Chlamydia Trachomatis CT849 Protein Induces Pro-Inflammatory Cytokines Secrection In Macrophages And Inhibits Apoptosis Of Macrophages

Objective:To investigate the mechanism of Chlamydial CT849 protein inducing pro-inflammatory cytokines secretion in macrophages,as well as the effect of CT849 protein on apoptosis of macrophages,and to provide theoretical basis for further elucidating C.trachomatis pathogenicity.Methods:The constructed pGEX-6p/ct849 vector was uesd to express in large quantities by 0.5mM IPTG at 37℃,and the protein was purified.CT849 protein was identified by SDS-PAGE and western blot after BCA assay detection.PreScission protease was used to remove endotoxin after cutting GST tags of CT849 protein.THP-1 cells were stimulated with 100 ng/ml PMA for 24 h to differentiate into adherent macrophages.The nuclear translocation of p65 and intracellular Phosphorylation of IκBα were detected by indirect immunofluorescence and western blot after stimulated by 10μg/mL CT849 for 0,30,60 and 120 min.After pretreatment with 10,20μM NF-κB inhibitor BAY11-7082 for 30 min and stimulated with 10 μg/mL CT849 protein,the secretion levels of TNF-α,IL-1β,IL-6 and IL-8 in the supernatant of the cells were determined by ELISA.After 0,5,10,15 μg/ml CT849 was stimulated for 60 min,the apoptosis rate was detected by flow cytometry,and the expression levels of Bax,Bcl-2 and H2AX were detected by western blot.The levels of Akt were detected after stimulated with 10μg/ml CT849 for 60min.After treatment with PI3K/AKT inhibitor LY294002,the apoptotic rate of macrophages was detected by flow cytometry.Results:(1)The purified protein with molecular weight of 44kDa was obtained by IPTG induced pGEX-6p/ct849 recombinant bacteria,and the target bands was markeble presente identified by western blot.(2)Western blot showed that the intracellular phosphorylation of IκBα was began at 30 min after stimulated,and reached the highest after stimulation for 120 min,and the nuclear translocation of p65 was significantly increased at 4 hours after stimulation with CT849.(3)After treatment with NF-κB inhibitor BAY11-7082,the nuclear translocation of p65 induced by CT849 protein in macrophages was significantly reduced,and the intracellular secretion levels of TNF-α,IL-1β,IL-6 and IL-8 were significantly reduced.(4)Flow cytometry results showed that the apoptosis rate was decreased significantly with the increase of protein stimulation concentration and time.In addition,the intracellular expression of Bax was also decreased significantly with the increase of stimulus concentration and duration,while Bcl-2 expression increased significantly.(5)Western blot resultes showed that after stimulation with 0,5,10 and 15μg/ml CT849 for 60 min,the phosphorylation of H2AX was decreased gradually with the increase of protein concentration.(6)After 0,5,10 and 15μg/ml CT849 protein stimulated for 60min,the intracellular Aktgradually increased and was maximized when stimulated by 15μg/ml CT849.However,after treatment with PI3K/AKT inhibitor LY294002,the rate of cell apoptosis was significantly increased compared with the control group without inhibitor treatment.Conclusions:Chlamydial CT849 protein could stimulate the secretion of TNF-α,IL-1β,IL-6 and IL-8 in macrophages via the NF-κB signaling pathway,and could inhibit the apoptosis of macrophages through the PI3K/AKT signaling pathway.