Study design and subjects
Subjects with AR based on criteria of the AR and its Impact on Asthma (ARIA) consensus statement [16] were recruited consecutively from the allergy-rhinology outpatient clinic of Beijing Tongren Hospital. On recruitment, each subject completed a questionnaire to record demographic data, nasal symptom severity, and history of asthma; and blood samples were collected from each subject for analysis of serum specific IgE antibodies. Peripheral mononuclear cells (PBMCs) were also prepared from blood samples of some healthy controls and HDM monosensitized AR patients. Because there was lack of a reliable validated assay, whether HDM monosensitized AR patients had IgE-reactivity to Der p10 were unknown. None of the subjects had received any allergen-specific immunotherapy or monoclonal antibody treatment. The study was approved by the Medical Ethics Committee of Beijing Tongren Hospital, and all patients provided written informed consent before entry into the study and collection of any samples.
Serum antigen-specific IgE measurements
The presence of IgE antibodies in blood was determined using a EUROLINE Atopy Screen (DP 3713 E; Lubeck Germany), which comprised two sets of allergens; one with a mix of aeroallergens [including tree mix (willow, poplar, elm), common ragweed, mugwort, house dust mite mix (Dermatophagoides pteronyssinus (Der p), Dermatophagoides farinae (Der f)), house dust, cat, dog, cockroach German, mould mix (Penicillium notatum, Cladosporium herbarum, Aspergillus fumigatus, Alternaria alternata) and hops], and one with a mix of food allergens [including egg white, cow’s milk, peanut, soybean, beef, mutton, sea fish mix (codfish, lobster, scallop), shrimp, and crab]. Furthermore, concentrations of Der f2 specific IgE, Der p1 specific IgE, and total IgE were also measured using the ImmunoCAP system (Immunodiagnostics; Thermo Fisher Scientific, Uppsala, Sweden). Allergen-specific IgE > 0.35 kU/L was considered as positive.
Mugwort protein analysis by mass spectrometry
Prior to analysis, 100 mg samples of mugwort (Artemisia sieversian (A. sieversian)) were separately prepared as peptide solutions by denaturing and treatment with protease trypsin according to the method described by León and colleagues [17]; and then analysed in a Triple-TOF 6600 mass spectrometer (Sciex, United States) fitted with a Nanospray III source (Sciex). The ion spray voltage was 2300 V, declustering potential 80 V, curtain gas 35 psi, nebulizer gas 5psi, and interface heater temperature at 150 °C. The peptides were introduced into the mass spectrometer via Nona 415 liquid chromatography column (Sciex) eluted with water/acetonitrile/formic acid (buffer B: 2/98/0.1%). In this regard, samples (4 μL) were injected onto a C18 desalted column (3 μm, 120 Å, 350 µm × 0.5 mm), and separated onto a C18 analysis column (3 μm, 120 Å, 75 µm × 150 mm) with gradients ranging from 5 to 16% buffer B in the first 25 min, from 16 to 26% buffer B in the next 20 min, from 26 to 40% buffer B in the following 3 min, from 40 to 80% buffer B in the next 5 min, and finally from 80 to 5% buffer B in the final 7 min; at a flow rate of 0.6 μL/min. Since the genome sequence annotation database of Artemisia sieversian are unavailable, the peptides presented in the samples were matched to the UniProt Artemisia carvifolia databases. All identified corresponding proteins in A. sieversian were separately listed in Additional file 1: Table S1.
Synthesizing tropomyosin peptide of mugwort
According to the result of mass spectrometry, two common repeat peptide sequences of tropomyosin protein from A. sieversian were synthesized from SynPeptide company (Shanghai, China) as follows: VGSPDESYEDFTNSLPSNECR; IEEQQVIVEK. Giving the preliminary data of ELISA inhibition experiment, basophil and PBMC activation experiments, synthetic peptide with sequence of VGSPDESYEDFTNSLPSNECR was chosen as the representative sequence for tropomyosin protein from mugwort.
