Figure 3

Activation of the innate immune response mediated by DNA-HSP65. (A) Expression of costimulatory molecules and HLA-DR on the surface of macrophages (Mφ) and DC stimulated with DNA vaccine. Cells were stimulated with DNA vaccine, DNA vector or LPS (positive control). After 48 h stimulation, the expression of surface molecules was evaluated by flow cytometry. Each column represents the mean percentage of Mφ or DC positive for CD80, CD86 or HLA-DR, or DC positive for CD83 ± SEM. Cells were obtained from 11 cultures of Mφ and 7–9 cultures of DC from different healthy individuals. (B) Mφ and DC were incubated for 48 h with DNA vaccine, DNA vector or LPS and the production of TNF-alpha, IL-6, IL-10 and IL-12p40 was evaluated. Each column represents the mean ± SEM of cytokine production detected in 6–8 Mφ cultures or 7–10 DC cultures obtained from healthy donors. * p < 0.05; ** p < 0.01; *** p < 0.001, in relation to non-stimulated Mφ. #p < 0.05; ##p < 0.01; ###p < 0.001, in relation to non-stimulated DC. (C) Intracellular growth of M. tuberculosis in Mφ or DCs stimulated with DNA-HSP65. Mφ and DCs were stimulated with DNA vaccine or DNA vector (both at 20 μg/mL) for 48 h and infected with M. tuberculosis at MOI = 1. CFU numbers were determined at 4 h (day 0) and 7 days (day 7) after infection. Results represent the mean ± SEM of five experiments (for DCs) or three experiments (for Mφ). * p < 0,05, when compared to CFU numbers recovered on days 0 and 7 postinfection.