The mAbs specific for CD80 (clone BB1) coupled to fluorescein isothiocyanate (FITC), CD86 (clone IT2.2), HLA-DR (clone G46-6), CD83 (clone HB15e), coupled to phycoerythrin (PE), CD11b (clone ICRF44), CD11c (clone B-ly6), and CD123 (clone 9F5) coupled to Cy-chrome, were purchased from BD (BD, San Diego, CA, USA). The mAbs specific for CD1c (clone AD5-8E7) and BDCA-4 (clone AD5-17F6) coupled to PE were obtained from Miltenyi Biotec (Auburn, CA, USA). The purified mAb TLR9 (26C593 clone) was obtained from Imgenex (San Diego, CA, USA), and the biotinylated anti-mouse IgG was obtained from Bioscience (Toronto, Canada).

DNA-HSP65 vaccine was derived from pVAX vector (Invitrogen, Carlsbad, CA, USA), which had previously been digested with BamHI and Not I (Invitrogen), and a 3.3-kb fragment (corresponding to the M. leprae HSP65 gene) was inserted. The vector pVAX was used as a control. Plasmids were replicated in DH5alpha Escherichia coli and purified with Endofree Plasmid Giga kit (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol. Endotoxin levels were determined using a QCL-1000 Limulus amoebocyte lysate kit (Cambrex Company, Walkersville, MD, USA), and were less than 0.1 endotoxin units (EU)/μg DNA.

The DNA vaccine was labeled with Alexa Fluor 594 or Alexa Fluor 488 by Universal Linkage System (ULS™) using the ULYSIS nucleic acid labelling kit (Invitrogen, Molecular Probe) as previously described 23. The conformation of labeled plasmid was not altered.

Peripheral blood mononuclear cells (PBMCs) were obtained from blood donated by healthy volunteers at the Fundação Hemocentro de Ribeirão Preto (Ribeirão Preto Haemocentre Foundation, Ribeirão Preto, Brazil). This work was approved by Comitê de Ética em Pesquisa do Hospital das Clínicas de Ribeirão Preto (Ethic Committee Research from Ribeirão Preto Clinical Hospital, Brazil). Mononuclear cells were separated by density gradient centrifugation using Ficoll-Paque (GE Life Sciences, Uppsala, Sweden). Monocytes were purified by density gradient centrifugation using Percoll (GE Life Sciences). Macrophages and DCs were differentiated by culturing monocytes in 24-well tissue culture plates (Corning, Corning, NY, USA) with 1 ng/mL of GM-CSF (BD) or with 14 ng/mL of IL-4 (BD) and 7 ng/mL of GM-CSF, respectively, for 7 days at approximately 1 × 10⁶ cells/mL in RPMI 1640 (Sigma-Aldrich) supplemented with 10% foetal bovine serum (FBS) (Invitrogen, Gibco), streptomycin/ampicillin (Invitrogen, Gibco) and gentamicin (Invitrogen, Gibco). Plasmacytoid dendritic cells (pDCs) were purified from PBMCs by positive selection with immunomagnetic microbeads (Miltenyi Biotec, Auburn, CA, USA) based on BDCA-4 expression.

An amount of 5 × 10⁴ macrophages and DCs were stimulated with 5 μg of Alexa Fluor 594-labeled DNA vaccine for 4 h for uptake assays. These cells were mounted on glass coverslips with Cell-tak (BD, New Bedford, MA, USA) with Fluormount-G (Electron Microscopy Sciences) and analysed with a Nikon Eclipse E800 fluorescence microscope (Nikon USA, Melville, NY). Images were acquired with a Nikon DXM-1200 digital camera (Nikon USA) connected to the microscope.

Macrophages, myeloid and plasmacytoid DCs were placed onto Cell-Tak-coated glass coverslips (BD Biosciences, New Bedford, MA, USA), fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA, USA) for 15 min at 37°C, and permeabilised with 0.3% Triton X-100 (Sigma-Aldrich) for 10 min at 25°C. The cells were washed with 0.1 M glycine (Sigma-Aldrich) for 5 min, and then labeled with purified mAb anti-TLR9 (5 μg/mL) (Imgenex) for 30 min at 4°C. Subsequently, the cells were incubated with biotinylated anti-mouse IgG (7.5 μg/mL; Bioscience) for 1 h at room temperature. Finally, cells were incubated with streptavidin conjugated to Alexa Fluor 488 (Molecular Probes, Eugene, OR, USA) for 30 min, mounted on glass slides with Fluormount (Electron Microscopy Sciences) and examined with a Leica TCS SP2 AOBS (Leitz, Manheim, Germany).

