Macrophage‐derived exosomes mediate silica‐induced pulmonary fibrosis by activating fibroblast in an endoplasmic reticulum stress‐dependent manner

Abstract Macrophages play a key role in silicosis, and exosomes are potent mediators of intercellular communication. This suggests that macrophage‐derived exosomes have a potential contribution to the pathogenesis of silicosis. To investigate whether macrophage‐derived exosomes promote or inhibit lung fibrosis, in vitro, silica‐exposed macrophage‐derived exosomes (SiO2‐Exos) were collected and cocultured with fibroblasts. The expression of collagen I and α‐SMA was evaluated. Furthermore, the endoplasmic reticulum (ER) stress markers BIP, XBP1s and P‐eIF2α were assessed after treatment with or without the ER stress inhibitor 4‐PBA. In vivo, mice were pre‐treated with the exosome secretion inhibitor GW4869 prior to silica exposure. After sacrifice, lung tissues were histologically examined, and the expression of proinflammatory cytokines (TNF‐α, IL‐1β and IL‐6) in bronchoalveolar lavage fluid (BALF) was measured. The results showed that the expression of collagen I and α‐SMA was up‐regulated after treatment with SiO2‐Exos, accompanied by increased expression of BIP, XBP1s and P‐eIF2α. Pre‐treatment with 4‐PBA reversed this effect. More importantly, an in vivo study demonstrated that pre‐treatment with GW4869 decreased lung fibrosis and the expression of TNF‐α, IL‐1β and IL‐6 in BALF. These results suggested that SiO2‐Exos are profibrogenic and that the facilitating effect is dependent on ER stress.


| INTRODUC TI ON
Silicosis is a traditional occupational disease with unfavourable prognosis, and it is usually caused by prolonged inhalation of free silica dust (SiO 2 ). The characteristics of silicosis include extensive silicon nodule formation, chronic inflammation, aberrant fibroblast activation and excessive extracellular matrix (ECM) deposition, which eventually lead to abnormal lung tissue repair and irreversible pulmonary failure. 1,2 Recent epidemiological work has shown that silicosis is still one of the severest occupational diseases around the world, especially in low-and middle-income countries. 3 China has the most patients with silicosis, with more than  4 Despite the multiple efforts made in recent years, treatments for silicosis are still not satisfactory. 2,3 Therefore, we need to further expand the understanding of silicosis at the cellular and molecular levels and improve therapies for this disease.
Alveolar macrophages and fibroblasts play crucial roles in the process of silicosis. Alveolar macrophages initially recognize and capture inhaled silica particles, and then, the process of inflammasome activation proceeds. 3,5,6 Various proinflammatory and profibrotic factors are released and activated, such as transforming growth factorβ (TGFβ), interleukin (IL)-6, IL-1β and tumour necrosis factorα (TNFα). 2,7-10 Then, these cytokines trigger the differentiation of fibroblasts into activated myofibroblasts, promote fibroblast recruitment and proliferation, and result in excessive accumulation of collagen fibres, eventually leading to pulmonary fibrosis. 2 To date, the crosstalk between macrophages and fibroblasts and how these cells communicate with each other to deal with stress from SiO 2 exposure in the pathogenesis of silicosis have not been fully elucidated. In addition, non-specific anti-inflammatory or antifibrotic therapy has not achieved a good therapeutic effect in clinical settings. 1,2 Exosomes are membrane-like vesicles with a diameter of 30-150 nm, and they are considered mediators of both local and distant intercellular communication. Exosomes contribute to biological processes by transporting a variety of cell-and cell-state-specific cargo, including nucleic acids, lipids and proteins. 11 Mounting evidence has demonstrated that exosomes widely participate in tumour progression, immune responses, heart failure, diabetes, etc [12][13][14] In addition, recent studies have revealed the potential contribution of exosomes to the pathogenesis of chronic lung diseases. 15,16 For example, the secretion level of exosomes into bronchoalveolar lavage fluid (BALF) is increased in human and experimental lung fibrosis, and exosomes found in increased amounts function as carriers of protein/miRNA that contribute to idiopathic pulmonary fibrosis (IPF) pathogenesis. 17,18 However, the secretion and potential contribution of alveolar macrophage-derived exosomes in silicosis remain largely unexplored. Cellular dysfunctions caused by disruption of endoplasmic reticulum (ER) homeostasis contribute to the development of fibrotic diseases. [19][20][21] The ER is a special organelle where proteins are folded, processed and quality-controlled to maintain proteostasis.
ER dysfunction can give rise to the accumulation of unfolded or misfolded proteins, resulting in homeostasis imbalance, a condition termed ER stress, that triggers unfolded protein response (UPR, a signalling cascade activated in response to ER stress) hyperactivation. 22 The UPR is mainly composed of three signalling pathways: (1) inositol-requiring enzyme-1α (IRE1α)-XBP1s, (2) protein kinase RNA (PKR)-like ER kinase (PERK)-P-eIF2α and (3) activating transcription factor 6 (ATF6). 23 The aim of UPR activation is to restore proteostasis; however, prolonged or overloaded ER stress may result in cell death. 24 Mounting evidence indicates that ER stress is involved in pulmonary fibrosis by regulating myofibroblast transdifferentiation, alveolar epithelial cell apoptosis, M2 macrophage polarization and epithelial-mesenchymal transition (EMT). 22 Inhibition of ER stress can effectively suppress TGF-β1-induced fibroblast activation and tissue fibrosis. 25 Additionally, a recent study showed that HeLa cell-derived exosomes disrupt vascular integrity by activating ER stress in vascular endothelial cells. 26 Consequently, we hypothesized that exosomes derived from SiO 2exposed macrophages (SiO 2 -Exos) may promote myofibroblast activation through ER stress.
In the present study, we revealed that SiO 2 -Exos could promote myofibroblast differentiation, proliferation and migration, and that inhibition of ER stress could reverse the fibrotic phenotype of activated myofibroblasts. Inhibition of exosome generation dampened SiO 2 -induced lung fibrosis and the inflammatory response in mice.  16 hours) for sterilization and inactivation of any endotoxin contamination. In this study, the SiO 2 dosage was 200 μg/mL. The conditioned medium of macrophages was harvested after treatment with SiO 2 for 48 hours. For an ER stress inhibition assay, fibroblasts were pre-treated with 4-phenylbutyric acid (4-PBA) (500 μmol/L, Sigma-Aldrich, Merck KGaA,) for 4 hours in growth medium without FBS. After washing with sterile phosphate-buffered saline (PBS) 3 times, these fibroblasts were treated with SiO 2 -Exos +4-PBA (500 μmol/L) for different time points in growth medium supplemented with 10% FBS.

