A total of 60 Specific pathogen-free (SPF) female BALB/c mice, 6–8 weeks of age, with an average weight of 20–25 g, were purchased from Slac Laboratory Animal Corporation and housed in SPF conditions (~19–27°C, 12-h light/dark cycle and ~40–70% humidity). All experimental protocols described in the present study were approved by the Institutional Animal Care and Use Committee of Shanghai Pudong Hospital and the procedures were approved by the Biological Research Ethics Committee of Shanghai Pudong Hospital.

IAL (cat. no. C15H20O2; MW, 232.31; purity >98%) was purchased from TargetMol; OVA (Grade V) was purchased from Sigma-Aldrich (Merck KGaA); Dulbecco's modified Eagle's medium (DMEM), antibiotics (10,000 units/ml penicillin and 10,000 µg/ml streptomycin), 0.25% trypsin and phosphate buffered saline (PBS) were purchased from Thermo Fisher Scientific, Inc.; Fetal bovine serum (FBS) was purchased from EVERY GREEN (Zhejiang Tianhang Biotechnology Co., Ltd.); and bicinchoninic acid (BCA) protein assay kit was purchased from Beyotime Institute of Biotechnology. The primary antibodies used in the present study were synthesized by Shanghai HuaGen Biotech Co., Ltd.. Phosphorylated (p)-STAT6 (Tyr641; cat. no. 56554), total (t)-STAT6 (cat. no. 5397), PPAR-γ (cat. no. 2443), KLF4 (cat. no. 12173) and β-actin (cat. no. 3700) were purchased from Cell Signaling Technology, Inc. The peroxidase AffiniPure goat anti-rabbit IgG (H + L; cat. no. 111-035-003) and peroxidase AffiniPure goat anti-mouse IgG (H + L) (cat. no. 115-035-003) secondary antibodies were purchased from Jackson ImmunoResearch Laboratories and diluted in EZ-Buffers (cat. no. C520011-0100; Sangon Biotech Co., Ltd.). Other chemical reagents were obtained from Sigma-Aldrich. Loading buffer was prepared by 1M TrisCl (cat. no. A610195; Sangon Biotech Co., Ltd.), 10% SDS (cat. no. B548118; Sangon Biotech Co., Ltd.), 7.5 mM bromophenol blue (cat. no. 60503ES08; Shanghai Yeasen Biotechnology Co., Ltd.) 25% glycerin (cat. no. A501745; Sangon Biotech Co., Ltd.) and 35.75 µM β-mercaptoethanol (cat. no. 200-464-6; Sigma-Aldrich; Merck KGaA).

Mice were randomly divided into three groups as follows: PBS + vehicle group, OVA + vehicle group and OVA + IAL group. IAL was dissolved in a vehicle comprising castor oil:ethanol: Physiological saline at a 1:1:8 ratio. For the OVA + vehicle group, mice were sensitized on days 0, 7 and 14 [40 µg OVA and 40 mg Al(OH)₃ in 0.2 ml PBS, intraperitoneal (i.p.)] and challenged at days 25, 26 and 27 [10 µg OVA in 0.2 ml PBS, intranasal (i.n.)]. For the OVA + IAL group, following sensitization, mice were administered IAL (20 mg/kg, i.p.) once daily from days 22 to 27, together with challenging at days 25, 26 and 27 (10 µg OVA in 0.2 ml PBA, i.n.). For the PBS + vehicle group, mice were injected with PBS (0.2 ml, i.p.) on days 0, 7 and 14 and the same volume of vehicle as the mice in the OVA + IAL group. Mice were anesthetized with sodium pentobarbital (50 mg/kg; i.p.) to collect samples on day 28 and blood, bronchoalveolar lavage fluid (BALF) and lung tissues were collected for further analysis (20).

The BALF was centrifuged at 500 × g, 4°C following collection. The supernatant was collected for analysis of protein concentration using the BCA protein assay kit according the manufacturer's protocols.

After fixing with 4% paraformaldehyde (Sangon Biotech Co., Ltd.) overnight, the left lung was embedded in paraffin and dehydrated using graded ethanol. Then the tissues were cut into 5 µm thick sections for haematoxylin and eosin (H&E) staining (Nanjing Jiancheng Bioengineering Institute). Images were captured using a microscope (RX51; Olympus Corporation).

