Fibrotic extracellular vesicles contribute to mechanical ventilation-induced pulmonary fibrosis development by activating lung fibroblasts via JNK signalling pathway: an experimental study

Animals

We obtained C57BL/6 male mice (6–8 weeks; 18–28 g) from Shanghai SLAC Laboratory Animals (China). There was no specific pathogen present in the housing environment, and the temperature was controlled (22–24°C), a 12-hour light/dark cycle was observed, and food and water were freely available to the animals.

Patient and public involvement

Patients and the public were not involved in this study’s design, recruitment and conduct.

Cell lines and culture

We obtained the human lung fibroblast MRC-5 cell line from the Cell Bank of the Chinese Academy of Sciences (China). MRC-5 cells were cultured in Dulbecco's Minimum Essential Medium from Gibco, USA, with 10% fetal bovine serum (FBS) from Gibco (USA), 100 IU/mL penicillin and streptomycin from Gibco. EV was removed from all FBS used for cell culture by ultracentrifugation at 100 000 g for 70 min.

Reagents and antibodies

The JNK signalling pathway inhibitor SP600125 (S1460) was purchased from Selleck (USA). The EV secretion inhibitor GW4869 (S7609) was purchased from Selleck (USA). The primary antibodies used in this study were rabbit anti-fibronectin (ab2413, Abcam, USA), mouse anti-α-SMA (ab5694, Abcam, USA), rabbit anti-p-JNK (#4668, CST, USA), rabbit anti-JNK (#9252, CST, USA), mouse anti-β-actin (#3700, CST, USA) and mouse anti-α-tubulin (#3873 CST, China). In addition, goat anti-mouse (#7076, CST, USA) and goat anti-rabbit (#7074, CST, USA) were used as secondary antibodies.

Experimental grouping and design of the mice

The mice were anaesthetised with ketamine (200 mg/kg) and xylazine (10 mg/kg) intraperitoneally. To establish the MVPF model, the mice were treated with MV for 2 hours with the following parameters: fractional inspired oxygen, 0.21; tidal volume, 20 mL/kg; and respiratory rate, 70 breaths/min.31 JNK inhibitor SP600125 and EV secretion inhibitor GW4869 were injected intraperitoneally before MV. On day 7 following MV, mice were euthanized with an overdose of euthanized to collect lungs, blood and BALF.

GW4869 (2.5 mg/kg in corn oil), an EV secretion inhibitor, was administered via intraperitoneal injection once a day for 3 consecutive days before MV.32 Mice were randomly assigned into sham (n=6), GW4869 (n=6), MV (n=6) and MV+GW4869 (n=6). Mice in the MV and MV+GW4869 groups received MV treatment for 2 hours.

SP600125 (15 mg/kg in corn oil), a JNK inhibitor, was administered via intraperitoneal injection once a day for 3 consecutive days before intubation.33 Mice were randomly assigned into sham (n=6), SP600125 (n=6), MV (n=6) and MV+SP600125 (n=6). Mice in the MV and MV+SP600125 groups received MV treatment for 2 hours.

Experimental grouping and design of MRC-5 cells

In 6-well culture plates, MRC-5 cells were plated at a density of 2×10⁴ per well. To starve the cells overnight, the medium was changed to serum-free minimum essential medium once the cells had adhered and reached 70% confluency. Then, the cells were incubated with BALF-EVs (MV-EVs or NC-EVs, 50 µg EVs protein/sample). An inhibitor of the JNK pathway, SP600125, was used to suppress JNK activity. Twenty-four hours after treatment, the cells and supernatants were collected.

Confluent MRC-5 cells were randomly assigned into the sham, SP600125, MV-EV and MV-EV+SP600125 groups. MRC-5 cells in the sham group received phosphate-buffered saline (PBS) treatment as a control; MRC-5 cells in the SP600125 group were cultured in a medium containing 20 µM SP600125 for 2 hours; MRC-5 cells in the MV-EV group received MV-EVs (50 µg EVs protein/sample) for 24 hours; and MRC-5 cells in the MV-EV+SP600125 group were cultured in a medium containing 20 µM SP600125 for 2 hours and received MV-EVs (50 µg EVs protein/sample) for 24 hours.

