Neutrophil Extracellular Traps Regulate the Pathogenesis of Pulmonary Fibrosis by Inducing Epithelial-Mesenchymal Transition

doi:10.21203/rs.3.rs-5279411/v1

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Research Article

Neutrophil Extracellular Traps Regulate the Pathogenesis of Pulmonary Fibrosis by Inducing Epithelial-Mesenchymal Transition

https://doi.org/10.21203/rs.3.rs-5279411/v1

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Background

Neutrophil extracellular traps released after neutrophil activation are associated with various diseases and redefine the biological functions of neutrophils. Studies have reported a significant increase in the number of neutrophils in the bronchoalveolar lavage fluid and lung tissue of patients with idiopathic pulmonary disease. However,neutrophil-mediated pathogenic mechanisms of idiopathic pulmonary disease remain unclear.

Methods

The biological functions of neutrophil extracellular traps were evaluated using proliferation,wound healing and transwell assays.The expression of different fibrosis factors was detected using western blot and immunohistochemistry staining in vivo and in vitro.

Results

Neutrophil extracellular traps promote the proliferation and migration of A549 and BEAS-2B cells by inducing epithelial-mesenchymal transition. Based on our current transcriptome RNA sequencing analysis, ELANE (encoding the neutrophil elastase gene) was a major differentially expressed gene, and the Wnt signaling pathway was the major pathway as demonstrated through Kyoto Encyclopedia of Genes and Genomes enrichment analysis. Neutrophil extracellular traps, through their protease neutrophil elastase interacting with β-catenin, trigger changes in the expression of markers of epithelial-mesenchymal transition, including E-cadherin and vimentin. Additionally, Sivelestat·Na disrupts the stability of neutrophil extracellular traps structures by inhibiting the activity of neutrophil elastase, thereby suppressing neutrophil extracellular traps-induced epithelial-mesenchymal transition, and alleviating acute lung injury and pulmonary fibrosis induced by bleomycin in mice.

Conclusions

Our results suggest that the neutrophil extracellular traps/Wnt axis promotes the progression of epithelial-mesenchymal transition and the progression of pulmonary fibrosis,recommending it a target for new therapeutic strategies for early-stage pulmonary fibrosis.

Pulmonary fibrosis

Neutrophil Extracellular Traps

Neutrophils

Sivelestat·Na

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease of unknown cause associated with complex biological processes.According to epidemiological data, there is a wide variation in the prevalence and incidence of IPF, primarily influenced by geographic factors (such as environmental exposure and healthcare resources).Race, age, sex, and genetic factors also play specific roles.Globally, the incidence of IPF ranges from 0.09 to 1.30 cases per 10,000 people, and the prevalence ranges from 0.33 to 4.51 cases per 10,000 people [1]. Researchers have found that the survival rate of patients with IPF is worse than that of patients with other types of cancers. It has a very high mortality rate, with a median survival time of only 3–5 years after diagnosis [2]. The poor prognosis of IPF indicates that researchers need a deeper understanding of its pathogenesis to develop treatment strategies to improve patient outcomes.

Neutrophils are the first line of defense against pathogen invasion. When recruited to an infection site, neutrophils recognize pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) and activate specific pattern-recognition receptors on their surfaces. PAMPs mainly originate from microorganisms, whereas DAMPs are endogenous factors released during tissue damage. Neutrophils enhance the immune response primarily through three mechanisms: phagocytosis, degranulation, and release of neutrophil extracellular traps (NETs) [3, 4]. After activation, neutrophils produce and release NETs during a programmed cell death, known as NET formation (NETosis). This form of death is distinct from necrosis and apoptosis and is associated with the pathogenesis of several diseases.NETosis is a comprehensive term associated with cellular and biological processes closely related to the occurrence and progression of various diseases. NETs are double-edged swords that can protect organisms from pathogen invasion through high levels of antimicrobial activity and phagocytosis and play a role in degrading virulence factors by forming fibrous net-like structures [5]. However, excessive production can damage organisms, and research has shown the occurrence and progression of various diseases such as severe infections, tumors, autoimmune diseases, connective tissue diseases, thrombotic diseases, arteriosclerosis, metabolic diseases, and coronavirus disease 2019 (COVID-19),are related to NETs [6–10].

Neutrophil elastase (NE) is a serine protease characterized by a serine-containing active tripeptide catalytic site and is the most abundant of serine protease in neutrophils. The other three serine proteases are proteinase 3, cathepsin G, and neutrophil serine protease 4 [11]. NE has a molecular weight of 29.5 kDa and is primarily located in the azurophilic granules of neutrophils under the influence of serglycin. NE triggers the release of NETs into the cytoplasm through various signals and releases them from azurophilic granules containing myeloperoxidase, after which they bind to and degrade F-actin [12]. NE translocates to the nucleus via passive diffusion and promotes chromatin decondensation by cleaving histone H4. Activated NE is a key factor in NET formation. Although research has shown that NE can be released from neutrophils independently of NETs, it is quickly deactivated by plasma antiproteases. However, NE associated with NETs appears to maintain its proteolytic activity for extended periods [13]. A literature review indicated that NE levels and activity reflect the status and severity of the disease, and that inhibiting NE may delay disease progression [14, 15].

