Study Of The Mechanism Of The Inflammation Induced By Chlamydia Trachomatis

ObjectiveInfection of Chlamydia trachomatis can lead to not only trachomatis but also sexually transmitted diseases such as cervicitis,salpingitis,pelvic inflammatory disease,subsequent tubal infertility,ectopic pregnancy, abortion et al.The inflammation induced by Chlamydia is contributed to the diseases,but the definite mechanism is unclear.In this report:①The signaling pathway mechanisms of chlamydial induction of proinflammatory factors including IL-1α,IL-6,IL-8 was explored.②The effects of IL-1αon chlamydial induction of IL-8 was studied.③The effects of caspase-1 on the resist to chlamydial infection and on the upper genital tract pathologies caused by Chlamydia were also studied.From all these the mechanisms of Chlamydia-induced diseases are tried to be found and these studies can establish the experimental foundation for therapy and prevention of Chlamydia.Methods1.To identify the production of proinflammatory cytokines such as IL-1α,IL-1β,IL-6,IL-8 et al induced by Chlamydia via ELISA and RT-PCR.To identify the production and location of intracellular IL-8 induced by Chlamydia via immunofluorescence assay.2.To identify the activation of different MAPK pathways(ERK or P38 or JNK)induced by Chlamydia via western-blot.And to identify the exact pathway responsible for the induction of different inflammatory cytokines such as IL-1α,IL-6 and IL-8 et al by using the chemical inhibitors for different signaling pathways via western-blot.3.To identify the role of IL-1αin Chlamydia-induced IL-8 via the methods of IL-1αsiRNA,infection experiment of IL-1αgene "knockout mice macrophage and infection experiment of different cell line which express different level of IL-1α.4.To identify the way that IL-1αparticipates in the Chlarnydia-induced IL-8 via the methods of IL-1Ra and IL-1αneutralization antibody blocking of the IL-1R I pathway,infection experiment of IL-1RⅠ^-/- mice macrophage.And to identify the way that IL-1αcontribute to the production of IL-8,localization of intracellular IL-1αthrough chlamydial infection experiment and transfection of pDsRed-pro-IL-1αplasmid into mammalian cells via immunoflourescence assay.5.To identify the activation of caspase-1 induced by Chlamydia via western-blot.Establishing the mouse genital C.muridarum infection model in order to observe the role of caspase-1 on the susceptibility to Chlamydia and on the ability of bacteria clearance between caspase-1^-/-mice and wild type through monitoring the genital shedding after chlamydial infection.And to assess the genital inflammation damage through pathological studies after primary and secondary C.muridarum infection.Result1.Chlamydia can induce inflammatory cytokines such as IL-1α, IL-1β,IL-6,IL-8 and is time-responsible.The production of extracellular IL-6,IL-8 is identical with the intracellular ones,while the extracellular IL-1αis 24 hours delayed after the detection of intracellular IL-1α.And Chlamydia-induced IL-8 is co-localized with Golgi via immunoflourescence.2.Chlamydia can induce the activation of MAPK/ERK and MAPK/P38 signaling pathways,and is time-responsible.The activation of MAPK/ERK and MAPK/P38 pathways is associated with the production of the proinflammmatory cytokines.Inhibiting different MAPK pathways by the chemical inhibitors,showed that MAPK/ERK pathway is contribute to the Chlamydia-induced IL-8 and MAPK/P38 pathway is contribute to the Chlamydia-induced IL-1α,IL-6 and IL-8.3.It showed that the IL-1αmRNA level and protein expression can not be detected in HT29 and HGF cell lines even after chlamydial infection.Compared with the cell lines such as SiHa,Hela229 and Hep2 which can express IL-1α,IL-8 can be detected by neither of these tow cell lines.To further study the role of IL-1αin Chlamydiainduced IL-8,experiments of IL-1αsiRNA and IL-1α^-/-mice macrophage infection were done.The studies showed that Chlamydia-induced IL-8 is dependent on IL-1α.4.Blocking of the IL-1RⅠpathway by IL-1Ra and IL-1αneutralization antibody showed that Chlamydia-induced IL-8 is not dependent on the IL-1RⅠpathway at 48h post infection,while is partially dependent on the IL-1RⅠpathway at 72h post infection.The studies showed that there is no significant difference between the production of IL-8 from Chlymadia-infected macrophage of IL-1RⅠ^-/-mice and of wild type mice.It also showed that Chlamydia can stimulate intracellular IL-1αto translocate into nuclear and IL-8 staining was found in Chlamydia-infected cells via immunoflourescence assay.And transfecting pDsRed-IL-1αinto mammalian cells showed that the fusion protein can move into nuclear and induce IL-8 production compared with the empty plasmid.5.Chlamydia trachomatis can activate caspsase-1 and processing IL-1β.Infection of the mouse genital tract with C.muridarum,showed that caspsae-1 had no effect on the susceptibility to Chlamydia and had no effect on the bacteria clearance through monitoring the genital shedding.The studies also showed that caspase-1 can significantly enhance the oviduct inflammation damages compared with the wild type mice(p＜0.05).Conclusion1.Activation of MAPK/ERK pathway is essential for the production of IL-8 induced by Chlamydia trchomatis,activation of MAPK/P38 pathway is essential for the production of IL-1α,IL-6 and IL-8 induced by Chlarnydia trchomatis.2.Intracellular interleukin-1αmediates interleukin-8 production induced by Chlamydia trachomatis infection via a mechanism independent of typeⅠinterleukin-1 receptor.3.Caspas-1 contributes to Chlamydia-induced upper urogenital tract inflammatory pathologies,but has no effect on the susceptibility to Chlamydia and on the ability of bacteria clearance.