C57BL/6J female mice (age, 6–8 weeks, body weight, 18–20 g) were purchased from the animal core facility of Nanjing Medical University. Colony-stimulating factor 3 deficient (Csf3 ^-/-) and peptidyl arginine deiminase 4 deficient (Padi4 ^-/-) C57BL/6J female mice were purchased from Cyagen Biosciences (Suzhou, China). Genotypes were identified by PCR analysis of tail DNA ( Supplementary Figures 1A, B ). These animals were housed in specific pathogen–free (SPF) conditions,12/12 h controlled light conditions with ad libitum access to water and food. All mice experiments were performed with approval from the Institutional Animal Care and Use Committee, Nanjing Medical University (reference number: IACUC-2208009). We guarantee that all experimental procedures were carried out in accordance with the guidelines established by the Institutional Animal Care and Use Committee, Nanjing Medical University. These experimental methods complied with Helsinki Declaration, and every effort was made to minimize suffering.

The mouse model was carried out with reference to the previous literature with slight modification (3, 18). The mice were randomly assigned to four groups: the phosphate buffered saline (PBS) group, the LPS group, the OVA group, and the LPS + OVA group. mice were intraperitoneally (i.p.) injected with 100μg OVA (grade V, Sigma-Aldrich, USA) and 50μl aluminum hydroxide gel (Thermo Fisher Scientific, USA) dissolved in 100μl PBS or 50μl aluminum hydroxide gel dissolved in 100μl PBS in non-OVA groups during the sensitization phase on days 0 and 7. From days 14 to 16, these mice were challenged by intratracheally (i.t.) instillation of OVA (25μg OVA dissolved in 50μl PBS) or PBS in non-OVA groups. Then, sacrificed the mice on day 17. In LPS and LPS+OVA groups,10μg LPS from Escherichia coli 0111: B4 (Sigma-Aldrich, USA) dissolved in 50μl of PBS was administered by intratracheal instillation on days 0 and 7.LPS was replaced by PBS in non-LPS groups. mice were lightly anesthetized with pentobarbital sodium when intratracheal intubation for drug administration was required.

Tracheas were cannulated and the right lung was lavaged slowly 3 times with 0.5ml PBS following the left lung ligation. The recovery rate of the BALF was greater than 60%. Each fluid was centrifuged and the supernatant was rapidly frozen at -80°C. The cells in BALF were resuspended in PBS and centrifuged in a cytocentrifuge, then stained with Wright−Giemsa (Baso, Zhuhai, China) and identified as macrophages, eosinophils, neutrophils and lymphocytes based on cellular morphology and staining characteristics ( Supplementary Figure 2A ). At least 200 cells were counted under x400 magnification.

After BALF was collected, the left lung tissues were completely removed, then fixed with 4% paraformaldehyde, embedded, processed into 4μm sections and stained with hematoxylin and eosin (HE) and periodic acid-Schiff (PAS). The infiltration of inflammatory cells and the level of mucus secretion in the airway was scored as previously described (19).

Pulmonary function was carried out by the AniRes2005 animal lung function analysis system (Bestlab High-Tech, Beijing, China). After system calibration, all mice were anesthetized with pentobarbital sodium, tracheostomized, and placed in a forced pulmonary maneuver system. Mice were administered methacholine chloride (Mch, Sigma-Aldrich, USA) with doses increasing in multiples via the jugular vein. RI and Cdyn were measured.

Concentrations of interleukin IL6 and IL8 (Mouse KC) in BALF were quantitated by enzyme-linked immunosorbent assay (ELISA) kit (YOBIBIO, Shanghai, China) according to the manufacturers’ instructions. The amount of dsDNA in the BALF was evaluated according to the instructions of the kit (Thermo Fisher Scientific, USA). The MPO-DNA complex was used as a quantified marker of NET release with a capture ELISA (YOBIBIO, Shanghai, China).

To identify NETs from lung tissues, the samples were first undergone antigen retrieval (citrate buffer, pH 6.0), blocking (5% BSA/0.3% Triton X-100), and then incubation with anti-citrullinated histone H3 (CitH3, 1:200, Abcam, ab5103) and anti-myeloperoxidase (MPO, 1:200, Abcam, ab300650) antibodies at 4°C overnight before treatment with secondary antibodies. After 4’,6-Diamidino-2-phenylindole (DAPI) staining, the sections were observed by a Leica SP8 confocal system, and images were captured randomly by independent experimenters blinded to group allocation throughout the study.