EV isolation, characterisation and labelling

BALFs were sequentially centrifuged one at 500 g, one at 2500 g and one at 12 000 g, and the supernatant was collected. A pellet was isolated and resuspended in 20 µL PBS for later use after being centrifuged at 100 000 g for 70 min.

Transmission electron microscope (TEM) was used to visualise the pellet after adsorption for 90 s onto a copper mesh coated with carbon. Uranyl acetate dye solution was used for the negative staining test for 30 s. Using a NanoSight NS300 instrument (Malvern Panalytica, UK), nanoparticle tracking analysis (NTA) was used to measure the diameter of the pellet. Western blotting (WB) was used to detect the surface marker proteins of the pellet. EVs were labelled with PKH-67 membrane dye (MIDI67, Sigma-Aldrich, USA) according to the manufacturer’s instructions.

Single-cell RNA-sequencing analysis

We used single-cell RNA sequencing to analyse mouse lung cells. Gene expression data have been deposited into the SRA database at the National Center for Biotechnology Information with accession number SUB11878630. A single-cell RNA sequence was used to analyse mice pulmonary cells from the MV and sham groups. The number and viability of prepared single cells were measured using the Rigel S2 (Countstar, China). Using the Chromium Single Cell 3’ V2 Chemistry Library Kit and Gel Bead & Multiplex Kit from 10x Genomics (Biomarker Technologies, China), single-cell libraries were constructed. Gel Bead-In-Emulsions were generated using cellular suspensions loaded onto the Chromium Controller (10x Genomics, Pleasanton). The Chromium Single Cell 3’ Reagent Kits V.3.1 (10x Genomics) were used to generate bar-coded sequencing libraries. Each sample was sequenced using paired-end sequencing on one lane of NovaSeq 6000 with 150 nt each end after library preparation. The 10x Genomics Cell Ranger pipeline was used to process raw reads. With Cell Ranger, the single cells can be clustered, the marker genes can be identified and the unique molecular identifiers can be exported. The R package Seurat (V.2.2) was used for further analysis. For most of the Seurat analyses, default parameters were used. The maximum cut-off for Feature Plot was 0.5.

Pulmonary histopathology

For collagen identification, the left lung was fixed in 4% paraformaldehyde (PFA) and stained with Masson’s trichrome. The Ashcroft fibrosis score was used to quantify PF.34 During the histological examination of the lungs, a pathologist blinded to the experimental groups performed the examination. In this study, pulmonary injury was graded on a scale of 0–4 (0, normal; 1, light; 2, moderate; 3, strong; 4, intense) using the following nine reference parameters: microscopic emphysema, perivascular haemorrhage, alveolar oedema, perivascular oedema, congestion, microscopic atelectasis, alveolar haemorrhage, alveolar and interstitial polymorphonuclear leucocyte infiltration, and hyaline membrane formation.35

Immunofluorescence staining

Formalin-fixed, paraffin-embedded sections of pulmonary tissue or cultured cells fixed with 4% PFA and permeabilised with 0.25% Triton X100 were stained with appropriate primary antibodies. Anti-mouse IgG (H+L), F(ab’)2 Fragment (Alexa Fluor 488 Conjugate) (#4408, CST, USA), anti-mouse IgG (H+L), F(ab’)2 Fragment (Alexa Fluor 594 Conjugate) (#8890, CST, USA), anti-rabbit IgG (H+L), F(ab’)2 Fragment (Alexa Fluor 488 Conjugate) (#4412, CST, USA) and anti-rabbit IgG (H+L), F(ab’)2 Fragment (Alexa Fluor 594 Conjugate) (#8889, CST, USA) were used as secondary antibodies. Cell nuclei were stained using 4’,6-diamidino-2-phenylindole (Santa Cruz Biotechnology, Heidelberg, Germany).

Cell viability assay (Cell Counting Kit-8 assay)

MRC-5 cells were cultured in 96-well plates. The Cell Counting Kit-8 (CCK-8) assay (Vazyme, China) was used to evaluate the viability of MRC-5 cells according to the manufacturer’s instructions. Briefly, after adding CCK-8 solution to each well, MRC-5 cells were incubated for 2 hours. The optical density of each well was measured using a DNM-9602 enzyme-labelled analyser (Beijing Perlong New Technology Co).

