MSC调控ARDS炎症反应的新机制——旁分泌TGF-β诱导Treg/Th17极化平衡

Acute respiratory distress syndrome (ARDS) is characterized by uncontrolled inflammation caused by the imbalance of anti-inflammation and pro-inflammation. Bone marrow derived Mesenchymal stem cells (MSCs) have the capacity to prevent this uncontrolled inflammation. However, the mechanism for it has not yet been clarified. We and other researchers found that Regulatory T cells (Treg) /T Helper type 17 (Th17) polarization plays an important role in the uncontrolled inflammation of ARDS. As we all know, both Treg and Th17 polarization need Transforming Growth Factor-β (TGF-β). MSC paracrines TGF-β and can regulate the polarization of Treg and Th17. What’s more, according to our preliminary trials, MSC can increase TGF-β level and Treg/Th17 ratio in the lung of ARDS mice. Therefore, we hypothesize that MSC inhibits inflammation of ARDS by inducing Treg/Th17 polarization balance mediated by paracrine TGF-β. In this study, we investigate the role of Treg/Th17 polarization induced by MSC in ARDS and identify its mechinism whether it is associated with the paracrine soluble factors TGF-β in vitro study. What’s more, in vivo mouse model, we inject MSCs through tail vein after LPS-induced lung injury, then we quantify both Treg and Th17 and estimate their function in regulating immune responses, assess the inflammation in lung tissue and also evaluate the injuried lung restoration to demostrate the protective effect of MSC paracrine TGF-β for ARDS.

抑炎和促炎反应失衡所致的炎症反应失控是ARDS发生发展的关键。MSC能有效控制ARDS失控的炎症反应，然而其具体作用环节和机制尚不清楚。Treg/Th17极化失衡在ARDS炎症反应失控中扮演着重要角色。MSC能调节Treg和Th17极化且能分泌二者极化的主要调节因子TGF-β。结合我们预实验发现MSC能上调ARDS小鼠肺组织中TGF-β水平和Treg/Th17计数比，推测旁分泌TGF-β诱导Treg/Th17极化平衡可能是MSC纠正ARDS炎症反应失衡的机制。本研究拟在MSC与Naïve CD4+T间接共培养和LPS诱导ARDS小鼠模型之上，利用基因工程上调或抑制MSC对TGF-β的分泌，体外以及在体实验证实ARDS时MSC通过旁分泌TGF-β能促进Naïve CD4+T向Treg极化而抑制其向Th17极化，诱导Treg/Th17极化平衡，从而纠正失控的炎症反应、减轻肺损伤并促进肺修复。

调节性T细胞（Treg）/辅助型T细胞（Th）17失衡是介导急性呼吸窘迫征（ARDS）免疫损伤的重要环节，是治疗ARDS的关键，间充质干细胞（MSC）可诱导CD4T细胞向Treg分化，结合我们前期发现MSC对ARDS早期炎症反应具有重要的调控作用，因而假设恢复Treg/Th17分化平衡可能是介导MSC调控ARDS失控的免疫炎症反应的重要路径。为证实该假设并进一步探讨ARDS时MSC调控Treg/Th17失衡的机制，（1）利用外周血样本，通过临床回顾性研究从转录水平证实ARDS患者存在Treg/Th17失衡；（2）通过LPS与低氧刺激，建立体外ARDS微环境模型，证实LPS或/与低氧刺激可引起Treg分化减少，Th17分化，即Treg/Th17分化失衡，同时细胞存活减少；（3）通过体外MSC与CD4+T细胞直接/间接共培养，发现MSC均可恢复ARDS微环境下Treg/Th17失衡，同时分泌抑炎型分泌因子增加，证实MSCs治疗作用不依赖细胞直接接触，给予TGF-β1中和阻断后，MSC治疗作用消失，证实旁分泌TGF-β1是MSC改善ARDS微环境下Treg/Th17失衡的重要环节；（4）建立慢病毒介导的TGF-β1高表达MSC，通过在体实验评价该细胞对LPS诱导的ARDS小鼠肺部Treg/Th17分化、炎症反应及病理损伤的影响，发现高表达TGF-β1MSC可进一步改善Treg/Th17分化平衡，抑制IL-17A表达，促进IL-10表达，改善肺毛细血管通透性，减轻病理损伤，同时不增加肺纤维化的发生。（5）体外MSC与CD4T细胞共培养基础上，通过生物信息学分析及miR-155表达检测，发现MSC可抑制LPS-低氧诱导下miR-155表达升高，TGF-β1中和后其作用消失，用时利用miR-155mimic转染上调细胞内miR-155水平后，促Treg型转录因子PTPN2、Foxp3基因表达降低，Treg分化减少，而Th17分化增加。综上可以证明：（1）MSC通过旁分泌TGF-β1 改善ARDS微环境下Treg/Th17失衡；（2）高表达TGF-β1MSC通过改善Treg/Th17失衡减轻ARDS小鼠肺部炎症反应及病理损伤；（3）抑制miR-155表达，释放对Treg型转录因子靶向结合是MSC旁分泌TGF-β1调控ARDS-Treg/Th17分化平衡的重要机制（之一）。