PM2.5 was collected using a 2033B high-volume air sampler (Qingdao Laoying Environmental Technology Co., Ltd.) at a flow rate of 100 l/min, 8 h/day for 12 months (between January and December 2018) in Yantai, Shandong, China. After sampling, the fiberglass filters were removed and cut into small pieces of 1×3 cm, the fibers were immersed in ultrapure water, ultrasonic vibrations were used to elute the PM2.5, which was concentrated by freeze-drying. The PM was mixed with saline to obtain a particle suspension prior to experimentation. The concentration of polycyclic aromatic hydrocarbons (PAHs) and metals in the samples were determined using high-performance liquid chromatography (HPLC; Hitachi Model 600 HPLC; Hitachi, Ltd.) and inductively coupled plasma atomic emission spectrometry (ICP-AES; 61E Trace and ICP-750; Thermo Jarrell-Ash) according to the manufacturer's protocol. HPLC was conducted at room temperature. PM2.5 samples (100 µl) were mixed with the same volume of Na₂SO₄, then added into 10 ml normal hexane/acetone (1:1). The samples were extracted by ultrasonication (20 kHz) for 20 min at room temperature and moved into the purification column (400×10 mm; cat. no. V0301050001; Shanghai Chubo Laboratory Equipment Co., Ltd.) at 1.0 ml/min. Ultrasonic extraction was performed for 5 min. PAHs were determined at 254 nm using PAH standards (cat. no. PAH525-1JM; Chemservice). ICP-AES was conducted at room temperature, the samples were added nitric acid to limit the acidity to 5%, the wavelength range was 165–872 nm, the power was 1100 W, and the analysis line and detection limits of each metal was as follows: Ca (317.933 nm; 0.003 mg/l), Fe (328.204 nm; 0.000 mg/l), Mn (257.610 nm; 0.000 mg/l), Zn (213.867 nm; 0.015 mg/l), K (766.490 nm; 0.12 mg/l), Ba (455.403 nm; 0.000 mg/l), Na (589.592 nm; 0.018 mg/l) and As (460.733 nm; 0.008 mg/l).

A total of 48 6-week-old male BALB/c mice (8–22 g in weight) were obtained from Ailingfei Animals Center (Nanjing, China). The animal experiments were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University (approval no. 20110217). Animals were anesthetized to minimize suffering. The mice were maintained under a 12 h light/dark cycle at a constant temperature (22±2°C) and a relative humidity of 55±10%; all animals had ad libitum access to food and water. The mice were randomly divided into four groups (n=12/group) as follows: i) Control group, ii) ovalbumin (OVA) group, iii) PM2.5 group and iv) OVA + PM2.5 group. A 26-day model was used based on our previous study (33). On days 0, 7 and 14, mice in the OVA and OVA + PM2.5 groups were intraperitoneally (i.p.) injected with 100 µg OVA (cat. no. A5503; Sigma-Aldrich; Merck KGaA), which was combined with an alum adjuvant (0.2 ml; cat. no. 77161; Thermo Fisher Scientific, Inc.); mice in the control and PM2.5 groups were administered an equivalent volume of PBS (i.p.). A total of 100 µg PM2.5 particles in 50 µl physiological saline was administered intranasally to mice in the PM2.5 and OVA + PM2.5 groups from days 16–20, an equivalent volume of PBS was administered intranasally to mice in the control and OVA group. Mice, except for those in the control group, were challenged with aerosolized 1% OVA (total volume, 100 ml) for 30 min from days 21–25. The detailed protocols are shown in Fig. 1.

Airway responsiveness to acetylcholine chloride (ACH) was assessed 24 h after the final OVA challenge using an AniRes 2005 animal lung function analysis system (Best Lab Solutions). After anesthetizing mice with sodium pentobarbital (70 mg/kg) via intraperitoneal injection, mice were intubated through the trachea for mechanical ventilation and the caudal vein for ACH administration. The mice were ventilated to measure airway resistance using progressively increasing doses of ACH (0, 10, 30, 90 and 180 µg/kg). All mice were ventilated at a breath rate set at 90 breaths/min and with the volume set as 6 ml/kg (34).

After sacrificing mice by cervical dislocation, blood was collected from the heart. The blood samples were centrifuged at 3,000 × g for 10 min at 4°C to obtain the serum. The serum levels of OVA-specific IgE were assessed using an ELISA kit (cat. no. ab157718; Abcam). The mice were subsequently intubated, the left main bronchus was ligated and the airway lumina of the right lungs were washed three times (0.4, 0.3 and 0.3 ml, respectively) with sterile saline. The BALF was centrifuged (12,000 × g at 4°C for 10 min) and ELISA kits were used to detect the levels of IL-4 (cat. no. 20186; Quanzhou Ruixin Biological Technology Co., Ltd.), IL-5 (cat. no. ab204523; Abcam), IL-13 (cat. no. ab219634; Abcam) and IFN-γ (cat. no. ab100689; Abcam), according to the manufacturer's protocol.