Macrophages and DCs were stimulated with 20 μg/mL of DNA vaccine or DNA vector over a 48 h period to evaluate cell surface phenotype. Additionally, 500 ng/mL of LPS (Salmonella typhimurium, Sigma) was used as positive control of cellular activation. To study the capacity of the cells to uptake DNA, macrophages and DCs were stimulated with Alexa Fluor 488-labeled DNA vaccine for 1 h. Then, the cells were analysed by flow cytometry (FACSort, Becton Dickinson, San Jose, CA, USA). A biparametric gate in the forward (FSC) and side scatter (SSC) dot plot was drawn around the macrophages or DCs populations. Approximately 4000 Mac-1⁺ (macrophages) or CD11c⁺ (DC) cells were acquired. The computer analysis was made using the Cell-Quest program (version 3.3).

Supernatants from macrophages and DCs cultures stimulated with DNA vaccine, DNA vector or LPS were harvested at 48 h after stimulation. Cytokine levels were determined by ELISA using recombinant cytokines for generating standard curves. Purified mAb anti-TNF-alpha (clone Mab1), anti-IL-6 (clone MQ2-13A5), anti-IL-10 (clone JES3-19F1), anti-IL-12p40 (clone C8.3), as well as biotinylated mAb anti-TNF-alpha (clone Mab11), anti-IL-6 (clone MQ2-39C3), anti-IL-10 (clone JES3-12G8), and anti-IL-12p40 (clone C8.6) were obtained from BD and used according to the manufacturer's instructions. Additionally, supernatants from cultures of monocyte-derived DCs and peripheral blood pDCs were assayed for IFN-alpha detection by Interferon-alpha ELISA Kit, (Immuno-Biological Laboratories, Minneapolis, MN, USA).

M. tuberculosis H37Rv (ATCC n° 27294) was obtained from an aliquot frozen at -70°C. Fifty microliters of this aliquot (viability greater than 85%) were cultured in Lowenstein-Jensen medium for 20–30 days at 37°C. M. tuberculosis was then added to 10 mL of 7H9 medium (Difco, BD, Detroit, USA) and incubated for 7–10 days at 37°C. After analysis of viability, the bacilli number was determined by optic density of the culture at 540 nm. The bacterial suspension was then centrifuged at 4000 × g for 20 min and the pellet was diluted in RPMI 1640 supplemented with 10% FBS and antibiotic-free. Macrophages and DCs were infected with M. tuberculosis with a multiplicity of infection (MOI) of 1 bacillus per 1 cell (MOI = 1). Four hours after infection the supernatants were removed, the cells were washed, centrifuged at 900 × g for 10 min and then lysed with a solution of 0,25% SDS-PBS (J.T. Baker, Phillipsburg, NJ, USA). Serial dilutions were plated in Middlebrook 7H11 agar medium. The same procedure was performed at 7 days after infection. The colony forming units (CFU) were counted after 20–30 days.

Total RNA was isolated from PBMCs by extraction in Trizol Reagent (Invitrogen) and alcohol precipitation, followed by an additional treatment with DNAse I amplification grade (Invitrogen) to avoid genomic and plasmid DNA contamination. Total RNA (1 μg) was reverse transcribed using oligo(dT) primers and reverse transcriptase (Invitrogen) according to the manufacturer instructions. The PCR amplification was carried out using 3 μL of cDNA preparation and specific primer pairs of M. leprae Hsp65 (sense 5'-TCAAGGTGGCGTTGGAAGC-3' and antisense 5'-CCGTGACCCACTGAAAGGTTA-3'; giving a 103-bp band). Samples were submitted to 35 cycles of amplification in a PTC-200 Peltier Thermal Cycler (MJ Research Inc., Watertown, MA, USA). In each cycle, denaturation was performed at 95°C for 45 sec, primers were annealed to target cDNA at 65°C for 40 sec, and extension was carried out at 72°C for 90 sec. Messenger RNA for beta-actin was detected by PCR using cDNA and beta-actin-specific primers (sense 5' ATGTTTGAGACCTTCAACA-3' and antisense 5'-CACGTCAGACTTCATGATGG-3'; giving a 495-bp band). The PCR products were visualised by ultraviolet illumination after electrophoresis on a 1% agarose gel containing ethidium bromide.

