间充质干细胞诱导 CD4CD25FOXP3 调节性 T 细胞与泛素化因子的调节和 TSDR 去甲基化有关。
Induction of CD4CD25FOXP3 regulatory T cells by mesenchymal stem cells is associated with modulation of ubiquitination factors and TSDR demethylation.
机构信息
Transplant Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
Department of Biology, Faculty of Science, Razi University, Kermanshah, Iran.
出版信息
Stem Cell Res Ther. 2018 Oct 25;9(1):273. doi: 10.1186/s13287-018-0991-1.
BACKGROUND
Mesenchymal stem cells (MSCs) are known for their ability to induce the conversion of conventional T cells (Tconvs) into induced regulatory T cells (iTregs) in specific inflammatory contexts. Stable Foxp3 expression plays a major role in the phenotypic and functional stability of iTregs. However, how MSCs induce stable Foxp3 expression remains unknown.
METHODS
We first investigated the role of cell-cell contact and cytokine secretion by bone marrow-derived MSCs (BM-MSCs) on the induction, stability, and suppressive functions of Tregs under various experimental conditions that lead to Foxp3 generation by flow cytometry and ELISA respectively. Second, we studied the effect of MSCs on TRAF6, GRAIL, USP7, STUB1, and UBC13 mRNA expression in CD4 T cells in correlation with the suppressive function of iTregs by real-time PCR; also, we investigated Foxp3 Treg-specific demethylated region (TSDR) methylation in correlation with Foxp3 stability by the high-resolution melting technique. Third, we studied the effect of ex-vivo-expanded BM-MSCs on the induction of transplant tolerance in a model of fully allogeneic skin transplantation. We further analyzed the cytokine secretion patterns in grafted mice as well as the mRNA expression of ubiquitination genes in CD4 T cells collected from the spleens of protected mice.
RESULTS
We found that in-vitro MSC-induced Tregs express high mRNA levels of ubiquitination genes such as TRAF6, GRAIL, and USP7 and low levels of STUB1. Moreover, they have enhanced TSDR demethylation. Infusion of MSCs in a murine model of allogeneic skin transplantation prolonged allograft survival. When CD4 T cells were harvested from the spleens of grafted mice, we observed that mRNA expression of the Foxp3 gene was elevated. Furthermore, Foxp3 mRNA expression was associated with increased TRAF6, GRAIL, UBC13, and USP7 and decreased STUB1 mRNA levels compared with the levels observed in vitro.
CONCLUSIONS
Our data suggest a possible ubiquitination mechanism by which MSCs convert Tconvs to suppressive and stable iTregs.
背景
间充质干细胞（MSCs）在特定炎症环境下能够将常规 T 细胞（Tconvs）诱导转化为诱导性调节性 T 细胞（iTregs），这一特性为人所熟知。Foxp3 的稳定表达在 iTregs 的表型和功能稳定性中起着重要作用。然而，MSCs 如何诱导 Foxp3 的稳定表达尚不清楚。
方法
我们首先通过流式细胞术和 ELISA 分别研究了骨髓来源的间充质干细胞（BM-MSCs）在各种导致 Foxp3 产生的实验条件下通过细胞间接触和细胞因子分泌对 Treg 的诱导、稳定性和抑制功能的作用。其次，我们通过实时 PCR 研究了 MSCs 对 CD4 T 细胞中 TRAF6、GRAIL、USP7、STUB1 和 UBC13 mRNA 表达的影响，以及与 iTreg 抑制功能的相关性；此外，我们通过高分辨率熔解技术研究了 Foxp3 Treg 特异性去甲基化区（TSDR）甲基化与 Foxp3 稳定性的相关性。第三，我们研究了体外扩增的 BM-MSCs 在完全同种异体皮肤移植模型中诱导移植耐受的作用。我们进一步分析了移植物中小鼠的细胞因子分泌模式以及来自受保护小鼠脾脏的 CD4 T 细胞中泛素化基因的 mRNA 表达。
结果
我们发现，体外 MSC 诱导的 Treg 表达高水平的泛素化基因，如 TRAF6、GRAIL 和 USP7，以及低水平的 STUB1。此外，它们具有增强的 TSDR 去甲基化。在同种异体皮肤移植的小鼠模型中输注 MSCs 可延长移植物存活时间。当从移植物小鼠脾脏中采集 CD4 T 细胞时，我们观察到 Foxp3 基因的 mRNA 表达升高。此外，与体外观察到的相比，Foxp3 mRNA 表达与 TRAF6、GRAIL、UBC13 和 USP7 的增加以及 STUB1 mRNA 水平的降低相关。
结论
我们的数据表明，MSCs 将 Tconv 转化为抑制性和稳定的 iTreg 可能存在一种泛素化机制。
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