The early stages of IPF are typically associated with chronic inflammation [16].The significant variation in the natural history and clinical course of patients with IPF suggest that multiple inflammatory pathways possibly trigger the disease. During the pathogenesis of IPF, the excessive release of cytokines, chemokines, and inflammatory mediators forms a complex network that promotes the recruitment of fibroblasts and the production of the extracellular matrix (ECM). The progressive fibrotic response in patients with IPF may be related to the persistent microinjury of epithelial cells and the poor effectiveness of anti-inflammatory treatments. The dysregulation of adaptive immune mechanisms and subsequent inflammatory responses may be essential factors in IPF progression [17]. Therefore, at least two distinct pathways (the inflammatory and the epithelial pathways) can lead to IPF [18, 19].A literature review revealed that compared with that of healthy control groups, the number of neutrophils in the bronchoalveolar lavage fluid (BALF) and lung tissues of patients with IPF significantly higher [20, 21]. However, whether neutrophil-mediated inflammatory responses are a driving factor in the progression of IPF or are merely a secondary phenomenon remains unclear.

Furthermore, researchers have shown that in a mouse model of lipopolysaccharide (LPS)-induced acute lung injury (ALI), NETs and their associated components, such as histones, can induce apoptosis in epithelial and endothelial cells [22]. Histones injection in mice leads to neutrophil margination, endothelial cell vacuolization, and intravascular thrombosis, which may be associated with IPF [23]. In addition to participating in innate immune responses, we speculate that NETs promote the pathogenesis and progression of IPF and may become a new target for IPF treatment.

Bleomycin (BLM)-induced lung injury is the most used experimental pulmonary fibrosis (PF) model and can simulate the pathogenesis of IPF. Studies have shown that signs of fibrosis were found 7 days after BLM administration, and a significant increase in collagen fiber deposition was observed on the 8th day, continuing until the 14th day [24, 25]. Therefore, we used a 14-day BLM administration model as an early PF model. First, we investigated the biological effects of NETs and their impact on PF mice in vivo and in vitro. NETs were cocultured with A549 cells, and RNA was extracted for transcriptome sequencing. Differentially expressed genes (DEGs) were identified, and the Wnt/β-catenin signaling pathway was the main pathway according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. By investigating the role of the Wnt/β-catenin signaling pathway in NET-induced epithelial-mesenchymal transition (EMT), this study explored the molecular mechanism by which NETs promote the pathogenesis of PF. Identifying DEGs revealed that ELANE (the gene encoding NE) was one of the major DEGs initiating EMT. We hypothesize that NE may act as a key protease for NETs to activate the Wnt/β-catenin signaling pathway.After intervention with the NE inhibitor sivelestat sodium salt hydrate (Sivelestat·Na), it was further verified that NE participates in the pathogenesis of PF by activating the Wnt/β-catenin signaling pathway both in vitro and in vivo.

Cell culture

The human leukemia 60 (HL-60) cell line used in this study was purchased from Shanghai Yihe Biotechnology.The A549 cell line (although A549 cells are a lung adenocarcinoma cell line, this cell line possesses the characteristics of type II alveolar epithelial cells and is widely used in studies of PF [26]) and human bronchial epithelioid cell (BEAS-2B) cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences in Shanghai. All cell lines were grown in RPMI-1640 medium (KeyGEN, China) supplemented with 10% fetal bovine serum (FBS, BI, Israel) and 1% penicillin and streptomycin (Beyotime, China).

Wright-Giemsa staining to detect the degree of differentiation of HL-60 cells

HL-60 cells in the logarithmic growth phase were removed and added to complete medium containing all-trans-retinoic acid (ATRA, Solarbio, China) at a final concentration of 1 µmol/L. The medium, which was freshly prepared with the same concentration of ATRA, was changed daily for 5 days until the target differentiation cycle was reached. The degree of differentiation of differentiated HL-60 (D-HL-60) cells was assessed using Wright-Giemsa staining.

Induction and purification of NETs

Five hundred microliters of the D-HL-60 cell suspension containing 5×10⁵ cells/mL was added to sterile 24-well plates. The cells were stimulated with phorbol 12-myristate 13-acetate (PMA; MedChemExpress, America; final concentrations of 0, 50, 100, 150, or 200 nmol/L) and then incubated at 37°C for 0, 1, 2, 3, or 4 hours. After stimulation, we carefully removed the supernatant, discarded as much of the PMA-containing medium as possible,and then added complete medium containing 20 gel units/mL micrococcal nuclease (MNase, Beyotime, China) at 37° C for 20 minutes. After the supernatant was collected and centrifuged, the concentration of cell-free DNA was determined using a Picogreen dsDNA assay kit.

Immunofluorescence staining

The cells were inoculated into a 24-well plate, and adherence was observed under a microscope. The cells were fixed with prechilled methanol for 20 minutes, followed by incubation with immunofluorescence blocking solution at 37 ℃ for 2 hours. Subsequently, the cells were incubated with antibodies against NE (1:50, Proteintech, China) and β-catenin (1:200, Proteintech, China) overnight at 4°C,then incubated with Alexa Fluor 594 or Alexa Fluor 488 (1:200, Abmart, China) for 1 hour at 37 ℃. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). Imaging was performed using laser-scanning confocal fluorescence microscope (Nikon,Tokyo,Japan) or an inverted fluorescence microscope (Nikon,Tokyo,Japan).

Cell proliferation assay

A549 and BEAS-2B cells were stimulated with freshly prepared NETs and divided into a control and NET treatment groups. Cell proliferation rates were assessed using Cell Counting Kit-8 (CCK-8) and plate colony formation assays.

