Wild-type BALB/c mice were obtained from Hangzhou Charles River Laboratories, China. All mice were housed in a specific pathogen-free environment, and female mice weighing 18-20 g were used for experiments. All experiments on animals were conducted in accordance with ethical protocols approved by the Laboratory Animal Ethics Committee of Wenzhou Medical University & Laboratory Animal Centre of Wenzhou Medical University.

All mice were randomly assigned to one of four groups: the control group, HDM group, HDM+SCIT group, and HDM+SCIT+GSI group. In this study, we established a model of HAAD and SCIT as described in Figure 1 and Supplementary Table 1 . Mice weighing 18-22 g received an intraperitoneal injection of 5 μg HDM (XPB91D3A25, Greer Laboratories, Lenoir, North Carolina, USA) adsorbed on 2.25 mg of aluminum hydroxide in 100 μl PBS on Days 1 and 15. The mice in the HDM+SCIT and HDM+SCIT+GSI groups were desensitized through subcutaneous injection of 250 μg HDM in 100 μl PBS on Days 29, 31, and 33 (36). The mice in the control group were injected with PBS. In the HDM+SCIT+GSI group, GSI L685, 458 (Merck, USA) (0.3 mg/kg) was given to the mice intratracheally 30 minutes (37–39) before each SCIT. Ultimately, the mice were challenged with 25 μg of HDM in 25 μl of PBS via intratracheal direct injection on Days 45, 47, and 49.

The ear swelling test (EST) (36) was measured to assess the early phase reaction to HDM before and after SCIT on Days 22 and 32, in order to evaluate the allergic sensitization of the mice. Following anesthesia with isoflurane/oxygen, mice were intradermally injected with 10 μl PBS containing 0.5 μg HDM in the right ear and subcutaneously with 10 μl PBS in the left ear as a control. After 2 hours, the thickness of the mouse ears was measured using a digimatic micrometer (Mitutoyo, Japan), and the thickness of the right ear was subtracted from the thickness of the left ear to calculate the degree of HDM-induced swelling.

After the final HDM challenge, mice were anesthetized via intraperitoneal injection of 1% pentobarbital sodium. Once the mice had completely ceased spontaneous respiration, they were immobilized and the trachea was surgically incised for tracheal intubation. Subsequently, the inserted tracheal tube was connected to SCIREQ Scientific Respiratory Equipment (FlexIVent, Montreal, Canada), and the intermittent ventilation mode (150 breaths/min, PEEP 2 cmH₂O) was selected. Baseline airway resistance (Rn) was measured at the stage of saline nebulizing. Then, the mice were challenged with an increasing dose (3.125, 6.25, 12.5, 25, and 50 mg/ml) of methacholine (MCh). And airway Rn was measured at every dose to calculate the percentage of each concentration to baseline.

After evaluating the lung function of the mice, they were cannulated, and the lungs were washed three times with 0.8 ml of cold PBS. The supernatant was collected by centrifuging the bronchoalveolar lavage (BAL) fluid for ELISA, and the cells were resuspended in cold PBS for counting and analyzed by flow cytometry (FCM).

The total protein in lung tissue was isolated and quantified. Protein lysates were electrophoresed using the PAGE Gel Fast Preparation Kit (Epizyme Biotech, China) and then electrotransferred to a polyvinylidene fluoride (PVDF) membrane. The resulting blots were blocked with 5% milk and incubated overnight with the following primary antibodies: anti-Notch1 (Abcam, UK), anti-Hes-1 (Cell Signaling Technology, USA), and anti-tubulin (Affinity Biosciences, USA). Subsequently, goat anti-rabbit IgG-HRP (Affinity, China) as the secondary antibody was incubated at room temperature for 30 minutes.

TRIzol (GlpBio, USA) was employed to extract total RNA from lung tissue. Next, cDNA was synthesized by reverse transcription using qRT Master Mix (TOROIVD, China) according to the manufacturer’s instructions. To examine gene mRNA expression, qPCR (Takara, Japan) was performed with SYBR Green PCR Master Mix. Supplementary Table 3 shows the primer sequences that were employed.

Mouse lung tissues were removed and cleaned in PBS to remove residual blood after right heart lavage with cold PBS under aseptic conditions. Fresh tissues were diced and digested in RPMI1640 medium containing 1 mg/ml Collagenase Type IV (Worthington, USA) and 0.2 mg/ml DNase I (Sigma, UAS) in a 37°C cell incubator for 1 h. Digested tissues were ground and filtered on a 70 μm filter and then further isolated and purified with Ficoll gradient (TBDscience, China). Naturally, the isolated cells were lysed by red blood cell lysis buffer to obtain a single-cell suspension of lung tissues for the following experiments.

