Neutrophil extracellular traps induce pyroptosis of pulmonary microvascular endothelial cells by activating the NLRP3 inflammasome

Abstract

Excessive formation of neutrophil extracellular traps (NETs) may lead to myositis-related interstitial lung disease (ILD). There is evidence that NETs can directly injure vascular endothelial cells and play a pathogenic role in the inflammatory exudation of ILD. However, the specific mechanism is unclear. This study aimed to investigate the specific mechanism underlying NET-induced injury to human pulmonary microvascular endothelial cells (HPMECs). HPMECs were stimulated with NETs (200 ng/ml) in vitro. Cell death was detected by propidium iodide staining. The morphological changes of the cells were observed by transmission electron microscopy (TEM). Pyroptosis markers were detected by western blot, immunofluorescence, and quantitative real-time polymerase chain reaction, and the related inflammatory factor Interleukin-1β (IL-1β) was verified by enzyme-linked immunosorbent assay (ELISA). Compared with the control group, HPMECs mortality increased after NET stimulation, and the number of pyroptosis vacuoles in HPMECs was further observed by TEM. The pulmonary microvascular endothelial cells (PMECs) of the experimental autoimmune myositis mouse model also showed a trend of pyroptosis in vivo. Cell experiment further confirmed the significantly high expression of the NLRP3 inflammasome and pyroptosis-related markers, including GSDMD and inflammatory factor IL-1β. Pretreated with the NLRP3 inhibitor MCC950, the activation of NLRP3 inflammasome and pyroptosis of HPMECs were effectively inhibited. Our study confirmed that NETs promote pulmonary microvascular endothelial pyroptosis by activating the NLRP3 inflammasome, suggesting that NETs-induced pyroptosis of PMECs may be a potential pathogenic mechanism of inflammatory exudation in ILD.

NETs promote human pulmonary microvascular endothelial cells pyroptosis in vitro . The pulmonary microvascular endothelial cells (PMECs) of the experimental autoimmune myositis mouse model also showed a trend of pyroptosis in vivo . NLRP3 inflammasome is activated during pyroptosis.

Graphical Abstract

Graphical Abstract.

Introduction

Interstitial lung disease (ILD) is a group of diseases characterized by interstitial inflammation or fibrosis of the lung, accompanied by damage to the alveolar epithelium and lung parenchyma [1], more commonly seen in connective tissue diseases. The pathogenic mechanism of ILD is very complex, including vascular injury, changes in the immune response, and activation of inflammation [2]. A study analyzing postmortem lung tissue from patients with scleroderma-associated ILD observed overactivation of microvascular endothelial cells [3]. Refractory hypoxemia in COVID-19 patients may be caused by a serious imbalance of the V/Q ratio due to vascular endothelial injury [4]. In addition, an ILD lung biopsy showed morphological evidence of microvascular injury [5]. These findings suggest that endothelial cell injury contributes to pathological changes in ILD patients to a certain extent. Endothelial cells are arranged throughout the vascular system, acting as the nexus of inflammatory responses and maintaining the dynamic balance between cells and tissues [6]. They play an important physiological function and are also the target of various damage stimuli [7, 8]. Endothelial cell dysfunction is an early pathogenic mechanism of many diseases [9], and studies have found that neutrophil extracellular traps (NETs) can damage vascular endothelial cells.

NETs are a fibrous network structure that exist outside the cell, mainly composed of genomic DNA skeletons, histones, myeloperoxidase (MPO), and antibacterial active proteins, such as neutrophil elastase (NE) and cathepsin G [10]. They were originally thought to limit infection by preventing the spread of pathogens, promoting coagulation, and inducing the production of proinflammatory cytokines. However, in recent years, our group has found that NETs were closely related to the progression of ILD in an experimental autoimmune myositis (EAM) mice model [11]. Our group also found that the abnormally increased low-density granulocytes (LDGs) in patients with idiopathic inflammatory myopathy (IIM)-ILD may exacerbate the abnormal formation of NETs [12], and the decrease of DNase I activity lead to the significant accumulation of NETs in the body [13]. Scholars have found that NETs can directly damage vascular endothelial cells in vitro and NETs from patients with IIM-ILD caused particularly significant damage to vascular endothelial cells. These evidence indicate that the abnormal formation of NETs may be involved in the pathogenesis of IIM-ILD, and may contribute to the inflammatory exudation of ILD.

