| Tuberculosis (TB) is one kind of zoonosis characterized by chronic respiratory infections mainly caused by Mycobacterium tuberculosis (MTB). As an ancient disease, TB in Egypt can be documented more than 5000 years ago, and it has been studied for more than 100 years since MTB was identified as its causative agent in the end of nineteenth century. However, there were more than one third of people over the world have infected MTB, and an estimated 8 million new TB cases per year as well as 1.3 million people died of TB. TB remains the single largest infectious disease causing high mortality in humans and acts as one of the most heaviest disease burden of the world.MTB as an intracellular bacteria causes chronic infections, and most infected individuals stay in a special process which termed as latent tuberculosis infection. The immune responses of the host to the pathogen determine the occurrence and development of the disease. Dendritic cells (DCs), which is the most powerful professional antigen presenting cell, play a key role and a bridge between natural immune responses and acquired immune responses against TB, through the functions of antigen presenting to the naive T lymphocytes as well as the secretion of cytokines such as IL-12 and so on. However, DCs are the highly heterogeneous population which composed of many subsets characterized with different phenotypes and functions. The conventional DC (cDC) and plasmacytoid DC (pDC) are the main DC subsets in murine spleen in the steady state, and the cDC subsets can be further divided into CD8+ cDC and CD8- cDC based on the expression of the CD8a molecule. The evaluation on the specific immunological functions of the different DC subsets in the immune responses of the host against TB will greatly promote the understanding of the immunological process of TB infection. However, there are little literatures focused on this subject, partly due to the rare number of DCs in vivo and the difficulties in the purification and preparation of the subsets of DCs.In the present study, the immunological characteristics of different murine DC subsets response to BCG infected in vitro and in vivo, and the gene expression profiles of these subsets infected with BCG in vitro have been evaluated to increase the knowledge on the immunological functions of different DC subsets in mycobacteria infection.1. Generation, identification and characterization of different bone marrow derived DC subsets of murine in vitroDC subsets of cDC and pDC were generated and identified from murine bone marrow hematopoietic stem cells cultured with the recombinant protein of Flt3 ligand (FL). The biological features, including cell morphology, expression of phenotype markers and co-stimulation molecules, cytokines production profile and stimulation of T cells proliferation have been evaluated by microscopy, flow cytometry (FCM) and enzyme linked immunosorbent assay (ELISA), respectively. FCM analysis showed that there was a more than 60% CDllc positive rate in the FL-DCs population after 9 days culture, and lots of cells showed the dendritic morphology characteristic observed by the optical microscopy after stimulation with LPS. The subset of CD11c+CD45RA-cDC, as well as CD11c+CD45RA+pDC which also bored with the mPDCA-1 molecule positive were presented in the generated FL-DCs population. Furthermore, two distinct CD24highCD11blow and CD24lowCDbhigh subsets were also identified from cDC subtype by FCM. Both cDC and pDC showed the significant up-regulated expression of both co-stimulation molecules such as CD40, CD80 and CD86 and molecules of MHC-Ⅰ and MHC-Ⅱ after stimulated with LPS. Both cDC and pDC showed the ability to produce high quantity of IL-6 and TNF-α while the disability of IL-10 and MCP-1 production in response to a range of TLR agonists. On the contrary, DCs generated from GM-CSF culture (GM-DC) produced higher levels of all the four cytokines or chemokines than both cDC and pDC subsets. Furthermore, pDC secreted more IFN-α than cDC and GM-DC when response to CpG while these three kinds of DC sub-population produced little IFN-γ in response to all TLR agonists. Although pDC showed weaker ability than cDC, both of them can stimulated CD4+T lymphocyte proliferation and production of IFN-γ and IL-4 after co-culture. These results demonstrated the good biological activities of FL-generated cDC and pDC subtypes were successfully obtained, which laid a foundation for the following studies of their roles against mycobacterial infection.2. Immunological characteristics analysis of the bone marrow derived cDC and pDC response to BCG infection in vitroThe immunological characteristics of cDC and pDC generated from FL culture response to BCG infection have been evaluated, including the infection rate, cellular activation and maturation, cytokines production and specific antigen presentation ability. The intracellular rBCG-GFP can be observed in both FL-cDC and FL-pDC by laser scanning confocal microscope, which demonstrated the ability of phagocytosis of the two DC subset to BCG However, most of the cells had no intracellular bacteria and FCM analysis also showed low GFP positive rates of the two DC subtypes, indicating the weak infection ability of BCG to DCs and their subsets. Even so, both cDC and pDC showed a significant up-regulated and a growing trend expression of molecules of CD40, CD80, CD86, MHC-Ⅰ and MHC-Ⅱ from 4 h to 12 h post of infection, suggesting the activating and maturation of the two subsets to BCG infection. Although high quantity of IL-6 and TNF-α have been detected in the cellular culture supernatant of the both DC subsets, FL-pDC secreted significant higher level of the cytokines than FL-cDC at 4 h post infection while FL-cDC showed a peak expression at 12 h post infection. In the antigen presentation assay in vitro, both FL-cDC and FL-pDC showed the ability to present the antigen of peptide devised from the Ag85A antigenic protein of BCG. But FL-pDC showed a generally weaker antigen presentation activity