Department of Immunology, IIS-Fundación Jiménez Díaz, Madrid, Spain.
CIBER de Enfermedades Respiratorias (CIBERES), Madrid, Spain.
Clin Exp Allergy. 2018 Sep;48(9):1173-1185. doi: 10.1111/cea.13122. Epub 2018 Mar 8.
Eosinophils, a central factor in asthma pathogenesis, have the ability to secrete exosomes. However, the precise role played by exosomes in the biological processes leading up to asthma has not been fully defined.
We hypothesized that exosomes released by eosinophils contribute to asthma pathogenesis by activating structural lung cells.
Eosinophils from asthmatic patients and healthy volunteers were purified from peripheral blood, and exosomes were isolated from eosinophils of asthmatic and healthy individuals. All experiments were performed with eosinophil-derived exosomes from healthy and asthmatic subjects. Epithelial damage was evaluated using primary small airway epithelial cell lines through 2 types of apoptosis assays, that is, flow cytometry and TUNEL assay with confocal microscopy. Additionally, the epithelial repair was analysed by performing wound healing assays with epithelial cells. Functional studies such as proliferation and inhibition-proliferation assays were carried out in primary bronchial smooth muscle cell lines. Also, gene expression analysis of pro-inflammatory molecules was evaluated by real-time PCR on epithelial and muscle cells. Lastly, protein expression of epithelial and muscle cell signalling factors was estimated by Western blot.
Asthmatic eosinophil-derived exosomes induced an increase in epithelial cell apoptosis at 24 hour and 48 hour, impeding wound closure. In addition, muscle cell proliferation was increased at 72 hours after exosome addition and was linked with higher phosphorylation of ERK1/2. We also found higher expression of several genes when both cell types were cultured in the presence of exosomes from asthmatics: CCR3 and VEGFA in muscle cells, and CCL26, TNF and POSTN in epithelial cells. Healthy eosinophil-derived exosomes did not exert any effect over these cell types.
Eosinophil-derived exosomes from asthmatic patients participate actively in the development of the pathological features of asthma via structural lung cells.
嗜酸性粒细胞是哮喘发病机制中的一个核心因素，具有分泌外泌体的能力。然而，外泌体在导致哮喘的生物学过程中的确切作用尚未完全确定。
我们假设嗜酸性粒细胞释放的外泌体通过激活结构肺细胞来促进哮喘发病机制。
从哮喘患者和健康志愿者的外周血中纯化嗜酸性粒细胞，并从健康和哮喘个体的嗜酸性粒细胞中分离出外泌体。所有实验均使用来自健康和哮喘个体的嗜酸性粒细胞衍生的外泌体进行。通过两种凋亡测定（即流式细胞术和 TUNEL 检测与共聚焦显微镜），评估上皮细胞损伤。另外，通过进行上皮细胞的伤口愈合实验分析上皮修复。在原代支气管平滑肌细胞系中进行增殖和抑制增殖等功能研究。还通过上皮细胞和肌肉细胞的实时 PCR 评估促炎分子的基因表达分析。最后，通过 Western blot 估计上皮细胞和肌肉细胞信号因子的蛋白表达。
哮喘嗜酸性粒细胞衍生的外泌体在 24 小时和 48 小时诱导上皮细胞凋亡增加，阻碍伤口闭合。此外，在外泌体添加后 72 小时，肌肉细胞增殖增加，并且 ERK1/2 的磷酸化增加。当两种细胞类型在存在哮喘患者外泌体的情况下培养时，我们还发现了几个基因的更高表达：肌肉细胞中的 CCR3 和 VEGFA，以及上皮细胞中的 CCL26、TNF 和 POSTN。健康嗜酸性粒细胞衍生的外泌体对这些细胞类型没有任何影响。
哮喘患者的嗜酸性粒细胞衍生的外泌体通过结构肺细胞积极参与哮喘病理特征的发展。
