人脐带间充质干细胞的外泌体通过重塑巨噬细胞极化来减轻重度激素抵抗性哮喘的炎症
2021/04/23
摘要
背景；严重的激素抵抗性哮喘（SSRA）是哮喘管理中的严重临床问题。受影响的患者具有严重的临床症状，生活质量恶化，并且对激素治疗（哮喘的主要治疗手段）无反应。因此，迫切需要有效的疗法。来源于间充质干细胞（MSC-Exo）的外泌体通过其免疫调节作用已成为肺部疾病治疗的有潜力的替代物。在这项研究中，我们探讨了MSC-Exo在SSRA中的治疗作用，并确定了MSC-Exo的治疗机制。
方法：分离人脐带间充质干细胞（hUCMSC）的外泌体，并通过透射电子显微镜，纳米粒子跟踪分析和流式细胞仪分析对其进行表征。评估了MSC-Exo对SSRA小鼠气道高反应性（AHR），炎症，组织病理学和巨噬细胞极化的影响。巨噬细胞的系统耗竭决定了巨噬细胞在小鼠SSRA的治疗作用中的作用。构建LPS刺激的RAW 264.7细胞模型，以确定MSC-Exo对巨噬细胞极化的潜在机制。进行了qRT-PCR，Western印迹，免疫荧光和流式细胞术以评估M1或M2标记的表达。应用串联质量标签（TMT）标记的定量蛋白质组学来研究MSC-Exo对巨噬细胞极化的调节作用过程中中心蛋白的作用。敲低和过度表达TRAF1被用来进一步阐明中央蛋白在巨噬细胞极化中的作用。
结果；我们成功地从hUCMSCs中分离并鉴定了外泌体。我们证实，气管内给予MSC-Exo可逆转SSRA小鼠的气道高反应性，组织病理学变化和炎症。系统性消耗巨噬细胞削弱了MSC-Exo的治疗效果。我们发现，MSC-Exo处理在LPS刺激的RAW 264.7细胞中抑制了M1极化并促进了M2极化。随后，肿瘤坏死因子受体相关因子1（TRAF1）被确定为中心蛋白，可能与TMT标记的定量蛋白质组学分析与巨噬细胞极化的调节密切相关。敲低和过度表达TRAF1表明，MSC-Exo处理对巨噬细胞极化，NF-κB和PI3K/AKT信号传导的影响取决于TRAF1。
结论：MSC-Exo可通过减轻炎症来改善SSRA，这是通过抑制TRAF1的表达重塑巨噬细胞极化来实现的。
Exosomes from human umbilical cord mesenchymal stem cells attenuate the inflammation of severe steroid-resistant asthma by reshaping macrophage polarization
Dong B, Wang C, Zhang J, et al.
Abstract
Background:Severe, steroid-resistant asthma (SSRA) is a serious clinical problem in asthma management. Affected patients have severe clinical symptoms, worsened quality of life, and do not respond to steroid, a mainstay steroid treatment of asthma. Thus, effective therapies are urgently needed. Exosomes derived from mesenchymal stem cell (MSC-Exo) has become attractive candidates for the lung inflammatory diseases through its immunomodulatory effects. In this study, we explored the therapeutic effects of MSC-Exo in SSRA and identified the therapeutic mechanism of MSC-Exo.
Methods:Exosomes from human umbilical cord mesenchymal stem cell (hUCMSC) were isolated and characterized by transmission electron microscopy, nanoparticle tracking analysis and flow cytometry analysis. Effects of MSC-Exo on airway hyper responsiveness (AHR), inflammation, histopathology, and macrophage polarization in SSRA in mice were evaluated. Systematic depletion of macrophages determined the role of macrophages in the therapeutic effect of SSRA in mice. LPS-stimulated RAW 264.7 cell model was constructed to determine the underlying mechanism of MSC-Exo on macrophage polarization. qRT-PCR, Western blotting, immunofluorescence, and flow cytometry were performed to evaluate the expression of M1 or M2 markers. Tandem mass tags (TMT)-labeled quantitative proteomics were applied to explore the central protein during the regulation effect of MSC-Exo on macrophage polarization. Knockdown and overexpression of TRAF1 were used to further clarify the role of the central protein on macrophage polarization.
Results:We successfully isolated and characterized exosomes from hUCMSCs. We verified that the intratracheal administration of MSC-Exo reversed AHR, histopathology changes, and inflammation in SSRA mice. Systematic depletion of macrophages weakened the therapeutic effect of MSC-Exo. We found that MSC-Exo treatment inhibited M1 polarization and promoted M2 polarization in LPS-stimulated RAW 264.7 cells. Subsequently, tumor necrosis factor receptor-associated factor 1 (TRAF1) was determined as the central protein which may be closely related to the regulation of macrophage polarization from TMT-labeled quantitative proteomics analysis. Knockdown and overexpression of TRAF1 demonstrated that the effect of MSC-Exo treatment on macrophage polarization, NF-κB and PI3K/AKT signaling was dependent on TRAF1.
Conclusion:MSC-Exo can ameliorate SSRA by moderating inflammation, which is achieved by reshaping macrophage polarization via inhibition of TRAF1.
