Increased Extracellular Vesicles Mediate WNT-5A Signaling in Idiopathic Pulmonary Fibrosis

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease characterized by lung epithelial cell injury, increased (myo)fibroblast activation and extracellular matrix deposition. Extracellular vesicles (EVs) regulate intercellular communication by carrying a variety of signaling mediators, including WNT proteins. The relevance of EVs in pulmonary fibrosis and their potential contribution to disease pathogenesis, however, remains unexplored.OBJECTIVE: To characterize EVs and study the role of EV-bound WNT signaling in IPF.METHODS: We isolated EVs from bronchoalveolar lavage fluid (BALF) from experimental lung fibrosis as well as samples from IPF, non IPF-ILD, non-ILD and healthy volunteers from two independent cohorts. EVs were characterized by transmission electron microscopy, nanoparticle tracking analysis and Western Blotting (WB). Primary human lung fibroblasts (phLFs) were used for EV isolation and analyzed by metabolic activity assays, cell counting, qPCR and WB upon WNT gain- and loss-of-function studies.MEASUREMENTS AND MAIN RESULTS: We found increased EVs, particularly exosomes, in BALF from experimental lung fibrosis as well as from IPF patients. WNT-5A was secreted on EVs in lung fibrosis and induced by TGF-β in primary human lung fibroblasts. The phLF-derived EVs induced phLF proliferation, which was attenuated by WNT-5A silencing and antibody-mediated inhibition and required intact EV structure. Similarly, EVs from IPF-BALF induced phLF proliferation, which was mediated by WNT-5A.CONCLUSIONS: Increased EVs function as carriers for signaling mediators, such as WNT-5A, in IPF and thus contribute to disease pathogenesis. Characterization of EV secretion and composition may lead to novel approaches to diagnose and develop treatments for pulmonary fibrosis.