Effect Of Treg Cells On Immune Response Mediated By M2 Macrophages In Mice With Periodontitis

ObjectiveIn this study,the animal model of periodontitis was established by infecting mouse oral cavity with high bacterial concentration of gingival porphyrin single cell(Porphvromonasgingivalis.P.gingivalis)W83.The function of Treg cells was blocked by intraperitoneal injection of GITR antibody,and the effect of Treg cells on M2 macrophage-mediated immune response in periodontitiswas observed.MethodsTwenty 7-week-old female SPF C57bl/6 mice were selected and randomly divided into 4 groups,5 in each group.Divided into normal control group,periodontitis group,Ig G group,and anti-GITR m Ab group.P.gingivalis W83(1x10~9/d) was applied intraorally for 7 consecutive days to establish periodontitis animal model,anti-GITR m Ab group.The mice were injected intraperitoneally with anti-GITR m Ab(100 mg/mouse) every 3 days on the first day of P.gingivalis W83 application.The mice in the Ig G group were intraperitoneally injected with the Ig G isotype control antibody(100 mg/mouse).Dynamically monitor the weight change of the 4 groups of mice,and take samples on the45th day.Observe the gingival index and tooth looseness of the 4 groups of mice under a dissecting microscope;measure from the cemento-enamel junction(CEJ)to the alveolar crest.(Alveolar bone crest,ABC)distance to evaluate alveolar bone resorption;Homatxylin and eosin(HE)staining to observe periodontal tissue infiltration of inflammatory cells,loss of connective tissue adhesion and alveolar bone resorption;Real-time quantitative RT-PCR was used to detect the expression of IL-10,TGF-β1,Foxp3 mRNA and M2 macrophage-related factor Arg-1 mRNA in Treg cells.Results1.The distance from(CEJ)to alveolar crest(ABC)in enamel bone boundary was observed under anatomical microscope.Compared with normal control group,12 sites on buccal and palatal side were measured.The results showed that the height of alveolar crest in simple periodontitis group,Ig G group and anti-GITR m Ab group decreased,while the distance from alveolar crest to enamel bone boundary in anti-GITR m Ab group was larger than that in simple periodontitis group and Ig G group.The degree of bone resorption increased significantly.2.The results of hematoxylin-eosin staining showed that the gingival epithelium of the normal control group was intact,there was a small amount of inflammatory cell infiltration in the subepithelial connective tissue,the attachment of the binding epithelium was located at the enamel-cementum boundary,and the periodontal ligament fibers were arranged neatly.the morphology and structure of alveolar bone was intact,and no alveolar bone resorption was found.In simple periodontitis group and Ig G group,the upper part of the epithelium was separated from the tooth surface,and the epithelium attached to the root of the enamel bone boundary.The collagen fibers of the periodontal ligament were disordered,dissolved,denatured,infiltrated by a large number of inflammatory cells in the lamina propria,and absorbed at the crest of the alveolar bone,and osteoclasts and bone resorption lacunae could be seen.In anti-GITR m Ab group,the conjugated epithelium migrated to the root,the periodontal pocket deepened,the collagen fibers beneath the conjugated epithelium degenerated,dissolved and lost,osteoclasts were active,inflammatory cells infiltrated,and the alveolar crest parietal bone resorption was obvious.Active osteoclastic resorption lacunae appeared in the alveolar bone.3.The results of real-time quantitative RT-PCR showed that compared with the normal control group,the expression of IL-10,TGF-β1 and Foxp3 mRNA of Treg cells in periodontitis group and Ig G group increased,while the expression of Arg-1 mRNA in M2 macrophages decreased,but there was no statistical significance.Compared with simple periodontitis group and Ig G group,the expression of IL-10 and TGF-β1 Foxp3 mRNA of Treg cells and the expression of M2 macrophage related factor Arg-1 mRNA in gingival tissue of anti-GITR m Ab group were significantly lower than those of simple periodontitis group and anti-GITR m Ab group(P<0 05).Conclusions1.The immune response mediated by M2 macrophages in periodontitis was weakened:compared with the normal control group,the expression of M2 macrophage-related cytokines Arg-1 mRNA in the gingival tissue of mice with simple periodontitis decreased.2.Treg cells can up-regulate the immune response mediated by M2 macrophages in periodontitis:after blocking the function of Treg cells with anti-GITR m Ab,compared with the simple periodontitis group,the destruction of periodontal tissue and bone resorption were aggravated in anti-GITR m Ab group,and the expression level of M2 macrophage-related factor Arg-1 mRNA was decreased.