Abstract：
BACKGROUND: The interplay between airway epithelium and macrophages plays a pivotal role in Chronic Obstructive Pulmonary Disease (COPD) pathogenesis. Exosomes, which transport miRNA cargo, have emerged as novel mediators of intercellular communication. MicroRNA-125a-5p (miR-125a-5p) has been implicated in macrophage polarization.This study aims to investigate the role of exosomal miR-125a-5p in the dysfunctional epithelium-macrophage cross-talk in cigarette smoke (CS)-induced COPD.
METHODS: In cell models, THP-1 monocytic cells were differentiated into macrophages (M0). Human bronchial epithelial cells treated with CS extract (CSE) were co-cultured with M0. Exosomes were isolated from culture media using commercial kits and characterized using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Exosomes labeled with PKH26 red fluorescent cell linker kits were incubated with macrophages. Luciferase reporter assay was used to confirm the target gene of miR-125a-5p. In mouse experiments, inhibiting miR-125a-5p was utilized to examine its role in macrophage polarization. Furthermore, the underlying mechanism was explored.
RESULTS: In vitro results indicated that CSE treatment led to upregulation of miR-125a-5p in HBE cells, and exosomes contained miR-125a-5p. PKH26-labeled exosomes were internalized by macrophages. Co-culture experiments between bronchial epithelial cells and miR-125a-5p mimic resulted in significant increase in M1 macrophage markers (TNF-α, iNOS-2, IL-1β) and decrease in M2 markers (IL-10 and Arg-1). In COPD mouse models, miR-125a-5p inhibitor reduced levels of TNF-α, IL-1β, and IL-6. Luciferase assays revealed that miR-125a-5p inhibitors enhanced the relative luciferase activity of IL1RN. Mechanistic experiments demonstrated that HBE-derived exosomes transfected with miR-125a-5p mimics promoted upregulation of MyD88, TRAF6, p65, iNOS-2, and downregulation of Arg-1.
CONCLUSIONS: This study suggests that exosomal miR-125a-5p may act as a mediator in the cross-talk between airway epithelium and macrophage polarization in COPD. Exosomal miR-125a-5p targeting IL1RN may promote M1 macrophage polarization via the MyD88/NF-κB pathway.
METHODS: In cell models, THP-1 monocytic cells were differentiated into macrophages (M0). Human bronchial epithelial cells treated with CS extract (CSE) were co-cultured with M0. Exosomes were isolated from culture media using commercial kits and characterized using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Exosomes labeled with PKH26 red fluorescent cell linker kits were incubated with macrophages. Luciferase reporter assay was used to confirm the target gene of miR-125a-5p. In mouse experiments, inhibiting miR-125a-5p was utilized to examine its role in macrophage polarization. Furthermore, the underlying mechanism was explored.
RESULTS: In vitro results indicated that CSE treatment led to upregulation of miR-125a-5p in HBE cells, and exosomes contained miR-125a-5p. PKH26-labeled exosomes were internalized by macrophages. Co-culture experiments between bronchial epithelial cells and miR-125a-5p mimic resulted in significant increase in M1 macrophage markers (TNF-α, iNOS-2, IL-1β) and decrease in M2 markers (IL-10 and Arg-1). In COPD mouse models, miR-125a-5p inhibitor reduced levels of TNF-α, IL-1β, and IL-6. Luciferase assays revealed that miR-125a-5p inhibitors enhanced the relative luciferase activity of IL1RN. Mechanistic experiments demonstrated that HBE-derived exosomes transfected with miR-125a-5p mimics promoted upregulation of MyD88, TRAF6, p65, iNOS-2, and downregulation of Arg-1.
CONCLUSIONS: This study suggests that exosomal miR-125a-5p may act as a mediator in the cross-talk between airway epithelium and macrophage polarization in COPD. Exosomal miR-125a-5p targeting IL1RN may promote M1 macrophage polarization via the MyD88/NF-κB pathway.
