Suppression of autophagy by extracellular vesicles promotes myofibroblast differentiation in COPD pathogenesis

Abstract

Extracellular vesicles (EVs), such as exosomes and microvesicles, encapsulate proteins and microRNAs (miRNAs) as new modulators of both intercellular crosstalk and disease pathogenesis. The composition of EVs is modified by various triggers to maintain physiological homeostasis. In response to cigarette smoke exposure, the lungs develop emphysema, myofibroblast accumulation and airway remodelling, which contribute to chronic obstructive pulmonary disease (COPD). However, the lung disease pathogenesis through modified EVs in stress physiology is not understood. Here, we investigated an EV-mediated intercellular communication mechanism between primary human bronchial epithelial cells (HBECs) and lung fibroblasts (LFs) and discovered that cigarette smoke extract (CSE)-induced HBEC-derived EVs promote myofibroblast differentiation in LFs. Thorough evaluations of the modified EVs and COPD lung samples showed that cigarette smoke induced relative upregulation of cellular and EV miR-210 expression of bronchial epithelial cells. Using co-culture assays, we showed that HBEC-derived EV miR-210 promotes myofibroblast differentiation in LFs. Surprisingly, we found that miR-210 directly regulates autophagy processes via targeting ATG7, and expression levels of miR-210 are inversely correlated with ATG7 expression in LFs. Importantly, autophagy induction was significantly decreased in LFs from COPD patients, and silencing ATG7 in LFs led to myofibroblast differentiation. These findings demonstrate that CSE triggers the modification of EV components and identify bronchial epithelial cell-derived miR-210 as a paracrine autophagy mediator of myofibroblast differentiation that has potential as a therapeutic target for COPD. Our findings show that stressor exposure changes EV compositions as emerging factors, potentially controlling pathological disorders such as airway remodelling in COPD.

Keywords: COPD; autophagy; exosome; extracellular vesicle; microRNA.

Fig. 1

Human bronchial epithelial cell-derived EVs are transported to primary lung fibroblasts (LFs). (a) Electron microscopy images of BEAS-2B- and HBEC-derived EVs, showing a size of approximately 50–150 nm in diameter. (b) Western blot of these cell-derived EVs for CD81, CD9, annexin A2 and HSP70. (c) HBECs (upper panel) and HBEC-derived EVs (lower panel) were analysed with a bioanalyser. Gels and electropherograms are shown. The left gel lane is the ladder standard, and the right lane is the total RNA from HBECs and HBEC-derived EVs. The y-axis of the electropherogram shows the signal intensities in arbitrary fluorescence units (FU), and the x-axis shows the size of the RNA in nucleotides (nt). (d) Schematic representation of the EV uptake experiment. BEAS-2B- and HBEC-derived EVs were labelled with PKH67 and incubated with primary LFs. (e) Purified BEAS-2B- and HBEC-derived EVs or vehicle PBS(-) as a control was labelled with PKH67 (green) and incubated with primary LFs. Nuclei were counterstained with DAPI (blue). Scale bar: 20 µm.

Fig. 2

CSE-induced HBEC-derived EVs promote a lung myofibroblast differentiation phenotype. (a) Schematic representation of the conditioned medium collected (on days 4 and 6) after HBECs (passage ≤ 4) was incubated with or without a low concentration of CSE (1.0%) for 2 days. MC: medium change. (b) Cell proliferation assay of HBECs with or without 1.0% CSE (days 0–2). (c and d) Nanoparticle tracking analyses of the particle size (c) and counts (d) in non-treated HBEC-derived EVs or CSE-induced HBEC-derived EVs. (e) Western blot of senescent markers and fibrotic markers by CSE-induced HBEC-derived EVs or non-treated HBEC-derived EVs in primary lung fibroblasts (LFs). (f) Immunofluorescence staining for collagen type I (red) and α-SMA (green) in primary LFs with CSE-induced HBEC-derived EVs or non-treated HBEC-derived EVs was evaluated by confocal microscopy. DAPI (blue) was used for nuclear staining. Scale bar: 20 µm.

