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A DNA vaccine against tuberculosis based on the 65 kDa heat-shock protein differentially activates human macrophages and dendritic cells.

Franco LH, Wowk PF, Silva CL, Trombone AP, Coelho-Castelo AA, Oliver C, Jamur MC, Moretto EL, Bonato VL - Genet Vaccines Ther (2008)

Bottom Line: As DNA vaccines are often less effective in humans, we aimed to find out how the DNA-HSP65 stimulates human immune responses.Our data suggest that the immune response is differently activated by the DNA-HSP65 vaccine in humans.These findings provide important clues to the design of new strategies for using DNA vaccines in human immunotherapy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Núcleo de Pesquisas em Tuberculose, Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo, Av, Bandeirantes, 3900, 14049-900, Ribeirão Preto, SP, Brasil. luishenrique@cpt.fmrp.usp.br

ABSTRACT

Background: A number of reports have demonstrated that rodents immunized with DNA vaccines can produce antibodies and cellular immune responses presenting a long-lasting protective immunity. These findings have attracted considerable interest in the field of DNA vaccination. We have previously described the prophylactic and therapeutic effects of a DNA vaccine encoding the Mycobacterium leprae 65 kDa heat shock protein (DNA-HSP65) in a murine model of tuberculosis. As DNA vaccines are often less effective in humans, we aimed to find out how the DNA-HSP65 stimulates human immune responses.

Methods: To address this question, we analysed the activation of both human macrophages and dendritic cells (DCs) cultured with DNA-HSP65. Then, these cells stimulated with the DNA vaccine were evaluated regarding the expression of surface markers, cytokine production and microbicidal activity.

Results: It was observed that DCs and macrophages presented different ability to uptake DNA vaccine. Under DNA stimulation, macrophages, characterized as CD11b+/CD86+/HLA-DR+, produced high levels of TNF-alpha, IL-6 (pro-inflammatory cytokines), and IL-10 (anti-inflammatory cytokine). Besides, they also presented a microbicidal activity higher than that observed in DCs after infection with M. tuberculosis. On the other hand, DCs, characterized as CD11c+/CD86+/CD123-/BDCA-4+/IFN-alpha-, produced high levels of IL-12 and low levels of TNF-alpha, IL-6 and IL-10. Finally, the DNA-HSP65 vaccine was able to induce proliferation of peripheral blood lymphocytes.

Conclusion: Our data suggest that the immune response is differently activated by the DNA-HSP65 vaccine in humans. These findings provide important clues to the design of new strategies for using DNA vaccines in human immunotherapy.

No MeSH data available.


Related in: MedlinePlus

Phenotypic characterization of monocyte-derived macrophages and DCs. (A) Expression of markers CD11b (Mac-1), CD86 and HLA-DR on the surface of macrophages, and (B) CD11c, CD86 and HLA-DR on the surface of DCs was evaluated by flow cytometry (all markers are indicated by solid lines). Dotted-line histograms indicate isotype control mAb. These results are representative of seven independent experiments. (C) Expression of CD1c, CD123 (IL-3 receptor) and BDCA-4 on the surface of DCs. (D) IFN-alpha production by monocyte-derived DC (mo-DC) and plasmacytoid DC (pDC). These results are representative of three independent experiments. (E) Intracellular expression of TLR9 by macrophages and DCs analysed by confocal microscopy.
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Figure 1: Phenotypic characterization of monocyte-derived macrophages and DCs. (A) Expression of markers CD11b (Mac-1), CD86 and HLA-DR on the surface of macrophages, and (B) CD11c, CD86 and HLA-DR on the surface of DCs was evaluated by flow cytometry (all markers are indicated by solid lines). Dotted-line histograms indicate isotype control mAb. These results are representative of seven independent experiments. (C) Expression of CD1c, CD123 (IL-3 receptor) and BDCA-4 on the surface of DCs. (D) IFN-alpha production by monocyte-derived DC (mo-DC) and plasmacytoid DC (pDC). These results are representative of three independent experiments. (E) Intracellular expression of TLR9 by macrophages and DCs analysed by confocal microscopy.

Mentions: 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.


