Inhibition of Pendrin by a small molecule reduces Lipopolysaccharide-induced acute Lung Injury

Rationale: Pendrin is encoded by SLC26A4 and its mutation leads to congenital hearing loss. Additionally, pendrin is up-regulated in inflammatory airway diseases such as chronic obstructive pulmonary disease, allergic rhinitis, and asthma. In this study, the effects of a novel pendrin inhibitor, YS-01, were investigated in an LPS-induced acute lung injury (ALI) mice model, and the mechanism underlying the effect of YS-01 was examined. Methods: Lipopolysaccharide (LPS, 10 mg/kg) was intranasally instilled in wild type (WT) and pendrin-null mice. YS-01 (10 mg/kg) was administered intra-peritoneally before or after LPS inhalation. Lung injury parameters were assessed in the lung tissue and bronchoalveolar lavage fluid (BALF). Pendrin levels in the BALF of 41 patients with acute respiratory distress syndrome (ARDS) due to pneumonia and 25 control (solitary pulmonary nodule) patients were also measured. Results: LPS instillation induced lung injury in WT mice but not in pendrin-null mice. Pendrin expression was increased by LPS stimulation both in vitro and in vivo. YS-01 treatment dramatically attenuated lung injury and reduced BALF cell counts and protein concentration after LPS instillation in WT mice. Proinflammatory cytokines and NF-κB activation were suppressed by YS-01 treatment in LPS-induced ALI mice. In BALF of patients whose ARDS was caused by pneumonia, pendrin expression was up-regulated compared to that in controls (mean, 24.86 vs. 6.83 ng/mL, P < 0.001). Conclusions: A novel pendrin inhibitor, YS-01, suppressed lung injury in LPS-induced ALI mice and our data provide a new strategy for the treatment of inflammatory airway diseases including sepsis-induced ALI.


Measurement of Cl -/SCNexchange
The human alveolar epithelial cells (hAEC) purchased from Science Cell (Catalog #3200), which consisted of alveolar type I and type II epithelial cells, lined more than 99% of the internal surface area of the lung. The hAEC from Science Cell research Laboratories were isolated from human lung tissues, cryopreserved at P0, and delivered frozen. The hAEC transient transfected human pendrin (PDS) and YFP-F46L/H148Q/I152L were plated in 96-well plate at a density of 2 × 10 4 cells per well and incubated for 48 h. Each well of the 96-well plate was washed two times with 200 µL of PBS, and it was filled with 100 µL of PBS. To measure the effect of YS-01 on hPDS-mediated Cl -/SCNexchange activity, cells were pre-treated with YS-01. After 10 min of incubation at 37°C, the 96-well plate was placed on the stage of an inverted fluorescence microscope (Nikon, Tokyo, Japan) equipped with a cooled charge-coupled device camera (Zyla sCMOS), image acquisition and analysis software (Meta Imaging Series 7.7). Each well was assayed individually for hPDS-mediated SCNinflux by recording YFP fluorescence continuously (2 s per point) for 4 s (baseline). Then, 100 µL of 140 mM SCNsolution was added at 4 s and then YFP fluorescence was recorded for 14 s. To investigate the effect of long-term treatment of YS-01 in LPS exposed hAEC, we treated the hAEC with LPS (10 μg/ml or 20 μg/ml) for 24 hours with or without YS-01 (20 μM/ml).

Transepithelial SCNtransport
Human nasal epithelial (HNE) cells were plated on transwell permeable supports and cultured at the air-liquid interface for 14 days. Complete differentiated HNE cells were incubated with LPS (10 μg/ml) or DMSO (control) for 24 hours with or without YS-01 (20 μM/ml). The basolateral side was treated with 1 mL of PBS containing 10 mM glucose and 5 μCi of S 14 CN (total concentration of SCN -: radioactivity.

Real-time RT-PCR analysis
Total messenger RNA (mRNA) was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) 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, CA, USA) and Thunderbird SYBR qPCR mix (Toyobo, Osaka, Japan). The thermal cycling conditions included an initial step of 95°C for 5 min followed by 40 cycles of 95°C for 10 s, 55°C for 20 s, and 72°C for 10 s in a 96-well reaction plate. The primer sequences are described in Table E3.

Western blotting
Lung tissues were harvested and lysed in homogenization buffer (PRO-PREP TM Extraction solution, iNtRON Biotechnology). The samples were centrifuged at 13000 g for 30 min at 4°C. Supernatant protein concentration was determined by BCA assay (Thermo Fisher Scientific). Equal amounts of protein were separated by SDS/PAGE and transferred to a nitrocellulose membrane. Membranes were blocked with 5% skim milk in TBS-T (TBS (170-6435, Bio-Rad Laboratories) and 1% Tween-20 (170-6531, Bio-Rad Laboratories) for 1 h at room temperature. Membranes were then incubated overnight with primary antibody diluted in 5% skim milk in TBS-T at 4°C. After washing with TBS-T, the blots were incubated with horseradish peroxidase-conjugated secondary antibodies and 5% skim milk in TBS-T for 1 h at room temperature, then developed using a Super-Signal West Pico chemiluminescence detection kit (Pierce). The antibodies used in the present study included rabbit SLC26A4 (ab98091, abcam), mouse phospho-IκB (9246, Cell Signaling Technology), mouse IκB (4814, Cell Signaling Technology), and rabbit α-tubulin (PA5-16891, Cell Signaling Technology).
Western blot quantification was conducted using ImageJ (Image Processing and Analysis in Java; NIH, USA) software.

IVIS (in vivo optical imaging)
Imaging of live animals and organs was performed using an IVIS kinetic imaging system (Caliper Life Sciences, Preston Brook Runcorn, UK). The IVIS system consisted of a cooled charge-coupled device camera mounted onto a light-tight specimen chamber. The fluorescent excitation light was provided by a halogen lamp in combination with appropriate excitation filters. Emission filters were placed in front of the camera aperture to allow recording of specific wavelengths of light, depending on the emission spectra of the FP examined. Fluorescence imaging was obtained with an excitation wavelength of 554 nm and emission wavelength of 581 nm (dTomato). Mice were divided into three groups for IVIS imaging, (DMSO + PBS, DMSO + LPS, and YS-01 (10 mg/kg, i.p) + LPS), and ex vivo lungs were aseptically removed 6 h after LPS treatment. When organs were imaged, they were placed as flat as possible to allow full and consistent light penetration in order to minimize potential variation in the measurements due to differing tissue thicknesses. Fluorescence was quantified using the region of interest tool in Living Image software (version 3.2, Caliper Life Sciences). Figure S1. NF-κB reporter mouse SUPPLEMENTARY    [4]. Sepsis is defined as SIRS and a known or suspected infection. ARDS was defined according to the Berlin Definition [5]. All ARDS patients were endotracheally intubated and mechanically ventilated using a low tidal volume ventilation strategy according to ARDSNet protocol [6].