN-γ and IL-10, and 62.5 pg/ml for IL-12, as determined by the manufacturer.

Additional lung sections were fixed in 10% buffered formalin and embedded in paraffin for cutting. Sections were stained by the Gomori-Grocott method and hematoxylin/eosin (HE).

Data shown are representative of at least two independent experiments. For in vivo experiments, a total of six animals per group were used. In vitro experiments were done in triplicates for each condition. Statistical analyses were performed using GraphPad Prism 5 software (San Diego, CA, United States). The results were expressed as mean values and standard deviations (SD) of the indicated values. Tukey’s significant difference test was employed for non-parametric data. p-values of ≤0.05 were used to indicate statistical significance.

Dendritic cells from BALB/c mice were analyzed by flow cytometry. Cell were first gated by granularity (SSC) and size (FSC) and then analyzed for CD11c and MHC-II expression. Double positive cells for CD11c and MHC-II were considered as differentiated DCs, reaching a frequency of appropriately 60% (Figure 1), as previously observed (Magalhães et al., 2012). Therefore, for in vivo experiments, each vaccine dose contained a total of 3 × 10⁵ cells, of which 1.8 × 10⁵ were DCs pulsed or not with P10.

We assessed the expression of MHC-II, CD80, and CD86 molecules within MHCII⁺/CD11c⁺ cell population after 2 h of stimulation with or without P10. According to the Median Fluorescence Intensity (MFI), we observed a decrease in MHC-II and increase of CD86 expression for P10-pulsed DCs and no significant changes for CD80 in comparison to control group (Figure 2A). However, the percentage of CD80⁺/CD86⁺ DCs showed a significant increase (Figure 2B), demonstrating that P10 might have a stimulatory effect for DCs activation.

Dendritic cells pulsed with P10 showed increased production of IL-10 and IL-12 compared with untreated DCs (Figure 3). P10 induced an increased production of these cytokines, which was even higher in DCs pulsed with IFN-γ, although these levels were higher in the setting of IFN-γ at 12 and 24 h.

Dexamethasone administered in water “ad libitum” to mice over a 65-day period significantly reduced the peripheral leukocyte counts in a sustained manner (Figure 4). Figure 5 depicts leukocyte counts on day 65, demonstrating decreased leukocyte counts in all groups in comparison with non-immunosuppressed mice (Sham). Leukocyte levels of immunosuppressed animals (DEX-only) were statistically different from all groups treated with DCs (DC, DC + TMP-SMX, DC-P10, DC + P10 + TMP-SMX). All of the DC administration conditions increased leukocyte levels in comparison to dexamethasone treated mice (DEX only). However, treatment with DCs did not significantly increase leukocyte levels in comparison to INFECT-only group.

The pulmonary fungal burden of immunosuppressed mice infected with P. brasiliensis was significantly reduced by administration of either TMP-SMX or unprimed DCs (Figure 6). The combination of unprimed DCs and TMP-SMX further reduced CFUs. However, the greatest reduction occurred when DCs primed with P10 were administered and this effect was further augmented when TMP-SMX was added to therapy.

IL-10, IL-12, IFN-γ, and TNF-α levels were measured by ELISA in the lungs of mice with different treatment regimens (Figure 7). A significant reduction of IL-10 was observed in all groups that received DCs with the greatest reduction occurring in mice that received P10-primed DCs together with TMP-SMX. Increased levels of IL-12 occurred in mice treated with P10-primed DCs with or without TMP-SMX treatment. Levels of TNF-α were reduced after the administration of DCs with TMP-SMX or primed DCs, but were only significantly reduced in the combination of primed DCs with TMP-SMX treatment. No significant differences were detected in IFN-γ levels, although there was a trend to lower levels in the groups treated with TMP-SMX combined with either P10-primed or unprimed DCs.

Lung sections were subjected to Gomori-Grocott and HE staining. Gomori-Grocott stained micrographics (Figure 8) depict the fungal burden, showing the presence of fungal cells in lung tissue. Figure 8A demonstrates uninfected immunosuppressed mice with an absence of fungal cells. Abundant fungal cells were present in tissues of immunosuppressed mice in the following groups: infected and not treated (Figure 8B), infected and treated with (Figure 8C) and non-pulsed DCs treated groups (Figure 8D). In contrast, lung sections from immunosuppressed and infected animals treated with P10-pulsed DCs (Figure 8E), non-pulsed DCs and TMP-SMX (Figure 8F) and P10-pulsed DCs and TMP-SMX (Figure 8G) had a significantly lower fungal burden. Moreover, the animals that received P10-pulsed DCs (Figures 8E,G) presented the lowest burden of fungal cells in lung tissue.

Hematoxylin/eosin staining (Figure 9) revealed normal tissue architecture in non-infected mice (Figure 9A). It was observed the presence of granulomas with fungal cells and intense inflammatory infiltrates in lung parenchyma of immunosuppressed and infected-only (Figure 9B), infected and treated with TMP-SMX (Figure 9C) and infected and treated with P10-pulsed DCs (Figure 9E) groups. Granuloma-like structures were also observed in the other infected groups, but inflammatory infiltrates were reduced in the non-pulsed DCs (Figure 9D), non-pulsed DCs and TMP-SMX (Figure 9F) and P10-pulsed DCs and TMP-SMX treated (Figure 9G) groups. Lung parenchyma was significantly best preserved and less compromised in P10-pulsed DCs and TMP-SMX treated (Figure 9G) group.