Extracellular vesicles from broncho-alveolar lavage act on cellular behavior during lung fibrosis.

Abstract

Introduction: Impaired intercellular crosstalk is an essential hallmark of idiopathic pulmonary fibrosis (IPF). WNT signaling, dysregulated in IPF, is based on extracellular ligands which can be secreted trough extracellular vesicles (EVs). Preliminary data from our lab suggests enhanced EVs accumulation during lung fibrosis. Our hypothesis is that EVs carry a WNT signal and contribute to fibrosis. Methods: EVs from BALF of mice injected with bleomycin (or NaCl) were isolated using sequential ultracentrifugation and characterized by immunoblotting, nanoparticle tracking analysis and electron microscopy. BALF EVs were subjected to an unbiased proteomic analysis and their effects were assessed in vitro on murine 3D lung tissue culture (LTC) and primary alveolar epithelial cells (ATII). Results: EVs accumulated in BALF after bleomycin compared to control, together with upregulated WNT5A. In LTCs, fibrotic mouse EVs increased the metabolic activity and enhanced myofibroblast markers Acta2 and Cola1a1. Fibrotic EVs affect ATII behavior with decreased E-cadherin or surfactant protein C mRNA expression along with preliminary data on WNT signaling alteration as measured by Axin2 and Nkd1 mRNA expression. Proteomic analysis of fibrotic or control BALF-EVs showed difference in components of TGF-beta signaling or involved in the complement system. Conclusion: EVs are increased in mouse BALF following bleomycin compared to control. Our data suggest that EVs isolated from bleomycin injected mice mediate pro-fibrotic effect. Using different approaches, we suggest that EVs could transport components of different developmental signaling, which will be further investigated.