Before staining with fluorochrome-conjugated antibodies, which are listed in Supplementary Table 2 , single-cell suspensions were blocked with anti-Fc receptor antibodies (anti-mouse CD16/32). For the flow cytometric fluorescence sorting (FACS), a cell viability dye (7-AAD, BD Bioscience, USA) was used to exclude dead cells. Afterward, fluorochrome-conjugated antibodies were used for surface dyeing (lineage antibodies, anti-CD45, anti-ST2, anti-CD127). We defined the population of cells expressing Lin^-CD45⁺CD127⁺ST2⁺ as ILC2s. The lineage antibodies we used included anti-CD3e, anti-CD11b, anti-CD45R/B220, anti-TER-119, anti-Ly-6G, and anti-Ly-6C, and then a MoFlo Astrios EQ (Beckman Coulter, USA) was employed for sorting. The purity of isolated ILC2s was up to 96.2%. For intracellular transcription factor staining, sorted ILC2s were handled with the Foxp3/Transcription Factor Staining Buffer Set (eBioscience, USA) according to instructions of the manufacturer after in vitro culture and stained with fluorochrome-conjugated antibodies anti-GATA3 antibodies at 4°C for 1.5 h.

To investigate the function of ILC2s in vitro, lung ILC2s were isolated from the HDM group and HDM+SCIT group, cultured on 96-well plates at a density of 5×10³ per well, and processed with GSI (20 μM/ml) or PBS in the presence of mouse IL-2 (10 ng/ml, PeproTech, USA) and IL-7 (10 ng/ml, PeproTech, USA). The intracellular transcription factor GATA3 of ILC2s was analyzed by FCM after stimulation for 16 h, while the cytokines in the culture supernatant were detected by ELISA after stimulation for 48 h.

The BAL fluid and serum from mice, as well as the supernatant from cultured ILC2s, were collected and stored at -80°C. Levels of the cytokines IL-4, IL-5, IL-13, and IgE were determined by ELISA as described by the manufacturer (Thermo Fisher, USA). To detect HDM-sIgE, we coated HDMs (10 μg/plate) on 96-well plates and incubated them overnight at 4°C. After washing with PBST, the coated 96-well plates were blocked with assay diluent buffer for 2 h at room temperature. Then, a 1:80 dilution of serum was added to the plate and incubated at room temperature for 2 h. Subsequently, after washing with PBST, the detection antibody (1:1000 diluted, Biotin Rat Anti-Mouse, BD Pharmingen, USA) was added to the plate and incubated at room temperature for 1 h. Next, 100 μl 1× streptavidin-horseradish peroxidase (HRP) was added to the 96-well plate and incubated for half an hour at room temperature. At last, 100 μl TMB was added to the plate and incubated for 15 min and then the reaction was stopped by adding stop solution. The absorbance of the plate was detected at 450 nm using an enzyme marker. The concentration of total IgE ranged from 4 to 250 ng/ml, with a detection range of 4-500 pg/ml for IL-4, IL-5, and IL-13.

Fresh lung tissue in 4% paraformaldehyde solution fixation for 48 h, followed by paraffin embedding. After tissue sectioning, hematoxylin-eosin (H&E) staining, Masson staining, and Alcian Blue Periodic acid Schiff (AB-PAS) staining were followed. Airway inflammation scores were referenced from our previous report (31, 37).

All data are expressed as the mean ± SEM and statistical significance was determined by one-way ANOVA or Student’s two-tailed t-test. GraphPad Prism 8.0 software was used for statistical analysis, and FlowJo 10.6.2 software was employed for FCM analysis. P<0.05 was recognized as a significant difference.

To investigate the potential impact of GSI on ILC2s function, we primarily evaluated its effect on the production of type 2 cytokines by HDM-activated ILC2s. To promote the expansion of ILC2s, BALB/c mice were pretreated with HDM, and lung ILC2s were isolated as Lin^-CD45⁺ST2⁺CD127⁺ cells ( Figure 2A ) using the sorting gating strategy (S3). We treated sorted lung ILC2s with GSI and observed a notable decrease in the levels of IL-13 and IL-5 in the supernatant of cultured ILC2s, as well as a reduction inintracellular GATA3 expression levels in ILC2s ( Figures 2B–E ).