| Exosome isolation, identification and treatment
Exosomes were isolated from conditioned medium of RAW264.7 or THP-1 cells by differential centrifugation (Beckman Coulter).

| Trafficking analysis of exosomes
To dynamically trace exosomes, the exosomes were labelled with a PKH26 fluorescent kit (Sigma-Aldrich, Merck KGaA). First, according to the manufacturer's directions, the purified exosomes were incubated with PKH26 dye. Next, the mixture was diluted in ice-cold PBS and then centrifuged at 110 000 × g at 4°C for 90 minutes to collect the PKH26-labelled exosome pellets, which were resuspended in 100 μL of sterile PBS and then added to coculture with HFL1 cells in complete growth medium for 24 hours. After washing 3 times with PBS, an inverted fluorescence microscope (Olympus) was used to detect the appearance of red fluorescence in HFL1 cells.

| Western blot analysis
Cell lysates and exosomal lysates were subjected to SDS-PAGE.
Western blot analyses were performed by using anti-collagen I

| Quantitative RT-PCR analysis of mRNA
Following the manufacturer's protocols, Trizol reagent (Thermo Scientific) was used to extract total RNA, and then, a NanoDrop 2000 spectrophotometer (Thermo Scientific) was used to evaluate RNA quality and quantity. Total RNA was reverse transcribed using a cDNA synthesis kit (Genecopoeia), and then, an ABI-7500 instrument (Applied Biosystems; Thermo Fisher Scientific Inc

| Proliferation and a wound closure assay
Following the manufacturer's instructions, cell proliferation was detected by Cell Counting Kit-8 (CCK-8 Kit; Dojindo Molecular Technologies). Cell migration was assessed by a wound closure assay in a 2D culture system. 1 × 10 5 HFL1 or NIH3T3 cells were inoculated into 24-well tissue culture plates. When the cells were approximately 70%-80% confluent, a cross-shaped scratch was carefully drawn in each well using a sterile 200-μL pipette tip. Each well was then washed 3 times to remove all the detached cells. Digital images were captured at different time points (0, 12 or 24 hours), and ImageJ software (National Institutes of Health) was used to quantitatively evaluate the gap width. (2.5 μg/g of bodyweight per day) was dissolved in PBS and preadministered intraperitoneally to each animal for 7 days before silica exposure. GW4869 was continuously administered to the mice until they were killed at 35 days for further study.

