Treponema Pallidum Flagellins Induces IL-1? And IL-18 Production In Macrophages Via The Activation Of NLRC4 Inflammasome

Objective:This research was to investigate the productions of interleukins such as IL-1?and IL-18 in macrophages derived from the THP-1 cells induced by recombinant Treponema pallidum flagellins FlaB1?Tp0868?,Fla B2?Tp0792?and FlaB3?Tp0870?,and to research the mechanisms of flagellins inducing macrophages to secrete IL-1?and IL-18 by activating NLRC4 inflammasome.Methods:The genes of tp0868,tp0792,and tp0870 was acquired from Gen Bank,and amplified by polymerase chain reactions.Then,subcloned the genes into the prokaryotic expression vector pET28a.The recombinant protein FlaB1,FlaB2 and FlaB3 was expressed in E.coli with the induction of IPTG and purified by Ni²⁺-NTA affinity chromotography.SDS-PAGE and Western blot were used to analysze and appraisal products expression.And then renatured the flagellin by dialysis renaturation method,removed the Endotoxin of recombinant FlaB1,FlaB2,and FlaB3 by Polymyxin B.Then,we measured the determination of protein by limulus reagent BCA.After that,we pretreated THP 1 cells with PMA for 12 h,and then stimulated by 0.1?g/mL to 10?g/mL recombinant protein FlaB3 and 25?g hemolytic chain bacterium hemolysin O?SLO?,after that,we collected the supernatant and cells at 0h,2h,4h,6h,12h and 24h.Then detected the expressions of NLRC4,ASC and caspase-1,IL-1?,IL-18 by RT-PCR,ELISA and WESTERN BLOT.Finally,we pretreated the macrophages with NLRC4-siRNA and caspase-1 inhibitor VX-765,and then dectected these factors.Results:?1?RecombinantplasmidspET28a-Tp0868,pET28a-Tp0792 and pET28a-Tp0870 were constructed successfully.The recombinant flagellin FlaB1,FlaB2,and FlaB3 was all expresssd as inclusion body,and its molecular weight were 36 kDa,35 kDa,and 34kDa,respectively.?2?Recombinant flagellins FlaB1,FlaB2 and FlaB3 were obtained by the Ni-NTA affinity chromatography column.Western blot identified those were the target proteins.BCA method showed that the concentration of purificated recombinant flagellins were 0.48 mg/mL,0.27 mg/mL,and 0.38 mg/mL,respectively.After removed the endotoxin with polymyxin B,the recombinant flagellins by Limulus reagent,and the concentration of bacterial endotoxin were 0.029 EU/mL,0.038 EU/mL,and 0.032 EU/mL respectively.?3?FlaB3 could stimulate macrophages to produce IL-1?and IL-18within a certain range of concentration and time dependence.?4?After stimulated macrophages with FlaB3,the NLRC4inflammasome was activated,and the production of caspase-1 increased.?5?After transfected the NLRC4 siRNA into macrophages,the expression of NLRC4 was inhibited,and causing a lower level of caspase1,IL-1?and IL-18.?6?After pretreated macrophages with the caspase-1 specific inhibitor vx-765,the NLRC4 expression was not significantly reduced,while caspase-1,IL-1?and IL-18 were significantly reduced.Conclusions:1.We constructed the recombinant plasmid pET28a-Tp0868,pET28a-Tp0792,pET28a-Tp0870 successfully.2.Tp recombinant flagellin FlaB3 could stimulate NLRC4inflammasome to induce macrophages to produce IL-1?and IL-18 by the activation of caspase-1.