Reagents and antibodies
Lipopolysaccharide (LPS) from Pseudomonas aeruginosa, dimethyl sulfoxide (DMSO), clarithromycin (CAM), Roxithromycin (RXM), dexamethasone (DEX), fluticasone propionate (FP), and inhibitors of ERK (U0126), p38 (SB203580), JNK (SP600125), Akt (LY294002) and nuclear factor-κB (NF-κB, BAY 11-7082) were all provided by Sigma-Aldrich Co. (St. Louis, MO). LPS from the photosynthetic bacterium Rhodobacter sphaeroides (LPS-RS, LPS inhibitor) was purchased from InvivoGen (Carlsbad, CA). Primary antibodies against TLR4, TSLP, phospho-ERK, total-ERK, phospho-p38, total-p38, phospho-JNK, total-JNK, phospho-Akt, total-Akt, p-p50, p50, and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary antibodies conjugated with horseradish peroxidase against anti-rabbit and anti-mouse were obtained from Vector Laboratories Inc. (Burlingame, CA).
Nasal inferior turbinate tissues were obtained from six patients with CRS (3 males and 3 females; mean age 41.2 ± 4.5 years) during endoscopic sinus surgery. The diagnosis of CRS was based on the history of patients and endoscopic/radiographic criteria, as well as CT findings of sinuses according to the 2012 European position paper on rhinosinusitis and nasal polyps (EPOS) guidelines. Nasal polyp tissues were obtained from the region of the middle meatus at the beginning of endoscopic sinus surgery in CRSwNP patients. None of the patients had taken oral steroids, non-steroidal anti-inflammatory drugs, antihistamines, or antibiotics for at least 4 weeks prior to endoscopic surgery.
All patients were recruited from the Department of Otorhinolaryngology, Korea University Medical Center, Korea. Informed consent was provided according to the Declaration of Helsinki. This study was approved by the Korea University Medical Center Institutional Review Board, which also authorized the research. The study was carried out in accordance with the guidelines of the Human Ethics Committee of Korea University Guro Hospital (KUGH14065-001).
Sinonasal fibroblast culture
Sinonasal tissues were isolated by enzymatic digestion with collagenase (500 U/mL; Sigma-Aldrich, St. Louis, MO), hyaluronidase (30 U/mL, Sigma-Aldrich), and DNase (10 U/mL, Sigma-Aldrich). Sinonasal fibroblasts were cultured in Dulbecco’s Modified Eagle Medium containing 10% heat-inactivated fetal bovine serum (Invitrogen, Carlsbad, CA), 1% 10,000 μg/mL streptomycin, and 1% 10,000 U/mL penicillin (Invitrogen). The purity of the sinonasal fibroblasts were confirmed by their characteristic spindle-shaped cell morphology and by flow cytometry.
Organ culture of nasal inferior turbinate
Nasal inferior turbinates were cut, using scissors, into 2 to 3mm3 pieces under sterile conditions. Tissue fragments were washed three times with phosphate buffered saline. The washed tissue fragments were placed on a pre-hydrated gelatin sponge (10 mm × 10 mm × 1 mm; Spongostan, Johnson & Johnson, San Angelo, TX) in 6 well plates. Well were filled with 1.5 ml of culture medium containing Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen,) and 2% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA) per well. The plates were placed in maintained at 37°C in 5% CO2.