Pyroptosis, as a programmed cell death mode accompanied by inflammatory reactions, has been found to play an important role in various pathological processes, such as tumors and inflammation, since its discovery in 1992 [14]. Morphologically, the typical characteristic of pyroptosis is the rupture of the plasma membrane, resulting in the release of cell contents, IL-1β and IL-18 [15, 16]. Pyroptosis can be caused by various pathological stimuli, such as oxidative stress, hyperglycemia, endotoxin, and fatty acids [17]. Several studies have confirmed that vascular endothelial cells are one of the target cells of pyroptosis [18]. In addition, the NLRP3 inflammatory pathway can sense high lipid or inflammatory mediators and trigger pyroptosis of vascular endothelial cells. A previous study found that compared with the healthy group, the expression level of NLRP3, caspase-1, IL-1β, and IL-18 in serum and muscle samples of patients with IIMs is significantly increased [19]. Activation of the NLRP3 inflammasome in lung tissue was found in a mouse model of pulmonary fibrosis [20–22]. These studies suggest that NLRP3-mediated pyroptosis may play a crucial role in the occurrence and development of IIM-ILD.

Therefore, it is speculated that NETs may mediate the development of IIM-ILD by activating the NLRP3 inflammasome and promoting the pyroptosis of vascular endothelial cells. To verify this hypothesis, we used human pulmonary microvascular endothelial cells (HPMECs) to test in vitro whether NET stimulation induces endothelial cell pyroptosis. An ILD mouse model was also established to determine whether there was pulmonary microvascular endothelial cell pyroptosis in mice. Exploring the mechanism of endothelial cell pyroptosis may bring new ideas for the treatment of ILD.

Materials and methods

Neutrophil isolation and NET extraction

Isolation of neutrophils

The whole blood of healthy volunteers was extracted into an ethylene diamine tetraacetic acid (EDTA) anticoagulant tube. Histopaque-1119 (sigma), Histopaque-1077 (Sigma), and whole blood were added sequentially in the ratio of 5:2:7 and centrifuged at 750 × g for 30 min. Neutrophil layers were put into a centrifuge tube containing 10 ml of phosphate buffer saline (PBS), mixed, and centrifuged at 450 × g for 10 min. Neutrophils were resuspended in 1640 medium containing 10% fetal bovine serum (Gibco, 10099-141, USA) and counted with a cell counter.

Phorbol 12-myristate 13-acetate induces the formation of NETs in vitro

Neutrophils were cultured in 1640 medium in a 6-well plate and incubated in a cell incubator at 5% CO₂ at 37°C. Then, 30 minutes later, phorbol 12-myristate 13-acetate (Solarbio, P6741-1mg, Beijing, China) at a final concentration of 100 nM was added to each well, gently shaken and mixed, and further incubated in a cell culture incubator. After 4 hours, the culture medium was discarded, and the cells were rinsed with 2 ml of calcium-magnesium-free PBS and centrifuged at 450 × g for 10 minutes. The supernatant of NETs without cell fragments was collected and stored at −80°C.

Cell culture and treatment

Primary HPMECs were purchased from CHI Scientific Inc, and the second or third-generation cells were used for the following experiments. In the control group, HPMECs were cultured in endothelial cell medium(ECM) (Sciencell, 1001, USA) supplemented with 5% serum and 1% double antibiotics. In the NET group, HPMECs were treated with the extracted NETs at a concentration of 200 ng/ml for 24 h. In the NETs + MCC950 group, HPMECs were pretreated with the NLRP3 inhibitor MCC950 (MedChemExpress, hy-12815a, USA) for 30 min and then cocultured with NETs for 24 h.

