Novel pendrin inhibitor attenuates airway hyperresponsiveness and mucin expression in experimental murine asthma

Abstract

The transmembrane anion exchanger pendrin (SCL26A4) exchanges Cl− with bases, such as HCO3−, I−, and SCN−, and is the most highly upregulated gene in endobronchial biopsies from patients with asthma.1Scott D.A. Wang R. Kreman T.M. Sheffield V.C. Karniski L.P. The Pendred syndrome gene encodes a chloride-iodide transport protein.Nat Genet. 1999; 21: 440-443Crossref PubMed Scopus (504) Google Scholar, 2Yick C.Y. Zwinderman A.H. Kunst P.W. Grunberg K. Mauad T. Dijkhuis A. et al.Transcriptome sequencing (RNA-Seq) of human endobronchial biopsies: asthma versus controls.Eur Respir J. 2013; 42: 662-670Crossref PubMed Scopus (61) Google Scholar Interestingly, patients with mutant pendrin have a low prevalence of asthma, and pendrin-null mice show reduced allergic airway inflammation.3Nakagami Y. Favoreto Jr., S. Zhen G. Park S.W. Nguyenvu L.T. Kuperman D.A. et al.The epithelial anion transporter pendrin is induced by allergy and rhinovirus infection, regulates airway surface liquid, and increases airway reactivity and inflammation in an asthma model.J Immunol. 2008; 181: 2203-2210Crossref PubMed Google Scholar, 4Nakao I. Kanaji S. Ohta S. Matsushita H. Arima K. Yuyama N. et al.Identification of pendrin as a common mediator for mucus production in bronchial asthma and chronic obstructive pulmonary disease.J Immunol. 2008; 180: 6262-6269Crossref PubMed Scopus (106) Google Scholar, 5Madeo A.C. Manichaikul A. Pryor S.P. Griffith A.J. Do mutations of the Pendred syndrome gene, SLC26A4, confer resistance to asthma and hypertension?.J Med Genet. 2009; 46: 405-406Crossref PubMed Scopus (26) Google Scholar These observations indicate that pendrin is a novel target for allergic asthma. In this study, we identified a novel pendrin inhibitor, YS-01 (2-(4-(tert-butyl)phenyl)-4-(thiophen-2-ylmethylene)oxazol-5(4H)-one), with strong therapeutic effects on allergic inflammation in a mouse model of ovalbumin (OVA)-induced asthma. YS-01 was identified by the high-throughput screening of 54,400 synthetic compounds (Fig 1, A). Detailed methodology is provided in the Methods section in the Online Repository at www.jacionline.org. YS-01 potently inhibited pendrin-mediated Cl−/SCN−, Cl−/I−, Cl−/HCO3−, and Cl−/OH− exchange activity in a dose-dependent manner (Fig 1, B and C; see Fig E1, A-C, in this article's Online Repository at www.jacionline.org). YS-01 showed no cytotoxicity up to 30 μM in NIH3T3 and CHO-K1 cells and more potently inhibited pendrin activity than the recently identified pendrin inhibitors, PDSinh-A01 and PDSinh-C01 (see Fig E1, D-I).6Haggie P.M. Phuan P.W. Tan J.A. Zlock L. Finkbeiner W.E. Verkman A.S. Inhibitors of pendrin anion exchange identified in a small molecule screen increase airway surface liquid volume in cystic fibrosis.FASEB J. 2016; 30: 2187-2197Crossref PubMed Scopus (29) Google Scholar YS-01 inhibited both human and mouse pendrin-mediated Cl−/I− exchange with nearly identical potency (see Fig E2, A and B, in this article's Online Repository at www.jacionline.org). YS-01 weakly inhibited Cl−/HCO3− exchange activities of SCL26A3 and SLC26A6 with IC50 greater than 100 μM and did not affect SCL26A7 and SLC26A9. Cystic fibrosis transmembrane conductance regulator, anoctamin-1, human ether-a-go-go-related gene channel, and 5-HT2A activities were not affected by YS-01 (see Fig E2, C-J). In primary cultures of human nasal epithelial (HNE) and human bronchial epithelial cells, IL-4 treatment strongly upregulated pendrin expression and pendrin-mediated Cl−/HCO3− exchange activity, and YS-01 potently inhibited pendrin-mediated Cl−/HCO3− exchange activity (Fig 1, D-F, and see Fig E3, A-C, in this article's Online Repository at www.jacionline.org). We observed the effect