After centrifugation (5,000 × g at 4°C for 10 min), part of the BALF sediment was resuspended in 1 ml PBS, BALF cytospins were stained at room temperature for 3 min using the Wright-Giemsa method (cat. no. RBR00502; Shanghai Rongbai Biotechnology Co., Ltd.), according to the manufacturer's protocol, and cells were counted using a BX53 light microscope (magnification, ×100; Olympus Corporation) and a hemocytometer. To avoid errors, each sample was counted by two independent researchers. The total number of cells and the number of four different types of inflammatory cells (macrophages, lymphocytes, eosinophils and neutrophils) were recorded.

The left lungs of the mice were prepared for histological analysis and fixed in 4% triformol for 48 h at room temperature. Paraffin-embedded sections were sliced into 5-µm thick sections for staining with the hematoxylin and eosin kit (Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 1 h to observe inflammatory changes using a BX53 light microscope (magnification, ×100; Olympus Corporation). Carl Zeiss Axiovision Viewer Image software (version 4.82.SP2 1CD; Carl Zeiss AG) were used to assess the mean liner intercept (MLI), matrix membrane layer and smooth muscle layer of the airways. Periodic Acid Schiff (PAS) (Beijing Solarbio Science & Technology Co., Ltd.) staining was used to assess mucus secretion. PAS⁺ cells were analyzed using Axiovision Viewer Image software and the ratio of PAS⁺ cells to bronchus was calculated.

The right lung was removed, and total protein was extracted using Total Extraction Sample Kit (Sigma-Aldrich; Merck KGaA) according to the manufacturer's protocols. Protein concentrations were determined using the BCA method (cat. no. 23225; Thermo Fisher Scientific, Inc.). Proteins (30 µg) were resolved by 10% SDS-PAGE, and then transferred to a PVDF membrane. Following blocking with TBST supplemented with 5% skim milk powder for 2 h at room temperature, the PVDF membranes were incubated with antibodies against GATA3 (1:1,000; cat. no. ab106625; Abcam), STAT6 (1:2,000; cat. no. ab32520; Abcam), T-bet (1:500; cat. no. ab91109; Abcam) or Runx3 (1:1,000; cat. no. ab135248; Abcam) at 4°C overnight. The membranes were subsequently incubated with HRP-conjugated goat anti-mouse secondary antibody (1:5,000; cat. no. ab205719; Abcam) the following day for 1 h at room temperature. Signals were visualized using ECL Chemiluminescence Detection kit (cat. no. SW2010; Beijing Solarbio Science & Technology Co., Ltd.). Protein expression levels were semi-quantified using Gel-Pro-Analyzer software (version 4.0; Media Cybernetics, Inc.) with β-actin as the loading control.

Right lung was removed, and total cellular RNA was extracted using TRIzol^® reagent (cat. no. 15596026; Invitrogen; Thermo Fisher Scientific, Inc.). A One-step RT-PCR kit (cat. no. 639504; Takara Bio, Inc.) was used according to the manufacturer's protocols to reverse transcribe RNA into cDNA. Subsequently, mRNA expression levels of cytokines and transcription factors were evaluated via RT-qPCR using BlazeTaq™ SYBR^® Green qPCR Mix (GeneCopoeia, Inc.) according to the manufacturer's protocol. The following thermocycling conditions were used for qPCR: 95°C for 30 sec; followed by 40 cycles at 95°C for 30 sec and at 60°C for 30 sec; and then at 72°C for 30 sec. Lightcycler software (version 480; Roche Molecular Diagnostics) was used to measure the Cq values and the results were expressed relative to GAPDH. Primers were selected from PrimerBank (35). The forward and reverse sequences of the primers used for amplification are presented in Table I. mRNA expression levels were quantified using the 2^−∆∆Cq method (36).

The spleen was taken to the ultra-clean table, ground using a cell strainer (mesh size, 100), and the cell suspension was obtained. After stimulation with phorbol myristate acetate (25 µg/ml) + lonomycin (20 µg/ml) at 37°C for 4 h (Beijing Solarbio Science & Technology Co., Ltd.), the cells were fixed using 4% paraformaldehyde at room temperature for 10 min. Cells were incubated with PerCP/cyanine5.5-conjugated anti-CD4 (cat. no. 45-0042-80; Thermo Fisher Scientific, Inc.), PE-conjugated anti-IL-4 (cat. no. 12-7041-41; Thermo Fisher Scientific, Inc.) and FITC-conjugated anti-IFN-γ (cat. no. 11-7311-41; Thermo Fisher Scientific, Inc.) antibodies for 20 min at room temperature. Th1 and Th2 cells were sorted using a FACSCanto™ flow cytometer (BD Biosciences). The corresponding percentage of Th1 cells, Th2 cells and Th1/Th2 ratio were calculated and analyzed using FlowJo software (version 10; BD Biosciences).

