Cell culture and inflammatory cell phenotypes

Single-cell suspensions of spleen tissue samples from TLR2^−/− or WT mice at the 4th wpi were aseptically prepared, diluted to a concentration of 2 × 10⁶ cells/mL, and dispensed into 48-well plates in a total volume of 500 μl of complete RPMI-1640 medium (1 × 10⁶ cells/well; Gibco) with or without L. infantum crude antigen (50 μg/mL). The cell culture supernatants were harvested after 72 h of culture at 37°C in 5% CO₂ and the IL-17, IFN-γ, and IL-10 levels in the supernatants were determined using commercial ELISA kits. For detection of IFN-γ and IL-17 in the liver, tissue samples were harvested by tissue trimmer, weighed, and titered in 1 mL of PBS Complete (Roche Diagnostics, Mannheim, Germany) containing protease inhibitor cocktail. The levels of cytokines were determined using commercial ELISA kits from R&D Systems for IL-10 and IL-17(catalog number DY417 and DY421, respectively) and BD Biosciences for IFN-γ (catalog number DY485).

For leukocyte identification, spleeny cells isolated from WT and TLR2^−/− were stained with fluorescent labeled monoclonal antibody that recognizes a target feature on or in the cell. The inflammatory cells were gated based on their characteristic size (FSC) and granularity (SSC), and the T lymphocytes (CD4⁺CD3⁺), dendritic cell activation markers (CD11c^highCD40⁺, CD11c^highCD86⁺, and CD11c^highMHC-II⁺) and neutrophils (Ly6G^highMHCII⁻) were identified individually. For intracellular staining, the cells were cultured with PMA (50 ng/mL) and ionomycin for 4 h in order to obtain the maximum cytokine production, permeabilized with a Cytofix/Cytoperm Kit (BD Biosciences) according to the manufacturer's guidelines and stained with anti-IFN-γ or anti-IL-17 conjugated to APC-Cy7 and Alexa Fluor 700 and with anti-CD3 and anti-CD4 for surface staining with FITC and PerCP, respectively. Rat IgG2b and IgG2a were used as the isotype controls. All the antibodies were supplied from BD Biosciences and eBiosciences (San Diego, CA, USA). Cell acquisition was performed using a FACSort flow cytometer. The data were plotted and analyzed using FlowJo software (Tree Star, Ashland, OR, USA). The total leucocyte counts were determined by measuring the relative expression of the leucocyte subpopulations stained with specific antibody in 300,000 acquired events proportional to the leukocyte number obtained in a Neubauer chamber.