ASK1-ER stress pathway-mediated fibrotic-EV release contributes to the interaction of alveolar epithelial cells and lung fibroblasts to promote mechanical ventilation-induced pulmonary fibrosis

www.nature.com/emm ARTICLE OPEN ASK1-ER stress pathway-mediated fibrotic-EV release contributes to the interaction of alveolar epithelial cells and lung fibroblasts to promote mechanical ventilation-induced pulmonary fibrosis Ri Tang 1,2 , Shuya Mei1,2, Qiaoyi Xu1, Jinhua Feng1, Yang Zhou1, Shunpeng Xing1, Zhengyu He 1✉ and Yuan Gao1 ✉ 1234567890();,: © The Author(s) 2022 Recent clinical research has revealed that mechanical ventilation (MV) can initiate pulmonary fibrosis and induce mechanical ventilation-induced pulmonary fibrosis (MVPF). However, the underlying mechanism remains largely uncharacterized. Based on a mouse model of MVPF and an alveolar epithelial cell cyclic strain model, the present study explores the possible mechanism of MVPF. Single-cell RNA-sequencing and EV RNA-sequencing analysis revealed that MV promoted apoptosis signal-regulating kinase 1 (ASK1)-mediated endoplasmic reticulum (ER) stress pathway activation and extracellular vesicle (EV) release from alveolar epithelial cells. Furthermore, the ASK1-ER stress pathway was shown to mediate mechanical stretch (MS)- or MV-induced EV release and lung fibroblast activation in vivo and in vitro. These processes were suppressed by ER stress inhibitors or by silencing ASK1 with ASK1- short hairpin RNA (shRNA). In addition, MVPF was suppressed by inhibiting ASK1 and ER stress in vivo. Therefore, the present study demonstrates that ASK1-ER stress pathway-mediated fibrotic-EV release from alveolar epithelial cells contributes to fibroblast activation and the initiation of pulmonary fibrosis during MV. The inhibited release of EVs targeting the ASK1-ER stress pathway might be a promising treatment strategy for MVPF. Experimental & Molecular Medicine; https://doi.org/10.1038/s12276-022-00901-1 INTRODUCTION In the development of acute respiratory distress syndrome (ARDS), pulmonary fibrosis (PF) plays an important pathological role. As a preferred treatment for respiratory failure, mechanical ventilation (MV) is widely used in the treatment of ARDS or PF. However, MV can cause ventilator-induced lung injury (VILI) and aggravate ARDS1. Furthermore, recent studies have revealed that MV can initiate lung fibrosis and induce mechanical ventilation-induced pulmonary fibrosis (MVPF)2,3. However, the underlying mechanism remains largely uncharacterized. In recent years, the influence of the pathological microenvironment on cell dysfunction has been widely recognized as essential to the pathogenesis of PF4. In addition to the effect of traditional biological or chemical factors on cell dysfunction, the effect of physical microenvironment changes caused by mechanical stretch (MS) on cellular dysfunction has recently been identified as a novel physical factor associated with MVPF pathogenesis3,5. An increasing number of studies have revealed that different types of cells interact with each other through extracellular vesicles (EVs) and thus form a cellular regulatory network in lung tissue to promote the progression of PF6. However, whether the EVmediated interaction between alveolar epithelial cells and lung fibroblasts under MS-induced microenvironment alteration plays a substantial role during the progression of MVPF is unclear. As a result of our single-cell analysis, MVPF was found to be associated with an increase in the expression of genes involved in ER stress in epithelial cells. EV bulk RNA sequencing implied that EV function was associated with the progression of pulmonary fibrosis. Therefore, we hypothesized that MV-induced fibrotic-EVs release from lung epithelial cells to facilitate the activation of lung fibroblasts and development of MVPF. Previous studies have shown that apoptosis signal-regulating kinase 1 (ASK1) is related to the ER stress pathway7, but the role of these processes in MVPF remains unclear. This study is based on a mouse model of MVPF and an alveolar epithelial cell cyclic strain model, and aims to clarify the role of the ASK1-ER stress pathway in crosstalk between epithelial cells and fibroblasts in MVPF. MATERIALS AND METHODS Ethics statement and animals C57BL/6 male mice (6–8 weeks old; 18–27 g) were acquired from Shanghai SLAC Laboratory Animal, China. The animals were maintained in a controlled environment with a temperature of 22–24 °C, a 12 h light/dark cycle, and free access to food and water. The Animal Care and Use 1 Department of Critical Care Medicine, Renji Hospital, School of Medicine, Shanghai Jiaotong University, 200127 Shanghai, China. 2These authors contributed equally: Ri Tang, Shuya Mei. ✉email: ; Received: 1 September 2022 Revised: 7 October 2022 Accepted: 11 October 2022 R. Tang et al. 2 Committee of Ren Ji Hospital, Shanghai Jiao Tong University School of Medicine approved all the experiments. Cell lines and culture The human lung fibroblast MRC-5 cell line and the murine alveolar epithelial cell line TC-1 were acquired from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The MRC-5 cells were cultured in minimum essential medium (MEM, HyClone, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 100 IU/ml penicillin, and 100 IU/ml streptomycin, while the TC-1 cells were cultured in RMPI 1640 medium (1640, Gibco, USA) supplemented with 10% FBS, 100 IU/ml penicillin, and 100 IU/ml streptomycin. To remove EVs from the FBS used in the cell cultures, the medium was ultracentrifuged at 100,000 × g for 70 min. MV model and animal procedures C57BL/6 male mice (6–8 weeks old; 18–27 g) were anesthetized with an intraperitoneal injection of 200 mg/kg ketamine and 10 mg/kg xylazine and randomly allocated to sham and MV group. The mice in the MV group were mechanically ventilated for 2 h using FiO2 0.21, VT 20 ml/kg, and RR 70 breaths/min8, while the sham group maintained spontaneous breathing after intubation. An intratracheal injection of ASK1 adeno-associated virus (AAV) to knockdown ASK1 expression in pulmonary tissue and intraperitoneal injection of the ER stress inhibitor 4-phenylbutyric acid (4-PBA) were administered to inhabit ER stress (Supplementary Fig. 1a). Observation was performed over a 7-day period following intubation of the animals, which had with free access to food and water, in the animal facility. To obtain samples of bronchoalveolar lavage fluid (BALF), 20 G intratracheal cannulas were used after the experiments were completed. To recover the BALF, the right lung was washed with 500 μL of cold PBS and then collected and centrifuged at 3000 × g for 10 mins. The total protein level of the BALF was measured, or the BALF was used for EV isolation. After BALF was collected, the sample from right lung tissue was used for western blotting (WB), PCR, and flow cytometry. Histopathology, immunohistochemistry, and transmission electron microscopy (TEM) were performed on left lung tissue, which was fixed in paraformaldehyde. ASK1 inhibition by knockdown AAV transfection Adeno-associated viruses (AAVs) were acquired from Genomeditech (...truncated)