Mice neutrophils were isolated from bone marrow. Briefly, the femurs and tibias were harvested and stripped of all muscle and sinew. The bone marrow was flushed out with 10 mL of RPMI medium containing 5% fetal bovine serum and 2mM EDTA on ice, then filtered (100um), and subjected to discontinuous Percoll gradient centrifugation (52%,65%,78%) at 500×g (30min, 4°C). The cells were gently aspirated from the neutrophil layer (65-78%), followed by deleting the erythrocytes and resuspension in Hanks’ Balanced Salt Solution (HBSS) containing 1% fetal bovine serum. HE and trypan blue staining was performed to identify the proportion and viability of neutrophils.

Neutrophil RNA was extracted with TRIzol (Invitrogen, USA). BGI DNBSEQ performed 150-bp paired-end sequencing. Low-quality reads (Q<15), adapters and poly-N were filtered. HISAT2 (v2.2.1) aligned clean reads to GRCm39, HTseq (v0.6.0) generated counts. DEGs were identified using the DEGseq method and screened with the criteria of Q value ≤ 0.05 and|log2FC|≥ 1. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis was also carried out with the online platform Dr. Tom (BGI Company, Shenzhen, China).

All statistics and graphs were performed using GraphPad Prism software v9.4.1 (San Diego, CA, USA). One-way ANOVA with Tukey’s post hoc method was used for multiple comparisons. Data are presented as means ± standard deviation (SD). Significant differences are shown as *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

To establish the mouse model of neutrophilic asthma, adult C57BL/6J mice were intraperitoneally (i.p.) sensitized with ovalbumin–aluminum hydroxide (OVA/alum) on days 0 and 7, and then challenged with OVA intratracheally (i.t.) on days 14, 15 and 16. LPS was instilled intratracheally (i.t.) during the sensitization phase ( Figure 1A ).

Compared with OVA group, the combination of LPS and OVA significantly increased the airway total cell counts, neutrophil count and proportion ( Figures 1B-D ), indicating that neutrophils were accumulating in airway. Intriguingly, despite the LPS+OVA group exhibiting a significantly lower proportion of eosinophil compared to the OVA group, their eosinophil counts were similar ( Figures 1C, D ). There was no notable difference in BALF cell count or neutrophil proportion between LPS and PBS groups. However, total cells, neutrophil counts and proportions in BALF of LPS group significantly increased by day 8 ( Supplementary Figures 3A-D ), suggesting that under non-allergic conditions, airway neutrophils had disappeared by day 17. Both HE and PAS scores were significantly elevated in LPS+OVA group. Compared with other groups, LPS group did not exhibited airway hypersecretion, indicating that LPS alone was insufficient to induce persistent mucin secretion ( Figures 1E-G ). Both airway resistance and lung dynamic compliance were more compromised in LPS+OVA group than in OVA group ( Figures 1H, I ). However, LPS intervention alone did not result in airway hyperreactivity.

Taken together, the LPS+OVA mouse model demonstrated features of neutrophilic asthma, including airway inflammation, hypersecretion, hyperresponsiveness, and significant infiltration of neutrophils.

Previous studies reported that IL6 and IL8 were associated with neutrophil recruitment (19, 20). Our findings showed elevated IL6 and IL8 levels in LPS+OVA group, aligning with increased airway neutrophil infiltration. ( Figures 2A, B ). The dsDNA concentration in the BALF of LPS+OVA group was much higher than in other groups, with no significant differences among the PBS, LPS, and OVA groups ( Figure 2C ). On day 8, dsDNA levels rose dramatically in LPS and LPS + OVA groups, consistent with BALF neutrophil counts ( Supplementary Figure 3E ). Given that dsDNA may originate from various cellular sources, we quantified myeloperoxidase-DNA (MPO-DNA). Results indicated that LPS treatment significantly elevated MPO-DNA levels in BALF, particularly when combined with OVA ( Figure 2D ).

Immunofluorescence staining of lung tissue sections was next conducted. As anticipated, in LPS+OVA group, there was a substantial co-localization of MPO and citrullinated histone H3 (CitH3), indicating the release of NETs by lung neutrophils ( Figure 2E ).