All experiments were performed at least three times. Data are presented as the mean ± standard deviation, and differences between four groups were analyzed using one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

The chemical constituents identified in PM2.5 samples are listed in Table II. Metals and PAHs accounted for the primary constituents of ambient PM2.5 samples collected in Yantai, China. The results showed that sodium, magnesium, aluminum, calcium and zinc were the primary metals found. Naphthalene, acenaphthene, phenanthrene, benzofluoranthene and benzopyrene were the primary PAHs in the ambient PM2.5. The results suggested that the metals and PAHs may account for the exacerbating effect of PM2.5 on the occurrence and prevalence of asthma.

The results of in vivo experiments showed that increased administration of ACH, OVA and PM2.5 co-exposure significantly increased the lung resistance compared with that observed in the groups treated with PM2.5 and OVA alone (Fig. 2). The airway resistance of mice in the PM2.5 group treated with ACH was significantly higher compared with the control group, but lower than that in the OVA group.

The levels of serum OVA-specific IgE increased significantly in the OVA and OVA + PM2.5 groups compared with the PM2.5 and control groups (Fig. 3A). Levels of OVA-specific IgE in the OVA + PM2.5 group were increased notably compared with the OVA group. However, there were no significant differences between the PM2.5 and control groups. OVA + PM2.5 significantly increased IL-4, IL-5 and IL-13 levels in BALF compared with the control, OVA and PM2.5 groups (Fig. 3B-D, respectively); OVA and PM2.5 alone significantly increased the levels of these cytokines in BALF compared with the control group. PM2.5 alone resulted in less secretion of these cytokines compared with the OVA mice. The changes in expression of IFN-γ in these four groups were opposite to that observed for IgE, IL-4, IL-5 and IL-13 (Fig. 3E).

PM2.5 exposure increased the total inflammatory cell count and the count of each type of inflammatory cell assessed, including macrophages, neutrophils, eosinophils and lymphocytes, in BALF sediments, particularly in the OVA + PM2.5 group (Fig. 4). The increase observed in the OVA group was significantly higher compared with the PM2.5 alone and control groups.

No pathological alterations were found in the lungs of the control group. PM2.5 alone caused a small degree of airway inflammation, whereas OVA exposure exacerbated airway inflammation, particularly in the OVA + PM2.5 group, OVA + PM2.5 exposure resulted in focal infiltration of inflammatory cells into the airway (Fig. 5A). PM2.5 increased mlI of mice, and a significant difference was observed between the asthmatic mice compared with the OVA group (Fig. 5B). PM2.5 increased matrix membrane layer thickness and smooth muscle layer thickness significantly (Fig. 5C and D, respectively), particularly in mice treated with both OVA and PM2.5. Furthermore, mucus secretion was significantly increased in the mice of the OVA + PM2.5 group; OVA and PM2.5 alone increased mucus secretion significantly compared with the control group, and the PM2.5 group showed fewer PAS⁺ cells compared with the OVA group (Fig. 5E).

The results of western blotting showed that a combination of OVA and PM2.5 significantly enhanced the protein expression levels of the GATA3 and STAT6 compared with the other groups (Fig. 6). The expression of GATA3 and STAT6 in the mice treated with PM2.5 alone was higher compared with the control group. Expression of the transcription factors in the OVA group were significantly higher compared with the PM2.5 group. Conversely, PM2.5 lowered the expression of Runx3, and the decrease was more pronounced in the OVA + PM2.5 group. Additionally, expression of Runx3 was significantly lower in the OVA group compared with the control group. No significant differences in Runx3 expression were observed between the OVA and PM2.5 groups. However, expression of T-bet in OVA and OVA + PM2.5 groups were significantly lower compared with all the other groups. PM2.5 alone decreased the expression of T-bet compared with the control mice, but no significant differences were observed in the expression levels of T-bet between the PM2.5 and control groups. These results suggested that transcription factor Runx3, but not T-bet served a vital role in PM2.5-induced asthmatic pathological processes.

The mRNA expression levels of inflammatory cytokines and transcription factors were measured by RT-qPCR. The variation in trends and mRNA ratios were the same as those observed for the changes in the protein expression levels (Fig. 7).

The proportion (%) of Th2 was significantly increased in the OVA group compared with the control group, as measured by flow cytometry (Fig. 8). PM2.5 exposure slightly decreased the proportion of Th1 cells and increased the proportion of Th2 cells, but no significant difference was observed compared with the control group. Co-exposure of OVA and PM2.5 significantly decreased the population of Th1 cells and increased the population of Th2 cells. In addition, PM2.5 significantly disturbed the balance between Th1/Th2 cells compared with the control group, OVA + PM2.5 co-exposure exhibited an enhanced effect, further disturbing the balance significantly compared with the OVA group.