The Effect Of Porphyromonas Gingivalis Infection On Th17/Treg Balance In The Progress Of Atherosclerosis

Porphyromonas gingivalis(P.gingivalis)is the main pathogen of chronic periodontitis.Increasing evidences have shown that there is a close relationship between P.gingivalis and atherosclerosis(AS).The immune balance between T helper 17(Thl7)cell and regulatory t cell(Treg)is critical in the autoimmune response of AS.However,it is still unclearly about the relation between PRgingivalis infection and Th17/Treg balance in the progress of AS.In this study,we will analyze the effect of P.gingivalis infection on Thl7/Treg balance to discuss the potential role of immune reaction in periodontitis-associated AS.Part I The characteristic of Th17/Treg balance inPorphyromonas gingivalis infected atherosclerotic patientsObjective To analyze the immune balance between Th17 and Treg of P.gingivalis infected AS patients and the association between P.gingivalis infection and Thl7/Treg balance.Methods 42 P.gingivalis infected coronary heart disease patients(Pg-AS),32 P.gingivalis infected patients(Pg)and 30 health controls(HC)were enrolled and divided into three group after clinical examination and experimental testing.Clinical risk factors and periodontal parameters were recorded carefully.The percentages of CD4+CD25+FOXP3+Treg and CD4+IL17+Th17 in peripheral blood were analyzed by flow cytometry(FCM).mRNA expression of Treg related factors FOXP3,TGF? and IL-10,and Th17 related factors RORrt and IL-17 was detected by qPCR assay.Furthermore,the concentrations of plasma IL-10,TGF? and IL18 were measured by ELISA.Results No significant difference was found in clinical risk factors among these groups.Periodontal parameters in P.gingivalis infected patients were higher than HC.Pg-AS patients had higher value of periodontal parameters(including plaque index,gingiva index and teeth losing)and elevated P.gingivalis IgG antibody than P.gingivalis-infected periodontitis patients and HCs.The percentages of CD4+CD25+FOXP3+Treg in Pg-AS group were decreased and the percentages of CD4+IL17+Th17 were increased when comparing with Pg and HC groups.No17 significant difference was found between Pg group and HC group.The concentration of plasma IL-10 and TGF? in Pg-AS patients is lower than that in Pg group and HC group.An increased level of plasma IL-17 was shown in Pg-AS group compared with Pg group and HC group.A positive correlation was shown between TGF?concentration,IL-10 concentration and the frequency of CD4+CD25+FOXP3+Treg.A negative relation was detected between IL-17 concentration and the percentage of CD4+IL17+Th17.Conclusion Thl7/Treg imbalance in P.gingivalis infected atherosclerotic patient is characterized with up-regulated Th17 and down-regulated Treg.Th17/Treg imbalance in atherosclerotic patients is associated with P.gingivalis infection.Part II Experimental study on the effect of P.gingivalis infection on Th17/Treg balance in ApoE-/-miceObjective To analyze the influence of P.gingivalis orally infection on Th17/Treg balance in the progress of AS in animal model.Methods ApoE-/-mice and wide-type(C57BL/6)mice were received a high-fat diet.Mice were inoculated with 10?8 CFU P.gingivalis orally.Mice in another two groups receive PBS as vehicle five times per week for nine weeks.Mice were anaesthetized five days after final inoculation.Alveolar bone loss of mandible was analyzed using microCT.AS lesion area was assessed in aortic root by HE staining and oil-red O staining.The stability of plaque was identified by Masson staining,CD68 and a-SMA immunohistochemical staining.Aorta was isolated and the expression of Th17-and Treg-related factors,as well as stability associated factors such as MCP-1,RNATES,COX-2,P-selectin,E-selectin and iNOS were determined by qPCR.Finally,we isolated splenocytes.The frequencies of Th17 and Treg were examined by FCM.The mRNA expression of Th17-and Treg-related factors was determined by qPCR.Results P.gingival is infection induced same alveolar bone resorption in both wide-type mice and ApoE-/-mice.P.gingivalis promoted AS lesion formation and increased the expression of macrophage and decreased the exression of aSMA.Collagen content showed no significant changes.However,P.gingivalis did not induce AS formation in wide-type mice.The expression of Th17 related cytokines were increased and Treg related cytokines were decreased in plaque.Furthermore,the mRNA expression of MCP-1,RNATES,COX-2,P-selectin,E-selectin and iNO is increased in P.gingivalis infected ApoE-/-mice.Thl7 and Treg in spleen showed the same tendency with that in arota.The frequencies of Thl7 were upregualted and the expression of Th17 related factors RORrt and IL-17 was increased.The frequencies of Treg were downregualted and the expression of Treg related factors FOXP3,TGF?and IL-10 was decreased.Conclusion P.gingivalis infection may promote AS formation and decreased AS stability through inducing Th17/Treg imbalance.Part III The effect of Porphyromonas gingivalis on T cell differentiationObjective To discuss whether P.gingivalis regulate T cell differentiation by alter the cytokine in micro-environment.Methods Splenic CD4+ T cells were isolated from ApoE-/-mouse with microbeads.CD4+T and CD4-(as APC)(1:3)were cocultured.Pingivalis was added to CD4+T cells and coculture cells for 48h.The frequencies of Treg and Th17 were determined by FCM.The expression of related transcriptional factors was analyzed by qPCR.The concentration of IL-6 in the supernatant of coculture cells was detected.Furthermore.FoxP3-expressing cells and IL 17-expressing cells were analyzed while IL-6 was neutralized with anti-IL-6 antibody in co-culture system to determine whether IL-6 was critical in P.gingivalis regulating T cells differentiation.Spleen cells were isolated from P.gingivalis infected ApoE-/-mice and co-cultured with aCD3 and aCD28 to determined IL-6.Bone marrow cells were isolated from 6-week-old ApoE-/-mice.DCs were induced with IL-4 and GM-CSF,and defined with anti-CD11c at 7d.DCs were infected with P.gingivalis at different MOI(0,1:10 and 1:100)for 12h.The release of IL-6 is determined by ELISA.The activation of p38 and Erk were analyzed by FCM.Results No significant difference was found in frequencies of Th17 cells and Tregs in CD4+T cells infected with P.Rgingivalis.The percentage of Th17 and RORrt expression was increased,and the percentage of Treg and FOXP3 expression was decreased in P.gingivalis-infected coculture cells.The concentration of IL-6 in the supernatant ofco-culture cells is increased.Importantly,blocking IL-6 increased FOXP3-expressing cells and decreased IL-17-expressing cells even in co-culture cells with P.gingivalis infection.Futhermore,the release of IL-6 in P.gingivalis infected ApoE-/-mice is increased compared with ApoE-/-mice.Bone marrow cells were stimulated with IL-4 and GM-CSF for 9 days.The purity of CD11c+ DC cells is about 85%.P.gingivalis infection promoted the release of IL-6 at a MOI dependent way.The expression of p-Erk and p-p38 were elevated.Conclusion P.gingivalis may influence Th17 and Tregs differentiation by altering the expression of IL-6 in cytokine environment,which may lead to Thl7/Treg imbalancein AS.