B cells expressing IL-10 mRNA modulate memory T cells after DNA-Hsp65 immunization

In DNA vaccines, the gene of interest is cloned into a bacterial plasmid that is engineered to induce protein production for long periods in eukaryotic cells. Previous research has shown that the intramuscular immunization of BALB/c mice with a naked plasmid DNA fragment encoding the Mycobacterium leprae 65-kDa heat-shock protein (pcDNA3-Hsp65) induces protection against M. tuberculosis challenge. A key stage in the protective immune response after immunization is the generation of memory T cells. Previously, we have shown that B cells capture plasmid DNA-Hsp65 and thereby modulate the formation of CD8+ memory T cells after M. tuberculosis challenge in mice. Therefore, clarifying how B cells act as part of the protective immune response after DNA immunization is important for the development of more-effective vaccines. The aim of this study was to investigate the mechanisms by which B cells modulate memory T cells after DNA-Hsp65 immunization. C57BL/6 and BKO mice were injected three times, at 15-day intervals, with 100 µg naked pcDNA-Hsp65 per mouse. Thirty days after immunization, the percentages of effector memory T (TEM) cells (CD4+ and CD8+/CD44high/CD62Llow) and memory CD8+ T cells (CD8+/CD44high/CD62Llow/CD127+) were measured with flow cytometry. Interferon γ, interleukin 12 (IL-12), and IL-10 mRNAs were also quantified in whole spleen cells and purified B cells (CD43−) with real-time qPCR. Our data suggest that a B-cell subpopulation expressing IL-10 downregulated proinflammatory cytokine expression in the spleen, increasing the survival of CD4+ TEM cells and CD8+ TEM/CD127+ cells.

B cells expressing IL-10 mRNA modulate memory T cells after DNA-Hsp65 immunization.

In DNA vaccines, the gene of interest is cloned into a bacterial plasmid that is engineered to induce protein production for long periods in eukaryotic cells. Previous research has shown that the intramuscular immunization of BALB/c mice with a naked plasmid DNA fragment encoding the Mycobacterium leprae 65-kDa heat-shock protein (pcDNA3-Hsp65) induces protection against M. tuberculosis challenge. A key stage in the protective immune response after immunization is the generation of memory T cells. Previously, we have shown that B cells capture plasmid DNA-Hsp65 and thereby modulate the formation of CD8+ memory T cells after M. tuberculosis challenge in mice. Therefore, clarifying how B cells act as part of the protective immune response after DNA immunization is important for the development of more-effective vaccines. The aim of this study was to investigate the mechanisms by which B cells modulate memory T cells after DNA-Hsp65 immunization. C57BL/6 and BKO mice were injected three times, at 15-day intervals, with 100 µg naked pcDNA-Hsp65 per mouse. Thirty days after immunization, the percentages of effector memory T (TEM) cells (CD4+ and CD8+/CD44high/CD62Llow) and memory CD8+ T cells (CD8+/CD44high/CD62Llow/CD127+) were measured with flow cytometry. Interferon γ, interleukin 12 (IL-12), and IL-10 mRNAs were also quantified in whole spleen cells and purified B cells (CD43-) with real-time qPCR. Our data suggest that a B-cell subpopulation expressing IL-10 downregulated proinflammatory cytokine expression in the spleen, increasing the survival of CD4+ TEM cells and CD8+ TEM/CD127+ cells.

Attenuation of experimental asthma by mycobacterial protein combined with CpG requires a TLR9-dependent IFN-γ-CCR2 signalling circuit.

BACKGROUND: Allergic asthma is a chronic pulmonary disease characterized by a Th2 inflammatory response. The modulation of a Th2 immune response based on immune deviation to a Th1 pattern or induction and migration of regulatory T cells to the lungs constitutes one of the major therapeutic approaches that is being investigated for the treatment of allergic asthma. The potentials of Mycobacterium leprae 65-kD heat-shock protein or Toll-like receptor 9 ligand (CpG oligodeoxynucleotides) as immune modulators for the treatment of airway allergic disease have been studied individually. OBJECTIVE: Mycobacterial protein combined with CpG was used as immunotherapy for airway allergy. METHODS: Using an ovalbumin-induced asthma model, mice were sensitized and challenged, and then treated with mycobacterial heat-shock protein (Hsp65) combined with CpG. RESULTS: The treatment of mice with established allergy led to the attenuation of eosinophilia, Th2 cytokines and airway hyperresponsiveness. Hsp65 plus CpG treatment also induced an increase in OVA-specific IFN-γ levels and in the frequency of lung inflammatory monocytes. Moreover, we show that the reduction of eosinophilia and the recruitment of inflammatory monocytes to the lungs required early triggering of TLR9, IFN-γ and CCR2 by immunotherapy components. CONCLUSION: In addition to immune deviation to a Th1 response in the modulation of Th2 allergic inflammation, our findings also attribute an important role to the innate response mediated by TLR9, associated with the recruitment of CCR2-dependent monocytes. CLINICAL RELEVANCE: Our findings show that the Hsp65/CpG treatment is a promising strategy for consideration in translational studies.

Recombinant DNA immunotherapy ameliorate established airway allergy in a IL-10 dependent pathway.

BACKGROUND: Previous studies have established that mycobacterial infections ameliorate allergic inflammation. However, a non-infectious approach that controls allergic responses might represent a safer and more promising strategy. The 60-65 kDa heat shock protein (Hsp) family is endowed with anti-inflammatory properties, but it is still unclear whether and how single mycobacterial Hsp control allergic disorders. OBJECTIVE: Therefore, in this study we determined whether the administration of Mycobacterial leprae Hsp65 expressed by recombinant a DNA plasmid could attenuate a previously established allergic response. METHODS: We used an experimental model of airway allergic inflammation to test the effects of immunotherapy with DNA encoding Hsp65. Allergic mice, previously sensitized and challenged with ovalbumin, were treated with tree intramuscular doses of recombinant DNA encoding Hsp65. After treatment, mice received a second allergen challenge and the allergic response was measured. RESULTS: We found that immunotherapy attenuated eosinophilia, pulmonary inflammation, Th2 cytokine and mucus production. Moreover, we showed that the inhibition of allergic response is dependent on IL-10 production. Both Hsp65 and allergen-specific IL-10-producing cells contributed to this effect. Cells transferred from DNA-immunized mice to allergic mice migrated to allergic sites and down-modulated the Th2 response. CONCLUSIONS AND CLINICAL RELEVANCE: Our findings clearly show that immunotherapy with DNA encoding Hsp65 can attenuate an established Th2 allergic inflammation through an IL-10-dependent mechanism; moreover, the migration of allergen- and Hsp65-specific cells to the allergic sites exerts a fundamental role. This work represents a novel contribution to the understanding of immune regulation by Hsp65 in allergic diseases.