Generation of bone marrow-derived dendritic cells and activation

The femur and tibia from C57BL/6, MyD88^-/- (B6.129P2(SJL)-MyD88^tm1.Defr/J), TLR2^-/- (B6.129-Tlr2^tmKir/J), and TLR4^-/- (B6.B10ScN-Tlr4^lps-del/JthJ) mice on C57BL/6J background (Jackson Research Laboratories, Bar Harbor, ME, USA) were used to generate bone marrow-derived DCs, as previously described [17]. Briefly, hematopoietic bone marrow stem cells were cultured in RPMI-1640 medium supplemented with 5% heat-inactivated fetal bovine serum, 10 mM HEPES, 2 mM L-glutamine, and 50 μM 2-mercaptoethanol (Gibco) and complemented with 20% granulocyte-macrophage colony-stimulating factor from mouse-transfected Ag8653 cells [46]. Cell purity was confirmed to be higher than 85% CD11c⁺ by flow cytometry as previously described [17]. Albeit this culture system cannot completely rule out the presence of other innate cells (such as macrophages), it represents an enriched source of DCs. Consequently, cytokines produced by contaminating cells would be minor [47]. Prior to infection, cells were resuspended at 1 x 10⁶ cells/mL in complete medium and stimulated with the different S. suis strains (1 x 10⁶ CFU/mL; initial MOI = 1). Conditions used were based on those previously published [17, 20]. Supernatants were collected 16 h following infection with S. suis, time at which secreted cytokine levels were maximal in the absence of S. suis-induced DC cytotoxicity as determined by lactate dehydrogenase release [17, 20]. Non-infected cells served as negative controls. Secreted levels of tumor necrosis factor (TNF), interleukin (IL)-6, C-C motif chemokine ligand (CCL) 2, CCL3, and C-X-C motif chemokine ligand (CXCL) 1 were quantified by sandwich ELISA using pair-matched antibodies from R&D Systems (Minneapolis, MN, USA) according to the manufacturer’s recommendations.