The Effect Of Mycobacterium Tuberculosis HtdY On Mouse Dendritic Cells Maturation And T Cell Polarization

Objective: To investigate whether HtdY can prompt DC mature and activate DC and whether HtdY can drive T cell polarization.Methods: The recombined Rv3389c-p ET32 a was transformed into E.coli BL21(DE3)for expression.HtdY protein was purified by Ni-NTA binding column then evaluated by SDS-PAGE gel and mass spectrum,the endotoxin of protein was removed by Triton X-114.As to investigate whether HtdY can prompt DC mature,we obtained DCs and Na(?)ve T cells from 7-weeks-old female mice,all of cells were cultured in RPMI1640 medium.The secretion level of the proinflammatory cytokines including IL-1β,IL-12,IL-6 and TNF-α from DCs culture supernatant were detected by ELISA.The expression level of cell surface molecule from DCs was analyzed by flow cytometry.To investigate the receptor HtdY recognised on DC,the TLR4 and TLR2 knock-out mice models were used to determine the recognition receptor of DC.The cell signal participate in DC was study by pharmacological inhibitors.And the IFN-γ secretion level of Na(?)ve T cell was detected by ELISA.Results: HtdY could prompt DC mature and activate DC by augmenting the expression of CD80,CD86 and MHC II molecules.Moreover,HtdY increased DCs secretion of the proinflammatory cytokines IL-1β,IL-12,IL-6 and TNF-α.Next,TLR4knock-out mice model revealed that HtdY was recognized by TLR4 on mouse DC.Pharmacological inhibitors showed that the maturation of DC was regulated by MAPK signal pathway.In addition,HtdY treated DCs accelerated the proliferation of Na(?)ve T cell,with increased level of IFN-γ.Conclusions: Our study provides evidence that HtdY is a novel DC maturation-inducing antigen through MAPK signal pathway that drives T cell immune responses toward Th1 polarization via TLR4,and is a potential target for the development of new immunological and vaccine strategies against tuberculosis.