The Effect Of CD4+ T Cell Plorizarion On The Expression Of RANKL During Development, Progress And Turnover Of Periodontitis

BackgroundAlveolar bone loss is a hallmark of periodontitis progression causing tooth mobility and loss,and its prevention is a key clinical challenge in periodontal disease treatment.The immune response mediated by CD4+ T helper(Th)cells plays an especially crucial role in bone damage during this process.In some inflammatory environments,these cytokines from CD4+ T cells play important roles in damage,repair and remodeling in bone by mediating the RANKL-OPG system.In the context of periodontitis,the relationship and interaction between CD4+ T cell polarization and RANKL-OPG system remains to be further explored.In the context of periodontal inflammation,the effect of RANKL-OPG system mediated by CD4+ T cells polarization on bone resorption is still not clear,and whether the inhibitory effect on bone resorption and inflammatory response could be explored by regulating the polarization reaction of CD4+ T cell remains to be further studied.Calcitriol plays a crucial role in the metabolism of bone tissues due to its immunoregulatory activities and the therapeutic role in the developmental process of periodontal disease remains unexplored.This study attempted to offer a new insight for inflammation control by exploring the effect of calcitriol on bone loss and CD4+ T cell polarizationn in vitro and in vivo,on the basis of defining the correlationship between the CD4 + T cell polarization and RANKL-OPG system.Aim1.To clarify the correlation between polarization of CD4+ T cells reflected in gingiva tissues and GCF and RANKL/OPG ratio in periodontitis by exploring the relationship between the polarization of CD4+ T cells and RANKL/OPG ratio in gingival tissue from patients with periodontitis and the effect of periodontal nonsurgical treatment on the relationship between the RANKL/OPG ratio and CD4+ T cells polarization of in GCF.2.To investigate the effect of CD4+ T cell polarization on RANKL expression and osteoclast activity in inflammatory environment by analyzing the effect of inflammatory stimulation on polarization and the expression level of RANKL in CD4+ T cells from mice spleen.3.To explore the effect of calcitriol on the CD4+ T cells polarization in the inflammatory environment and further to assess the effect of CD4+ T cells on the osteoclast activity after calcitriol intervention in vitro by discussing effect of calcitriol intervention on polarization and the expression of RANKL in CD4+ T cells from mice spleen in inflammatory stimulation.4.To verify the inhibitory effect of calcitriol on bone resorption and immune response in LPS-induced alveolar bone damage in rats.Method1.Gingival crevicular fluid(GCF)and gingival tissues were obtained from periodontally healthy individuals(PH group)and patients with advanced chronic periodontitis(CP group)in this study.GCF levels of IFN-?,IL-4,IL-17 and IL-10 closely linked to T-helper cell polarization toward the Th1,Th2,Th17 and Treg subsets,respectively,were determined by enzyme-linked immunosorbent assay(ELISA).The expression levels of these cytokines and the polarized T-helper cells in gingival tissues were assessed based on immunohistochemical and immunofluorescence assays.Finally,RANKL and OPG expression levels in gingival tissues were detected by immunohistochemical assay,and linear regression analysis was used to identify the potential relationship between T-helper cell polarization and the RANKL/OPG ratio.2.Demographic information,periodontal clinical parameters(baseline)and GCF(baseline)were collected respectively.Then the patients in the CP group received periodontal treatment,and periodontal clinical parameters and GCF were collected at 2nd,4th and 6th month after treatment.The expression levels of RANKL,OPG and CD4+ T cells cytokines in GCF were further analyzed by ELISA.Finally,the relationship between the CD4+ T cell cytokines and the RANKL/OPG ratio was analyzed using a linear relationship in different groups respectively.3.After separation of CD4+ T cells and DCs(dendritic cells)from mice spleen,CD4+ T cells received inflammatory co-stimulus using LPS and DCs.Then,the CD4+ T cells polarization was analyzed by flow cytometry in the co-cultured system after inflammatory stimulation and expression levels of transcription factors and cytokines related to polarization were evaluated by PCR and Western blot assay.Further,RANKL expression level was detected by flow cytometry,PCR and Western blot assay.Finally,in the co-culture assay of CD4+ T cells and RAW264.7 cells,the effect of CD4+ T cells on osteoclast activity of RAW264.7 cells was explored by TRAP(tartrate-resistant acid phosphatase)staining and expression levels of NFATC1 and MMP-9 were