Porphyromonas Gingivalis Infects Human Extravillous Trophoblasts In Vitro

Part 1.Porphyromonas gingivalis induces IL-8 and IFN-y secretion and apoptosis in human extravillous trophoblast derived HTR-8/SVneo cellsObjective:Preterm birth is a major cause for infant mortality and morbidity.A large number of studies have suggested a link between periodontitis and preterm birth.The purpose of this study was to investigate the interaction between a periodontopathic bacterium Porphyromonas gingivalis and human extravillous trophoblast derived HTR-8/SVneo cells.Methods:Production of cytokines in HTR-8/SVneo cells was measured via ELISA.Annexin V/PI flow cytometry was performed to assess apoptosis.Protein expression was measured by western blot.Results:Porphyromonas gingivalis invades and survives within HTR-8/SVneo cells.HTR-8/SVneo cells released significant amounts of IL-8 and IFN-y during exposure to Porphyromonas gingivalis.The IL-8 level started to rise at 6 h,peaked at 24 h and declined at 48 h,while the IFN-y level began to increase at 24 h and maximized at 48 h.Meanwhile,the percentages of both early and late apoptotic cells and the expression of active caspase-3 increased significantly in response to Porphyromonas gingivalis.Additionally,both heat-killed Porphyromonas gingivalis and Porphyromonas gingivalis lipopolysaccharide significantly induced IL-8 production,and conditioned medium significantly induced IL-8 production and apoptosis.Conclusions:Porphyromonas gingivalis invades HTR-8/SVneo cells and induces inflammation and apoptosis in HTR-8/SVneo cells.The abnormal regulation of inflammation and apoptosis in human trophoblasts by Porphyromonas gingivalis infection may give new insights into how maternal periodontitis and periodontal pathogens might be linked to preterm birth.Part 2.Role of MAPK and NF-κB pathways in Porphyromonas gingivalis-induced inflammation and apoptosis in HTR-8/SVneo cellsObjective:This study aims to analyze whether mitogen-activated protein kinase(MAPK)and nuclear factor-κB(NF-κB)pathways are involved in Porphyromonas gingivalis-induced inflammation and apoptosis.Methods:Production of cytokines in HTR-8/SVneo cells was measured via ELISA.Annexin V/PI flow cytometry was performed to assess apoptosis.Protein expression was measured by western blot.Specific pharmacological inhibitors were used to inactivate relevant signaling pathways(p38 MAPK,SB203580;ERK1/2,U0126;JNK,SP600125;NF-κB,JSH-23)to determine their roles in inflammation and apoptosis.Results:Each inhibitor significantly blocked the release of at least one proinflammatory cytokine by HTR-8 cells with the addition of Porphyromonas gingivalis.Specifically,the most significant effect on cytokine release was found using SB203580 and U0126,followed by SP600125 and JSH-23.It should be noted that the proinflammatory response to Porphyromonas gingivalis could not be completely blocked through the inhibition of a single pathway,suggesting that the production of IL-8 and IFN-y by HTR-8/SVneo cells in response to Porphyromonas gingivalis might involve multiple pathways.Moreover,neither SP600125 nor JSH-23 affected the apoptosis effect of Porphyromonas gingivalis.However,U0126 and SB203580 both partially but significantly suppressed the apoptosis,especially U0126.Conclusions:This study demonstrated that activation of ERK1/2 and p38 MAPK pathways participates in Porphyromonas gingivalis-induced inflammation and apoptosis..Part 3.IFN-γ and Fas are involved in Porphyromonas gingivalis-induced apoptosis in HTR-8/SVneo cells.Objective:The present study aims 1)to investigate the role of inflammation(IFN-γ)in porphyromonas gingivalis-induced apoptosis,and 2)to investigate the role of the Fas/FasL pathway in porphyromonas gingivalis-induced apoptosis and its correlation with the ERK1/2 pathway.Methods:Cell apoptosis,cell viability,protein expression,and cytokine production in HTR-8/SVneo cells were measured via flow cytometry,CCK-8 assay,western blot,and ELISA,respectively.Results:Porphyromonas gingivalis decreased cell viability and increased cell apoptosis and Fas expression in HTR-8/SVneo cells.The ERK1/2 inhibitor U0126 and the FasL neutralizing antibody NOK1 that blocks the FasL/Fas interaction both significantly suppressed Porphyromonas gingivalis-induced apoptosis.Treatment with recombinant IFN-γ also significantly decreased the number of viable HTR-8/SVneo cells and increased Fas expression,and IFN-y-induced effect was significantly inhibited by NOK1,suggesting that IFN-y may play an important role in Porphyromonas gingivalis-induced apoptosis of HTR-8/SVneo cells,at least partially through regulation of Fas expression.Moreover,U0126 also inhibited both IFN-y secretion and Fas expression close to the control levels.Conclusions:This study demonstrated that Porphyromonas gingivalis induces IFN-y secretion and Fas expression in HTR-8/SVneo cells in an ERK1/2-dependent manner,that IFN-y and Fas are involved in Porphyromonas gingivalis-induced apoptosis,and that IFN-γpromotes apoptosis at least partially through regulation of Fas expression.