背景；严重的激素抵抗性哮喘（SSRA）是哮喘管理中的严重临床问题。受影响的患者具有严重的临床症状，生活质量恶化，并且对激素治疗（哮喘的主要治疗手段）无反应。因此，迫切需要有效的疗法。来源于间充质干细胞（MSC-Exo）的外泌体通过其免疫调节作用已成为肺部疾病治疗的有潜力的替代物。在这项研究中，我们探讨了MSC-Exo在SSRA中的治疗作用，并确定了MSC-Exo的治疗机制。
方法：分离人脐带间充质干细胞（hUCMSC）的外泌体，并通过透射电子显微镜，纳米粒子跟踪分析和流式细胞仪分析对其进行表征。评估了MSC-Exo对SSRA小鼠气道高反应性（AHR），炎症，组织病理学和巨噬细胞极化的影响。巨噬细胞的系统耗竭决定了巨噬细胞在小鼠SSRA的治疗作用中的作用。构建LPS刺激的RAW 264.7细胞模型，以确定MSC-Exo对巨噬细胞极化的潜在机制。进行了qRT-PCR，Western印迹，免疫荧光和流式细胞术以评估M1或M2标记的表达。应用串联质量标签（TMT）标记的定量蛋白质组学来研究MSC-Exo对巨噬细胞极化的调节作用过程中中心蛋白的作用。敲低和过度表达TRAF1被用来进一步阐明中央蛋白在巨噬细胞极化中的作用。
结果；我们成功地从hUCMSCs中分离并鉴定了外泌体。我们证实，气管内给予MSC-Exo可逆转SSRA小鼠的气道高反应性，组织病理学变化和炎症。系统性消耗巨噬细胞削弱了MSC-Exo的治疗效果。我们发现，MSC-Exo处理在LPS刺激的RAW 264.7细胞中抑制了M1极化并促进了M2极化。随后，肿瘤坏死因子受体相关因子1（TRAF1）被确定为中心蛋白，可能与TMT标记的定量蛋白质组学分析与巨噬细胞极化的调节密切相关。敲低和过度表达TRAF1表明，MSC-Exo处理对巨噬细胞极化，NF-κB和PI3K/AKT信号传导的影响取决于TRAF1。
结论：MSC-Exo可通过减轻炎症来改善SSRA，这是通过抑制TRAF1的表达重塑巨噬细胞极化来实现的。
（中日友好医院呼吸与危重症医学科 李春晓 摘译 林江涛 审校）
（Stem Cell Res Ther, 2021, 12(1): 204）
（Stem Cell Res Ther, 2021, 12(1): 204）
Exosomes from human umbilical cord mesenchymal stem cells attenuate the inflammation of severe steroid-resistant asthma by reshaping macrophage polarization
Dong B, Wang C, Zhang J, et al.
Abstract
Background:Severe, steroid-resistant asthma (SSRA) is a serious clinical problem in asthma management. Affected patients have severe clinical symptoms, worsened quality of life, and do not respond to steroid, a mainstay steroid treatment of asthma. Thus, effective therapies are urgently needed. Exosomes derived from mesenchymal stem cell (MSC-Exo) has become attractive candidates for the lung inflammatory diseases through its immunomodulatory effects. In this study, we explored the therapeutic effects of MSC-Exo in SSRA and identified the therapeutic mechanism of MSC-Exo.
Methods:Exosomes from human umbilical cord mesenchymal stem cell (hUCMSC) were isolated and characterized by transmission electron microscopy, nanoparticle tracking analysis and flow cytometry analysis. Effects of MSC-Exo on airway hyper responsiveness (AHR), inflammation, histopathology, and macrophage polarization in SSRA in mice were evaluated. Systematic depletion of macrophages determined the role of macrophages in the therapeutic effect of SSRA in mice. LPS-stimulated RAW 264.7 cell model was constructed to determine the underlying mechanism of MSC-Exo on macrophage polarization. qRT-PCR, Western blotting, immunofluorescence, and flow cytometry were performed to evaluate the expression of M1 or M2 markers. Tandem mass tags (TMT)-labeled quantitative proteomics were applied to explore the central protein during the regulation effect of MSC-Exo on macrophage polarization. Knockdown and overexpression of TRAF1 were used to further clarify the role of the central protein on macrophage polarization.
Results:We successfully isolated and characterized exosomes from hUCMSCs. We verified that the intratracheal administration of MSC-Exo reversed AHR, histopathology changes, and inflammation in SSRA mice. Systematic depletion of macrophages weakened the therapeutic effect of MSC-Exo. We found that MSC-Exo treatment inhibited M1 polarization and promoted M2 polarization in LPS-stimulated RAW 264.7 cells. Subsequently, tumor necrosis factor receptor-associated factor 1 (TRAF1) was determined as the central protein which may be closely related to the regulation of macrophage polarization from TMT-labeled quantitative proteomics analysis. Knockdown and overexpression of TRAF1 demonstrated that the effect of MSC-Exo treatment on macrophage polarization, NF-κB and PI3K/AKT signaling was dependent on TRAF1.
Conclusion:MSC-Exo can ameliorate SSRA by moderating inflammation, which is achieved by reshaping macrophage polarization via inhibition of TRAF1.
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严重中性粒细胞性哮喘患者痰中ACE2，TMPRSS2和FURIN基因表达
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IL-33/IL-1RL1通路在哮喘中的关键作用：从发病机制到治疗干预
