Division of Pulmonary and Critical Care Medicine, Department of Medicine, School of Medicine, Boston University, Boston, Massachusetts 02118, USA.
Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, New Jersey 08854, USA.
Toxicol Sci. 2022 Apr 26;187(1):162-174. doi: 10.1093/toxsci/kfac025.
Ozone is a ubiquitous air pollutant that causes lung damage and altered functioning. Evidence suggests that proinflammatory macrophages contribute to ozone toxicity. Herein, we analyzed the role of extracellular vesicles (EVs) and microRNA (miRNA) cargo in ozone-induced macrophage activation. Exposure of mice to ozone (0.8 ppm, 3 h) resulted in increases in bronchoalveolar lavage fluid EVs, which were comprised predominantly of microvesicles (MVs). NanoFACS analysis revealed that MVs generated following both air and ozone exposure was largely from CD45+ myeloid cells; these MVs were readily taken up by macrophages. Functionally, MVs from ozone, but not air treated mice, upregulated mRNA expression of inflammatory proteins in macrophages including inducible nitric oxide synthase (iNOS), CXCL-1, CXCL-2, and interleukin (IL)-1β. The miRNA profile of MVs in bronchoalveolar lavage fluid (BALF) was altered after ozone exposure; thus, increases in miR-21, miR-145, miR320a, miR-155, let-7b, miR744, miR181, miR-17, miR-92a, and miR-199a-3p were observed, whereas miR-24-3p and miR-20 were reduced. Ingenuity pathway analysis revealed that these miRNAs regulate pathways that promote inflammatory macrophage activation, and predicted that let-7a-5p/let-7b, miR-24-3p, miR-21-5p, miR-17, and miR-181a-5p are key upstream regulators of inflammatory proteins. After ozone exposure, miR-199a-3p, but not precursor miR-199a-3p, was increased in lung macrophages, indicating that it is derived from MV-mediated delivery. Furthermore, lung macrophage mRNA expression of IL-1β was upregulated after administration of MVs containing miR-199a-3p mimic but downregulated by miR-199a-3p inhibitor. Collectively, these data suggest that MVs generated following ozone exposure contribute to proinflammatory macrophage activation via MV-derived miRNAs including miR-199a-3p. These findings identify a novel pathway regulating macrophage inflammatory responses to inhaled ozone.
臭氧是一种普遍存在的空气污染物，会导致肺部损伤和功能改变。有证据表明，促炎巨噬细胞有助于臭氧毒性。在此，我们分析了细胞外囊泡 (EVs) 和 microRNA (miRNA) 货物在臭氧诱导的巨噬细胞激活中的作用。将小鼠暴露于臭氧 (0.8 ppm，3 h) 导致支气管肺泡灌洗液 EVs 增加，其主要由微泡 (MVs) 组成。NanoFACS 分析显示，空气和臭氧暴露后生成的 MVs 主要来自 CD45+髓样细胞；这些 MVs 很容易被巨噬细胞摄取。功能上，来自臭氧而非空气处理的小鼠的 MVs 上调了巨噬细胞中炎症蛋白的 mRNA 表达，包括诱导型一氧化氮合酶 (iNOS)、CXCL-1、CXCL-2 和白细胞介素 (IL)-1β。支气管肺泡灌洗液 (BALF) 中 MVs 的 miRNA 谱在臭氧暴露后发生改变；因此，观察到 miR-21、miR-145、miR320a、miR-155、let-7b、miR744、miR181、miR-17、miR-92a 和 miR-199a-3p 增加，而 miR-24-3p 和 miR-20 减少。通路分析表明，这些 miRNA 调节促进炎症性巨噬细胞激活的途径，并预测 let-7a-5p/let-7b、miR-24-3p、miR-21-5p、miR-17 和 miR-181a-5p 是炎症蛋白的关键上游调节剂。臭氧暴露后，肺巨噬细胞中 miR-199a-3p 而非前体 miR-199a-3p 增加，表明其来自 MV 介导的递送。此外，MV 中含有 miR-199a-3p 模拟物的肺巨噬细胞 mRNA 表达上调，而 miR-199a-3p 抑制剂下调 IL-1β。总的来说，这些数据表明，臭氧暴露后产生的 MVs 通过包括 miR-199a-3p 在内的 MV 衍生 miRNA 促进促炎巨噬细胞激活。这些发现确定了一种调节巨噬细胞对吸入臭氧炎症反应的新途径。
