Recombinant ifn γ

The concentrations of IL-12p40, IL-12p70, IL-4, and IL-17 in the culture supernatants were measured in triplicate using ELISA kit (eBioscience, San Diego, CA). The level of IFN-γ was quantified with specific murine anti-IFN-γ HB170 coating and biotinylated anti-IFN-γ XMG1.2 mAbs and the standard curve was generated using recombinant IFN-γ (BD Bioscience). The wells were finally washed again with PBST (0.05% Tween-20 in PBS) and o-phenylenediamine (OPD) containing citrate and H₂O₂ was added to each well and incubated for 20 min at room temperature. To stop the reaction, 2 N H₂SO₄ was added to each well. Developed colors were detected on a VMax kinetic microplate reader at 490 nm. The measurement was performed in triplicate.

Autophagy was triggered by treatment with recombinant IFN-γ (10 ng/mL; BD Pharmingen) or rapamycin (200 ng/mL; Assay Designs) for 18 h at 37°C in full nutrient medium [13 (link),20 (link)]. Alternatively, autophagy was induced by amino acid and serum starvation through incubation of cells in PBS at 37°C for 18 h [10 (link),20 (link)].

Bone marrow-derived macrophages (BMDM) were prepared as described previously (31 (link)). Bone marrow was collected from femurs and tibiae of 8- to 10-week-old mice, passed through a cell strainer, and centrifuged at 400 × g for 5 min at 4°C. Cells were suspended in BMDM medium (Dulbecco modified Eagle medium [DMEM], 10% FCS, 20% L929 conditioned medium as a source of colony-stimulating factor 1, 0.1% gentamicin, 1% sodium pyruvate) and seeded at a density of 5 × 10⁶ cells in 10 ml BMDM growth medium into 10-mm² tissue culture dishes. An additional 5 ml BMDM medium was added 3 days later. At 7 days postseeding, the fully differentiated and adherent macrophages were washed with DMEM, removed by scraping, and plated into 6-well plates (1 × 10⁶ cells/well). BMDM were confirmed to be >90% F480⁺and CD11b⁺ cells by FACS analysis. Cells were treated with a mixture of poly(I)·poly(C) [poly(I·C)] (5 μg) (high molecular weight; InvivoGen, San Diego, CA) and FuGENE 6 transfection reagent (Promega, Madison, WI) in DMEM following the manufacturer's protocol. After 4 or 12 h of poly(I·C) treatment, poly(I·C) was removed and 50 ng/ml recombinant IFN-γ (BD Pharmingen) was added to cultures and left for 18 h. After stimulation, cells were lysed directly in 800 μl TRIzol (Invitrogen) and stored at −70°C for RNA isolation as described above.

Thioglycollate-induced peritoneal macrophages were isolated by adherence (2 h at 37°C in 5% CO₂) to plastic-bottom tissue-culture plates (1×10⁶ cells/well in 24 well plates). Macrophages were washed to remove nonadherent cells and cultivated overnight with fresh complete medium (DMEM, Dulbecco's Modified Eagle's Medium, Sigma, containing 10% fetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin) in the presence or absence of recombinant IFN-γ (20 ng/ml in culture medium, BD-Pharmingen) and/or 1MT (1-methyl-d,l-tryptophan, Sigma-Aldrich). 1MT was used in the concentration of 1 mM that was previously shown to efficiently inhibit the IDO activity of macrophages [6] (link). Macrophage cultures were infected or not with P.brasiliensis yeasts in a macrophage∶yeast ratio of 25∶1 and cocultivated for 4 h. This ratio was previously determined and was shown to be non-deleterious to macrophage cultures and adequate for killing assays [38] (link). The monolayers were then washed to remove nonadherent cells and incubated for an additional 48 h period in the presence or absence of IFN-γ (20 ng/ml in culture medium, BD Biosciences) and/or 1MT.