The Role Of CPAF?STING And MyD88 In The Process Of Chlamydia Muridarum Infectinon In Mice

Chlamydia trachomatis is one of the common causes of sexually transmitted diseases,C.trachomatis is an obligate intracellular parasite.Chlamydial Protease-Like Activity Factor CPAF is a most important protease-like factor after invading host.It can help Ct escape the immunity survillance of host contributing to the proliferation of Ct in epithelial cells.Stimulator of interferon genes(STING)and myeloid differentiation factor 88(MyD88)are two important intracellular signaling pathways adaptor protein.Both share the downstream of the signal pathway associated with chlamydial infection.So we deduce these immunity molecules play critical roles in anti-chlamydia pathogenises.Basing on the similar genotype composition and structure and pathological manifestations of MoPn(Chlamydia muridarum,mouse pneumonitis agent)in mice as to Ct in human genital system the mode made by MoPn is used to studyof Ct.Objective:In this research,we first test the possible mechanism of CPAF assisting MoPn escape host immunity survillance.Secondly,mice deficient in STING,MyD88 and wild type was used to innoculate MoPn by different routes to built mode.Numbers of Chlamydia trachomatisl were compared with wild type mice to invetigate the role of STING and MyD88 played in the genital barrier of MoPn infection.Methods:1)Simulate the antigen presentation process by T cells,DC(dendritic cell)and OT1 or OT2 antigen.In this process,add different concentrations CPAF(0/1/10/100 including g/ml)in each group.Detect the quantity of IL-2 in the supernatant fluid which released by T cells.Protein electrophoresis separation and kamath bright blue method were uesed to test the stripe width change after seven kinds of chlamydial antigens were treated or untreated by CPAF.Select one chlamydial antigen which could be enzymolysised by CPAF and one could not to instead of OT1 and OT2 to repeat the process of antigen presentation.Test the quantity of IL-2 in the supernatant fluid by ELISA that released byT cells.2)Innoculate 2×105 IFUs(inclusion forming unit)MoPn from different ways(vagina,uterus,distal segment of the uterus and ovary bursa)in wild-type,STING-/-and MyD88-/-mice.Sacrifice mice in different time points.After scoring,part of the mice reproductive tracts were taken pictures and then comparing the chlamydia content and the mononuclear cell infiltration by H&E staining and cell or tissue immunofluorescence technique(immunofluorescence technique,IF).3)Different parts of mice reproductive tract tissue homogenated to detect the content of chlamydia by IF technology to compare the difference of two sides of the uterotubal junction barrier and cervical barrier.Results:1)CPAF enzyme OT2 selectively to restrain antigen presentation mediated by MHC II not MHC I.And chlamydial antigens were used instead of OT1 and OT2 to repeat antigen presented process.CPAF selectively enzyme chlamydial antigen of T cells as well(***p<0.001).2)In the transcervical group,STING-/-group is more severe in general pathological changes and the inflamation scores under microscope(**p<0.01).Showing that STING deficiency may inhibit U-T barrier function inhibiting chlamydia,and has nothing to do with cervical barrier function.3)There is significant difference of chlamydia sustaining time in lower genital tract between MyD88-/-and wild type mice(**p<0.01).MyD88-/-mice is more severe in general pathological changes and the inflamation scores under microscope innoculated transvaginal or transcervical(**p<0.01).Showing that MyD88 deficiency may inhibit U-T barrier and cervical barrier function inhibiting chlamydia.4)Innoculated C57BL/6J mice from distal segment of the uterus or ovary bursa,there is significant differences of chlamydia quantity between two sides of U-T structur(*p<0.05).However,there is no significant difference between two sides of U-T structure innoculating chlamydia by two innoculation routes in STING-/-and MyD88-/-mice(p>0.05).Showing that STING or MyD88 both play critical roles in U-T structure function.Conclusions:1)CPAF selectively digests chlamydial T cell cntigens assisting Ct escape host immunity survillance.2)The U-T barrier effect is dependent on STING signal pathway innate immunity for preventing spread of C.muridarum organisms across the mouse genital tract.3)The U-T and cervical barrier effects are dependent on Myd88 signal pathway innate immunity for preventing spread of C.muridarum organisms across the mouse genital tract.