The Effect Of Chlamydial Plasmid-encoded Protein PGP3 Binding With LL-37 On The Inflammatory Response

[Objective]To detect whether LL-37 could induce inflammatory cytokines IL-6and IL-8 in human vaginal epithelial cells MS74 and whether the chlamydial plasmid-encoded protein pGP3 could block this process.To detect whether pGP3could induce inflammatory cytokine IL-6 and IL-8 in neutrophils that prepared from C3H/HeJ mice and whether LL-37 could enhance this process.To investigate the effect of pGP3 binding to LL-37 on inflammatory response.[Methods]（1）Recover the human vaginal epithelial cells MS74.The cells were cultured in DMEM medium at 37℃,5%CO₂,supplemented with 10%fetal calf serum,100U/ml penicillin and 100μg/ml streptomycin.Set the MS74 cells up at1.5×10⁵ cell/ml/well in a 24 well cell culture plate.The MS74 cells were treated with or without 12μmol/L of LL-37,pGP3 or pGP3/LL-37 complexes for 24h at 37℃.Collect the supernatants to measuring IL-6 and IL-8 and the cytokine concentrations were calculated from 3 independent experiments.The pGP3/LL-37 complexes were formed by pre-incubating the two together each at 12mmo/L in cell culture medium for 30min at 37℃before applying the co-incubation mixtures to the cell culture wells.ELISA kits were used for measuring cytokines.（2）Bone marrow cells were harvested from tibiae and femurs of C3H/HeJ mice which have resistance to lipopolysaccharide due to a spontaneous mutation in toll-like receptor 4 gene.Neutrophils isolated from the bone marrow cells with kit.After lysing red blood cell,neutrophils were cultured in RPMI-1640 medium in 10cm dish at 37℃,5%CO₂,supplemented with 2mmol/L L-glutamine,10%fetal calf serum,100U/ml penicillin and 100μg/ml streptomycin.Every 3 days,the floating cells were harvested and re-cultured in fresh growth medium.Bone marrow-derived neutrophils made from C3H/HeJ mice were set up at 1×10⁶ cell/ml/well in a 24 well plate.The cells were treated with or without 10ng/ml LPS（TLR4 ligand as a negative control）,0.1μg/ml Pam3（TLR2 ligand as positive control）,LL-37,pGP3 or LL-37/pGP3 complexes at 0.05,0.25 and 1.25μmol/L at 37℃for 24h.The supernatants were collected for measuring both IL-6 and IL-8 and the cytokine concentrations were calculated from 3 independent experiments.ELISA kits were used for measuring cytokines.（3）statistical method:Wilcoxon rank sum test were used for quantitative data.[Results]Although LL-37 induced significant levels of IL-6 and IL-8,the LL-37proinflammatory activity was blocked by forming complex with pGP3(^**Wilcoxon rank sum test).pGP3 but not LL-37 alone stimulated minimal levels of cytokines and the proinflammatory activity of pGP3 was significantly enhanced by forming complexes with LL-37(^**Wilcoxon rank sum test).[Conclusion]LL-37 induction of inflammatory cytokines in human vaginal epithelial cells MS74 is inhibited by chlamydial plasmid-encoded protein pGP3.pGP3 induction of cytokine production in neutrophils is enhanced by forming complexes with LL-37.Thus,pGP3/LL-37 interaction may benefit chlamydial survival in the genital tract epithelial tissue especially during the early stage of chlamydial infection and activate myeloid cell-mediated inflammation in the later period which may promote chlamydial dissemination.Tubal inflammatory responses induced by chlamydial organisms can lead to long-lasting fbrosis,which may be the basis of Chlamydia-induced sequelae in the female genital tract.