Treponema Pallidum Flagellar Filament Outer Layer Protein Tp0664 Induces THP-1 Cells To Express IL-6 Via TLR2 Pathway

Objective: This study is to investigate the mechanisms by which Treponema pallidum flagellar filament outer layer protein Tp0664 induces proinflammatory cytokine IL-6 in human THP-1 cells.Methods: The tp0664 gene was ampli?ed by PCR and then subcloned into the prokaryotic expression vector pET28 a. The recombinant protein Tp0664 was expressed in E. coli by the induction of IPTG and purified by Ni2+-NTA affinity chromotography. Protein concentration was determined by using a bicinchoninic acid protein assay kit. Polymixin B was used to remove the endotoxin in the recombinant protein. Human THP-1 cells were cultured for transient transfection with dominant negative(DN) plasmids or siRNA of TLR2, TLR4, TLR5,TLR6 and MyD88, or pretreated with the ERK1/2 inhibitor PD98059,JNK inhibitor SP600125, p38 inhibitor SB203580, and NF-κB inhibitor BAY11-7082, and then stimulated with recombinant protein Tp0664 for indicative time intervals. After incubation with recombinant protein Tp0664, cells were collected and disrupted, before being tested for the transcription of IL-6 mRNA, expression of IL-6, and the phosphorylation of several signaling molecule using RT-PCR, ELISA, and Western bloting,respectively.Results: Recombinant plasmid pET28a-Tp0664, confirmed by colony PCR and sequence, was successfully constructed. The recombinant protein was expressed as supernatant with a molecularweight of 34 kDa. After Ni-NTA affinity purification, the purity of the recombinant protein reached up to 95%. Western bloting showed the molecular weight was consistent with the predicated. After processed with polymixin B, the endotoxin in the recombinant protein was found to be less than 0.04 EU/mL. When THP-1 cells were stimulated by recombinant protein Tp0664 ranging from 0.1μg/mL to 10μg/mL, the transcription of IL-6 mRNA and the expression of IL-6 significantly increased. When THP-1 cells were pretreated with the inhibitors of ERK1/2, p38, and NF-κB, the transcription of IL-6 mRNA and the production of IL-6 decreased significantly. However, inhibition of JNK was insufficient to attenuate IL-6 mRNA transcription and IL-6expression. Moreover, Tp0664-induced IL-6 mRNA transcription and IL-6 expression were also abrogated by transfection with DN-TLR2 and DN-TLR6. There are significant statistical differences between group transfected with DN-TLR2 and control groups(P < 0.05), but no statistical significance between group transfected with DN-TLR6 and control groups(P>0.05).Conclusions:(1) Treponema pallidum flagellar filament outer layer protein Tp0664 could induce THP-1 cells to express IL-6;(2) TLR2,ERK1/2, p38, and NF-κB are involved in the IL-6 expression by THP-1cells under the induction of recombinant protein Tp0664.