Cell migration assay

A549 and BEAS-2B cells were cocultured with NETs for 48 hours and seeded onto transwell plates to observe cell migration. In the lower chamber of the 24-well plate, 650 µL of culture medium containing 10% FBS was added. In the upper chamber, 150 µL of cell suspension without FBS was added. The plates were incubated at 37° C for 48 hours. After fixation with 4% paraformaldehyde for 20 minutes, the cells were stained with 0.1% crystal violet for 15 minutes, and photographs were taken using a smart biological image navigator. For the wound healing assay, FBS-free culture medium and supernatants containing different concentrations of NETs were added to A549 and BEAS-2B cells, respectively. Images were captured at 0, 24, 48, and 72 hours using an inverted fluorescence microscope, and the migration distance of the cells in each group was calculated.

Western Blotting Analysis

The separating and stacking gels for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were prepared according to the molecular weight of the antibodies.The proteins were subjected to SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane. After blocking at 37 ℃ for 2 hours, the PVDF membrane was incubated overnight at 4°C on a shaker with antibodies against β-actin (1:20000, Proteintech, China), β-catenin (1:5000, Proteintech, China), E-cadherin (1:20000, Proteintech, China), N-cadherin (1:5000, Proteintech, China), vimentin (1:2000, Proteintech, China), Snail1 (1:1000, Proteintech, China), and NE (1:1000, Proteintech, China). The next day, the PVDF membrane was incubated with horseradish peroxidase-conjugated secondary antibodies specific for rabbit or mouse primary antibodies (1:5000, Proteintech, China) at 37 ℃ for 1 hour. Membranes were then visualized using enhanced chemiluminescence detection reagents on a gel imaging analysis system.

Transient Transfection

A549 and BEAS-2B cells were seeded on 6-well plates at a density of approximately 50–60%. The cells were transfected with small interfering RNA (siRNA) (RiboBio, China) using Lipofectamine™ 2000 Transfection Reagent (Invitrogen, USA) according to the manufacturer's instructions.Proteins were extracted for western blot analysis 48 hours after cell transfection.

Co-immunoprecipitation (Co-IP)

Proteins were collected from the IP cell lysates. Equal amounts of anti-IgG and anti-β-catenin antibodies were added to protein G/A magnetic beads in tris-buffer saline, which were placed on a rotary mixer and mixed for 1 hour. Protein G/A magnetic beads were collected, and an equal volume of protein solution was added. The mixture was placed on a rotating mixer at 4°C overnight. The next day, the magnetic beads were collected, and an appropriate amount of radio immunoprecipitation assay lysis buffer was added. The cells were lysed on ice for 15 minutes and boiled in a 100°C metal bath for 10 minutes to denature the proteins, followed by SDS-PAGE.

BLM Mouse Modeling and Sampling

Animal experiments were approved by the Ethics Committee of the Second Affiliated Hospital of Harbin Medical University. Male C57BL/6J mice aged 8–10 weeks (weight, 26–30 g) were housed under specific pathogen-free conditions following the institutional regulations. Mice were anesthetized using 2,2,2-tribromoethanol (200 mg/kg, Sigma, Germany) by intraperitoneal injection illuminated by a halogen fiber optic cold light source, revealing the vocal cords that open and close with breathing.A24G catheter needle along the vocal cords into the trachea; the needle core was removed. A solution of BLM was adminstered (3.5 mg/kg, MedChemExpress, USA) into the trachea, while the control group received an equivalent volume of phosphate buffered saline (PBS) solution. Two hours after BLM administration, the mice were immediately given a solution of Sivelestat·Na (30 mg/kg, Sigma, Germany) by intraperitoneal injection. The control group was given an equivalent volume of saline. Mice were administered Sivelestat·Na or saline at the same time every day, and their condition was observed for signs of illness, such as respiratory rate, oral cyanosis, weight loss, and abnormal behavior. The mice were euthanized on day 14th, and BALF, serum, and lung tissues were collected.

Enzyme-linked immunosorbent assay (ELISA)

Krebs von den Lungen-6 (KL-6), surfactant-associated protein-D (SP-D), and NE levels in mouse BALF and serum were measured using KL-6, SP-D, and NE ELISA Kits, respectively.

Immunohistochemistry (IHC)

Lung tissues fixed in 4% paraformaldehyde were embedded in paraffin and subjected to hematoxylin and eosin (H&E), Masson’s trichrome, and IHC staining as described previously [27]. The structure of the lung tissue was meticulously assessed according to the modified Ashcroft scoring criteria [28], and the PF index was calculated.

Transcriptomic RNA sequencing analysis

A549 cells were seeded into 6-well plates and divided into control (Ctrl) and NETs groups. Forty-eight hours later, the cells were washed three times with sterile PBS and TRIzol lysis solution was added to collect the samples. The samples were placed on dry ice at Sangon Biotech Co. Ltd.(Shanghai, China) for transcriptomic RNA sequencing. DEGs were identified using the limma package in R software (version 4.3.1) based on the sequenced gene matrix with the following criteria: |log2FoldChange|>1 and false discovery rate (FDR) < 0.05. Heatmaps were generated using the pheatmap package, and volcano plots were generated using the dplyr, ggplot2, and ggrepel packages. KEGG enrichment analyses were performed for the DEGs using the colorspace, stringi, ggplot2, digest, GOplot, org.Hs.eg.db, DOSE, clusterProfiler, and enrichplot packages.

Statistical analysis

The experimental data are presented as the mean ± standard deviation (mean ± SD). Statistical analysis for comparisons among multiple groups was conducted using one-way analysis of variance (ANOVA) or two-way ANOVA, with Bonferroni correction for repeated measurements. Ashcroft scores were analyzed using the Kruskal-Wallis rank sum test. P < 0.05 was considered to indicate statistical significance (ns, not significant; P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). After quantification using ImageJ, statistical analysis and graphing were performed using GraphPad Prism 9.5 software. Bioinformatics analysis was carried out using R software (version 4.3.1), with P < 0.05 considered to indicate statistical significance.