than FL-cDC, in spite of the similar dynamic trend of presentation, the strongest presentation at 12 h for example. These results demonstrated that both of the FL-cDC and FL-pDC possess the ability of phagocytosis followed with maturation, cytokines production and antigen presentation in the immune responses to the infection of BCG.3. The immunological functions of different splenic DC subsets of murine in response to BCG infection in vivoThe immune response properties of splenic cDC and pDC as well as CD8+cDC and CD8-cDC subtypes have been evaluated in C57BL/6 mice which were intravenously injected with BCG The intracellular survival of BCG in cDC and pDC and the possible anti-mycobacterial functions also have been analyzed in this study. The immunological characteristics of cDC and pDC in BCG short-term infection (48 h) have been evaluated firstly, including the infection rate, cellular activation and maturation, and specific antigen presentation ability. FCM analysis showed that the GFP positive rate of pDC was significantly higher than that of cDC, indicating a more sensitivity response to BCG infection. In the first 24 h after infection, there was a similar infection rate between the CD8+cDC and CD8-cDC subsets, however, the infection rate of CD8+cDC was gradually decreased while the rate in CD8- cDC was increased in the following 24 h after infection. Compared with the uninfection control group, the splenic pDC as well as CD8+ and CD8- cDC subsets prepared from BCG infected mice showed a increased positive rate of cells which expressed CD40, CD80, CD86, and MHC-Ⅱ at 4 h after immunization, respectively. However, further analysis showed that only subsets of pDC and CD8+cDC but not CD8- cDC also had the increased expression intensity of the above molecules on the cellular surface of the positive cells, respectively. Results of antigen presentation assay ex vivo showed that both splenic cDC and pDC can presented the antigen devised from the protein of Ag85A to the antigen specific T cell hybridoma. In addition, both cDC and pDC showed the strongest presentation activity at 4 h after infection, although pDC showed a generally weaker antigen presentation activity than cDC. Meanwhile, splenic cDC secreted significantly hgher level of IL-12 than pDC, whereas pDC secreted more TNF-α than cDC. During long term infection of BCG, the GFP positive rate of splenic pDC and CD8+cDC showed a gradually declining trend and a value similar to the background after 15 d post infection, while the GFP positive rate of CD8" cDC kept stable at about 1% during at least 2 months after infection. Results of CFU counting to the survival intracellular BCG also showed the similar trend with the GFP positive rate of splenic cDC and pDC, i.e., the BCG numbers in pDC decreased gradually while the numbers in cDC kept stable and significantly higher than pDC since 15 d after infection. In addition, both GFP positive cDC and pDC showed the significant higher expression intensity of CD40, CD80, CD86 and MHC-Ⅱ molecules than that of GFP negative in the long term infection of BCG, indicating that the intracellular survival of BCG may maintain the maturation and functions of the infected DCs during the long term infection. The splenic cDC showed a higher concentration of total intracellular nitric oxide but a less amount of autophagy associated proteins of LC3-II and p62 than pDC during different states of the long term infection, indicating that the main bactericidal mechanism employed by the DCs may differ among subsets.4. The gene expression profiles of the bone marrow derived cDC and pDC infected with BCG in vitroThe cDNA microarray (gene chip) was used to identify the differentially expressed (DE) gene profiles of FL-cDC and FL-pDC subtypes at 4 h and 24 h after BCG infection. In this study, genes with the statistical P value less than or equal to 0.05 and Fold Change (FC) greater than or equal to 2 were identified as up regulated expression, while genes with the P value less than or equal to 0.05 and FC less than or equal to 0.5 were identified as down regulated expression. Data analysis of the chips showed that,1974 DE genes were detected in the group of 4c/0c including 1305 up regulated expression and 669 down regulated,1402 DE genes were detected in the group of 24c/0c including 765 up regulated expression and 637 down regulated,1100 DE genes were detected in the group of 4p/0p including 736 up regulated expression and 364 down regulated,1299 DE genes were detected in the group of 24p/0p including 873 up regulated expression and 426 down regulated. By DAVID analysis of online software, according the Gene Ontology (GO) annotation, the molecular function classification associated to these DE genes are mainly focus on the GO terms of binding of different materials, transcription factor activity, cytokine and chemokine activity, cytokine and chemokine receptor activity and so on. And it is worth noting that there are wide range of differences between different groups in both GO terms and their enrichment of P values. Results analyzed by DAVID based on the KEGG database showed that the DE genes mainly involved in the signal pathways including cytokine-cytokine receptor interaction, ECM-receptor interaction, focal adhesion, Toll-like receptor signaling pathway, NOD-like receptor signaling pathway, and some other pathways associated with cancer and so on. There are also many differences in the pathway items and their enrichment of P values between different groups. Further analysis to the genes which refer to cytokine-cytokine receptor activity showed that both cDC and pDC had a large amount of up regulated expression genes of cytokines and chemokines but most receptors genes of them are down regulated expression in the early infection groups of 4c/0c and 4p/0p. Among these DE genes, tumor necrosis factor super family and their receptor super family may play an important immune regulatory role in the process of infection. |
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