The Role Of Notch Signaling Pathways Regulating Th1/Th2 Balance In Mycobacterium Tuberculosis Infection

Backgroud and objective:Tuberculosis(TB)is a chronic infectious disease caused by mycobacterium tuberculosis,is still one of the important public health problems.Host immunity plays an important role in anti-tuberculosis,many studies have shown that Th1/Th2 imbalance involved in the development of tuberculosis(TB),and multiple signaling pathways play a vital role in mycobacteria persistent infection in the host.Notch signaling pathway is an evolutionary conservative signal mechanism,a large number of studies have shown that it involved in regulating T cell function and Th cell differentiation,and different ligands induced CD4~+T cell different direction,but there has been controversial.Therefore,this study forced on expression of Notch receptors,ligands and target genes Hes1 and whether related with Th1/Th2 imbalance in TB patients.?-secretase inhibitors(DAPT),specific RNA interference or NICD1 gene expression plasmid regulated Notch signaling pathways,in order to study expression of Th1 cells,Th2 cells,related-transcript factors and cytokines,cell apoptosis and proliferation,and understand the role of Notch signaling pathway in the development of TB.By analyzing whether Jagged2 ligands on DCs regulating T cells response,we discussed blockage of Notch signaling pathway will be feasible in the adjuvant treatment of tuberculosis(TB),provides new ideas for targeted therapy in TB,and experimental basis for its become latent TB treatment strategies.Methods:1)Quantitative real-time PCR(qRT-PCR)is used to detect expression of Notch 1-4,Jagged1-2,and downstream target genes Hes1 mRNA in TST-,LTBI and TB groups PBMCs.2)Expression of Notch1,Jagged2 and Hes1 protein in PBMCs of TST-,LTBI groups by Western Blotting(WB).3)Expression of Notch1,Jagged2 and downstream target genes Hes1 protein and their location in lung tissue in healthy controls(HC)and TB groups by using immune histochemical method analysis.4)Th1 and Th2 cells were detected by flow cytometry(FCM)in TST-,LTBI and TB groups,and the correlation between Notch1,Jagged2 or Hes1 and Th1 or Th2 cells.5)qRT-PCR detected expression of Notch1,Jagged2 and Hes1 Mrna in TB patients with and without treatment.6)Different concentrations(0,2.5,5,10 and 20?mol/L)DAPT treated PBMCs in TST-,LTBI and TB groups,qRT-PCR and WB detected expression of Hes1.7)Analysis of Th1and Th2 cells by flow cytometry method with and without DAPT treatment;qRT-PCR detected expression of T-bet and GATA3 mRNA,CBA method analysed supernatant of IFN-?,IL-10 and IL-4.8)Cells number was counted by microscope,cell apoptosis by flow cytometry and proliferation by CCK8 kits with and without DAPT;9)siRNA-Notch1 or NICD1 gene expression plasmid regulated Notch signaling pathway,and expression of IL-4 in supernatant;10)DCs in TB patients were separated by human DCs enrichment,qRT-PCR detected expression of Jagged1 and Jagged2 mRNA with and without ESAT-6;11)CD4~+T cells were cocultured with DCs pre-sitimulated by ESAT-6,then GSI blocked Notch signaling pathways,CBA method detected supernatant of Th1and Th2 related cytokine.12)CD4~+T cells were co-cultured with Jagged2-Fc,Th1 and Th2 related cytokine was detected by CBA;expression of GATA3 and T-bet mRNA by qRT-PCR.13)Down regulated Jagged2 expression on DCs by RNA interference,using qRT-PCR and WB method detected expression of Jagged2 mRNA and protein.14)CD4~+T cells were co-culture with siRNA-Jagged2,Th1 and Th2 cells were detected by FCM,expression of GATA-3 and T-bet mRNA.15)Statistical methods:Rank-sum test was used for ranked data.?~2 test was used for emumeration data.Rank-sum test was used for paired comparison of multiple groups and statistical analysis of ranked data.Kruskal-wallis test,a nonparametric test,was used for paired comparison of multiple groups of data.Nonparametric Spearman rank correlation method was used for correlation analysis.SPSS19.0 statistical software was used.?