A DNA vaccine against tuberculosis based on the 65 kDa heat-shock protein differentially activates human macrophages and dendritic cells.

Franco LH, Wowk PF, Silva CL, Trombone AP, Coelho-Castelo AA, Oliver C, Jamur MC, Moretto EL, Bonato VL - Genet Vaccines Ther (2008)

Phenotypic characterization of monocyte-derived macrophages and DCs. (A) Expression of markers CD11b (Mac-1), CD86 and HLA-DR on the surface of macrophages, and (B) CD11c, CD86 and HLA-DR on the surface of DCs was evaluated by flow cytometry (all markers are indicated by solid lines). Dotted-line histograms indicate isotype control mAb. These results are representative of seven independent experiments. (C) Expression of CD1c, CD123 (IL-3 receptor) and BDCA-4 on the surface of DCs. (D) IFN-alpha production by monocyte-derived DC (mo-DC) and plasmacytoid DC (pDC). These results are representative of three independent experiments. (E) Intracellular expression of TLR9 by macrophages and DCs analysed by confocal microscopy.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2267464&req=5

Figure 1: Phenotypic characterization of monocyte-derived macrophages and DCs. (A) Expression of markers CD11b (Mac-1), CD86 and HLA-DR on the surface of macrophages, and (B) CD11c, CD86 and HLA-DR on the surface of DCs was evaluated by flow cytometry (all markers are indicated by solid lines). Dotted-line histograms indicate isotype control mAb. These results are representative of seven independent experiments. (C) Expression of CD1c, CD123 (IL-3 receptor) and BDCA-4 on the surface of DCs. (D) IFN-alpha production by monocyte-derived DC (mo-DC) and plasmacytoid DC (pDC). These results are representative of three independent experiments. (E) Intracellular expression of TLR9 by macrophages and DCs analysed by confocal microscopy.
Mentions: 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.

Bottom Line: As DNA vaccines are often less effective in humans, we aimed to find out how the DNA-HSP65 stimulates human immune responses.Our data suggest that the immune response is differently activated by the DNA-HSP65 vaccine in humans.These findings provide important clues to the design of new strategies for using DNA vaccines in human immunotherapy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Núcleo de Pesquisas em Tuberculose, Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo, Av, Bandeirantes, 3900, 14049-900, Ribeirão Preto, SP, Brasil. luishenrique@cpt.fmrp.usp.br

ABSTRACT

Background: A number of reports have demonstrated that rodents immunized with DNA vaccines can produce antibodies and cellular immune responses presenting a long-lasting protective immunity. These findings have attracted considerable interest in the field of DNA vaccination. We have previously described the prophylactic and therapeutic effects of a DNA vaccine encoding the Mycobacterium leprae 65 kDa heat shock protein (DNA-HSP65) in a murine model of tuberculosis. As DNA vaccines are often less effective in humans, we aimed to find out how the DNA-HSP65 stimulates human immune responses.

Methods: To address this question, we analysed the activation of both human macrophages and dendritic cells (DCs) cultured with DNA-HSP65. Then, these cells stimulated with the DNA vaccine were evaluated regarding the expression of surface markers, cytokine production and microbicidal activity.

Results: It was observed that DCs and macrophages presented different ability to uptake DNA vaccine. Under DNA stimulation, macrophages, characterized as CD11b+/CD86+/HLA-DR+, produced high levels of TNF-alpha, IL-6 (pro-inflammatory cytokines), and IL-10 (anti-inflammatory cytokine). Besides, they also presented a microbicidal activity higher than that observed in DCs after infection with M. tuberculosis. On the other hand, DCs, characterized as CD11c+/CD86+/CD123-/BDCA-4+/IFN-alpha-, produced high levels of IL-12 and low levels of TNF-alpha, IL-6 and IL-10. Finally, the DNA-HSP65 vaccine was able to induce proliferation of peripheral blood lymphocytes.

Conclusion: Our data suggest that the immune response is differently activated by the DNA-HSP65 vaccine in humans. These findings provide important clues to the design of new strategies for using DNA vaccines in human immunotherapy.

No MeSH data available.


Related in: MedlinePlus