The Effects Of Stem Cells From Apical Papilla On DNA Methylation Of Foxp3 Locus In CD4~+T Cells:An In Vitro Study

Objective:Stem cells from apical papilla(SCAP),which are derived from developing apical papilla tissue,have superior potential in terms of self-renewal,multilineage differentiation and immunoregulation.Our previous study found that SCAP could up-regulate the expression level of forkhead box protein-3(Foxp3)in CD4~+T cells and promote their conversion to Tregs in an in vitro co-culture system using transwells.Regulatory T cells(Tregs)are a subset of CD4~+CD25~+Foxp3~+T cells that can improve the local immune microenvironment of the host by suppressing excessive immune responses,thus promote the repair and regeneration of damaged tissues.Foxp3is the key factor in the development and maturation of Tregs.Recently,many studies have found that the hypomethylation status of the Foxp3 locus is required to maintain the stable expression of Foxp3 and obtain the complete immunosuppressive phenotype of Tregs.Accordingly,the purpose of this study was to explore the effects of SCAP on the methylation level of the Foxp3 locus,the expression level of DNA methyltransferase 1(DNMT1)and ten-eleven translocation(TET)family in CD4~+T cells.This study aims to clarify the role of SCAP in regulating the conversion of Tregs,thus providing the foundation for further studying of related mechanisms.Methods:(1)Primary extraction of SCAP from the apical papilla tissue was performed through enzymatic digestion followed by cell culture.Stem cell surface markers and the osteogenic/adipogenic differentiation potential of the SCAP were determined.(2)Mouse spleen T lymphocytes were isolated,and then CD4~+T cells were purified by magnetic activated cell sorting and assessed for purity using flow cytometry.(3)SCAP and CD4~+T cells were co-cultured in vitro at a ratio of 1:5 for 72 h using transwells(co-culture group),while CD4~+T cells cultured alone served as the control group.Flow cytometry was used to detect the proliferation level of CD4~+T cells and the ratio of Tregs.(4)MethylTarget?technology was used to measure the methylation level of the Foxp3 locus in CD4~+T cells.The level of 5-hydroxymethylcytosine(5hmC)of the Foxp3 locus in CD4~+T cells was evaluated using hydroxymethylated DNA immunoprecipitation-polymerase chain reaction(hMeDIP-PCR).(5)The mRNA expression level of DNMT1 and Tet1,Tet2 and Tet3 was detected by real time-PCR.Western blot was used to detect the protein expression levels of DNMT1,Tet1 and Tet2.The experimental results were statistically analysed using SPSS 20.0 software and P values less than 0.05 were considered statistically significant.Results:(1)Primary cultured SCAP were adherent cells with spindle shapes,which expressed the mesenchymal stem cell surface markers CD105,CD90,CD44 and CD29,and failed to express the hematopoietic stem cell markers CD45 and CD34.Following osteogenic induction,alizarin red S staining showed that the SCAP formed mineralized nodules.Lipid droplets were observed using oil red O staining after adipogenic induction.As a result,the SCAP conformed to the standard for stem cell identification proposed by the International Society for Stem Cell Research.(2)The purity of the CD4~+T cells was 97.27±2.30(%)after magnetic activated cell sorting.(3)No significant difference in the proliferation rate of CD4~+T cells between the co-culture group(SCAP+CD4~+T cells)and the control group(CD4~+T cells)was identified(P>0.05).Compared with the control group,the expression level of Foxp3was up-regulated,thus the ratio of Tregs was increased(P<0.05).(4)The methylation level of Foxp3 locus was decreased and the 5hmC level of Foxp3 locus was elevated in CD4~+T cells in the co-culture group compared with the control group(P<0.05).(5)The expression level of DNMT1 was down-regulated and Tet2 was up-regulated in the co-culture group compared to the control group(P<0.05),but the expression levels of Tet1 and Tet3 underwent no obvious change(P>0.05).Conclusions:It was found that SCAP down-regulated the methylation level of the Foxp3 locus in CD4~+T cells to maintain stable Foxp3 expression by paracrine factors.Furthermore,the mechanisms through which SCAP induced demethylation of the Foxp3 locus may be associated with the suppression of DNMT1 expression and an increase in Tet2expression.