The following reagents were purchased from Biolegend (San Diego, CA, USA): human RBC lysis buffer, Brilliant Violet 510™ (BV510)-conjugated donkey anti-rabbit IgG polyclonal antibody, PE/Cy7-conjugated mouse anti-human CD14 monoclonal antibody, BV510-conjugated rat anti-mouse Gr-1 monoclonal antibody, BV510-conjugated rat anti-mouse F4/80 monoclonal antibody, PerCP-conjugated rat anti-mouse CD11b monoclonal antibody, human Fc receptor blocking solution, rat anti-mouse CD16/32 antibody, Brefeldin A, Zombie NIR™ Fixable Viability Kit, and Zombie Green™ Fixable Viability Kit. Mouse IL-18BPd DuoSet ELISA kit, APC-conjugated mouse anti-human IL-18Rα monoclonal antibody, PE-conjugated mouse anti-human IL-18 monoclonal antibody, APC-conjugated rat anti-mouse IL-18Rα monoclonal antibody and their respective isotype controls (catalog #: IC002A, IC002P, IC005A) were supplied by R&D Systems (Minneapolis, MN, USA). Rabbit anti-human IL-18BPa and rabbit IgG isotype control were obtained from Novus Biologicals (Minneapolis, MN, USA). IFNγ was supplied by PeproTech (Rocky Hill, NJ, USA). Trypan blue dye and Ovalbumin (OVA, Grade V) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Human IL-18BPa and IL-18 ELISA kits were from ImmunoWay Biotechnology Company (Plano, TX, USA) and ExCell Bio (Shanghai, China), respectively. Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit was bought from BD Biosciences (Beldford, MA, USA). Fetal bovine serum (FBS, Hyclone), penicillin-streptomycin antibiotic mixture and RPMI 1640 medium were obtained from Gibco BRL (Grand Island, NY, USA). Alhydrogel® adjuvant was bought from InvivoGen (San Diego, CA, USA). RBC Lysis Buffer (Multi-species), mouse IL-18 ELISA kit and TRIzol reagent were purchased from Invitrogen (Carlsbad, CA, USA). anti-human CD14 MicroBeads and autoMACS Running Buffer–MACS Separation Buffer, and Lymphoprep™ were obtained from Miltenyi Biotec (Bergisch Gladbach, Germany) and AXIS-SHIELD PoC AS (N-0504 Oslo, Norway), respectively. Dermatophagoides pteronyssinus allergen extract (DPAE) was bought from Greer Laboratories, Inc. (Lenoir, NC, USA). Artemisia sieversiana wild allergen extract (ASWAE) and Platanus pollen allergen extract (PPAE) were purchased from Macro Union Pharmaceutical Co. Ltd (Beijing, China). Allergens for skin prick tests were supplied by ALK-Abelló, Inc. (Denmark). Most of the general-purpose chemicals such as salts and buffer components were of analytical grade.
Subjects And Animals
General characteristics of 33 patients with perennial allergic rhinitis (pAR), 9 patients with sAR and 25 healthy control subjects (HC) recruited in this study were summarized in Supplement table 1. The diagnosing criteria of pAR and sAR were conformed to the Chinese Society of Allergy Guidelines for Diagnosis and Treatment of Allergic Rhinitis .
The experiment was approved by the ethical committees of the First Affiliated Hospital of Jinzhou Medical University and the General Hospital of Northern Military Area. 5 mL of peripheral blood samples were drawn into K2EDTA containing tubes after obtaining written informed consent from each participant, and centrifuged at 450 × g for 10 min. The cells and plasma were collected separately for flow cytometric and ELISA analysis. For CD14+ monocytes isolation study, approximately 180 mL of peripheral blood was taken from each donor.
Five-week-old female BALB/c mice, weighting 18–22 g, were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China), and maintained in specific pathogen-free facilities with free access to standard rodent chow and water, at a constant temperature 23–28°C and relative humidity of 60–75% of the First Affiliated Hospital of Jinzhou Medical University maintained as described previously . The animal experiment procedures were authorized by the Animal Care Committee at Jinzhou Medical University.
Isolation Of Cd14 Monocytes And Allergen Challenge Test
CD14+ cells were enriched by density gradient centrifugation and positive selection on magnetic cell sorting (MACS) according to the manufacturer’s instructions. Purities of recovered CD14+ cells were evaluated by flow cytometry with PE/Cy7-conjugated anti-human CD14 antibody.
To further investigate the direct action of allergen on the expression of IL-18, IL-18BP and IL-18R in monocytes, the isolated monocytes at a density of 1 × 106 per mL were cultured in RPMI 1640 medium containing 3% FBS and 100 U/ml penicillin/streptomycin in a 12-well cell culture plate (Nest, Wuxi, China) in the presence or absence of ASWAE, PPAE, DPAE (all at a concentration of 1.0 µg/mL) or IFNγ (as positive control) at 5 ng/mL for 10, 30 and 60 min, respectively at 37 °C in a 5% (v/v) CO2, water-saturated atmosphere. Brefeldin A at 2 µg/mL was added in wells for detecting the intracellular expression of IL-18 and IL-18BP before stimulation. Cells were then harvested and centrifuged at 450 × g for 10 min at 4 °C. Cell pellets containing approximately 0.5 × 106 and 1 × 106 cells were resuspended in PBS for flow cytometric analysis and in TRIzol reagent for RT-PCR, respectively.
