Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, 1909 Skip Bertman Drive, Baton Rouge, LA, 70803, USA.
Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, 70803, USA.
Sci Rep. 2021 Dec 1;11(1):23203. doi: 10.1038/s41598-021-02256-5.
Lung epithelial lining fluid (ELF) harbors a variety of proteins that influence homeostatic and stress responses in the airspaces. Exosomes, nano-sized extracellular vesicles, contain many proteins that vary in abundance and composition based on the prevailing conditions. Ozone causes inflammatory responses in the airspaces of experimental animals and humans. However, the exosomal protein signatures contained within the ELF from ozone-exposed lung airspaces remain poorly characterized. To explore this, we hypothesized that ozone triggers the release of exosome-bound inflammatory proteins from various cells that reflect mucoobstructive lung disease. Accordingly, we repetitively exposed adult male and female C57BL/6 mice to HEPA-filtered air (air) or 0.8 ppm ozone (4 h per day) for 14 days (five consecutive days of exposure, 2 days of rest, five consecutive days of exposure, 2 days of rest, four consecutive days of exposure). Exosome-bound proteomic signatures, as well as the levels of soluble inflammatory mediators in the bronchoalveolar lavage fluid (BALF), were determined 12-16 h after the last exposure. Principal component analyses of the exosome-bound proteome revealed a clear distinction between air-exposed and ozone-exposed mice, as well as between ozone-exposed males and ozone-exposed females. In addition to 575 proteins that were enriched in both sexes upon ozone exposure, 243 and 326 proteins were enriched uniquely in ozone-exposed males and females, respectively. Ingenuity pathway analyses on enriched proteins between ozone- and air-exposed mice revealed enrichment of pro-inflammatory pathways. More specifically, macrophage activation-related proteins were enriched in exosomes from ozone-exposed mice. Cytokine analyses on the BALF revealed elevated levels of G-CSF, KC, IP-10, IL-6, and IL-5 in ozone-exposed mice. Finally, the histopathological assessment revealed significantly enhanced intracellular localization of mucoinflammatory proteins including MUC5B and FIZZ1 in ozone-exposed mice in a cell-specific manner indicating the cellular sources of the proteins that are ferried in the exosomes upon ozone-induced lung injury. Collectively, this study identified exosomal, secretory, and cell-specific proteins and biological pathways following repetitive exposure of mice to ozone.
肺上皮衬液 (ELF) 中含有多种影响气道稳态和应激反应的蛋白质。外泌体是纳米大小的细胞外囊泡，其中包含的蛋白质根据流行情况在丰度和组成上存在差异。臭氧会引起实验动物和人类气道的炎症反应。然而，臭氧暴露的气道中 ELF 中的外泌体蛋白特征仍未得到很好的描述。为了探究这一点，我们假设臭氧会触发各种细胞释放外泌体结合的炎症蛋白，这些蛋白反映了黏液阻塞性肺病。因此，我们反复将成年雄性和雌性 C57BL/6 小鼠暴露于高效空气过滤器过滤的空气（空气）或 0.8ppm 臭氧（每天 4 小时）中 14 天（连续 5 天暴露，休息 2 天，连续 5 天暴露，休息 2 天，连续 4 天暴露）。在最后一次暴露后 12-16 小时，测定外泌体结合的蛋白质组学特征以及支气管肺泡灌洗液（BALF）中可溶性炎症介质的水平。外泌体结合蛋白质组的主成分分析显示，空气暴露组和臭氧暴露组以及臭氧暴露的雄性和雌性之间存在明显差异。除了在臭氧暴露时在两性中均富集的 575 种蛋白质外，分别在臭氧暴露的雄性和雌性中还富集了 243 种和 326 种蛋白质。臭氧暴露和空气暴露小鼠之间富集蛋白的 Ingenuity 途径分析显示促炎途径的富集。更具体地说，在臭氧暴露的小鼠的外泌体中富集了巨噬细胞激活相关蛋白。BALF 的细胞因子分析显示，臭氧暴露的小鼠中 G-CSF、KC、IP-10、IL-6 和 IL-5 的水平升高。最后，组织病理学评估显示，臭氧诱导的肺损伤时，MUC5B 和 FIZZ1 等黏蛋白炎症蛋白在臭氧暴露的小鼠中细胞内定位显著增强，表明这些蛋白的细胞来源是臭氧诱导肺损伤时携带在囊泡中的。总的来说，这项研究确定了小鼠反复暴露于臭氧后，外泌体、分泌和细胞特异性蛋白质和生物学途径。