HDM-specific IgE blockage by synthetic peptide of mugwort tropomyosin
Serum samples of 15 HDM+AR patients with a high or low level of HDM-specific IgE were used to assess whether the IgE can be pre-blocked by synthetic peptides of mugwort tropomyosin. Briefly, 200 μL of serum from HDM+AR patients were incubated with or without synthetic mugwort tropomyosin peptides (1000 ng/mL for each) for 1 h at room temperature, and at the end of incubation the serum samples were analysed for the concentrations of HDM-specific IgE using the ImmunoCAP system.
Basophil activation test
PBMCs isolated from non-allergic donors (5 \(\times\) 10^5 cells) were stripped in 2 mL ice cold lactic acid buffer (0.13 M KCl, 0.05 M NaCl, 0.01 M lactic acid, pH = 3.9) for 30 s as described before [18]. After washing 3 times by PBS, cells were pre-incubated with sera from HDM-allergic individuals for 1 h at 37 °C. And then, cells were stimulated by different concentrations of synthetic mugwort tropomyosin or Derp 10 (50, 500 ng/mL) in hepes buffer containing IL-3 (R&D, Minneapolis, Minnesota, USA). In the meanwhile, cells exposed to FLMP (Sigma, St. Louis, USA)) were taken as a positive control. The reaction was stopped by EDTA buffer (20 mM). In the end, PBMCs were stained with basophil surface markers: CD123BV650, CCR3-APC-fire750 and CD63-PE (BioLegend, San Diego, CA, USA), and the percentages of CD63+CD123+CCR3+ cells were analysed by Flowjo software.
ELISA inhibition experiment
Plates were pre-coated with Der p10 obtained from CUSABIO (Wuhan, China) overnight at 4 °C, then incubated with PBS supplemented with 1% BSA and 0.05% tween 20 for 6 h at room temperature to reduce non-specific binding. Inhibition was performed by adding sera from HDM monosensitzed patients with synthetic mugwort tropomyosin peptides (50, 500 ng/mL), and sera without peptides were taken as non-inhibition conditions. Anti-human IgE (2 μg/mL, NOVUS, USA) were added, followed by streptavidin-HRP conjugated secondary antibodies (diluted 1:2000; EasyBio, Beijing, China). Absorbance was determined using an ELISA reader (BioTek, Vermont, USA) at 405 nm. All experiments were performed in duplicate. Percent inhibition was calculated using the following equation: percent inhibition = 100 − [(OD of serum with tropomyosin peptide/OD of serum without peptide) × 100].
Stimulation of PBMCs ex vivo
PBMCs were isolated from the blood of 6 healthy donors, 16 HDM+AR patients and 1 mugwort+ AR patient using Ficoll-Hypaque density gradient centrifugation according to the standard protocol (Lymphoprep™, Nycomed Pharma, Oslo, Norway). Cells were plated at a density of 1 × 106 cells/well in a 24-well plate in 0.5 mL RPMI 1640 (Gibco, USA) culture medium containing either HDM (Der p1 extract; 0.2, 1, 5 μg/mL; GREER Laboratories, Lenoir, NC, USA), mugwort (1, 10, 100, 1000 ng/mL; A. sieversian locally prepared in Beijing Tongren Hospital), or synthetic peptides of mugwort tropomyosin, and then incubated at 37 °C in 5% CO2 for 48 h. Cells incubated with RPMI 1640 medium alone were used as controls. After incubation, the cell suspensions were collected and the supernatants were assessed for IL-5, IL-17, and IFN-γ using Luminex xMAP suspension array technology in a Bio-Plex 200 system (Bio-Rad, MI). All cytokine kits were purchased from R&D Company and the results were expressed as pg/mL.
Statistical analysis
Statistical analysis was performed using the SPSS version 22.0 software package (IBMCorp, Armonk, NY, USA). Categorical variables were described using frequencies and/or percentages and continuous variables were presented as mean ± standard deviation (SD). Multiple logistic regression was used to analyse the possible risk factors for polysensitized HDM+AR patients. The influence of polysensitization on asthma development was assessed by the Chi squared test. The prevalence of different allergens in HDM+AR patients was estimated using Fisher’s exact test and logistic regression. The Wilcoxon test was used for paired comparisons of the effect of specific antigen stimulation on the release of cytokines from PBMCs, and the effect of synthetic mugwort peptides on blocking HDM specific IgEs between groups. P values of less than 0.05 were regarded as statistically significant.