Mice were sacrificed by cervical dislocation following anesthesia as aforementioned. Mouse bone marrow-derived macrophages (BMDMs) were isolated from BALB/c mice and incubated with DMEM supplemented with 10% FBS, 1% penicillin/streptomycin at 37°C and 10 ng/ml macrophage colony-stimulating factor (M-CSF; PeproTech, Inc.) for 6 days. BMDMs were incubated at 37°C, 5% CO₂ and stimulated with mouse 10 ng/ml IL-4 (mIL-4; PeproTech, Inc.) and 0, 3, 10, or 30 µM IAL. Cells and culture supernatants were collected for RNA and protein expression analyses.

Western blotting was conducted as previously described (9). Briefly, BMDMs were plated in 6-well plates (1×10⁶/well) and incubated overnight at 37°C, 5% CO₂. BMDMs were pre-treated with IAL (0, 3, 10, or 30 µM) for 30 min and challenged with mIL-4 (10 ng/ml) for 30 min or 24 h, with dimethyl sulfoxide (DMSO) as a solvent control. Cells were washed twice with PBS, harvested using RIPA lysis buffer (Beyotime Institute of Biotechnology) and protein concentration was determined using the BCA method. Subsequently, the samples were loaded on 10% SDS/PAGE gels with 20 µg protein loaded per lane, transferred to nitrocellulose filter membranes and blocked with 5% milk at room temperature for 2 h. The membrane was incubated with primary antibodies (1:1,000) overnight at 4°C. Blots were then incubated with secondary antibodies (1:10,000) for 1 h at room temperature. The antibody-specific proteins were visualized using ECL western blotting substrate (Thermo Fisher Scientific, Inc.) β-actin was used as loading control. Quantification of western blots was performed using ImageJ software 1.4.3.67 (National Institute of Mental Health).

Total RNA was extracted from BMDMs and lung tissues using TRIzol reagent (Thermo Fisher Scientific, Inc.). cDNA was prepared using the ReverTra Ace RT kit (Toyobo Life Science) according to the manufacture's protocol. Gene expression values were quantified by PCR using StepOne Plus (Thermo Fisher Scientific, Inc.). The following thermocycling conditions were used for PCR: Initial denaturation at 94°C for 30 sec; 35 cycles of 94°C for 30 sec, 55°C for 30 sec and 72°C for 2 min; and a final extension at 72°C for 3 min and stored at 4°C. The primers were synthesized by Huagene Biosciences. Relative mRNA levels were normalized to GAPDH mRNA levels. The quantification of RT-qPCR was based on the ΔΔCq (21). The sequences of primers used for each gene were as follows: Arginase-1 (Arg-1) (forward, 5′-CAATGAAGAGCTGGCTGGTGT-3′, reverse, 5′-GTGTGAGCATCCACCCAAATG−3′), Ym-1 (forward, 5′-TACTCACTTCCACAGGAGCAGG-3′, reverse, 5′-CTCCAGTGTAGCCATCCTTAGG-3′), CCL17 (forward, 5′-CGAGAGTGCTGCCTGGATTACT-3′, reverse, 5′-GGTCTGCACAGATGAGCTTGCC-3′), CCL22 (forward, 5′-GTGGAAGACAGTATCTGCTGCC-3′ reverse, 5′-AGGCTTGCGGCAGGATTTTGAG-3′) and GAPDH (forward, 5′-CATCACTGCCACCCAGAAGACTG-3′ reverse, 5′-ATGCCAGTGAGCTTCCCGTTCAG-3′).

After blocking Fc receptors with Fc blocking anti-mouse CD16/32 antibody (BD Biosciences), cells were collected from BALF and incubated with phycoerythrin-conjugated anti-Siglec-F antibodies (BD Biosciences) and allophycocyanin-conjugated anti-CD11c antibodies (BioLegend) at 4°C for 30 min. Samples were analyzed using a flow cytometer (LSRFortessa; BD Biosciences) and FlowJo v10 software (FlowJo LLC).

The concentrations of IL-4 (cat. no. M4000B), IL-5 (cat. no. M5000), CCL17 (cat. no. MCC170) and CCL22 (cat. no. MCC220) were quantified using ELISA kits from R&D Systems, Inc. and OVA-specific IgE (cat. no. 88-50460-88) was quantified using an ELISA kit from Thermo Fisher Scientific, Inc., according to the manufacturer's instructions.

