| Tuberculosis(TB)caused by Mycobacterium tuberculosis(Mtb)remains to be a serious infectious disease worldwide and increases the burden of global public health.Bacille Calmette-Guérin(BCG)is the current TB vaccine while providing limited protection,therefore developing more effective TB vaccines is the key to preventing and controlling TB.Studies have found that in the dormant period of Mtb,Rv2029c which is encoded by the dormant survival regulator(DosR)genes has great potential to become a novel TB vaccine-associated antigen,but the immune response and related response mechanisms have not yet been fully elucidated.Objective:Exploring the role and mechanism of DosR antigen Rv2029c in the regulation of innate immunity and adaptive immunity.Methods:(1)The recombinant protein Rv2029c(rRv2029c)was obtained through genetic engineering technology,the C57BL/6 mice peritoneal macrophages and THP-1cells were treated with rRv2029c.The expression of toll-like receptor 2(TLR2)and phosphorylated nuclear factor?B(NF-?B)of mouse macrophages stimulated by rRv2029c were detected by Western Blot.The expression of co-stimulatory molecules CD80,CD86 and CD40 on the surface of macrophages were detected by flow cytometry.(2)The C57BL/6 mice peritoneal macrophages and THP-1 cells were treated with rRv2029c.The apoptosis of macrophages stimulated by rRv2029c were detected by flow cytometry;the expression of caspase-8,caspase-3,Bax,Bcl-2 mRNA of THP-1cells was detected by qRT-PCR.(3)The C57BL/6 mice were immunized with rRv2029c at week 0,2,and 4.Mice were infected by 5×10~8 CFU BCG via the nasal route after 3weeks of the last immunization.After three weeks,the lung inflammation was evaluated by pathological detection,the expressions of Interferon-?(IFN-?),interleukin-4(IL-4),IL-10,transforming growth factor-?(TGF-?)in CD4~+T cells and CD8~+T cells in lungs,lymph nodes(LNs)and spleens were detected by flow cytometry.Results:(1)Compared with the control group,rRv2029c enhanced the expression of TLR2 in macrophages of the C57BL/6 mice in a dose-dependent manner(p<0.01),and the expression of NF-?B(p-p65)was up-regulated when the dose of rRv2029c is100 ng/ml(p<0.05).rRv2029c induced the expression of co-stimulatory molecules CD40 and CD80 on the surface of the mouse macrophages compared with the control group(p<0.05).THP-1 cells that were treated with rRv2029c at the dose of 100,500,1000 ng/ml induced the expression of MHC-II,and rRv2029c also enhanced the expression of CD40,CD80 and CD86 compared with the control group(p<0.05).(2)Compared with the control group,rRv2029c-treated(12 h,24 h)mouse macrophages showed much higher apoptotic rations(p<0.05).THP-1 cells that were treated with rRv2029c at the dose of 100 ng/ml for 6 h,24 h increased the apoptotic rations compared with the control group(p<0.05).Moreover,rRv2029c stimulated THP-1 cells to express caspase-8,caspase-3 and Bax at mRNA level,while while the expression of Bcl-2 was reduced(p<0.05).(3)Compared with the control group,rRv2029c-immunized mice showed lower lung inflammation(p<0.05).The number of IL-4 in CD4~+T cells and CD8~+T cells was decreased in lungs,LNs and spleens(p<0.05),while the number of TGF-?was increased in CD8~+T cells in LNs and in CD4~+T cells and CD8~+T cells in spleens(p<0.05).Conclusion:The DosR antigen Rv2029c might exert immunoprotective effects,and its possible pathways include activation of macrophages,induction of macrophage apoptosis,and balancing of Th1/Th2 cellular immune responses. |
PDF Full Text Request