Additional methodological details are available in the online supplement.
Information on 17 subjects with AR enrolled in this study and the exclusion criteria are described in the online data supplement. Participation was voluntary, and written informed consent was obtained from all subjects. The Institutional Review Board of Seoul National University College of Medicine (No. 1709-049-883) and Gyeongsang National University Hospital (No. 2019-05-004) approved the protocol for this study.
Mucus and/or nasal mucosa from the middle turbinate of human subjects was collected and assessed for quality as described in the online data supplement (supplementary video file).
The characterization of staphylococcus species in human allergic nasal mucosa
To isolate bacterial colonies, the mucus was placed in lysogeny broth (LB) plates. After 1 day of incubation, bacterial colonies were obtained from LB plates. The species of each colony was identified using GS-FLX 454 pyrosequencing with 16S rRNA gene amplification, as described previously . Staphylococcus epidermidis and S. aureus strains from five individual subjects with AR were used in the study.
Normal human nasal epithelial (NHNE) and allergic rhinitis nasal epithelial (ARNE) cells were each cultured from five subjects using an air-liquid interface method. Cells were used 14 days after creation of the air-liquid interface (ALI). Isolated AR-SA and AR-SE were inoculated as described previously . Details are available in the online supplement.
Murine inoculation model
Experiments with four-week-old female wild type (WT) BALB/c mice (Orient, Gyeonggi, Republic of Korea) were carried out according to guidelines approved by the Institutional Animal Care and Use Committees of Seoul National University Hospital (No. 2016-1470). Microbiome depletion, AR-SA and AR-SE inoculation, nasal lavage (NAL) sample collection, and nasal tissue harvesting are described in the online supplement.
Mice were divided into four groups depending on S. aureus inoculation. The negative control group (WT) was sensitized and challenged with phosphate-buffered saline (PBS), and the positive control group (AR-OVA) was sensitized and challenged with ovalbumin (OVA). Both WT mice and AR-OVA mice were inoculated with human nasal microbiome S. aureus by intranasal delivery. The schematic figure for allergen sensitization, intranasal challenge, and S. aureus inoculation is shown in Fig. 4a. Mice were euthanized and sacrificed by cervical dislocation and by intramuscular injection of high dose of a mixture of 10mg/kg xylazine (Bayer, Puteaux, France) and 5mg/kg ketamine (Merial, Lyon, France) according to the reviewed protocol. Death was verified when no heartbeat was detected. There were no mice without euthanasia. When mice were euthanized by injection, a cervical dislocation was also performed to ensure that the mice were dead.
Serum levels of total and OVA-specific IgE
AR mouse serum samples were stored at -70℃ before use for measurements of total IgE and OVA specific IgE, as described previously . Details are available in the online supplement.
Mice heads were fixed in 10% formalin, decalcified, and embedded in paraffin wax. Nasal tissues were sectioned and stained with hematoxylin and eosin (H&E) for inflammatory cell counting, Sirius red stain for eosinophil counting, and periodic acid-Schiff (PAS) stain to indicate secretory cells of the nasal epithelium.
Bacterial samples were serially diluted ten-fold with phosphate-buffered saline (PBS). Next, 10 μl of each diluted sample was plated on an LB agar plate. The plates were incubated for 24 h at 37°C. The numbers of AR-SA and AR-SE colonies growing on the agar surface were counted manually. Bacterial growth was reported based on the colony-forming units (CFUs) for each sample.
Real-time PCR and RNA preparation
Levels of transcripts encoding human IL-33 and TSLP, mouse IL-4, IL-5, IL-13, and IL-33, or femAs specific for S. aureus (femA-SA) and S. epidermidis (femA-SE) were determined using real-time PCR (RT-PCR), as described in the online supplement.
Protein isolation and Western blot
IL-33 levels were assessed by western blotting using human IL-33 antibody (AF3625, R&D Systems, Minneapolis, MN, USA). Details are available in the online supplement.
Quantification of secreted cytokines
Secreted mouse IL-33 (DY3626) was quantified using the DuoSet® ELISA kit from R&D Systems according to the manufacturer’s instructions. To quantify IL-33, nasal lavage (NAL) fluid of a murine model was collected. The working range of the assay was 15.6-1000 pg/mL.
At least three independent experiments were performed with cultured cells from each donor, and data are expressed as mean ± standard deviation (SD) of three independent experiments. Groups were compared by the Mann-Whitney U test, and differences among treatment groups were evaluated by analysis of variance (ANOVA) with a post hoc test. A P-value of < 0.05 was considered statistically significant. All statistical analyses were performed with GraphPad Prism software (version 5; GraphPad Software, Inc., La Jolla, CA, USA).