Cellular immunofluorescence

HPMECs were cultured on sterilized round slides at a density of 3 × 10⁴/ml and stimulated with the extracted NETs for 24 hours after cell adhesion. After treatment, 4% paraformaldehyde was used for fixation, and 0.1% Triton X-100 was used for permeability. The Quick Block immunostaining blocking solution was sealed at room temperature for 1 h, and NLRP3 (Immunoway, YT5382, USA, 1:200), gasdermin D (GSDMD; Proteintech, 20770-1-AP, Wuhan, China, 1:200), and apoptosis-associated speck-like protein containing CARD (ASC; Proteintech, 10500-1-AP, 1:200) antibodies were dripped onto the slides and incubated overnight in a wet box at 4°C. On the second day, the corresponding fluorescent secondary antibody was used for labeling, and an anti-fluorescence quenching agent containing 4',6-diamidino-2-phenylindole (DAPI) was used for sealing. The images were acquired using a fluorescence microscope (Olympus, BX53 + DP74, Japan).

Cell and tissue transmission electron microscopy

Mouse lung tissue samples were prepared and examined by the transmission electron microscopy (TEM) at the Cuiying Biomedical Research Center, Lanzhou University Second Hospital. Lung tissues were fixed in a 2.5% glutaraldehyde solution in 0.1 M phosphate buffer at 4°C for 24 hours, and then were stained and observed under the TEM (Hitachi, HT7800). Cells for TEM were cultured in 10-cm dishes. When the cells grew to 80% density, they were stimulated with NETs for 24 hours. Trypsin digestion was performed, and the cells were collected in a 1.5-ml centrifuge tubes and then centrifuged at 214 x g for 10 minutes. The supernatant was discarded, and fixing solution (2.5 glutaraldehyde) was slowly added along the tube wall to resuspend the cells at room temperature. The cells were transferred to a 1.5-ml centrifuge tube and centrifuged at 9503 x g for 10–12 minutes. The supernatant was discarded with a pipette, and room temperature or 4°C precooled fixing solution was slowly added along the tube wall. The sample sections were stained and observed under a TEM.

Western blot

Protein was extracted from HPMECs using radio-immunoprecipitation assay (RIPA) buffer (Solarbio, R0010) containing protease inhibitors. The protein concentration was measured by the bicinchoninic acid (BCA) method. The protein lysate was mixed with the sample loading buffer solution and boiled at a high temperature for 10 minutes. The proteins were then transferred to an activated polyvinylidene fluoride (PVDF) membrane by wet transfer in a polyacrylamide gel (sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) and sealed with 5% skim milk at room temperature for 1 hour. The primary antibodies against recombinant glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Proteintech, 60004-1-lg, 1:10 000), NLRP3 (Immunoway, YT5382, 1:1000), cleaved caspase-1 (Immunoway, YC0002, 1:1000), GSDMD (Proteintech, 20770-1-AP, 1:750), N-gasdermin D (N-GSDMD) (Abcam, ab215203, England, 1:1000), and ASC (Proteintech, 10500-1-AP, 1:2000) were added and incubated overnight at 4°C. Tris-buffered salineand tween20 (TBST) was used to wash the membrane, and goat anti-rabbit (Proteintech, SA00001-2, 1:10 000) and goat anti-mouse (Proteintech, SA00001-1, 1:10 000) secondary antibodies were incubated with the membrane at room temperature for 1 hour. After washing the membrane, enhanced chemiluminescence (ECL) color solution was added and exposed for photography.