Detection and identification of NETs

After 5 days of induction with a final concentration of 1 µmol/L ATRA, Wright-Giemsa staining revealed that the cytoplasm of D-HL-60 cells was light pink, with vacuolar-like changes visible in the cytoplasm and many fine granules distributed throughout. The nuclei appeared lobulated or horseshoe-shaped, indicating successful induction of differentiation into neutrophil-like cells (Fig. 1A). To assess the ability of D-HL-60 cells to release NETs, we stimulated D-HL-60 cells with various concentrations of PMA.We used a Picogreen dsDNA assay kit to measure the concentration of cell-free DNA in the supernatant at 0, 1, 2, 3, and 4 hours, thereby performing a quantitative analysis of NETs. The results showed that under PMA stimulation, the concentration of cell-free DNA gradually increased over time in a dose-dependent manner. The optimal stimulation concentration and time point were 200 nmol/L and 4 hours, respectively (Fig. 1B). Immunofluorescence staining was performed on PMA-stimulated D-HL-60 cells to observe the expression of NE and the net-like structure of NETs. Confocal microscopy revealed that, compared to the Ctrl group, a significant number of net-like structures were released extracellularly in the PMA group, as the expression of NE colocalized with the expression of cell-free DNA. This phenomenon was positively correlated with the concentration of PMA (Fig. 1C). These results suggest that D-HL-60 cells serve as a neutrophil-like model for studying NETs and related diseases.

To further evaluate the formation of NETs, D-HL-60 cells were pretreated with the specific NE inhibitor Sivelestat·Na for 30 minutes before stimulation with PMA, while deoxyribonuclease I (DNase I) was used as a positive control. Using the Picogreen dsDNA assay kit, it was found that the concentration of cell-free DNA in both the PMA + Sivelestat·Na group and the PMA + DNase I group was significantly lower than that in the PMA group (Fig. 1D). Similarly, immunofluorescence revealed a reduction in the generation of extracellular net-like structures (Fig. 1E). These results indicate that Sivelestat·Na may disrupt the stability of NETs by inhibiting the activity of NE, as demonstrated by in vitro experiments showing that Sivelestat·Na can reduce the release of NETs.

Biological functions of NETs

To observe the effects of NETs on alveolar epithelial type II (AT-II) cells, we cocultured NETs with A549 and BEAS-2B cells. Using the CCK-8 assay to measure cell proliferation, the results showed that at 24, 48, and 72 hours, the optical density at 450 nm (OD450) was significantly greater in the NETs group than in the Ctrl group, indicating a positive correlation with the concentration of NETs. However, at 96 hours, the OD450 of BEAS-2B cells in the 200 ng/mL NET group did not significantly differ from that of the Ctrl group (Fig. 2A). Subsequently, a plate colony formation assay confirmed that the proliferation of A549 and BEAS-2B cells stimulated with NETs was significantly accelerated and positively correlated with NET concentration (Fig. 2B-C).

To assess the effect of NETs on the migratory ability of AT-II cells, we conducted a wound healing assays on A549 and BEAS-2B cells after stimulation with NETs. Compared to the Ctrl group, the migration ability of the NETs group was significantly greater, and the ability was positively correlated with the concentration of NETs (Fig. 2D-E). AT-II cells cocultured with NETs were seeded into transwell chambers, and NETs promoted the migration of A549 and BEAS-2B cells in a concentration-dependent manner (Fig. 2F-G).

NETs promoted the progression of BLM-induced PF in mice

To investigate the effects of NETs on PF, we established a mouse model of PF by intratracheal instillation of BLM or PBS. Two hours later, a solution of Sivelestat·Na was injected intraperitoneally, while the Ctrl group received an equivalent volume of saline. Sivelestat·Na and saline were administered daily at fixed intervals. Figure 3A shows a schematic of the mouse model process. On the 14th day, the mice were euthanized, and lung tissues were collected. Figure 3B shows that the lung tissue structure of mice in both the Ctrl group and the Sivelestat·Na group was full and had a healthy reddish color; in contrast, mice in the BLM group showed signs of lung tissue atrophy and a darker red color change. Mice in the BLM + Sivelestat·Na group also exhibited signs of PF but to a significantly lesser extent than that in the BLM group.

To measure the levels of NETs in the BALF and serum of mice from each group, we used a Picogreen dsDNA assay kit to measure the concentration of cell-free DNA. The results revealed that the concentrations of cell-free DNA in both the BALF and serum of mice in the BLM group were significantly greater than those in the Ctrl and the Sivelestat·Na groups. In contrast, the concentrations of cell-free DNA in the BALF and serum of mice in the BLM + Sivelestat·Na group were significantly lower than those in the BLM group (Fig. 3C). Immunofluorescence staining also showed the release of many NETs in the lung tissues of the BLM group mice, with colocalization observed between the expression of NE and cell-free DNA. In contrast, the NETs in the lung tissues of mice in the BLM + Sivelestat·Na group were significantly reduced compared with those in the BLM group (Fig. 3D). These findings confirmed that Sivelestat·Na can inhibit the release of NETs in vivo.

