Induction Of Cytokines In Dendritic Cells By M. Bovis And BCG

Tuberculosis is a chronic consumptive zoonosis casued by Mycoabcterium tuberculosis complex. One third of the world population is currently estimated to be infected with M. tuberculosis, although less than 10% of those infected show clinical signs of infection. This is mainly due to the robust immune response of the hosts that is initiated by the bacillus, which effectively contains the infection and allows the hosts to exist in equilibrium with a subclinical infection.Bovine tuberculosis is a chronic consumptive zoonosis casued by Mycoabcterium bovis (M. bovis), and is fixed in the list of notifiable diseases by OIE (World Organisation for Animal Health). The disease caused about 3 billion dollars of direct economic loss all over the world. According to statistics, more than 10% of the human tuberculosis is caused by Mycobacterium bovis. Bovine tuberculosis exerts a tremendous influence on the development of animal husbandry and human health, and is also a worldwide public health problem.M. bovis bacillus Calmette-Gue’rin (BCG) is an attenuated strain from virulent Mycobacterium bovis which lost its virulence with a deletion of 5 regions after 230 passages during 13 years. It is the current unique TB vaccine in human and can induce effective immune response, although the contradictory views on its exits. To utilize BCG and Mycobacterium bovis as model bacteria will be helpful to find the pathogenesis of tuberculosis. Furthermore, since identity of the genome sequence between M.tb and M. bovis was over 99.95% and there are no genes unique to M. bovis compared to M.tb, the findings from the pair of BCG and virulent M. bovis can lend support to understand the mechanism of M.tb pathogenesis.Cytokines and chemokines play an extremely important role in immune regulation to resist TB infection. Cytokine can extensive regulate immune response, hematopoietic function and involved in the inflammatory injury process. Chemokines stimulate leukocyte chemotaxis, attracting neutrophils, monocytes/macrophages and other inflammatory cells to move to the inflammatory lesions, and enhanced killing effect of phagocytosis of inflammatory cells, promote the release of inflammatory mediators, direct involved in the inflammatory process.In this study, we infected dendritic cells with BCG or M. bovis at the multiplicity of infection (MOI) of 10. The supernatant of DCs culture with or without infection of M. bovis or BCG was collected at 6h,12h and 24h postinfection. There were 8 kind of cytokine and chemokine in the culture supernatants were measured by sandwich enzyme-linked immunosorbent assay (ELISA); Detection of chemokine and cytokine after blocking of TLR2, TLR4 and NF-κB; qRT-PCR and western blot to detect the relative expression of some key molecules of TLR/NF-κB signaling pathway. Finally, this study analyzed the difference induction of cytokine and chemokine by BCG and M. bovis and to explore its regulatory pathways, the main results are as follows:1. The difference induction of cytokines and chemokines in DCs by BCG and M. bovis.The supernatant of DCs culture with or without infection of M. bovis or BCG was collected at 6h,12h and 24h postinfection. There are 8 kinds of cytokines and chemokines in DCs culture were detected by ELISA kits. These are IL-1β, IL-4, IL-6, IL-12, IL-23, TNF-α, RANTES, MCP-1. The final results showed that the secretion of IL-4 in uninfected DC was very low which was 31.2±0.2 pg/ml. After infected by either BCG or M. bovis, it caused a slowly increase as the time of infection. However, the overall concentration of IL-4 was still very lowindicating that humoral immunity did not play a key role in defense against M. bovis. For other cytokines and chemokines (IL-1(3, IL-6, IL-12, TNF-a, RANTES, MCP-1), there was a very sharp increase after infected by BCG or M. bovis in DCs. And compared to M. bovis, BCG induced higher concentrations of IL-6, IL-12, TNF-a, RANTES and MCP-1, while M. bovis induced more higher concentrations of IL-1(3 and IL-23. These results suggest that M. bovis mainly induces inflammatory reaction, while BCG protective immune response.2. qRT-PCR to detect the relative expression of some key molecules of TLR/NF-κB signaling pathwayFive critical genes TLR2, TLR4, Myd88, NF-κB p50 and p65 subunit were detected in DCs infected with M. bovis and BCG by qRT-PCR. The results shows BCG induced higher level of transcription of TLR2, TLR4, Myd88, NF-κB p50 and p65 subunit than M. bovis in DCs.3. Western blot to detect the expression of some key molecules of NF-κB sinaling pathwayResults of Western blot shows that compared with M. bovis infection, the expression of phos-NF-KB p65 subunit was significantly higher in BCG infected DCs. And BCG infected DCs shows a higher level of degradation of I-κB. This suggests that the activation of NF-κB is significantly higher in BCG infected DCs. After addition of NF-κB inhibitor PDTC, the concentration of phop-NF-KB p65 decreased in both BCG and M. bovis infected DCs, it indicate that PDTC can inhibit the activation.of NF-κB.4. To detect the secretion of cytokine and chemokine after blocking of TLR2, TLR4 and NF-κB IL-1β, IL-6, RANTES and MCP-1 production in BCG or M. bovis infected DCs was significantly inhibited by TLR2 mAb and PDTC, but not by TLR4 mAb. This suggests that the secretion of IL-1β, IL-6, MCP-1 and RANTES are regulated by TLR2/NF-κB pathway.