A total of 40 subjects (> 18 years old) included 20 AR and 20 controls were enrolled in our study. The inclusion criteria for AR patients included: (1) confirmed diagnosis of AR as described by the Allergic Rhinitis and its Impact on Asthma (ARIA) guideline, (2) at least 1 year history, (3) typical symptoms such as sneezing, blocked nose, itchy nose and runny nose, (4) allergic to at least one inhalant allergen (dust mites, pets, molds, cockroach, etc.) confirmed by specific IgE measurement (Phadia ImmunoCAP, Sweden). The exclusion criteria included: (1) the presence of other allergic diseases, such as asthma, dermatitis, (2) acute or chronic rhinosinusitis, (3) pregnancy or breastfeeding, (4) previous treatment with immunotherapy, (5) with immunologic disease, tumors, or chronic infection, (6) use of systemic corticosteroids or anti-histamine drugs in the past 2 weeks, and (7) with diabetes, hypertension, myxedema, hypothyroidism, obesity, liver and kidney diseases and other diseases which may affect blood lipid levels. Normal subjects had no history of allergy and positive allergen test. The exclusion criteria for controls were similar to that of AR. Our study obtained approval and written informed consent from local ethics committee boards.
Total nasal symptom scores
The nasal symptoms severity questionnaire was completed. The nasal symptoms including sneezing, runny nose, itchy nose, and blocked nose were rated between 0 and 3 scale (0, none; 1, mild; 2, moderate; and 3, severe).
Blood lipid measurement
Venous fasting blood were obtained for assaying serum total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein, and triglyceride (TG) levels. The blood lipids were determined using a Cobas Integra 400 (Roche, Switzerland).
Effects of HDL on ILCs
Peripheral blood mononuclear cells (PBMCs) were purified using density-gradient centrifugation from of AR and controls. PBMCs (1.0 × 106 cells/mL) were incubated in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum and treated by human HDL-DiI (Biotrend, FL) for 24 h at 37 °C.
For determination of ILC2s, lineage markers-FITC, FceRI-APC, CD45-APC/Cy7, CRTH2-PE, CD127-PE-Cy7 antibody (BD Bioscience, NJ) were used for staining. Lin− FceRI CD45+ CRTH2+CD127+ cells were defined as ILC2s. The proliferation of ILC2 was determined using tritiated thymidine incorporation.
Total RNA was extracted from stimulated PBMCs by TRIzol (Invitrogen, US). RNA was reverse-transcribed for cDNA synthesis. PCR reaction was performed using real-time PCR detection system (BioRad). The relative levels of target genes was normalized to GAPDH housekeeping gene using 2-ΔΔCt method. The primers were listed as follows: GATA3 sense, 5ʹ-GCGGGCTCTATCACAAAATGA-3ʹ, antisense, 5ʹ-GCTCTCCTGGCTGCAGACAGC-3ʹ; RORα sense, 5-AAGGAGCCAGAAGGGATGAAC-3ʹ, antisense, 5ʹ-GGAACA ACAGACGCCAGTAAG-3ʹ; GAPDH sense, 5ʹ- AGCCACATCGCTCAGACAC-3ʹ, antisense, 5ʹ- GCCCAATACGACCAAATCC -3ʹ.
Enzyme-linked immunosorbent assay (ELISA)
The concentration of IL-5, IL-13 were examined using ELISA kits (R&D systems, USA). The sensitivity of cytokines was: IL-5, 3.9 pg/mL, IL-13, 125 pg/mL.
Prism (GraphPad Software 8.0) were used for analysis with P values lower than 0.05 as statistically significant in all analyses. The Kruskal–Wallis H test or nonparametric Mann–Whitney U test was done. Spearman rank was done for correlation analysis.
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