Cell proliferation assay (EDU assay)

MRC-5 cells were cultured in 6-well plates. The EDU Alexa Fluor 594 imaging kit (Epizyme, China) was used to evaluate MRC-5 cell proliferation according to the manufacturer’s instructions.

Briefly, after adding EDU solution to each well, the MRC-5 cells were incubated for 2 hours. Incubated MRC-5 cells were fixed in 75% ethyl alcohol for 24 hours at 20°C and penetrated with 0.3% PBS with Tween (PBST) for 30 min. Three washings in 0.1% PBST for 15 min were followed by 30 min incubation in EDU click solution (with Alexa Fluor 594) away from light.

Wound healing migration assay

MRC-5 cells were cultured in 6-well plates. The plates were scratched using a 200 µL pipette tip led by a scratch ruler after the MRC-5 cells were cultured to 80% confluence. An inverted light microscope (magnification: ×100, Olympus, Japan) was used to capture images of each same place 0 and 24 hours after treatment and cultivation.

Western blotting

We separated proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred them to polyvinylidene difluoride membranes. The membranes were incubated with primary and secondary antibodies. Enhanced ECL Chemiluminescent Substrate Kit (Vazyme, China) was used to detect blots using Image Lab (Bio-Rad, USA).

Statistical analysis

We display the data as means±SEM. Multiple comparisons with group and time factors were performed using a two-way analysis of variance, followed by the Bonferroni test. For the in vitro experiments, a non-parametric test was performed to compare cells with and without stretch. GraphPad Prism V.5.0 (GraphPad Software, La Jolla, California, USA) was used for all analyses, and p values of <0.05 were used to denote statistical significance.

MV initiated the process of PF

To create the MVPF model in vivo, mice were observed 7 days after MV at 20 mL/kg for 2 hours. Histologically, pulmonary injury, interstitial leucocyte infiltration, alveolar oedema and haemorrhage were more obvious in the MV group than in the sham group. Masson staining displayed an increase in collagen deposition in the pulmonary interstitium after MV. The total pulmonary injury and fibrosis scores were increased in the MV group (figure 1A).

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Consistent with the histological observations, MV significantly increased the protein levels of fibronectin and α-SMA in the lungs (figure 1B). MV was also associated with an upregulation of fibronectin+/collagen I+ in total cells, compared with the sham group (figure 1C).

Inhibition of EV secretion alleviates the severity of MVPF

Studies revealed that EVs were associated with organ fibrosis. We isolated and purified EVs from BALF. TEM and NTA determined the structure and size of the particles isolated from BALF. The BALF-EV markers CD63 was detected using WB (figure 2A). To investigate whether the inhibition of EV release in vivo affects MVPF development and progression, mice were intraperitoneally pretreated with GW4869 (EV secretion inhibitor). As shown in figure 2B, the severity of pulmonary histopathology could be attenuated by GW4869 pretreatment.

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Immunofluorescence staining showed that MV significantly increased the percentage of fibronectin+ and α-SMA+ in total cells (figure 2C), whereas WB showed upregulated protein levels of fibronectin and α-SMA in pulmonary tissue (figure 2D). Furthermore, these responses could be alleviated by pretreating with GW4869 (figure 2B–D). Therefore, we speculated that inhibiting EV secretion contributes to the alleviation of MVPF.

MV-EVs induce lung fibroblast activation

To investigate the role of EVs, we next used MRC-5 cells (human lung fibroblasts) as the target cells to evaluate the functions of NC-EVs (EVs attracted from BALF in the sham group) and MV-EVs (EVs attracted from BALF in the MV group). After treating MRC-5 cells with PKH-67-labelled EVs (green) (50 µg EVs protein/sample), figure 3A shows that the EVs were taken up by MRC-5 cells.