Based on the above results, we speculate that Sivelestat·Na may play an essential role in the early inflammatory stage of BLM-induced PF in mice. Subsequently, we used ELISA to measure the levels of fibrotic factors (MUC1/KL-6 and SP-D) in the BALF and serum of mice. Many biomarkers, including KL-6/MUC1 and SP-D, are associated with alveolar epithelial cell damage during IPF pathogenesis. KL-6/MUC1 is a glycoprotein expressed on the surface of alveolar and bronchial epithelial cells and is closely associated with the severity and prognosis of IPF [29]. SP-D is a lipoprotein complex produced and secreted by alveolar epithelial cells and plays a crucial role in maintaining lung tissue homeostasis. According to the literature, SP-D can serve as an independent predictive factor for the prognosis of patients with IPF [30]. This study revealed that the levels of KL-6 and SP-D in the BALF and serum of the BLM group were significantly higher than those in the Ctrl group. After treatment with Sivelestat·Na, the levels of the fibrosis factors in the BALF and serum of PF mice were significantly lower than those in BLM mice, indicating that Sivelestat·Na can effectively inhibit the release of fibrosis factors in mice with PF, thereby exerting anti-inflammatory and antifibrotic effects (Fig. 3E).

We found that Sivelestat·Na can inhibit the release of NETs by neutrophils both in vivo and in vitro. Subsequently, HE and Masson’s trichrome were performed to observe histopathological changes in the lung tissues of the mice. HE staining revealed that the lung tissue structure in both the Ctrl and Sivelestat·Na groups was largely intact. In the BLM group, the lung tissue structure was significantly damaged, with the alveolar spaces almost disappearing, septa thickening, extensive infiltration of inflammatory cells, and congestion of the lung blood vessels (Fig. 3F). Based on the modified Ashcroft scoring criteria for quantifying fibrosis in HE-stained lung tissue sections, the Ashcroft score of the BLM group was significantly greater than that of the Ctrl group and the Sivelestat·Na group (Fig. 3G). Masson staining revealed a large amount of collagen fiber deposition in the alveolar septa of mice in the BLM group, with a significant increase in ECM. After treatment with Sivelestat·Na, there were still minor fibrotic changes in the alveolar septa. However, the extent of these changes was significantly reduced compared with that in the BLM group (Fig. 3H). In summary, these results suggest that NETs promote the progression of BLM-induced PF in mice. Sivelestat·Na may alleviate BLM-induced ALI and PF in mice by inhibiting NE, thereby reducing the release of NETs.

NETs promoted EMT in AT-II cells by activating the Wnt signaling pathway

Transcriptome RNA sequencing of A549 cells treated with NETs was performed, and DEGs were identified using the limma package in R software. We identified 1061 upregulated and 968 downregulated genes (|log2FoldChange|>1, FDR<0.05). The results were visualized using volcano plots (Fig. 4A). KEGG analysis revealed the enrichment of pathways related to the cell cycle, DNA replication, and Wnt signaling pathway (Fig. 4B). This suggests that A549 cells stimulated by NETs may undergo phenotypic transformation associated with the activation of the Wnt signaling pathway.

The induction of the canonical Wnt/β-catenin signaling pathway centers on the accumulation of β-catenin in the cytoplasm, followed by its translocation into the nucleus, where it is activated. β-catenin forms a transcription factor complex by binding to TCF/LEF-legless-PYGO DNA. To explore the mechanisms underlying phenotypic transformation in AT-II cells, we cocultured NETs with A549 and BEAS-2B cells. Seventy-two hours later, we observed the expression of β-catenin using immunofluorescence staining. In the Ctrl group, β-catenin was mainly expressed in the cytoplasm and on the cell membrane. In contrast, in the NETs group, β-catenin was expressed in the nucleus (Fig. 4C). This indicates that in A549 and BEAS-2B cells, NETs can trigger the activation of the Wnt/β-catenin signaling pathway, leading to the nuclear translocation of β-catenin. Previous studies have shown that NETs promote the proliferation and migration of AT-II cells. The expression of EMT markers was assessed through western blot analysis and compared with that in the Ctrl group; the expression level of the epithelial marker (E-cadherin) significantly decreased in the NETs group, while the expression level of the mesenchymal marker (vimentin) increased, which was related to the concentration of NETs (Fig. 4D). These results suggest that NETs trigger the initiation of downstream EMT after activating the Wnt/β-catenin signaling pathway.

NETs promoted EMT through interactions between NE and β-catenin

To further validate the role of the Wnt/β-catenin signaling pathway in NET-induced EMT, three CTNNB1 siRNAs were transiently transfected into A549 and BEAS-2B cells. The efficiency of CTNNB1 silencing was confirmed using western blotting (Fig. 5A). Based on the analysis of the proteins, the siRNA with the highest silencing efficiency was selected for subsequent experiments. After transfecting A549 and BEAS-2B cells with CTNNB1 siRNA, the cells were cocultured with NETs, and the expression of EMT-related proteins was analyzed by western blotting. The results showed that in the NETs group, after transfection with CTNNB1 siRNA, the expression level of E-cadherin increased. In contrast, the expression level of vimentin decreased, indicating that silencing β-catenin inhibited NET-induced EMT (Fig. 5B).

Based on the transcriptome RNA sequencing data, the top 30 DEGs were identified and are displayed using a heatmap (Fig. 5C). These genes include ELANE (encoding the NE gene), indicating that NE plays a vital role in NETs intervention in A549 cells. Co-IP assays revealed an interaction between β-catenin and the key protease NE on NETs (Fig. 5D), suggesting that NE might be the hub protein responsible for activating the Wnt/β-catenin signaling pathway to induce EMT.

