PORF5Plasmid Protein Of Chlamydia Trachomatis Induces Proinflammatory Cytokines Response In THP-1Cells Through MAPK Pathways Activated By Toll-like Receptor

Objective: Chlamydia trachomatis(Ct) is an obligate, intracellular pathogen that is a majorcause of trachoma, urethritis and pelvic inflammatory disease worldwide. It has been provedthat immuno-pathological damage caused by excessive inflammation is the importantpathogenic mechanism responsible for chlamydial diseases. However, the mechanism remainsunknown. Herein, the purpose of the present study was to investigate whetherproinflammatory cytokines (including IL-8, IL-1β, TNF-α) were induced in THP-1cellstreated with pORF5plasmid protein through MAPK pathways activated by Toll-like receptor2(TLR2).Methods：pGEX-6p-1/pORF5recombination plasmid was transformed into E.coli XL1-bluehost cells to express GST-pORF5fusion protein induced by isopropylβ-D-1-thiogalactopyranoside (IPTG). After purified with glutathione-Sepharose4B, thepORF5fusion protein was cleaved by PreScission Protease to get the pORF5target proteinwithout GST tag. BCA method was employed to detect the concentration of pORF5;SDS-PAGE and Western blotting analysis were used to characterize pORF5target protein;After endotoxin removal with polymyxin B, different concentrations of pORF5protein wereused to incubated with THP-1cells in vitro respectively, and the proinflammatory cytokinesIL-8,IL-1β and TNF-α induced by pORF5protein were detected by the quantitative ELISAtechnique at the time of0h,6h,12h,24h,36h;Western blotting was applied to detectphosphorylated level of p38, JNK and ERK1/2; THP-1cells were preincubated with differentconcentrations(1，10，30μM) of p38specific inhibitor SB203580,ERK1/2specific inhibitor PD98059and TLR2antibody, and then6μg/mL of pORF5protein was added to the mediumfor another12h, the proinflammatory cytokines was detected by ELISA.Results:(1) GST-pORF5fusion protein with a molecular weight about54KD was successfullyexpressed in XL1-blue host cells habouring pGEX-6p-1/pORF5recombinant. And theGST tag from GST-pORF5fusion protein was cleaved by PreScission protease.the purityof pORF5protein was more than95%.(2) pORF5protein induced THP-1cells to produce IL-8, IL-1β and TNF-α, and theproduction of the proinflammatory cytokines was found to be dose-dependent in a rangefrom2μg/mL to6μg/mL of pORF5protein, but when the concentration of pORF5wasmore than6μg/mL, the amount of IL-8、IL-1β and TNF-α reduced significantly; Thelevels of TNF-α reached its peak at6h and lasted for12h, but the peaks arrived at12h forIL-1β and IL-8.(3) Western blotting showed that pORF5protein activated ERK1/2/MAPK and p38/MAPKin THP-1cells, the intensity of the signaling pathways to phosphorylated ERK1/2/MAPK and p38/MAPK increased as the incubation time proceeds, the peak ofphosphorylated ERK1/2arrived at15min, and the peak of phosphorylated p38/MAPKarrived at30min, and lasted for more than60min. But the intensity of the phosphorylatedSAPK/JNK1/2didn’t changed.(4) ELISA showed that the specific inhibitors were able to decrease the levels of IL-1β, IL-8and TNF-α. IL-8decreased to57%and58.8%respectively,IL-1β decreased to22%and45%respectively,TNF-α decreased to22.4%and16.3%respectively.(5) When TLR2antibody was preincubated with THP-1cells for30min and then stimulatedwith6μg/mL of pORF5protein, ELISA results showed that the amount of IL-1β、IL-8and TNF-α decreased9.8%,32.7%and25.3%respectively. Conclusions:(1) Ct pORF5protein could induce THP-1cells to produce inflammatory cytokines IL-8,IL-1β and TNF-α;(2) ERK/MAPK and p38/MAPK pathways were activated by pORF5protein in THP-1cells.The activated p38/MAPK and ERK/MAPK pathways were involved in theproduction of IL-8, IL-1β and TNF-α,this is maybe related to TLR2activation.