of long-term treatment with YS-01 on the functional expression of pendrin and other ion channels involved in airway surface liquid (ASL) regulation. Interestingly, the IL-4–induced upregulation of Cl−/HCO3− exchange activity and pendrin protein expression levels were strongly decreased by long-term treatment with YS-01, without changes in mRNA expression levels, but the IL-4–induced upregulation of anoctamin-1 was not affected (Fig 1, G-I). The mRNA expression levels and ion channel activities of anoctamin-1, cystic fibrosis transmembrane conductance regulator, and epithelial sodium channels (ENaC) were not altered by long-term treatment with YS-01 (see Fig E3, D-F). Pretreatment with YS-01 significantly attenuated OVA-induced airway hyperresponsiveness (Fig 2, A) and reduced the numbers of eosinophils and neutrophils in the bronchoalveolar lavage fluid of OVA-sensitized mice. A histological analysis showed that the increased inflammatory infiltration and epithelial thickness and the increased number of goblet cells in the central airways were attenuated by treatment with YS-01 in OVA-challenged mice. The average inflammation score of OVA-challenged mice was significantly lower in the YS-01–treated group than in the untreated group, but the serum levels of OVA-specific IgE were not altered by YS-01, indicating that YS-01 does not act on the general allergic response mechanism (see Fig E4 in this article's Online Repository at www.jacionline.org). Recent studies have shown that nuclear factor kappa B (NF-κB) activation by increased hypothiocyanite (OSCN−) production via the upregulation of pendrin, peroxidases, and dual oxidase (Duox1/Duox2) in the airway epithelium is involved in allergic airway inflammation.7Suzuki S. Ogawa M. Ohta S. Nunomura S. Nanri Y. Shiraishi H. et al.Induction of airway allergic inflammation by hypothiocyanite via epithelial cells.J Biol Chem. 2016; 291: 27219-27227Crossref PubMed Scopus (16) Google Scholar, 8Izuhara K. Suzuki S. Ogawa M. Nunomura S. Nanri Y. Mitamura Y. et al.The significance of hypothiocyanite production via the pendrin/DUOX/peroxidase pathway in the pathogenesis of asthma.Oxid Med Cell Longev. 2017; 2017: 1054801Crossref PubMed Scopus (8) Google Scholar The SCN− concentration at the apical surface was significantly increased by IL-4 treatment and inhibited by YS-01 (Fig 2, B). Real-time PCR analyses showed that IL-4 treatment significantly increased the mRNA expression of Duox1 but did not affect Duox2, and YS-01 did not affect the mRNA expression of Duox1 or Duox2 (Fig 2, C). Pretreatment with YS-01 significantly inhibited the IL-4–induced activation of NF-κB in HNE cells (Fig 2, D). Notably, the intranasal administration of NaSCN significantly blocked the inhibitory effect of YS-01 on airway hyperresponsiveness and abolished the protective effect of YS-01 on lung injury in OVA-challenged asthmatic mice (Fig 2, E and F). In addition, YS-01 significantly blocked the NF-κB activation in the lungs of OVA-treated mice, and treatment of SCN− inhibited the protective effect of YS-01 in a transgenic NF-κB reporter mice (see Fig E4, G). Application of YS-01 to an established model of allergic asthma significantly decreased OVA-induced airway hyperresponsiveness and periodic acid-Schiff–positive cells in airway epithelium (see Fig E5 in this article's Online Repository at www.jacionline.org). These results suggest that YS-01 may be useful in both preventing and treating allergic asthma. Pendrin is associated with the airway inflammation-mediated upregulation of MUC5AC in the airway epithelium.4Nakao I. Kanaji S. Ohta S. Matsushita H. Arima K. Yuyama N. et al.Identification of pendrin as a common mediator for mucus production in bronchial asthma and chronic obstructive pulmonary disease.J Immunol. 