Jiangsu Provincial Key Laboratory of Critical Care Medicine, Department of Critical Care Medicine, School of Medicine, Zhongda Hospital, Southeast University, 87 Dingjiaqiao Road, Nanjing, 210009, People's Republic of China.
Stem Cell Res Ther. 2020 Jul 22;11(1):311. doi: 10.1186/s13287-020-01826-0.
T helper 17 cells (Th17)/regulatory T cells (Treg), as subtypes of CD4 T cells, play an important role in the inflammatory response of acute respiratory distress syndrome (ARDS). However, there is still a lack of effective methods to regulate the differentiation balance of Th17/Treg. It was proven that mesenchymal stem cells (MSCs) could regulate the differentiation of CD4 T cells, but the mechanism is still unclear. TGFβ1, a paracrine cytokine of MSCs, could also regulate the differentiation of Th17/Treg but is lowly expressed in MSCs. Therefore, mouse MSCs (mMSCs) overexpressing TGFβ1 were constructed by lentivirus transduction and intratracheally transplanted into LPS-induced ARDS mice in our study. The aim of this study was to evaluate the therapeutic effects of mMSCs overexpressing TGFβ1 on inflammation and immunoregulation by impacting the Th17/Treg balance in LPS-induced ARDS mice.
mMSCs overexpressing TGFβ1 were constructed using lentiviral vectors. Then, mouse bone-marrow-derived MSCs (mBM-MSC) and mBM-MSC-TGFβ1 (mBM-MSC overexpressing TGFβ1) were transplanted intratracheally into ARDS mice induced by lipopolysaccharide. At 3 and 7 days after transplantation, the mice were sacrificed, and the homing of the mMSCs was assayed by ex vivo optical imaging. The relative numbers of Th17 and Treg in the lungs and spleens of mice were detected by FCM. IL-17A and IL-10 levels in the lungs of mice were analysed by western blot. Permeability and inflammatory cytokines were evaluated by analysing the protein concentration of BALF using ELISA. Histopathology of the lungs was assessed by haematoxylin and eosin staining and lung injury scoring. Alveolar lung fibrosis was assessed by Masson's trichrome staining and Ashcroft scoring. The mortality of ARDS mice was followed until 7 days after transplantation.
The transduction efficiencies mediated by the lentiviral vectors ranged from 82.3 to 88.6%. Overexpressing TGFβ1 inhibited the proliferation of mMSCs during days 5-7 (p < 0.05) but had no effect on mMSC differentiation or migration (p > 0.05). Compared to that in the LPS + mBM-MSC-NC group mice, engraftment of mMSCs overexpressing TGFβ1 led to much more differentiation of T cells into Th17 or Treg (p < 0.05), improved permeability of injured lungs (p < 0.05) and ameliorative histopathology of lung tissue in ARDS mice (p < 0.05). Moreover, IL-17A content was also decreased while IL-10 content was increased in the LPS + mBM-MSC-TGFβ1 group compared with those in the LPS + mBM-MSC-NC group (p < 0.05). Finally, mMSCs overexpressing TGFβ1 did not aggravate lung fibrosis in ARDS mice (p > 0.05).
MSCs overexpressing TGFβ1 could regulate lung inflammation and attenuate lung injuries by modulating the imbalance of Th17/Treg in the lungs of ARDS mice.
辅助性 T 细胞 17 细胞（Th17）/调节性 T 细胞（Treg）作为 CD4 T 细胞的亚群，在急性呼吸窘迫综合征（ARDS）的炎症反应中发挥重要作用。然而，目前仍然缺乏有效调节 Th17/Treg 分化平衡的方法。有研究证明间充质干细胞（MSCs）可以调节 CD4 T 细胞的分化，但具体机制尚不清楚。MSCs 的旁分泌细胞因子 TGFβ1 也可以调节 Th17/Treg 的分化，但在 MSCs 中的表达水平较低。因此，本研究通过慢病毒转导构建了过表达 TGFβ1 的小鼠 MSCs（mMSCs），并将其气管内移植入脂多糖诱导的 ARDS 小鼠中。本研究旨在通过影响 LPS 诱导的 ARDS 小鼠中 Th17/Treg 平衡来评估过表达 TGFβ1 的 mMSCs 对炎症和免疫调节的治疗作用。
通过慢病毒载体构建过表达 TGFβ1 的 mMSCs。然后，将小鼠骨髓来源的 MSCs（mBM-MSCs）和 mBM-MSC-TGFβ1（过表达 TGFβ1 的 mBM-MSCs）通过气管内移植入脂多糖诱导的 ARDS 小鼠中。在移植后 3 和 7 天，处死小鼠，通过离体光学成像检测 mMSCs 的归巢情况。通过 FCM 检测小鼠肺和脾中 Th17 和 Treg 的相对数量。通过 Western blot 分析小鼠肺中 IL-17A 和 IL-10 水平。通过 ELISA 分析 BALF 中蛋白浓度评估通透性和炎症细胞因子。通过苏木精和伊红染色和肺损伤评分评估肺组织的病理变化。通过 Masson 三色染色和 Ashcroft 评分评估肺泡肺纤维化。观察 ARDS 小鼠的死亡率，直到移植后 7 天。
慢病毒载体介导的转导效率范围为 82.3%至 88.6%。过表达 TGFβ1 在第 5-7 天抑制 mMSCs 的增殖（p<0.05），但对 mMSC 分化或迁移没有影响（p>0.05）。与 LPS+mBM-MSC-NC 组小鼠相比，过表达 TGFβ1 的 mMSCs 移植后导致 T 细胞向 Th17 或 Treg 的分化更多（p<0.05），改善了受损肺的通透性（p<0.05），并改善了 ARDS 小鼠的肺组织病理变化（p<0.05）。此外，与 LPS+mBM-MSC-NC 组相比，LPS+mBM-MSC-TGFβ1 组的 IL-17A 含量降低，IL-10 含量增加（p<0.05）。最后，过表达 TGFβ1 的 mMSCs 并没有加重 ARDS 小鼠的肺纤维化（p>0.05）。
过表达 TGFβ1 的 MSCs 可以通过调节 ARDS 小鼠肺部 Th17/Treg 的失衡来调节肺部炎症和减轻肺损伤。