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To further elucidate the effects of EVs on MRC-5 cells, the differentiation, proliferation and migration abilities of MRC-5 cells were measured. Using the CCK-8 and EDU assays, our data showed that when MRC-5 cells took up MV-EVs, the cell viability and proliferation ability were increased compared with those of the MRC-5 cells that took up NC-EV or the sham group (figure 3B,D), indicating that MV-EVs increased the proliferation ability of MRC-5 cells. Similarly, using a wound healing assay, our data showed that the cell migration ability was increased when the MRC-5 cells took up MV-EVs compared with that in the MRC-5 cells that took up NC-EV or the sham group (figure 3C). The WB and immunofluorescence staining were both performed to evaluate the expression of differentiation-related proteins. Fibronectin and α-SMA were chosen to reflect the ECM secretion and the differentiation level. Our data showed that after treatment with MV-EVs, the expression of differentiation-related proteins was increased in MRC-5 cells, whereas the NC-EVs and sham groups did not exert significant changes (figure 3E,F). Our findings showed that MV-EVs induced lung fibroblast activation.

Single-cell RNA-sequencing analysis revealed that the JNK pathway is involved in the fibrosis process after MV in fibroblast cluster

To detect the mechanism of fibroblast differentiation, we performed single-cell RNA sequencing to analyse pulmonary cells from mice from the sham and MV groups. Raw reads were processed using a 10x Genomics Cell Ranger pipeline. With Cell Ranger, the single cells can be clustered, and the marker genes can be identified. The UMAP plot was used to display all cell types (figure 4A). As the core effector cells of PF, fibroblast cluster marker genes are shown in figure 4B.

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With the FindMarkers R packages, differentially expressed genes (DEGs) of the fibroblast clusters between the sham and MV groups were detected. By Gene Ontology enrichment analysis of DEGs in fibroblasts, we found significantly changed processes associated with fibrosis, such as ‘ECM’ and ‘focal adhesion’ (figure 4C). By Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs in fibroblasts, we found that the mitogen-activated protein kinase (MAPK) pathway might be a possible mechanism involved in fibroblast activation (figure 4D). The mammalian MAPK family consists of extracellular signal-regulated kinase (ERK), p38 and JNK (also known as stress-activated protein kinase).36 According to the results of the Gene Set Enrichment Analysis (figure 4E), we speculated that the JNK pathway is involved in the fibrosis process after MV in fibroblast cluster.

Inhibition of the JNK pathway inhibits MV-EV-induced lung fibroblast activation

According to our analysis of our single-cell data, we sought to demonstrate whether JNK pathway activation was involved in MV-EV-induced MRC-5 cell activation.

As shown in figure 5A, WB indicated that the protein level of JNK was not changed, whereas the protein level of phosphorylated JNK (p-JNK) was upregulated in MRC-5 cells after treatment with MV-EVs.

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Because SP600125 could suppress the JNK pathway, we explored whether suppressing the JNK pathway affects the activation of MRC-5 cells. WB and immunofluorescence staining demonstrated that MV-EVs increased the differentiation ability in MRC-5 cells, which could be suppressed by pretreating with SP600125 (figure 5B,C). Using the wound healing assay, our data showed that the cell migration ability was increased when MRC-5 cells took up MV-EVs; these processes could be suppressed by pretreating with SP600125 (figure 5D). Similarly, using the EDU assay, figure 5E shows that by pretreating with SP600125 before taking up MV-EVs in MRC-5 cells, the increased proliferation ability in the MV-EV group would be suppressed. Therefore, it is reasonable to imply that the inhibition of the JNK pathway could reduce MV-EV-induced lung fibroblast activation.

Inhibition of the JNK pathway alleviated the severity of MVPF

To elucidate the effects of a JNK inhibitor in vivo, mice with MVPF were used to observe the effects of SP600125. The mice were injected intraperitoneally with 15 mg/kg concentrations of SP600125 before MV. After 7 days of MV, lung tissue samples from these mice were separated for WB, histopathology and immunofluorescence staining.

As shown in figure 6A, the increased protein level of p-JNK/JNK could be decreased in the SP600125-treated group compared with that in the MV group. Through H&E and Masson staining, our data showed that pulmonary histopathology could be decreased in the MV+SP600125 group compared with that in the MV group (figure 6B). Immunofluorescence and WB were performed to show the expression of fibronectin and α-SMA in pulmonary tissue. Our data showed that both fibronectin and α-SMA were reduced in the MV+SP600125 group compared with those in the MV group (figure 6C,D). Certainly, SP600125-treated mice without MV did not show obvious differences from those in the sham group. Our findings showed that SP600125 reduced the expression of fibrosis-related genes and histopathology changes in mice treated with MV, indicating that the inhibition of the JNK pathway reduced the severity of MVPF.

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