A total of 2 × 10⁵ PBMCs were stained with 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester (CFSE; Invitrogen, Molecular Probes) and plated in 96 well round bottom culture plate (Corning) in RPMI 1640 (Sigma-Aldrich) supplemented with 10% autologous serum, streptomycin/ampicillin (Invitrogen, Gibco) and gentamicin (Invitrogen, Gibco). PBMCs were cultured during 7 days with recombinant Hsp65 or during 12 days with DNA vaccine or vector. Additionally, PBMC were cultured with recombinant Hsp65 plus DNA vaccine or vector during 12 days. The cells were then harvested and evaluated for their CFSE content by flow cytometry. As positive control, PBMCs were stimulated with phytohemagglutinin. A gate in FSC and SSC dot plot was drawn around the lymphoblast population and the frequency of CFSE-containing cells was determined.

Data are expressed as means ± SEM. Statistical significance of differences was determined by the unpaired Student's t-test. Differences which provided P < 0.05 were considered to be statistically significant. Statistical analyses were performed by using PRISM software (version 4.0; GraphPad, San Diego, CA, USA).

Freshly isolated monocytes cultured with GM-CSF differentiate into macrophages, whereas those cultured with GM-CSF plus IL-4 differentiate into DCs. As expected, macrophages and DCs differed morphologically. Macrophages were characterized as large and adherent cells, while DCs were round, smaller than macrophages and presented cytoplasmic extensions (dendrites) (data not shown). Macrophages and DCs were characterized as CD11b⁺ and CD11c⁺ cells, respectively. Both CD11b⁺ and CD11c⁺ cells constitutively expressed CD86 and HLA-DR molecules (Figures 1A and 1B). We further observed that 17% of DCs were characterized as CD11c⁺CD1c⁺ and 98% were CD11c⁺ BDCA-4⁺. CD123, a receptor exclusively expressed by plasmacytoid dendritic cells (pDCs), was not detected on the surface of either CD1c⁺ or BDCA-4⁺ cells (Figure 1C). Moreover, we also evaluated the IFN-alpha production by these cells. An experimental control was performed with pDCs. The pDCs stimulated with DNA vaccine secreted higher levels of IFN-alpha in comparison to the unstimulated pDCs (Figure 1D). On the other hand, monocyte-derived DCs did not secrete IFN-alpha.

In order to analyse the expression of TLR9 by macrophages and DCs, confocal microscopy was used (Figure 1E). It was found that monocyte-derived macrophages and DCs displayed strong cytoplasmic staining for TLR9, indicating its presence in intracellular compartments. pDCs isolated from peripheral blood were stained and used as positive control for TLR9 expression. These results were confirmed by flow cytometry analyses (data not shown).

To determine whether human macrophages and DCs would be able to taken up naked DNA-HSP65, we stimulated cells with fluorescent-labeled DNA vaccine. Four hours after stimulation with naked DNA-HSP65, fluorescent endocytic vesicles were observed in the cytoplasm of macrophages and DCs (Figure 2A), suggesting that the plasmid was taken up by these cells during this period. Flow cytometry analyses showed that macrophages and DCs had different ability to taken up naked DNA vaccine (Figure 2B). The DNA vaccine or vector was uptaken by almost 100% of macrophages CD11b⁺ and by approximately 85% of DCs CD11c⁺. The analysis of median fluorescence intensity, which indicates the ability to take up DNA on a per-cell basis, show that DCs behaved with a bimodal pattern: while a subpopulation of CD11c+ cells displayed low uptake rates, the other one presented high uptake capacity. These values were similar when the cells were stimulated with either vaccine or vector. These results suggest that the uptake of DNA-HSP65 vaccine or vector by DCs was higher than that observed in macrophages.