=0.05 was significant level(bilateral).Results:1)Expression of Notch1-4,Jagged2 and Hes1 mRNA in TB PBMCs was higher than that in LTBI and TST-groups respectively,while Jagged1 expression among three groups hade no statistical significance,expression of Notch1 in TB group was higher than the other three receptors.2)Expression of Notch1,Jagged2 and Hes1 protein by WB method verified the results of mRNA level.3)Immunohistochemistry results showed that Notch1 and Jagged2 were mainly expressed in lymphocytes cytoplasm in human lung tissue,and their expression in TB group was obviously stronger than those in healthy controls(HC);Hes1 is mainly expressed in lymphocyte cell nucleus and cytoplasm in TB group,and healthy control group had only a small amount of expression.Notch1,Jagged2 and Hes1 expression were higher than those in healthy controls.4)Th2cells increased and Th1 cells were no change in TB patients,there was imbalance of Th1/Th2,and positive correlation between Notch1,Jagged2 or Hes1,and is not related with Th1 cells.5)Compared with before treatment,expression of Notch1,Jagged2 and Hes1 reduced dramatically after treatment of TB patients.6)Expression of Hes1 gene and protein reduced after DAPT treatment in PBMCs in TST-,LTBI and TB groups,it showed that DAPT can block Notch signaling pathway,laying a foundation for further study.7)After DAPT treatment,the percentage of Th2 cells reduced with dose dependence,Th1 cells did not change significantly,DAPT reduced the abnormal rise of Th2 cells and restored Th1/Th2 balance in TB patients.8)Before treatment,IL-4increased significantly in TB patients,while IL-10 and IFN-?little changed.After treatment,IL-4 reduced with a dose dependence,it showed that DAPT reduced expression of IL-4,but not IFN-?and IL-10 in TB patients in cell culture supernatant.9)Expression of GATA3 mRNA raised in TB patients before treatment,GATA3 and GATA3/T-bet mRNA reduced with a dose dependence after treatment,it further proved that the percentage of Th2 cells and IL-4 levels are associated with GATA3 in the presence of DAPT in TB patients.10)Cells number reduced in a dose dependent in TST-and LTBI groups,while when DAPT was 10 mumol/L,cells decreased significantly in TB patients,cells increased significantly in 20?mol/L DAPT.In a word,DAPT can increase cell apoptosis,not proliferation.11)SiRNA interference or NICD1 gene expression plasmid down-or up-regulated Notch signal pathway,further confirmed that Notch signaling pathway involved in regulation of Th2 cells.12)Jagged2 express increased significantly on DCs stimulated by ESAT-6 from TB,when DAPT blocked Notch Pathways,IL-4,IL 5 and IL-13 reduced in supernatant of CD4~+T cells co-cultured with DCs,while IFN-?was not obvious.13)Jagged2-Fc affected CD4~+T cells response in TB patients,increased expression of Th2-related cytokines and GATA3,but Th1-related cytokines and T-bet were not obvious.14)SiRNA interfered Jagged2 on DCs,reducing Th2 cells,IL-4 and GATA3,but Th1 cells,IFN-?and T-bet.Conclusion:1)Notch signal pathway was activated in TB patients,and which decreased after treatment,suggesting that Notch signaling pathways play a role in the development of TB,and is associated with Th1/Th2 imbalance in TB patients,particularly related with Th2 cells.2)?-secretase inhibitors(DAPT)blocked Notch signaling pathways to restore Th1/Th2 balance in TB patients,and suppressed Th2 cells depends on GATA3 and IL-4,up-regulating Notch signal pathway further confirmed that Notch signaling pathway involved in regulation of Th2 cells.3)Expression of Th2related factor increased or decreased by up-or down-regulating Jagged2 expression,but no effect on Th1 related factors obviously,Jagged2 on DCs participated in Th2 immune response.Based on the above experimental results,blocking Notch signal pathway delays the progress of TB,which offers a new way for TB target therapy.