Materials
The following reagents were used in this study: purified HDM extract from dermatophagoides pteronyssinus was purchased from LSL (Tokyo, Japan); SRT1720 was from Selleck Chemicals (Houston, TX); GB83 was from Axon Medchem (Groningen, Netherlands); Lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS) was from Invivogen (San Diego, CA); Dexamethasone, E64, 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), and mouse monoclonal anti-β-actin antibody were from Sigma (St Louis, MO). Protein block, a blocking reagent, was from Dako (Kyoto, Japan); Rabbit polyclonal anti-PGC-1α antibody, rabbit monoclonal anti-TFAM antibody, rabbit monoclonal anti-E-cadherin antibody, rabbit polyclonal anti-ZO-1 antibody, FITC-conjugated goat anti-rabbit secondary antibody, and Alexa Fluor 647-conjugated goat anti-rabbit secondary antibody were from Abcam (Cambridge, MA); horseradish peroxidase–conjugated secondary antibodies were from Santa Cruz Biotechnology (Dallas, TX); MitoTracker Red probe and Hoechst 33342 were from Invitrogen Life Technologies (Eugene, OR); Keratinocyte-SFM (serum-free medium) and Human Keratinocyte Growth Supplements were from Gibco (Grand Island, NY). Bronchial epithelial growth medium (BEGM) bullet kit including bronchial epithelial basal medium (BEBM) and subculture reagents were purchased from Lonza Japan Bioscience (Tokyo, Japan)
Cell culture
BEAS-2B cells, a human bronchial epithelial cell line, were obtained from American Type Culture Collection (Manassas, VA). The BEAS-2B cells were cultured in Keratinocyte-SFM medium supplemented with Keratinocytes Supplements at 37 ℃ in a humidified atmosphere of 5 % CO2 and passaged in 75 cm2 flasks. Normal primary human bronchial epithelial cells (pHBEC) were obtained from Lonza (Wokingham, UK) and cultured in BEGM. To prepare BEGM, BEBM was supplemented with human recombinant epidermal growth factor (0.5 ng/ml), insulin (5 μg/ml), transferrin (10 μg/ml), hydrocortisone (0.5 μg/ml), triiodothyronine (6.5 μg/ml), epinephrine (0.5 μg/ml), retinoic acid (50 nM), gentamycin and amphotericin-B (50 μg/ml) and bovine pituitary extract (35 mg/ml).
Animals
8-week-female C57BL/6J mice were purchased from SEIMI (Sendai, Japan). All mice were housed in a specific pathogen-free facility and maintained under constant temperature (24°C), humidity (40%), and light cycle (8:00 A.M. to 8:00 P.M.), with food and water provided ad libitum. The mice were exposed to HDM (30 μg/body, 50 μl) intranasally every other day as described previously. 20 The control mice received PBS. The mice were euthanized on day 15, and the trachea was cannulated after the pulmonary circulation was perfused and free of blood. The lung was inflated with 10% paraformaldehyde for 10 min at 25 cm H2O and subsequently removed and fixed in 10% paraformaldehyde for 24 h at room temperature. Samples were then dehydrated in ethanol and xylene, embedded in paraffin, cut in 5-μm sections. All experiments were approved by the Tohoku University Animal Experiment Ethics Committee and performed in accordance with the Regulations for Animal Experiments and Related Activities at Tohoku University.
Western blotting
The expression of markers of mitochondrial biogenesis (PGC-1α and TFAM) and junctional proteins (E-cadherin and ZO-1) in BEAS-2B cells was analyzed by western blotting. Cells (2 × 105 cells/ml) were seeded in 6-well culture plates and grown to 80-90 % confluence. Then the cells were maintained in Keratinocytes Supplements free medium for 24 h and stimulated with or without HDM (100 μg/ml). 24 h after HDM stimulation, the cells were homogenized in cell lysis buffer (0.05 % TritonX, 3 mM Tris-HCl, pH7.4 0.4 mM EGTA, 10 mM MgCl2, 1 μM phenylmethylsulfonyl fluoride, 100 μg/ml aprotinin, 1 μg/ml leupeptin) on ice and harvested for western blotting. SRT1720 (1 μM), a PGC-1α activator, was added to the culture medium at 6 h before HDM stimulation. Protease activated receptor (PAR) 2 inhibitor GB83, Toll-like receptor (TLR) 4 inhibitor LPS-RS, cysteine peptidase inhibitor E64, and serine peptidase inhibitor AEBSF were added at 30 min before HDM stimulation. Synthetic glucocorticoid dexamethasone was added at 24 h before HDM stimulation. The whole cell lysates were homogenized for several seconds using an ultrasonic homogenizer (BANDELIN, Berlin, Germany). The samples were solubilized in SDS-PAGE sample buffer (Bio-Rad, Hercules, CA). Equal amounts of protein were loaded and separated by electrophoresis on 12% SDS polyacrylamide gels. After electrophoresis, the separated proteins were transferred to polyvinylidene difluoride membranes (Millipore, Darmstadt, Germany). The membranes were blocked with a blocking reagent. Rabbit polyclonal anti-PGC-1α antibody (1:5000), rabbit monoclonal anti-TFAM antibody (1:5000), rabbit monoclonal anti-E-cadherin antibody (1:10000), rabbit polyclonal anti-ZO-1 antibody (1:2000), and mouse monoclonal anti-β-actin antibody (1:5000) were used as primary antibodies to detect the target proteins. Horseradish peroxidase–conjugated secondary antibodies (1:5000) were detected using ECL-prime Western Blotting Reagent (Amersham Biosciences, Buckinghamshire, UK) and visualized with a chemiluminescence imaging system (LAS-4000 mini, FUJIFILM, Tokyo, Japan). Each band intensity was quantified using ImageJ (National Institutes of Health, Bethesda, MD, USA).