Sivelestat·Na can inhibit the Wnt pathway activated by NE to alleviate PF

To investigate the effects of NETs treated with Sivelestat·Na on the phenotype of AT-II cells, we divided the D-HL-60 cells into six groups: the Ctrl group, DNase I group, Sivelestat·Na group, PMA group, PMA + DNase I group, and PMA + Sivelestat·Na group. DNase I was used as the positive control. Supernatants from each group of cells were collected and cocultured with A549 and BEAS-2B cells for 72 hours. The expression levels of the EMT biomarkers E-cadherin and vimentin were assessed through western blot assays. Compared with those of the PMA group, the expression levels of E-cadherin were increased in both the PMA + Sivelestat·Na group and the PMA + DNaseI group, while the expression levels of vimentin were decreased (Fig. 6A). The results demonstrated that the NE inhibitor Sivelestat·Na can inhibit NET-induced EMT.

To demonstrate that NE is a key protease in the pathogenesis of PF induced by NETs, we conducted studies using Sivelestat·Na to treat BLM-induced mice. Initially, the activity of NE in the BALF and serum of mice from each group was assessed using ELISA. The results revealed that the NE activity in the BLM group was significantly greater than in the Ctrl and the Sivelestat·Na groups. After treatment with Sivelestat·Na, the activity of NE significantly decreased, indicating that it is an effective inhibitor of NE in vivo (Fig. 6B). To further clarify the impact of Sivelestat·Na on BLM-induced mice, the expression levels of fibrosis markers were assessed via western blotting. The results showed that the levels of the key EMT-driving transcription factor Snail1, fibrosis markers (β-catenin, N-cadherin, vimentin), and NE in the BLM group were significantly greater than those in the Ctrl and the Sivelestat·Na groups, while the levels of E-cadherin were lower (Fig. 6C). These results confirm that Sivelestat·Na improves PF in mice by inhibiting NE, which in turn blocks the Wnt/β-catenin signaling pathway. Furthermore, the IHC results validated the above findings, showing that the expression levels of β-catenin and NE were increased in the BLM group. After treatment with Sivelestat·Na, the expression of β-catenin and NE was significantly reduced (Fig. 6D). Therefore, we speculate that NE might be an essential target in NET-related PF and that intervening in the NE-Wnt/β-catenin axis could become an effective strategy for treating PF.

Our study elucidated that NETs facilitate the progression of PF by triggering EMT via the regulation of the Wnt/β-catenin signaling pathway by NE. Sivelestat·Na effectively alleviated BLM-induced ALI and PF in mice by inhibiting NE activity and destabilizing the structure of NETs (Fig. 7).

Our study initially established a neutrophil-like model by inducing the differentiation of HL-60 cells with ATRA for 5 days. Subsequently, the cells exhibited neutrophil-like characteristics. Neutrophils are crucial cells in innate immunity, but studying their functions is challenging because of their short half-life of only 6–10 hours after entering the bloodstream. HL-60 cells are human myeloid leukemia cells that can undergo granulocytic differentiation when stimulated with drugs such as ATRA or dimethyl sulfoxide (DMSO) [31]. When HL-60 cell maturation is inhibited, ATRA can induce and promote differentiation. ATRA-induced granulocytes differentiation mainly occurs through the peptidylarginine deiminase 4/SOX4/PU.1 signaling pathway [32]. Previous research has indicated that the phagocytic function of cells differentiated by various compounds shows distinct characteristics. For instance, compared with D-HL-60 cells treated with DMSO, D-HL-60 cells treated with ATRA exhibit greater maturity. This effect is primarily manifested due to stronger chemotaxis and actin polymerization in cells stimulated with interleukin (IL)-8 or PMA [33]. Manda-Handzlik reported that compared with DMSO-treated cells, D-HL-60 cells treated with ATRA exhibited greater nicotinamide adenine dinucleotide phosphate oxidase activity after PMA stimulation and greater phagocytic efficiency [31]. Overall, HL-60 cells differentiated through ATRA induction exhibited characteristics of neutrophils and can be widely employed in research related to neutrophils.

Numerous studies have indicated that NETs released by neutrophils trigger for many diseases, and the exploration of NETs constitutes a vital component of research into the biological functions of neutrophils. Therefore, to investigate the biological functions of NETs, we cocultured NETs with A549 and BEAS-2B cells. Compared to the control group, NETs promoted the proliferation of AT-II cells. Wound healing and transwell assays demonstrated a significant enhancement in the migratory ability of AT-II cells, suggesting that A549 and BEAS-2B cells may have lost their original epithelial cell polarity and acquired mesenchymal cell characteristics, initially displaying EMT features. These results suggest that NETs may alter the phenotypic functions of alveolar epithelial cells; however, further research is needed to confirm these findings.

BLM-induced PF in mice is a commonly used experimental animal model with pathological features similar to human IPF. BLM can induce epithelial cell death through mitochondrial activation and DNA damage [34]. HE staining revealed infiltration of many inflammatory cells in the BLM group. Masson’s staining revealed a significant number of fibrotic lesions in the model group, consistent with the pathological changes observed in IPF. These findings indicated that the BLM group exhibited significantly enhanced inflammatory and fibrotic responses, demonstrating successful model establishment. ELISA was used to detect the biomarkers KL-6 and SP-D in IPF. The results showed that the serum and BALF levels of KL-6 and SP-D in the BLM group were significantly higher than those in Ctrl group. Additionally, we found that the levels of cell-free DNA in the lung tissue, BALF, and serum of mice in the BLM group were significantly higher than those in the Ctrl group, suggesting that NETs may play a role in PF pathogenesis. After treatment with Sivelestat·Na, the levels of cell-free DNA in the mice lung tissue, BALF, and serum in the early 14-day model group were significantly lower than those in the BLM group. These findings demonstrated that Sivelestat·Na can inhibit the formation of NETs in vivo. These results indicated that NETs may facilitate the progression of BLM-induced ALI and PF in mice. By inhibiting NETs, Sivelestat·Na could serve as a therapeutic drug for PF.

