BALB/c mice were sensitized with ovalbumin with/without polyinosinic-polycytidylic acid (poly(I:C)) from week 6 (A6 mice) or week 12 (A12 mice) after birth. cytokine, and OPN levels in BALF and the expression of Lck inhibitor 2 phosphorylated Smad3, TGM2, and in the lungs. OPN brought on TGF-1/Smad3 signaling in the lungs, which was suppressed by dexamethasone and anti-IL5 antibody. In conclusion, aging and exposure to viral infections may induce OPN release and consequently modulate inflammation and TGF-1/Smad3-related remodeling, contributing to the development of LOA. (Der F(Der P) and spp. [Bencard Co., Bredford, UK]). Patients with asthma underwent spirometry (FEV1%, FVC% predicted values) and methacholine (Mct) challenge tests to evaluate airway hyperresponsiveness (AHR) according to the European Respiratory Society standard26. The concentration of Mct required to produce a 20% decrease in FEV1 from baseline (MctPC20) was recorded. Severe asthma was defined according to the American Thoracic Society/European Respiratory Society guidelines27. Serum samples from patients and HCs were collected, stored at Sirt6 ?70?C and thawed before use. Total IgE levels in serum were measured by the ImmunoCAP system (Thermo Fisher Scientific, Waltham, MA, USA) in the detection range of Lck inhibitor 2 2C5000?kU/L. Classification of asthma phenotype LOA and EOA were defined when asthma had been diagnosed at the age of 40 years and 40 years, respectively28. To identify eosinophilic asthma, we used blood eosinophil counts with the cutoff at 300 cells/l as previously described29. HAEC cultures and treatment HAECs, including A549 cells and primary small airway epithelial cells (SAECs), were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA). A549 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, penicillin G sodium (100?UI/mL) and streptomycin sulfate (100?g/mL) (all from Gibco, Grand Island, NY, USA). SAECs were cultured in basal medium Lck inhibitor 2 supplemented with a bronchial epithelial cell growth kit (ATCC), penicillin G sodium (10?UI/mL), streptomycin sulfate (10?g/mL) (Gibco), and amphotericin B (25?ng/mL) (Sigma Aldrich, St. Louis, MO, USA) according to the manufacturers protocol. Cells were produced at 37?C in humidified air with 5% CO2. For treatment, cells (2??105) were seeded onto a 12-well plate and stimulated with polyinosinic:polycytidylic acid (poly(I:C)) (Sigma Aldrich) at 1 and 10?g/mL. After 24-h incubation, the supernatant was collected; cells were lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitor (Thermo Fisher Scientific) and stored Lck inhibitor 2 at ?70?C for further experiments. Establishment of an LOA mouse model Female BALB/c mice at 6 and 12 weeks aged (weight 20??2 and 21??2?g, respectively) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA), housed under specific pathogen-free conditions, maintained on a 12-h light/dark cycle and fed ad libitum. Asthma was induced at two time points, altered from a previous protocol30. Briefly, on days 0 and 14, mice were intraperitoneally sensitized with ovalbumin (OVA)/aluminum hydroxide (Alum) answer at 10?g/1?mg. On days 28C30, the mice were challenged with 2% OVA for 30?min using an ultrasonic nebulizer (NE-SM1; Ktmed Inc., Seoul, South Korea). To establish the mouse model of virus-induced asthma exacerbation, mice were administered intranasal poly(I:C) (10?g/mouse) prior to sensitization/challenge. To investigate the effects of OPN on asthma, mice were treated intranasally with 4?g of mouse recombinant OPN Lck inhibitor 2 protein (rOPN, 763606, Biolegend, San Diego, CA, USA) for 1?h prior to sensitization on days 0 and 14. In some experiments, mice were given dexamethasone 21-phosphate disodium salt (D1159) (Dex, 1?mg/kg), montelukast sodium hydrate (Mon, 10?mg/kg) or anti-IL-5 antibody (504302) (anti-IL5, 20?mg/kg) for 3 consecutive days prior to the challenge. Mice were assayed at 24?h after the last challenge. All animal experiments were approved by the Institutional Animal Care and Use Committee of Ajou University (IACUC 2018-0041). OVA, Dex and Mon were from Sigma Aldrich, Alum was from Thermo Fisher Scientific, and the anti-IL-5 antibody was from Biolegend. Measurement of AHR AHR to acetyl–methylcholine chloride was recorded using the FlexiVent system (Scireq, Montreal, QC, Canada). Mice were anesthetized with pentobarbital sodium, intubated with a cannula and ventilated with.