| Bovine tuberculosis, mainly caused by Mycobacterium bovis (M.bovis), is a chronic consumptive zoonosis which has a great harm to cow industry. The main host of the disease are the cattle, sick animals can also cause the infection of wild animals and humans. The prevalence of bovine tuberculosis not only seriously affected the healthy and sustainable development of animal husbandry, but also caused tremendous obstacles to human health and the prevention and control of tuberculosis. BCG is an attenuated strain of high virulent Mycobacterium bovis through 230 passages, BCG vaccine has been used for decades in the history, but still displayed some inadequate effect.Innate immunity and specific cellular immunity are the main mechanism on the resistance to Mycobacterium tuberculosis infection. From the perspective of individual development, Innate immunity plays a primordial role in protection when pathogens invade the organism, and then the specific immunity produces. Thus, innate immunity is the basis for all capability of immune protection.Dendritic cells (DCs) are the most powerful professional antigen presenting cells (APC),and the only APC to activate naive T cells, which are also the host cells of Mycobacterium tuberculosis, So it is of great significance on the protection and immunity.IFN-y is a kind of critical Thl-type cytokines to control tuberculosis infection. IFN-y can induce ThO cells differentiation to Th1 cells, activate more cytotoxic T cells (CTL) and effector cells which are very important cells to kill or inhibit the Mycobacterium tuberculosis, increase the capacity of macrophage to kill and inhibit M. tuberculosis, IFN-y is considered to be the main innate immune molecule to activate macrophages in resistance to intracellular pathogens.PTX3 is a very important innate humoral immune component in innate immunity, is a multifunctional soluble pattern recognition receptors, plays an important role in the inflammatory response and defense of certain pulmonary pathogens. Varieties of microbial components can active the dendritic cells to induce PTX3 production through corresponding signal transduction pathway recognized by Toll-like receptor.This study is trying to adopt Mycobacterium bovis and BCG as model pathogens, use dendritic cells as an infection cell model in vitro to explore the different immunological mechanism of innate immune molecules IFN-y and PTX3 in dendritic cells infected by Mycobacterium bovis with different virulence, to finally explain the mechanism of Mycobacterium bovis persistent infection and the protective role or insufficient protection of vaccine BCG. The main contents are as follows:1. The effect of IFN-y on cytokines secretion between DCs infected by M. bovis with different virulenceIL-4 level is detected by ELISA, in BCG group, IFN-y had no effect of IL-4 secretion. In M.bovis infection group,12 hours later, the secretion of IL-4 is significantly inhibited by IFN-y. Study of IL-10 level found that in BCG group, At all time points, IFN-y could significantly inhibit IL-10 secretion, but in M.bovis group, IFN-y has little effect on the secretion of IL-10. ELISA test of IL-12 find that in BCG group, IFN-y can reduce the secretion of IL-12, especially at the time points of 12 hours and 24 hours, but in M.bovis group, IFN-y could significantly increase the secretion of IL-12 at the three time points. ELISA test of TNF-a find IFN-y can significantly promote the secretion of TNF-a of both the two infected groups at the three time points.2. Differencial secretion of PTX3 in DCs infected by M.bovis with different virulence and its signaling pathwaysIt was found that compared with the control group, BCG and M. bovis can both significantly induce the secretion of PTX3. However, the induction of PTX3 in M. bovis group was significantly less than the BCG infection group. Study found that in BCG group, block of TLR2, TLR4 and NF-κB significantly inhibited PTX3 protein secretion, but in M.bovis group, TLR2 and TLR.4 blockade did not impede the secretion of PTX3; NF-κB blocking effect was as BCG group, which have been significantly reduced, indicated that the secretion of PTX3 in BCG infection group is regulated by TLR2 and TLR4/NF-κB signaling pathways, in M.bovis infection group, the secretion of PTX3 is significantly inhibited after NF-κB is blocked. Blocking study by using PTX3 monoclonal antibody have found that the differentiation and maturation of DCs were inhibited,indicating that PTX3 can promote the differentiation and maturation of DCs.3. The different effect of IFN-y, IL-lβ, TNF-a, PGE2 on the secretion of PTX3In BCG group, IFN-y could mainly inhibit PTX3 mRNA transcription, whereas in M.bovis group it was found that IFN-y shows a great distinct impact on transcription levels of PTX3 mRNA, at 12 hours and 24 hours, PTX3 mRNA transcription levels were significantly induced. Pretreatment of IL-1βfound that in the BCG group, IL-1βcould induce the secretion of PTX3 in a concentration dependent manner, whereas in M.bovis group IL-1βcan up-regulate PTX3 when the concentrations is low, but high level of 1L-1βshowed an inhibition effect. Pretreatment of TNF-a found that in the BCG group, TNF-a can induce PTX3 in a concentration-dependent manner when the concentrations is low, but high level of TNF-αshows an inhibition effect. In M. bovis group an inhibition effect on PTX3 was found by a concentration dependent manner. PGE2 pretreatment experiment found that when the concentration of PGE2 reaches a certain level (200ng/mL), it inhibit PTX3 secretion in a concentration dependent manner. It was also found that the role of PGE2 on the secretion of PTX3 is related with the time to stimulate DCs, at 24 h, PGE2 inhibited the secretion of PTX3 in a concentration-dependent manner, but when the effect time was modulated up to 36 hours, different concentrations of PGE2 all significantly inhibited the secretion of PTX3 protein to a consistent low level. |
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