2008; 180: 6262-6269Crossref PubMed Scopus (106) Google Scholar, 9Lee H.J. Yoo J.E. Namkung W. Cho H.J. Kim K. Kang J.W. et al.Thick airway surface liquid volume and weak mucin expression in pendrin-deficient human airway epithelia.Physiol Rep. 2015; 3: e12480Crossref PubMed Scopus (19) Google Scholar Notably, our results showed that treatment with YS-01 significantly decreased the IL-4–induced MUC5AC transcript (see Fig E6, A, in this article's Online Repository at www.jacionline.org). In addition, pretreatment with YS-01 attenuated IL-4– and IL-13–induced goblet cell hyperplasia in HNE cells (see Fig E6, B). ASL thickness in IL-13–treated primary cultures of airway epithelial cells is significantly higher in pendrin-null mice and deaf patients carrying a mutant SLC26A4 gene compared with controls.3Nakagami Y. Favoreto Jr., S. Zhen G. Park S.W. Nguyenvu L.T. Kuperman D.A. et al.The epithelial anion transporter pendrin is induced by allergy and rhinovirus infection, regulates airway surface liquid, and increases airway reactivity and inflammation in an asthma model.J Immunol. 2008; 181: 2203-2210Crossref PubMed Google Scholar, 9Lee H.J. Yoo J.E. Namkung W. Cho H.J. Kim K. Kang J.W. et al.Thick airway surface liquid volume and weak mucin expression in pendrin-deficient human airway epithelia.Physiol Rep. 2015; 3: e12480Crossref PubMed Scopus (19) Google Scholar In HNE cells expressing wild-type pendrin, IL-4 and IL-13 treatment strongly increased pendrin protein expression and decreased the total volume of ASL and fluid meniscus compared with those of controls, and YS-01 treatment almost completely rescued the IL-4– and IL-13–induced decreases in the total volume of ASL and fluid meniscus. However, the total volume was not altered by IL-4 and YS-01 in HNE cells expressing mutant pendrin (see Fig E6, C-F). This result is consistent with that of Haggie et al,6Haggie P.M. Phuan P.W. Tan J.A. Zlock L. Finkbeiner W.E. Verkman A.S. Inhibitors of pendrin anion exchange identified in a small molecule screen increase airway surface liquid volume in cystic fibrosis.FASEB J. 2016; 30: 2187-2197Crossref PubMed Scopus (29) Google Scholar who found that PDSinh-A01 significantly increased ASL depth in IL-13–treated human bronchial epithelial cells. MUC5AC inhibition and an increase in ASL thickness by YS-01 might provide additional beneficial effects on airway inflammatory diseases, such as chronic obstructive pulmonary disease, cystic fibrosis, and asthma. There is a possibility that alterations in hearing or thyroid hormone level may be induced by pendrin inhibition because the patients carrying SLC26A4 gene mutations are associated with prelingual deafness and goiter.1Scott D.A. Wang R. Kreman T.M. Sheffield V.C. Karniski L.P. The Pendred syndrome gene encodes a chloride-iodide transport protein.Nat Genet. 1999; 21: 440-443Crossref PubMed Scopus (504) Google Scholar However, hearing threshold and plasma levels of T3 and T4 were not changed after treatment of YS-01 (10 mg/kg/d) for 1 week (see Fig E7, A-C, in this article's Online Repository at www.jacionline.org). In addition, YS-01 did not affect tracheal smooth muscle contraction (see Fig E7, D). In summary, YS-01 reduced airway hyperresponsiveness and airway inflammation via the inhibition of the SCN−/NF-κB pathway. In addition, YS-01 showed protective effects against IL-4– and IL-13–induced goblet cell hyperplasia and ASL depletion (see Fig E8 in this article's Online Repository at www.jacionline.org). These results indicate that pendrin inhibitors are promising candidates for the treatment of allergic asthma. We gratefully acknowledge Korea Mouse Phenotyping Center for technical support of mouse phenotyping. CHO-K1 cells expressing human wild-type pendrin and YFP-F46L/H148Q/I152L were plated in 96-well microplates at a density of 2 × 104 cells per well and incubated for 48 hours. Each well