PORF5Plasmid Protein Of Chlamydia Trachomatis Induces IL-1β And IL-18Production In THP-1Cells Through Activation Of NALP3Inflammasome

Objective:Chlamydia trachomatis(Ct) is an important pathogen which can contribute totrachoma and sexually transmitted disease. Inflammation plays a key role in Ctinfection, but the exact mechanisms remain unclear. pORF5plasmid protein is theonly secreted protein which encoded by Ct plasimid gene. NALP3inflammasome is akind of multi-protein complexes located in the nucleus which may inducsesinflammatory cytokines. Herein, the aim of the present study was to investigatewhether pORF5plasmid protein could induce THP-1cells to secrete IL-1β and IL-18by activating NALP3inflammasome and the relationship between NALP3inflammasome and p38MAPK signaling pathway.Methods:The pGEX-6p-1/pORF5recombinant plasmid was transformed into XL1-bluebacteria to express pORF5fusion protein by inducing IPTG(0.2mM). pORF5fusionprotein was purified by Glutathione Sepharose4B and and cleaved by PreScissionProtease to prepare pORF5plasmid protein without GST-tagged protein.Concentration of pORF5plasmid protein was detected by BCA assay. Thecharacteristic of pORF5plasmid protein was analyzed by SDS-PAGE and Westernblotting. Different concentrations of pORF5plasmid protein(range of3to36μg/mL)were used to stimulate THP-1cells treated by160nmol/L of PMA, then theinflammatory cytokines IL-18and IL-1β were detected by ELISA at the time of0h,8h,16h,24h,36h; The mRNA expression of NALP3inflammasome and IL-1β were detected by real time-PCR; Phosphorylated level of p38MAPK was detected byWestern blotting. RNA interference technology was used to silence NALP3and ASCgene, then the mRNA of NALP3and ASC, the level of IL-1β and IL-18, and the effecton p38MAPK signaling pathway were observed. After pretreated with Caspase-1inhibitor(Z-YVAD-FMK) and p38MAPK inhibitor (SB202190) for30min,24μg/mLof pORF5plasmid protein was used to stimulate THP-1cells respectively for24h and30min, then inflammatory cytokines IL-1β and IL-18were analyzed by ELISA.Results:1. The pGEX-6p-1/pORF5recombinant plasmid was transformed into XL1-bluebacteria. After induced by IPTG, pORF5fusion protein with a molecular weightabout54KD was expressed. The pORF5fusion protein was cleaved byPreScission protease to get pORF5plasmid protein without GST tag with amolecular size of28KD.2. The plasmid pORF5protein induced THP-1cells to secrete IL-1β and IL-18by adose and time-dependent manner.24μg/mL of pORF5plasmid protein was usedto stimulate THP-1cells, the level of IL-1β reached its peak at24h, the level ofIL-18reached peak at16h.3. Real time-PCR analysis showed that pORF5plasmid protein induced mRNAexpressions of NALP3inflammasome and IL-1β in THP-1cells,Western blottingshowed that pORF5plasmid protein activated p38.4. After transfected with NALP3-siRNA and ASC-siRNA, the mRNA expression ofNALP3and ASC were reduced by31.5%and48%, the levels of IL-1β and IL-18ingroup transfected with NALP3-siRNA were reduced by27.7%and21%, the levelsof IL-1β and IL-18in group transfected with ASC-siRNA were reduced by44.1%and40.1%.Western blotting showed that phosphorylation level of p38was notsignificantly different in pretreated group compared with control.5. Caspase-1inhibitor was able to decrease the production of IL-1β and IL-18, thelevels of IL-1β and IL-18were reduced by41.9%and37.2%; After inhibited by p38inhibitor, the levels of IL-1β and IL-18were reduced by29.6%and37.8%,themRNA expression of NALP3, ASC, Caspase-1and IL-1β were reduced by23%、31%、13%and23%respectively in THP-1cells pretreated with p38MAPKinhibitor.Conclusion:1. Ct pORF5plasmid protein could induce THP-1cells to secrete IL-1β and IL-18byactivating NALP3inflammasome.2.p38MAPK signal pathway activated by pORF5plasmid protein could influenceNALP3inflammasome.