Neutrophil extracellular traps amplify neutrophil recruitment and inflammation in neutrophilic asthma by stimulating the airway epithelial cells to activate the TLR4/ NF-κB pathway and secrete chemokines

NETs are generated in a mouse model of NA

A mouse model of NA was generated to investigate the role of NETs in the aggravation of neutrophil recruitment and inflammation (Figure 1A). HE staining showed that robust neutrophil inflammation was located around the bronchovascular bundle of the NA mice (Figure 1B). Total cell count, especially of neutrophils, and total protein in the BALF of NA mice were significantly increased (Figure 1C–1E). Compared to the control group, airway resistance was significantly increased in NA mice after induction with a higher dose of methacholine (Mch) nebulization (Figure 1F). Western blotting and immunofluorescence showed that specific NET markers, Cit-H3 and MPO, were highly expressed in this mouse model of NA (Figure 1G and 1H). Additionally, dsDNA levels in BALF of NA mice were robustly higher than in the control group (Figure 1I). Thus, the results showed that higher inflammation levels, neutrophil counts, and NETs existed in the airway of NA mouse model.

Figure 1. NETs were generated in a mouse model of neutrophilic asthma (NA). (A) Flow chart for generation of NA mice. (B) HE staining of lungs showed prominent inflammatory cell infiltration around the bronchi and blood vessels of NA mice (200X). (C–E) Total cells, differential cell count, and total protein in the BALF supernatant of NA mice were measured. (F) Airway resistance was measured in NA mice after treatment with different concentrations of methacholine. (G) Cit-H3 expression was measured by western blotting. (H) Cit-H3 and MPO expression was measured by immunofluorescence (400X). (I) dsDNA concentration in the BALF supernatant of NA mice was detected by PicoGreen analysis. *: P<0.05, **: P<0.01, ***: P<0.001 vs control group.

Preventing NET formation can alleviate lung neutrophilic inflammation, without the structural integrity of NETs having any effect

The NA mice were then treated with sivelestat (selective neutrophil elastase inhibitor) and DNase I to evaluate the role played by NETs in neutrophilic airway inflammation (Figure 2A). Sivelestat was added to prevent NET formation. DNase I was added to deconstruct the backbone of NETs in the NA mice. HE stain results showed that sivelestat treatment prevented neutrophil recruitment, relieved airway inflammation, reduced the BALF cells and neutrophil count, increased the BALF total protein content, and alleviated airway resistance, while DNase I treatment had no effect on these (Figure 2B–2F). It was also confirmed that sivelestat and DNase I were both able to eliminate NETs according to the immunofluorescence, western blotting, and BALF PicoGreen analysis results (Supplementary Figure 1). Furthermore, the expression of CXCL1, CXCL2, and CXCL8 was significantly increased in NA mice as compared to control mice, but declined after sivelestat treatment; DNase I treatment had no obvious effect in the NA mice (Figure 2G–2H). It is worth noting that chemokines were mainly expressed in HBEs and HAEs (Figure 2H). These results indicate that NETs promote airway inflammation in the NA model independently of their structural integrity.

Figure 2. Inhibition of NET formation alleviated neutrophilic airway inflammation in NA model, rather than degrading the NETs DNA. (A) Flow chart for sivelestat or DNase I administration intraperitoneally. (B) HE staining results showed that sivelestat could alleviate inflammation while DNase I could not (200X). (C–E) Total cells, differential count of cells, and total protein were measured after treatment with sivelestat or DNase I in the BALF of NA mice. (F) Airway resistance was measured after methacholine treatment. (G) Concentration of CXCL1, CXCL2 and CXCL8 in the BALF supernatant of NA mice was measured by ELISA. (H) CXCL1, CXCL2, and CXCL8 expression was measured by Immunohistochemistry (200X). #: P<0.05, ##: P<0.01, ###: P<0.001, ####: P<0.0001 vs control group. *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001 vs NA group. ns = no statistical difference vs NA group.