of the 96-well plate was washed 2 times with 200 μL of PBS, and it was filled with 50 μL of HEPES-buffered solution. Test compounds were added at 50 μM. After 10 minutes of incubation at 37°C, the 96-well plate was placed on the FLUOstar Omega Microplate Reader (BMG Labtech, Ortenberg, Germany) for a fluorescence assay. The compound collection used for screening (55,000 synthetic small molecules) was purchased from ChemDiv (San Diego, Calif). Each well was assayed individually for pendrin-mediated I− influx by recording fluorescence continuously (400 ms per point) for 1 second (baseline). Then, 50 μL of NaI-substituted HEPES-buffered solution (NaI replacing NaCl) was added using a liquid injector at 1 second and YFP fluorescence was recorded for 5 seconds. The initial iodide influx rate was determined from the initial slope of fluorescence by nonlinear regression, after the infusion of iodide. Glycine (150 mg, 2 mmol) was dissolved in 1.5 mL of 10% sodium hydroxide solution. Then, 4-tert-butylbenzoyl chloride (0.34 mL, 2.4 mmol) was added at room temperature under stirring for 6 hours. After acidification until pH 2 by HCl, the produced mixture was filtered with eluding water. Then, the precipitate was filtered and washed once more with methylene chloride to obtain (4-(tert-butyl)benzoyl)glycine (450 mg, 95.7%, white solid). 1H NMR (400 MHz, DMSO-d6) δ 12.54 (s, 1H), 8.72 (t, 1H, J = 5.8 Hz), 7.77 (d, 2H, J = 8.4 Hz), 7.46 (d, 2H, J = 8.8 Hz), 3.88 (d, 2H, J = 6.0 Hz), 1.26 (s, 9H); ESI (m/z) 236 (MH+) (4-(tert-butyl)benzoyl)glycine (450 mg, 1.91 mmol) and 1-ethyl-3-(3-dimethylamino) propyl carbodiimide hydrochloride (550 mg, 2.87 mmol) were sequentially added to 0.1 mol methylene chloride (19.1 mL) at room temperature under stirring for 12 hours. Then, furfural (0.17 mL, 2.10 mmol) and triethylamine (0.53 mL, 3.83 mmol) were added at room temperature under stirring for 12 hours. The mixture was distilled off under reduced pressure. After dissolving with methanol, the precipitate was filtered to obtain YS-01 (480 mg, 85%, yellow solid). 13C NMR (400 MHz, DMSO-d6) δ 166.7, 162.1, 157.2, 137.6, 136.9, 136.6, 130.7, 128.6, 128.1(2), 126.6(2), 124.9,122.8, 35.5, 31.2(3) 1H NMR (400 MHz, DMSO-d6) δ 8.02 (d, 1H, J = 5.2 Hz), 7.96 (d, 2H, J = 7.6 Hz), 7.80 (d, 1H, J = 3.6 Hz), 7.66 (s, 1H), 7.61 (d, 2H, J = 7.6 Hz), 7.22 (t, 1H, J = 4.4 Hz), 1.29 (s, 9H); ESI (m/z) 296 (MH+) Snapwell inserts containing anoctamin-1 (ANO1)- and cystic fibrosis transmembrane conductance regulator (CFTR)-expressing Fischer rat thyroid (FRT) and primary cultures of HNE cells were mounted on Ussing chambers (Physiologic Instruments, San Diego, Calif). HNE cells were obtained as previously described.E1Yoon J.H. Kim K.S. Kim H.U. Linton J.A. Lee J.G. Effects of TNF-alpha and IL-1 beta on mucin, lysozyme, IL-6 and IL-8 in passage-2 normal human nasal epithelial cells.Acta Otolaryngol. 1999; 119: 905-910Crossref PubMed Scopus (54) Google Scholar For the measurement of short-circuit current in HNE cells, the apical and basolateral bath were filled with symmetrical HCO3−-buffered solution containing (in mM) 120 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 d-glucose, 5 HEPES, and 25 NaHCO3 (pH 7.4). For ANO1- and CFTR-expressing FRT, the apical bath was filled with half-Cl− solution containing (in mM) 60 NaCl, 60 Na-gluconate, 5 KCl, 1 MgCl2, 1 CaCl2, 10 d-glucose, 5 HEPES, and 25 NaHCO3 (pH 7.4) and the basolateral bath was filled with the HCO3−-buffered solution. The basolateral membrane was permeabilized with 250 μg/mL of amphotericin B to measure the apical membrane currents of ANO1 and CFTR. All cells were bathed for a 20-minute stabilization period and aerated with 95% O2/5% CO2 at 37°C. Short-circuit currents and apical membrane currents were measured with an EVC4000 Multi-Channel V/I Clamp (World