摘要：
背景：气道上皮和巨噬细胞之间的相互作用在慢性阻塞性肺疾病（COPD）的发病机制中起着关键作用。外泌体,运输miRNA货物，已经成为细胞间通讯的新型介体。MicroRNA-125a-5p(miR-125a-5p)与巨噬细胞极化有关。本研究旨在探讨外泌体miR-125a-5p在香烟烟雾（CS）诱导的COPD中功能失调的上皮-巨噬细胞串扰中的作用。
方法：在细胞模型中，THP-1单核细胞分化为巨噬细胞(M0)。将用CS提取物(CSE)处理的人支气管上皮细胞与M0共培养。使用商业试剂盒从培养基中分离外泌体,并使用纳米颗粒跟踪分析(NTA)和透射电子显微镜(TEM)表征。用PKH26红色荧光细胞接头试剂盒标记的外泌体与巨噬细胞一起孵育。荧光素酶报告基因测定用于确认miR-125a-5p的靶基因。在老鼠实验中,抑制miR-125a-5p被用于检查其在巨噬细胞极化中的作用。此外，探讨了潜在的机制。
结果：体外结果表明，CSE处理导致HBE细胞中miR-125a-5p上调，外泌体含有miR-125a-5p。PKH26标记的外泌体被巨噬细胞内化。支气管上皮细胞和miR-125a-5p模拟物之间的共培养实验导致M1巨噬细胞标记物显着增加（TNF-α，iNOS-2，IL-1β）和M2标志物（IL-10和Arg-1）的减少。在COPD小鼠模型中,miR-125a-5p抑制剂降低TNF-α水平，IL-1β,IL-6荧光素酶测定显示miR-125a-5p抑制剂增强了IL1RN的相对荧光素酶活性。机制实验表明，用miR-125a-5p模拟物转染的HBE衍生的外泌体促进MyD88，TRAF6，p65，iNOS-2的上调和Arg-1的下调。
结论：这项研究表明，外泌体miR-125a-5p可能在COPD患者气道上皮和巨噬细胞极化之间的相互作用中起中介作用。靶向IL1RN的外泌体miR-125a-5p可能通过MyD88/NF-κB通路促进M1巨噬细胞极化。
方法：在细胞模型中，THP-1单核细胞分化为巨噬细胞(M0)。将用CS提取物(CSE)处理的人支气管上皮细胞与M0共培养。使用商业试剂盒从培养基中分离外泌体,并使用纳米颗粒跟踪分析(NTA)和透射电子显微镜(TEM)表征。用PKH26红色荧光细胞接头试剂盒标记的外泌体与巨噬细胞一起孵育。荧光素酶报告基因测定用于确认miR-125a-5p的靶基因。在老鼠实验中,抑制miR-125a-5p被用于检查其在巨噬细胞极化中的作用。此外，探讨了潜在的机制。
结果：体外结果表明，CSE处理导致HBE细胞中miR-125a-5p上调，外泌体含有miR-125a-5p。PKH26标记的外泌体被巨噬细胞内化。支气管上皮细胞和miR-125a-5p模拟物之间的共培养实验导致M1巨噬细胞标记物显着增加（TNF-α，iNOS-2，IL-1β）和M2标志物（IL-10和Arg-1）的减少。在COPD小鼠模型中,miR-125a-5p抑制剂降低TNF-α水平，IL-1β,IL-6荧光素酶测定显示miR-125a-5p抑制剂增强了IL1RN的相对荧光素酶活性。机制实验表明，用miR-125a-5p模拟物转染的HBE衍生的外泌体促进MyD88，TRAF6，p65，iNOS-2的上调和Arg-1的下调。
结论：这项研究表明，外泌体miR-125a-5p可能在COPD患者气道上皮和巨噬细胞极化之间的相互作用中起中介作用。靶向IL1RN的外泌体miR-125a-5p可能通过MyD88/NF-κB通路促进M1巨噬细胞极化。
