Study On The Effect Of The Downstream Anti-tuberculosis Immune Response By Downregulating DC-SIGN

Objective To investigate the effect of the downstream anti-tuberculosis immune response by downregulating dendritic cell - specific ICAM - 3 grabbing nonintergrin,and then to explore the effect of DC-SIGN in anti-tuberculosis immune response.Methods : 1. The effect of ManLAM on DCs'mature and DCs'function: The peripheral blood mononuclear cells were obtained from healthy individuals by density gradient centrifugation, cells were adhered 2h, the suspension cells were removed. The adherent cells were cultured in medium with GM-CSF and IL-4. The sixth day,the cells were divided into two groups, such as DCs group and ManLAM group, LPS and ManLAM were added to stimulate DCs. The Seventh day, the cells were collected, the expression levels of DC-SIGN,CD83,CD86 and HLA-DR on the surface of DCs were detected by flow cytometry. The levels of IL-12 and IL-10 in the DCs culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA).2. To research the effect of DCs' maturity after downregulating DC-SIGN:①To detect the effect of RNAi: The cells were divided into three groups, such as DCs group,RNAi group and negative control group, According to the experimental design, the medium was changed 12 hours later. Efficiency of infection was calculated by fluorescence microscope and fluorescence intensity was observed 60 hours later. The sixth day, LPS were added to stimulate DCs. The Seventh day, the cells were collected, and DC-SIGN mRNA level was detdected by RT-PCR. The level of DC-SIGN were detdected by Western Blot.②To research the effect of surface molecule and cytokine expressed by transfection DCs: The cells were divided into four groups, such as DCs group, RNAi group, ManLAM group and ManLAM+RNAi group, The levels of surface molecules of DCs were detected by flow cytometry. The supernatant IL-12 and IL-10 levels were measured by ELISA.3. To research the effect of na?ve T cells activation and proliferation in downstream immunity after downregulating DC-SIGN: The naive CD4~+CD45RA~+T cells which were separated by immunomagnetic beads together with allogeneic DCs mix-cultured for 48 hours. Subsequently the supernatant IFN-γlevel in different groups were measured by ELISA. Proliferation induction of DCs to CD4~+T lymphocytes was measured by mixed lymphocyte reaction(MLR).4. To research the effect of macrophage'activation and the ability of killing MTB after Downregulating DC-SIGN: Monocytes were cultured in medium with GM-CSF. The ninth day, macrophages were harvested. Macrophages and CD4~+T cells were mix-cultured for 48 hours. Subsequently, the supernatant TNF-αlevel was measured by ELISA in different groups, NO level was measured by NO detection kit in different groups. MTB was phagocytized by Macrophages observed by transmission electron microscope. Break up Macrophages, and then cultured MTB in Lowenstein-Jensen culture, then counted it one week later.Results: 1. DCs which were induced from the peripheral blood mononuclear cells appeared typical morphology characteristic in the seventh day. The levels of DC-SIGN,CD83 and CD86 in ManLAM group was lower than that in DCs group (p<0.05), IL-10 level in ManLAM group was higher than that in DCs group (p<0.05). IL-12 level in ManLAM group was lower than that in DCs group (p<0.05).2.①Green fluorescence was appeared on the cells surface in each transfection group observed by fluorescent microscope. When multiplicity of infection(MOI) was 20, the efficiency of infecting virus of DCs was 85%. Compared with the other two groups, the expression of DC-SIGN mRNA in RNAi group was much lower (p<0.05). The expression level of DC-SIGN protein in RNAi group was much lower measured by Western Blot (p<0.05).②The positive rate of DC-SIGN on the surface of DCs in RNAi group was lower, so were ManLAM group and ManLAM+RNAi group(p<0.05). Expressions of HLA-DR were no significant difference among the four groups (p>0.05). The positive rates of CD83 and CD86 on the surface of DCs in ManLAM group were lower(p<0.05). IL-10 level in ManLAM group was higher than that in other groups (p<0.05). Compare with DCs group and RNAi group IL-10 level in ManLAM+RNAi group was much higher ( p<0.05 ) . The rest groups were no significant difference (p>0.05).IL-12 level In ManLAM group was lower than in the other groups (p<0.05). The rest groups were no significant difference(p>0.05).3. Compare with the other groups,IFN-γlevel in the co-culture solution of DCs and na?ve T cells in ManLAM group was much lower (p<0.05). IFN-γlevel in ManLAM+RNAi group was lower than that in DCs group(p<0.05).The rest groups were no significant difference (p>0.05). The potency of proliferation induction of DCs to T lymphocytes in ManLAM group was lower than that in the other three groups (p<0.05). The rest groups were no significant difference(p>0.05).4. TNF-αlevel in ManLAM group was lower than that in DCs group and in ManLAM+RNAi group (p<0.05). Compare with DCs group and ManLAM+RNAi group,NO level in ManLAM group was much lower (p<0.05).Viable count which was phagocytized by macrophage in ManLAM group was higher than that in DCs group and in ManLAM+RNAi group(p<0.05).Conclusion:1. The maturation of DCs could be inhibited by ManLAM,the proliferation of T cells and the activation of na?ve CD4+CD45RA~+T cells into Th1 cells which were induced by DCs could also be inhibited by ManLAM, and then the potency of macrophage activation was depressed, MTB could not be killed efficiently.2. The expression of DC-SIGN on the surface of DCs could be inhibited effectively by lentivirus-mediated RNA interference, but the maturity and the function of DCs were not influenced .3. The downstream anti-tuberculosis immune function which was inhibited by ManLAM could be recoverd completely or partly after downregulating DC-SIGN, and the potency of macrophage killing MTB could be recovered too.