Precision Instruments, Sarasota, Fla) and PowerLab 4/35 (AD Instruments, Castle Hill, Australia). The data were recorded and analyzed using Labchart Pro 7 (AD Instruments). The sampling rate was 4 Hz. For Cl−/HCO3− and Cl−/OH− measurements, intracellular pH (pHi) was measured in wild-type pendrin-expressing CHO-K1, HNE, and human bronchial epithelial cells using the pH sensor SNARF5-AM (Molecular Probes, Eugene, Ore). The cells were loaded with 5 μM SNARF5-AM for 30 minutes, and then mounted in a perfusion chamber on the stage of an inverted fluorescence microscope (Nikon, Tokyo, Japan) equipped with a cooled charge-coupled device camera (Zyla sCMOS; Andor Technology, Belfast, United Kingdom) and image acquisition and analysis software (Meta Imaging Series 7.7; Molecular Devices, Biberach, Germany). The cells were perfused with the HCO3−-buffered solution. To measure Cl−/HCO3− exchange activity, the HCO3−-buffered solution was changed to Cl−-free HCO3−-buffered solution containing (in mM) 120 Na-gluconate, 5 K-gluconate, 1 MgCl2, 1 CaCl2, 10 d-glucose, 5 HEPES, and 25 NaHCO3 (pH 7.4). Applying external Cl−-free HCO3−-buffered solution increases the efflux of Cl− and the influx of HCO3− via pendrin. To maintain the pH of HCO3−-buffered solutions, the solutions were continuously gassed with 95% O2 and 5% CO2. For Cl−/OH− exchange measurement, the HEPES-buffered solution was replaced with a Cl−-free HEPES-buffered solution for the generation of a Cl− gradient driving cytosolic Cl− efflux and OH− influx through pendrin. SNARF5 fluorescence was recorded at an excitation wavelength of 515 ± 10 nm and emission wavelength of 640 ± 10 nm, and intracellular pH calibration was performed with solutions containing 145 mM KCl, 10 mM HEPES, and 5 μM nigericin, with the pH adjusted to 6.2 to 7.6. For Cl−/I− and Cl−/SCN− measurements, YFP fluorescence was measured in pendrin and YFP (a halide sensor)-expressing CHO-K1 cells. Cl−/I− exchange activity was measured as described earlier for cell-based screening. To measure Cl−/SCN− exchange activity, HEPES-buffered solution was replaced with NaSCN-substituted HEPES-buffered solution (with NaSCN replacing NaCl) for the generation of an SCN− gradient driving SCN− influx through pendrin. YFP fluorescence changes by SCN− influx were monitored using the FLUOstar Omega Microplate Reader (BMG Labtech) and MARS Data Analysis Software (BMG Labtech). HEK-293T cells were stably transfected with human ether-a-go-go-related Gene (hERG) and seeded at a density of 7 × 104 cells per well in poly-l-lysine–coated 96-well plate, and the cells were incubated for 48 hours. Four hours before the assay, the cells were switched from 37°C to 28°C for enhanced membrane expression of hERG channel. After 4 hours, the medium was replaced with 80 μL/well of the FluxOR (Invitrogen, Carlsbad, Calif) loading buffer and incubated for 1 hour at 37°C in the dark. The loading buffer was removed and 100 μL of assay buffer was added to each well. To measure the effect of YS-01 on hERG channels, the cells were pretreated with YS-01 for 10 minutes. FluxOR fluorescence (excitation/emission: 490/525 nm) was recorded for 4 seconds before addition of 20 μL of the stimulus buffer containing thallium ions, and the fluorescence was monitored for an additional 56 seconds. FluxOR fluorescence was recorded and analyzed using FLUOstar Omega Microplate Reader (BMG Labtech) and MARS Data Analysis Software (BMG Labtech). FRT cells expressing human 5-HT2A, ANO1(abc), and YFP-F46L/H148Q/I152L were plated in a 96-well plate at a density of 2 × 104 cells per well and incubated for 48 hours. Each well of the 96-well plate was washed 2 times with 200 μL of PBS, and it was filled with 100 μL of PBS. To measure the effect of YS-01 on 5-HT2A channels, cells were pretreated with YS-01. After 10 minutes of incubation at 