Real-time fluorescence quantitative polymerase chain reaction

Total RNA was extracted from HPMECs using TRIzol reagent (AgBio, HuNan, China) according to the manufacturer’s instructions. The purity and concentration were determined using a spectrophotometer, and then reverse transcribed into cDNA. By using the SYBR Green Kit, the 20-μl real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) reaction consisted of 10 μl of 2× PCR premix, 0.8 μl of 10 μM primer, 7.2 μl of RNase-free water, and 2 μl of reverse transcription template. qRT-PCR amplification conditions were as follows: 95°C for 30 seconds, 95°C for 5 seconds, and 60°C for 30 seconds, for a total of 40 cycles. The human GAPDH was used as the internal control. The sequence of the PCR primers was as follows: NLRP3: 5ʹ-TGGCATCGTGAAGTGGTTGT-3ʹ, 5ʹ-AGCCAAATGCTTACCAGAAAGT-3ʹ; IL1B: 5ʹ-GGACAGGATATGGAGAGCAACAAGTGG-3ʹ, 5ʹ-TCATCTTTCAACACGCAGGACAGG-3ʹ; GSDMD: 5ʹ-CCAGaAGaAGACGGTCACCATCC-3ʹ, 5ʹ-TGGAACGCTTGTGGCCTGTC-3ʹ; ASC: 5ʹ-CTCCTCAGTCGGCAGCCAAG-3ʹ, 5ʹ-AGCATCCAGCAGCCACTCAAC-3ʹ; GAPDH: 5ʹ-ATCACCATCTTCCAGGAGCGA-3ʹ, 5ʹ-CCTTCTCCATGGTGGTGAAGAC-3ʹ. After the reaction, based on the amplification curve, the relative expression levels of each target gene were calculated using the 2^−ΔΔCt method.

Enzyme-linked immunosorbent assay

According to the instructions of the ELISA kit, the levels of IL-1β (Jianglai, Shanghai, China) were determined from the cell supernatant.

Cell death propidium iodide staining

The cells were inoculated in 24-well plates and divided into different groups. Then, 2 µg/ml propidium iodide (PI; Solarbio, CA1120) and 5 µg/ml Hoechst were added to stain at room temperature for 10 minutes. The cells were observed under an inverted fluorescence microscope. The dead cells showed red fluorescence and blue fluorescence.

Construction of the EAM mouse model

BALB/C wild-type mice (aged 6–8 weeks) were purchased from Lanzhou Veterinary Research Institute. All mice were raised at the SPF Laboratory of the Lanzhou University Second Hospital with no restrictions on water and food supply. Rat skeletal muscle homogenate with a protein content of 30 mg/ml was prepared, mixed with 0.25 ml with an equal volume of complete Freund’s adjuvant (CFA), and injected subcutaneously into both sides of the mouse’s back. The mice were immunized with 0.25 ml on each side. Immunizations were performed 5 times on days 0, 7, 14, 21, and 28. Pertussis toxin (2 μg) was injected intraperitoneally on days 0 and 7. On day 35, the mice were sacrificed and lung tissue samples were collected. All animal experiments were approved by the Ethics Committee of the Second Hospital of Lanzhou University (Project number: D2022-061).

Histopathological staining

After fixing the lung tissue in 4% formalin, the sections were embedded in paraffin. After antigen repair, all slides were incubated with rabbit anti-Cit-H3 (Abcam, ab5103, 1:1000) at 4°C for 12 hours. After incubation with horseradish peroxidase coupled with a second antibody, diaminobenzidine (DAB) staining was performed, and the slides were sealed and observed for positive expression under a microscope.

Data analysis

The measured values of data were from three independent experiments and are expressed as the mean ± SD. Statistical analysis was conducted with GraphPad Prism 8.0. To determine data normality, the Shapiro–Wilk normality test was used. Differences between mean values of normally distributed data were analyzed by a t-test or ANOVA, and P < 0.05 was statistically significant.

Results

The death rate of HPMECs increased in the NETs group

To verify the effect of NETs on endothelial cells, we used PI staining to detect cell death in different groups. When a cell dies and its membrane ruptures, PI can pass through the broken cell membrane and bind to DNA in the nucleus, emitting red fluorescence. The results showed that compared with the control group, the NETs group showed a significant increase and enhancement of red fluorescence in the cells (Fig. 1A), indicating that NETs could lead to the death of pulmonary microvascular endothelial cells.

Figure 1.

NET-induced death of pulmonary microvascular endothelial cells. (A) Compared with the control group, the NETs group showed significantly increased cell death (200×). (B) Quantitative analysis showed that the percentage of cell death increased in the NETs group. *P < 0.05.