Real-time polymerase chain reaction was used to evaluate the mRNA levels in nasal fibroblasts and inferior turbinate tissues. Total RNA was extracted using TRIzol reagent (Invitrogen). Reverse transcription was performed using MMLV reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. RT-PCR was performed using the following primers: TSLP (sense sequence, 5’- TAT GAG TGG GAC CAA AAG TAC CG-3’; anti-sense sequence, 5’- GGG ATT GAA GGT TAG GCT CTG G-3’), TLR4 (sense sequence, 5’-TGA GCA GTC GTG CTG GTA TC-3’; anti-sense sequence, 5’-CAG GGC TTT TCT GAG TCG TC-3’), GAPDH (sense sequence, 5’-GTG GAT ATT GTT GCC ATC AAT GAC C-3’; anti-sense sequence, 5’-GCC CCA GCC TTC TTC ATG GTG GT-3’). RT-PCR was performed with Quantstudio3 (Applied Biosystems, Foster City, CA) using Power SYBR Green PCR Master Mix (Applied Biosystems). The delta-delta Ct (2-ddCt) method was used to analyze relative gene expression levels. Experiments were repeated at least three times, and GAPDH was used as the internal control.
Nasal fibroblasts were seeded into 60 mm culture dishes at a density of 5×105 cells/mL. Sinonasal fibroblasts were lysed in RIPA buffer (Sigma-Aldrich) with protease inhibitors (Sigma-Aldrich) and phosphatase inhibitors (Sigma-Aldrich). Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinyl difluoride membranes (Merck Millipore, Billerica, MA). Membranes were blocked with 5% skim milk. The blots were incubated with primary antibodies against TLR4, phospho-Akt, total-Akt, β-actin (Santa Cruz Biotechnology, Inc., Dallas, TX), phospho-ERK, ERK, phospho-p38, p38, phospho-JNK, JNK, p-p50, and p50 (Millipore Inc., Billerica, MA). Next, the blots were visualized with HRP-conjugated secondary antibodies and an ECL system (Pierce, Rockford, IL). Images were analyzed using ImageJ software (NIH, Rockville, MD). Protein expression was normalized to β-actin or total protein in the case of phosphorylated proteins.
Enzyme-linked immunosorbent assay (ELISA)
The concentration of TSLP production within the supernatants was determined by using an ELISA kit (R&D Systems, Minneapolis, MN) according to manufacturer’s instructions. The optical densities of the standards and samples were measured at 450nm using a microplate reader (Bio-Rad, Hercules, CA).
Sinonasal fibroblasts were seeded on 8-well culture slides (SPL Life Sciences, Korea) at 5×104 cells/ml per well and grown to 60% - 70% confluence. Sinonasal fibroblasts were fixed with 4% paraformaldehyde for 10 minutes and treated with 0.01% Triton X-100 (Sigma-Aldrich) for permeabilization. Blocking was performed by adding 3% bovine serum albumin for 1 hour. Sinonasal fibroblasts were stained with the following primary antibodies overnight at 4℃: anti-TSLP, TLR4 (1:1,000, Santa Cruz Biotechnology, Inc.) or p-p50 (Millipore Inc). The sinofibroblasts were then incubated with anti-mouse Alexa 488 (Invitrogen) or anti-rabbit Alexa 555 (Invitrogen) secondary antibodies for 1 hour. Counterstaining was performed using 4'-6-diamidino-2-phenylindole (Sigma-Aldrich). Image acquisition and processing were performed using a confocal laser scanning microscope (LSM700; Zeiss, Oberkochen, Germany).
NF-κB luciferase reporter gene constructs (luc2P/NF-κB-RE/Hygro and hRluc/TK; Promega Co., Madison, WI) were transiently transfected into nasal fibroblasts by using fetal bovine serum and antibiotic-free DMEM containing 5 μL of FuGENE transfection reagent (Promega Co). After 5 hours of incubation, the medium was replaced with DMEM containing 10% fetal bovine serum. Luciferase assays were performed to determine the firefly luciferase activity relative to the Renilla luciferase activity in the cell lysate using a luminometer (Promega Co.).
Statistical analysis of the differences between control and experimental data was performed with unpaired t-test or one-way analysis of variance followed by Tukey’s test (GraphPad, version 7, GraphPad Software, Inc., La Jolla, CA). Significance was established at the 95% confidence level. P values less than 0.05 were accepted as statistically significant. Results were obtained from at least three independent replicate experiments.