In this study, A549 cells stimulated with NETs were subjected to transcriptomic RNA sequencing. KEGG enrichment analysis revealed that the Wnt/β-catenin signaling pathway might be necessary for A549 cells cocultured with NETs. Immunofluorescence staining revealed that in NET-stimulated A549 and BEAS-2B cells, β-catenin translocated from the cell membrane and cytoplasm to the nucleus, demonstrating the activation of the Wnt/β-catenin signaling pathway. Subsequently, the expression level of E-cadherin, a downstream factor of the Wnt/β-catenin signaling pathway, decreased. In contrast, the expression level of vimentin increased, as demonstrated by western blot analysis, indicating the initiation of the EMT process. The expression levels of EMT-related proteins induced by NETs are positively correlated with the concentration of NETs, indicating that this process can only be observed when the concentration of NETs is high enough to exceed the phagocytic capacity of epithelial cells. ECM remodeling is associated with both cancer and fibrosis, and various ECM components serve as signals for impaired immune system function. A literature review revealed that proteases attached to NETs can inhibit immune responses, stabilizing cancer cells and promoting metastasis [35]. Although the mechanisms by which NET-associated proteases participate in ECM remodeling of the ECM have not been fully elucidated, the relationships between ECM components, proteases, and host immune evasion underscore the existence of this effect. The occurrence of distant tumor metastasis and PF are closely linked to changes in the tissue-specific microenvironment, and the prerequisite for changes in different microenvironments primarily involves the remodeling of the ECM, with the most common form being the deposition of collagen. Recently, various studies have emphasized that NETs drive EMT. In the context of breast cancer, NETs can upregulate transcription factors (ZEB1 and Snail) involved in EMT, demonstrating that NETs drive the occurrence of EMT [36]. Another study on patients with COVID-19 found that during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the formation of NETs facilitated the activation of dormant cancer cells and favored the occurrence of metastasis [37]. These studies suggested that NETs could become a new target in EMT process, potentially addressing issues related to fibrosis or cancer metastasis.

Regarding the role of Wnt/β-catenin in EMT, we found that silencing CTNNB1 reversed NET-induced EMT. Heuberger et al. discovered that the C-terminal tail of E-cadherin can bind to β-catenin, which was the first demonstration of the important role of β-catenin in cell adhesion junctions [38]. E-cadherin also negatively regulates the Wnt signaling pathway by inhibiting β-catenin [39]. However, E-cadherin does not activate β-catenin transcription in the nucleus. Instead, it disrupts by disrupting other parts of the complex (such as adenomatous polyposis coli or AXIN), thereby activating β-catenin [40]. In the cytoplasm, the E-cadherin/β-catenin complex maintains a link between epithelial cells. During EMT, disruption of this system leads to barriers in the connections between epithelial cells. Transcription factors that mediate cell adhesion (such as connexin 43), transcription factors for E-cadherin (such as twist), and transcription factors involved in cell migration (such as fascin) play crucial roles in regulating the occurrence of EMT [41–43]. The Wnt signaling pathway can regulate the activity of transcription factors that are significant in the initial transdifferentiation process of EMT. For example, in Wnt signaling activation, silencing E-cadherin in colon cancer cells increases β-catenin-mediated transcription [44]. Furthermore, phosphorylation at the C-terminal end of β-catenin leads to its degradation, while phosphorylation at the N-terminal end changes its affinity for other proteins, inducing β-catenin nuclear translocation[45].

Phosphorylation of proteins within the cytoplasm and nucleus, mediated by various kinases, can disrupt the E-cadherin/β-catenin complex and activate β-catenin in diverse ways to promote EMT. In both the in vivo and in vitro model groups, we observed a decrease in the expression levels of E-cadherin, suggesting that NE may activate β-catenin by cleaving the E-cadherin/β-catenin complex, leading to the loss of epithelial cell polarity and impaired barrier function, which are critical factors in triggering EMT. For example, in the pathological tissues of ovarian cancer, there is increased neutrophil infiltration, elevated NE expression, and significantly reduced E-cadherin expression[46]. NE induces the activation of the Wnt/β-catenin signaling pathway, possibly through the following mechanisms: direct action on β-catenin, inducing a conformational change by cleaving the E-cadherin/β-catenin complex to release β-catenin, or through other unknown pathways that still require further research.

