Cyclic GMP-AMP Synthase Pathway-mediated Immune Responses Of Murine Bone Marrow Derived Dendritic Cells During Mycobacterium Bovis Infection

Mycobacterium bovis(M.bovis),the causative agent of tuberculosis(TB)in cattle,is a classic intracellular pathogen that can cross species,and trigger disease in humans and other mammals.It have caused great economic loss and severely threatened public health on a global scale.Recently,the research group of Chinese American scientists Chen Zhi-jian professor found a new DNA sensor cGAS(cyclic GMP-AMP synthase),cGAS belong to the family of nucleotide transferase which can recognize dsDNA in cytoplasm,and catalyzed synthesis second messenger cGAMP.Further,it depends on STING signal pathway and triggers type IIFN production.Interaction between M.bovis and macrophages have been extensively characterized in various studies,while similar analyses in dendritic cell(DC)were often overlooked.With the physiological function gradually clear in DC,the effect of DC have been paid more and more attention in M.bovis infection.This study performed to investigate the expression of cGAS and downstream related protein during M.bovis infected murine bone marrow derived dendritic cell(BMDC).And then we explored the regulatory effect of cGAS signal pathway for BMDC on maturation and activation.Further,we researched the immune response of CD4+ T cells after M.bovis infection and clarified the correlative mechanism.1.This study was based on the primary BMDC culture and the purity above 90%.Western blot detected the expression of cGAS and downstream protein,immunofluorescence analyzed the phenomena of IRF3 enter the nucleus.The detection result showed the expression of cGAS and p-TBK1 were increased and STING showed two bands during M.bovis infection,and the phenomena of IRF3 enter the nucleus was obvious.Whereas in the group of cGAS were knockdown by siRNA,the expression of cGAS signal pathway were inhibited and the phenomena of IRF3 enter the nucleus was weak.2.This study was based on the flow cytometry and IFNAR1 mAb,we detected the surface markers in BMDC such as CD86,CD80,CD40,MHC-?,and ELISA analyze the main cytokines production in BMDC supernatant,such as IFN-?,IL-12p70,IL-10,IL-6,TNF-?.The result showed the expression of surface markers were up-regulated significantly after M.bovis infection,but in silent group and block group the expression were restrained.The production of cytokines had similar trend,however TNF-?was not affected by IFNAR1 mAb.The exogenous IFN-? promoted the level of TNF-??IL-12p70,while IL-10?IL-6 had no significant change.3.This study was based on the immune-magnetic beads to isolate CD4+ T cells from murine spleen and added fluorescent tracer tag,establish co-culture system between BMDC and CD4+ T cells.Flow cytometry analyzed the proliferation of CD4+ T cells and ELISA detected the expression of IFN-?.The result showed the M.bovis can induce mass propagation of CD4+ T cells,but IFNAR1 mAb inhibited the ability of proliferation.In infection group the production of IFN-? was significant increase,whereas in block group it was restrained.In conclusion,this research confirmed the cGAS signal pathway exist in BMDC,during M.bovis infection cGAS and downstream related protein can be activated.And cGAS pathway and IFNAR1 contribute to phenotypic maturation and functional activation of BMDC,promote the progress of antigen presentation.In addition,in CD4+ T cells the proliferation and IFN-? expression were significantly enhanced after M.bovis infection,further induce the adaptive immune response.