Profiling small extracellular vesicles (EVs) in idiopathic pulmonary fibrosis (IPF)

Abstract

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease that affects 3 million people worldwide. Senescence and small extracellular vesicles (EVs) have both been implicated in the pathogenesis of IPF, although how EVs promote disease remains unclear. Here, we profile EVs from bronchial epithelial cells and determine potential regulatory smRNA content. EVs were isolated from conditioned media collected from normal human bronchial epithelial cells (NHBEs) and IPF diseased human bronchial epithelial cells (IPF DHBEs). Increased EV release from IPF DHBEs compared to NHBEs (n=6; p<0.001) was detected by nanoparticle tracking analysis (NTA). These particles were of correct EV size (50-200nm) and were successfully characterised by blotting for common EV proteins such as CD81 and CD63. Uptake of fluorescently tagged EVs by NHBEs showed maximal incorporation by 24 hours. NHBEs co-cultured with IPF DHBE-derived EVs for 72 hours expressed higher levels of SpiderGAL, γH2AX, p16, p21 and TP53 and higher protein release of IL6 and IL8 (all n=6; p<0.05). EVs were also co-cultured with healthy ALI cultures, as a more physiologically relevant system. Increases were observed in p21 and p16 gene expression and IL6 and IL8 (basal and apical) secretion when co-cultured with IPF DHBE-derived EVs (n=6; p<0.05). RNA-seq showed 19 significantly differentially expressed miRNA in IPF DHBE-derived EVs as opposed to healthy. 5 of these miRNA were validated by qPCR; miRNA-411, miRNA-137, miRNA-195, miRNA-7 and miRNA-320a, all of which are being further investigated (all n=5; p<0.05). This data demonstrates that IPF DHBE-derived EVs have the potential to transfer senescence to neighbouring healthy cells via miRNA cargo.