The Effect Of Mycophenolic Acid On Expression Of Autoimmune Related Genes Through Epigenetic Modifications In CD4~+T Cells From Patients With Systemic Lupus Erythematosus

Objective:Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease characterized by uncontrolled lymphocyte autoreactivity and overproduction of autoantibodies. Mycophenolate mofetil(MMF) is a new type of immunosuppresive agent, which is transformed into its active metabolite form mycophenolic acid (MPA) in vivo. It is widely used in the treatment of patients with SLE, particularly those with nephritis. In this study, we investigated whether MPA could change the aberrant epigenetic modifications in SLE CD4+T cells and whether this regulation contributes to therapeutic activity of MPA.Methods:T cells were isolated by positive selection using CD4beads. Lupus CD4+T cells were exposed to MPA at concentration of luM or methanol for48hours. The global DNA methylation and global histone modification status were performed following the protocol of Epigentek Assay Kit in MPA and methanol groups. Then related epigenetic modification enzymes, such as histone acetyltransferases(HATs) and histone deacetylases(HDACs), were measured by reverse transcription-quantitative PCR(RT-qPCR). We also examined the expression levels of autoimmune related genes, such as CD40L, CD11a and CD70, by RT-qPCR and flow cytometric analysis(FCM). Pyrosequencing was used to test the DNA methylation status and the chromatin immunoprecipitation(ChIP) assay was used to investigate the histone modification status in CD40L promoter region. Production of IgG was quantified by ELISA.Results:No significant change in global DNA methylation status was found between MPA treated CD4+T cells and methanol treated CD4+T cells(P=0.2701). Compared with methanol treated CD4+T cells, the global histone H3/H4acetylation was up-regulated(P=0.0335,0.0241), while global histone H3K4/H3K9methylation had no significant changes(P=0.5407,0.3868) in MPA treated CD4+T cells. The mRNA expression levels of histone deacetylases(HDAC2, HDAC7and SIRT1)were decreased(P=0.0199,0.0425,0.0225), while the mRNA expression levels of histone acetyltransferases (CREBBP and PCAF) were increased in the MPA treated CD4+T cells(P=0.0153,0.0201). Compared with the methanol group, CD40L expression was inhibited in MPA group(P=0.0032). However, the expression of CD11a and CD70 showed no significant changes after the MPA treatment(P>0.05). Compared with methanol treated CD4+T cells, the production of IgG was decreased in MPA-treated lupus CD4+T cells when cocultured with autologus CD19+B cells(P=0.0013). Furthermore, the DNA methylation status at CD40L promoter region showed no significant changes after the MPA treatment(P>0.05). The ChIP results showed that histone H4acetylation level and histone H3K4tri-methylation levels of CD40L promoter region were decreased(P<0.05) while there was no significant change in histone H4acetylation and H3K9methylation level of CD40L promoter region(P>0.05) in MPA-treated CD4+T cells comparing with control group.Conclusions:MPA could reverse the abnormal status of global histone hypoacetylation by regulating histone acetylation related enzymes in SLE CD4+T cells. MPA could reduce CD40L expression and inhibit the autoreactivity of CD4+T cells by down-regulating histone H4acetylation and histone H3K4tri-methylation in CD40L promoter region.