Flow cytometric analysis of IL-18, IL-18BP and IL-18R in human peripheral blood monocytes and isolated CD14 + monocytes
The procedures for detecting IL-18, IL-18BP and IL-18R expression in human peripheral blood monocytes were mainly adopted from a previous study by Zhang et al . Briefly, whole blood cells were challenged with or without ASWAE, PPAE, or DPAE (all at a concentration of 1.0 µg/mL) for 1 h.
For cell surface molecules, whole blood cells were preincubated with human Fc receptor blocking solution and Zombie Green dye , then stained with PE/Cy7 conjugated anti-human CD14 and APC conjugated anti-human IL-18Rα antibodies. Following erythrocyte lysis, leukocytes were analyzed with FACSVerse flow cytometer (BD Biosciences, San Jose, CA, USA). An irrelevant isotype- and concentration-matched antibody of anti-human IL-18Rα was used for fluorescence minus one (FMO) control. Dead cells and doublets were discriminated by SSC-A–live/dead cell dye and FSC-H–FSC-A gating strategies. As for isolated monocytes, cells were processed as above.
For intracellular molecules, resultant leukocytes prepared as above were fixed and permeabilized, and stained with anti-human IL-18BP and PE conjugated anti-human IL-18 antibodies, which was followed by the addition of BV510-conjugated donkey anti-rabbit polyclonal antibody. These procedures were applicable to isolated monocytes.
Establishment Of Mouse Ar Model
A total of 14 mice were randomly divided into control and AR groups. OVA-induced AR mouse model was mainly adopted from a previous study by Mo JH et al . Briefly, mice were sensitized on days 0, 7 and 14 by intraperitoneal injection of 25 µg OVA emulsified in 1 mg of alhydrogel. On days 21–27 mice were challenged by intranasal instillation with 500 µg of OVA dissolved in PBS (10 µL/nostril) once daily. For control experiments, control mice received vehicle only instead of OVA solution. At 24 h following the last OVA challenge, mice were sacrificed by eyeball enucleation, blood and nasal lavage fluid (NLF) samples were thereafter collected. Total cells were determined and centrifuged as above, amongst cells were used for flow cytometric analysis, plasma and NLF supernatant was aliquoted and frozen at -80°C for ELISA assay.
To evaluate allergic symptoms, numbers of sneezing and nasal-rubbing motions of all survived mice during the first 15 min after each challenge were recorded, and compared with healthy control mice (HM) by observers blinded to the study. As presented in Supplement Fig. 1A and Fig. 1B, the numbers of nasal rubbing and sneezing motion in AR mice (n = 7) were substantially higher than that in HM (n = 7) during a 7-day observation period.
Flow cytometric analysis of IL-18R expression in mouse blood monocytes and NLF macrophages
To detect IL-18R expression in mouse blood monocytes, whole blood cells were preincubated with anti-mouse CD16/32 antibody and Zombie NIR dye . Each labeled monoclonal antibody including BV510-conjugated anti-mouse Gr-1, PerCP-conjugated anti-mouse CD11b and APC-conjugated anti-mouse IL-18R was added into tubes before lysing erythrocytes. Finally, cells were processed as for human blood samples and analyzed using flow cytometry.
To detect IL-18R expression in NLF macrophages, cells were first incubated with anti-mouse CD16/32 antibodies and Zombie NIR dye, subsequently incubated with BV510-conjugated anti-mouse F4/80 and APC conjugated anti-mouse IL-18R antibodies, and analyzed as above.
Real-time PCR for IL-18, IL-18BP and IL-18R in isolated CD14 + monocytes
cDNA generated from total RNA of isolated human blood CD14+ cells was used as templates, and qPCR assay was performed as described by Zhang et al  using specific primers of IL-18, IL-18BP and IL-18R as listed in Supplement table 2. Each measurement of a sample was conducted in duplicate.
Measurement of cytokine levels in plasma and NLF supernatant, and calculation of molar concentration ratio of plasma IL-18BP/IL-18
Levels of total IL-18 (tIL-18) and IL-18BP (tIL-18BP) in plasma or NLF supernatant were determined by using ELISA kit according to the manufacturer’s instructions. Molar concentration of human IL-18 and IL-18BP, and free IL-18 (fIL-18) and IL-18BP (fIL-18BP) were calculated as described by Novick D et al .
Human bio-plex panel (Bio-Rad Laboratories, California, USA) was employed to detect plasma levels of IL-1β and TNF-α. The detection ranges for IL-1β and TNF-α were 0.24–3994 pg/mL and 0.57–9270 pg/mL, respectively.
Statistical analyses were performed by SPSS software (version 21.0, IBM Corporation). Data were displayed as a boxplot, which indicates the median, interquartile range, the largest and smallest values for indicated experiments. Where Kruskal–Wallis analysis indicated significant differences between groups, a pairwise test was used for multiple comparisons between the groups. Correlations were determined using Pearson’s correlation or Spearman rank correlation analysis. For all analyses, P < 0.05 was considered statistically significant.