The TLR4/NF-κB pathway is activated in this NA model and is responsible for airway inflammation

The role of NET-induced neutrophilic inflammation via TLR4/NF-κB pathway by treatment with TAK-242, a TLR4 inhibitor, was evaluated in NA mice (Figure 3A). The p-p65 NF-κB expression was found to be obviously increased with the transfer of NF-κB from cytoplasm to nucleus in the airway cells of NA mice, while TAK-242 treatment inhibited p-p65 NF-κB expression and NF-κB transfer (Figure 3C and 3D). Additionally, TAK-242 treatment significantly alleviated neutrophilic inflammation, reduced the BALF cells and neutrophil count, increased the BALF total protein content, alleviated airway resistance, and decreased the CXCL1, CXCL2, and CXCL8 expression (Figure 3B, 3E–3H, and Supplementary Figure 1). As expected, the TLR4/NF-κB pathway was activated in the airway epithelial cells of NA mice and was responsible for airway inflammation.

Figure 3. Blocking the TLR4/NF-κB pathway with TAK-242 reduced the expression of chemotactic factors and alleviate airway inflammation in NA mice. (A) Flow chart for intraperitoneal administration of TAK-242. (B) HE staining results showed that TAK-242 treatment could alleviate inflammation (200X). (C) p65 NF-κB and p-p65 NF-κB expression was measured by western blotting. (D) p65 NF-κB expression was measured by immunohistochemistry (200X). (E–G) Total cells, differential cells, and total protein were measured in the BALF of NA mice. (H) Airway resistance was measured after methacholine treatment. (I) CXCL1, CXCL2, and CXCL8 expression in the BALF of NA mice was measured by ELISA. (J) CXCL1, CXCL2, and CXCL8 expression was measured by Immunohistochemistry in the lung tissues of NA mice. #: P<0.05, ##: P<0.01, ###: P<0.001, ####: P<0.0001 vs control group. *: P<0.05, **: P<0.01: ***: P<0.001, ****: P<0.0001 vs NA group. ns = no statistical difference vs NA group.

NETs stimulate HBEs and A549 to express neutrophilic chemokines in vitro

Twenty-four hours after HBEs or A549 cells were treated with NETs, cell culture supernatant was collected for Luminex and transwell analyses. CXCL1, CXCL2, and CXCL8 expression was significantly increased in the HBE and A549 culture supernatants, with an exception for CXCL1 in HBEs, in the NETs treated group as compared to the control group (Figure 4A and 4B). Additionally, the cell culture supernatant in the NET-treated group was able to promote neutrophil migration, whereas the effect was reversed with the addition of the neutralizing CXCL1, CXCL2, and CXCL8 antibodies, especially neutralizing CXCL8 (Figure 4C and 4D). These results showed that airway epithelial cells could recruit neutrophils under stimulation of NETs.

Figure 4. NETs stimulate HBEs and A549 to express neutrophilic chemokines. (A and B) CXCL1, CXCL2, and CXCL8 expression in the supernatant was analyzed by Luminex and RT-qPCR, after HBEs (or A549) were treated with NETs co-cultured for 24 h. (C and D) Neutrophil migration was analyzed by transwell after cell culture supernatant was induced for 48 h (200X). NET treated supernatant was added to the lower chamber. #: P<0.05, ##: P<0.01, ###: P<0.001, ####: P<0.0001 vs control group. *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001 vs NA group. ns = no statistical difference vs NETs group.

Degrading NETs by DNase I cannot eliminate chemokine stimulating effects in vitro

To assess the effect of NETs on the stimulation of airway epithelial cells expressing chemokines and recruiting neutrophils in vitro, NETs were generated by using 500 μM PMA to treat fresh neutrophils [19]. Next, the effect of in vitro degradation of the backbone structure of NETs was evaluated using DNase I which could reverse the chemokine stimulating effects of directly treated NETs. HBEs or A549 cells were treated with DNase I and NETs simultaneously as described previously [19]. Similar to the results obtained in the mice NA model, DNase I treatment had no effect on CXCL1, CXCL2, or CXCL8 expression or neutrophil migration (Figure 5). These results showed that destroying the structural integrity of NETs could not eliminate its stimulatory effects.