To examine the expression of Notch in the HAAD model, lung tissues were harvested and assessed using western blotting and qPCR. As shown in Figure 3 , the mRNA expression of Notch1 was significantly higher in the lung tissue of the HDM group compared to the control group. Interestingly, the protein expression of Notch1 and its downstream target Hes-1 was dramatically elevated in the lung tissue of HDM mice compared to the control group. These findings indicate that HDM induces an increase in Notch signaling expression in the lung tissue of asthmatic mice.

To begin with, we evaluated the extent of ear swelling in mice before and after SCIT treatment ( Figure 4A ). We observed that ear swelling was significantly more severe in the asthma group of mice compared to the control group (P<0.001), but it was reduced after SCIT treatment compared to the asthma group (P<0.001). This indicates that SCIT therapy suppresses early allergic reactions in mice. Subsequently, we measured total serum IgE and HDM-specific IgE (HDM-sIgE) levels in mice post-SCIT and challenge ( Figures 4B, C ). The total serum IgE levels in the SCIT group post-SCIT and challenge were not statistically significant compared to the asthma group. However, serum HDM-sIgE levels in the SCIT group were significantly lower post-challenge than those in the asthma group (P<0.001). These results suggest a successful SCIT model. Airway resistance (Rn) was evaluated after the last challenge. The results ( Figures 4D–H ) showed that airway resistance was higher in the HDM group of mice compared to the control group (P<0.5), while SCIT significantly reduced Rn (P<0.5). Interestingly, the decrease in airway resistance was more pronounced in the HDM+SCIT+GSI group mice compared to the HDM+SCIT group(P<0.5). These observations suggest that GSI in combination with SCIT can attenuate HDM-induced AHR and early allergic reactions in mice.

The lung histopathological sections were stained with H&E( Figures 5A–C ). As expected, bronchial wall thickness decreased after SCIT and inflammatory cell infiltration in lung tissue was alleviated (P<0.5). The total cell count and eosinophil count of BAL fluid were significantly reduced ( Figures 5F–H ). More significantly, inflammatory cell infiltration in the lungs of mice in the HDM+SCIT+GSI group was considerably lower than in the HDM+SCIT group ( Figures 5A–C ). Similarly, the HDM+SCIT+GSI group showed the most prominent decrease in BALF total cell count and eosinophil count compared to the HDM+SCIT group ( Figures 5F–H ).

Afterward, to further investigate the effect of GSI combined with SCIT on asthma, lung tissues of mice were examined using Masson staining ( Figures 5A, D ) and AB-PAS staining ( Figure 5E ). We found that SCIT dramatically reduced peritracheal collagen deposition (P<0.5) and decreased airway mucus secretion. Unexpectedly, the percentage of peritracheal collagen deposition in mice in the HDM+SCIT+GSI group (P<0.5) decreased obviously compared with that in mice in the HDM+SCIT group, and airway mucus secretion was also reduced. Overall, these results indicate that GSI combined with SCIT can synergistically alleviate airway inflammation and inhibit airway collagen fibrosis and mucus secretion.

Asthma is closely correlated with the type 2 cytokines interleukin (IL)-4, IL-5, and IL-13 (40). Therefore, we measured IL-4, IL-5, and IL-13 levels in serum and BALF using ELISA. The results ( Figure 6 ) showed that IL-4, IL-5, and IL-13 levels were substantially increased in the HDM group compared to the control group (P<0.5), while the HDM+SCIT group (P<0.5) showed a significant decrease compared to the HDM group. IL-4, IL-5, and IL-13 levels were further reduced by combining SCIT with GSI treatment (P<0.5). These results indicate that combining SCIT and GSI can effectively reduce inflammatory cytokines in BAL fluid and serum.

Next, we investigated the effect of SCIT in combination with Notch signaling pathway inhibitors on ILC2s. We first evaluated the ratio of lung ILC2s in the HAAD and SCIT models ( Figures 7A, B ). Using FCM, we found that the percentage of ILC2s decreased after SCIT compared to the HDM group (P<0.5). When SCIT and GSI were combined (P<0.5), the ILC2s ratio decreased significantly compared to SCIT alone. Furthermore, ILC2s were extracted by FACS from lung tissue of mice in the HDM+SCIT group and cultured in vitro ( Figures 7C–G ). It was discovered that inhibiting the Notch signaling pathway decreased the MFI of intracellular GATA3 in ILC2s and reduced IL-13 and IL-5 production. Overall, SCIT and GSI treatments have the potential to synergistically suppress the activation of ILC2s in mice with HAAD.