NETs-induced pyroptosis of HPMECs

The effects of different forms of cell death on the disease varied greatly. To further clarify the form of cell death, TEM was used to observe the ultrastructure of cells. After HPMECs were stimulated by NETs for 24 hours, the cells in each group were collected and fixed. TEM results showed that the cell membrane of the control group was intact, and no significant abnormalities were observed in various organelles in the cytoplasm. A large number of vacuoles were observed in the cells stimulated by NETs (Fig. 2A). Immunofluorescence staining showed an increased expression of the pyroptosis indicator (GSDMD) in the NETs group (Fig. 2B). It suggested that NETs can stimulate HPMECs to undergo pyroptosis. To explore the effect of NETs on the expression of pyroptosis-related indicators in HPMECs, total protein and mRNA were extracted, and the pyroptosis markers were determined by western blot (WB) (Fig. 2C), qRT-PCR (Fig. 2D), and ELISA (Fig. 2E). The results indicated that the expression of pyroptosis-related markers, such as GSDMD and N-GSDMD, in the NETs group was enhanced. The levels of IL-1β in cell supernatant were increased significantly in the NETs group (Fig. 2E). These results suggest that NETs can induce pyroptosis in HPMECs.

Figure 2.

NET-induced pyroptosis of human pulmonary microvascular endothelial cells. (A) The organelle structure of cells was observed by TEM. Compared with the normal organelle structure in the control group, a large number of vacuoles were observed in the NETs group (4000×, 6000×). (B) Immunofluorescence staining showed that the expression of GSDMD in the cytoplasm was significantly enhanced after NET stimulation as compared with the control group (400×). (C) The WB results showed that the expression of GSDMD and N-GSDMD was significantly increased in the NET-stimulated group. (D) qRT-PCR further confirmed that GSDMD mRNA levels were increased. (E) Compared with the control group, the levels of IL-1β in cell supernatant were increased significantly. *P < 0.05 vs. NC. **P < 0.005 vs. NC. TEM, transmission electron microscopy; GSDMD, gasdermin D.

NET infiltration was found in lung interstitial lesions in EAM mouse models

Lung H&E staining results showed that EAM mice had pulmonary interstitial changes (Fig. 3A). The lung tissues of the control group were structurally intact, with no cellular infiltration and stroma increase in the lung interstitium. Significant interstitial thickening and a large number of inflammatory cells were observed in the lung tissues of the EAM group mice. Immunohistochemical staining of lung tissue confirmed significant infiltration of NETs in the diseased lung tissue (Fig. 3B). Cit-H3 is the most specific protein representing the formation of NETs. Immunohistochemical staining results showed that compared with the control group, Cit-H3 and MPO were positively expressed in the EAM group, suggesting that NETs were associated with ILD in the EAM mouse model.

Figure 3.

Increased expression of NETs in EAM lung tissue. (A) H&E staining, scale = 200 μm/50 μm. In the control group, the alveolar structure of the mice was intact. In the EAM group, inflammatory cell infiltration was observed in the alveolar interstitium, with varying sizes of alveolar cavities and significantly thickened alveolar septum. (B) Immunohistochemical staining of lung tissues, scale = 100 μm/50 μm. The control group had intact lung lobe structure, normal alveolar septum, and no Cit-H3 and MPO positive staining. In the EAM group, there were a large number of inflammatory cells infiltrating the alveolar septum, with Cit-H3 and MPO positive expression. EAM, experimental autoimmune myositis; Cit-H3, Citrullinated histone 3; MPO, Myeloperoxidase.

Pyroptosis of PMECs was found in the EAM mouse model

In immunohistochemical staining, NLRP3 and GSDMD were positively expressed in the lung interstitium of EAM mice (Fig. 4A). To further investigate whether pyroptosis of PMECs occurs in EAM, TEM was used to observe the ultrastructure of lung tissue. It was found that the structure of PMECs was normal, and the cell membrane was intact in the control group mice, while vacuoles around PMECs were visible in EAM mice (Fig. 4B). According to the results shown in the figure, it is confirmed that PMECs pyroptosis occurred during the development of EAM in vivo.