NE is a proteolytic enzyme with broad substrate specificity that plays a significant role in inflammatory and degenerative diseases [47, 48]. It is involved in cellular apoptosis and fibroproliferative responses through various signaling pathways, making it a pathogenic factor in several lung diseases [49–51]. We found that a specific inhibitor of NE, Sivelestat·Na, has protective effects on general conditions and histopathological changes in mice with PF. Sivelestat·Na treated with mice showed significantly reduced pulmonary inflammation and fibrosis. Furthermore, the expression levels of fibrosis-related factors (KL-6 and SP-D) and proteins (N-cadherin, vimentin, and Snail1) were also significantly decreased. These findings suggest that Sivelestat·Na could be an effective drug for the treatment of early-stage PF. Early research in Japan confirmed that Sivelestat·Na can reduce pulmonary vascular permeability, inhibit the secretion of mucus by the epithelial layer, decrease the production of inflammatory cytokines in the body (such as IL-1β, IL-6, and tumor necrosis factor-alpha), and prevent lung damage induced by ischemia-reperfusion[52–55]. In February 2022, the "Clinical Application Consensus of Sivelestat·Na" was published, and Sivelestat·Na was approved for treating patients infected with SARS-CoV-2 [56]. In animal models infected with severe acute respiratory syndrome and Middle East respiratory syndrome coronavirus, researchers observed that excessive inflammation and immune-mediated "cytokine storms" can induce apoptosis in epithelial and endothelial cells. This is followed by an increase in vascular permeability and an abnormally enhanced reaction of T cells and macrophages, leading to rapid progression to ALI/acute respiratory distress syndrome (ARDS) [57]. In a study of 167 patients with sepsis with ARDS or disseminated intravascular coagulation (DIC), those admitted to the intensive care unit were continuously administered Sivelestat·Na for 5 days. The results indicated that compared with those in the Ctrl group, the lung injury score, oxygenation index, DIC score, and survival rate of the Sivelestat·Na group patients was better [58]. This suggests that for patients with severe infections and high-risk factors for progression to PF, Sivelestat·Na can serve as a new therapeutic option. Clinical research on Sivelestat·Na has primarily focused on its role in inhibiting NE. However, studies have also shown that Sivelestat·Na can improve respiratory dysfunction in patients with ALI induced by extracorporeal circulation by inhibiting NE and IL-8 [59]. Additionally, due to the different targets regulated by Sivelestat·Na, its effects may vary accordingly. Therefore, determining the optimal timing for initiating or discontinuing Sivelestat·Na treatment is a clinical issue that requires further evaluation in additional clinical trials.

Treatment options for patients with IPF are limited. Traditional immunosuppressive therapies, such as systemic corticosteroids or azathioprine, have failed to demonstrate significant therapeutic effects.IPF patients with a high percentage of neutrophils in their BALF exhibit resistance to corticosteroid treatment [60]. Early application of Sivelestat·Na significantly mitigated the progression of BLM-induced PF in mice, highlighting the potential of using nontraditional methods to inhibit fibroblast proliferation and function. Sivelestat·Na holds promise as a new therapeutic approach for patients with IPF. However, due to the lack of evidence based on evidence-based medicine, more basic experiments or clinical studies should be conducted to identify the groups that benefit from Sivelestat·Na, identify efficacy evaluation indicators, and establish more accurate personalized treatment strategies.

In conclusion, our research has preliminarily explored the specific molecular mechanisms by which NETs promote the onset and progression of PF, elucidating the significant role of the Wnt signaling pathway in the pathogenesis of PF. The key protease NE on NETs may activate β-catenin by cleaving the E-cadherin/β-catenin complex, which initiates downstream EMT as a crucial effector. Sivelestat·Na disrupts the stability of NETs by inhibiting the activity of NE, thereby alleviating the onset and progression of PF. These findings provide critical molecular targets and a theoretical basis for the early diagnosis and precise treatment of PF in the future.

General authorship contribution statement

For research articles with several authors, a short paragraph specifying their individual contributions must be provided. The following statements should be used “Conceptualization, WCS and SDJ; methodology, YS and ZHL; software,YY; formal analysis, SS;data curation, LJL; writing—original draft preparation, WCS; writing—review and editing, YKL; funding acquisition, SDJ and HC.

ALI

acute lung injury

BALF

bronchoalveolar lavage fluid

DEGs

differentially expressed genes

ECM

extracellular matrix

EMT

epithelial mesenchymal transition

IPF

idiopathic pulmonary fibrosis

ILD

interstitial lung disease

KEGG

kyoto encyclopedia of genes and genomes

KL-6

krebs von den Lungen-6

neutrophil elastase

NETs

neutrophil extracellular traps

SP-D

surfactant-associated protein-D

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethics approval and consent to participate

The animal experiments were conducted in accordance with the principles of the National Institutes of Health and were approved by the Animal Research Committee of the Second Affiliated Hospital of Harbin Medical University (SYDW2023-079).

Funding

This research was supported in part by the Key Research and Development Program of Heilongjiang (GZ20210158) and Special Subsidized Research Program of the Fourth Affiliated Hospital of Harbin Medical University (HYDSYTB202303).

Data availability declaration

The datasets supporting the conclusions of this article are included within the article and its additional files.

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No competing interests reported.

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Neutrophil Extracellular Traps Regulate the Pathogenesis of Pulmonary Fibrosis by Inducing Epithelial-Mesenchymal Transition

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Abstract

Figures

Introduction

Materials and Methods

Cell culture

Wright-Giemsa staining to detect the degree of differentiation of HL-60 cells

Induction and purification of NETs

Immunofluorescence staining

Cell proliferation assay

Cell migration assay

Western Blotting Analysis

Transient Transfection

Co-immunoprecipitation (Co-IP)

BLM Mouse Modeling and Sampling

Enzyme-linked immunosorbent assay (ELISA)

Immunohistochemistry (IHC)

Transcriptomic RNA sequencing analysis

Statistical analysis

Results

Biological functions of NETs

NETs promoted the progression of BLM-induced PF in mice

NETs promoted EMT in AT-II cells by activating the Wnt signaling pathway

NETs promoted EMT through interactions between NE and β-catenin

Sivelestat·Na can inhibit the Wnt pathway activated by NE to alleviate PF

Discussion

Abbreviations

Declarations

Ethics approval and consent to participate

References

Additional Declarations

Supplementary Files

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