人气道上皮细胞外囊泡miRNA特征随哮喘发展改变
2020/02/11
摘要
背景:miRNA是哮喘关键信号通路的主调节因子，并在细胞间在细胞外囊泡(EV)内传递。我们的目的是研究哮喘发生时，原发正常人支气管上皮细胞(NHBE)分泌的EV的miRNA含量是否发生改变。
方法:NHBE细胞在气-液界面培养，用白细胞介素(IL)-13诱导哮喘样表型。通过从基础培养基或根尖表面洗涤的沉淀分离EV，通过纳米颗粒跟踪分析、透射电镜和Western Blot对其进行了表征，并通过基于RT-qPCR的分析鉴定了EV相关的miRNA。通过大小排除层析法从轻度至中度哮喘(n=8)或严重哮喘(n=9)儿童和健康对照组(n=9)的鼻腔灌洗液中分离得到的EVs中确认了重要的候选对象。
结果:NHBE细胞向顶面和基底面分泌细胞外囊泡。细胞外囊泡中有47个miRNA表达，其中16个在IL-13处理后基础的EV中显著改变。mirna的表达可以在人鼻腔灌洗的细胞外囊泡中得到证实。值得注意的是，miR-92b、miR-210和miR-34a水平与儿童肺功能参数显著相关(FEV1 FVC%pred和FEF25-75%pred)，因此，鼻灌洗中低水平的EV-miRNA与气道阻塞相关。随后的独创性途径分析预测这些miRNA可以调节Th2极化和树突状细胞成熟。
结论:我们的数据表明，来自气道上皮细胞在细胞外囊泡内miRNA的分泌，尤其是miR-34a、miR-92b和miR-210，可能参与气道和哮喘中Th2反应的早期发展。
Human airway epithelial extracellular vesicle miRNA signature is altered upon asthma development.
Bartel S, et al. Allergy. 2019 Aug 6.
Abstract
BACKGROUND:miRNAs are master regulators of signalling pathways critically involved in asthma and are transferred between cells in extracellular vesicles (EV). We aimed to investigate if the miRNA content of EV secreted by primary normal human bronchial epithelial cells (NHBE) is altered upon asthma development.
METHODS:NHBE cells were cultured at air-liquid interface and treated with Interleukin (IL)-13 to induce an asthma-like phenotype. EV isolations by precipitation from basal culture medium or apical surface wash were characterized by Nanoparticle Tracking Analysis, Transmission Electron Microscopy and Western Blot, and EV-associated miRNAs were identified by a RT-qPCR -based profiling. Significant candidates were confirmed in EVs isolated by size exclusion chromatography from nasal lavages of children with mild-to-moderate (n=8) or severe asthma (n=9), and healthy controls (n=9).
RESULTS:NHBE cells secrete EVs to the apical and basal side. 47 miRNAs were expressed in EVs and 16 thereof were significantly altered in basal EV upon IL-13 treatment. Expression of miRNAs could be confirmed in EVs from human nasal lavages. Of note, levels of miR-92b, miR-210 and miR-34a significantly correlated with lung function parameters in children (FEV1 FVC%pred and FEF25-75%pred ), thus lower EV-miRNA levels in nasal lavages associated with airway obstruction. Subsequent Ingenuity Pathway Analysis predicted the miRNAs to regulate Th2 polarization and dendritic cell maturation.
CONCLUSION:Our data indicate that secretion of miRNAs in EVs from the airway epithelium, in particular miR-34a, miR-92b and miR-210, might be involved in the early development of a Th2 response in the airways and asthma.
背景:miRNA是哮喘关键信号通路的主调节因子，并在细胞间在细胞外囊泡(EV)内传递。我们的目的是研究哮喘发生时，原发正常人支气管上皮细胞(NHBE)分泌的EV的miRNA含量是否发生改变。
方法:NHBE细胞在气-液界面培养，用白细胞介素(IL)-13诱导哮喘样表型。通过从基础培养基或根尖表面洗涤的沉淀分离EV，通过纳米颗粒跟踪分析、透射电镜和Western Blot对其进行了表征，并通过基于RT-qPCR的分析鉴定了EV相关的miRNA。通过大小排除层析法从轻度至中度哮喘(n=8)或严重哮喘(n=9)儿童和健康对照组(n=9)的鼻腔灌洗液中分离得到的EVs中确认了重要的候选对象。
结果:NHBE细胞向顶面和基底面分泌细胞外囊泡。细胞外囊泡中有47个miRNA表达，其中16个在IL-13处理后基础的EV中显著改变。mirna的表达可以在人鼻腔灌洗的细胞外囊泡中得到证实。值得注意的是，miR-92b、miR-210和miR-34a水平与儿童肺功能参数显著相关(FEV1 FVC%pred和FEF25-75%pred)，因此，鼻灌洗中低水平的EV-miRNA与气道阻塞相关。随后的独创性途径分析预测这些miRNA可以调节Th2极化和树突状细胞成熟。
结论:我们的数据表明，来自气道上皮细胞在细胞外囊泡内miRNA的分泌，尤其是miR-34a、miR-92b和miR-210，可能参与气道和哮喘中Th2反应的早期发展。
（中国医科大学附属第一医院 李文扬 摘译 杨冬 审校）
（Bartel S, et al. Allergy. 2019 Aug 6.）
（Bartel S, et al. Allergy. 2019 Aug 6.）
Human airway epithelial extracellular vesicle miRNA signature is altered upon asthma development.
Bartel S, et al. Allergy. 2019 Aug 6.
Abstract
BACKGROUND:miRNAs are master regulators of signalling pathways critically involved in asthma and are transferred between cells in extracellular vesicles (EV). We aimed to investigate if the miRNA content of EV secreted by primary normal human bronchial epithelial cells (NHBE) is altered upon asthma development.
METHODS:NHBE cells were cultured at air-liquid interface and treated with Interleukin (IL)-13 to induce an asthma-like phenotype. EV isolations by precipitation from basal culture medium or apical surface wash were characterized by Nanoparticle Tracking Analysis, Transmission Electron Microscopy and Western Blot, and EV-associated miRNAs were identified by a RT-qPCR -based profiling. Significant candidates were confirmed in EVs isolated by size exclusion chromatography from nasal lavages of children with mild-to-moderate (n=8) or severe asthma (n=9), and healthy controls (n=9).
RESULTS:NHBE cells secrete EVs to the apical and basal side. 47 miRNAs were expressed in EVs and 16 thereof were significantly altered in basal EV upon IL-13 treatment. Expression of miRNAs could be confirmed in EVs from human nasal lavages. Of note, levels of miR-92b, miR-210 and miR-34a significantly correlated with lung function parameters in children (FEV1 FVC%pred and FEF25-75%pred ), thus lower EV-miRNA levels in nasal lavages associated with airway obstruction. Subsequent Ingenuity Pathway Analysis predicted the miRNAs to regulate Th2 polarization and dendritic cell maturation.
CONCLUSION:Our data indicate that secretion of miRNAs in EVs from the airway epithelium, in particular miR-34a, miR-92b and miR-210, might be involved in the early development of a Th2 response in the airways and asthma.
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氯暴露通过固有淋巴细胞和CD11c中间型巨噬细胞加重哮喘炎症
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