| Haematoxylin and eosin, Masson's trichrome and immunohistochemical staining
Lungs were fixed overnight with 4% paraformaldehyde at a constant pressure to ensure that the lungs were totally submerged, and then, the lungs were dehydrated and embedded in paraffin. The sections were deparaffinized and stained with Masson's trichrome or haematoxylin and eosin (H&E) (Sigma-Aldrich, Merck KGaA).
Immunohistochemical staining was performed with an antiα-SMA antibody (1:200; 14395-1-AP; Proteintech) as described previously. 28 Digital images of the area of positive staining were captured by using a light microscope attached to an image-analysis system (K-Viewer, KFBIO).

| Statistics
GraphPad Prism 5 (GraphPad Software) was used for statistical analysis. Numerical data were compared using Student's t test (unpaired, two-tailed) or two-way ANOVA, and the statistical significance was set at P < .05.  Figure 1D), which showed that the sizes of exosomes were mostly between 30 and 200 nm (peak: l44.5 nm). In addition, we found that the total protein content of SiO 2 -Exos increased significantly compared with NC-Exos ( Figure 1E, exosome total μg of protein/mL: NC 129.8 ± 13.63, SiO 2 253.7 ± 17.92, P = .0006).

| SiO 2 -Exos promote myofibroblast differentiation in vitro
Previous studies have shown that the activation of macrophage induced by SiO 2 initiates pulmonary fibrosis in silicosis, followed by myofibroblast activation and aberrant collagen deposition. 2 32 Additionally, recent studies have indicated that exosomes play a crucial role in fibrotic diseases as carriers of intercellular communication. 15,16 The number of exosomes in the BALF of IPF patients is increased, and these exosomes promote lung fibroblast proliferation. 17 Given that high secretion of exosomes is related to SiO 2 -induced pulmonary fibrosis, we explored whether exosomes derived from macrophages mediate myofibroblast activation. First, we investigated whether macrophage-secreted exosomes can be ingested by fibroblasts. SiO 2 -Exos were labelled with the red fluorescent dye PKH26 (Figure 2A), and then, these exosomes were incubated with HFL1 cells for 24 hours. The red fluorescence in HFL1 cells showed that these exosomes had been effectively phagocytized by the cells (Figure 2A). These results indicate that exosomes derived from macrophages can be ingested by fibroblasts.
Next, we evaluated whether exosomes derived from macrophages can induce myofibroblast differentiation. HFL1 cells and NIH-3T3 cells were treated with PBS, NC-Exos, SiO 2 -Exos or exosomes from macrophages treated with SiO 2 +GW4869 (SiO 2 +GW4869-Exos). Notably, treatment with SiO 2 -Exos resulted in significantly increased expression of collagen I and α-SMA in HFL1 cells ( Figure 2B) and NIH-3T3 cells ( Figure 2C), and these effects were attenuated by pre-treatment of macrophages with GW4869 ( Figure 2B,C), which inhibits exosome generation. 27 Consistent with these observations, the mRNA expression levels of collagen I and α-SMA were also increased in HFL1 cells ( Figure 2D,E) and NIH-3T3 cells ( Figure 2F,G) treated with SiO 2 -Exos, and these effects could be reversed by GW4869 pre-treatment.

| SiO 2 -Exos promote myofibroblast proliferation and migration
Activated myofibroblasts proliferate and migrate to form fibrotic foci and produce excessive ECM components, which are characteristics of fibrotic diseases. 33

