Eight BALB/c mice were exposed to saline (control) or ovalbumin (OVA; OVA/OVA) and treated with NAC or dexamethasone (Fig. 1A). We evaluated the impact of NAC and CLDN18 on the levels of cytokines, CLDNs, and signal-pathway proteins. The patient study was approved by the Soonchunhyang University Institutional Review Board (SCHBC-2014-10-011). Written informed consent was obtained from all patients. The animal study was approved by the Soonchunhyang University Institutional Animal Care and Use Committee (SCHBC-animal-2014-13).

Biospecimens and clinical data were provided by the biobank of Soonchunhyang University, Bucheon Hospital, a member of the Korea Biobank Network. All subjects had a clinical diagnosis of asthma according to Global Initiative for Asthma (GINA) guidelines [13] with modification. All subjects underwent standardized assessments that included complete blood cell and differential counts, immunoglobulin E (IgE) quantification, chest posteroanterior radiography, skin prick allergy testing, and spirometry (Table 1). All data were collected at the time of diagnosis, before administration of asthma medication. Normal control subjects were recruited from among spouses of the subjects or members of the general population.

All subjects had a clinical diagnosis of asthma that was supported by one or more of the following criteria: variability in the maximum diurnal peak expiratory flow greater than 20% over the course of 14 days; an increase in the forced expiratory volume in 1 second (FEV₁) greater than 15% after inhalation of 200 to 400 μg albuterol; or a positive provocative concentration 20 (PC20) response, defined as a 20% reduction in FEV₁ in response to an inhalation of < 10 mg/mL methacholine. Asthma exacerbation was defined, based on the GINA guidelines [13], as episodes of progressive increases in shortness of breath, cough, wheezing, or chest tightness; some combination of these symptoms, accompanied by decreases in expiratory airflow and use of systemic corticosteroids (tablets, suspension, or injection); or an increase from a stable maintenance dose for at least 3 days and a hospitalization or emergency department visit because of asthma, requiring systemic corticosteroids.

All data were collected at the time of diagnosis, before the administration of asthma medication, and during the period of exacerbation. Asthmatics from the hospital cohort were matched to healthy subjects according to age, sex, and body mass index. These healthy control subjects were included based on the following criteria: negative responses on a screening questionnaire for respiratory symptoms and other allergic diseases, FEV₁ values greater than 80% of predicted values, PC20 methacholine values > 10 mg/mL, and normal findings in simple chest radiographs. The exclusion criteria included respiratory infection during sputum induction, chronic obstructive pulmonary disease, vocal cord dysfunction, obstructive sleep apnea, Churg-Strauss syndrome, cardiac dysfunction, allergic bronchopulmonary aspergillosis, or poor compliance with treatment.

All experimental animal methods followed a protocol approved by the Institutional Animal Care and Use Committee of the Soonchunhyang University Bucheon Hospital. Female BALB/c mice (6 weeks old) were sensitized and challenged with OVA. Airway responsiveness was evaluated, bronchoalveolar lavage fluid (BALF) was collected, and lung tissue was harvested for protein extraction, hematoxylin and eosin (H&E) staining and immunohistochemistry.

NAC (100 mg/kg) (Boryung, Seoul, Korea) inhalation treatment was performed 1 hour after each challenge with OVA for 6 consecutive days. Additional groups of mice were treated with intraperitoneally dexamethasone (3 mg/kg) (Sigma-Aldrich, St. Louis, MO, USA) 1 hour before each OVA challenge daily for 3 days.

For Western blot analysis, Protein concentration in the homogenates of lung tissue was determined using a BCA Protein Assay Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Proteins were separated and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% bovine serum albumin in 0.1% Tween 20 in Tris-buffered saline, and incubated with anti-CLDN7 and CLDN18 antibodies (1:200, Abcam, Cambridge, MA, USA) (4°C, overnight) followed by the horseradish peroxidase-conjugated secondary antibody. Bands were visualized using the WESTZOL plus Western blotting Detection System (iNtRon, Seongnam, Korea). The relative abundance of proteins was determined by quantitative densitometry with normalization to β-actin (Sigma-Aldrich).