Figure 4.

Pyroptosis of pulmonary microvascular endothelial cells was observed in EAM model mice. (A) Immunohistochemical staining of lung tissue, scale = 100um/50um. NLRP3 and GSDMD were positively expressed in the lung interstitium of EAM mice. (B) In the control group, the pulmonary microvascular endothelial cell structure was normal, and the cell membrane was intact (5000×/10 000×), The formation of vacuoles around pulmonary microvascular endothelial cells indicates pyroptosis vacuoles in EAM mice (4000×/7000×).

The activation of NLRP3 inflammasome in NET-stimulated HPMECs was increased

We further explored the mechanism of pyroptosis. Immunofluorescence staining showed that the expression of NLRP3 and junctional protein ASC was enhanced and localized in the cytoplasm in the NETs group (Fig. 5A and B). qRT-PCR results further confirmed that the expression level of NLRP3 and ASC was significantly increased in the NETs group (Fig. 5C and D). NLRP3 inflammasome expression was determined by WB and the results showed that the expression of NLRP3, ASC, and caspase-1 was significantly enhanced after HPMECs were stimulated with NETs (Fig. 5E). These results showed that NET stimulation can induce the activation of NLRP3 inflammasome in HPMECs.

Figure 5.

Expression of the NLRP3 inflammasome increased in NET-stimulated HPMECs. (A, B) Immunofluorescence staining showed that the expression of NLRP3 and ASC in the cytoplasm was significantly increased after NET stimulation as compared with the control group (A, 200×; B, 400×). (C, D) qRT-PCR further confirmed the increase of NLRP3 and ASC mRNA levels in the NET-stimulated group. (E) The expression of related indicators was detected by WB. Compared with the control group, the expression of NLRP3, ASC, and caspase-1 was significantly increased. *P < 0.05 vs. NC, ***P < 0.001 vs. NC. NLRP3, NOD-like receptor thermal protein domain associated protein 3; ASC, Apoptosis-associated speck-like protein containing CARD.

Inhibition of NLRP3 significantly reduced pyroptosis of HPMECs

To further verify the relationship between NLRP3 inflammasome and pyroptosis of HPMECs, cells were pretreated with the NLRP3 inhibitor MCC950 for half an hour, followed by stimulation with 200 ng/ml NETs for 24 hours. The expression levels of pyroptosis-related indicators were verified by WB (Fig. 6A) and qRT-PCR (Fig. 6B). The results showed that the expression of NLRP3, GSDMD, N-GSDMD, caspase-1, ASC, and other indicators was significantly inhibited in the group pretreated with MCC950 as compared with the group stimulated with NETs. These results suggested that NETs mediate pyroptosis of HPMECs through the classical pyroptosis pathway by activating NLRP3 inflammasome.

Figure 6.

Inhibition of NLRP3 significantly reduced pyroptosis of HPMECs. (A) After pretreatment with NLRP3 inhibitor (MCC950), HPMECs was stimulated with NETs. Compared with the NETs group, the expression of pyroptosis-related indicators, including NLRP3, GSDMD, N-GSDMD, caspase-1, ASC, and other markers were significantly inhibited in the MCC950 pretreatment group. (B) qRT-PCR further verified that the mRNA levels of GSDMD, ASC, and IL-1B in the MCC950-pretreated group were significantly decreased compared with the NETs group. **P < 0.005 vs. NC, ****P < 0.0001 vs. NC, ^#P < 0.05 vs. NETs, ^##P < 0.005 vs. NETs, and ^####P < 0.0001 vs. NETs.

Discussion

Pulmonary interstitial exudation is a remarkable characteristic of IIM-ILD, and endothelial injury may play a key role in the exudation. In this study, significant interstitial lesions were found in the lung tissue of EAM mice, accompanied by NETs infiltration. In addition, PMECs were observed to undergo pyroptosis in the EAM model mice in vivo and after stimulated with NETs in vitro. Activation of NLRP3 inflammasomes is indispensable in the process of pyroptosis of PMECs in vitro. Pyroptosis was significantly relieved in cells pretreated with the NLRP3 inhibitor MCC950. These results suggested that NETs induce pyroptosis of PMECs through the activation of NLRP3 inflammasome, which may be a potential mechanism for inflammatory exudation in ILD.

