Heat Shock Proteins Of Chlamydophila Pneumoniae Stimulate Cytokine Production Through ERK And NF-κB Signal Pathways In HUVEC

Chlamydophila pneumoniae (C. pneumoniae) is an obligate intracellularpathogen which spread worldwidely. It is responsible for approximately10%of casesof community acquired pneumonia and5%of cases of bronchitis among adults. It cancause a pharyngitis, bronchitis, sinusitis and possibly atherosclerosis. The infection iscommon with200,000~300,000new cases reported annually, mostly in young adults.The seroprevalence in the adult population is about50~70%and nearly everybody hasbeen infected at least once during their lifetime. The asymptomatic infection whichoften be neglected and untreated usually causes chronic persistent infection andsevere complications. Therefore, research on the pathogenic factor and thenosogenesis has to be of great value in diagnosis and prevention/control theinfection.The C. pneumoniae-related disease is characterized by the inflammations whichinduced with the bacteria, and the HSPs may play a key role in inducing it. In order toclarify the pathogenesis of the C. pneumoniae HSPs (CHSPs) in inflammatory/immunopathologic lesions, and identify the receptors as well as the signal pathwaysinvolved in it, we applied the celluar microbiology to analysing the interaction ofTLR2/4, ERK and NF-κB signal pathway with CHSPs inducing inflammatoryreactions.ObjectivesThe research is carried out to investigate①the functions of CHSPs in inducing thepro-inflammtory cytokines including interleukin-lβ (IL-1β) and IL-6in HUVEC;②the correlation between activation of ERK and NF-κB signal pathways withproduction of cytokines;③the role of TLR2and TLR4involving in activation ofERK and NF-κB signal pathways with CHSPs stimulation. These laid the foundationfor the pathogenesis research of C. pneumoniae. MethodsThe genetic sequence of CHSP60and CHSP70obtained from Genbank, and thespecific primers designed were used to amplifie the targeted genes from C.pneumoniae complete genome by polymerase chain reactions (PCR). The PCRproducts were ligased into the prokaryotic expression vector pGEX-6p-2to constructthe recombinant plasmid, and then were transformed into E. coli XL1-Blue. Afterinduced with IPTG, the three recombinant proteins were expressed and identified withwestern-blot, and purified using affinity chromatography. The purified proteins wereconcentrated into1mg/mL, and were excluded the possible contaminated endotoxinby using polymyxin B agarose or50μg/mL polymyxin B, then the samples weredetermined the endotoxin activity with Limulus amoebocyte lysate (LAL) test kit.HUVEC were cultivated in24well plates and stimulated with purified samplesobtained above, the stimulus concentrations of CHSPs varied from0.5to30μg/mL(0.5、1、5、10、15、20、30μg/mL) and the secretion of IL-1β and IL-6aredose-dependent, the stimulus time of CHSPs varied from2to60h (2、6、12、24、36、48、60h) and the secretion of IL-1β and IL-6are time-dependent. To furtherconfirmed the ability of CHSPs inducing cytokines, the purified sample wereperformed by heating at56℃for30min or100℃for20min, or deproteinizingtreatments, respectively, and then were used to stimulate the cells for detection of thelevels of cytokines.HUVEC were incubated with CHSPs in different time (2、15、30、45、60min).The cells lysate were collected to react with anti-phospho-ERK and anti-ERKantiboties in order to determine the activation of ERK signal pathway by western-blot.LPS and C. pneumoniae stimulation were used as positive controls to compare themanner in activating ERK pathway, and PBS and GST were used as negative controls.The ability in activating ERK pathway between CHSPs were compared. ERKinhibitor U0126were used to preincubated with cells, the level of IL-1β, IL-6and theactivation of ERK pathway were determined after CHSPs stimulation.HUVEC were collected after30min,60min and120min incubation of CHSPs.Nuclear protein extracts were mixed with the labeling DNA probe and the activatedtranscription factors that bind DNA will migrate differently than free DNA. In electrophoretic mobility shift assay (EMSA), PBS and GST were used as negativecontrols to evaluate the ability of CHSPs in activating NF-κB by observingDNA-NF-κB interactions. Supershift assay were performed to further analyze thespecific p65antigen in extracts. NF-κB inhibitor PDTC were used to preincubatedwith cells, the level of IL-1β, IL-6and the activation