Fig. 3

HBEC-derived EV miR-210 promotes lung myofibroblast differentiation. (a) A heat map of the EV miRNA microarray analysis revealed differentially expressed miRNAs (change >1.5-fold) in CSE-induced HBEC-derived EVs or non-treated HBEC-derived EVs. (b) qRT-PCR validation of EV miRNAs from the 2 EV groups. miR-16 was used as an internal control. (c) qRT-PCR analyses of miR-210 expression levels in non-smoker or smoker lungs (including non-COPD smokers and COPD patients; P=0.049). (d) miR-210-specific probe, scramble control probe and β-actin were hybridized in situ with normal lung tissue. Original magnification, 200×. (e) A transwell co-culture assay with transfected HBECs (top well) and primary lung fibroblasts (LFs) (bottom well). A 0.4-µm porous membrane is between the 2 wells, inhibiting cell–cell contact. (f) A co-culture assay to study the miRNA cargo from HBECs to primary LFs. HBECs were transfected with a Cy3-labelled miRNA (red) or a control precursor miRNA (non-labelled). Nuclei were counterstained with DAPI (blue). Scale bar: 50 µm. (g) miR-210 expression in LFs after 72 h co-culturing with HBECs transfected with miR-210 mimic (pre-miR-210) or miR-NC. RNU6B was used as an internal control. (h) Western blot of fibrotic markers in primary LFs (the bottom well) in the co-culture assay. HBECs were transfected with a precursor of miR-210 (pre-miR-210) or a control precursor miRNA (pre-miR-NC) and co-cultured with primary LFs for 72 h. *P<0.05. NC: negative control.

Fig. 4

miR-210 directly targets ATG7 in lung fibroblasts (LFs), leading to a modulating autophagy process. (a) Western blot of ATG7 in LF homogenates from non-COPD (n=5) and COPD patients (n=4). The right panel shows the average taken from 3 independent experiments shown as the relative expression of ATG7 as compared with that of β-actin. (b) Western blot of LC3 in LF homogenates from non-COPD (n=5) and COPD patients (n=4) in the presence of protease inhibitors (E64d and pepstatin A). The right panel shows the average taken from 3 independent experiments shown as the relative expression of LC3-II as compared with that of β-actin. (c) The relationship between relative ATG7 expression normalized to β-actin and the percentages of FEV1/FVC (n=9). (d) The relationship between relative ATG7 expression normalized to β-actin and miR-210 expression normalized to RNU6B (n=9). (e) Western blotting of ATG7 and fibrotic markers in primary LFs transfected with ATG7-siRNA or siRNA-NC. (f) Western blot of ATG7 and fibrotic markers in primary LFs transfected with pre-miR-210 or pre-miR-NC. (g) Schematic miR-210 putative target sites in the 3′ UTRs of ATG7 and the sequence of mutant UTRs. (h) The effect of co-transfection of pre-miR-210 with wild-type (Wt) and mutant (Mut) psiCHECK2 vectors with each gene construct in MRC5 cells was measured using luciferase reporter assays. (i) LC3 western blotting of primary LFs transfected with pre-miR-210 or pre-miR-NC in the presence or absence of protease inhibitors (E64d and pepstatin A). (j) Fluorescence microscopic detection of pEGFP-LC3 dot formation in primary LFs. The right panel is the percentage of positive cells with more than 5 dot formations. (k) LC3 western blotting of primary LFs cultured with CSE-induced HBEC-derived EVs or non-treated HBEC-derived EVs in the presence or absence of protease inhibitors (E64d and pepstatin A). *P<0.05. **P<0.01. NC: negative control.

Fig. 5

Proposed novel airway remodelling model for bronchial epithelial cells and fibroblast crosstalk in COPD.