GraphPad Prism 5 (GraphPad Software, Inc.) was used to perform the data analysis. All data, which were obtained from at least three independent experiments, were evaluated with one-way ANOVA and Tukey's test was used to determine the difference between two groups. P<0.05 was considered to indicate a statistically significant difference.

To investigate the effect of IAL on asthmatic inflammation, an OVA-induced mouse asthma model (Fig. 1A) was established as shown in Fig. 1, inflammatory cell infiltration was induced in OVA-induced asthmatic inflammation. However, IAL pretreatment significantly reduced cell infiltration (Fig. 1B), indicating that IAL relieved OVA-induced asthmatic inflammation. In addition, treatment with IAL did not result in an increase in serum OVA-specific IgE, whereas OVA treatment significantly increased the expression of OVA-specific IgE (Fig. 1C). Furthermore, IAL significantly reduced the protein concentration in BALF caused by OVA treatment (Fig. 1D). These results indicated that IAL had an inhibitory effect on asthmatic inflammation.

In the process of asthma, lung tissues are destroyed by the infiltration of inflammatory mediators and inflammatory cells, especially eosinophils, which are significantly increased in BALF. Therefore, the number of eosinophils in BALF indicates the severity of asthma (22). Thus, the accumulation of eosinophils in BALF was measured to determine the therapeutic effect of IAL on inflammatory cell infiltration. As shown in Fig. 2, the proportion of eosinophils increased significantly following OVA induction, while it was significantly reduced following IAL treatment, from 98.3 to 37.6% (Fig. 2A). In addition, the number of total cells in the BALF significantly increased following OVA induction. Following IAL treatment, the total number of cells in the BALF was significantly lower (Fig. 2B). Consistent with this, the proportion of eosinophils and the number of eosinophils in BALF were significantly decreased following IAL treatment (Fig. 2C and D). Furthermore, the absolute number of macrophages in BALF was detected and it was found that the total macrophage number in BALF was enhanced following challenge with OVA. However, IAL treatment did not have an effect on the OVA-induced number of macrophages (Fig. 2E). These results indicated that IAL inhibited OVA-induced eosinophil accumulation.

Asthma is generally associated with enhanced type 2 immune responses, including increased production of IL-4, IL-5, IL-13, CCL17 and CCL22 (23–25). Therefore, the generation of IL-4, IL-5, IL-13, CCL17 and CCL22 in BALF was detected. The results demonstrated that compared to the control group, the expression of IL-4, IL-5, IL-13, CCL17 and CCL22 was significantly increased following OVA treatment and was significantly reduced by IAL treatment (Fig. 3). These results indicated that IAL ameliorated OVA-induced asthmatic inflammation and Th2 cytokine production.

It is well established that alternatively activated M2 macrophages, induced by IL-4, serve an important role in asthmatic inflammation (24). Arg-1 and Ym-1 are expressed by alternatively activated macrophages during asthmatic inflammation (26) and CCL17 and CCL22 are the main chemokines involved in eosinophil recruitment (7). To determine the effect of IAL on macrophage M2 polarization, BMDMs were pretreated with IAL (0, 3, 10, or 30 µM) for 30 min and stimulated with IL-4 (10 ng/ml) for 24 h. The mRNA and protein expression levels of CCL17 and CCL22 were detected. Although IL-4 treatment induced the expression of Arg-1, Ym-1, CCL17 and CCL22 in BMDMs, they were significantly decreased in a dose-dependent manner following treatment with IAL (Fig. 4). These results suggested that IAL inhibited IL-4-induced M2 macrophages.

To determine how IAL inhibits alternatively activated macrophages, BMDMs were pre-treated with IAL (0, 3, 10, or 30 µM) and BMDMs challenged with IL-4. p-STAT6 and PPAR-γ and KLF4 were detected by western blotting. As shown in Fig. 5, the phosphorylation of STAT6 and the expression of PPAR-γ and KLF4 were induced by IL-4 treatment, while IAL inhibited their expression in a dose-dependent manner (Fig. 5). Taken together, these results suggested that IAL inhibited IL-4-mediated activation of STAT6/PPAR-γ/KLF4.