Cell culture

A normal human bronchial epithelial cell line (BEAS-2B) was purchased from American Type Culture Collection (ATCC, USA). The cells were cultured in complete RPMI-1640 medium (Gibco, Thermo Fisher Scientific, USA) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% streptomycin/penicillin (Gibco) in a humidified incubator containing a 5% CO2 atmosphere at 37 °C. The inhibition of JAK/STAT3 and ERK1/2 signaling pathway were referenced previous methods [28, 29]. In short, cells were pretreated with tofacitinib (50 μM, a JAK inhibitor, MCE), PD98059 (20 μM, a ERK1/2 inhibitor, MCE) or DMSO (dilution ratio equal to specific inhibitors) for 1 h, and then exposed to rhIL-24 (100 ng/ml) for 24 h or 48 h. All cell experiments were repeated independently three times.

Cell viability assay

To determine the cytotoxic effect of IL-24 in BEAS-2B cells, a Cell Counting Kit-8 assay (Beyotime, Shanghai, China) was performed according to the manufacturer’s instructions. Cells were seeded in a 96-well plate (1 × 104/well) and treated with various concentrations of IL-24 (0.1–100 ng/ml, cat# 200-35-20, PeproTech, USA) at 37 °C. After incubation for 24 h, 10 μl CCK-8 solution (Beyotime) was added to each well, and then the cells were incubated for 2 h at 37 °C. Finally, the absorbance at 450 nm was measured using a microplate reader (BioTek, USA). We set five replicate wells per group, and each experiment was repeated three times.

Cell apoptosis assay

For apoptosis analysis, the cells (5 × 105/well) were seeded in a 6-well plate and treated with 10 or 100 ng/ml IL-24 for 24 h. The single-cell suspensions were collected and incubated with 3 μl Annexin V-FITC and propidium iodide (PI) antibodies (Beyotime) for 15 min at room temperature in the dark for apoptosis analysis using a flow cytometer (BD Biosciences, USA) according to the kit’s procedures. The data represent three independent experiments.

Cell cycle analysis

For cell cycle analysis, cells (5 × 105/well) were seeded in 6-well plates and treated with 10 or 100 ng/ml IL-24. After 24 h, the cells were harvested and fixed with ice-cold 70% ethanol overnight at 4 °C. The cells were washed twice in ice-cold phosphate-buffered saline (PBS) and subsequently incubated with RNase A/PI solution (Beyotime) for 30 min at room temperature after centrifugation and removal of ethanol. The DNA contents were carried out by flow cytometry. Histograms of DNA were analyzed using ModFit LT software (Verity Software House, USA). The data represent three independent experiments.

Quantitative real-time-PCR (RT-qPCR)

Total RNA from cultured cells was extracted by RNAiso Plus reagent (Takara, Dalian, China) according to the manufacturer’s protocol. Then, the RNA sample was reverse transcribed into cDNA by PrimeScript RT Master Mix (Takara). RT–qPCR was performed using TB Green Premix Taq (Takara) following the standard procedure on an ABI PRISM 7500 sequence detector (Applied Biosystems, USA). RT–qPCR conditions were as follows: 30 s at 95 °C, followed by 40 cycles of 5 s at 95 °C and 34 s at 60 °C. The relative levels of target genes were compared with the internal reference GAPDH and calculated by the 2−ΔΔCt method. The primers are shown in Table 1. All RT-qPCR experiments were repeated independently three times (n = 3 repeated wells).

Table 1 Primer sequences used in this studyWestern blot

The proteins of cells or murine right lung tissues were extracted by ice-cold RIPA lysis buffer containing protease and phosphatase inhibitor cocktails (Beyotime) on ice. Protein concentrations were determined with a BCA protein quantification kit (DingGuo, China). The samples (40–70 μg total protein/well) were loaded onto SDS–PAGE gels and then transferred onto PVDF membranes. After blocking with 5% bovine serum albumin (5% BSA, Sigma-Aldrich, USA) for 1 h, the bands were incubated with the appropriate primary antibody (dilution, 1:1000) overnight at 4 °C. Next day, the secondary antibody of goat anti-rabbit or goat anti-mouse IgG linked with HRP (dilution, 1:3000) was incubated for 1 h at room temperature. The bands were visualized with enhanced chemiluminescence (ECL) solution (Merck Millipore, Germany), and the density was quantified using ImageJ software. The relative expression levels were analyzed with β-actin as a loading control. The antibodies were used as follows: anti-E-cadherin (cat# 14472, Cell Signaling Technology, USA), anti-vimentin (cat# 5741, CST), anti-α-SMA (cat# ab32575, Abcam), anti-p-STAT3(cat# 9145, CST), anti-STAT3(cat# 12,640, CST), anti-p-ERK1/2 (cat# 4073, CST), anti-ERK1/2 (cat# 4695, CST), anti-p-p38MAPK (cat# 9215, CST), anti-p38MAPK (cat# 8690, CST), anti-p-NF-κb p65 (cat# 3033, CST), anti-NF-κb p65 (cat# 8242, CST), anti-p-JNK (cat# 4668, CST), anti-JNK(cat# 9252, CST), β-actin (cat# 20536-1-AP, Pepro Tech), anti-rabbit IgG HRP-linked antibody (cat# 7074, CST), and anti-mouse IgG HRP-linked antibody (cat# 7076, CST).