Figure 5. Degradation of NETs by DNase I has no effect on chemokine expression and neutrophil migration in vitro. (A, B) After treatment of HBEs and A549 cells with DNase I and NETs simultaneously, CXCL1, CXCL2, and CXCL8 levels in the supernatant were analyzed by Luminex and RT-qPCR. (C and D) After treatment of HBEs and A549 cells with DNase I and NETs simultaneously, the supernatant was collected and added into the lower chamber. Neutrophils were cultured in the upper chamber and neutrophil migration was analyzed by transwell after cell culture supernatant was induced for 48 h (200X). #: P<0.05, ##: P<0.01, ###: P<0.001, ####: P<0.0001 vs control group. *: P<0.05, **: P<0.01, ***: P<0.001: ****, P<0.0001 vs NA group. ns = no statistical difference vs NA or control group.

Neutrophilic chemotaxis by stimulation of NETs via the TLR4/NF-κB pathway in vitro

The direct activation of TLR4/NF-κb pathway by NETs in vitro was evaluated. HBEs and A549 cells were treated with TAK-242 and NETs simultaneously. After treatment for 48 h, p-p65 NF-κB expression was obviously inhibited in the combined TAK-242 and NETs groups but not in in the NETs group (Figure 6A). Additionally, CXCL1, CXCL2, and CXCL8 expression was significantly decreased in HBEs and A549s culture supernatants, with an exception for CXCL1 in HBEs, in the combined TAK-242 and NETs groups rather than in the NETs group (Figure 6B and 6C). Additionally, cell culture supernatants in both TAK-242 and NETs groups resulted in a significant reduction of neutrophil migration compared to the NETs group (Figure 6D and 6E).

Figure 6. Inhibition of the TLR4/NF-κB pathway reduced chemokine expression and neutrophil migration in vitro after stimulation of HBEs and A549 by NETs. (A) After treatment of HBEs and A549 cells with TAK-242 and NETs simultaneously, p-p65 NF-κB and p65 NF-κB expression was measured by western blot. (B and C) After treatment of HBEs and A549 cells with TAK-242 and NETs simultaneously, CXCL1, CXCL2, and CXCL8 levels in the supernatant were analyzed by Luminex and RT-qPCR. (D and E) After treatment of HBEs and A549 cells with TAK-242 and NETs simultaneously, the supernatant was collected and added into the lower chamber. The neutrophils were then cultured in the upper chamber and neutrophil migration was analyzed by transwell after cell culture supernatant was induced for 48 h (200X). #: P<0.05, ##: P<0.01, ###: P<0.001, ####: P<0.0001 vs control group. *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001 vs NA group. ns = no statistical difference vs NA or control group.

Mouse model of NA

Wild type male C57BL6 mice were purchased from the Experimental Animal Center of Central South University (Changsha, China). All mice were aged 6-8 weeks and weighed between 18 g and 22 g. All mice were kept in specific pathogen-free environment with freely available standard food and water. All animal experiments were approved by the Animal Care and Use Committee of Central South University and were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. The LPS/OVA administration into the NA mouse model was performed in accordance with Wilson’s protocol [44]. Briefly, on day 0 and day 6, mice were anesthetized by pentobarbital administration and were administered 100 μg low-endotoxin ovalbumin (OVA, Grade V, Sigma-Aldrich, St. Louis, MO, USA) and 15 μg LPS (Sigma-Aldrich) dissolved in 50 μl PBS (delivery vehicle) intratracheally (i.t.), followed by 200 μl of air, on day 13, all mice were challenged with a single nebulization comprising of an aerosol of 1% OVA (Grade III, Sigma-Aldrich) in saline for 40 minutes.

