Erratum: Roles of 1,25(OH)2D3 and Vitamin D Receptor in the Pathogenesis of Rheumatoid Arthritis and Systemic Lupus Erythematosus by Regulating the Activation of CD4+ T Cells and the PKCδ/ERK Signaling Pathway

Background/Aims: The study aims to elucidate the roles of 1,25(OH)2D3 and vitamin D receptor (VDR) in the pathogenesis of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) by regulating the activation of CD4+ T cells and the PKCδ/ERK signaling pathway. Methods: From January 2013 to December 2015, a total of 130 SLE patients, 137 RA patients and 130 healthy controls were selected in this study. Serum levels of 1,25(OH)2D3 and VDR mRNA expression were detected by ELISA and real-time fluorescence quantitative PCR (RT-qPCR). Density gradient centrifugation was performed to separate peripheral blood mononuclear cells (PBMCs). CD4+ T cells were separated using magnetic activated cell sorting (MACS). CD4+T cells in logarithmic growth phase were collected and assigned into 9 groups: the normal control group, the normal negative control (NC) group, the VDR siRNA group, the RA control group, the RA NC group, the VDR over-expressed RA group, the SLE control group, the SLE NC group, and the VDR over-expressed SLE group. The mRNA and protein expressions of VDR, PKCδ, ERK1/2, CD11a, CD70 and CD40L were detected by RT-qPCR and Western blotting. Bisulfite genomic sequencing was conducted to monitor the methylation status of CD11a, CD70 and CD40L. Results: Compared with healthy controls, serum 1,25(OH)2D3 level and VDR mRNA expression in peripheral blood were decreased in SLE patients and RA patients. With the increase of concentrations of 1,25(OH)2D3 treatment, the VDR mRNA expression and DNA methylation levels of CD11a, CD70 and CD40L were declined, while the expressions of PKCδ, ERK1/2, CD11a, CD70 and CD40L were elevated in SLE, RA and normal CD4+T cells. Compared with the SLE contro, RA control, SLE NC and RA NC groups, the expressions of PKCδ, ERK1/2, CD11a, CD70 and CD40L decreased but DNA methylation levels of CD11a, CD70 and CD40L increased in the VDR over-expressed SLE group and VDR over-expressed RA group. However, compared with the normal control and normal NC groups, the expressions of PKCδ, ERK1/2, CD11a, CD70 and CD40L increased, but DNA methylation levels of CD11a, CD70 and CD40L decreased in the VDR siRNA group. Compared with the normal control group, the expressions of PKCδ, ERK1/2, CD11a, CD70 and CD40L increased, but DNA methylation levels of CD11a, CD70 and CD40L decreased in the SLE control and RA control groups. Conclusion: Our study provide evidence that 1,25(OH)2D3 and VDR could inhibit the activation of CD4+ T cells and suppress the immune response of SLE and RA through inhibiting PKCδ/ERK pathway and promoting DNA methylation of CD11a, CD70 and CD40L.

背景与目的：本研究旨在阐明1,25(OH)2D3与维生素D受体（vitamin D receptor, VDR）在类风湿关节炎（rheumatoid arthritis, RA）及系统性红斑狼疮（systemic lupus erythematosus, SLE）发病机制中的作用，通过调控CD4+ T细胞活化与PKCδ/ERK信号通路实现。方法：本研究于2013年1月至2015年12月期间，共纳入130例SLE患者、137例RA患者及130名健康对照者。采用酶联免疫吸附试验（enzyme linked immunosorbent assay, ELISA）检测血清1,25(OH)2D3水平，通过实时荧光定量PCR（real-time fluorescence quantitative PCR, RT-qPCR）检测VDR mRNA的表达水平。采用密度梯度离心法分离外周血单个核细胞（peripheral blood mononuclear cells, PBMCs），并利用磁激活细胞分选（magnetic activated cell sorting, MACS）纯化CD4+ T细胞。收集处于对数生长期的CD4+ T细胞，分为9组：正常对照组、正常阴性对照（negative control, NC）组、VDR小干扰RNA（small interfering RNA, siRNA）组、RA对照组、RA NC组、VDR过表达RA组、SLE对照组、SLE NC组及VDR过表达SLE组。采用RT-qPCR与蛋白质印迹（Western blotting）检测VDR、PKCδ、ERK1/2、CD11a、CD70及CD40L的mRNA与蛋白表达水平。通过亚硫酸氢盐基因组测序（Bisulfite genomic sequencing）监测CD11a、CD70及CD40L的甲基化状态。结果：与健康对照者相比，SLE患者与RA患者的血清1,25(OH)2D3水平及外周血VDR mRNA表达均显著降低。随着1,25(OH)2D3处理浓度的升高，SLE、RA及正常CD4+ T细胞中的VDR mRNA表达水平与CD11a、CD70、CD40L的DNA甲基化水平均呈下降趋势，而PKCδ、ERK1/2、CD11a、CD70及CD40L的表达则显著升高。与SLE对照组、RA对照组、SLE NC组及RA NC组相比，VDR过表达RA组与VDR过表达SLE组中的PKCδ、ERK1/2、CD11a、CD70及CD40L的表达均下调，而CD11a、CD70、CD40L的DNA甲基化水平则上调。与之相反，与正常对照组及正常NC组相比，VDR siRNA组中的PKCδ、ERK1/2、CD11a、CD70及CD40L的表达均上调，而CD11a、CD70、CD40L的DNA甲基化水平则下调。与正常对照组相比，RA对照组与SLE对照组中的PKCδ、ERK1/2、CD11a、CD70及CD40L的表达均上调，而CD11a、CD70、CD40L的DNA甲基化水平则下调。结论：本研究证实，1,25(OH)2D3与VDR可通过抑制PKCδ/ERK信号通路、促进CD11a、CD70及CD40L的DNA甲基化，进而抑制CD4+ T细胞活化，缓解SLE与RA的免疫应答。