Wound healing (scratch) assay

Cells were plated on 6-well plates (2 × 105 cells/well) in complete culture medium until 90% confluence. The straight monolayers were scratched with a 10 μl pipette tip on the bottom of the plate and washed three times with PBS. IL-24 (100 ng/ml, PeproTech) with or without IL-37 (100 ng/ml, cat# 200–39-25, PeproTech) was added to the medium and cultured at 37 °C. The area of the scratch was recorded at 0 h, 12 h, and 24 h under an inverted microscope and then quantified using ImageJ software. All wound healing assay was repeated three times. At least three randomly selected fields were calculated, and the average closure area rates were presented.

Cell migration assay

Cell migration assays were conducted using 24-well Transwell chambers (8.0 μm; Corning, USA). In brief, 600 μl medium (supplemented with 10% FBS) was added to the lower chamber, and 100 μl of serum-free cell suspension (1 × 105 cells) was added to the upper chamber. After IL-24 (100 ng/ml, PeproTech) with or without IL-37 (100 ng/ml, PeproTech) was added to the lower chamber as a chemoattractant, the chambers were incubated at 37 °C for 24 h. After the cell suspension of the upper chamber was removed, the lower chambers were fixed with 4% paraformaldehyde (Biosharp, China) for 1 h and stained with 0.5% crystal violet for 15 min. The migrated cells to the bottom surface were captured using an inverted microscope (Nikon, Japan), and the average number of migratory cells in five randomly selected fields of each well was calculated using ImageJ software. Representative histogram represents three duplicate experiments.

Immunofluorescence

The treated BEAS-2B cells or murine lung sections were fixed with 4% paraformaldehyde and then permeated with 0.2% Triton X-100 (Solaibio, China) for 30 min. After blocking with 5% BSA for 1 h at room temperature, the cells or slides were incubated with anti-vimentin (1:200, CST), anti-α-SMA (1:200, Abcam), anti-p-STAT3 (1:400, CST), anti-p-ERK1/2 (1:400, CST), anti-IL-1R8(1:50, cat# PA5-20,078, Invitrogen), and anti-IL-18Ra (1:50, cat# MAB840, RD) primary antibodies overnight at 4 °C. The second day, the cells were incubated with Alexa Fluor 488-labeled goat anti-rabbit IgG (1:1000, cat# 4412, CST), Alexa Fluor 488-labeled mouse anti-rabbit IgG (1:1000, cat# 4408, CST), Alexa Fluor 555-labeled goat anti-rabbit IgG (1:1000, cat# 4409, CST) and Alexa Fluor 555-labeled goat anti-mouse IgG (1:1000, cat# 4413, CST) for 1 h in the dark and subsequently counterstained with DAPI (Beyotime) for nuclear staining. Images were captured under a fluorescence microscope (Nikon, Japan). Five random fields of each slide were selected for quantification of fluorescence intensity.

Animal experiments

Wild-type (WT) SPF BALB/c mice (female, 6–8 weeks, 20–25 g) were purchased from Yancheng Biotechnology Co., Ltd [Guangzhou, China, license number: SCXK (liao) 2020-0001], and maintained under SPF conditions with regular food and water supplementation under a 12 light/dark cycle at 22 ± 2 °C. Animal experimental procedures were approved by the Ethics Committee of Animal Experiments of the Third Affiliated Hospital of Sun Yat-sen University.