Evaluation of mitochondrial mass and immunochemical localization of E-cadherin and ZO-1 in BEAS-2B cells
BEAS-2B cells were seeded in 8-well chamber slides at a density of 1 × 105 cells/ml and cultured for 24 h, and then the media were replaced with the keratinocyte supplement-free medium for 24 h. After incubation with 1 µM SRT1720 for 6 h, the cells were stimulated with HDM (100 μg/ml) for 24 h. The mitochondrial mass was evaluated using MitoTracker Red probe. MitoTracker Red was added to the existing medium to a final concentration of 200 nM and cells were incubated for 30 min at 37 ℃, 5 % CO2. After staining, the cells were washed once with PBS. Then, the slides were fixed with freshly prepared 4 % paraformaldehyde in PBS for 30 min at room temperature. The slides were permeabilized with 0.1 % Triton X-100 in PBS for 10 min at room temperature. The slides were blocked with a blocking solution for 30 min at room temperature. The slides were then incubated overnight with rabbit monoclonal anti-E-cadherin antibody (1:500), rabbit polyclonal anti-ZO-1 antibody (1:200) at 4℃. The slides were incubated with the appropriate FITC-conjugated secondary antibody (1:3000)for 1 h at room temperature. The nuclei of the cells were stained with Hoechst 33342 (1:200). Fluorescent images were obtained using a confocal laser scanning microscope system (Nikon ECLIPSE Ti-E, C2si; Nikon, Tokyo, Japan). The mean fluorescence intensity of E-cadherin, ZO-1, MitoTracker were quantified by using ImageJ.
Immunochemical localization of PGC-1α, TFAM and E-Cadherin in lung tissues from mice
Tissue samples were fixed in 10% formalin and embedded in paraffin. 5 μm thick serial tissue sections were obtained and mounted in Superfrost/Plus glass slides (Fischer Scientific). Deparaffinization was performed by washing three times for 5 minutes in xylene, then washing in 100%, 95%, 80%, 70% ethanol three times for 5 minutes and, finally, rinsing with distilled water. Slides were washed in PBS for 5 min and processed for antigen retrieval using citrate buffer. The sections were permeabilized with Triton-X 100 for 15 min. Sections were then washed in PBS, blocked using blocking reagent (Dako) for 60 min at room temperature. Sections were incubated overnight at 4 °C with rabbit polyclonal anti-PGC-1α antibody (1:1000), rabbit monoclonal anti-TFAM antibody (1:10000), rabbit monoclonal anti-E-Cadherin antibody (1:10000) or nonspecific polyclonal rabbit IgG as a negative control at 4°C overnight. After being washed, the samples were incubated with Alexa Fluor 647-conjugated goat anti-rabbit secondary antibody (1:3000) for 60 min at room temperature. After washing, the nuclei of the cells were stained with Hoechst 33342 (1:200). Fluorescent images were obtained by microscopy (BX53-33-SDO; Olympus, Tokyo, Japan) and photographed with a digital camera (DP71-SET; Olympus). The mean fluorescence intensities of PGC-1α, TFAM, and E-cadherin were quantified by using ImageJ.
Transepithelial electrical resistance measurements (TEER)
Primary human bronchial epithelial cells were seeded onto collagen type I coated 12-well transwell inserts (transparent, 0.4 µm; Greiner) at 1 × 105 cells/cm2 with 500 µl apical and 1500 µl basolateral volumes, maintained in BEGM, and incubated until the formation of cell monolayers. Then, SRT1720 (1 µM) was added to the apical medium. After 24 h incubation with SRT1720, the cells were stimulated with HDM (100 ng/ml) or DMSO (vehicle) for 24 h. Then, TEER was monitored using a Millicell ERS-2 voltohmmeter (Millipore, Billerica, MA).
Statistical Analysis
Results are presented as mean ± SD. Comparisons among groups were performed by One-way analysis of variance followed by the Tukey's multiple comparison test or unpaired t-test. p<0.05 was considered statistically significant.