Division of Rheumatology, Department of Medicine, Saban Research Institute, Children's Hospital Los Angeles, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America.
PLoS One. 2011;6(9):e24590. doi: 10.1371/journal.pone.0024590. Epub 2011 Sep 13.
It has been documented all-trans retinoic acid (atRA) promotes the development of TGF-β-induced CD4(+)Foxp3(+) regulatory T cells (iTreg) that play a vital role in the prevention of autoimmune responses, however, molecular mechanisms involved remain elusive. Our objective, therefore, was to determine how atRA promotes the differentiation of iTregs.
METHODOLOGY/PRINCIPAL FINDINGS: Addition of atRA to naïve CD4(+)CD25(-) cells stimulated with anti-CD3/CD28 antibodies in the presence of TGF-β not only increased Foxp3(+) iTreg differentiation, but maintained Foxp3 expression through apoptosis inhibition. atRA/TGF-β-treated CD4(+) cells developed complete anergy and displayed increased suppressive activity. Infusion of atRA/TGF-β-treated CD4(+) cells resulted in the greater effects on suppressing symptoms and protecting the survival of chronic GVHD mice with typical lupus-like syndromes than did CD4(+) cells treated with TGF-β alone. atRA did not significantly affect the phosphorylation levels of Smad2/3 and still promoted iTreg differentiation in CD4(+) cells isolated from Smad3 KO and Smad2 conditional KO mice. Conversely, atRA markedly increased ERK1/2 activation, and blockade of ERK1/2 signaling completely abolished the enhanced effects of atRA on Foxp3 expression. Moreover, atRA significantly increased histone methylation and acetylation within the promoter and conserved non-coding DNA sequence (CNS) elements at the Foxp3 gene locus and the recruitment of phosphor-RNA polymerase II, while DNA methylation in the CNS3 was not significantly altered.
CONCLUSIONS/SIGNIFICANCE: We have identified the cellular and molecular mechanism(s) by which atRA promotes the development and maintenance of iTregs. These results will help to enhance the quantity and quality of development of iTregs and may provide novel insights into clinical cell therapy for patients with autoimmune diseases and those needing organ transplantation.
已有文献证明全反式维甲酸（atRA）可促进 TGF-β诱导的 CD4+Foxp3+调节性 T 细胞（iTreg）的发育，而 iTreg 在预防自身免疫反应中起着至关重要的作用，但涉及的分子机制仍不清楚。因此，我们的目标是确定 atRA 如何促进 iTreg 的分化。
方法/主要发现：在 TGF-β存在的情况下，将 atRA 添加到抗 CD3/CD28 抗体刺激的幼稚 CD4+CD25-细胞中，不仅增加了 Foxp3+iTreg 的分化，而且通过抑制细胞凋亡来维持 Foxp3 的表达。atRA/TGF-β 处理的 CD4+细胞表现出完全无能，并显示出增强的抑制活性。与 TGF-β 单独处理的 CD4+细胞相比，输注 atRA/TGF-β 处理的 CD4+细胞可对抑制慢性 GVHD 小鼠的症状和保护其生存产生更大的效果，且这些小鼠表现出典型的狼疮样综合征。atRA 对 Smad3 KO 和 Smad2 条件性 KO 小鼠分离的 CD4+细胞中 Smad2/3 的磷酸化水平没有显著影响，但仍可促进 iTreg 的分化。相反，atRA 显著增加 ERK1/2 的激活，而 ERK1/2 信号通路的阻断完全消除了 atRA 对 Foxp3 表达的增强作用。此外，atRA 显著增加了 Foxp3 基因座启动子和保守非编码 DNA 序列（CNS）元件内的组蛋白甲基化和乙酰化，以及磷酸化 RNA 聚合酶 II 的募集，而 CNS3 中的 DNA 甲基化没有明显改变。
结论/意义：我们已经确定了 atRA 促进 iTreg 发育和维持的细胞和分子机制。这些结果将有助于提高 iTreg 的数量和质量，并为自身免疫性疾病患者和需要器官移植的患者的临床细胞治疗提供新的见解。
