Antigen Presenting Characteristic And Mechanism Of Murine Dendritic Cells And Macrophages Induced By BCG

Tuberculosis remains a major health problem worldwide, which causes 8.6 million new cases and 1.3 million deaths annually. BCG, a live-attenuated bacterial vaccine developed in the 1920s, is still used to vaccinate children worldwide.A major fraction of the secreted proteins in the culture filtrate of M. tuberculosis, M. bovis is formed by the Ag85 complex, the Ag85 complex consists of Ag85A, Ag85B, and Ag85C, which secreted at a constant ratio of 3:2:1 throughout the culture period. The Ag85 complex has a mycolyl transferase enzyme activity required for the biogenesis of cord factor, and plays an important role in cell wall biosynthesis. As documented, Ag85A can induce stronger CD4+T cells and CD8+CTL response, and it is one of the most promising candidate antigens for the development of vaccines against tuberculosis.Both dendritic cell (DC) and macrophage (Mφ) have been suggested as being important in inducing the immune response against pathogen infection. They phagocytose infected or invading pathogens; they identify pathogens or cellular stress using pattern recognition receptors; they shape inflammatory processes by secreting cytokines and other factors; and they play an essential role in adaptive immunity by presenting antigen to T cells. However, the immune responses and mechanisms of dendritic cell and macrophage against mycobacteria need to be investigated.In this study, the immunogenicity M. bovis Ag85A protein and BCG was evaluated in C57BL/6 mice. Then the protective immune responses of dendritic cells and macrophages against tuberculosis, such as specific antigen presentation activity against Ag85A, were explored in vitro and in vivo, respectively. At last, the expression profiles of 84 genes of murine lymph node DC and MO infected with BCG using the RTZ Profiler PCR Array, in order to analyze cellular and molecular mechanisms of dendritic cell and macrophage immune responses induced by mycobacteria.1. Solubly prokaryotic expressing and immunogenicity of M. bovis BCG Ag85A proteinIn this study, the cold expressed system and chaperone competent cells BL21 were combined to solubly express M. bovis BCG Ag85A protein, then the protein was purified with affinity chromatography and identified by Western blot analysis, which provided an important biological material for the further research and application. The immunogenicity of the purified Ag85A protein was evaluated in C57BL/6 mice, the results showed that high level of specific IgG was elicited in the serum, and the splenocytes and lymph node cells of immunized mice could produce Thl cytokines, such as IFN-y and TNF-a, after stimulated by specific antigen. It indicated that the Ag85A protein can induce strong specific humoral and cellular immune responses.2. Evaluation of Ag85A specific immune responses induced by BCG, the recombinant BCG overexpressing Ag85AThe specific immune responses against Ag85A protein were evaluated in C57BL/6 mice, the results showed that after BCG subcutaneous injection, a high level of Ag85A specific IgG was detected in the serum, and their splenocytes and lymph node cells can produce Thl cytokines, such as IFN-y and TNF-a. It indicated that the BCG can induce strong Ag85A-specific humoral and cellular immune responses. In the present study, a recombinant BCG strain overexpressing the immunodominant Ag85A antigen was constructed and evaluated. The results showed that high levels of Ag85A-specific IgG were tested in the sera of mice infected with rBCG or BCG, and higher level of IFN-y was found in the culture supernatants of lymphocytes obtained from rBCG-immunized mice compared with that of the BCG-immunized, all these results suggesting that rBCG may induce stronger immune protection than BCG in mice. It demonstrated that rBCG could have potential as a vaccine candidate against tuberculosis.3. Antigen presenting characteristic of murine dendritic cells and macrophages against BCG in vitroThe murine splenic dendritic cells and macrophages were both infected with rBCG-GFP, then the infection ratio was detected by FACS, the results showed that the infection ratio of Mφs was higher than that of DCs, it means that Mφs has more strong phagocytic activity against BCG than DCs. During BCG infection, splenic DCs mainly secreted IL-6, IFN-y and TNF-a after BCG infection, while the splenic Mφs produced high level of TNF-a and MCP-1. Moreover, BCG-infected splenic Mφs also produce the immunosuppressive cytokine IL-10. The level of ROS, iNOS and NO in murine splenic Mφs were all higher than DCs after BCG infection, which seemed that Mφs also kill the BCG more effectively. The Ag-presenting activity was further investigated in vitro. The splenic DCs and Mφs of C57BL/6 were sorted and treated with Ag85A peptide, Ag85A protein and BCG, and then cocultured with T hybridoma DE10, specific for a dominant Ag85A peptide p241-160. The formation of Ag85A peptide/MHC complexes was successfully detected through measuring IL-2 in culture supernatants. It confirmed that both DC and Mφ had capacity for presenting Ag85A peptide to specific T hybridoma in vitro.4. Antigen presenting characteristic of murine dendritic cells and macrophages against BCG in vivoAfter infected with rBCG-GFP, the infection ratio of murine splenic and lymph node DCs and Mφs was detected by FACS, the results showed that the infection ratio of Mφs was higher than DCs. The CFU count of BCG in Mφs was higher than DCs, but it decreased in Mφs after 7 days. It confirmed that Mφs has more strong phagocytic activity against mycobacteria than DCs, and Mφs also kill the mycobacteria more effectively. The expression of MHCII, and CD40, CD80, CD86 costimulatory molecules of DCs and Mφs required for T cell activation were analyzed. Up-regulation of MHCII, and CD40, CD80, CD86 costimulatory molecules occurred on DCs, and the level of all these molecules of DCs was higher than that of Mφs . These results indicate that DCs, but not Mφs, undergo functional maturation. The Ag-presenting activity was investigated ex vivo by harvesting DCs and Mφs of lymph node from infected mice. After i.v. administration of Ag85A protein, the formation of Ag85A peptide/MHC complexes was detected at 4h after Ag85A protein administered but was barely detectable at 48h. When Ag85A protein was injected s.c., Ag-presenting activity was detected ex vivo in draining lymph nodes from 24h. And when BCG was injected s.c., high level of Ag85A peptide/MHC complexes was detected, but direct stimulation of T hybridoma DE10 was hardly detectable in Mφs. The results showed that only DCs are able to initiate the primary T cell responses in the early period of BCG infection.5. Transcriptional profiles of murine dendritic cell and macrophage after BCG infectionIn order to analyze cellular and molecular mechanisms of dendritic cell and macrophage immune responses induced by BCG, we studied the expression profiles of 84 genes of murine lymph node DC and Mφ infected with BCG using the RT2 Profiler PCR Array. In general, the number of genes upregulated (fold change≥2) were more than gene downregulated in both DCs and Mφs, and the change fold decreased from 12h to 48h. As expected, among the commonly upregulated genes in DCs were some important molecular associated with antigen presentation and T cell activation. However, the upregulated genes in Mφs included inflammatory cytokine, cell surface Fc receptor and Toll-like receptor. The results confirmed that DC and MO may have different roles during Mtb infection, macrophages secrete proinflammatory cytokines and induce inflammatory response, whereas DC are primarily involved in inducing antimycobacterial T cell immune response. The searching of some differentially expressed genes in DCs and Mφs may serve as a guide for the molecular mechanism research of antigen presenting function.