Department of Rheumatism and Immunology, Peking University Shenzhen Hospital, Shenzhen, Guangdong, China.
Shenzhen Key Laboratory of Immunity and Inflammatory Diseases, Shenzhen, Guangdong, China.
Front Immunol. 2023 Feb 9;14:1104881. doi: 10.3389/fimmu.2023.1104881. eCollection 2023.
Smad7 is protective in a mouse model of rheumatoid arthritis. Here we investigated whether Smad7-expressing CD4 T cells and the methylation of gene in CD4 T cells contribute to the disease activity of RA in patients.
Peripheral CD4 T cells were collected from 35 healthy controls and 57 RA patients. Smad7 expression by CD4 T cells were determined and correlated with the clinical parameters of RA including RA score and serum levels of IL-6, CRP, ESR, DAS28-CRP, DAS28-ESR, Swollen joints and Tender joints. Bisulfite sequencing (BSP-seq) was used to determine the DNA methylation in Smad7 promoter (-1000 to +2000) region in CD4 T cells. In addition, a DNA methylation inhibitor, 5-Azacytidine (5-AzaC), was added to CD4 T cells to examine the possible role of Smad7 methylation in CD4 T cell differentiation and functional activity.
Compared to the heath controls, Smad7 expression was significantly decreased in CD4 T cells from RA patients and inversely correlated with the RA activity score and serum levels of IL-6 and CRP. Importantly, loss of Smad7 in CD4 T cell was associated with the alteration of Th17/Treg balance by increasing Th17 over the Treg population. BSP-seq detected that DNA hypermethylation occurred in the Smad7 promoter region of CD4 T cells obtained from RA patients. Mechanistically, we found that the DNA hypermethylation in the Smad7 promoter of CD4 T cells was associated with decreased Smad7 expression in RA patients. This was associated with overreactive DNA methyltransferase (DMNT1) and downregulation of the methyl-CpG binding domain proteins (MBD4). Inhibition of DNA methylation by treating CD4 T cells from RA patients with 5-AzaC significantly increased Smad7 mRNA expression along with the increased MBD4 but reduced DNMT1 expression, which was associated with the rebalance in the Th17/Treg response.
DNA hypermethylation at the Smad7 promoter regions may cause a loss of Smad7 in CD4 T cells of RA patients, which may contribute to the RA activity by disrupting the Th17/Treg balance.
Smad7 在类风湿关节炎的小鼠模型中具有保护作用。在这里，我们研究了 Smad7 表达的 CD4 T 细胞和 CD4 T 细胞中 基因的甲基化是否有助于患者 RA 的疾病活动。
从 35 名健康对照者和 57 名 RA 患者中收集外周血 CD4 T 细胞。测定 CD4 T 细胞中 Smad7 的表达，并将其与 RA 的临床参数（包括 RA 评分和血清中 IL-6、CRP、ESR、DAS28-CRP、DAS28-ESR、肿胀关节和压痛关节）相关联。使用亚硫酸氢盐测序（BSP-seq）来确定 CD4 T 细胞中 Smad7 启动子（-1000 至+2000）区域的 DNA 甲基化。此外，向 CD4 T 细胞中添加 DNA 甲基化抑制剂 5-氮杂胞苷（5-AzaC），以检查 CD4 T 细胞分化和功能活性中 Smad7 甲基化的可能作用。
与健康对照组相比，RA 患者 CD4 T 细胞中的 Smad7 表达显著降低，且与 RA 活性评分以及血清中 IL-6 和 CRP 水平呈负相关。重要的是，Smad7 在 CD4 T 细胞中的缺失与 Th17/Treg 平衡的改变有关，通过增加 Th17 细胞超过 Treg 细胞群。BSP-seq 检测到来自 RA 患者的 CD4 T 细胞中 Smad7 启动子区域发生了 DNA 高甲基化。从机制上讲，我们发现 CD4 T 细胞中 Smad7 启动子的 DNA 高甲基化与 RA 患者 Smad7 表达降低有关。这与过度活跃的 DNA 甲基转移酶（DNMT1）和甲基化胞嘧啶结合域蛋白（MBD4）的下调有关。用 5-AzaC 处理 RA 患者的 CD4 T 细胞抑制 DNA 甲基化，可显著增加 Smad7 mRNA 表达，同时增加 MBD4 表达，减少 DNMT1 表达，从而与 Th17/Treg 反应的再平衡有关。
Smad7 启动子区域的 DNA 高甲基化可能导致 RA 患者 CD4 T 细胞中 Smad7 的缺失，通过破坏 Th17/Treg 平衡可能导致 RA 活性增加。