Based on pathological and imaging characteristics, IIM-ILD is mainly classified into nonspecific interstitial pneumonia (NSIP), common interstitial pneumonia, organized interstitial pneumonia, and acute interstitial pneumonia (AIP) [23]. Among them, NSIP and AIP show obvious exudation, suggesting that endothelial cell injury was a pathogenic mechanism. In this study, the lung tissue of EAM mice showed extensive inflammatory exudation similar to NSIP and AIP, and NETs were infiltrated in the ILD region. TEM revealed pyroptosis of PMECs in vivo and in vitro. These results suggested that NETs may be related to the pyroptosis of PMECs.

NETs are thought to be a new type of damage-associated molecular pattern (DAMP), abnormal aggregation can activate the innate immune system, leading to more production of inflammatory mediators, forming a vicious circle. Several studies have shown that NETs can cause damage to a variety of cells [24–26], and Anti-neutrophil cytoplasmic antibodies (ANCA)-related endothelial injury has also been shown to be mediated by NETs [27]. The mechanism of cell death induced by NETs was further explored. Isolated NETs were used to interact with alveolar epithelial cells and endothelial cells, and it was found that the injury to cells was dose-dependent, and the induced cytotoxicity could directly cause cell death. The results showed that most of the proteins in NETs are involved in the NET-mediated cytotoxicity against epithelial and endothelial cells [28]. Previous studies by our group have shown that NETs abnormal regulation was involved in IIM-ILD, and NETs promote fibroblast proliferation [12–14]. The present study confirms that NETs participate in the mechanism of endothelial cell injury by inducing endothelial cell pyroptosis, which further enriches the study of the mechanism of NETs involved in the pathogenesis of IIM-ILD.

There are two key roles of endothelial cell junctions in inflammation: regulation of the migration of white blood cells out of the vascular lumen and regulation of the permeability of blood vessels to macromolecules. Endothelial cells line the inner walls of all blood vessels which are essential for maintaining the barrier function of the vascular system, but are vulnerable to a variety of biochemical insults. When stimulated by lipopolysaccharide (LPS) in vitro, the proliferation of PMECs is inhibited, cell pyroptosis, apoptosis, inflammation, and oxidative stress are significantly increased, accompanied by an enhancement of the endothelial permeability [29].

Increased expression of the NLRP3 inflammasome, cleaved GSDMD fragments, and release of the proinflammatory cytokine IL-1β were further demonstrated in vivo lung injury mouse models exposed to LPS. The NLRP3 inhibitor, MCC950, significantly reduced the levels of the inflammasome and pyroptosis-related markers in LPS-treated lung vascular endothelial cells and LPS-induced ALI mice [30]. A recent study in a diabetic kidney injury model demonstrated that the damage of renal endothelial cells induced by NETs is pyroptosis, and the clearance of NETs significantly delayed the progression of kidney injury [31]. This study focused on the relationship between NET-induced PMEC injury and pyroptosis, and it was confirmed in vitro that NET-treated pulmonary microvascular endothelial cells undergo pyroptosis. The previous research by our group confirmed that the common phenotypes of EAM are very similar to PM/DM-ILD, and NETs mediate the occurrence of EAM, suggesting that EAM can serve as an ideal model for studying PM/DM-ILD [32]. Moreover, we found that there was significant infiltration of NETs in the lung tissue of EAM model mice, and there was pyroptosis of PMECs. We speculated that NETs may be involved in the pathogenesis of ILD by inducing pyroptosis of PMECs. The exploration of the mechanism of endothelial cell pyroptosis may bring new ideas for the treatment of ILDs.