In order to study the activation of innate immune response mediated by DNA-HSP65, human macrophages and DCs stimulated with the DNA vaccine were evaluated regarding the cytokine production, expression of surface markers and microbicidal activity. In relation to cellular phenotype, we did not observe any variation in the number of DNA vaccine-stimulated macrophages expressing HLA-DR, CD80 or CD86 molecules (Figure 3A) or changes in the median fluorescence intensity (data not shown). Conversely, the stimulation of DCs with DNA vaccine resulted in an up-regulation of CD80, CD86 and CD83 (a maturation marker) expression (Figure 3A). After stimulation with DNA vaccine or DNA vector, no difference was seen in the number of DCs expressing HLA-DR. In all experiments LPS was used as positive control of cellular activation.

Regarding the cytokines production, macrophages stimulated with DNA vaccine secreted levels of TNF-alpha, IL-6 and IL-10 significantly higher than those of the unstimulated cells. Similar levels of these cytokines were secreted by vector-stimulated cells (Figure 3B). Notably, vaccine-stimulated macrophages did not produce either IL-12p40 (Figure 3B) or IL-12p70 (data not shown). In contrast, vaccine or vector-stimulated DCs provided significantly higher levels of TNF-alpha and IL-12p40 than those provided by the unstimulated cells. The production of TNF-alpha by DCs was also observed in experiments that were carried out with immunostimulatory CpG, an additional control (data not shown). DCs produced lower levels of TNF-alpha, IL-6 and IL-10 compared to macrophages.

To evaluate whether the activation induced by DNA-HSP65 could increase the microbicidal capacity of macrophages and DCs against M. tuberculosis, these cells were stimulated with DNA vaccine or vector and then were infected. Figure 3C shows that unstimulated macrophages were more permissive to M. tuberculosis growth when compared to macrophages that had been stimulated with DNA vaccine or DNA vector. On day 7 after infection, the bacterial load of the unstimulated infected macrophages differed significantly from that recovered on day 0 (after 4 h). However, the number of CFU recovered from macrophages that had previously been stimulated with DNA vaccine or vector was similar at 0 and 7 days postinfection. When we analysed the mycobacterial growth in cultures of DCs that had previously been stimulated with DNA vaccine or DNA vector, we observed that the CFU number was similar to that detected in unstimulated DC cultures. It is interesting to note that bacilli growth was higher in unstimulated macrophages than in unstimulated DCs, despite the fact that a similar number of bacilli were detected within both cells after 4 h of infection (12,1 × 10⁴ ± 5,9 × 10⁴ and 10,7 × 10⁴ ± 6,1 × 10⁴ CFU, respectively). These data show that while macrophages secreted high levels of TNF-alpha, IL-6 and IL-10 after DNA-HSP65 stimulation, DCs secreted IL-12 and up-regulated the expression of CD80, CD86 and CD83. Moreover, the stimulation of human macrophages with DNA-HSP65 seems to improve its microbicidal potential against M. tuberculosis, since we did not find significant difference between CFU numbers after 4 h and 7 d of infection. On the other hand, DCs were unable to kill intracellular M. tuberculosis after being stimulated with DNA-HSP65.

To investigate the ability of DNA-HSP65 to activate adaptive immune response, the proliferation of PBMC induced by the DNA vaccine was determined. For this purpose, CFSE-labeled PBMC were stimulated with DNA vaccine or vector and analysed by flow cytometry. As positive control of specific proliferation, PBMC were stimulated with recombinant Hsp65 protein (rHsp65). The mRNA for Hsp65 was detected in monocytes cultured for 96 h with DNA-HSP65 (Figure 4A). DNA-HSP65 induced significant proliferation of PBMC compared to unstimulated cells. On the other hand, DNA vector was unable to induce a significant proliferation of PBMC (Figure 4B). Recombinant Hsp65 protein did not exhibit an additional effect on PBMC proliferation induced by DNA-HSP65. Figure 4C shows the histograms and is representative of one experiment. These data indicate that DNA-HSP65 is also able to activate the adaptive immune response leading to a Hsp65-specific cell proliferation.