37°C, the 96-well plate was placed on the FLUOstar Omega Microplate Reader for YFP fluorescence measurement. Each well was assayed individually for 5-HT2A–mediated I− influx by recording YFP fluorescence continuously (800 ms per point) for 2 seconds (baseline). Then, 100 μL of 140 mM I− solution containing 20 μM 5-HT (Sigma-Aldrich, St Louis, Mo) was added at 2 seconds and then YFP fluorescence was recorded for 10 seconds. CHO-K1, HNE cells were lysed with cell lysis buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, and protease inhibitor mixture). Whole-cell lysates were centrifuged at 15,000g for 10 minutes at 4°C to remove cell debris, and equal amounts (50 μg of protein or 5 μg protein/lane) of supernatant protein were separated on a 4% to 12% Tris-glycine precast gel (KOMA BIOTECH, Seoul, Korea) and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, Mass). The membrane was blocked with 5% nonfat skim milk in TBS including 0.1% Tween 20 (TBST) or 5% BSA in TBST for 1 hour at room temperature. The membrane was then incubated overnight with a primary antibody for pendrin (sc-50346; Santa Cruz Biotechnology, Santa Cruz, Calif), NF-κB p65 (4764S; Cell Signaling Technology, Danvers, Mass), p-NF-κB p65 (3033S; Cell Signaling Technology), IkBα (9242S; Cell Signaling Technology), p-IkBα (9141S; Cell Signaling Technology), β-actin (sc-47778; Santa Cruz Biotechnology), or ANO1 (ab64085; Abcam, Cambridge, Mass) at 4°C. After washing with TBST, the membrane was incubated with secondary antibody for 60 minutes at room temperature, and then washed 3 times with TBST for 5 minutes. The membrane was visualized using the ECL Plus western blotting detection system (GE Healthcare Amersham, Piscataway, NJ). Total mRNA was extracted using TRIzol reagent (Invitrogen) and reverse-transcribed using random hexamer primers, an oligo (dT) primer, and SuperScript III Reverse Transcriptase (Invitrogen). Quantitative real-time PCRs were performed using the StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, Calif) and Thunderbird SYBR qPCR mix (Toyobo, Osaka, Japan). The thermal cycling conditions included an initial step of 95°C for 5 minutes followed by 40 cycles of 95°C for 10 seconds, 55°C for 20 seconds, and 72°C for 10 seconds in a 96-well reaction plate. The primer sequences are listed in Table E1. Total volume of ASL and fluid meniscus, a fluid along with the cell culture insert wall, was measured by fluid absorption through filter paper. Whatman filter paper (10 mm diameter circle; GE Healthcare) was placed on the apical side of a 12-well Transwell insert (Costar, Cambridge, Mass) for 10 seconds. The absorption of ASL and fluid meniscus was determined from the weight changes in the filter paper. The weight of the filter paper was measured using an analytical balance (Sartorius BP61S; Sartorius AG, Göttingen, Germany). The experiments were performed using 8-week-old BALB/c mice. The study protocols were approved by the Institutional Animal Ethics Committee of Yonsei University. Mice were bred and maintained under standard laboratory conditions (12/12-hour light/dark cycle, controlled temperature [21°C ± 2°C] and humidity [55% ± 5%], and ad libitum access to food and water) in the Animal Facility. All experiments were performed according to the guidelines of the Yonsei University Committee on Animal Research. Mice were divided into 3 groups: a vehicle group (PBS-sensitized and -challenged mice treated with vehicle), the OVA group (OVA-sensitized and -challenged mice treated with vehicle), and the OVA + YS-01 group (OVA-sensitized and -challenged mice treated with YS-01). Age- and sex-matched 8-week-old mice were sensitized by intraperitoneal injection of OVA (Sigma-Aldrich) on days 0 and 14. The sensitizing emulsion consisted of 50 μg of OVA and 2 mg of aluminum potassium sulfate in 200 