of NF-κB pathway weredetermined after CHSPs stimulation. Dual-Luciferase reporter system was set up bytransfection with pNF-κB-luc and pRL-TK, the expression of luciferase in HUVECwere measured quantitatively to confirm the transcriptional activation induced withNF-κB.HEK293cells were cotransiently transfected with or without different amountsof plasmids including human TLR2, TLR4as well as CD14and MD2according toresearch design, together with pNF-kB-luc and pRL-TK for normalization. Aferstimulation with CHSPs, the expression of luciferase in HUVEC were measuredquantitatively to confirm the TLR2/4involvment in activating signal pathway andinducing cytokines.ResultsThe recombinant plasmids of targeted gene with pGEX-6p-2vector wereconstructed successfully and expressed in E. coli which induced with IPTG.Restriction enzyme digestion analysis and sequencing suggested the insertedfragments were targeted gene, which show100%similarity with sequence reported inGenbank. The results obtained by western blot analysis showed a band at the expectedmolecular weight, confirming correct insertion of the encoding gene. Purified proteinswere obtained with affinity chromatography and then concentrated into1mg/mLsamples. After removing the possible effects of LPS with polymyxin B agarose, theendotoxin activity of purified samples was determined <0.04EU/μg by using the LALassay kit.Dose-dependent curve was observed in presence of various concentrations ofCHSPs (0.5,1,5,10,15,20,30μg/mL) in production of inflammatory cytokines IL-1β and IL-6in HUVEC. Low concentrations of prepared protein induced lowlevels of cytokine secretion and high concentrations induced high levels of cytokineproduction. Of increasing concentrations tested,20or30μg/mL CHSPs was thestronger inducers of cytokine production in HUVEC compared with0.5,1,5,10,15μg/mL protein. The pro-inflammatory activity was also time-dependent. From2to60h, the amounts of IL-6and IL-1β peaked at12h after stimulation with CHSP10, whilepeaked at24h after stimulation with CHSP60or CHSP70. Among CHSPs, the abilityof CHSP60inducing cytokines is strongest, CHSP10triggered low synthesis ofinflammatory cytokines comparable with CHSP60and CHSP70.After stimulation with CHSPs, the phosphorylated ERK were tested time-dependently and the peak occurred at30min, which is consistent with thecircumstance of C. pneumoniae. The phosphorylated ERK peaked after15minstimulation with LPS and then decreased. Compared with other two CHSPs, theactiviation of CHSP60to ERK pathway is the strongest, while CHSP10and CHSP70is lower than it. U0126can not only inhibit the phosphorylation of ERK but alsodecreased the secretion of IL-1β and IL-6.In EMSA assay, after incubated with CHSPs, the cell lysates can react with DNAprobes and show DNA-protein complex which migrate differently than free DNA.The activated NF-κB is the strongest which induced with CHSP60and peaked at60min with all the CHSPs. In addition to specific migrant band of DNA-proteincomplex, supershift test showed another supershift band of p65antigen-antibodycomplex. PDTC can not only inhibit the activation of NF-κB but also decreased thesecretion of IL-1β and IL-6. The reportor gene also comfirmed the activation ofNF-κB which induced with CHSPs in HUVEC.After transfection with different amounts of indicated plasmids including TLR2or TLR4, CD14and MD2, the phosphorylated ERK increased in TLR2and/or TLR4transfected cells with CHSPs incubation. Dual luciferase reporter assay system thattransfected HEK293cells together with pNF-κB-luc and pRL-TK was to analyze therole of TLRs in activation of NF-κB pathway. It showed the effect of TLR4whichinvolved in activating NF-κB in HEK293cells was better than TLR2, and the effect of two TLRs was more effective than transfection of one TLR.Conclusion1. Prokaryotic expression vector of CHSPs were successfully constructed, and thesoluble purified proteins with molecular weight about15kDa,86kDa,98kDawere obtained.2. CHSPs can induce secretion of proinflammation cytokines IL-1β and IL-6inHUVEC dose-dependently and time dependently, and may be implicated in thepathogenesis of inflammation which induced with C. pneumoniae.3. CHSPs can activate ERK signal pathway to produce inflammatory cytokines inHUVEC.4. CHSPs can activate NF-κB signal pathway to produce inflammatory cytokines inHUVEC.5. TLR2and TLR4may be involved in activation of ERK and NF-κB signalpathways induced with CHSPs.