To explore the role of neutrophils in neutrophilic asthma, we conducted experiments using colony-stimulating factor3 (Csf3) deficient mice with significantly reduced neutrophils in vivo. In Csf3 ^-/-mice, OVA could induce allergic asthma characterized by eosinophilic airway inflammation and airway hyperresponsiveness ( Figures 3A-H ). In LPS+OVA group, Csf3 ^-/- mice had significantly lower total cell, neutrophil counts and neutrophil proportion in BALF compared to WT mice, while eosinophil levels were similar in both groups ( Figures 3A-C ). Additionally, the pulmonary resistance in Csf3 ^-/- mice in the LPS+OVA group was significantly lower than that in WT mice and even lower than in the OVA group of WT mice. Notably, it was comparable to the OVA group in Csf3 ^-/- mice ( Figure 3D ). The lung compliance results mirrored those of lung resistance ( Figure 3E ), indicating that neutrophils may play a crucial role in the development of airway hyperresponsiveness. Similar to WT mice, LPS intervention did not exacerbate lung function ( Supplementary Figures 4A, C ). In LPS+OVA group, Csf3 ^-/- mice exhibited reduced levels of airway inflammatory infiltration and mucus hypersecretion compared to WT mice ( Figures 3F-H ). The neutrophil related inflammatory indicators such as IL6, IL8 and dsDNA, MPO-DNA which represented levels of NETs were analyzed, similar results were obtained in LPS+OVA group between Csf3 ^-/- mice and WT mice ( Figure 3I ). Immunofluorescence staining conducted on lung tissues demonstrated a significant reduction in NETs co-localizing with MPO and citH3 in LPS+OVA group of Csf3 ^-/- mice ( Figure 4A ).

To further investigate the mechanism by which neutrophils contribute to asthma exacerbation, we isolated bone marrow-derived neutrophils from the OVA and LPS+OVA groups of WT mice and performed transcriptome sequencing analysis. This analysis identified 1,625 DEGs, with 872 genes up-regulated and 753 genes down-regulated ( Figure 4B ; Supplementary Figure 5A ). Among these, 184 DEGs were related to immune function. We conducted Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses on the DEGs involved in immune pathways. The KEGG analysis indicated significant enrichment of DEGs in the neutrophil extracellular trap formation pathway ( Figure 4C ). GO analysis revealed that DEGs were significantly enriched in processes related to neutrophil chemotaxis, aggregation, and activation ( Figure 4D ). A heatmap was generated to visualize the expression levels of 26 DEGs and 179 genes associated with the NETs pathway ( Figure 4E ; Supplementary Figure 5B ). It was observed that, compared to the OVA group, the DEGs and genes in LPS+OVA group exhibited significantly higher expression levels. These findings suggested that neutrophils and NETs may be involved in neutrophilic asthma at the transcriptome level. Interestingly, KEGG pathway analysis of immune-related DEGs in LPS group versus PBS group did not show enrichment in the NETs pathway ( Supplementary Figures 5C, D ), implying that under allergic conditions, neutrophils and their NETosis function may be activated for a prolonged period.

To further explore the role of NETs in neutrophilic airway inflammation, we conducted experiments utilizing peptidyl arginine deiminase 4 (Padi4) deficient mice. Padi4 participated in the formation of NETs by catalyzing the citrullination of arginine residues in histones. Knocking out the Padi4 gene significantly inhibited the generation of NETs. In Padi4 ^-/-mice, we established a conventional model of allergic asthma via OVA sensitization and challenge, characterized by inflammatory cell infiltration, goblet cell hypersecretion, and airway hyperresponsiveness. Treatment with OVA in conjunction with LPS led to an increase in neutrophil count and proportion, but did not exacerbate the aforementioned asthma-related parameters ( Figures 5A-F ). Compared to the WT+LPS+OVA group, the total cell count, neutrophil count, and proportion in the BALF of the Padi4 ^-/-+LPS+OVA group were significantly reduced. However, there was no significant difference in the proportion and count of eosinophils ( Figure 5A ). In Padi4^-/- mice, lung resistance and dynamic compliance in LPS+OVA group were also significantly improved compared to those in WT mice ( Figure 5C ). Similar to WT and Csf3^-/- mice, LPS intervention did not exacerbate lung function ( Supplementary Figures 4B, D ). Surprisingly, the lung resistance in LPS+ OVA group of Padi4 ^-/- mice was significantly higher than that of Csf3 ^-/-mice ( Supplementary Figure 4E ), suggesting that, beyond NETs, additional neutrophil functions may contribute to airway hyperresponsiveness. As expected, Inhibiting NETs formation improved inflammatory infiltration and mucus hypersecretion ( Figures 5D-F ). After knocking out Padi4, the combination of LPS and OVA did not exacerbate the production of IL6 and IL8 ( Figures 5G, H ), nor did it increase the production of dsDNA and MPO-DNA ( Figures 5I, J ). The co-localized MPO-CitH3 immunofluorescence further confirmed a significant reduction of NETs generation in LPS+OVA group ( Figure 5K ). In other groups, there were no significant differences among mice with different genotypes ( Supplementary Figure 2B ).