analyzed by PCR and Western blot assay.4.Calcitriol intervention was performed when CD4+ T cells were co-stimulated by LPS and DCs.The effect of calcitriol on the polarization of CD4+ T cells in inflammatory environment was analyzed(flow cytometry,PCR and Western blot)and the effect of CD4+ T cells with calcitriol intervention on osteoclast activity of RAW264.7 was further evaluated.5.The influence of calcitriol intervention on the development of LPS-induced periodontitis in rats was determined in terms of bone loss(micro-CT analysis),local inflammatory infiltration levels and the number of osteoclasts(hematoxylin and eosin staining)and the level of osteoclastogenesis TRAP staining.Furthermore,immunohistochemistry was used to assess the expression levels of RANKL and OPG as well as the cytokine levels of IFN-?,IL-4,IL-17 and IL-10,throughout the LPS-injected region.Finally,the polarization potential of Th cells in peripheral blood was analyzed using flow cytometry.Result1.In total,22 individuals(PH group)and 35 patients(CP group)were enrolled in the present study.In both GCF and gingival tissues,increased IL-17 level and inferior IL-4 and IL-10 levels were detected in the CP group.When polarized T-helper cells were identified in gingival tissues,more Th1 and Th17 cells were found in the CP group,while more Th2 and Treg cells were found in the PH group.Although there was no significant difference in OPG expression between the two groups,the RANKL/OPG ratio in the CP group was higher than that in the PH group(p < 0.05).Linear regression analysis showed that the presence of more Th1 and Th17 cells correlated with a higher RANKL/OPG ratio(r = 0.71 and r = 0.75,respectively),while the presence of more Th2 cells correlated with a lower RANKL/OPG ratio(r =-0.48).2.In total,22 healthy volunteers and 21 patients with periodontitis were enrolled in the present study.Clinical examination showed that periodental non-surgical treatment improved clinical parameters of patients;The results of ELISA in GCF showed that RANKL/OPG ratio was decreased by periodontal treatment(p < 0.01).At the same time,the expression levels of IFN-? and IL-17 were decreased(p < 0.01),while the expression levels of IL-4 and IL-10 were increased(p < 0.01).However,linear regression analysis showed that there was still a linear relationship between RANKL/OPG ratio and IL-4 and IL-17 after periodontal non-surgical treatment(p < 0.05).3.CD4+ T cells and DCs could be separated from mice spleen using immunomagnetic bead sorting kit.In vitro inflammatory stimulation model(CD4+ T cells co-cultured with DC cells and inflammatory factor LPS),the downregulated Th2 polarization(p < 0.01),upregulated Th17 polarization(p < 0.01)and increased Th2/Th17 ratio were detected.Meanwhile,the Th2 response transcription factors GATA3,STAT5 and cytokine IL-4were decreased(p < 0.01),while the Th2 response transcription factors ROR?T,STAT3 and cytokine IL-17 were increased(p < 0.01).In addition,RANKL of CD4+ T cells inflammatory stimulation was highly expressed,and the osteoclast activity of RAW264.7 cells was significantly promoted.4.When calcitriol was added to intervene inflammatory stimulation model of CD4+ T cells in vitro(together with the inflammatory factor LPS),upregulated Th2 polarization(p < 0.01),downregulated Th17 polarization(p < 0.01)and upregulated the Th2/Th17 ratio were observed(p < 0.01).Meanwhile,the transcription factors and cytokine related to Th2 polarization were up-regulated(p < 0.01)and the transcription factors and cytokine related to Th17 polarization were downregulated(p < 0.01).In addition,calcitriol intervention significantly reduced the expression level of RANKL in CD4+ T cells in the co-culture system(p < 0.01),and inhibited the osteoclast activity of RAW264.7 cells.5.Calcitriol intervention decreased alveolar bone loss in response to LPS injection and inflammatory cell infiltration.Analysis of the number of osteoclasts and the expression of RANKL and OPG showed that bone resorption activity was largely suppressed in response to calcitriol administration,along with decreased IL-17 levels but increased IL-4 and IL-10 levels in periodontal tissues(the LPS-injected region).Similarly,the percentages of Th2 and Treg cells in peripheral blood increased but the percentages of Th1 and Th17 cells decreased in rats receiving calcitriol.ConclusionThe polarization of CD4+ T cells plays a crucial role in the development and progression of periodontitis.That RANKL-OPG system regulated by polarized CD4+ T cells and their secreted cytokines is largely involved in bone resorption in periodontitis.The intervention of CD4+ T cell polarization-regulated drugs(such as calcitriol)is expected to be a new method for periodontal immunotherapy,which is worthy of further study.