Immune regulatory effect of pHSP65 DNA therapy in pulmonary tuberculosis: activation of CD8+ cells, interferon-gamma recovery and reduction of lung injury

Abstract

A DNA vaccine based on the heat-shock protein 65 Mycobacterium leprae gene (pHSP65) presented a prophylactic and therapeutic effect in an experimental model of tuberculosis. In this paper, we addressed the question of which protective mechanisms are activated in Mycobacterium tuberculosis-infected mice after immune therapy with pHSP65. We evaluated activation of the cellular immune response in the lungs of infected mice 30 days after infection (initiation of immune therapy) and in those of uninfected mice. After 70 days (end of immune therapy), the immune responses of infected untreated mice, infected pHSP65-treated mice and infected pCDNA3-treated mice were also evaluated. Our results show that the most significant effect of pHSP65 was the stimulation of CD8+ lung cell activation, interferon-gamma recovery and reduction of lung injury. There was also partial restoration of the production of tumour necrosis factor-alpha. Treatment with pcDNA3 vector also induced an immune stimulatory effect. However, only infected pHSP65-treated mice were able to produce significant levels of interferon-gamma and to restrict the growth of bacilli.

Figure 1

Lung lymphocyte, macrophage (Mac-1⁺) and dendritic cell (CD11c⁺) numbers obtained from uninfected mice (PBS), 30-day infected mice (RV-30 days), 70-day infected mice (RV-70 days), infected pcDNA-3-treated mice (pcDNA3-70 days) and infected pHSP65-treated mice (pHSP65-70 days). Results are expressed as the mean of CD4⁺, CD8⁺, (a), Mac-1⁺, or CD11c⁺, (b), cell numbers of 10–12 mice per group, evaluated individually, obtained from three independent experiments. Statistical significance: *P < 0·05, **P < 0·01, ***P < 0·001.

Figure 2

CD18 and CD28 expression in CD4⁺ and CD8⁺ lung lymphocytes obtained from uninfected mice (PBS), 30-day infected mice (RV-30 days), 70-day infected mice (RV-70 days), infected pcDNA-3-treated mice (pcDNA3-70 days) and infected pHSP65-treated mice (pHSP65-70 days). Results are expressed as the mean of the percentage of CD28⁺ population, (a), or as the mean of the median of fluorescence intensity (MFI) for the CD18 receptor, (b), on CD4⁺ and CD8⁺ cells obtained from 10–12 mice per group, evaluated individually, from three independent experiments. Statistical significance: *P < 0·05, **P < 0·01, ***P < 0·001.

Figure 3

Percentage of CD95L⁺ in CD4⁺ and CD8⁺ lung lymphocytes and CD95 expression in lung CD4⁺, CD8⁺, Mac-1⁺ (macrophages) and CD11c⁺ (dendritic cells) cells obtained from uninfected mice (PBS), 30-day infected mice (RV-30 days), 70-day infected mice (RV-70 days), infected pcDNA-3-treated mice (pcDNA3-70 days) and infected pHSP65-treated mice (pHSP65-70 days). Results are expressed as percentage of CD95L⁺ population in CD4⁺ and CD8⁺ cells, (a), or as the mean of median of fluorescence intensity (MFI) for CD95 receptor on CD4⁺, CD8⁺, (b), Mac-1⁺ and CD11c⁺, (c), cells obtained from 10–12 mice per group, evaluated individually, from three independent experiments. Statistical significance: *P < 0·05, **P < 0·01, ***P < 0·001.

Figure 4

Production of regulatory cytokines IL-12, (a), IFN-γ, (b), IL-4, (c), and IL-10, (d), in the lungs of uninfected mice (PBS), 30-day infected mice (RV-30 days), 70-day infected mice (RV-70 days), infected pcDNA3-treated mice (pcDNA3-70 days) and infected pHSP65-treated mice (pHSP65-70 days). Results are expressed as the mean of cytokine concentration detected in lung homogenates obtained from 10 to 12 mice per group, evaluated individually, from three independent experiments. Statistical significance: *P < 0·05, **P < 0·01.

Figure 5

Production of inflammatory cytokines TNF-α, (a), and IL-6, (b), and chemokines MCP-1, (c), and IP-10, (d), in the lungs of uninfected mice (PBS), 30-day infected mice (RV-30 days), 70-day infected mice (RV-70 days), infected pcDNA-3-treated mice (pcDNA3-70 days) and infected pHSP65-treated mice (pHSP65-70 days). Results are expressed as the mean of cytokine concentration detected in lung homogenates obtained from 10 to 12 mice per group, evaluated individually, from three independent experiments. Statistical significance: *P < 0·05, **P < 0·01, ***P < 0·001.

Figure 6

Progression of Mycobacterium tuberculosis infection in mice submitted to DNA therapy or no therapy. (a) Growth of M. tuberculosis in lungs of 30-day infected mice (RV-30 days), 70-day infected mice (RV-70 days), infected pcDNA3-treated mice (pcDNA3-70 days) and infected pHSP65-treated mice (pHSP65-70 days) was followed by a count of the colony-forming units (CFU). The data are expressed as the mean ± standard deviation of CFU of 10 mice per group, evaluated individually, from three independent experiments. (b) Growth of M. tuberculosis in the lungs of infected untreated mice, infected pcDNA3-treated mice and infected pHSP65-treated mice 85 days after the end of immune therapy. The data are expressed as the mean ± standard deviation of CFU of five mice per group. *P < 0·05, **P < 0·01.

Figure 7

Histological representation of lung sections from uninfected mice (a), 30-day infected mice (b), 70-day infected mice (c), infected pcDNA-3-treated mice (d) and infected pHSP65-treated mice (e). Formalin-fixed lung tissue was prepared and sectioned for light microscopy. The tissue was stained with haematoxylin & eosin. Magnification is ×10. Images are representative of three independent experiments.