| Inhibition of exosome secretion blocks SiO 2induced proinflammatory cytokine production and pulmonary fibrosis in mice
Next, we investigated whether exosomes contributed to the progression of fibrosis in a silicosis model. To determine the exosome-mediated promotive effects on pulmonary fibrosis in silicosis, GW4869 was used to block the generation of exosomes.
We constructed a silicosis pulmonary fibrosis model given various treatments ( Figure 4A). Either PBS or GW4869 (2.5 μg/g per day) was used to pre-treat mice for 7 days prior to silica suspension exposure; the silica suspension (100 mg/kg) was then administered by intrabronchial injection. GW4869 was continuously administered to the mice until they were killed on day 35 ( group compared with the SiO 2 group ( Figure 4D). Furthermore, the results of an analysis of proinflammatory cytokines in the BALF revealed significant increases in the levels of TNFα ( Figure 4E), IL-1β ( Figure 4F) and IL-6 ( Figure 4G) in the SiO 2 group compared with the control group. Pre-treatment with GW4869 resulted in a significant decrease in the production of TNFα, IL-1β and IL-6 in BALF induced by SiO 2 (Figure ). In addition, we observed that the average bodyweight was decreased in the SiO 2 group compared with the control group and increased in the SiO 2 +GW4869 group compared with the SiO 2 group ( Figure 4H).

| SiO 2 -Exos regulate myofibroblast activation through ER stress
ER stress and the UPR are known to contribute to the development of pulmonary fibrosis by regulating myofibroblast differentiation. [20][21][22]36 Additionally, recent studies have shown that exosomes modulate apoptosis through ER stress. [37][38][39] Consequently, we hypothesized that SiO 2 -Exos may regulate myofibroblast activation through ER stress. First, to investigate whether ER stress is involved in regulating myofibroblast differentiation induced by SiO 2 -Exos, we examined the expression levels of several protein markers related to these processes. SiO 2 -Exos (± 4-PBA) were used to treat fibroblasts, and the expression levels of UPR-related genes (XBP1s and P-eIF2α), ER stress marker (BIP) and myofibroblast differentiation markers (collagen I and α-SMA) were measured. These results showed that after treatment with SiO 2 -Exos, the expression levels of BIP, XBP1s and P-eIF2α were up-regulated over time ( Figure 5A,B). Similarly, the expression of collagen I and α-SMA in HFL1 cells ( Figure 5A) and NIH-3T3 cells ( Figure 5B) was also increased over time. When SiO 2 -Exos and the

| Inhibition of ER stress attenuates fibroblast proliferation and migration induced by SiO 2 -Exos
We next examined whether ER stress is involved in fibroblast

| D ISCUSS I ON
Silicosis is a lethal pneumoconiosis and used to be a disease of miners. However, because of poor surveillance and a lack of effective protection in contemporary industrial manufacturing, such as the sand blasting process, silicosis is re-emerging around the world. 2 To date, no proven curative treatment for silicosis exists, and treatment options are limited. Hence, it is necessary to further explore the pathogenesis of silicosis and improve the effects of therapies.
After silica dust exposure, alveolar macrophages are the first line of defence and initially activate the inflammatory response by producing various kinds of chemokines, cytotoxic oxidants, proteases and cytokines that stimulate fibroblasts to produce excessive ECM, eventually resulting in lung remodelling and fibrogenesis. 40,41 However, the cell-to-cell communication between macrophages and fibroblasts has not been fully elucidated. Exosomes are secreted membranous nanoparticles and mediators that facilitate EMT in pulmonary fibrosis. 25,49 Consequently, we hypothesized that ER stress may contribute to SiO 2 -Exo-induced myofibroblast activation. Our study revealed that fibroblast activation induced by SiO 2 -Exos was dependent on ER stress activation. However, we have not yet determined which of the molecular and cellular cargo components in exosomes contribute to the fibrotic pathomechanism in silicosis. Moreover, the mechanism of ER stress activation induced by SiO 2 -Exos will also be the focus of our further research.
In this study, we demonstrate that SiO 2 -Exos are profibrogenic and contribute to pulmonary fibrosis and inflammation during silicosis. Inhibition of exosome generation can dampen pulmonary inflammation and attenuate pulmonary fibrosis. In addition, recent studies have shown that exosomes have great potential in the treatment of pulmonary fibrosis. [50][51][52] Therefore, macrophage-derived exosome may be a therapeutic target for silicosis.

ACK N OWLED G EM ENTS
We appreciate Dr Junpu Wang (Xiangya Hospital, Central South University, Changsha, China) for performing transmission electron microscopy analysis. This work was supported by grants from the National Natural Science Foundation of China (No: 81673120).

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

AUTH O R CO NTR I B UTI O N S
Xiaofeng Qin: Investigation (equal); Writing-original draft (equal).

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are included within the article.