Antigen challenged protocol

Mice were randomly assigned to five groups (n = 8/group): (1) PBS group (control group); (2) HDM group (asthma group); (3) HDM plus si-IL-24 group (asthma + si-IL-24 group); (4) HDM plus si-NC group (asthma + si-negative control group); and (5) HDM plus IL-37 group (asthma + IL-37 group). To establish a chronic allergic asthma model, the mice were delivered intranasally with 25 μg of house dust mite (HDM) extract (cat. No. XPB82D3A2.5, Geer) dissolved in 10 μl PBS, 5 days per week for five consecutive weeks, as previously described [27]. From the third week, mice in group 3 or group 4 were treated with si-IL-24 or si-NC (1 nmol/mouse/day, RiboBio Biotechnology, Guangzhou, China), respectively, intranasally 1 h prior to HDM challenge, three times per week for 3 weeks. The mice in group 5 received rhIL-37 intranasally (1 μg/mouse/day, Pepro Tech) 1 h prior to HDM challenge, three times per week for 3 weeks. Apart from that, the control group mice were administered an equal amount of PBS at the same time as the negative control. All mice were euthanized 24 h after the last exposure. The small interfering (si)RNA sequence for silencing IL-24 in vivo: 5′-GACCUGGAUGCAGAAAUUCUAtt-3′, 5′-UAGAAUUUCUGCAUCCAGGUCtt-3′.

Assessment of airway hyperresponsiveness (AHR)

Airway resistance of mice was measured using a Buxco® FinePointe™ RC system (DSI, USA) within 24 h after the last HDM challenge. Mice were anesthetized with 1% pentobarbital sodium (60 mg/kg, intraperitoneal injection) and intubated intratracheally to connect to an animal ventilator (120 breaths/min) following exposure to aerosolized saline or increasing doses of methacholine (6.25–50 mg/ml, Sigma–Aldrich). Mice were nebulized for 180 s at each dose and for 30 s intervals in whole-body plethysmography. Then, the average Penh values reflecting airway resistance were detected and analyzed from each group (8 mice per group).

Analysis of bronchoalveolar lavage fluid (BALF)

The tracheas were flushed with 0.5 ml precooled PBS three times after tracheal cannula. The supernatant of BALF was harvested and centrifuged at 3000 r/min for 5 min at 4 °C, followed by storage at -80 °C for further ELISA analysis. After lysis of RBCs, the total number of cells was counted with a hemocytometer and then the remaining suspension was prepared onto slide preparations by a centrifugal machine for further Diff-Quick staining (TBD, Tianjin, China). At least 400 cells/slide were counted to analyze the percentage of macrophages, eosinophils, neutrophils and lymphocytes under a light microscope at × 200 magnification. For ELISA, the level of active TGF-β1 in BALF was measured by commercial ELISA kits (cat#70-EK981-96, Multisciences Biotech, China) according to the manufacturer’s protocol.

Histopathological analysis

Mouse lung sections (5 μm) were deparaffinized and rehydrated, and then hematoxylin and eosin (H&E) staining, periodic acid-Schiff (PAS) staining and Masson’s trichrome (Masson) staining were performed to assess the extent of inflammation, mucus production and collagen deposition in murine lung tissues according to the manufacturer’s instructions. The images were captured with a light microscope. The airway inflammation score was quantified according to previously published methods [30]. The quantification of PAS staining and Masson staining were performed by ImageJ software.

Immunohistochemistry (IHC)

IHC was performed on paraffin-embedded sections (5 μm) from murine left lung tissues. After deparaffinization and rehydration, the slides were incubated in 3% H2O2 for inactivation of endogenous peroxide and permeabilized with 1% Triton-X (Solarbio, China). The slides were preincubated in citrate buffer and heated for 15 min in a pressure cooker for antigen retrieval. After blocking with 5% BSA, the tissue sections were incubated with anti-rabbit IL-24 antibody (1:100, Pepro Tech) and anti-mouse E-cadherin antibody (1:400, CST) at 4 °C overnight. Next, the slides were incubated with the appropriate HRP-labeled goat anti-rabbit/anti-mouse IgG for 1 h. Brown staining was visualized using diaminobenzidine (DAB) solution (Solarbio). Finally, the slides were counterstained with hematoxylin and dehydrated by xylene as well as a grade alcohol series. The slides were sealed and visualized by a light microscope. The average positive brown area of each slice was analyzed through five randomly selected HPF fields using ImageJ software.

Statistical analysis

Data are presented as the mean ± standard deviation (SD) and were analyzed in triplicate or more. Statistical analysis was performed by GraghPad Prism 9.0. Student’s t test and one-way ANOVA were used for significant differences between two or multiple groups, respectively. LSD-t test was used for comparison between groups. In all cases, P < 0.05 was considered statistically significant.