In vivo DNase I treatment

Mice were administered 3 mg/ml DNase I (5 mg/kg, Roche, USA) or vehicle control (8.77 mg.ml NaCl, 0.15 mg/ml CaCl₂ DNase I working solution) i.t. right before and 6 hours after OVA challenging.

In vivo treatment with neutrophil elastase inhibitor (NEi) and TLR4 inhibitor

In vivo NE and TLR4 inhibition were accomplished by intraperitoneally injecting 10 mg/kg of an NEi, sivelestat sodium tetrahydrate (Selleck, USA) every day for 4 days before harvest and a TLR4 inhibitor, TAK-242 (Selleck, USA) right before nebulization and 6 hours after nebulization. The corresponding control group received an equivalent PBS dose.

Hematoxylin-eosin staining

For histopathological analysis, the right lung lobes were fixed in 4% PFA and embedded in paraffin. Sections (4 μm) were placed onto glass slides and stained with hematoxylin and eosin (HE).

Quantification of extracted double-stranded DNA and total protein

Double-stranded DNA extracted from the BALF supernatant was quantified by a Quant-iT PicoGreen assay kit in accordance with the manufacturer’s instructions (Invitrogen, Burlington, ON, Canada). The dsDNA extracted from NETs of treated cells were quantified using a Nanodrop2000 spectrophotometer (ThermoFisher Scientific). The total protein in the BALF supernatant was quantified using an Enhanced BCA Protein Assay Kit (Beyotime, Beijing, China), following the manufacturer’s protocol.

Western blotting

Protein detection for whole cell lysates of mice lung tissue and in vitro cultured cells was conducted using western blotting. PVDF membranes were incubated overnight with Cit-H3 (1:500, Abcam, USA) and NF-κB (1:500, ABclonal Technology, China) antibodies. Anti-GAPDH antibody (1:5000, Proteintech, USA) was used as the internal control.

Immunohistochemistry and immunofluorescence

Immunohistochemistry was used to detect CXCL1, CXCL2, and CXCL8 levels in airway cells of the mouse model. Immunofluorescence was used to detect Cit-h3 (Abcam) and MPO (Abcam) expression in vivo and in vitro. Paraffin-embedded mouse lungs were sectioned at 4 μm thickness and mounted on anti-off slides. After dewaxing and hydration, slides were immersed for 20 minutes in boiling Tris-EDTA antigen retrieval solution (pH9.0, ServiceBio, China). Antigen blocking was performed by coating the slides with 10% normal serum and incubating for 1 hour at room temperature followed by incubation with primary antibody overnight at 4°C for antigen detection. Immunohistochemistry was performed using a 2-step detection kit (ZSGB-Bio, China) for DAB reaction, in accordance with the manufacturer’s instructions. Immunofluorescence was performed using Alexa Fluor 488 donkey anti-rabbit (Abcam) and Alexa Fluor 647 donkey anti-goat (Abcam) as secondary antibodies and incubating for 1 hour at room temperature to detect primary antibodies. Fluoroshield mounting media with DAPI (Abcam) was used for mounting and DNA detection. The protocol for detection of NETs in PMA stimulated neutrophils was similar for the paraffin-embedded slides, but the samples on carry sheet glass were fixed by methyl alcohol for 20 minutes without the dewaxing and the hydration processes.

Enzyme-linked immunosorbent assay (ELISA)

CXCL1, CXCL2, and CXCL8 levels in mouse BALF supernatant were measured using commercial mouse enzyme-linked immunosorbent assay (ELISA) kits (RayBiotech for CXCL1 and CXCL2, USA; Neobioscience for CXCL8, China). The assays were performed following the manufacturer’s instructions.