Lung sections were deparaffinized, rehydrated in an ethanol series, and stained with H&E. For detection of CLDNs, Lung sections were incubated with anti-CLDN7 and CLDN18 antibodies (1:200, Abcam) at 4°C overnight. Next, the sections were incubated with an avidin and biotinylated horseradish peroxidase macromolecular complex (Vector Laboratories, Burlingame, CA, USA). Color was developed by staining with liquid DAB + substrate kit (Golden Bridge International Inc., Mukilteo, WA, USA). After immunohistochemical staining, the slides were counterstained with Harris’s hematoxylin for 1 min. Images were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

The levels of IL-1α, IL-10, and tumor necrosis factor-α (TNF-α) in BALF from mice were determined using the Multi Analyte ELISArray Kit (Qiagen, Valencia, CA, USA) and CLDN18 levels in human plasma were measured using an enzyme-linked immunosorbent assay (ELISA) (USCN, Wuhan, China). The lower detection limit was set at 32.5 pg/mL (IL-1α), 59.4 pg/mL (IL-10), 30.5 pg/mL (TNF-α), or 0.055 ng/mL (CLDN18), respectively, based on the manufacturer’s recommendations.

Data are expressed as means ± standard errors of the mean (SEMs), and the Mann-Whitney U test was used to evaluate the significance of differences between groups. A p < 0.05 was deemed to indicate statistical significance.

OVA-sensitized/challenged mice had a significantly greater airway hyperresponsiveness (AHR) compared to sham mice (Fig. 1B), which was significantly reduced by treatment with NAC or dexamethasone. The numbers of total inflammatory cells, macrophages, eosinophils, and neutrophils in BALF were significantly increased in OVA/OVA mice compared to sham mice (p < 0.05) (Fig. 1C) and were significantly reduced by NAC or dexamethasone.

Next, we evaluated the levels of mononuclear phagocyte-produced cytokines in BALF. IL-1α levels tended to be higher in OVA/OVA mice compared to sham mice and were reduced by NAC or dexamethasone (Fig. 2). IL-10 and TNF-α levels tended to be higher in OVA/OVA compared to sham mice and were reduced by dexamethasone (Fig. 2).

CLDN18 levels were significantly higher in the lungs of OVA/OVA mice than in sham mice and were increased further by NAC or dexamethasone (Fig. 3A). Histopathologic examination revealed epithelium loss and increased infiltration of inflammatory cells in the lungs of OVA/OVA mice, which were decreased by NAC or dexamethasone. CLDN18 levels were increased in the lung tissue of OVA/OVA mice compared to control mice and were increased further by NAC (Fig. 3B).

Activation of Akt and phosphorylated-pyruvate dehydrogenase kinase 1 (p-PDK1) was assessed by Western blotting (Fig. 4). Treatment with NAC increased and decreased the p-PDK1 and p-Akt/Akt protein levels, respectively. NAC and dexamethasone suppressed the phosphorylation of Akt in lungs.

In total, 20 patients with stable asthma (median age [range], 49 years [23 to 74]), 17 patients with exacerbated asthma (63 years [42 to 83]), and 15 control subjects (69 years [56 to 78]) were recruited (Table 1). Initial FEV₁% and FEV₁/forced vital capacity (FVC) were significantly lower in patients with asthma than in controls. Total IgE levels and amount of smoking (pack-years) were significantly higher in patients with asthma than in controls. The body mass index was similar between patients and controls. FEV₁% and FVC% were significantly lower in patients with exacerbated asthma than in those with stable asthma.

Plasma levels of CLDN18 were lower in asthma patients than in healthy controls (p < 0.05). The levels tended to be lower in patients with exacerbated asthma than in those with stable asthma (p > 0.05) (Fig. 5), and were correlated with the percentage of neutrophils, FEV₁/FVC%, and FEV₁% (neutrophils, ρ = 0.286, p = 0.040; FEV₁/FVC%, ρ = –0.463, p = 0.040; FEV₁%, ρ = –0.576, p = 0.019) (Fig. 6).