Dental Follicle Stem Cells Ameliorate Lipopolysaccharide-Induced Inflammation by Secreting TGF-β3 and TSP-1 to Elicit Macrophage M2 Polarization
Dental Follicle Stem Cells Ameliorate Lipopolysaccharide-Induced Inflammation by Secreting TGF-β3 and TSP-1 to Elicit Macrophage M2 Polarization
复制标题
牙囊干细胞通过分泌 TGF-β 3 和 TSP-1 引发巨噬细胞 M2 极化来改善脂多糖诱导的炎症
DOI：
10.1159/000495873
复制
发表时间：
2018-01-01
影响因子：
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通讯作者：
Wei, Xi
中科院分区：
文献类型：
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作者：
Chen, Xiaochuan;Yang, Bo;Wei, Xi
背景/目的：越来越多的证据表明间充质干细胞（MSCs）在免疫治疗中的新作用。然而，获取这些细胞的困难和可能的伦理问题限制了它们的应用。最近，我们从牙囊中分离出了一个独特的MSC群体，具有有效的干细胞样特性。这项研究的重点是牙囊干细胞（DFSC）对巨噬细胞活化和极化的影响，以确定它们在免疫调节中的作用，并测试DFSC是否是基于MSC的免疫治疗的有前途的细胞来源。研究方法：采用脂多糖（LPS）诱导的大鼠急性肺损伤（ALI）模型，观察DFSC/DFSC-CM的体内免疫调节作用。通过左上肺叶的干/湿重量比确定肺渗透性。肺组织病理学损伤按0至4+量表分级。采用ELISA法检测支气管肺泡灌洗液（BALF）中炎性细胞因子的含量。建立了LPS诱导的体外巨噬细胞炎症模型。在DFSC-CM处理后，通过RT-qPCR、ELISA、蛋白质印迹和流式细胞术分析测量巨噬细胞中的炎性细胞因子产生和极化标志物表达。DFSC-CM中的旁分泌因子通过RayBiotech蛋白阵列显示。此后，进行中和研究以确认DFSC-CM中的潜在免疫调节剂。结果如下：DFSC/DFSC-CM不仅减轻了组织病理学损伤和肺通透性，而且下调了BALF中的促炎细胞因子MCP-1、IL-1、IL-6和TNF-α，并上调了抗炎细胞因子IL-10。免疫荧光染色显示巨噬细胞M2标志物Arg-1表达增加。进一步的体外研究显示，当与DFSC-CM共培养时，巨噬细胞转变为抗炎M2表型，其特征在于抑制促炎细胞因子MCP-1、IL-1、IL-6、TNF-α和M1极化标记物iNOS和CD 86的产生;以及增加抗炎细胞因子IL-10和M2极化标记物Arg-1和CD 163的表达。RayBiotech蛋白质阵列显示，与无血清Ham's F-12 K培养基相比，DFSC-CM中有42种差异表达（> 2倍）的旁分泌因子，其中TGF-β 3和血小板反应蛋白-1（TSP-1）分别上调18倍和105倍。中和研究证实了TGF-β 3和TSP-1在巨噬细胞活化和极化中的免疫调节作用。结论：这些结果表明，DFSCs可以将巨噬细胞重编程为抗炎的M2表型，旁分泌因子TGF-β 3和TSP-1可能是其机制之一。这项研究支持了DFSC有希望用于基于MSC的免疫治疗的假设。(C)2018作者（S）由S发布。Karger AG，巴塞尔
Background/Aims: Increasing evidence has demonstrated the novel roles of mesenchymal stem cells (MSCs) in immunotherapy. However, difficulty in acquiring these cells and possible ethical issues limited their application. Recently, we have isolated a unique MSC population from dental follicles with potent stem cell-like properties. This study focused on the effects of dental follicle stem cells (DFSCs) on macrophage activation and polarization to determine their role in immunomodulation and to test if DFSCs are a promising cell source for MSC-based immunotherapy. Methods: Rat acute lung injury (ALI) models induced by Lipopolysaccharide (LPS) were applied to test the immune-modulatory effects of DFSC/DFSC-CM in vivo. The pulmonary permeability was determined by the dry / wet weight ratios of the left upper lung lobe. The lung histopathological damage was graded on a 0 to 4+ scale. And the inflammatory cytokines in bronchoalveolar lavage fluid (BALF) were tested by ELISA. Then we established LPS-induced inflamed macrophage models in vitro. Inflammatory cytokine production and polarization marker expression were measured by RT-qPCR, ELISA, western blot and flow cytometric analysis in macrophages following DFSC-CM treatment. The paracrine factors in DFSC-CM were revealed by a RayBiotech Protein Array. Thereafter, neutralization studies were performed to confirm the potential immune regulators in DFSC-CM. Results: The DFSC/DFSC-CM not only attenuated histopathological damage and pulmonary permeability, but also downregulated pro-inflammatory cytokines MCP-1, IL-1, IL-6 and TNF-alpha, and upregulated anti-inflammatory cytokine IL-10 in BALF. Immunofluorescence staining revealed the increased expression of macrophage M2 marker Arg-1. Further in vitro study revealed that macrophages switched to an anti-inflammatory M2 phenotype when co-cultured with DFSC-CM, characterized by suppressed production of pro-inflammatory cytokines MCP-1, IL-1, IL-6, TNF-alpha and M1-polarizing markers iNOS and CD86; and increased expression of the anti-inflammatory cytokine IL-10 and the M2-polarizing markers Arg-1 and CD163. A RayBiotech Protein Array revealed 42 differentially expressed (> 2-fold) paracrine factors in DFSC-CM compared with the serum-free Ham's F-12K medium, among which TGF-beta 3 and Thrombospondin-1 (TSP-1) were upregulated by 18- and 105-fold, respectively. Neutralization studies confirmed the immunoregulatory roles of TGF-beta 3 and TSP-1 in macrophage activation and polarization. Conclusion: These results indicated that DFSCs can reprogram macrophages into the anti-inflammatory M2 phenotype, the paracrine factors TGF-beta 3 and TSP-1 may be one of the underlying mechanisms. This study supports the hypothesis that DFSCs are promising for MSC-based immunotherapy. (C) 2018 The Author(s) Published by S. Karger AG, Basel