μL of saline. On days 21, 22, and 23, the sensitized mice were lightly anesthetized by isoflurane inhalation and challenged with 100 μg of OVA in 30 μL of saline administered intranasally. Control mice were treated in the same way with PBS. YS-01 (10 mg/kg) was administered 12 hours before each intranasal OVA challenge by intraperitoneal injection. Airway responses to methacholine were measured 24 hours after the last OVA exposure using the FlexiVent system (Scireq, Montreal, QC, Canada). Mice were anesthetized by the intraperitoneal injection of a mixture of Zoletil (30 mg/kg; Virbac Laboratories, Carros, France) and Rompun (10 mg/kg; Bayer, Leverkusen, Germany), then tracheostomized and connected to the FlexiVent. Baseline airway resistance was measured after the nebulization of saline for 10 seconds using an Aeroneb ultrasonic nebulizer (SCIREQ, Montreal, Quebec, Canada). After baseline measurements, mice were exposed to increasing concentrations of nebulized methacholine (0, 1.56, 3.13, 6.25, 12.5, and 25 mg/mL). Whole-body plethysmography (Buxco, Miami, Fla) was also used to measure bronchial airway responsiveness. Enhanced pause (Penh) was used as the main index of airway responsiveness. Penh was measured for 2 minutes in baseline conditions. Mice were then exposed to the inhalation of PBS or methacholine (25 mg/mL) for 2 minutes. Penh results are expressed as absolute values. Bronchoalveolar lavage was performed after the assessment of airway responses. Cells were pelleted by centrifugation and resuspended in PBS to obtain cell counts. Cytospins were prepared with a cytocentrifuge (Shandon Cytospin 4 cytocentrifuge; Thermo Scientific, Waltham, Mass) and were stained with the Diff-Quik Stain Set (Dade Behring, Newark, Del) to assess inflammation. For the assessment of airway inflammation, the left lungs were fixed overnight in 4% paraformaldehyde and embedded in paraffin. Histological slides were prepared from 5-μm sections and stained with hematoxylin and eosin. The inflammatory score indicating the severity of peribronchial inflammation was evaluated by 3 blinded observers using a previously reported method.E2Nakagami Y. Favoreto Jr., S. Zhen G. Park S.W. Nguyenvu L.T. Kuperman D.A. et al.The epithelial anion transporter pendrin is induced by allergy and rhinovirus infection, regulates airway surface liquid, and increases airway reactivity and inflammation in an asthma model.J Immunol. 2008; 181: 2203-2210Crossref PubMed Scopus (86) Google Scholar To assess goblet cell hyperplasia, sections were stained with periodic acid-Schiff using a periodic acid-Schiff Staining Kit (Sigma-Aldrich) according to the manufacturer's protocol. The harvested nasal mucosa and HNE cells on the Transwell inserts were gently washed with PBS and fixed with 4% paraformaldehyde. After deparaffinization and hydration, the slides were immersed in periodic acid solution for 5 minutes at room temperature. After being rinsed in distilled water, the slides were immersed in Schiff's reagent for 15 minutes at room temperature and then washed in running tap water for 5 minutes. OVA-specific IgE in the serum was measured using the Anti-Ovalbumin IgE (mouse) ELISA Kit (Cayman Chemical, Ann Arbor, Mich) according to the manufacturer's instructions. To measure OVA-specific IgE levels in the serum, diluted serum was added to an anti-IgE antibody–precoated 96-well plate, followed by incubation with the OVA-biotin conjugate. The bound biotinylated OVA was detected with streptavidin-horseradish peroxidase using 3,3′,5,5′-tetramethylbenzidine as a substrate. Nasal epithelial cells were plated on Transwell permeable supports and cultured at the air-liquid interface for 14 days. Complete differentiated epithelial cells were incubated with IL-4 (Invitrogen, 10 ng/mL) for 48 hours, and then YS-01 (30 μM) or vehicle was