Twenty-eight children with exacerbated asthma and without using any glucocorticoids were recruited from the pediatric department of the First Affiliated Hospital of Guangxi Medical University (Nanning, Guangxi, China). The subjects had no respiratory infection in the past 4 weeks. The diagnosis and severity assessmentof asthma were performed according to Global Initiative for Asthma (GINA) guidelines. Spirometry (MasterScreen, JAEGER, Germany) was performed as recommended by the GINA guidelines. Ten healthy children with no respiratory symptoms and normal lung function were recruited as the healthy control group (HC group) (n = 10). All the participants were given informed consent prior to their inclusion in the study. The study was approved by the institutional ethics review board of the First Affiliated Hospital of Guangxi Medical University.
Sputum Induction And Processing
Sputum was induced using hypertonic saline (4.5% for mild-moderate asthma and controls; 0.9% for severe asthma) delivered via an ultrasonic nebuliser as previously described. Induced sputum was selected from saliva, and processed within 2 hours for the preparation of cell suspension by adding four volumes of phosphate-buffered saline solution ( PBS) and mixed by rotating for 15 min at 22 °C. Then, the cell suspension was centrifuged at 400 g for 10 min. The supernatant was aspirated and stored at -80 °C for cytokine assays, and the cell pellet was resuspended by adding a volume of 0.1% dithiothreitol and mixed by rotating for 15 min at 22 °C. The suspension was then filtered, and a total cell count and viability were determined. The remaining sample was centrifuged and the cell pellet was diluted with PBS to 1 × 106 cells/mL. Slides were prepared by using cytospin (Cytopro 7620 ,Wescor, USA) and stained with Diff-Quick stain for differential cell counts, 400 nonsquamous cells were counted. Results were expressed as a percentage of total non-squamous cells.
Classification Of Asthma Phenotypes
Based on previous studies[15, 16], subjects with ≥ 2% sputum eosinophils and < 61% sputum neutrophils were classified as eosinophilic asthma (EA), and those with > 61% sputum neutrophils and < 2% sputum eosinophils were classified as neutrophilic asthma (NA). Subjects with ≤ 61% sputum neutrophils and < 2% sputum eosinophils were classified as paucigranulocytic asthma (PGA). Twenty-eight children with exacerbated asthma in this study were divided into three groups: EA group (n = 12), NA group (n = 10) and PGA group (n = 6) according to the induced sputum cytology.
Heparinized blood samples from asthmatic patients and healthy controls were obtained and erythrocytes were lysed by BD Pharm Lyse™ Lysing Buffer (BD Biosciences, USA). The cells (1 × 106 cells/ml) were thereafter incubated in 24-well cell culture plates in RPMI 1640 cell culture medium (Gibco, USA) containing 10% heat-inactivated FBS, 2 mM L-glutamine, 20 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin, and activated with phorbol-12-myristate-13-acetate (PMA) (25 ng/ml) (Sigma, USA) and ionomycin calcium salt (1 µg/ml) (Sigma) for 1 h at 37 °C in a humidified 5% CO2 atmosphere, then stimulated for additional 4 h in the presence of protein transport inhibitor (0.7 µl /ml) (BD GolgiStop™, BD Biosciences, USA). Th17, Th2 cells and nuclear-associated antigen Ki-67expressed in Th17 cells were detected by surface antigen and intracellular cytokine staining using flow cytometry. The cells were washed in ice-cold PBS followed by staining with APC-labeled anti-human CD4 (BD Biosciences) and PerCP-Cy™ 5.5-labeled anti- human CD8 (BD Biosciences) for 30 min at 4 °C in the dark. Samples were washed twice in PBS before fixation and permeabilization using Transcription Factor Buffer Set (BD Biosciences) as recommended by the manufacturer. Intracellular staining was performed at 4 °C for 50 minutes in the dark with PE-labeled anti-human cytokine antibodies (IL17, IL4, BD Biosciences) and Alexa Fluor 488-labeled anti-human cytokine antibodies (Ki-67, BD Biosciences). PE-conjugated Mouse IgG1κ and Alexa Fluor 488-conjugated Mouse IgG1κ (BD Biosciences) were used as isotype controls. Four-color flow cytometry was performed by a FACS Calibur System (Becton Dickinson, USA). Results were analyzed by FCS Express 4 software (De Novo Software, Canada).
Peripheral Blood Mononuclear Cell (PBMC) Culture And Stimulation
Blood samples from patients and healthy controls were collected in acid citrate dextrose vacutainer tubes (Becton Dickinson, USA) and used for plasma selection and PBMC isolation. The cells were isolated by density gradient centrifugation on the lymphocyte separation medium Lymphoprep with a density of 1.077 g/ml, PBMCs were collected and washed twice with PBS. The cells (1 × 106 cells/ml) were cultured for 5 hours in 24-well cell culture plates in complete medium in presence of PMA (25 ng/ml) (Sigma) and ionomycin calcium salt (1 µg/ml) (Sigma) at 37 °C in a humidified 5% CO2 atmosphere. The culture supernatant and plasma were collected and stored at -80 °C for cytokine assays. The cells were processed for real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis.
Total RNA was extracted from PBMCs using a E.Z.N.A.® Total RNA Kit I (OMEGA, USA), then the cDNAs were synthesized using a PrimeScript™ RT reagent Kit (TaKaRa, RR037S, Dalian, China), following the instructions provided by the manufacturer. Based on mRNA sequences of RORγt in Genbank, the primers were designed with primer premier 5.0 software and synthesized by Sangon Biotech Co.,Ltd (Shanghai, China). The following primers were used: RORγt, 5’-CCTGGGCTCCTCGCCTGACC-3’ (forward primer) and 5’-TCTCTCTGCCCTCAGCCTTGCC-3’ (reverse primer) with the amplicon size of 169 bp, β-actin, 5’ -CACGAAACTACCTTCAACTCC-3’ (forward primer) and 5’ -CATACTCCTGCTTGCTGATC-3’ (reverse primer) with the amplicon size of 262 bp. The qRT-PCR was performed with a 7500 Real Time PCR system (Applied Biosystems, USA) using FastStart Universal SYBR Green Master (ROX) (Roche, Swiss) in a 20 µl reaction volume containing 200 nM primers and 5 ng cDNA. Thermal cycling was initiated with a 10-min denaturation at 95 °C, followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s. The relative RORγt mRNA expressions were normalized to the level of β-actin (the housekeeping gene) transcripts and quantified by the 2∆∆Ct method using 7500 System Sequence Detection software (Applied Biosystems).
The levels of IL-17A in sputum supernatant, culture supernatant from PMA-stimulated PBMCs as well as plasma, and the concentrations of IL-8, IL-5 in sputum supernatant were measured using commercial enzyme-linked immunosorbent assay (ELISA) kit (Boster, China) according to the manufacturer’s instructions. The lower detection limits were 1 pg/ml for IL-17A, 1 pg/ml for IL-8 and 1 pg/ml for IL-5, respectively.
Statistical Package for Social Sciences version 16.0 software was used for data analysis. Normal distribution data are summarized as mean and standard deviation (SD), and those of non-normal distribution are summarized as median and interquartile range (IQR). Differences between groups were determined using one-way analysis of variance (ANOVA) with Student- Newman-Keuls post hoc test for parametric data or Kruskall Wallis test for nonparametric data. Associations between variables were examined using Pearson’s correlation coefficients. A p-value of < 0.05 (two-tailed) was considered statistically significant.