In vitro generation of neutrophils and NETs

Polymorphonuclear neutrophils (PMNs) were obtained by performing density gradient centrifugation. Briefly, whole blood was diluted 1:1 diluted in sterilized PBS, loaded on a human lymphocyte separating media followed by density gradient centrifugation (800 g, 21°C, without break), leaving RBCs and PMNs at the bottom of the centrifugation tube, which were then mixed with 6% dextran 40. Following 30 minutes of sedimentation, the supernatant was centrifugated at 1000 g for 5 minutes. Red blood cell lysing solution (Geneview, China) was used to resuspend the neutrophilic rich pellet. This was then recentrifuged and resuspended in RPMI1640 culture media with 10% FBS. The purity of the neutrophils obtained (consistently > 95%) was verified by Giemsa staining, and cell viability (> 95%) was verified by trypan blue staining. PMNs were counted and loaded into 6-well cell culture plates (10⁶/well). Phorbol 12-myristate 13-Acetate (PMA, Sigma-Aldrich) at 500 nM was added to the PMNs and incubated for 4 hours at 37°C with 5% CO₂. After incubation, the supernatant was aspirated by gently washing the wells with cold PBS and all the PBS solution was collected. This solution was then centrifuged at 400 g at 4°C to separate the remaining neutrophils, and the supernatant was divided into 1.5 ml micro-centrifuge tubes and centrifuged at 18000 g at 4°C. The pellet was resuspended using cold PBS and the DNA concentration was adjusted for subsequent experiments. 24-well cell culture plates and carry sheet glass was used for NETs detection by immunofluorescence.

Airway resistance

After anesthetizing with pentobarbital (125 mg/kg i.p.), the mice were intubated and connected to a modular and invasive airway resistance detection system (Buxco). Mice were ventilated and a steady baseline of airway pressure was generated. Next, graded doses of methacholine (0, 3, 6, 12, 25 mg/ml, Sigma-Aldrich) were administered to mice via a nebulizer, and the changes in airway resistance were recorded.

Cells and treatments

HBEs and HAEs (A549) were obtained from cell repository of Advanced Research Center, Central South University, and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) or Roswell Park Memorial Institute 1640 (RPMI1640) with 10% FBS and 1% penicillin-streptomycin. The cells were treated with human-derived NETs (DNA concentration at 5 μg/ml), TAK-242 (3 μM/L, Selleck USA), or DNase I (3 mg/ml, ThermoFisher) for 24 h after the cells achieved a growth confluence of 80%.

Quantitative PCR analysis

Total RNA was extracted from matured cells using RNeasy Mini Kit (Qiagen, Germany) while cDNA was synthesized using PrimeScript RT reagent Kit with gDNA Eraser (Takara, Japan). Quantitative real-time polymerase chain reaction (RT-qPCR) was performed with a total volume of 10 μl by using specific primers (Table 1) and TB Green Premix Ex Taq II kit (Takara, Japan) in the ViiA™ 7 Real-Time PCR System (Applied Biosystems, USA). All analyses were performed following the manufacturer’s instructions.

Migration assay

Cell culture supernatant was collected for the transwell migration assay. Fresh human neutrophils were separated as per the previously mentioned method. Transwell plates were incubated in RPMI1640 culture media for 1 hour at 37°C before loading the cells. Neutrophils were resuspended in 10⁵/ml of media and 10⁴ cells were loaded into the upper chamber of the transwell systems while 600 μl cell culture supernatant was loaded into the lower chamber of the transwell systems. The lower chamber was exposed to 0.1% crystal violet and methyl alcohol for 30 minutes for fixing and staining.

Additionally, neutralizing antibodies were added to the lower chamber right before incubation to neutralize CXCL1 (1:200, Arigo, China), CXCL2 (1:500, GeneTex, USA) and CXCL8 (1:100, Arigo, China).

Detection of cytokine concentration using Luminex assays

Luminex assays were performed in accordance with the instruction provided in the kit (LXSAHM-03, R&D systems, USA) and the instrument (X-200, Luminex, USA). The concentration was detected by Univ bio-technology corporation (China).

Statistical analyses

All data were recorded as the mean ± standard deviation (SD). Differences between more than two sets of data were evaluated by one-way ANOVA and Tukey’s multiple-comparisons test. P < 0.05 (two-tailed) was considered statistically significant.