Heat-killed M. pachydermatis suspension
M. pachydermatis (standard strain CBS-1696 isolated from dog) was maintained in Sabouraud medium incubated at 32ºC for 5 to 7 days at the Universidade Paulista - Unip, Molecular Biology Laboratory. The fungus extract was made with a suspension containing 5 yeasts for each macrophage (5:1), diluted in PBS and boiled at 120ºC for 30 minutes and then filtered with a 0.20 µm filter to remove debris.
E. cuniculi spores
Spores of E. cuniculi (genotype I) (from Waterborne Inc., New Orleans, LA, USA) that were used in this experiment were previously cultivated in a rabbit kidney cell lineage (RK-13, ATCC CCL-37) in RPMI medium supplemented with 10% of fetal calf serum (FCS- Sigma-Aldrich, St. Louis, MO, USA), pyruvate, nonessential amino acids (Sigma-Aldrich, St. Louis, MO, USA), and gentamicin at 37 °C in a humidified atmosphere with 5% CO2. The spores were purified by centrifugation and cellular debris was excluded by 50% Percoll.
Macrophage cultures
RAW 264.7 macrophages were maintained in RPMI (Sigma-Aldrich, St Louis, MO, USA) supplemented with 10% FCS (R10 at 37 °C in a humidified atmosphere with 5% CO2. The maintenance of the cultures to obtain the macrophages was made with periodic changes of the medium R10, respecting the necessity of the culture. The medium was changed every 2 days, until 70 to 80% of confluence was reached.
Macrophages treatment and infection
Macrophages were platted in 24-well plates with 3x105 cells/well in 300 µl of R10 and incubated overnight at 37 °C in a 5% CO2 atmosphere. Heat-killed M. pachydermatis suspension were added for 1 hour to the macrophages. After removal of supernatants, the cultures were infected with E. cuniculi spores at a 2:1 concentration (2 E. cuniculi spores for each macrophage) for 15, 30, 60 minutes and 48 hours. The experiments were done in triplicate. After the established periods, the supernatants were removed and stored at -80ºC for subsequent measurement of cytokines. To investigate to visualize the spores inside the phagocytic cells, 10 μl of Calcofluor (Sigma-Aldrich, St. Louis, USA) was added per cell cultures. Phagocytic capacity and index were calculated according to the formula: PC = number of phagocytes containing at least one ingested spore/100 phagocytes and PI = total number of phagocytic spores/100 phagocytes containing spores.
Quantification of Cytokines
Cytokines were measured in culture supernatants using CBA Mouse Th1/Th2/Th17 Cytokine Kit (BD Biosciences, CA, EUA), according to the manufacturer’s instructions. A mixture of 25 μl of each sample, 25 μl of mixed beads containing specific capture antibodies, and 25 μl of PE-conjugated detection antibody was incubated for 2 h at room temperature in the dark. Subsequently, the samples were centrifuged, washed, and resuspended for analysis by two-color flow cytometry using C6 Accuri (BD Biosciences, Mountain View, CA, USA). All the analyses were done using the software FCAP Array 3.0.
Transmission Electron Microscopy (TEM)
Macrophages was adjusted to 1×107 cells, transferred to 25 cm2 bottles and incubated overnight in R10 medium at 37 °C with 5% CO2 atmosphere. Then, heat-killed M. pachydermatis suspension were added for 1 hour to the macrophages, respecting the 5:1 ratio. After removal of the extract, the cultures were infected with E. cuniculi spores at a 2:1 concentration for 15, 60 minutes and 48 hours. The cultures were collected and fixed using 2% glutaraldehyde in 0.2 M cacodylate buffer (pH 7.2) at 4 °C for 10 h. They were then post-fixed in 1% OsO4 buffer for 2h. Semi-thin sections stained with toluidine blue were made for visualization by light microscope, and ultrathin sections were made for TEM analysis (LEO EM 906E).
Statistical Analysis
Prism 5 (GraphPad Software Inc, La Jolla, CA, EUA) using a one-way analysis of variance (ANOVA) followed by Bonferroni test post hoc analysis was used to compare samples with untreated macrophage cells. All results are presented as mean ± SEM. A p value of 0.05 or less was considered significant.