added for 30 minutes. The Transwell inserts with cells were washed with PBS and the basolateral side was treated with 1 mL of PBS containing 10 mM of glucose and 5 μCi of S14CN (total concentration of SCN−: 86 μM). The apical side was filled with 0.5 mL of PBS containing 5 μM of amiloride to block epithelial sodium. Subsequently, the apical fluid was collected every 5 minutes and placed in scintillation vials for the evaluation of radioactivity. Female balb/c mice were dosed intraperitoneally with 10 mg/kg of YS-01 every 24 hours over a 7-day period to test the negative effects of YS-01. Twenty-four hours after the last administration, the hearing of the mice was measured and the plasma was obtained. Total plasma levels of triiodothyronine (T3) or thyroxine (T4) were assayed using a mouse ELISA kit (Calbiotech, San Diego, Calif; T3043T-100 or T4044T-100) according to the manufacturer's instructions. Hearing levels were determined in each mouse by measuring the auditory brainstem response (ABR) thresholds using an auditory-evoked potential workstation and the BioSig software (Tucker-Davis Technologies, Alachua, Fla). The output from the speakers was calibrated by using a PCB 377C10 microphone (PCB Piezotronics, Inc, New York, NY) and was found to be within ±4 dB for the frequency range tested. Mice were anesthetized, following which each ear was stimulated with an ear probe sealed inside the ear canal. Body temperature of the mice was maintained at 38°C with an isothermic heating water-pad. The intensity of clicking sounds was decreased from 70 dB SPL to 10 dB SPL in 5-dB decrements. The average values of ABR were calculated and the hearing threshold was defined as the lowest recognizable ABR response until wave I of the ABR could no longer be visually discerned. Eight-week-old NF-κB luciferase-dTomato reporter mice were sensitized by intraperitoneal injection of OVA (50 μg, Sigma-Aldrich) and 2 mg of aluminum potassium sulfate on days 0 and 14. On days 21, 22, and 23, the sensitized mice were anesthetized by isoflurane inhalation and challenged with 150 μg of OVA in 30 μL of saline administered intranasally. Control mice were treated in the same way with PBS. YS-01 (10 mg/kg) was administered 12 hours before each intranasal administration. NF-κB expression in the lung of NF-κB reporter mice was compared using the IVIS system. The lungs were isolated from the mice and fluorescently imaged (Ex 570, Em 620) using the IVIS spectrum. The average fluorescent density of images was analyzed using Living Image 4.3.1 software (PerkinElmer, Waltham, Mass). The experiments were performed using 4-week-old SD male rat. The study protocols were approved by the Institutional Animal Ethics Committee of Yonsei University. The rats were sacrificed and the trachea strips were isolated. After separation of connective tissues, the trachea was cut into rings of 3 mm length. Tracheas were mounted for tension recording under a 1 g tension and were allowed to equilibrate for 1 hour in organ bath containing 25 mL of an oxygenated physiological solution. The solutions were continuously gassed with 95% O2 and 5% CO2 at 37°C. After 1 hour of stabilization, 0.3 μM of carbachol was used to induce the sustained contractile response in organ bath. Once the sustained tension was established, YS-01 (30 μM), forskolin (10 μM), and IBMX (100 μM) were applied to the bath. Tracheal contraction was measured with an FORT 10G transducer (World Precision Instruments, Sarasota, Fla) and PowerLab 4/35 (AD Instruments, Castle Hill, Australia). The data were recorded and analyzed using Labchart Pro 7 (AD Instruments). The results of multiple experiments are presented as means ± SE. Statistical analyses were performed with Student t tests or ANOVAs, as appropriate. A value of P less than .05 was considered statistically significant .