Treponema Pallidum Membrane Lipoproteins Tp0663, Tp0821 And Tp0971 Induce Macrophages To Produce Proinflamatory Cytokines Via TLR2 Pathway

Objective This study was designated to investigate the production of proinflamatory cytokines IL-6 and IL-1βfrom macrophages induced by Treponema pallidum membrane lipoproteins Tp0663, Tp0821 and Tp0971, and the correlation between signal transduction pathway mediated by TLR2, CD14 and NF-κB and production of proinflamatory cytokines induced by Tp lipoproteins Tp0663, Tp0821 and Tp0971.Methods The sequences of gene Tp0663, Tp0821 and Tp0971 from Genbank were amplified from T. pallidum Nichols strain by polymerase chain reactions, then sucloned into the prokaryotic expression vector pET28a (+) to construct the recombinant plasmid pET28a (+) / Tp0663, pET28a (+) / Tp0821, and pET28a (+) / Tp0971. After identification by analysis of restriction enzyme digestion, PCR and DNA sequencing, the recombinant plasmids were transfected into E.coli Rosseta strain to express their proteins by IPTG induction. The expression products were purified by Ni-NTA affinity chromatography, and the concentration was determinated by BCA method. Detoxi-Gel was used to remove endotoxin contamination in during the protein preparation. After induced by PMA, THP-1 cells were incubated with various concentrations of Tp0663, Tp0821 and Tp0971 recombinant proteins. Levels of IL-1βor IL-6 were detected by the quantitative"sandwich"ELISA technique. Pretreated with anti-TLR2, anti-CD14 or pyrrolidine dithiocarbamate, THP-1 cells were stimulated with Tp0663, Tp0821 and p0971, respectively for evaluation of the role of TLR2 ,CD14, and NF-κB in the production of cytokines by ELISA.Results Tp0663, Tp0821 and Tp0971 genes were amplified successfully by PCR, and the recombinant plasmids were confirmed by enzyme digestion and sequencing. SDS-PAGE results showed three recombinant proteins were expressed as the soluble or inclusion bodies with a relative molecular weight of 34kDa (Tp0663), 36kDa (Tp0821) and 29kDa (Tp0971) in bacterial cells, their purity reached to 95% after Ni-NTA affinity purification. TAL showd the endotoxin level was less than 0.04EU/mL after processed with endotoxin removal gel. 0.5μg/ml～10μg/ml of proteins could stimulate macrophages to produce IL-6 and IL-1β. However, when the concentration of proteins increased to 3μg/ml for Tp0663 and 5μg/ml for Tp0821 and Tp0971, the produciton of IL-6 and IL-1βdecreased. Anti-TLR2 antibody inhibited Tp0663, Tp0821 and Tp0971 to induce IL-6 of production to 66%, 64% and 60%, and IL-1βproduction to 63.0%,62.0% and 62.0%, repectively. Anti-CD14 antibody inhibited 71.0%, 73.0% and 69.0% of IL-6 production for Tp0663, Tp0821 and Tp0971, and decrease IL-1βproduction to 69.0%, 70.0% and 66.0% ,repectively. The inhibit rates of IL-6 reached to 54.0%, 55.0% and 56.0% when combined use of anti-TLR2 and anti-CD14 antibodies, repectively, and the inhibit rates of IL-1βreached to 51.0%, 50.0% and 52.0%. PDTC inhibited 20% of IL-6 and IL-1βproduction. There are statistical significant differences between groups treated with antibodies or PDTC and control groups.Conclusion1 Tp0663, Tp0821 and Tp0971 are able to induce macrophages to produce proinflamatory cytokines IL-6 and IL-1β.2 Production of IL-6 and IL-1βinduced by Tp0663, Tp0821 and Tp0971 may be related with signal transduction pathway mediated by TLR2, CD14 s and NF-κB.