2.1 | Patients and tissue samples
We diagnosed and classified CRS according to the EPOS 2012 guidelines. CRSwNP was further diagnosed by bilateral, endoscopically visualized polyps in middle meatus, which had been confirmed by postoperative pathology. Patients with fungal rhinosinusitis, cystic fibrosis, primary ciliary dyskinesia or severe pulmonary and cardiac diseases were excluded from this study. Besides, minors and pregnant women were also excluded. None of the enrolled patients had histories of surgery or the use of corticosteroids or antibiotics within the month prior to surgery. Subjects did not have acuter infection within a month prior to the sampling. Polyps from patients diagnosed with CRSwNP were obtained along with uncinate process and sinus mucosal tissues from patients with CRSsNP; control tissues of the inferior turbinate or uncinate process were obtained from patients without sinonasal disease during other rhinologic surgeries, such as pituitary adenoma, orbital decompression or nasoseptal deviation surgery. A part of the samples was stored at -80 °C, and subsequently used for studying histologic changes, RNA and protein isolation, and immunofluorescence. The rest was freshly submerged in PBS supplemented with 1% antibiotic-antimycotic (Invitrogen, California, USA) for subsequent HNECs culture. More details can be found in the supplementary material.
2.2 | Human nasal epithelial cells culture
Human nasal tissues were cut into 1×1-mm fragments, digested in Dispase II (50 mg/mL, Sigma-Aldrich, St. Louis, MO, USA) overnight at 4 °C, followed by further digestion in trypsin for 15 min at 37 °C; the digestion was terminated by adding complete dulbecco’s modified eagle medium (DMEM) (90% DMEM containing 10% FBS). The digested nasal tissue pieces were then filtered by a 100-mm cell strainer to obtained cells. Cells were then suspended in complete DMEM and cultured in an incubator for 30 min to remove fibroblasts. Then the obtained HNECs were cultured in bronchial epithelial growth medium (BEGM) (Lonza, Basel, Switzerland) added with 1% antibiotic-antimycotic (Invitrogen, California, USA) at a density of (3-5)×105 cells/cm2 at 37℃ in an atmosphere of 5% CO2 and 95% relative humidity. BEGM was refreshed every two days, after 10-14 days incubation, HNECs were stimulated and collected for subsequent experiments.
2.3 | Real-time quantitative PCR (RT-qPCR)
RT-qPCR was performed, as described previously, to detect the mRNA level of target genes. More details can be found in online supplementary material.
2.4 | ELISA
Supernatant of HNECs was collected and examined by MMP-9 ELISA kits (CUSABIO, Wuhan, China). All procedures followed the manufacturers’ instructions.
2.5 | Western blot
Western blotting was performed as reported previously. The PVDF membranes were first incubated with primary antibodies MMP-9 (1:1000; Abcam, Cambridge, UK), pERK, ERK, pIκBα, IκBα (1:1000; Cell Signaling Technology, Boston, USA), and GAPDH (1:3000; PeproTech, Rocky Hill, USA) overnight at 4 °C separately, and then incubated with HRP-conjugated secondary antibody (Bioworld, Nanjing, China). Color development was achieved by ECL reagents. More information can be found in the online supplementary material.
2.6 | Immunofluorescence (IF) staining and confocal microscopy
The frozen sections from human nasal tissue were blocked with 10% goat serum for 30 min and stained first with IL-19 (1:50; Abcam, Cambridge, UK) and MMP-9 antibodies (1:50; Abcam) overnight at 4 °C, separately. HNECs were fixed with 4% paraformaldehyde, blocked in 1% BSA for 30 min, and then stained first with MMP-9 antibodies or p65 (1:500; Cell Signaling Technology, Boston, USA) overnight at 4 °C. Subsequently, the frozen tissue sections and HNECs were incubated with Alexa Fluor 594–/or 488–conjugated secondary antibodies (1:1000; Invitrogen, California, USA) for 1 h at room temperature. Finally, the slices were stained with DAPI (1:30000; Biolegend, California, USA) to mark the nuclei, and sealed with antifade mountant (Invitrogen, California, USA). Immunofluorescence was examined at 400× magnification under a microscope (Leica, Wetzlar, Germany), or at 630× magnification in a confocal microscope (Carl Zeiss, Jena, Germany).
2.7 | siRNA transfection and ERK and NF-kB pathway inhibition
Human IL-20R1 siRNA (5′-CUUACACUGUGCAGUAUUUUU-3′) was synthesized by IGE Biotechnology company (Guangzhou, China). For siRNA transfection, HNECs were transfected for 48 h using Lipofectamine™ 3000 Reagent (Invitrogen, California, USA), in line with the recommended protocol. Subsequently, HNECs were stimulated with 100 ng/mL IL-19 (or without) for 24 h. For pathway inhibition, ERK and NF-kB pathways were suppressed by inhibitors PD98059 or BAY 11-7082 (MedChem Express, Monmouth Junction, NJ, USA) for 1 h, respectively. Similarly, HNECs were stimulated with IL-19 for 24 h.
2.8 | Statistical analysis
IBM SPSS 20 (SPSS, Chicago, IL, USA) was used for statistical analyses. When data were normally distributed, they were presented as mean ± SEM; otherwise, they were presented as median (25–75 percentiles). One-way ANOVA or Kruskal-Wallis test was applied to analyze significance across groups, for comparative study, Student’s t or Mann-Whitney U test (2-tailed) was then applied for comparisons across groups. Paired Student’s t or Wilcoxon matched-pair signed rank test was used when suitable. P<0.05 was regarded as significant.