The NLRP3 inflammatory vesicles are expressed in a wide range of cells and serve as a sensor for the innate immune system to recognize various stimuli [33]. When stimulated, the NLRP3 inflammatory complex cleaves pro-caspase-1 to form a mature caspase-1 fragment. Activated caspase-1 can cleave pro-IL-1β and pro-IL-18. At the same time, GSDMD is cleaved into N-GSDMD, forming pores on the cell membrane, leading to cell pyroptosis [34, 35]. It has been demonstrated that the process of LPS-induced endothelial cell pyroptosis relies on a nonclassical pyroptosis pathway, in which LPS can enter the cell through bacterial microbubbles or by disrupting the plasma membrane of the endothelial cell. Then, LPS directly induces the activation of caspases-4/5/11, which triggers pyroptosis through the cleavage of GSDMD [36, 37]. In this study, we demonstrated that the mechanism of NET-induced pyroptosis is different from the nonclassical pyroptosis pathway induced by LPS. After NET stimulation, the expression of NLRP3 in endothelial cells was significantly increased. In addition, the expression of the downstream joint protein ASC, activated caspase-1 fragment, pyroptosis protein GSDMD, and related inflammatory factor IL-1β was significantly increased. In the group pretreated with the NLRP3 inhibitor MCC950, the expression of downstream pyroptosis proteins was significantly decreased, suggesting that the pyroptosis induced by NETs occurred through the classical pyroptosis pathway.

Current studies also have some limitations, as they mainly focus on in vitro cell experiments. Although it was observed that the presence of NETs infiltration was accompanied by the pyroptosis of pulmonary microvascular endothelial cells in the EAM mouse model, it is unclear if pyroptosis is caused by the accumulation of pulmonary NETs. As such, the specific pathogenic mechanism still needs to be further explored. In addition, the specific components of NETs that can activate the NLRP3 inflammasome to induce endothelial cell pyroptosis also need to be explored.

Conclusion

NETs, serving as a novel type of DAMP, may participate in the progression of IIM-ILD by activating NLRP3 to induce endothelial cell pyroptosis. In this study, NETs were associated with pulmonary microvascular endothelial cell pyroptosis, which enriched the research on the pathogenesis of NETs in IIM-ILD. In addition, targeting pyroptosis may provide a new idea for the treatment of IIM-ILD.

Glossary

Abbreviations

AIP

acute interstitial pneumonia

ASC

apoptosis-associated speck-like protein containing CARD

Cit-H3

citrullinated histone 3

EAM

experimental autoimmune myositis

GSDMD

gasdermin D

HPMECs

human pulmonary microvascular endothelial cells

IIM

idiopathic inflammatory myopathy

ILD

interstitial lung disease

LDGs

low-density granulocytes

MPO

myeloperoxidase

NE

neutrophil elastase

NETs

neutrophil extracellular traps

NLRP3

NOD-like receptor thermal protein domain associated protein 3

NSIP

nonspecific interstitial pneumonia

PI

propidium iodide

PMECs

pulmonary microvascular endothelial cells

qRT-PCR

real-time fluorescence quantitative polymerase chain reaction

TEM

transmission electron microscopy

Ethical approval

The procedures involving animals and their care were conducted in accordance with ARRIVE guidelines and were approved by the Animal Ethics Committee of the Second Hospital of Lanzhou University (D2022-061).

Conflict of interests

The authors declare that they have no potential cnflicts of interest.

Funding

This study was sponsored by the National Natural Science Foundation of China (grant No. 82060302 and grant No. 82260325), the Medical Innovation and Development Project of Lanzhou University (lzuyxcx-2022-168), the Cuiying Scientific and Technological Innovation Program of Lanzhou University Second Hospital (CY2021-MS-A04 and CY2021-QN-B02), the Education technology innovation project of Gansu Province (2022B-051), and the National College Student Innovation and Entrepreneurship Training Program of Lanzhou University (202310730203).

Data availability

The data underlying this article will be shared on reasonable request to the corresponding author.

Author contributions

S.Z. and J.Z. participated in the design of the study. P. Z., L.B., and W.M. carried out the experiments, analyzed the data, and wrote the manuscript. F.L., L.Z., and C.Z. participated in the design of the study, carried out the experiments, and analyzed the data. All authors read and approved the final manuscript.